Changes of enzyme activity and gene expression in embryonic zebrafish co-exposed to beta-cypermethrin and thiacloprid

Journal Pre-proof Changes of enzyme activity and gene expression in embryonic zebrafish co-exposed to beta-cypermethrin and thiacloprid Yanhua Wang, Xinfang Li, Guiling Yang, Hongbiao Weng, Xinquan Wang, Qiang Wang PII:

S0269-7491(19)33032-5

DOI:

https://doi.org/10.1016/j.envpol.2019.113437

Reference:

ENPO 113437

To appear in:

Environmental Pollution

Received Date: 9 June 2019 Revised Date:

18 October 2019

Accepted Date: 18 October 2019

Please cite this article as: Wang, Y., Li, X., Yang, G., Weng, H., Wang, X., Wang, Q., Changes of enzyme activity and gene expression in embryonic zebrafish co-exposed to beta-cypermethrin and thiacloprid, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113437. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Graphical abstract

Synergism (1+1 > 2)

1

Changes of enzyme activity and gene expression in embryonic

2

zebrafish co-exposed to beta-cypermethrin and thiacloprid

3 4

Yanhua Wang, Xinfang Li, Guiling Yang, Hongbiao Weng, Xinquan Wang, Qiang

5

Wang*

6

7

State Key Laboratory for Quality and Safety of Agro-products / Key Laboratory for

8

Pesticide Residue Detection of Ministry of Agriculture / Laboratory (Hangzhou) for

9

Risk Assessment of Agricultural Products of Ministry of Agriculture, Institute of

10

Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences,

11

Hangzhou 310021, Zhejiang, China

12 13 14

*

15

Agro-products, Institute of Quality and Standard for Agro-products, Zhejiang

16

Academy of Agricultural Sciences, Hangzhou 310021, Zhejiang, China; E-mail:

17

[email protected] (Q Wang)

Correspondence author. Address: State Key Laboratory for Quality and Safety of

18 19 20 21 22

1

23

Abstract

24

Pesticides often occur as mixtures of complex compounds in water environments,

25

while most of studies only focus on the toxic effects of individual pesticides with little

26

attention to the joint toxic effects. In the present study, we aimed to the mixture

27

toxicity of beta-cypermethrin (BCY) and thiacloprid (THI) to zebrafish (Danio rerio)

28

employing multiple toxicological endpoints. Results displayed that the 96-h LC50

29

values of BCY to D. rerio at various developmental stages ranged from 2.64×10

30

(1.97×10~3.37×10) to 6.03×103(4.54×103~1.05×104) nM, which were lower than

31

those

32

(2.19×105~5.87×105) nM. Mixtures of BCY and THI exhibited synergistic response in

33

embryonic zebrafish. Meanwhile, the enzyme activities of antioxidants (CAT and

34

SOD) and detoxification enzyme (CarE), endogenous T-GSH and MDA contents, as

35

well as gene expressions (tsh, crh, cxcl and bax) involved in oxidative stress, cellular

36

apoptosis, immune system and endocrine system were obviously changed in the

37

mixture exposure compared with the respective BCY or THI treatment. Consequently,

38

the increased toxicity of pesticide mixture suggested that the toxicological data

39

acquired from individual pesticide tests might underrate the toxicity risk of pesticides

40

that actually arise in the real environment. Taken together, our present study provided

41

evidence that mixture exposure of BCY and THI could induce additional toxic effect

42

compared with their respective individual pesticides on D. rerio, offering valuable

43

insights into the toxic mechanism of pesticide mixture.

of

THI

ranging

from

2.97×104

44 2

(1.96×104~4.25×104)

to

2.86×105

45

Capsule: Synergistic effects and underlying mechanism of beta-cypermethrin and

46

thiacloprid on zebrafish.

47 48

Keywords: Mixture toxicity; Danio rerio; Synergistic effect; Beta-cypermethrin;

49

Thiacloprid

50

3

51

1. Introduction

52

Different pesticides are frequently co-applied for their high efficiency,

53

convenience and fast actions, and such applications are becoming a pyramidal trend in

54

modern agriculture (Hernández et al., 2017). However, the practice makes them likely

55

to coexist in the aquatic ecosystem through drift or run-off from agricultural fields

56

(Schreiner et al., 2016; Dupraz et al., 2019). Since the pesticides often occur as

57

mixtures of complex chemicals in water environments, extra effects may be induced

58

on aquatic organisms compared with single substances (Sanches et al., 2017). It is

59

now generally accepted that environmental chemical toxicity results from exposure to

60

compounds in mixtures instead of individual compounds (Belden and Brain, 2018).

61

Therefore, it is crucially important to include the joint effects when determining the

62

ecological risk of pesticides on water ecosystem (Levine and Borgert, 2018).

63

Fish play vital functions in aquatic food chain, and become sentinels for the

64

quality of waters that serve as sources of drinking water for humans (Faggio et al.,

65

2014; Shukla et al., 2017; Burgos-Aceves et al., 2018a, b, 2019; Hong et al., 2018).

66

Consequently, a fish bioassay is an ordinary approach to assess the side effects of

67

pesticides on aquatic environment (Mu et al., 2016). Zebrafish (Danio rerio) is rapidly

68

turning into important model fish species in toxicological evaluation due to its

69

inherent characteristics, such as low cost, short reproductive cycle and synchronously

70

developing embryos and so on (Crosby et al., 2015; Icoglu and Ciltas, 2018).

71

Although massive ecotoxicological assays employing zebrafish have been performed 4

72

in the past decade, most of them have focused on effects of individual pesticides (Ge

73

et al., 2015; Mu et al., 2016; Maharajan et al., 2018). Nevertheless, the combined

74

effects of pesticide mixtures on zebrafish are still poorly unexplored (Rizzati et al.,

75

2016; Levine and Borgert, 2018).

