Uptake, Translocation, and Biotransformation of Organophosphorus

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Uptake, Translocation, and Biotransformation of Organophosphorus Esters in Wheat (Triticum aestivum L.) Weining Wan, Honglin Huang, Jitao Lv, Ruixia Han, and Shuzhen Zhang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b01758 • Publication Date (Web): 10 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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Environmental Science & Technology

1

Uptake, Translocation, and Biotransformation of Organophosphorus Esters in

2

Wheat (Triticum aestivum L.)

3

Weining Wan,† ,‡ Honglin Huang,† Jitao Lv,† Ruixia Han,† ,‡ Shuzhen Zhang,*,†,‡

4

† State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

5

Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box

6

2871, Beijing 100085, China

7

‡ University of Chinese Academy of Sciences, Beijing 100049, China

8 9

ACS Paragon Plus Environment


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ABSTRACT: The uptake, translocation and biotransformation of organophosphate

11

esters (OPEs) by wheat (Triticum aestivum L.) were investigated by a hydroponic

12

experiment. The results demonstrated that OPEs with higher hydrophobicity were

13

more easily taken up by roots, and OPEs with lower hydrophobicity were more liable

14

to be translocated acropetally. A total of 43 metabolites including dealkylated,

15

oxidatively dechlorinated, hydroxylated, methoxylated, and glutathione- and

16

glucuronide- conjugated products were detected derived from eight OPEs, with

17

di-esters formed by direct dealkylation from the parent tri-esters as the major products,

18

followed with hydroxylated tri-esters. Molecular interactions of OPEs with plant

19

biomacromolecules were further characterized by homology modeling combined with

20

molecular docking. OPEs with higher hydrophobicity were more liable to bind with

21

TaLTP1.1, the most important wheat non-specific lipid transfer protein, consistent

22

with the experimental observation that OPEs with higher hydrophobicity were more

23

easily taken up by wheat roots. Characterization of molecular interactions between

24

OPEs and wheat enzymes suggested that OPEs were selectively bound to TaGST4-4

25

and CYP71C6v1 with different binding affinities, which determined their abilities to

26

be metabolized and form metabolite products in wheat. This study provides both

27

experimental

28

biotransformation of OPEs in plants.

and

theoretical

evidence for

the

uptake,

29 30 31 32 33 34 35


accumulation

and

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36 37 38 39 40 41 42 43 44 45

TOC

46



47

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INTRODUCTION

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Except the use as insecticides in history, organophosphate esters (OPEs) are

49

widely used as flame retardants and plasticizers in a variety of industrial applications

50

and household products, particularly after the implementation of regulations phasing

51

out the production and usage of polybrominated diphenyl ethers in many countries in

52

recent years.1 OPEs are mainly used as non-reactive additives in these products and

53

are liable to leach out to the environment. Among the OPEs, tris(1-chloro-2-propyl)

54

phosphate

55

tri(2-butoxyethyl) phosphate (TBEP) have been suspected to be carcinogenic,2-4 while

56

triethyl-chloro-phosphate (TCEP), tributyl phosphate (TnBP) and tri-phenyl

57

phosphate (TPhP) have been observed to have neurotoxic effects,5,6 which may pose

58

threats to human health. Because of their increasing consumption volumes and

59

potentially adverse effects, the behaviors of OPE plasticizers and flame retardants in

60

the environment have attracted increasing attention from both the public and

61

researchers.2 Due to the particular purpose of this study, we will use the term OPEs

62

exclusively

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organophosphate pesticides.

