Polychlorinated Diphenylsulfides Activate Aryl Hydrocarbon Receptor

Subscriber access provided by Kaohsiung Medical University

Ecotoxicology and Human Environmental Health

Polychlorinated Diphenylsulfides Activate Aryl Hydrocarbon Receptor 2 in Zebrafish Embryos: Potential Mechanism of Developmental Toxicity Rui Zhang, Xiaoxiang Wang, Xuesheng Zhang, Chao Song, Robert J. Letcher, and Chunsheng Liu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00366 • Publication Date (Web): 09 Mar 2018 Downloaded from http://pubs.acs.org on March 9, 2018

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

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35

Environmental Science & Technology

1

Polychlorinated Diphenylsulfides Activate Aryl

2

Hydrocarbon Receptor 2 in Zebrafish Embryos: Potential

3

Mechanism of Developmental Toxicity

4 5

Rui Zhang,†,ǁ Xiaoxiang Wang,‡,§,ǁ Xuesheng Zhang,⊥ Chao Song,⎕,# Robert J.

6

Letcher,¶ Chunsheng Liu*,∇

7 8

†

9

China

School of Resources and Environment, University of Jinan, Jinan 250022, P. R.

10

‡

11

Environment, Nanjing University, Nanjing 210023, P. R. China

12

§

13

Limburgerhof 67117, Germany

14

⊥School

15

230601, P. R. China

16

⎕Freshwater

17

214081, P. R. China

18

#

19

Environmental Factors (Wuxi), Ministry of Agriculture, Wuxi 214081, P. R. China

20

¶

21

5B6, Canada

22

∇College

23

ǁ

State Key Laboratory of Pollution Control and Resources Reuse, School of the

Association of Chinese Chemists and Chemical Engineers in Germany,

of Resources and Environmental Engineering, Anhui University, Hefei

Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi

Laboratory of Quality & Safety Risk Assessment for Aquatic Products on

Departments of Chemistry and Biology, Carleton University, Ottawa, Ontario K1S

of Fisheries, Huazhong Agricultural University, Wuhan 430070, P. R. China

The authors contribute equally.

24 25

*

26

College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China

27

Tel: 86 27 87282113, Fax: 86 27 87282114, E-mail: [email protected]

Author for correspondence:

ACS Paragon Plus Environment


28

ABSTRACT

29

It is hypothesized that polychlorinated diphenyl sulfides (PCDPSs) induce lethal

30

toxicity in zebrafish which is mediated by aryl hydrocarbon receptor 2 (Ahr2)

31

activation. In this study an assay was developed based on in vivo exposure of

32

wild-type and Tg(cyp1a:gfp) transgenic zebrafish embryos/larvae to PCDPS

33

congeners (i.e. six dichloro- to heptachloro-diphenyl sulfides) coupled with a

34

zebrafish Ahr2-luciferase reporter gene (LRG) expression. Waterborne PCDPSs were

35

found to be accumulated in zebrafish larvae and exposure to PCDPSs led to a

36

significant increase in mortality and cyp1s mRNA expression. Furthermore, treatment

37

with PCDPSs caused a significant induction of Ahr2-LRG activity in COS-7 cells,

38

and extremely significant correlations were observed between the in vivo median

39

lethal concentrations and the levels of cyp1s mRNA expression and Ahr2 activation.

40

Molecular dynamics simulations indicated the interaction between dioxins/dioxin-like

41

compounds (DLCs) and six key amino acid residues in the ligand-binding domain of

42

Ahr2 probably determined the susceptibility to dioxins/DLCs in zebrafish. These

43

results strongly support the hypothesis that early life-stage mortality of zebrafish is

44

initiated and mediated by Ahr2 activation.

45 46 47


Page 2 of 35

Page 3 of 35


48

INTRODUCTION

49

Polychlorinated diphenyl sulfides (PCDPSs) are a group of chlorinated aromatic

50

compounds comprising 209 theoretical congeners like polychlorinated biphenyls

51

(PCBs). They have been extensively employed as high-temperature resistant

52

lubricants,1 additives in flame retardants and insulating media,2 as well as being

53

widely used as acaricides on fruit and tea crops (known as Tetrasul, mainly

54

2,4,4’,5-TCDPS) in many countries like China and the USA.3-7 Due to their

55

considerable persistence and environmental mobility properties,8, 9 PCDPSs have been

56

frequently found in various environmental matrices, such as dust from metal recycling

57

plants,10 gas and fly ash from waste incineration (tri- and tetra-CDPSs),11 wastewater

58

from pulp and paper mill (tri-CDPS isomers),11 fruits (2,4,4’,5-TCDPS),5 tea leaves

59

(2,4,4’,5-TCDPS),6 and water and sediment samples from the Elbe river in Germany

60

(4,4’-DCDPS)12 and the Yangtze river in China (mono- to hepta-CDPSs, total

61

concentrations ranging from 0.18 to 2.03 ng/L and 0.10 to 6.90 ng/g in surface water

62

and sediment, respectively).13 The bioconcentration and bioaccumulation potentials of

63

PCDPSs were also revealed by modeling calculations.8, 9 These findings have raised

64

concerns about their potential adverse effects on human and ecosystem health.

65

Increasing evidence has shown that exposure to PCDPSs can elicit a number of

66

adverse effects in various vertebrates like rat, pig, rabbit, fish and chicken, including

67

mortality,14-16 growth retardation,14 hepatic oxidative stress15,

68

reproductive performance.14

17, 18

and diminished

69

Several PCDPS congeners can be categorized as dioxin-like compounds (DLCs),

70

as our previous studies have demonstrated they are active via binding to mammalian19

71

and avian20 aryl hydrocarbon receptor (AHR) with subsequent gene expression in

72

vitro. This raises the question of whether the adverse biological outcomes in

73

vertebrates are elicited due to the activation via the AHR by PCDPSs. Addressing this

74

question can obviously promote understanding about the mechanisms of toxicity of

75

PCDPSs.

76

The zebrafish (Danio rerio) represents a superb model for vertebrate

77

development, disease processes and toxicological mechanism research.21-23 Therefore,



Page 4 of 35

78

the primary goal of the present study is to test the hypothesis that some PCDPS

79

congeners are DLCs and induce lethal toxicity in vertebrates that is initiated and

80

mediated by the activation of AHR using zebrafish as a representative species. The

81

specific objectives were four-fold: (1) characterize the concentration-dependent lethal

82

effect and bioconcentration potential of six PCDPS congeners ranging from dichloro-

83

to heptachloro- diphenyl sulfides in early life stages of zebrafish; (2) develop a

84

luciferase reporter gene (LRG) assay to verify Ahr2, which is the dominant isoform

85

responsible for dioxin-like toxicity in zebrafish,24 was activated to different extent in

86

zebrafish embryos/larvae following in vivo congener-specific exposure to PCDPSs; (3)

87

compare in vivo median lethal concentration (LC50) values with LRG assay-derived

88

Ahr2 activation levels to demonstrate that PCDPS-induced lethality in vivo is initiated

89

and mediated by Ahr2 activation in zebrafish; (4) gain a further understanding of the

90

mechanism underlying the level differences of Ahr2 activation by different

91

dioxins/DLCs at the atomic level using homology modeling and molecular dynamics

92

(MD) simulations. The results provide new information regarding the mechanisms of

93

the PCDPS congener-specific toxicity, and support our understanding of the

94

mechanism underlying the differences in the level of Ahr2 activation by different

95

dioxins/DLCs in zebrafish at molecular and atomic levels.

96 97

MATERIALS AND METHODS

98

Chemicals and Reagents. The six PCDPS congeners, including 4,4’-DCDPS,

99

2,2’,4-Tris-CDPS, 2,4,4’,5-TCDPS, 2,2’,3’,4,5-Penta-CDPS, 2,3,3’,4,5,6-Hexa-CDPS

100

and

2,2’,3,3’,4,5,6-Hepta-CDPS,

101

palladium-catalyzed carbon-sulfur bond formation method.25 The purities were more

102

than 99% with no detectable dioxin-like PCDDs/Fs present as described previously.20

103

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, CAS number 1746-01-6; >98% purity),

104

1,2,3,7,8-pentachlorodibenzo-p-dioxin

105

40321-76-4;

106

(1,2,3,7,8,9-HxCDD,

107

2,3,7,8-tetrachlorodibenzofuran (2,3,7,8-TCDF, CAS number 51207-31-9; >98%

>98%

were

(1,2,3,7,8-PeCDD,

purity), CAS

synthesized

number

previously

CAS

by

the

number

1,2,3,7,8,9-hexachlorodibenzo-p-dioxin 19408-74-3;


>98%

purity),

Page 5 of 35


108

purity),

1,2,3,7,8-pentachlorodibenzofuran

(1,2,3,7,8-PeCDF,

CAS

number

109

57117-41-6; >98% purity), 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PeCDF, CAS

110

number 57117-31-4; >98% purity) and 3,3’,4,4’,5-pentachlorobiphenyl (PCB126,

111

CAS number 57465-28-8; >99% purity) were purchased from AccuStandard (New

112

Haven, CT, USA). All chemical stock solutions and serial dilutions were prepared in

113

dimethyl sulfoxide (DMSO, CAS number 67-68-5; >99.7% purity; Sigma-Aldrich,

114

St. Louis, MO, USA). MS-222 (3-aminobenzoic acid ethyl ester, methane sulfonate

115

salt, CAS number 886-86-2; >98% purity) was purchased from Sigma-Aldrich (St.