76

The pyrethroid insecticide beta-cypermethrin (BCY) and neonicotinoid

77

insecticide thiacloprid (THI) are widely used in both rural and urban areas worldwide

78

(Regan et al., 2017; Zanuzo et al., 2017). Moreover, the two pesticides are usually

79

applied together as tank mixes, leading to their coexistence in the same environmental

80

sample (Wei et al., 2017; Belden and Brain, 2018). Up to now, little knowledge is

81

available about the mechanistic basis responsible for the joint effects on D. rerio

82

between them (Osterauer and Köhler, 2008). The environmental concentrations range

83

from 3.08×10-3 to 2.36×10 nM for BCY, and from 5.03×10-3 to 7.69×102 nM for THIy

84

(Grung et al., 2015; Schreiner et al., 2016; Li et al., 2018b). Therefore, we aimed to

85

examine the potential threats of these two pesticides to the zebrafish, with special

86

attention to the lethal toxicity, enzymatic activity and gene transcription. Such

87

systematic test provided a foundation for further studies on toxicological mechanism

88

of pesticide mixtures to aquatic organisms.

89 90

2. Materials and methods

91

2.1. Chemicals and reagents

92

BCY (purity of 97%) was donated by Jiangsu Yangnong Chemical Group Co.,

93

Ltd. (Yangzhou, Jiangsu, China). THI (purity of 98%) was obtained from Rudong 5

94

Zhongyi Chemical Industrial Group (Nantong, Jiangsu, China).

95

Stock solutions of pesticides (1.20×108 nM for BCY and 2.40×108 nM for THI)

96

were prepared with dimethyl sulfoxide and Tween-80 and then preserved at 4°C

97

before further analyses. All stock solutions were further diluted to requested

98

concentrations utilizing standard water containing 2 mmol L-1 Ca2+, 0.5 mmol L-1

99

Mg2+, 0.75 mmol L-1 Na2+ and 0.074 mmol L-1 K+ (ISO, 1996).

100 101

2.2. Zebrafish husbandry and egg collection Adult zebrafish (AB strain) were employed as breeding stocks. The stocks were

102

maintained in a flow-through system (26 ± 1

, 12-h light:12-h dark), fed ad libitum

103

twice a day with a commercial fish diet Tetramin (Tetra, Melle, Germany), and

104

replenished once a day with live brine shrimp (Artemia spp.) (Binzhou Haifa

105

Biological Technology Co., Ltd., Shandong, China). For reproduction, female and

106

male fish were transferred to spawning boxes with a ratio of 1:2 overnight. Spawning

107

was prompted in the following morning when the light was turned on and completed

108

within 30 min. All rearing and treatment protocols were conducted according to the

109

ethical guidelines of Zhejiang Academy of Agricultural Sciences for the care and use

110

of laboratory animals.

111

2.3. Individual pesticide toxicity assays

112

Acute toxicity assays of individual pesticide to zebrafish at multiple life stages

113

(embryonic, larval, juvenile and adult stages) were conducted in accordance with

114

OECD guidelines 203 and 236 (OECD, 1992, 2013). The exposure solutions were

115

renewed every 12 h in order to keep the suitable concentration of pesticide and water 6

116

quality. The external conditions (temperature and light cycle) were maintained the

117

same as the rear surroundings during exposure period. Details in test procedure of

118

pesticides to the animals at different life stages were provided in the supplemental

119

information.

120

2.4. Mixture toxicity assay

121

Mixture toxicity of pesticides was investigated with embryonic zebrafish. The

122

toxicities of individual pesticides were directly compared with their mixtures. To

123

examine the mixture toxicity of BCY and THI, embryonic zebrafish were exposed to

124

serial dilutions of each pesticide with a fixed constant equitoxic ratio based on the

125

determined individual LC50 values. The total concentration of each mixture was

126

systematically varied, while all the above-mentioned ratios remained unchanged for

127

building the relationship of concentration-response. All measurements were carried

128

out in triplicate for each concentration.

129

2.5. Biochemical and molecular assays

130

2.5.1. Sampling

131

Embryos at about 2 hpf were arbitrarily shifted into 500-mL beakers containing

132

test solutions. The contents were selected according to the results of embryo acute

133

toxicity. Concentrations of 1/320, 1/80 and 1/20 of 96-h LC50 for each pesticide were

134

set as the low, middle and high contents, respectively. Correspondingly, low, middle

135

and high contents in the joint treatment of BCY+THI (JOI) were mixtures of both

136

BCY and THI at the low, middle and high contents, respectively. Each beaker

137

consisted of 500 mL test solution and 250 embryos, and three beakers were set up for 7

138

each concentration. Pesticide solution was refreshed every 12 h. After treatment for 96

139

h, hatched larvae (150 for antioxidant index determination; and 40 for RNA extraction)

140

from each treatment were gathered and rinsed twice with reconstituted water. The

141

collected larvae were reserved at -80°C until further tests.

142

2.5.2. Biochemical assays

143

About 150 larvae from each beaker were homogenized (1:20, w/v) using an

144

electric homogenizer in 50 mM potassium phosphate buffer (pH 7.0) consisting of 0.5

145

mM EDTA. The homogenate was centrifuged at 12,000 rpm for 30 min at 4 , and the

146

supernatant was collected for the analysis of biochemical parameters.

147

The contents of oxidative stress malonaldehyde (MDA), total glutathione

148

(T-GSH), oxidized glutathione (GSSG) and reactive oxygen species (ROS), as well as

149

the activities of antioxidant enzymes [catalase (CAT), total superoxide dismutase

150

(T-SOD), Cu/Zn superoxide dismutase (Cu/Zn-SOD) and peroxidase (POD)],

151

apoptotic enzymes [caspase 3 and caspase 9] and detoxification enzymes

152

[glutathione-S-transferase (GST), carboxylesterase (CarE) and cytochrome P450

153

(CYP450)] were detected employing commercially available kits (Nanjing Jiancheng

154

Bioengineering Institute, Nanjing, China) as previously described (Wang et al., 2018).

155

Protein determinations were conducted by the Bradford method using bovine serum

156

albumin (BSA) as a standard (Bradford, 1976). Additionally, the levels of vitellogenin

157

(VTG) and thyroid hormones (THs), such as triiodothyronine (T3), were determined

158

using

159

manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing,

enzyme-linked

immunosorbent

quantification

8

kits

according

to

the

160

China).

