Characterization of Synergistic Embryotoxicity of ... - ACS Publications

Subscriber access provided by University Libraries, University of Memphis

Article

Characterization of Synergistic Embryotoxicity of Nickel and Buprofezin in Zebrafish Tingting Ku, Wei Yan, Wuyao Jia, Yang Yun, Na Zhu, Guangke Li, and Nan Sang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es506293t • Publication Date (Web): 19 Mar 2015 Downloaded from http://pubs.acs.org on March 23, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Environmental Science & Technology 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 33

Environmental Science & Technology

1

Title page

2 3 4 5

Characterization of Synergistic Embryotoxicity of Nickel and Buprofezin in

6

Zebrafish

7 8

Tingting Ku, Wei Yan, Wuyao Jia, Yang Yun, Na Zhu, Guangke Li, Nan Sang*

9

College of Environment and Resource, Research Center of Environment and Health, Shanxi University,

10

Taiyuan, Shanxi 030006, PR China

11 12

* Corresponding author. Tel.: +86-351-7011932

13

Fax: +86-351-7011932

14

E-mail: [email protected]

15

Mailing address: Nan Sang

16

College of Environment and Resource,

17

Shanxi University, Taiyuan, Shanxi 030006

18

People’s Republic of China

19 20 21 22 23 24 25 26 27 28

ACS Paragon Plus Environment


29

30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45


Page 2 of 33

Page 3 of 33


46

ABSTRACT: Multiple pollutants, usually at low levels, co-exist and may interact in the environment. It

47

is therefore important to analyze the toxicity of mixtures of co-existing chemicals to evaluate the

48

potential ecological risk. Concern regarding the co-occurrence and combined bio-effects of heavy

49

metals and organic insecticides in aquatic settings has existed for many years, but a clear

50

understanding of the interactions between and potential combined toxicity of these chemicals remains

51

elusive. In the present study, the combined effects of the heavy metal nickel (NiSO4) and insect growth

52

regulator buprofezin on the induction of embryo toxicity in zebrafish were assessed. By applying

53

nonlinear regression to the concentration-response data with each of the chemicals using the Hill and

54

Langmuir functions and computing the predictions using the model of concentration addition (CA), we

55

confirmed that NiSO4 and buprofezin acted together to produce synergistic embryotoxicity in zebrafish.

56

Subsequently, we further found that the combination of NiSO4 and buprofezin formed a complex that

57

facilitated the uptake of nickel (Ni) and buprofezin by the embryos. Following this, we clarified that an

58

oxidative mechanism of the complex might underlie the synergistic embryotoxicity of NiSO4 and

59

buprofezin.

60 61 62 63 64 65 66 67



68

INTRODUCTION

69

Multiple pollutants, usually at low levels, coexist and may interact in the environment. For this

70

reason, studies of the concentration-response relationship of individual chemicals cannot reflect the

71

actual exposure risk to organisms. Therefore, a key question in the risk assessment for co-exposures is

72

whether mixture effects occur when chemicals are combined at low doses that individually do not

73

induce observable effects. Awareness of the need to address combined exposures is growing among

74

experts, and mounting evidence indicates that there is a combined toxicity as a result of simultaneous

75

co-exposure to chemicals. This is especially true for the combined bio-effects of heavy metals and

76

organic insecticides in aquatic settings, although studies of these interactions are not new. However,

77

current reports regarding harmful chemicals appear regularly in media outlets, and communication to

78

the public of the risks of these interactions and the potential combined effects at low concentrations is

79

still a significant problem.

80

Buprofezin (2-tert-butylimino-3- isopropyl-5-phenyl-1,3,5-thiadiazinan-4-one) is an insect growth

81

regulator characterized by a broad-spectrum, specific mode of action and a good record of

82

environmental safety and has risen in popularity for agriculture use.

83

buprofezin technical is approximately 4,500 tons/year. Due to its long-term retention, buprofezin

84

residues can be detected in fruits, vegetables, crops, soil and aquatic environments,4-6 and their

85

presence in the environment is a potential problem. Nickel (Ni) is a naturally occurring metallic element

86

in the earth's crust, and its concentration in surface water and groundwater ranges between 1 and 10

87

µg/L. Because Ni and its compounds are widely used in industry for various applications such as

88

electroplating, welding, flame cutting, flame spraying and mold making,7 they are inevitably found at

89

elevated levels in sediments, the water column, and biota, with concentrations of up to 100 to 1000

1-3


Currently, Chinese production of

Page 4 of 33

Page 5 of 33


90

µg/L in freshwater and estuarine areas surrounding heavily developed urban areas.8 In addition, dietary