116

Louis, MO, USA). The actual concentrations of PCDDs/Fs and PCB126 were

117

determined using isotope dilution following US EPA method 1613 by high-resolution

118

gas chromatography high-resolution mass spectrometry (HRGC/HRMS).26 For in vivo

119

waterborne exposure, the serially diluted solutions of the PCDPSs were diluted again

120

with embryonic rearing water (60 mg/L instant ocean salt in aerated distilled water) to

121

the desired test concentrations immediately before use. The final concentration of

122

DMSO in the exposure solutions was 0.5% (v/v). For in vitro reporter gene assays,

123

test solutions of PCDPSs, PCDDs/Fs and PCB126 were prepared by dissolving

124

serially diluted solutions with cell culture medium. The final in-well concentration of

125

DMSO in 96-well plates was 0.5%.

126

Animals and Waterborne Exposure Experiment. Adult wild-type zebrafish (AB

127

strain, 7-month old) and Tg(cyp1a:gfp) transgenic zebrafish (9-month old)27 were

128

maintained based on the standard zebrafish protocols.28 All fertilized eggs used in the

129

present study were obtained by artificial fertilization to ensure consistency in

130

developmental stages.29 The eggs were examined under a stereomicroscope (M205FA,

131

Leica Microsystems, Wetzlar, Germany) and only normally developed embryos were

132

selected for subsequent experiments. All work was approved by the Institutional

133

Animal Care and Use Committee of the Huazhong Agricultural University. All

134

animals were treated humanely and with regard for the alleviation of suffering.

135

The exposure experiment included two parts. First, wild-type zebrafish embryos

136

were randomly distributed into glass beakers and exposed to each of the 6 individual

137

PCDPS congeners (4, 20, 100, 500, 2500 or 5000 nM) or DMSO from 2 h ACS Paragon Plus Environment


Page 6 of 35

138

post-fertilization (hpf) to 120 hpf to study the early life-stage mortality induced by

139

PCDPSs. These exposure concentrations were set to be distributed as evenly as

140

possible across a range, which is from non-observed lethal concentration to median or

141

even 100% lethal concentration based on the results of preliminary experiments and

142

the requirements of acute lethal toxicity. Three replicate beakers per dilution of the

143

PCDPSs or DMSO were included, each containing 100 mL of exposure solution and

144

100 embryos. For each treatment, 50% of the exposure solution was replaced by

145

freshly prepared exposure solution on a daily basis. The embryos/larvae were kept

146

under 28 ± 0.5 °C with a 12:12 light/dark cycle. Hatching rates were monitored at 48,

147

54, 60 and 72 hpf. Mortalities were recorded at 24, 48, 72, 96 and 120 hpf. Mortality

148

was identified by embryonic coagulation and missing heartbeat (Leica M205FA, 8×).

149

At 120 hpf, larvae were anesthetized with 0.03% MS-222 (Sigma-Aldrich) and 25

150

individuals from each beaker (20, 100 and 500 nM) were randomly sampled,

151

immediately frozen in liquid nitrogen, and stored at -80 °C until subsequent

152

quantitative real-time polymerase chain reaction (qRT-PCR) assays. In the second part

153

of the experiment, based on the results of acute lethal toxicity from the first part,

154

Tg(cyp1a:gfp) transgenic zebrafish embryos were exposed to each of the six

155

individual PCDPS congeners (20, 100 or 500 nM) or DMSO with 50% exposure

156

solution renewed each day from 2 to 120 hpf to visually characterize and confirm the

157

overexpression of cyp1a gene in live zebrafish induced by congener-specific PCDPS

158

exposure. The fertilized eggs in this part experiment were produced by in vitro

159

artificial fertilization using eggs from three 7-month-old female Tg(cyp1a:gfp)

160

transgenic zebrafish and semen from three male wild-type adult zebrafish. Each

161

treatment contained 10 mL exposure solution and 10 embryos. After exposure for

162

about 120 hpf, Tg(cyp1a:gfp) larvae were first anaesthetized with 0.03% MS-222

163

(Sigma-Aldrich)

164

stereomicroscope with the same setting at a magnification of 2.76× (Leica M205FA).

165

The images of Tg(cyp1a:gfp) larvae were converted to 8-bit grayscale images using

166

ImageJ30 (developed by Wayne Rasband; available at https://imagej.nih.gov/ij/), an

167

open code Java-based image processing software. A threshold range was set to 33-255

and

then

observed

and

imaged


using

a

fluorescence

Page 7 of 35


168

in gray values to distinguish the regions of interest (ROI, namely specific organ

169

regions with strong fluorescence signals, such as kidney, liver and gut indicated in a

170

previous publication27) from the background. Then the “Analyze Particles” feature of

171

ImageJ was used to determine the maximum gray value of pixels and total area of

172

ROI. The data was transferred into SPSS 12.0 (SPSS Inc., Chicago, IL, USA) for

173

statistical analysis.

174

Quantification of PCDPS congeners and QA/QC. In the acute toxicity test, 1 mL of

175

exposure solution of the individual PCDPS congeners from each of 3 replicate

176

beakers were collected in 4-nM (the lowest exposure concentration) treatment groups

177

at 96 hpf (after renewing of exposure solutions), and 120 hpf to check if degradation

178

had occurred during exposure. Three replicate 20-, 100- and 500-nM exposure

179

solutions were also sampled at 120 hpf for determination of the actual waterborne

180

concentrations. In addition, 25 larvae from each replicate in 20-, 100- and 500-nM

181

treatment groups were collected at 120 hpf to analyze congener-specific PCDPS

182

bioconcentration in the early life stages of zebrafish. Twenty larvae from each

183

replicate of the 500 nM, 2,2’,3’,4,5-Penta-CDPS treatment group were also collected

184

at 72 and 96 hpf to determine the time-course of accumulation. Detailed protocols for

185

extraction, clean up, GC-MS quantification, and quality assurance and quality control

186

(QA/QC) were provided in Supporting Information (Supplementary Methods and

187

Tables S1 and S2).

188

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Assay. PCR

189

primer sets for a select group of genes involved in dioxin-like toxicity in zebrafish,

190

including cyp1a1, cyp1b1, ahr2, ahrra, ahrrb and arnt1, are shown in Table S3. The

191

primer sequences for arnt1 were designed using NCBI/Primer-BLAST software. The

192

other primer sequences were obtained in our previous publications.31, 32 The mRNA

193

expression of each selected gene was normalized to the geometric mean of that of the

194

housekeeping gene ribosomal protein l8 (rpl8) whose expression was unaffected by

195

any exposure condition in the present study. Detailed methods for total RNA isolation,

196

reverse transcription and qRT-PCR analysis are presented in Supporting Information.

197

COS-7 Cell Culture, Transfection, and Zebrafish Ahr2-LRG Assay. The culturing ACS Paragon Plus Environment


Page 8 of 35

198

of COS-7 cells, transfection of constructs, and the LRG assay were performed in

199

96-well plates according to methods described previously31,

200

modifications. A firefly luciferase reporter vector (pGudLuc6.1) under the control of a

201

mouse mammary tumor virus promoter that is regulated by four aryl hydrocarbon

202

response elements (AHREs) from the murine cyp1a1 promoter, was a generous gift

203

from Dr. Michael S. Denison (University of California, Davis, CA, USA).35,

204

Zebrafish pBKCMV-zfAhr2 and pBKCMV-zfARNT1c expression constructs were

205

generously donated by Dr. Richard E. Peterson (University of Wisconsin-Madison

206

Madison, WI, USA).37 The optimized amounts of transfected DNA per 6 µL of

207

transfection mixture were 8 ng of pBKCMV-zfAhr2, 1.5 ng of pBKCMV-zfARNT1c,

208

8 ng of pGudLuc6.1, 0.75 ng of Renilla luciferase vector (Promega, Madison, WI,

209

USA), and 31.75 ng of salmon sperm DNA (Invitrogen, Burlington, ON, Canada).

210

COS-7 cells were dosed 5 h after transfection with DMSO (solvent control) or DMSO

211

solutions of the five DLCs (namely 1,2,3,7,8-PeCDD, 1,2,3,7,8,9-HxCDD,

212

2,3,7,8-TCDF, 1,2,3,7,8-PeCDF and PCB126) or PCDPS congeners with nominal

213

concentrations ranging from 0.1 to 6000 nM (no cytotoxic effect could be observed by

214

MTS cytotoxicity assay). A positive control (300 nM TCDD) was also included when

215

cells were dosed with other tested chemicals and DMSO. LRG activity was measured

216

20 h after dosing using Dual-Glo luciferase assay kits (Promega) in a Synergy H4

217

Hybrid Multi-Mode Microplate reader (BioTek Instruments, Winooski, VT, USA).