161

2.5.3. Gene expression analysis

162

Gene expressions at the mRNA level were determined by quantitative real-time

163

PCR (qRT-PCR) as previously described (Wang et al., 2018). Total RNA was isolated

164

from samples using RNAiso Plus (TaKaRa, Dalian, China) in accordance to the

165

manufacturer’s protocols. Besides, β-actin gene was chosen as the housekeeping gene.

166

The primer sequences were provided in Table S1. The expression levels of target

167

genes were determined with the 2-△△Ct method (Livak and Schmittgen, 2001).

168

2.6. Statistical analysis

169

A probit analysis was performed to evaluate the acute toxicity of pesticides to D.

170

rerio using a program developed by Chi (Chi, 1997). The significant level of mean

171

separation (P < 0.05) was tested according to the lack of overlap between the 95 %

172

confidence interval of two LC50 values. The interaction pattern of pesticide mixtures

173

was judged on the basis of an additive index method (Su et al., 2016). The one-way

174

ANOVA statistical discrepancies were assessed by a Dunnett's post-hoc test

175

performed with SPSS software (SPSS version 18.0, USA).

176 177

3. Results

178

3.1. Individual pesticide toxicity assays

179

Acute toxicity data of BCY and THI to D. rerio at different life stages were

180

presented in Table 1. Results displayed that different pesticides varied widely in their

181

toxic selectivity to zebrafish, and each pesticide elicited distinct toxicities to different 9

182

life stages of D. rerio. Between these tested insecticides, BCY exhibited a greater

183

toxicity to various life stages of zebrafish with 96-h LC50 values ranging from

184

2.64×10 (1.97×10~3.37×10) to 6.03×103(4.54×103~1.05×104) nM. In contrast, THI

185

displayed a lesser toxicity to the animals with 96-h LC50 values ranging from

186

2.97×104 (1.96×104~4.25×104) to 2.86×105 (2.19×105~5.87×105) nM.

187

Based on the 96-h LC50 values, the descending toxicity order of two evaluated

188

pesticides for multiple life stages of zebrafish was ranked as follows: adult fish >

189

juvenile fish > larval fish > embryos for BCY; adult fish > juvenile, larval fish >

190

embryos for THI. Overall, zebrafish embryos were the most tolerant to the tested

191

pesticides, while adults were the most sensitive.

192

3.2. Mixture toxicity assays

193

To explicit the mixture toxicity of BCY and THI towards embryos of D. rerio, the

194

LC50 values of their mixtures after treatment for 96 h were examined (Table 2). The

195

mixture of BCY and THI showed synergistic responses with an AI value of 1.49, 2.43,

196

2.84 and 3.05 after treatment for 24, 48, 72 and 96 h, respectively. Furthermore, the

197

AI value for the mixture was increased with the extension of treatment time, implying

198

that the mixture toxicity was positively correlated with treatment period.

199

3.3. Analysis of biochemical parameters

200

3.3.1. Oxidative stress determination

201

MDA content was significantly increased in the BCY and THI treatments (with

202

the exception of the low content of BCY and high content of THI treatments)

203

compared with the control. Besides, significant increase was also found in the low 10

204

content of JOI treatment compared with the control and the corresponding BCY or

205

THI treatment (Fig. 1A). CAT activity was distinctly induced in the BCY and JOI

206

treatments (with the exception of the middle content of JOI treatment) compared with

207

the control. In contrast, distinct inhibition was discovered in the JOI treatment

208

compared with the corresponding BCY treatment (Fig. 1B). T-SOD activity was

209

obviously enhanced in the BCY treatment compared with the control. Additionally,

210

obvious enhancement was also detected in the low content of THI and JOI treatments

211

compared with the control. Nevertheless, its activity was obviously weakened in the

212

low content of JOI treatment compared with the corresponding THI treatment (Fig.

213

1C).

214

Cu/Zn-SOD activity was markedly up-regulated in response to the high content of

215

BCY and THI treatments compared with the control. However, marked

216

down-regulation was monitored in the low content of JOI treatment compared with

217

the control and the corresponding BCY or THI treatment. Additionally, its activity

218

was also markedly down-regulated in the high content of JOI treatment compared

219

with the corresponding THI treatment (Fig. 1D). T-GSH level was noticeably

220

evaluated in all the contents of BCY, THI and JOI treatments (with the exception of

221

the low content of BCY and THI treatments) compared with the control. Furthermore,

222

noticeable elevation was found in the low content of JOI treatment compared with the

223

corresponding BCY or THI treatment. On the contrary, its level was noticeably

224

diminished in the middle and high contents of JOI treatment compared with the

225

corresponding BCY treatment (Fig. 1E). GSSG content was not significantly changed 11

226

in the JOI treatment compared with the corresponding BCY or THI treatment (Fig.

227

1F).

228

POD activity was distinctly increased in the BCY, THI and JOI treatments (with

229

the exception of the high content of BCY and THI treatments) compared with the

230

control. Besides, its activity was also distinctly increased in the high content of JOI

231

treatment compared with the corresponding THI treatment. However, distinct

232

decrease was observed in the middle content of JOI treatment compared with the

233

corresponding BCY treatment (Fig. 1G). ROS level was pronouncedly induced in the

234

BCY, THI and JOI treatments (with the exception of the high content of THI

235

treatment) compared with the control. Additionally, pronounced induction was also

236

detected in the high content of JOI treatment compared with the corresponding THI

237

treatment. In contrast, its level was pronouncedly inhibited in the low content of JOI

238

treatment compared with the corresponding BCY or THI treatment (Fig. 1H).

239

3.3.2. Apoptotic enzyme activities

240

Caspase3 activity was significantly inhibited in the BCY treatment compared with

241

the control and the corresponding JOI treatment. Conversely, significant induction

242

was found in the middle content of THI treatment compared with the control and the

243

corresponding JOI treatment (Fig. 2A). Similar to Caspase3 activity, Caspase9

244

activity was also obviously weakened in the BCY treatment compared with the

245

control and the corresponding JOI treatment (with the exception of the low content of

246

JOI treatment). Besides, obvious weakening was also monitored in the middle content

247

of JOI treatment compared with the corresponding THI treatment (Fig. 2B). 12

248

3.3.3. Detoxification enzyme activities

249

CYP450 activity was markedly down-regulated in the high content of BCY

250

treatment compared with the control. Moreover, marked down-regulation was also

251

found in the middle content of THI treatment compared with the control and the

252

corresponding JOI treatment (Fig. 3A). CarE activity was noticeably elevated in most

253

of the BCY and THI treatments (with the exception of the middle content of BCY and

254

the low content of THI treatments) compared with the control. However, JOI

255

treatment noticeably diminished CarE activity at high content compared with the

256

corresponding BCY or THI treatment (Fig. 3B). GST activity was not obviously

257

different in the BCY, THI and JOI treatments compared with to the control (Fig. 3C).