91

intake through contaminated foodstuffs is another primary route of Ni exposure.9

92

Environmental surveys have shown the co-occurrence of Ni and buprofezin in aquatic

93

environments. In the Mekong River Delta of Vietnam, buprofezin in soils and sediments was

94

omnipresent, and it was detected in all samples during both rice seasons with concentrations of up to

95

521 µg/kg dm. Furthermore, buprofezin is the second most frequently detected insecticide in surface

96

water and occasionally occurs in drinking water, with a quantification frequency of 58.7 % and 4.8 %

97

and a median concentration of 0.19 and 0.12 µg/L, respectively. In contrast, in the Mekong River delta

98

and its associated coastal zone, Ni has been detected as one of the trace elements in the dissolved

99

phase with concentrations of 7.8 and 8.4 nM and has been found in suspended matter and superficial

100

sediments in concentrations of 32 and 18 µg/g.10-12 In addition, preliminary studies have indicated that

101

buprofezin and Ni co-exist in food and drink. Since buprofezin was recommended for use on exportable

102

grapes in 2006, its residues have frequently been detected in wine. Whereas, Ni is typically one of the

103

ultra microelements detected in wines from different regions.13,14 Among foodstuffs, relatively high

104

concentrations of Ni are present in the leaves of the tea plant Camellia sinensis, whereas buprofezin

105

residue has been found in tea samples.15,16 Furthermore, considering that Ni is an essential

106

micronutrient and consistently found in organisms at low levels, even in the absence of elevated Ni

107

exposure,

108

hydrophobicity and stability in acid and alkali environments (the half-life is up to approximately 36−104

109

days ), it is reasonable for us to expect that there is an increase in opportunities for co-exposure to

110

buprofezin and Ni, and with this increase, the environmental safety of these chemicals, especially at

111

low concentrations, are challenged.

17-19

and buprofezin residues are commonly detected in plants, soil and water

5,6

with high

20



Page 6 of 33

112

Despite the coexistence of these two substances in the environment, their ecological and health

113

risk assessments have been primarily on the basis of the toxicological effects of the individual

114

substances. The toxic effects of Ni in aquatic organisms, including oligochaetes, mollusks, fish and sea

115

urchins,21-23 have been evaluated. These effects primarily include respiratory injuries to freshwater fish

116

involving impaired gas exchange due to severely limiting the diffusive capabilities of the gills,

117

increased morphological abnormality and mortality. Previous studies of buprofezin toxicity have mainly

118

focused on the resultant dose-dependent reductions in the body length and spawning rates of Daphnia

119

and decreases in the hatching and survival rates of African catfish embryos.

120

interaction between Ni and buprofezin has not been investigated thus far, and their combined toxicity at

121

low doses and the relevant mechanisms for such toxicity remain unclear.

122

27,28

24-26

and

However, the

In the present study, the combined effects of NiSO4 and buprofezin on the induction of embryo

123

toxicity

124

concentration-response analyses with the individual chemicals and computing the prediction of the

125

effects of a 1:1 mixture of the two chemicals by concentration addition (CA) model, we confirmed that

126

NiSO4 and buprofezin acted together to cause synergistic embryo toxicity in zebrafish, even when each

127

one existing at level below its no-observed-effect-concentration (NOEC). Subsequently, we used

128

fluorescence spectrometry to investigate the interaction of these two chemicals and found that NiSO4

129

and buprofezin formed a Ni-buprofezin complex. This complex facilitated uptake of Ni and buprofezin

130

by the embryos after exposure to both and contributed to the combination effects. Finally, using

131

pharmacological interference, we showed that the oxidative mechanism of the complex might underlie

132

the synergistic embryotoxicity of NiSO4 and buprofezin.

133

MATERIALS AND METHODS

in

zebrafish

were

assessed.

Applying

nonlinear

regression


to

the

extensive

Page 7 of 33


134

Chemicals. Nickel sulfate (NiSO4, purity>97%) and buprofezin (purity>99%) were purchased from

135

Sigma-Aldrich Company (Saint Louis, Missouri, America) and Zhenbo Company (Jiangyin City, Jiangsu,

136

China), respectively. All other chemicals used were commercially available appropriate grades. The

137

buprofezin stock solution was prepared by dissolving a weighed amount of buprofezin in dimethyl

138

sulfoxide (DMSO), and then, different exposure concentrations for the embryonic bioassay were

139

obtained by diluting the buprofezin stock solutions into culture medium. The NiSO4 test solution was

140

prepared by dissolving NiSO4 in distilled water to obtain a stock solution and then diluting the stock

141

solution into culture medium immediately before use.