218

Triplicate concentration−response curves were obtained from three independent

219

experiments for each chemical treatment, with four technical replicates per dilution of

220

tested chemicals. The normalization of luciferase ratio (firefly to Renilla) was

221

performed according to the protocol described in OECD guideline 455.38

33, 34

with minor

36

222

The data were fitted to a four-parameter logistic model using GraphPad Prism

223

5.0 software (GraphPad Prism Software Inc., San Diego, CA, USA).39 When

224

concentration-response curves did not reach a plateau or a plateau could not be

225

estimated accurately by curve fitting, EC50 values could not be calculated and were

226

therefore not presented. In addition, the highest observed response was reported as the

227

maximal response in these instances. The concentrations of chemicals tested that ACS Paragon Plus Environment

Page 9 of 35


228

elicited a response equal to 10%, 20%, 50%, and 80% of the positive control response

229

were referred to as PC10, PC20, PC50 and PC80 values. These PCs, EC50, and maximal

230

response values were determined for each replicate concentration-response curve

231

using the logistic curve fitting, and were presented as the mean ± standard error (SE).

232

Relative potency (ReP) values were calculated as follows: EC50, PC10, PC20, PC50, or

233

PC80 of TCDD ÷ EC50, PC10, PC20, PC50, or PC80 of chemicals tested.

234

Molecular Dynamics Simulations. The MD simulations were carried out with the

235

GROMACS 5 package40 on an International Business Machines (IBM) blade cluster

236

system. Prior to simulation, initial structures for simulation needed to be prepared,

237

and the process of preparation is presented in the Supporting Information. The

238

CHARMM 27 force field41 was applied to all structural models. Parameters of the

239

force field for ligands were obtained from SwissParam42 (http://www.swissparam.ch/).

240

The protein-ligand complex was solvated in a box with TIP4P water molecules,43

241

keeping the boundary of the box at least 1 nm away from all protein atoms. Three

242

sodium ions were subsequently added for charge neutralization. The whole system

243

was then energetically minimized by the steepest-descent method.44 The minimized

244

systems were then gradually heated from 0 to 310.15 K at a constant volume. The

245

heated systems at 310.15 K were equilibrated for 200 ps with position restraints for

246

ligands and for 1 ns without restraints at 1 bar and 310.15 K. The MD simulations

247

were then performed in the NPT ensemble with periodic boundary conditions. Both of

248

electrostatic interactions and van der Waals interactions were calculated using the

249

particle mesh Ewald (PME) algorithm.45 All simulations were carried out for at least

250

50 ns using a 2 fs time step, and snapshots for analysis were saved every 2 ps. The

251

trajectories obtained from MD simulations were used for binding free energy

252

calculations by molecular mechanics Poisson-Boltzmann surface area (MM-PBSA)

253

method.46, 47 In brief, the binding free energy ∆G can be defined (Equation 1).

254

∆G = G − (G + G )

(1)

255

where G , G and G are total free energies of the receptor-ligand

256

complex, receptor and ligand in solvent, respectively. The G value for each term (G ) ACS Paragon Plus Environment


257

Page 10 of 35

can be calculated (Equation 2). G = E − TS + G

258

(2)

259

where E is the molecular mechanics energy; TS denotes the entropic contribution

260

where T and S refer to the temperature and entropy, respectively; G is the

261

solvation free energy. More details about this method were given in our previous

262

study.48 The E values between compounds tested and each amino acid residue in

263

Ahr2-ligand binding domain (LBD), which indicates the strength of interaction

264

between the compounds and corresponding residue, were recorded for further

265

analysis.

266

Statistical Analysis. Data normality and homogeneity were examined by

267

Kolmogorov-Smirnov and Levene’s test respectively using SPSS 12.0 (SPSS Inc.,

268

Chicago, IL, USA) before statistical procedures were applied. If normality and equal

269

variance were achieved, significant differences between mortality percentage,

270

maximal response, EC50, PC10, PC20, PC50, and PC80 values for different compounds

271

tested, actual and nominal exposure concentrations, and gene expression difference

272

and fluorescence signal difference between different treatments were examined by a t

273

test or a one-way ANOVA followed by Tukey’s multiple comparison test. A value of p

274

< 0.05 was considered statistically significant. When one of these assumptions was

275

violated, the non-parametric Kruskal–Wallis test was used followed by Dunn’s

276

multiple comparison test. For mortality, data were presented as proportions, but they

277

were square root arcsine-transformed for analysis of variance. Classification and

278

visualization of the PCDPSs based on similarities of differentially expressed genes

279

involved in dioxin-like toxicity in zebrafish was accomplished by use of ToxClust49 in

280

R software version 3.4.2 (R Core Team, Vienna, Austria).

281 282

RESULTS AND DISCUSSION

283

Time- and Concentration-Dependent Accumulation in Larvae of Waterborne

284

PCDPSs. As shown in Figure S1, no significant concentration change of any of the

285

six PCDPS congeners was observed in the 4-nM exposure solutions (the lowest

286

exposure concentration) at 120 hpf compared with those at 96 hpf (after renewing the ACS Paragon Plus Environment

Page 11 of 35


287

exposure solutions). This indicated that no significant congener-specific degradation

288

of PCDPSs occurred in the exposure solutions that were renewed on a daily basis. A

289

time-dependent concentration increase was observed in larvae exposed to 500-nM

290

2,2’,3’,4,5-Penta-CDPS during 72 to 120 hpf (Table S4), suggesting that waterborne

291

PCDPSs could be accumulated in zebrafish larvae in a time-dependent manner. In

292

addition, the measured concentrations for all the PCDPSs tested in zebrafish larvae

293

increased with increasing concentrations of waterborne PCDPSs solution at 120 hpf

294

(Table S4), suggesting that waterborne PCDPSs could be accumulated in zebrafish

295

larvae in a concentration-dependent manner. Bioconcentration factors (BCFs) for each

296

of the PCDPS tested were also calculated as the ratio of measured concentration in

297

zebrafish larvae (µg/g, ww) to that in exposure medium (µg/mL) at 120 hpf. As

298

shown in Table S4, the BCF values ranged from 169 to 1513 among the PCDPS

299

congeners. Moreover, a higher BCF value seemed to be always followed by a lower

300

waterborne exposure concentration for a given PCDPS congener. These results

301

indicated that the potential health and ecological risk of PCDPSs cannot be ignored

302

even at low environmental concentrations.

303

Early Life-Stage Mortality of Zebrafish Exposed to PCDPSs. As shown in the

304

concentration-response curves (Figure 1A), mortality percentages of zebrafish

305

embryos/larvae were increased significantly upon exposure to 1000 nM of

306

2,3,3’,4,5,6-Hexa-CDPS, 2,2’,3’,4,5-Penta-CDPS or 4,4’-DCDPS at 120 hpf. This

307

was especially evident for 2,3,3’,4,5,6-Hexa-CDPS, where the 1000 nM exposure

308

concentration caused almost 100% mortality.

309

(2,2’,3,3’,4,5,6-Hepta-CDPS, 2,2’,4-Tris-CDPS and 2,4,4’,5-TCDPS), no significant

310

effects on the mortality were observed following exposure to 1000 nM or lower

311

exposure concentrations at 120 hpf. In contrast, the mortality percentages reached or

312

approached 100% by 120 hpf exposure to only a 5 times greater concentration (5000

313

nM), indicating a relatively narrow lethal concentration range. Moreover, the lethality

314

occurred mainly (81-99%) during 72 to 120 hpf at each exposure concentration,

315

which significantly increased the mortality (Figure S2). This was probably related to

316

the sustained toxic effects along with continuous accumulation of PCDPSs. Besides

For


the

other 3

PCDPSs


317

mortality, the classical dioxin-like effects, such as spinal curvature, tail malformation,

318

yolk sac edema and pericardial edema, following exposure to PCDPSs were also

319

observed for all the PCDPSs tested (Table S5).

320

Significant Relationships between CYP1s mRNA Expression and Lethal Toxicity.

321

The mRNA expressions of 6 genes involved in dioxin-like toxicity in zebrafish were

322

examined after exposure to 20, 100 or 500 nM of each PCDPS tested. The mRNA

323

expressions were altered significantly in a concentration-dependent manner (Figure

324

1B). Clustering of PCDPSs on the basis of the concentration-dependent mRNA

325

expression indicated that highly chlorinated PCDPS congeners tended to cause a

326

greater change of mRNA expression level involved in dioxin-like toxicity in zebrafish.