258

3.3.4. T3 level and VTG content

259

T3 level and VTG content were not significantly changed in all the contents of

260

BCY, THI and JOI treatments compared with the control. However, JOI treatment

261

significantly increased T3 level at the low content compared with the corresponding

262

THI treatment (Fig. 4). VTG content was significantly increased in the high content of

263

JOI treatment compared with the corresponding BCY treatment (Fig. S1).

264

3.4. Analysis of gene expression

265

3.4.1. Effect on the expressions of genes involved in endocrine system

266

The expression of TRα was distinctly inhibited in the high content of BCY

267

treatment compared with the control. Conversely, distinct induction was detected in

268

the middle content of JOI treatment compared with the control and the corresponding

269

BCY or THI treatment (Fig. 5A). The expression of TRβ was markedly diminished in 13

270

the low and high contents of BCY treatment compared with the control. However,

271

marked elevation was monitored in the high content of JOI treatment compared with

272

the corresponding BCY treatment (Fig. 5B). The expression of tsh was significantly

273

weakened in the low content of BCY treatment compared with the control. However,

274

its expression was significantly enhanced in all the contents of THI treatment

275

compared with the control. Significant enhancement of tsh expression was also found

276

in the low and middle contents of JOI treatment compared with the corresponding

277

BCY treatment (Fig. 5C).

278

The ERα expression in the low and high contents of BCY treatment was markedly

279

down-regulated compared with the control and the corresponding JOI treatment. In

280

contrast, marked up-regulation was observed in the middle content of BCY treatment

281

compared with the control and the corresponding JOI treatment (Fig. 5D). The

282

expression of cyp19a in all the contents of THI treatment was noticeably elevated

283

compared with the control and the corresponding JOI treatment. Besides, noticeable

284

elevation of its expression was also detected in the low content of JOI treatment

285

compared with the control (Fig. 5E). The expression of crh was significantly induced

286

in the middle content of THI treatment, and the middle and high contents of JOI

287

treatment compared with the control. Significant induction was also monitored in the

288

high content of JOI treatment compared with the corresponding BCY or THI

289

treatment (Fig. 5F).

290

3.4.2. Effects on the expressions of genes involved in immunosuppression and

291

anti-oxidative stress 14

292

The expression of Tnf was markedly induced in all the contents of THI treatment

293

compared with the control. Moreover, marked induction was also found in the low

294

and high contents of JOI treatment compared with the control and the corresponding

295

BCY treatment (Fig. 6A). The expression of cxcl was apparently increased at the

296

middle and high contents of JOI treatment compared with the control. Besides,

297

significant increase was also observed in the JOI treatment compared with the

298

corresponding BCY or THI treatment (Fig. 6B). The expression of IL was

299

pronouncedly weakened in the low and middle contents of BCY and the middle

300

content of JOI treatment compared with the control (Fig. 6C).

301

The expression of cat was markedly induced in the low content of BCY treatment

302

compared with the control. Conversely, its expression was markedly inhibited in the

303

high content of BCY and JOI treatments compared with the control. Additionally,

304

marked down-regulation of cat expression was also monitored in the low content of

305

JOI treatment compared with the corresponding BCY treatment (Fig. 6D). Similar to

306

cat, the expression of Cu/Zn-sod was noticeably elevated in the low content of BCY

307

treatment compared with the control. In contrast, noticeable diminishment of

308

Cu/Zn-sod expression was found in the high content of BCY treatment compared with

309

the control. Its expression was also noticeably diminished in the high content of JOI

310

treatment compared with the control and the corresponding THI treatment. Besides,

311

the expression of Cu/Zn-sod was also noticeably diminished in the low and middle

312

contents of JOI treatment compared with the corresponding BCY treatment (Fig. 6E).

313

The expression of Mn-sod was significantly increased in the middle content of BCY 15

314

and JOI treatments compared with the control. Moreover, significant increase was

315

observed in the high content of JOI treatment compared with the corresponding BCY

316

treatment. In contrast, its expression was significantly decreased in the high content of

317

BCY treatment compared with the control (Fig. 6F).

318

3.4.3. Effects on the expressions of genes involved in cell apoptosis

319

The expression of bax was obviously weakened in the BCY and THI treatments

320

compared with the control. Obvious enhancement was also detected in all the contents

321

of JOI treatments compared with the corresponding BCY or THI treatment (Fig. 7A).

322

The P53 expression was distinctly elevated in the middle content of BCY treatment

323

compared with the control and the corresponding JOI treatment. In contrast, distinct

324

down-regulations were monitored in the low and high contents of THI treatment and

325

the high content of BCY treatment compared with the control (Fig. 7B).

326

The expression of cas8 was significantly diminished in the BCY treatment

327

compared with the control. Besides, significant elevation was also found in all the

328

contents of JOI treatment compared with the corresponding BCY treatment (Fig. 7C).

329

Similar to cxcl, significant increase of cas9 expression was also detected in the high

330

content of JOI treatment compared with the control, and the corresponding BCY or

331

THI treatment (Fig. 7D).

332 333

4. Discussion

334

Lethal toxicity tests are generally the first step in assessing the detrimental effect

335

of pesticides on aquatic organisms (Jia et al., 2018; Ni et al., 2019). Results from this 16

336

study exhibited that BCY had a greater toxicity to different life stages of zebrafish

337

compared with THI. Previous studies have indicated that the 96-h LC50 values of BCY

338

and THI to adult zebrafish are 1.88×10 and 4.74×104 nM, respectively, which is

339

consistent with our present study (Osterauer and Köhler, 2008; Wang et al., 2016).