142

Zebrafish Embryo Collection and Exposure. Mature wild-type (AB strain) zebrafish

143

(approximately 8 months old) were maintained at 28 ± 0.5 ℃ in a 14 h light /10 h dark cycle in a

144

continuous flow-through system in charcoal-filtered tap water. The fish were fed twice daily with

145

Artemia nauplii. Zebrafish eggs were obtained from the spawning adults in groups of approximately 20

146

males and 10 females held in tanks overnight. The zebrafish eggs were collected within 4 h of

147

spawning from several breeding tanks, pooled, washed, and then randomly transferred into glass

148

beakers. At 24 h post-fertilization (hpf), we randomly exposed normally developed embryos to Ni at 10,

149

20, 40, 80, 200, 300, 400 and 600 mg/L or buprofezin at 2.5, 10, 40, 200, 300, 400, 500 and 600 mg/L,

150

respectively. The exposure solution contained 294.0 mg/L CaCl22H2O, 123.3 mg/L MgSO47H2O, 63.0

151

mg/L NaHCO3, and 5.5 mg/L KCl.

152

buprofezin were both at 5, 10, 20, 40, 80 and 100 mg/L. To inhibit reactive oxygen species (ROS)

153

production, the zebrafish embryos were exposed to Ni and buprofezin both at 10 mg/L in the absence

154

and presence of a 2 h pre-treatment with the antioxidant agent Vitamin C (Vc, 250 µmol/L). Embryos

155

were exposed to different treatments in a 24-well multi-plate at 3 embryos/well with 2 mL test solution,

29

In the co-exposure treatments, the concentrations of Ni and



Page 8 of 33

156

and the plates were kept in a humidified incubator at 28 ± 0.5 ℃ under controlled lighting conditions.

157

Both the vehicle control and the treated embryos received the maximum amount of 0.2 % DMSO (v/v),

158

and three independent experiments for each treatment concentration were conducted in triplicate. For a

159

single experiment, each concentration group contained 216 normally developed embryos. The

160

embryos were examined at the end of the exposure period, the survival rate was recorded as the

161

toxicity end point, and mortality was identified by a missing heartbeat, coagulation of the embryos,

162

non-detached tail and failure to develop somites, as previously reported by Du et al.30

163

Detection of ROS Production. According to a previously reported protocol,

31

200 zebrafish

164

embryos were washed twice with cold PBS (pH = 7.4) and then homogenized in 800 µL cold buffer.

165

The homogenate was centrifuged at 12,000 × g for 10 min at 4 ℃, and the supernatant was collected.

166

Next, 20 µL of the homogenates were added to the wells of a black 96-well plate(Costar 3925, Corning

167

Incorporated, USA) and incubated at room temperature for 5 min. After the incubation, 100 µL PBS and

168

8.3 µL dichlorofluorescein-diacetate (DCF-DA) stock solution (dissolved in DMSO, 10 mg/mL) were

169

added to each well. The plate was incubated at 37 ℃ for 30 min in the dark. The fluorescence intensity

170

was measured using a microplate reader (Varioskan Flash, Thermo Scientific, America) with excitation

171

and emission at 485 and 530 nm, respectively. The ROS concentration was expressed in arbitrary units

172

(fluorescence units/mg protein).

173

Lipid Peroxidation and Protein Assay. Lipid peroxidation was detected by spectrofluorometric

174

analysis as malondialdehyde (MDA) reacting with thiobarbituric acid to form a colored complex. The

175

embryos were homogenized in cold PBS, and the homogenate was centrifuged at 12,000 × g for 10

176

min at 4 ℃ to precipitate insoluble material. The supernatant was collected to analyze the MDA content

177

using the corresponding reagent kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The


Page 9 of 33


178

level of MDA is expressed as nanomoles per milligram protein. Protein levels were estimated by

179

Bradford's method using bovine serum albumin as a standard.32

180

Lactate Dehydrogenase (LDH) Release Assay. The culture medium was used to assess LDH

181

release, and the activity was measured according to the protocol of Fontella et al.33 In detail, 100 µL of

182

the medium was transferred to the reaction medium containing 7.5 mmol/L NAD , 260 mmol/L lactate,

183

and

184

1,10-phenanthroline was converted to a colored complex by reacting to NADH, which was determined

185

at 490 nm.

+

1.6

mmol/L

1,10-phenanthroline.