327

Moreover, cyp1a1, cyp1b1 and ahrra were the most significant up-regulated genes,

328

with the maximum fold-change of 109 for cyp1a1 in 500 nM 2,3,3’,4,5,6-Hexa-CDPS

329

exposure group. The induction of cyp1a expression is an associative event and

330

common marker of AHR activation.50 Thus, it indicated that Ahr2 might be activated

331

by congener-specific PCDPS exposure. In addition, extremely significant linear

332

relationships (P < 0.0001) were observed between PCDPS-induced cyp1s mRNA

333

expression and the median lethal concentration (LC50) values at 120 hpf (Figure 1C

334

and Table S6) with R2 higher than 0.900, indicating PCDPS-induced lethal toxicity

335

might be mediated by Ahr2 activation, while no significant change of Ahr2 expression

336

was observed. To further confirm that the lethal toxicity is indeed mediated by the

337

Ahr2 activation, a zebrafish Ahr2-LRG assay, which was developed in the present

338

study, was then performed.

339

Induction of Ahr2-LRG Activity in COS-7 Cells. The concentration-dependent

340

effects of TCDD, the five DLCs and the six PCDPSs on LRG activity in COS-7 cells

341

transfected with zebrafish Ahr2 construct were shown in Figure 2A. LRG activity

342

induced by TCDD reached a plateau, indicating that the 300 nM of TCDD was an

343

appropriate positive control for the normalization of LRG activity data in the

344

zebrafish Ahr2-LRG assay. All of the tested chemicals including the six PCDPSs

345

induced significant LRG activity in zebrafish Ahr2-transfected cells with different

346

EC50, PCx, maximal responses and ReP (Tables 1 and S7). Only the ACS Paragon Plus Environment

Page 12 of 35

Page 13 of 35


347

concentration-response curves of 2,3,7,8-TCDF, 1,2,3,7,8,9-HxCDD, PCB126 and

348

2,3,3’,4,5,6-Hexa-CDPS achieved a plateau besides TCDD. In addition, the efficacies

349

(or the maximal response) induced by 1,2,3,7,8-PeCDD and 2,3,3’,4,5,6-Hexa-CDPS

350

were greater than or equal to that of TCDD, which resembles that of

351

2,3,3’,4,5,6-Hexa-CDPS in avian Ahr2-LRG assays.20 In the Ahr2-LRG assay,

352

reporter controls which were COS-7 cells transfected with all the expression vectors

353

except the zebrafish Ahr2 expression construct, were also included for each PCDPS.

354

No significant induction of LRG activity was observed after exposure to TCDD,

355

DLCs or the PCDPSs tested (data no shown). Overall, these results indicated that

356

zebrafish Ahr2 was activated by PCDPS exposure, and PCDPS-induced cyp1s

357

expression was mediated indeed by Ahr2.

358

Relative Potency (ReP) of PCDPS Congeners in the Zebrafish Ahr2 Construct. It

359

was demonstrated that the developed zebrafish Ahr2-LRG assay could be used to

360

predict dioxin-like potencies of compounds which are mediated by Ahr2 activation in

361

early life stages of zebrafish, and the extent of Ahr2 activation is reflected by the

362

induction of LRG activity by use of five DLCs. More details are presented in the

363

section of “ReP Values of the Five DLCs Tested” and Table S8 in Supporting

364

Information. Based on the ReP values (Tables 1 and S7), visual inspection of the

365

concentration-response curves (Figure 1A), and the results of statistical analyses

366

between endpoints in the LRG assays (Table 1), the rank order of PCDPS potency

367

was

368

2,2’,3’,4,5-Penta-CDPS > 2,4,4’,5-TCDPS ≥ 2,2’,4-Tris-CDPS ≥ 4,4’-DCDPS in

369

the zebrafish Ahr2 construct. The rank order of congener-specific PCDPS potency

370

was generally consistent with that of RePLC50 values observed in zebrafish embryos

371

exposed to PCDPSs in vivo until 120 hpf (Tables 1 and S7). Furthermore, the

372

differences between the in vitro and in vivo assays-derived ReP values for the

373

PCDPSs were within 1 order of magnitude (2- to 4-fold difference between

374

LRG-RePavg and RePLC50 values). These results demonstrated that the ReP values

375

derived from zebrafish Ahr2-LRG assays could give reasonable ReP estimates for the

376

early life-stage mortality of PCDPS exposed zebrafish.

2,3,3’,4,5,6-Hexa-CDPS

>

2,2’,3,3’,4,5,6-Hepta-CDPS


≥


377

The data gap of fish-specific ReP values of PCDPSs was filled by including the

378

LRG-RePavg values for zebrafish, which is the least sensitive fish tested to date, to

379

TCDD-induced early life-stage toxicity.54 The LRG-RePavg values of the PCDPSs

380

tested were equal to or higher than the WHO-TEFs of 1,2,3,6,7,8-HexaCDD,

381

1,2,3,7,8,9-HexaCDD, 1,2,3,4,6,7,8-HeptaCDD, OctaCDD, 1,2,3,4,6,7,8-HeptaCDF,

382

1,2,3,4,7,8,9-HeptaCDF, OctaCDF, and most PCBs for fish,51 which highlights the

383

potential high dioxin-like toxicity might be caused by PCDPSs. Moreover, there is an

384

upward trend for their dioxin-like potencies with an increase in the number of

385

substituted Cl atoms on PCDPSs for zebrafish Ahr2 construct (Tables 1 and S7),

386

which is consistent with that for avian AHR1 constructs20 and rat hepatoma H4IIE-luc

387

cell line.19 ReP values of some PCDPSs for three major vertebrate groups including

388

mammals,16 birds17 and fish are now available. The relatively high ReP values

389

highlight the potential health and ecological risk induced by PCDPSs, especially the

390

ecological risk for fish because it seems to be more responsive to PCDPSs based on

391

the higher ReP values than mammals and birds.

392

Correlations between Results of In Vivo (Early Life-Stage Mortality) and In Vitro

393

(Dioxin-Like Activity) Studies of PCDPSs. The LC50 values of PCDPSs derived

394

from the in vivo experiment were compared to the PC10, PC20, PC50 and PC80 values

395

of PCDPSs determined in zebrafish Ahr2-LRG assays. As shown in Figures 2B and

396

S3, there were extremely significant correlations between in vivo LC50 values and in

397

vitro PC10, PC20, PC50 and PC80 values. A significant correlation was also observed

398

between in vivo RePLC50 values and in vitro LRG RePavg values of PCDPSs either

399

including or excluding 2,3,3’,4,5,6-Hexa-CDPS (Figure 2C). These results suggested

400

the lethal toxicity of PCDPSs increased with the increasing level of Ahr2 activation in

401

zebrafish larvae, thereby further supporting the hypothesis that most, if not all,

402

PCDPS-induced lethal toxicity in early life stages of zebrafish was initiated and

403

mediated by the activation of Ahr2. It has been demonstrated that the zebrafish Ahr2

404

morpholino did not protect embryos from TCDD-induced mortality,24 although the

405

early life-stage mortality caused by TCDD is initiated and mediated by AHR

406

activation in vertebrates.55 This may be due to the high efficiency of Ahr2 in binding ACS Paragon Plus Environment

Page 14 of 35

Page 15 of 35


407

ligands and mediating toxicity probably resulting from fast signaling transduction and

408

protein biosynthesis in zebrafish, as observed in the present study that ahr2 was only

409

upregulated about 2-fold even though cyp1a1 was upregulated more than 100-fold

410

(Figure 1B). Thus, we can reasonably speculate that Ahr2 knockdown would also be

411

ineffective in protecting embryos from TCDD-induced mortality in zebrafish, thereby

412

indicating that developing a knockout model is needed to fully support our conclusion.

413

Thus, developing an Ahr2-homozygous (Ahr2−/−) mutant may be a more reliable

414

method to determine the relationship between early life-stage mortality of zebrafish

415

induced by PCDPS exposure and Ahr2 activation. However, it is time- and

416

labor-consuming. Given that COS-7 cells express no endogenous AHR and very little

417

ARNT,56, 57 and four expression plasmids are just needed to transiently transfected in

418

COS-7 cells, the zebrafish Ahr2-LRG assay thus may be a basically equally reliable,

419

and more cost-effective, time- and labor-saving alternative method to Ahr2 knockout.

420

There is certainly a possibility that the mortality is mediated by some other

421

mechanism or combination of mechanisms.

422

Dose-Dependent GFP Induction in Tg(cyp1a:gfp) Zebrafish Larvae. To visually

423

characterize the dioxin-like activity induced by PCDPS exposure in live zebrafish

424

larvae, we carried out dosage-dependent tests in Tg(cyp1a:gfp) zebrafish larvae with 4

425

to 500 nM PCDPSs. An apparent dosage-dependent increase of GFP expression was

426

observed in the treated larvae after about 120 h of exposure from 2 hpf (Figures 3 and

427

S4) for all PCDPSs tested, which was consistent with that of the total fluorescent area

428

and the maximum fluorescent value represented by the maximum gray value and total

429

area of ROI respectively (Table S9). In addition, GFP was induced mainly in the

430

kidney, intestine and liver and weakly in several other tissues such as olfactory, gills

431

and skin in Tg(cyp1a:gfp) zebrafish larvae. These results further demonstrated the

432

dioxin-like activity of PCDPSs induced in live zebrafish larvae intuitively.