(TCPP),

tri(1,3-dichloro-2-propyl)

for OPE flame

retardants

and

phosphate

(TDCPP)

plasticisers, and

and

not include

64

Contamination by OPEs has been detected in various environmental matrices such

65

as soil,7-8 water,9-10 and air,11-12 and soil is one of their major sink in the

66

environment.1,13-14 OPEs have been detected in soils, and high concentrations were

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found in soils at sites near contamination sources.7,8 For example, tri-phenyl

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phosphate (TPhP) was detected at concentrations of 11-3300 ng/g dry weight (dw) in

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the surface soils around an e-waste recycling workshop in northern Vietnam.7 In our

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recent work in North China, we detected eight OPEs in the soils not only at plastic

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waste treatment sites, with total concentrations of 38-1250 ng/g dw, but also in the


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nearby farmlands, with the total concentration range of 38-162 ng/g dw.8 The uptake

73

and accumulation of OPEs by crops were also evidenced in this field investigation,

74

with all the eight OPEs detected in wheat tissues. Therefore, plant uptake and

75

accumulation may provide the potential for OPEs to transfer into the food chain and

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cause a threat to human health. However, to date, studies on plant uptake of OPE

77

plasticizers and flame retardants are scarce. There have been only two works

78

demonstrating the uptake and translocation of OPEs by plants.15-17 Nevertheless, both

79

of these works involved very limited OPE congeners. The factors that determine plant

80

uptake, translocation and accumulation are far from clear.

81

Transformation of organic contaminants in the environment may bring additional

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adverse influences to bear on the environment and human health due to the fact that

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the biological effects of metabolites may be different compared with precursor

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compounds. Therefore, transformation of organic contaminants in biota and the

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environment has attracted increasing research attention. Several studies have detected

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the metabolites of OPEs in human urine samples, with di-esters as the major

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degradation products from the parent tri-esters,18,19 and Reemtsma et al.20 have also

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found mono-esters generated by further hydrolysis of di-esters. So far only a few

89

experiments have been performed on in vitro or in vivo biotransformation of

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OPEs.21-25 Wang et al.21 observed metabolism of TPhP by zebrafish, with di-phenyl

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phosphate (DPhP) as the major product in the liver and intestine. Bis(2-chloroethyl)

92

phosphate (BCEP) and bis(1-chloro-2-propyl) phosphate (BCPP) were recently

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reported as important metabolites of TCEP and TCPP in human liver microsomes and

94

S9 fractions.22,23 However, these studies mainly focused on humans or animals.

95

Biotransformation of OPE plasticizers and flame retardants in plants has not yet been

96

reported.



97

OPEs all contain the same phosphate base unit. The chemical and physical

98

properties vary extensively depending on the specific moiety of each OPE.

99

Differences in the polarity and functional groups of OPEs have the key influences on

100

their interaction with organisms.2,26,27 Therefore, it is expected that information on the

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interaction between plant biomacromolecules and OPEs would be very helpful in

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elucidating the molecular mechanisms of accumulation and transformation of OPEs in

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plants. Plant non-specific lipid transfer proteins (nsLTPs) are biomacromolecules in

104

all terrestrial plants and provide the key sites for lipid binding.28,29 They have been

105

also reported to function in binding of hydrophobic xenobiotics in plants.30,31 The

106

isozymes glutathione-S-transferase (GST) and cytochrome P450 monooxygenases

107

(CYP450) have been demonstrated to play the key roles in plant metabolization of

108

organic pollutants.32-34 In wheat, TaLTP1.1 has been reported to be the most important

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nsLTP,16 and TaGST4-4 and CYP71C6v1 are isozymes confirmed to take part in the

110

metabolism of herbicides.35,36 Homology modeling can construct three-dimensional

111

(3D) models of unknown proteins, which combined with molecular docking has been

112

successfully applied to characterize the interactions of some organic pollutants, such

113

as polychlorinated biphenyls and pesticides, with isozymes.37-39 The function of

114

enzymes can be predicted by their 3-D structures, and the enzyme active sites, binding

115

modes and binding affinities between contaminants and enzymes can be characterized

116

by molecular docking. This modeling is a valuable way to apply such information to

117

clarify the molecular mechanisms of accumulation and biotransformation of OPEs in

118

plants.

119

Therefore, the objectives of this study were to investigate the uptake, translocation

120

and biotransformation of OPEs in wheat by a hydroponic experiment. Attempts were

121

then made to clarify the molecular interactions between OPEs and wheat nsLTP and


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the isozymes TaGST4-4 and CYP71C6v1 using homology modeling combined with

123

molecular docking. The theoretical results obtained were further used to explain the

124

uptake and transformation of different OPEs in wheat in vivo. This study represents a

125

thorough investigation on uptake and transformation of OPEs in plants.