340

BCY is highly toxic to fish due to high rates of gill absorption, slow hydrolytic

341

detoxification and hypersensitivity of the piscine nervous system (Zhang et al., 2018).

342

Neonicotinoid insecticides normally exhibit low toxicity to fish (Casida, 2018; Hladik

343

et al., 2018). Nevertheless, among neonicotinoids examined so far, THI has been

344

discovered to display a comparatively high toxicity to fish. Consequently, the usage of

345

BCY and THI poses potential risks to aquatic organisms. However, previous studies

346

on BCY and THI have mostly focused on their single toxicity, while their possible

347

mixture toxicity has been seldom investigated (Osterauer and Köhler, 2008; Wang et

348

al., 2016; Zhang et al., 2018).

349

As complex mixtures of pesticides are often found in aquatic environment, the

350

ecological risk of pesticide in practical water samples cannot be precisely predicted by

351

estimates on the basis of individual compounds. The understanding of mixture effects

352

between pesticides is very important for the restriction of using defined mixture with

353

negative effects. A strong synergistic response was elicited by mixture of BCY and

354

THI on the embryonic zebrafish in a time-dependent mode, implying a greater

355

toxicity of mixture compared with their individual pesticides (Belden and Brain,

356

2018). Similar results using the additive index method have demonstrated that both

357

triazophos in combination with imidacloprid and cyprodinil in combination with 17

358

kresoxim-methyl exhibit synergistic response on D. rerio (Wang et al., 2018; Wu et al.,

359

2018). Since most compounds are assumed to have additive toxicity, the synergistic

360

response of pesticide mixtures can generate severe side-effects on fish population,

361

threatening the normal function of aquatic ecosystems. Our results indicated that it

362

was urgently necessary to explore the mixture toxicity of pesticides to zebrafish

363

because the risk assessment of pesticides towards aquatic organisms is usually

364

conducted only on individual pesticides, which might lead to underestimated toxicity

365

under realistic conditions (Lanteigne et al., 2015; Rizzati et al., 2016).

366

Because of increasing ethical attentions worldwide, numerous studies have

367

shown that toxicity assay on zebrafish embryo can be an accepted alternative for adult

368

toxicity test in the risk assessment of chemicals (Glaberman et al., 2017; Icoglu and

369

ciltas, 2018). Moreover, various practical benefits, such as relatively easy

370

maintenance, the transparent embryos and full development to most organ systems

371

within 96 hpf, make them a perfect vertebrate model system (Belanger et al., 2013).

372

With the ubiquitous co-occurrence of pesticides in aquatic ecosystem, it is very

373

important to understand the detoxification mechanism of pesticide mixtures by

374

aquatic organisms (Chen et al., 2016). CYP450 is one of important detoxification

375

enzymes in many organisms (Yang et al., 2016). Results from this study showed that

376

diminished CYP450 activity could be the toxic mechanism of BCY and THI to D.

377

rerio. On the contrary, we discovered that the CYP450 activity was markedly elevated

378

in the middle content of JOI treatment compared with the respective THI treatment,

379

which might lead to the active metabolism of THI in zebrafish embryos. 18

380

CarE is frequently implicated in the detoxification to pesticides mainly through

381

up-regulation (Wheeloch et al., 2005). The present results exhibited that CarE played

382

the detoxification role in response to treatments of BCY, THI and their mixture in

383

zebrafish embryos. However, its activity was pronouncedly diminished in the high

384

content of JOI treatment compared with the respective BCY or THI treatment, which

385

might result in the increased mixture toxicity. Therefore, the enhanced CYP450 and

386

decreased CarE activities were primarily attributed to the synergistic effects of BCY

387

and THI on D. rerio.

388

SOD catalyzes superoxide anion radical into H2O2, in which CAT is responsible

389

for the subsequent degradation of H2O2, creating not-toxic H2O (Liu et al., 2013;

390

Faggio et al., 2016; Gobi et al., 2018; Freitas et al.., 2019). The present study

391

exhibited that an antioxidant status was elevated to neutralize oxidative stress.

392

However, significant reduction of Cu/Zn-SOD activity was observed in the low

393

content of JOI treatment compared with the control group, which was probably

394

attributed to the excessive production of free radicals (Ni et al., 2019). T-GSH and

395

GSSG are direct oxyradical scavengers, and they play decisive roles in the cellular

396

regulation of detoxification (Paravani et al., 2019). Our results implied that the T-GSH

397

biosynthesis was enhanced to preserve the zebrafish embryos from oxidative stress.

398

The level of MDA is usually used to measure lipid peroxidation, which has been

399

known as a major contributor to the damage of cell function under oxidative stress

400

(Ge et al., 2015; Burgos-Aceves et al., 2018a). The current study suggested that BCY, 19

401

THI and JOI treatments could produce severe oxidative stress (Ni et al., 2019).

402

significant inductions of ROS were observed in the BCY, THI and JOI treatments

403

(with the exception of the high content of THI treatment) compared with the control

404

group, which might be attributed to that the antioxidant systems in zebrafish embryos

405

could not thoroughly remove excess ROS from the body. Therefore, the dynamic

406

equilibrium between ROS level and the antioxidant defense system was undermined,

407

ultimately generating oxidative stress (Wang et al., 2019). Based on the

408

above-mentioned results, we deduced that BCY, THI and JOI treatments elevated the

409

ROS production in zebrafish embryos and afterward activated antioxidant defense,

410

while antioxidant responses could not entirely clear up the excess ROS, leading to

411

oxidative impairment.

412

Examining the expressions of antioxidative genes can be beneficial in assessing

413

anti-oxidant ability (Liu et al., 2013). Pronounced up-regulation of cat and Cu/Zn-sod

414

in the low content of BCY treatment as well as Mn-sod in the middle content of BCY

415

and JOI treatments was observed compared with the control group. On the contrary,

416

we found that the cat and Cu/Zn-sod expressions in the high content of both BCY and

417

JOI treatments as well as the Mn-sod expression in the high content of BCY treatment

418

were significantly down-regulated compared with the control group. More

419

interestingly, no distinct changes in cat, Cu/Zn-sod and Mn-sod expressions were

420

detected in the THI treatment compared with the control group. The mispairing

421

between activities of anti-oxidant enzymes and the transcriptional levels of their

422

coding genes might also be attributed to the existence of multiple gene copies in 20

423

zebrafish, a time-lag effect between transcription and translation, and post-translation

424

modifications (Li et al., 2018a).