433

MD Simulations: Level of Ahr2 Activation May be Determined by Interactions

434

between the Ligand and Six Key Amino Acid Residues in Ahr2-LBD. MD

435

simulations were performed to understand what factors affect the ability of



436

compounds to activate Ahr2 in zebrafish at atomic level. The ∆G between the

437

biological macromolecule and small ligands is an index describes the binding affinity

438

between ligands and receptors.58 In principle, there is a significantly positive

439

relationship between ln (−∆G ) and RePavg from LRG assay if the potency is

440

determined by the free energy.59 We calculated ∆G between each compound

441

tested and zebrafish Ahr2-LBD. The values of ln (−∆G ) for the compounds

442

tested here were plotted against RePavg in Figure 4A. No significant relationship was

443

observed between these two parameters, which suggested ∆G is not the

444

determinant factor of ReP.

445

Previously, various molecular docking and MD simulation studies on different

446

proteins showed that some specific amino acid residues are particularly important for

447

the biological functions of proteins.60-62 Accordingly, we conjecture that ReP values

448

are determined by the interaction between the compounds tested (ligands) and some

449

specific amino acid residues in Ahr2-LBD. The E between TCDD and all amino

450

acid residues were calculated and shown in Figure 4B. The average values of each

451

E is about -0.8 kJ/mol. The amino acid residues whose E with TCDD are

452

lower than -4 kJ/mol (5 times of the average value) were identified as the key amino

453

acid residues for TCDD, namly Phe300, Tyr327, Ile330, Cys338, His342 and Leu353.

454

Especially, Phe300 has the highest value of E (= -13.04 kJ/mol). As observed in

455

the structure of the complex of compounds tested and zebrafish Ahr2-LBD (Figure

456

4C), all of the ligands locate in the central part of the pocket of Ahr2-LBD. This

457

structural information implies all chemicals share a similar mechanism of activating

458

Ahr2. Binding to the Ahr2 in zebrafish is the common molecular initiating event of

459

dioxin/DLC-induced toxicity that triggers subsequent effects at higher levels of

460

biological organization.51 Thus, the six amino acid residues were reasonably assumed

461

to be also important for other DLCs. The E values between other compounds

462

tested and these residues were then calculated. The total value of E between each

463

compound and the six key amino acid residues (E# ) was shown in Table S10, and


Page 16 of 35

Page 17 of 35


464

plotted against the ln$LRG ReP ) in Figure 4D. A note is that a lower value of

465

E# represents a stronger interaction (e.g. the interaction for -32.13 kJ/mol is larger

466

than the interaction for -7.81 kJ/mol). A linear fitting formula was obtained as

467

followed.

468

ln$LRG ReP ) = -0.29×E# -10

(3)

469

The corresponding R* and P value were 0.79 and 0.0001, respectively. The

470

result suggested the ReP may be mainly determined by the interaction between

471

chemicals and the six key amino acid residues mentioned above. A stronger

472

interaction between ligand and the key residues leads to a higher ReP value.

473

Obviously, this finding from MD simulations should be further examined by

474

experiments. Fraccalvieri et al. have used site-directed mutagenesis and functional

475

analysis to reveal that 3 residues (Tyr296, Thr386, and His388) in zebrafish Ahra1

476

reduce the amount of internal space available to TCDD in cavity and lead to that

477

zebrafish Ahra1 does not bind to TCDD.63 The significant role of Leu 302, Leu309

478

and Leu 347 in mouse AHR has also been proved by site-directed mutagenesis and

479

AHR functional analysis.64 Based on sequence alignment, site-directed mutagenesis

480

and LRG assays, Manning et al. found that amino acid residues 324 and 380 in the

481

AHR1-LBD are the major determinants of avian species sensitivity to PCBs.65 Thus,

482

site-directed mutagenesis and functional analysis would be helpful to examine and

483

determine whether the several specific amino acids in zebrafish Ahr2-LBD are

484

important in ligand binding and ligand-dependent Ahr2 activation, and the studies

485

mentioned above provided paradigms for our future research. Overall, our simulations

486

promote the understanding of the mechanism underlying differences in the level of

487

Ahr2 activation among different dioxins/DLCs in zebrafish at atomic level, and can

488

also help people to predict the ReP value of compounds when experimental data are

489

unavailable or prior to performing experiments. Moreover, the results of MD

490

simulations also supported the hypothesis that PCDPS can bind Ahr2 to trigger the

491

expression of downstream genes.

492

This study strongly supports the hypothesis that the early life-stage mortality of



493

zebrafish induced by waterborne exposure to PCDPSs was mediated by Ahr2

494

activation. Furthermore, the level of Ahr2 activation by the different dioxins/DLCs

495

appear to be due to the differences of interaction between dioxins/DLCs and six key

496

amino acid residues (Phe300, Tyr327, Ile330, Cys338, His342 and Leu353) in

497

Ahr2-LBD based on homology modeling and MD simulations. In addition, the

498

present study fills the data gap of fish-specific ReP of PCDPSs, which seems to

499

increase with an increasing number of substituted Cl atoms. To our knowledge, this is

500

a first report revealing PCDPS-induced lethal toxicity in early life stages of

501

vertebrates is mediated by AHR activation, thereby supporting that PCDPSs should be

502

considered to be DLCs. Furthermore, our findings from MD simulations support the

503

understanding of the mechanism underlying differences in the level of Ahr2 activation

504

among different dioxins/DLCs in zebrafish at atomic level. In addition, from a

505

methodological point of view, the zebrafish Ahr2-LRG assay developed here and the

506

linear model found in MD calculations provide novel useful tools for determination of

507

the ReP of DLCs on activation of Ahr2 in zebrafish.

508 509

SUPPORTING INFORMATION

510

Supporting Information includes further details on preparation of models for

511

simulation, analytical methods, results and discussion on ReP values of the five DLCs

512

tested, Figures S1-S4 and Tables S1−S10 as noted in the text. This material is

513

available free of charge via the Internet at http://pubs.acs.org.

514 515

ACKNOWLEDGMENTS

516

This research is financially supported by the Natural Science Foundation of

517

Shandong Province (No. ZR2016BB19), Natural Science Foundation of University of

518

Jinan (No. XKY1628), National Natural Science Foundation of China (No.

519

21607001), Anhui Provincial Natural Science Foundation (No. 1608085QB45),

520

Science Research Project of Anhui Education Department (No. KJ2015A090),

521

Central Public-interest Scientific Institution Basal Research Fund, CAFS (No.

522

2017HY-ZD0208). Xiaoxiang Wang acknowledges the support from China ACS Paragon Plus Environment

Page 18 of 35

Page 19 of 35


523

Scholarship Council (CSC, 201406190170).

524 525

REFERENCES

526

(1) Nakanishi, H.; Umemoto, N. Thermal-resistant lubricating oil composition for

527

gas turbines or jet engines. 13, JP 2002003878 A. 2002.

528

(2) Munch, R. H.; Thompson, Q. E. Electrical Devices containing readily

529

biodegradable dielectric fluids. 29, CA 1081937. 1980.

530

(3) Pscheidt, J. W.; Peachey, R. E.; Castagnoli, S. 2015 Peach Pest Management

531

Guide for Oregon; Corvallis, Or.: Extension Service, Oregon State University: 2015.

532

(4) Mitchell, L. R. Collaborative study of the determination of endosulfan,

533

endosulfan sulfate, tetrasul, and tetradifon residues in fresh fruits and vegetables. J.

534

Assoc. Off. Anal. Chem. 1976, 59 (1), 209-212.

535

(5) Yang, X.; Zhang, H.; Liu, Y.; Wang, J.; Zhang, Y. C.; Dong, A. J.; Zhao, H. T.;

536

Sun, C. H.; Cui, J. Multiresidue method for determination of 88 pesticides in berry

537

fruits using solid-phase extraction and gas chromatography–mass spectrometry:

538

Determination of 88 pesticides in berries using SPE and GC–MS. Food Chem. 2011,

539

127 (2), 855-865.

540

(6) Fan, C. L.; Li, Y.; Chang, Q. Y.; Pang, G. F.; Kang, J.; Cao, J.; Zhao, Y. B.; Li, N.;

541

Li, Z. Y. High-throughput analytical techniques for multiresidue, multiclass

542

determination of 653 pesticides and chemical pollutants in tea–Part IV: Evaluation of

543

the ruggedness of the method, error analysis, and key control points of the method. J.

544

AOAC Int. 2015, 98 (1), 130-148.

545

(7) Dorweiler, K. J.; Gurav, J. N.; Walbridge, J. S.; Ghatge, V. S.; Savant, R. H.