126

MATERIALS AND METHODS

127

Chemicals and Reagents. Standards including TCEP, TCPP, TDCPP, TBEP, TnBP,

128

TPhP and EHDPP, and surrogate standards d27-TnBP, d15-TPhP, and d10-BDCPP

129

were purchased from Wellington Laboratories, Inc. (Guelph, Ontario, Canada). The

130

standard TiBP was obtained from AccuStandard (New Haven, USA). OPE metabolite

131

standards BCEP, BCPP, BDCPP, BBEP, DnBP, DiBP, MnBP and the surrogate

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standard d10-DPhP were custom synthesized by Toronto Research Chemicals

133

(Toronto, Ontario, Canada). Metabolite standards MPhP and DPhP were purchased

134

from Tokyo Chemical Industry Development Co., Ltd. (Shanghai, China).

135

Pentafluorobenzyl bromide (PFBBr) was purchased from Aldrich Chemical Co. Inc.

136

(Steinheim, Germany). Purities of all the standards were higher than 97%. The

137

chemical names, abbreviations, formulae and properties of OPEs and metabolites are

138

listed in Table S1 of the Supporting Information (SI). Solvents, including

139

dichloromethane, n-hexane, ethyl acetate, methanol, acetonitrile, acetone and toluene,

140

were of HPLC grade (Thermo fisher, MA, USA). Deionized water (18.2 MΩ) was

141

prepared using a Milli-Q Advantage water purification system (US Millipore, Bedford,

142

MA, USA). All other chemicals used were of analytical grade (Sinopharm Chemical

143

Reagent Co., Ltd, Beijing, China). Anhydrous sodium sulfate (Na2SO4) (60 mesh),

144

neutral silica gel, Florisil and alumina (100-200 mesh) were purchased from Fisher

145

Scientific Inc. (Fair Lawn, NJ).

146

Plant Exposure Experiment. A hydroponic experiment was conducted to investigate



147

the uptake, translocation and biotransformation of organophosphate esters (OPEs) by

148

wheat (Triticum aestivum L.). Wheat seeds were obtained from the Chinese Academy

149

of Agricultural Sciences, Beijing, China. Prior to germination, seeds of similar size

150

were selected and surface-sterilized in 3% (v/v) H2O2 for 15 min, followed by

151

thorough washing with sterilized deionized water, and subsequently germinated on

152

moist filter paper in the dark at 25 °C. After germination, seedlings of uniform size

153

were transferred to glass containers containing autoclaved half-strength Hoagland

154

nutrient solution for cultivation in a controlled environment growth chamber for five

155

days. All the containers, paper and deionized water were autoclaved to prevent any

156

possible introduction of foreign microorganisms.

157

Batches of fifteen uniformly sized wheat seedlings were transferred to autoclaved

158

150 mL glass-stoppered flasks for exposure. The mouth of the containers was covered

159

with aluminum foil and sealed with parafilm. The containers were wrapped with

160

light-proof paper to prevent the photolysis of OPEs. Standard solutions of OPEs

161

dissolved in methanol were added to the sterile nutrient solution to prepare the

162

exposure solutions at nominal concentration of 75 µg/L, which exceeded their

163

environmentally relevant concentration, but was necessary for the determination of

164

their accumulation and particularly their metabolism in plant tissues. The initial

165

concentrations of TCEP, TCPP, TDCPP, TBEP, TnBP, TiBP, TPhP and EHDPP

166

determined in the spiked exposure solutions were 76.2 ± 1.5, 72.5 ± 2.6, 73.8 ± 2.2,

167

71.4 ± 2.4, 69.6 ± 1.8, 74.0 ± 3.1, 75.8 ± 2.7 and 71.0 ± 2.3 µg/L (n = 3), respectively.