425

Apoptosis is related to development and growth of aquatic organisms, and

426

pathogenesis in response to stimuli in different systems (Wang et al., 2019). This

427

study proved that exposure to BCY significantly diminished caspase 3 and caspase 9

428

activities and the expressions of bax and cas8 compared with the control group.

429

Nevertheless, the bax and P53 expressions were distinctly inhibited in most of the

430

THI treatments. It was noteworthy that no significant changes of caspase 3 and

431

caspase 9 activities were detected in JOI treatment compared with the control group.

432

On the other hand, we found that the bax expression in the middle content of JOI

433

treatment, the cas8 expression in the low and high contents of JOI treatments and the

434

cas9 expression in the high content of JOI treatment were also pronouncedly elevated

435

compared with the control group. Oxidative stress may produce injury to cellular

436

constituents, and accordingly lead to apoptosis (Yang et al., 2018). We deduced that

437

JOI treatment-prompted oxidative stress in zebrafish embryos might underlie the

438

mechanism of its apoptotic effect (Wu et al., 2018).

439

Oxidative stress can also change immune competence and thus has been

440

considered as a mechanism for pesticide-induced immunotoxicity (Wang et al., 2019).

441

These present results implied that the JOI treatment of BCY and THI caused a

442

stronger inflammatory reaction compared with their individual compound treatments.

443

In contrast, the expression of IL was significantly inhibited in the low and middle

444

contents of BCY treatments and the middle content of JOI treatment compared with 21

445

their respective control groups. These variations exhibited that the defense of immune

446

system was assaulted when the embryonic zebrafish were exposed to BCY, THI and

447

their mixture.

448

Extensive studies have suggested that some pesticides can generate harmful

449

effects on the development of aquatic vertebrates by disturbing their endocrine system

450

(Burgos-Aceves et al., 2016; Zhang et al., 2017). The expression modes of genes

451

related to the hypothalamic-pituitary-gonadal/thyroid (HPG/HPT) axis were also

452

examined to reveal the potential mechanism of endocrine disruption caused by

453

exposure to BCY, THI and their mixtures (Walter et al., 2019). THs controlled by

454

HPT axis play vital functions in the adjustment of development and growth in fish

455

(Wu et al., 2018). Results from this study showed that the TRɑ and tsh expressions

456

were significantly elevated in the middle content of JOI and THI treatments,

457

respectively, while such treatments had no effects on the T3 level. Moreover, distinct

458

changes were discovered in the TRɑ, TRβ and tsh expressions after exposure to BCY

459

and JOI compared with the control group and the respective individual pesticides,

460

respectively. The variation of mRNA expressions in the HPT axis implied that BCY

461

and THI had a potential to cause thyroid disruption, whereas the JOI treatment elicited

462

more serious ventures compared with their individual pesticides (Wang et al., 2018).

463

In oviparous vertebrates, the female-specific yolk protein precursors VTGs act to

464

transport nutrients into oocytes during the maturation of oocytes, and the synthesis of

465

VTG is modulated through 17β-estradiol activation of estrogen receptors (ERs)

466

(Yilmaz et al., 2018). HPG axis modulates sex hormones, which are closely correlated 22

467

to reproduction of fish (Cao et al., 2019). The ERɑ expression was diminished in the

468

low and high contents of BCY treatments compared with the control group, indicating

469

that a potential estrogenic effect was prompted by BCY (Bertotto et al., 2019). In

470

contrast, significant variations were also observed in the expressions of ERɑ, cyp19a

471

and crh in the JOI treatment compared with the respective BCY or THI treatment,

472

indicating that BCY, THI and JOI treatments could result in impaired reproduction of

473

zebrafish.

474

Organisms are equipped with interdependent cascades of enzymes, which can

475

alleviate oxidative stress and repair damaged macromolecules during normal

476

metabolism or by exposure to environmental toxicants (Ge et al., 2015; Chen et al.,

477

2016). Gene expression is involved in the initial stages of stress responses compared

478

with more traditional toxicological endpoints, and it is useful supplements to protein

479

examinations for evaluating the potential toxic effects and further mechanisms (Liu et

480

al., 2013; Li et al., 2018a; Wang et al., 2019). Both enzymatic activities and gene

481

response profiles in D. rerio showed different patterns for pesticide mixture compared

482

with the individual compounds, suggesting that the mode of action at the both

483

biochemical and molecular levels was quite different between pesticide mixture and

484

the individual chemicals.

485 486

5. Conclusions

487

BCY elicited greater toxicity than THI to different life stages of D. rerio. Mixtures

488

of BCY and THI showed synergistic response to zebrafish embryos. Significant 23

489

variations of CAT, SOD and CarE activities, as well as the expressions of four genes

490

(tsh, crh, cxcl and bax) were detected in JOI treatment compared with the respective

491

BCY or THI treatment. Findings from this study employing multiple endpoints

492

provided valuable insights into the overall joint toxic effects and their underlying

493

mechanism caused by BCY, THI and JOI treatment on zebrafish.

494

495

Acknowledgments

496

The authors acknowledge the technical assistance of Xing Wang and Jian Li

497

(Zhejiang Academy of Agricultural Sciences). The research was supported by the

498

National

499

2018YFC1603004), Zhejiang Provincial Natural Science Foundation (Grant No.

500

LY18C030004) and the Special Fund for Agro-scientific Research in the Public

501

Interest (Grant No. 201503107).

Key Research

and

Development

Program

of China

(Grant No.