Following

the

conversion

of

lactate

to

pyruvate,

186

Fluorescence Spectral Analysis. A stock solution of buprofezin (10 g/L) was made by dissolving

187

buprofezin in DMSO, sonicated for 5 min, and diluted by double distilled water. For the quenching

188

experiments, the diluted buprofezin solutions and NiSO4 at various concentrations were applied into a

189

measuring flask; and then the system was thoroughly mixed and quantified to 5 mL with double distilled

190

water. The fluorescence intensity of mixtures was detected at 570-630 nm, with excitation wavelength

191

of 300 nm (Cary Eclipse, Agilent Technologies, Australia).

192

Determination of the Ni and Buprofezin Content of Embryos.

For Ni analysis, embryos of

193

each treatment group were washed repeatedly with distilled water and digested overnight. The

194

digestion was completed by microwave-assisted digestion at 185℃ for 20 min (Multiwave 3000, Anton

195

Paar, Australia). After cooling, the residues were transferred to 50 mL flasks and diluted with deionized

196

water. Before analysis, the samples were filtered through a 0.45 µm membrane filter.34 The Ni contents

197

were eventually measured by flame atomic absorption spectrometry (FAAS, AA240FS, Varian, USA).

198

For the buprofezin determination, a gas chromatograph-mass spectrometer (GC-MS) was applied

199

(TRACE-DSQ, Thermo Scientific, America). A DB-5 MS column of 30 m × 0.25 mm I.D × 0.25 µm film



200

thickness was programmed at 100 ℃ for 1 min, and then the temperature was elevated to 300 ℃ at

201

25 ℃/min and kept at 300 ℃ for 4 min. The temperatures of injection port and transfer line from GC to

202

MS were kept at 250 ℃. The samples were automaticly introduced in splitless mode (t = 1 min), with a

203

helium carrier gas flow of 1 mL/min. The EI was operated at 70 eV. The full scan mode ranged from m/z

204

40 to 400. The ion source temperature was 250 ℃. The embryos were ultrasonically extracted (VWR

205

ultrasonic bath, Filtrafine, Spain) with chloroform, and the extract was concentrated for 5 min in a rotary

206

evaporator. Following this, the extract was dissolved in 1 mL of chloroform and dried under a gentle

207

stream of N2. Finally, the extract was dissolved in 10 mL of chloroform, and 1 µL was injected into the

208

GC-MS. Based on the data from the FAAS and GC-MS, we obtained the concentration of Ni and

209

buprofezin of total embryos. The mass of Ni and buprofezin contained in each embryo was calculated

210

from the measured concentration of the aliquots and the dilution factor according to previous

211

literature.35 In detail, the Ni or buprofezin content of the embryo was calculated using the following

212

equation: M

213

were the concentration of the Ni or buprofezin of total embryos from FAAS and GC-MS, the final

214

volume and the total number of embryos, respectively.

215

= CV N . In this equation, M was the Ni or buprofezin content of the embryo; C, V, and N

Concentration-response Analysis. Based on the scatter plots of embryo mortality ratio versus 36

216

concentration and previously reported “best fit” approach,

217

analyses using suitable regression models, such as Hill and Langmuir models, etc. Also, we calculated

218

the 95% confidence intervals of the estimated mean effects. Table S1 showed a complication of the

219

nonlinear regression models applied in present study, and the NOEC of the individual chemicals and

220

mixtures were estimated using the Dunnett test.36

221

we conducted concentration-response

Calculation of Predicted Mixture Effects. We prepared mixtures at a mixture ratio of 1:1 and


Page 10 of 33

Page 11 of 33


222

then treated the embryos with serial dilutions of a master solution. Then, we calculated the predicted

223

combination effects over a range of concentrations using CA model. The model assumes that the

224

combination effect of a mixture with n components is concentration additive, therefore, the following

225

formula is used for calculating the mixture effects.37-42

226

ECxmix = [∑ pi / ECxi ]−1 [1]

227

In this equation, pi is the relative proportion of substance i expressed as a fraction of the total

228

concentration of substances in the mixture. The effect concentrations ECxi were computed using the

229

inverse expression of the proper regression functions from the concentration response function of

230

individual components i (Table S1).

231

Statistical Analysis. A one-way analysis of variance (ANOVA) was used to analyze the mean

232

differences among groups compared to the vehicle control. A two-tailed Student’s t-test was used to

233

analyze experimental data between two groups. The data are presented as the mean ± SE. p

Characterization of Synergistic Embryotoxicity of ... - ACS Publications

Recommend Documents