168

The volume of methanol in the exposure solutions was less than 1‰ (v/v), and the pH

169

was adjusted to 6.5. Then 120 mL of the exposure solutions of each OPE were added

170

individually to each of the containers. Pots were kept in a controlled environment

171

growth chamber at a light intensity of 250 µmol/m-2s-1 provided by supplementary


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illumination with a photoperiod of 14 h each day. The day/night temperature regime

173

was 22/20 °C and the relative humidity was 70%. Approximately 20 mL/d of sterile

174

half-strength Hoagland nutrient solution saturated with oxygen was injected into each

175

container to compensate for transpiration losses. The containers were positioned

176

randomly and re-randomized every day. Plants were harvested at the intervals of 12,

177

24, 36, 48, 72, 96, 144, 192 and 240 h. At the end of the exposure, unplanted,

178

untreated and root exudate controls were also sampled. All the treatments were

179

performed in triplicate.

180

Three types of controls were included. An untreated plant control without OPEs in

181

the nutrient solution was used to monitor possible cross-contamination due to

182

volatilization of OPEs. OPE-spiked nutrient solution controls without plants were set

183

as unplanted controls to monitor any possible volatilization or abiotic degradation of

184

OPEs. Root exudate controls were carried out mainly to explore the combined roles of

185

root exudates and microbes, with details supplied in SI.

186

Sample Preparation. Roots were thoroughly rinsed with sterilized deionized water,

187

and the rinse water was collected and combined with the exposure solutions for the

188

analysis of OPEs and their metabolites in solutions. Wheat seedlings were separated

189

into roots and shoots for the subsequent analysis. All the harvested plant samples were

190

freeze-dried at -50 °C for 48 h in a lyophilizer (FD-1, Boyikang Instrument Ltd,

191

Beijing, China), weighed and finely chopped, then stored at -20 °C for analysis. The

192

solution samples were extracted immediately after sampling. The extraction, cleanup,

193

and analysis procedures were based on our previous method.8 Briefly, 0.3-0.8 g (dry

194

weight) of plant samples or 50 mL of exposed solutions were spiked with 10 ng of

195

surrogates d27-TnBP and d15-TPhP for OPEs, and d10-BDCPP and d10-DPhP for

196

metabolites. Then the plant samples were subjected to Soxhlet extraction with a



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197

n-hexane/DCM mixture [1:1 (v/v)], and solution samples were extracted with Oasis®

198

HLB SPE cartridges (Waters, Milford, MA). Details of extraction and cleanup of plant

199

and solution samples are provided in the SI.

200

Instrumental Analysis. OPEs were separated with a HP-5MS column (30 m × 0.25

201

mm × 0.25 µm) (Agilent J&W Scientific, USA) and analyzed by an Agilent 7890

202

chromatography-mass spectrometry system (5975 Inert) (Agilent, Palo Alto, USA).

203

The MS system was operated in selected ion monitoring (SIM) mode under EI

204

ionization for quantitation. Helium was used as the carrier gas at a flow rate of 1.5

205

mL/min. The oven temperature program was as follows: 1 min at 50 °C, first ramp at

206

10 °C/min to 200 °C (1 min hold), second ramp at 3 °C/min to 260 °C (5 min hold).

207

The injected volume was 1 µL, and the injection-port temperature was 280 °C in the

208

splitless mode. The electron energy was set at 70 eV, and the ion source temperature

209

was 230 °C.

210

Quantification of the metabolites including BCEP, BCPP, BDCPP, BBEP, DiBP,

211

DnBP, DPhP, MnBP and MPhP was performed by GC-MS (Agilent J&W Scientific,

212

USA) under the SIM mode with a DB-35MS column (60 m × 0.25 mm × 0.25 µm).

213

The oven temperature program was as follows: 1 min at 70 °C, first ramp at 5 °C/min

214

to 110 °C (3 min hold), second ramp at 10 °C/min to 290 °C (3 min hold). Other

215

instrument conditions were the same as the ones for the parent compounds. Possible

216

metabolites of OPEs having no corresponding commercial standards were identified

217

by UPLC coupled with electrospray ionization quadrupole time-of-flight tandem mass

218

spectrometry

219

Chromatographic separation of the metabolites was achieved using a BEH C18 column

220

(50 mm × 2.1 mm × 1.7 µm, Waters) with 5 mM ammonium acetate and methanol as

221

mobile phases. The flow rate was 0.15 mL/min and the injection volume was 20 µL.