502 503

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690

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33

691

Table 1 Acute toxicity of beta-cypermethrin and thiacloprid to various developmental stages of zebrafish. LC50 (95% CI) a nM

Pesticide

Beta-cypermethrin Thiacloprid 692

a

Embryo 3

3

Larvae 4

6.03×10 (4.54×10 ~1.05×10 ) 1.40×105(8.90×104~1.90×105)

2

Juvenile

2

2

2.89×10 (2.19×10 ~3.85×10 ) 2.86×105(2.19×105~5.87×105)

CI confidence interval

693 694 695 696 697 698 699 700 701 702

34

2

Adult 2

1.49×10 (9.13×10~1.97×10 ) 1.13×105(5.72×104~1.56×105)

2.64×10 (1.97×10~3.37×10) 2.97×104(1.96×104~4.25×104)

703

Table 2 Joint toxic effects of beta-cypermethrin and thiacloprid on the zebrafish embryos. LC50 (95% CI)a nM LC50 (95% CI)b nM Exposure time (h) Beta-cypermethrin Thiacloprid Beta-cypermethrin Thiacloprid 24 48 72 96

704 705

a

706

c

b

6.23×104(4.26×104~1.72×105) 4.23×104(3.15×104~6.66×104) 2.34×104(1.22×104~3.30×104) 6.03×103(4.54×103~1.05×104)

1.24×106(8.81×105~3.06×105) 8.50×105(6.47×105~1.28×106) 5.51×105(3.16×105~7.98×105) 1.40×105(8.90×104~1.90×105)

1.25×104(8.99×103~1.66×104) 6.18×103(4.23×103~1.04×104) 3.05×103(1.97×103~4.16×103) 7.45×102(4.33×102~1.39×103)

The LC50 (95% confidence interval) for Beta-cypermethrin or thiacloprid individually. The LC50 (95% confidence interval) for Beta-cypermethrin or thiacloprid in the mixture. AI additive index.

35

2.48×105(1.64×105~3.42×105) 1.24×105(7.62×104~1.83×105) 7.15×104(4.50×104~1.06×105) 1.73×104(9.05×103~2.61×104)

AIc value 1.49 2.43 2.84 3.05

707

Figure captions

708

Fig. 1. The oxidative responses in zebrafish embryos exposed to BCY, THI and their

709

mixtures. Each bar represents as the means ± standard deviation of three triplicates. *

710

p < 0.05, significant difference compared with the control; # p < 0.05, significant

711

difference compared with the BCY treatment group at corresponding concentration; ☆

712

p < 0.05, significant difference compared with the THI treatment group at

713

corresponding concentration. BCY = beta-cypermethrin; THI = thiacloprid. L = low

714

concentration of BCY and THI (1.88×10 and 4.38×102 nM); M = medium

715

concentration of BCY and THI (7.54×10 and 1.75×103 nM); H = high concentration

716

of BCY and THI (3.02×102 and 7.00×103 nM).

717

Fig. 2. The apoptotic enzyme activities in zebrafish embryos exposed to BCY, THI

718

and their mixtures. Each bar represents as the means ± standard deviation of three

719

triplicates. * p < 0.05, significant difference compared with the control; # p < 0.05,

720

significant difference compared with the BCY treatment group at corresponding

721

concentration; ☆ p < 0.05, significant difference compared with the THI treatment

722

group at corresponding concentration. BCY = beta-cypermethrin; THI = thiacloprid. L

723

= low concentration of BCY and THI (1.88×10 and 4.38×102 nM); M = medium

724

concentration of BCY and THI (7.54×10 and 1.75×103 nM); H = high concentration

725

of BCY and THI (3.02×102 and 7.00×103 nM).

726

Fig. 3. The detoxification enzyme activities in zebrafish embryos exposed to BCY,

727

THI and their mixtures. Each bar represents as the means ± standard deviation of

728

three triplicates. * p < 0.05, significant difference compared with the control; # p < 36

729

0.05, significant difference compared with the BCY treatment group at corresponding

730

concentration; ☆ p < 0.05, significant difference compared with the THI treatment

731

group at corresponding concentration. BCY = beta-cypermethrin; THI = thiacloprid. L

732

= low concentration of BCY and THI (1.88×10 and 4.38×102 nM); M = medium

733

concentration of BCY and THI (7.54×10 and 1.75×103 nM); H = high concentration

734

of BCY and THI (3.02×102 and 7.00×103 nM).

735

Fig. 4. T3 level in zebrafish embryos exposed to BCY, THI and their mixtures. Each

736

bar represents as the means ± standard deviation of three triplicates. * p < 0.05,

737

significant difference compared with the control; # p < 0.05, significant difference

738

compared with the BCY treatment group at corresponding concentration; ☆ p < 0.05,

739

significant difference compared with the THI treatment group at corresponding

740

concentration. BCY = beta-cypermethrin; THI = thiacloprid. L = low concentration of

741

BCY and THI (1.88×10 and 4.38×102 nM); M = medium concentration of BCY and

742

THI (7.54×10 and 1.75×103 nM); H = high concentration of BCY and THI (3.02×102

743

and 7.00×103 nM).

744

Fig. 5. Effects on expressions of genes involved in the endocrine system in zebrafish

745

embryos exposed to BCY, THI and their mixtures. Each bar represents as the means ±

746

standard deviation of three triplicates. * p < 0.05, significant difference compared

747

with the control; # p < 0.05, significant difference compared with the BCY treatment

748

group at corresponding concentration; ☆ p < 0.05, significant difference compared

749

with

750

beta-cypermethrin; THI = thiacloprid. L = low concentration of BCY and THI

the

THI treatment

group

at

corresponding

37

concentration.

BCY =

751

(1.88×10 and 4.38×102 nM); M = medium concentration of BCY and THI (7.54×10

752

and 1.75×103 nM); H = high concentration of BCY and THI (3.02×102 and 7.00×103

753

nM).

754

Fig. 6. Effects on expressions of genes involved in the immunology and anti-oxidative

755

systems in zebrafish embryos exposed to BCY, THI and their mixtures. Each bar

756

represents as the means ± standard deviation of three triplicates. * p < 0.05,

757

significant difference compared with the control; # p < 0.05, significant difference

758

compared with the BCY treatment group at corresponding concentration; ☆ p < 0.05,

759

significant difference compared with the THI treatment group at corresponding

760

concentration. BCY = beta-cypermethrin; THI = thiacloprid. L = low concentration of

761

BCY and THI (1.88×10 and 4.38×102 nM); M = medium concentration of BCY and

762

THI (7.54×10 and 1.75×103 nM); H = high concentration of BCY and THI (3.02×102

763

and 7.00×103 nM).