546

Determination of stability from multicomponent pesticide mixes. J. Agric. Food

547

Chem. 2016, 64 (31), 6108-6124.

548

(8) Mostrag, A.; Puzyn, T.; Haranczyk, M. Modeling the overall persistence and

549

environmental mobility of sulfur-containing polychlorinated organic compounds.

550

Environ. Sci. Pollut. Res. 2010, 17 (2), 470-477.

551

(9) Shi, J.; Zhang, X.; Qu, R.; Xu, Y.; Wang, Z. Synthesis and QSPR study on

552

environment-related properties of polychlorinated diphenyl sulfides (PCDPSs). ACS Paragon Plus Environment


553

Chemosphere 2012, 88 (7), 844-854.

554

(10)Sinkkonen, S.; Vattulainen, A.; Aittola, J. P.; Paasivirta, J.; Tarhanen, J.; Lahtiperä,

555

M. Metal reclamation produces sulphur analogues of toxic dioxins and furans.

556

Chemosphere 1994, 28 (7), 1279-1288.

557

(11) Sinkkonen, S.; Kolehmalnen, E.; Laihia, K.; Koistinen, J.; Rantio, T.

558

Polychlorinated diphenyl sulfides: preparation of model compounds, chromatography,

559

mass spectrometry, NMR, and environmental analysis. Environ. Sci. Technol. 1993,

560

27 (7), 1319-1326.

561

(12) Schwarzbauer, J.; Littke, R.; Weigelt, V. Identification of specific organic

562

contaminants for estimating the contribution of the Elbe river to the pollution of the

563

German Bight. Org. Geochem. 2000, 31 (12), 1713-1731.

564

(13) Zhang, X.; Qin, L.; Qu, R.; Feng, M.; Wei, Z.; Wang, L.; Wang, Z. Occurrence of

565

polychlorinated diphenyl sulfides (PCDPSs) in surface sediments and surface water

566

from the Nanjing section of the Yangtze River. Environ. Sci. Technol. 2014, 48 (19),

567

11429-11436.

568

(14) Verschuuren, H. G.; Kroes, R.; Van Esch, G. J. Toxicity studies on tetrasul I.

569

Acute, long-term and reproduction studies. Toxicology 1973, 1 (1), 63-78.

570

(15) Li, Y.; Li, M.; Shi, J.; Yang, X.; Wang, Z. Hepatic antioxidative responses to

571

PCDPSs and estimated short-term biotoxicity in freshwater fish. Aquat. Toxicol. 2012,

572

120–121 (0), 90-98.

573

(16) Smyth, H. F.; Carpenter, C. P.; Well, C. S.; Pozzani, U. C.; Striegel, J. A.

574

Range-finding toxicity data: List VI. Am. Ind. Hyg. Assoc. J. 1962, 23 (2), 95-107.

575

(17) Zhang, X.; Liu, F.; Chen, B.; Li, Y.; Wang, Z. Acute and subacute oral toxicity of

576

polychlorinated diphenyl sulfides in mice: Determining LD50 and assessing the status

577

of hepatic oxidative stress. Environ. Toxicol. Chem. 2012, 31 (7), 1485-1493.

578

(18) de Tonkelaar, E. M.; van Esch, G. J. No-effect levels organochlorine pesticides

579

based on induction of microsomal liver enzymes in short-term toxicity experiments.

580

Toxicology 1974, 2 (4), 371-380.

581

(19) Zhang, J.; Zhang, X.; Xia, P.; Zhang, R.; Wu, Y.; Xia, J.; Su, G.; Zhang, J.; Giesy,

582

J. P.; Wang, Z.; Villeneuve, D. L.; Yu, H. Activation of AhR-mediated toxicity ACS Paragon Plus Environment

Page 20 of 35

Page 21 of 35


583

pathway by emerging pollutants polychlorinated diphenyl sulfides. Chemosphere

584

2016, 144 (Supplement C), 1754-1762.

585

(20) Zhang, R.; Zhang, X.; Zhang, J.; Qu, R.; Zhang, J.; Liu, X.; Chen, J.; Wang, Z.;

586

Yu, H. Activation of avian aryl hydrocarbon receptor and inter-species sensitivity

587

variations by polychlorinated diphenylsulfides. Environ. Sci. Technol. 2014, 48 (18),

588

10948-10956.

589

(21) Stegeman, J. J.; Goldstone, J. V.; Hahn, M. E. Perspectives on zebrafish as a

590

model in environmental toxicology. In Fish Physiology, Perry, S. F.; Ekker, M.;

591

Farrell, A. P.; Brauner, C. J., Eds. Academic Press: 2010; Vol. 29, pp 367-439.

592

(22) Dai, Y. J.; Jia, Y. F.; Chen, N.; Bian, W. P.; Li, Q. K.; Ma, Y. B.; Chen, Y. L.; Pei,

593

D. S. Zebrafish as a model system to study toxicology. Environ. Toxicol. Chem. 2014,

594

33 (1), 11-17.

595

(23) Perkins, E. J.; Ankley, G. T.; Crofton, K. M.; Garcia-Reyero, N.; Lalone, C. A.;

596

Johnson, M. S.; Tietge, J. E.; Villeneuve, D. L. Current perspectives on the use of

597

alternative species in human health and ecological hazard assessments. Environ.

598

Health Perspect. 2013, 121 (9), 1002-1010.

599

(24) Prasch, A. L.; Teraoka, H.; Carney, S. A.; Dong, W.; Hiraga, T.; Stegeman, J. J.;

600

Heideman,

601

2,3,7,8-tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. Toxicol. Sci.

602

2003, 76 (1), 138-150.

603

(25) Zhang, X.; Shi, J.; Sun, L.; Wang, Z. Palladium-catalyzed carbon-sulfur bond

604

formation to synthesize polychlorinated diphenyl sulfides. Chinese J. Org. Chem.

605

2011, 31 (7), 1107-1113.

606

(26) U.S.EPA. Method 1613 tetra- through octa-chlorinated dioxins and furans by

607

isotope dilution HRGC/HRMS. U. S. Environmental Protection Agency Office of

608

Water. 1994.

609

(27) Xu, H.; Li, C.; Li, Y.; Ng, G. H. B.; Liu, C.; Zhang, X.; Gong, Z. Generation of

610

Tg(cyp1a:gfp) transgenic zebrafish for development of a convenient and sensitive in

611

vivo assay for aryl hydrocarbon receptor activity. Mar. Biotechnol. 2015, 17 (6),

612

831-840.

W.;

Peterson,

R.

E.

Aryl

hydrocarbon


receptor

2

mediates


Page 22 of 35

613

(28) Westerfield, M. The zebrafish book. A guide for the laboratory use of zebrafish

614

(Danio rerio). 4th edition. Eugene: Univ. of Oregon Press. 2000.

615

(29) Liu, C.; Yan, W.; Zhou, B.; Guo, Y.; Liu, H.; Yu, H.; Giesy, J. P.; Wang, J.; Li, G.;

616

Zhang, X. Characterization of a bystander effect induced by the endocrine-disrupting

617

chemical 6-propyl-2-thiouracil in zebrafish embryos. Aquat. Toxicol. 2012, 118

618

(Supplement C), 108-115.

619

(30) Abràmoff, M. D.; Magalhães, P. J.; Ram, S. J. Image processing with ImageJ.

620

Biophotonics international 2004, 11 (7), 36-42.

621

(31) Liu, C.; Wang, Q.; Liang, K.; Liu, J.; Zhou, B.; Zhang, X.; Liu, H.; Giesy, J. P.;

622

Yu, H. Effects of tris(1,3-dichloro-2-propyl) phosphate and triphenyl phosphate on

623

receptor-associated mRNA expression in zebrafish embryos/larvae. Aquat. Toxicol.

624

2013, 128-129, 147-157.

625

(32) Zhu, Y.; Ma, X.; Su, G.; Yu, L.; Letcher, R. J.; Hou, J.; Yu, H.; Giesy, J. P.; Liu, C.

626

Environmentally

627

tris(1,3-dichloro-2-propyl) phosphate inhibit growth of female zebrafish and decrease

628

fecundity. Environ. Sci. Technol. 2015, 49 (24), 14579-14587.

629

(33) Farmahin, R.; Wu, D.; Crump, D.; Hervé, J. C.; Jones, S. P.; Hahn, M. E.;

630

Karchner, S. I.; Giesy, J. P.; Bursian, S. J.; Zwiernik, M. J.; Kennedy, S. W. Sequence

631

and in vitro function of chicken, ring-necked pheasant, and Japanese quail AHR1

632

predict in vivo sensitivity to dioxins. Environ. Sci. Technol. 2012, 46 (5), 2967-2975.

633

(34) Abnet, C. C.; Tanguay, R. L.; Heideman, W.; Peterson, R. E. Transactivation

634

activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed

635

in COS-7 cells: Greater insight into species differences in toxic potency of

636

polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners. Toxicol.

637

Appl. Pharmacol. 1999, 159 (1), 41-51.