(ESI-Q-TOF-MS)

(Agilent

Technologies,


Santa

Clara,

CA).

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222

The products observed were assigned according to their [M-H]- m/z and retention

223

time. It was assumed that the response factors for the isomers of metabolites were

224

equal to their corresponding parent compounds based on several studies,40,41 and

225

concentrations were semi-quantified by using peak areas. Details of the analysis of

226

OPEs and metabolites are provided in the SI, and the mass spectrometer operational

227

parameters are available in Table S2.

228

Quality Assurance and Quality Control. Quality control was performed by regular

229

analysis of the procedural blanks, blind duplicate samples, and random injection of

230

solvent blanks and standards. Recoveries were obtained by the addition of 20.0, 100.0

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and 500.0 ng of OPE standards, surrogate standards and metabolites in methanol at

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concentration of 1 mg/mL individually to the freeze-dried and pulverized untreated

233

control plant samples and nutrient solutions before extraction and analysis. The

234

average recoveries (n = 3) of different OPEs ranged from 75.6% to 90.0% for plant

235

samples, and from 80.2% to 104.5% for solution samples, respectively. The average

236

recoveries (n = 3) of the metabolites in plant and solution samples were 71.3%-82.4%

237

and 77.9%-92.1%, respectively. Recoveries of surrogate standards were as follows:

238

77.4-86.5% for d27-TnBP, 79.7-89.6% for d15-TPhP, 76.2-85.7% for d10-BDCPP

239

and 73.5-82.8% for d10-DPhP, respectively (Table S3). The limits of detection (LOD)

240

for OPEs and metabolites, defined as a signal-to-noise ratio (S/N) of 3, were in the

241

range of 0.06-0.35 ng/g for plant tissues and 1.2-75 ng/L for solutions, respectively.

242

Details of quality assurance and quality control are provided in SI.

243

Enzyme and Gene Expression Assays. Wheat roots were carefully rinsed with

244

sterilized deionized water and finely chopped. Approximately 1 g of fresh root sample

245

was used for enzyme assays. The protein level was measured by the method of

246

Bradford42 using bovine serum albumin as the standard. GST activity was measured



247

using the method of Habig et al.,43 and CYP450 activity was assayed with the Plant

248

CYP450 ELISA Kit (Dongge Biotechnology Co. Ltd, Beijing, China). All the enzyme

249

activities were determined spectrophotometrically at 25 °C and expressed as U·mg−1

250

protein. Methods in detail are available in the SI.

251

Total RNA was isolated from frozen root samples (100 mg), followed by

252

precipitation with lithium chloride and then isopropanol.35 mRNA was isolated from

253

the total RNA using a QuickPrep Micro mRNA Purification Kit (Amersham

254

Pharmacia Biotech) as outlined in the manufacturer’s directions. qRT-PCR assays

255

were conducted in a Bio-Rad iQ5 System (Bio-Rad Company, CA, USA). The details

256

in procedure are provided in the SI.

257

Homology Modeling and Molecular Docking. TaLTP1.1, TaGST4-4 and

258

CYP71C6v1 in wheat were chosen to investigate the interaction between OPEs and

259

biomacromolecules. The 3-D structures of the proteins TaLTP1.1 and TaGST4-4 are

260

available in the Protein Data Bank (PDB) with entry codes 1CZ2 and 1GWC,

261

respectively. The 3-D structure of CYP71C6v1 was constructed by homology

262

modeling. The primary sequences of CYP71C6v1 are available at the National Center

263

for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih.gov). The

264

template protein 3PM0 (Human CYP1B1, resolution: 2.70 Å) identified by the

265

BLAST program (http://blast.ncbi. nlm.nih.gov/Blast.cgi) was employed as the

266

template for CYP71C6v1. The sequence identity was 25% for CYP71C6v1 to

267

CYP1B1 (Figure S1A). The homology modeling and the subsequent molecular

268

docking were based on the method of Li et al.38 with some modification, and the

269

details are given in the SI. Results showed that the percentage of the residues in the

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core Ramachandran region was 91.8% for CYP71C6v1 (Figure S1B), and residues

271

located in the unfavorable regions were far from the substrate-binding domain,


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implying negligible influence on the ligand-protein binding simulations. This suggests

273

that the models of the plant enzymes are of reasonable quality compared with the

274

crystal structures of the templates. TaLTP1.1 contained four α helices and random

275

coils. TaGST4-4 contained nine α helices, four β sheets and random coils (Figure

276

S1C). CYP71C6v1 contained nine α helices, nine β sheets and random coils.