764

Fig. 7. Effects on expressions of genes related to cell apoptosis in zebrafish embryos

765

exposed to BCY, THI and their mixtures. Each bar represents as the means ± standard

766

deviation of three triplicates. * p < 0.05, significant difference compared with the

767

control; # p < 0.05, significant difference compared with the BCY treatment group at

768

corresponding concentration; ☆ p < 0.05, significant difference compared with the

769

THI treatment group at corresponding concentration. BCY = beta-cypermethrin; THI

770

= thiacloprid. L = low concentration of BCY and THI (1.88×10 and 4.38×102 nM); M

771

= medium concentration of BCY and THI (7.54×10 and 1.75×103 nM); H = high

772

concentration of BCY and THI (3.02×102 and 7.00×103 nM). 38

Fig. 1.

B *

#☆

*

*

*

1.0

*

2.4 * * *

*

1.8 #☆

* *

0.5

#

* # *

#☆#

0.6

0

0

80

60 C

D

48

60 ** *

40

*

*

*

36

☆

*

☆

#☆

24

*

20

12

0

0

75

18

60

E

F *

45

*

30

15 12 9

#☆

*

*

*

# #

* *

6

15

3 0

0 2.4 POD (U/mg prot)

1.2

280 G

H

1.8 1.2

210 *

*

**

* *#

* ☆

* *

* *

*

* #

*

140

*

0.6

70

0 BCY — THI —

#☆ *

☆

0 L M H —— —

— — — L M H

L M H L M H

— —

774

39

L M H —— —

— — — L M H

L M H BCY L M H TH I

Cu/Zn-SO D (U/mg prot)

A 1.5

CAT (U/ mg prot )

3.0

GSSG (µmol/ g prot)

2.0

RO S (percent o f contro l, %)

T-GSH (µmol /g prot)

T-SOD (U/mg prot)

MDA (nmol/mg prot)

773

Fig. 2.

Caspase3 (U/mg prot)

250 200

250

A

B *

150 100

*

*

200 #

#

#

#☆

#☆

*

*

100

**

50

50

0 B CY — THI —

150

0 L M H — — —

— — — L M H

L M H L M H

— —

776 777 778 779 780 781 782 783 784 785 786 787 788 789 790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 40

L M H — ——

— —— L M H

L M H B CY L M H THI

Caspase9 (U/mg prot)

775

Fig. 3. 1.2

3.0

A

B

0.9

*

0.6

*

*

*

1.8

*

0.3

#☆ 1.2

0 — —

100 GST (U /mg prot)

*

0.6

0

80

2.4

*

☆

C

60 40 20 0 BCY — THI —

L M H — ——

— — — L M H

L M H L M H

809

41

L M H — ——

—— — L M H

L M H B CY L M H THI

CarE (U/mg prot)

CY P4 50 (nM /mg/min )

808

810

Fig. 4.

5 .2 T3 (ng/ mg )

☆

3 .9 2 .6 1 .3 0

811

BC Y — THI —

L M H — — —

— — — L M H

812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835 836 837 838 839 840 42

L M H L M H

Fig. 5.

A: TRα α

B: TRβ β

#☆

2.0

*

# 1.5

1.8 #

1.2

1.0

*

*

0.6

*

0.5

0

0 4.0

Relative mRNA Leve l

3.5 2.8

D: ERα α

C: tsh

**

2.1

2.4

* # #☆

1.4 0.7

3.2

*

#

☆

*

*

#

#

1.6 0.8

* 0

0 12 Relative mRNA Level

Relative mRNA Level

2.4

2.5

10 E: cyp19a

#☆

F: crh

*

3.3

**

6

*

4

5.5

* 4.4

8

* ☆

*

2

#*

* 2.2 1.1

☆ ☆

0

0 BCY — TH I —

Relative mRNA Level

Relative mRNA Leve l

3.0

L M H — — —

—— — L M H

— —

L M H L M H

842

43

L M H —— —

— — — L M H

L M H BCY L M H THI

Relative mRNA Level

841

Fig. 6.

A: Tnf

B: cxcl

#☆

#☆

*

#☆

* 4.5

*

6

*

3.0

**

4

#

*

#☆ *

2

1.5

0

0

Rela tive mRNA L eve l

2.4

2.5 C: IL

D: cat

1.8

2.0

*

1.5

#

1.2

☆

*

0.6

*

*

*

*

1.0 0.5

0

0 2.4

2.4 Rela tive mRNA L eve l

Rela tive mRNA L eve l

8

6.0

Relative mRNA Le ve l

Relative mRN A Level

10

E: Cu/Zn-sod

F: Mn-sod 1.8

1.8

*

*

1.2

# #

*

* #

☆

*

*

0.6

0.6

0

0 BC Y — TH I —

1.2

L M H — — —

— — — L M H

L M H L M H

— —

844

44

L M H —— —

— — — L M H

L M H B CY L M H TH I

Rela tive mRNA L evel

843

Fig. 7.

1.6

4.5

A: bax

B: p53 *

#☆ #☆ * #☆

1.2

** *

0.8

***

3.6 2.7

#

1.8 ☆

0.4

*

0

*

#

#

*

0 2.5

Rela tive mRNA L evel

2.8

C: cas8 *

1.4

D: cas9

#

2.1

* *

#☆ 2.0

*

#

#

0.9

#

*

1.5 1.0

***

0.7

0.5 0

0 BCY — THI —

L M H ———

—— — LM H

— —

L M H L M H

847

45

L M H —— —

Re lative mRNA Le ve l

Relative mRN A Le ve l

2.0

—— — L M H

L M H BCY L M H THI

Re lative mRNA Le ve l

845 846

848

46

Highlight: > Beta-cypermethrin exerted greater toxicity than thicloprid to zebrafish. > Mixtures of beta-cypermethrin and thicloprid had synergistic effect on zebrafish. > Expressions of 4 genes exerted greater changes in pesticide mixtures. > Mixture effects should be considered in the ecological risks of pesticide.

Conflict of interest The authors declare that they have no conflict of interest.