638

(35) Han, D.; Nagy, S. R.; Denison, M. S. Comparison of recombinant cell bioassays

639

for the detection of Ah receptor agonists. Biofactors 2004, 20 (1), 11-22.

640

(36) Rushing, S. R.; Denison, M. S. The silencing mediator of retinoic acid and

641

thyroid hormone receptors can interact with the aryl hydrocarbon (Ah) receptor but

642

fails to repress Ah receptor-dependent gene expression. Arch. Biochem. Biophys. 2002,

relevant

concentrations

of


the

flame

retardant

Page 23 of 35


643

403 (2), 189-201.

644

(37) Prasch, A. L.; Tanguay, R. L.; Mehta, V.; Heideman, W.; Peterson, R. E.

645

Identification of zebrafish ARNT1 homologs: 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

646

toxicity in the developing zebrafish requires ARNT1. Mol. Pharmacol. 2006, 69 (3),

647

776-787.

648

(38)Organisation for Economic Co-operation and Development (OECD). Test No. 455:

649

The Stably Transfected Human Estrogen Receptor-á Transcriptional Activation Assay

650

for Detection of Estrogenic Agonist-Activity of Chemicals. OECD Publishing. OECD

651

Guideline for the Testing of Chemicals, Section 4: Health Effects. 2009.

652

(39) Head,

653

ethoxyresorufin-O-deethylase activity and cytochrome P4501A mRNA abundance in

654

chicken embryo hepatocytes. Anal. Biochem. 2007, 360 (2), 294-302.

655

(40) Abraham, M. J.; Murtola, T.; Schulz, R.; Páll, S.; Smith, J. C.; Hess, B.; Lindahl,

656

E. GROMACS: High performance molecular simulations through multi-level

657

parallelism from laptops to supercomputers. SoftwareX 2015, 1, 19-25.

658

(41)Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S. A.;

659

Karplus, M. CHARMM: a program for macromolecular energy, minimization, and

660

dynamics calculations. J. Comput. Chem. 1983, 4 (2), 187-217.

661

(42) Zoete, V.; Cuendet, M. A.; Grosdidier, A.; Michielin, O. SwissParam: a fast force

662

field generation tool for small organic molecules. J. Comput. Chem. 2011, 32 (11),

663

2359-2368.

664

(43) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L.

665

Comparison of simple potential functions for simulating liquid water. The Journal of

666

chemical physics 1983, 79 (2), 926-935.

667

(44) Garrett, B. C.; Redmon, M. J.; Steckler, R.; Truhlar, D. G.; Baldridge, K. K.;

668

Bartol, D.; Schmidt, M. W.; Gordon, M. S. Algorithms and accuracy requirements for

669

computing reaction paths by the method of steepest descent. J. Phys. Chem 1988, 92

670

(1476), e1488.

671

(45) Darden, T.; York, D.; Pedersen, L. Particle mesh Ewald: An N⋅log (N) method for

672

Ewald sums in large systems. The Journal of chemical physics 1993, 98 (12),

J.

A.;

Kennedy,

S.

W.

Same-sample


analysis

of


673

10089-10092.

674

(46) Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Electrostatics

675

of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci.

676

U. S. A. 2001, 98 (18), 10037-10041.

677

(47) Kumari, R.; Kumar, R.; Lynn, A.; Consortium, O. S. D. D. g_mmpbsa–A

678

GROMACS tool for high-throughput MM-PBSA calculations. J. Chem. Inf. Model

679

2014, 54 (7), 1951-1962.

680

(48) Chen, Q.; Wang, X.; Shi, W.; Yu, H.; Zhang, X.; Giesy, J. P. Identification of

681

thyroid hormone disruptors among HO-PBDEs: in vitro investigations and coregulator

682

involved simulations. Environ. Sci. Technol. 2016, 50 (22), 12429-12438.

683

(49) Zhang, X.; Wiseman, S.; Yu, H.; Liu, H.; Giesy, J. P.; Hecker, M. Assessing the

684

toxicity of naphthenic acids using a microbial genome wide live cell reporter array

685

system. Environ. Sci. Technol. 2011, 45 (5), 1984-1991.

686

(50) Denison, M. S.; Fisher, J. M.; Whitlock, J. P. Inducible, receptor-dependent

687

protein-DNA interactions at a dioxin-responsive transcriptional enhancer. Proc. Natl.

688

Acad. Sci. U. S. A. 1988, 85 (8), 2528-2532.

689

(51) Van den Berg, M.; Birnbaum, L.; Bosveld, A.; Brunström, B.; Cook, P.; Feeley,

690

M.; Giesy, J. P.; Hanberg, A.; Hasegawa, R.; Kennedy, S. W.; Kubiak, T.; Larsen, J. C.;

691

van Leeuwen, F. X.; Liem, A. K.; Nolt, C.; Peterson, R. E.; Poellinger, L.; Safe, S.;

692

Schrenk, D.; Tillitt, D.; Tysklind, M.; Younes, M.; Waern, F.; Zacharewski, T. Toxic

693

equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

694

Environ. Health Perspect. 1998, 106 (12), 775-792.

695

(52) Seok, S. H.; Baek, M. W.; Lee, H. Y.; Kim, D. J.; Na, Y. R.; Noh, K. J.; Park, S.

696

H.; Lee, H. K.; Lee, B. H.; Park, J. H. In vivo alternative testing with zebrafish in

697

ecotoxicology. J. Vet. Sci. 2008, 9 (4), 351-357.

698

(53) Ton, C.; Lin, Y.; Willett, C. Zebrafish as a model for developmental neurotoxicity

699

testing. Birth Defects Res. A Clin. Mol. Teratol. 2006, 76 (7), 553-567.

700

(54) Doering, J. A.; Giesy, J. P.; Wiseman, S.; Hecker, M. Predicting the sensitivity of

701

fishes to dioxin-like compounds: possible role of the aryl hydrocarbon receptor (AhR)

702

ligand binding domain. Environ. Sci. Pollut. Res. 2013, 20 (3), 1219-1224. ACS Paragon Plus Environment

Page 24 of 35

Page 25 of 35


703

(55) Ankley, G. T.; Bennett, R. S.; Erickson, R. J.; Hoff, D. J.; Hornung, M. W.;

704

Johnson, R. D.; Mount, D. R.; Nichols, J. W.; Russom, C. L.; Schmieder, P. K.;

705

Serrrano, J. A.; Tietge, J. E.; Villeneuve, D. L. Adverse outcome pathways: A

706

conceptual framework to support ecotoxicology research and risk assessment. Environ.

707

Toxicol. Chem. 2010, 29 (3), 730-741.

708

(56) Ema, M.; Matsushita, N.; Sogawa, K.; Ariyama, T.; Inazawa, J.; Nemoto, T.; Ota,

709

M.; Oshimura, M.; Fujii-Kuriyama, Y. Human arylhydrocarbon receptor: functional

710

expression and chromosomal assignment to 7p21. The Journal of Biochemistry 1994,

711

116 (4), 845-851.

712

(57) Ema, M.; Ohe, N.; Suzuki, M.; Mimura, J.; Sogawa, K.; Ikawa, S.;

713

Fujii-Kuriyama, Y. Dioxin binding activities of polymorphic forms of mouse and

714

human arylhydrocarbon receptors. J. Biol. Chem. 1994, 269 (44), 27337-27343.

715

(58) Wlodawer, A. Rational approach to AIDS drug design through structural biology.

716

Annu. Rev. Med. 2002, 53 (1), 595-614.

717

(59) Sharp, K. A. Statistical thermodynamics of binding and molecular recognition

718

models. Prot.-Lig. Interact 2012, 3.

719

(60) Oshima, A.; Doi, T.; Mitsuoka, K.; Maeda, S.; Fujiyoshi, Y. Roles of Met-34,

720

Cys-64, and Arg-75 in the assembly of human connexin 26 Implication for key amino

721

acid residues for channel formation and function. J. Biol. Chem. 2003, 278 (3),

722

1807-1816.

723

(61) Szklarz, G. D.; He, Y. A.; Halpert, J. R. Site-directed mutagenesis as a tool for

724

molecular modeling of cytochrome P450 2B1. Biochemistry 1995, 34 (44),

725

14312-14322.

726

(62) Mukrasch, M. D.; Markwick, P.; Biernat, J.; von Bergen, M.; Bernadó, P.;

727

Griesinger, C.; Mandelkow, E.; Zweckstetter, M.; Blackledge, M. Highly populated

728

turn conformations in natively unfolded tau protein identified from residual dipolar

729

couplings and molecular simulation. J. Am. Chem. Soc. 2007, 129 (16), 5235-5243.

730

(63) Fraccalvieri, D.; Soshilov, A. A.; Karchner, S. I.; Franks, D. G.; Pandini, A.;

731

Bonati, L.; Hahn, M. E.; Denison, M. S. Comparative analysis of homology models of

732

the Ah receptor ligand binding domain: verification of structure–function predictions ACS Paragon Plus Environment


733

by site-directed mutagenesis of a nonfunctional receptor. Biochemistry 2013, 52 (4),

734

714-725.

735

(64) Motto, I.; Bordogna, A.; Soshilov, A. A.; Denison, M. S.; Bonati, L. New aryl

736

hydrocarbon receptor homology model targeted to improve docking reliability. J.

737

Chem. Inf. Model. 2011, 51 (11), 2868-2881.

738

(65) Manning, G. E.; Farmahin, R.; Crump, D.; Jones, S. P.; Klein, J.; Konstantinov,

739

A.; Potter, D.; Kennedy, S. W. A luciferase reporter gene assay and aryl hydrocarbon

740

receptor 1 genotype predict the LD50 of polychlorinated biphenyls in avian species.

741

Toxicol. Appl. Pharmacol. 2012, 263 (3), 390-401.


Page 26 of 35

Page 27 of 35


FIGURE LEGENDS

742 743 744

Figure 1. (A): Concentration-dependent mortality of zebrafish embryos/larvae

745

following waterborne exposure to different concentrations of PCDPS congeners from

746

2 to 120 hpf. Points represent the mean percent mortality of three replicates. Bars

747

represent standard error. (B): Clustering of the concentration-dependent expression of

748

the 6 genes involved in dioxin-like toxicity in zebrafish following exposure to PCDPS

749

congeners from 2 to 120 hpf. Classification and visualization of the gene expression

750

were derived by use of ToxClust.49 The dissimilarity between genes was calculated by

751

the Manhattan distance between the mRNA expression at every concentration. The

752

fold change of gene expression was indicated by color gradient. Gene expression was

753

determined at three exposure concentrations (20, 100 and 500 nM), and displayed

754

from left to right in the rectangle area labeled by each PCDPS tested. (C): Linear

755

regression analyses comparing mRNA expressions of cyp1a1/cyp1b1 with the in vivo

756

log-transformed LC50 values of PCDPSs in zebrafish embryos/larvae following

757

exposure to PCDPSs. Points represent the average of mRNA expression of three

758

biological replicates with the mean LC50 value for each PCDPS tested. Bars represent

759

standard error.

760 761

Figure 2. (A): Concentration-dependent effects of TCDD, the five DLCs and the six

762

PCDPS congeners on Ahr2-mediated LRG activity in COS-7 cells transfected with

763

zebrafish Ahr2 construct. Data are presented as percent response values relative to

764

that of a 300 nM TCDD positive control. Points represent mean, positive

765

control-normalized luciferase ratios obtained from 3 independent experiments, each

766

with 4 technical replicates per concentration. Bars represent standard error. (B):

767

Linear regression analysis comparing in vivo LC50 values of PCDPSs in zebrafish

768

embryos/larvae with PC50 values determined in zebrafish Ahr2-LRG assays. Points

769

represent the average of three replicates. Bars represent standard error. (C): Linear

770

regression analysis comparing ReP values derived from in vivo LC50 values of

771

PCDPSs in zebrafish embryos/larvae with the average ReP values determined in ACS Paragon Plus Environment


772

zebrafish Ahr2-LRG assays.

773 774

Figure 3. Dose-dependent green fluorescent protein (GFP) induction in Tg(cyp1a:gfp)

775

transgenic zebrafish larvae by 2,3,3’,4,5,6-Hexa-CDPS at 120 hpf. GFP expression

776

was photographed under a fluorescent microscope. Scale bar: 1 mm in

777

27.6×magnification.

778 779

Figure 4. The energetic and structural information from MD simulations. (A) The

780

relationship between RePavg values of the compounds tested (namely TCDD, the five

781

DLCs and the six PCDPS congeners) derived from zebrafish Ahr2-LRG assays and

782

ln (−∆G ). Points represent the average ReP value calculated from EC50-, PC10-,

783

PC20-, PC50- and PC80-based ReP values with the average ln (−∆G )

784

determined in MD simulations. Bars represent standard error. (B) The molecular

785

mechanics energy between TCDD and different amino acid residues in the

786

ligand-binding domain (LBD) of Ahr2 in zebrafish. (C) The structure of

787

ligands-Ahr2-LBD complex. α-helixes are shown in salmon, β-sheets in light orange,

788

loops in white, and ligands (namely TCDD, the five DLCs and the six PCDPSs) in

789

grey. Six key residues are shown in purple (Phe300), cyan (Tyr327), yellow (Ile330),

790

blue (Cys338), green (His342) and red (Leu353), respectively. (D) Linear regression

791

analysis between ln (LRG ReP ) and E# . Points represent the average ReP value

792

calculated from EC50-, PC10-, PC20-, PC50- and PC80-based ReP values. Bars represent

793

standard error.

794


Page 28 of 35

Page 29 of 35

795 796


Figure 1



797 798

Figure 2


Page 30 of 35

Page 31 of 35


799 800

Figure 3



801 802 803

Figure 4


Page 32 of 35

Page 33 of 35


TABLE LEGENDS

804 805 806

Table 1. Endpoints determined for Ahr2-mediated LRG Activity in COS-7 cells

807

transfected with the zebrafish Ahr2 construct and LC50 values determined in zebrafish

808

embryos/larvae

809

2,3,7,8-TCDF, 1,2,3,7,8-PeCDF, PCB126 or each PCDPS congener. EC50, PC10, PC20,

810

PC50, PC80 and maximal response values represent the average of three replicates ±

811

standard error (SE) obtained from three 96-well plates for each compound. LRG

812

activity values were normalized to responses of 300 nM TCDD positive control (PC).

813

Maximal response values were obtained from the curve fit, unless otherwise indicated.

814

LC50 values of TCDD and PCDPSs represent the average of three replicates ± SE

815

obtained from three beakers per treatment. The average relative potency (RePavg)

816

values were calculated from EC50-, PC10-, PC20-, PC50- and PC80-based ReP values

817

(Table S7, Supporting Information). The RePLC50 values were calculated based on

818

LC50 values from in vivo bioassays.

exposed

to

TCDD,

1,2,3,7,8-PeCDD,


1,2,3,7,8,9-HxCDD,


819

820 821 822 823 824 825

Page 34 of 35

Table 1 Compound

EC50±SE (nM)

PC10±SE (nM)

PC20±SE (nM)

PC50±SE (nM)

PC80±SE (nM)

Max. response±SE (% PC)

LC50±SE (nM)

RePavg

RePLC50

1,2,3,7,8-PeCDD 1,2,3,7,8,9-HxCDD 2,3,7,8-TCDF 1,2,3,7,8-PeCDF PCB126 TCDD 2,3,3’,4,5,6-Hexa-CDPS 2,2’,3,3’,4,5,6-Hepta-CDPS 2,2’,3’,4,5-Penta-CDPS 2,4,4’,5-TCDPS 2,2’,4-Tris-CDPS 4,4’-DCDPS

NC 74.4±4.4 10.9±0.66 NC 266±35 6.76±0.53a 158±2.1b NC NC NC NC NC

0.297±0.011 2.30±0.33 1.83±0.097 3.41±0.75 10.5±7.7 1.02±0.26a 41.1±2.1a 191±10b 203±35b 330±14c 435±8.0d 462±9.4d

0.723±0.0075 7.19±0.59 3.94±0.10 6.27±1.1 33.1±22 1.94±0.34a 64.0±2.2b 401±16c 443±38c 617±6.1d 719±9.0e 825±3.0f

2.70±0.075 45.0±2.0 16.8±1.1 16.9±1.7 225±184 5.55±0.48a 131±1.4a 1229±163b 1391±32b 1673±81c 1628±30c 2213±33d

6.30±0.11 197±14 564±2.8 38.5±2.4 1305±64 13.6±1.7a 234±6.9a 2613±364b 2852±111bc 3633±77d 3186±94cd NE

139±11† 108±1.5 81.0±0.59 106±5.1† 106±18 114±14a 120±1.8a 107±5.7†ab 107±0.95†ab 90.9±2.8†bc 95.5±4.2†b 76.4±2.5†c

NA NA NA NA NA 38.9±13‡a 475±14a 2226±55b 2287±665bc 2973±98c 2862±57bc 4029±159d

2.6 0.20 0.41 0.32 0.043 1.0 0.040 5.0×10-3 4.5×10-3 3.3×10-3 3.2×10-3 2.4×10-3

NA NA NA NA NA 1.0 0.082 0.017 0.017 0.013 0.014 0.010

Superscript letters indicate significant differences among TCDD and PCDPSs treatments (p < 0.05). NC: Not calculated because a plateau was not reached. NA: No published data available. NE: Not estimated because the maximum observed response was below 80% of positive control response. † A plateau was not reached. Values represent the highest observed response. ‡ It was estimated based on two published data.52, 53


Page 35 of 35

826


TOC ART

827 828


Polychlorinated Diphenylsulfides Activate Aryl Hydrocarbon Receptor

Recommend Documents