277

Molecular docking was carried out to simulate the detailed binding modes of each

278

OPE congener to biomacromolecules using Autodock 4.2. The MOLDOCK score

279

algorithm was employed to calculate the energy-based evaluations and to select the

280

best orientation with the highest docking score. The lowest energy docking models

281

were selected from the fine grid docking.

282

Data Analysis. Statistical analysis was performed using Microsoft Excel 2010 and

283

Origin 8.0 (OriginLab Corporation) software. Means and standard deviations were

284

calculated from triplicate measurements. All of the results are expressed on a dry

285

weight basis. A p-value of 0.05 was taken as statistically significant.

286

RESULTS AND DISCUSSION

287

Uptake and Translocation of OPEs by Wheat Seedlings. After 10 d, 91.8-96.9% of

288

the initial dose of OPEs in the solutions remained in the unplanted controls (Table S5),

289

indicative of negligible dissipation of OPEs derived from their adsorption to the

290

reactors, evaporation to the air and metabolization. Concentrations of OPEs in the

291

exposure solution were time-dependent due to their plant uptake and metabolism. All

292

the eight OPEs were detected in wheat roots. Concentrations of the OPEs in the roots

293

increased first, reaching the peak levels at 3.50, 6.72, 13.8, 17.7, 16.9, 13.0, 23.6, and

294

31.7 µg/g (dw) for TCEP, TCPP, TDCPP TBEP, TnBP, TiBP, TPhP and EHDPP,

295

respectively (Figure 1), at different exposure times, and then decreased afterwards. To

296

compare the uptake abilities of OPEs, root-to-solution ratios of OPEs were calculated



297

for each exposure time point. Both the mean and the maximum values of log

298

root-to-solution ratios were in the following order: EHDPP > TPhP > TBEP > TnBP >

299

TiBP > TDCPP > TCPP > TCEP. A significantly positive linear relationship was found

300

between the maximum and the average log root-to-solution values and logKow of

301

OPEs (Figure 2A and Figure S2A, P < 0.001), demonstrating that partitioning was the

302

major pathway for root uptake of OPEs from the exposure solutions.

303

The concentrations of OPEs in shoots of the blank controls after exposure are

304

shown in Table S5. Only TCPP and TBEP were detected in one shoot sample at

305

concentrations of 1.89 and 1.02 ng/g, respectively, which accounted for less than

306

0.1% of the corresponding congeners accumulated in shoots of the exposed plants.

307

This implied that the contribution from foliar uptake to the accumulation of OPEs in

308

shoots was negligible under the present experimental conditions. All the OPEs were

309

detected in shoot samples after exposures, with the maximum concentrations at 2.03,

310

1.90, 1.42, 1.95, 1.82, 1.24, 1.07, and 0.59 µg/g (dw) for TCEP, TCPP, TDCPP, TBEP,

311

TnBP, TiBP, TPhP and EHDPP, respectively (Figure 1), much lower than their root

312

concentrations. Transpiration stream concentration factors (TSCFs), estimated as the

313

ratios between the chemical concentrations in the transpiration stream and the

314

exposure solution, have been widely used as the descriptor of acropetal translocation

315

of organic.4-46 The TSCFs for OPEs were calculated for different exposure intervals.

316

Both the mean and the maximum TSCF values were found to be negatively correlated

317

with their logKow values (Figure 2B and Figure S2B, P < 0.005), suggesting that OPEs

318

with lower hydrophobicity were more prone to be translocated from roots to shoots. In

319

particular, the relationship between the maximum TSCF versus logKow followed a

320

nearly sigmoidal curve (Table S6), similar to the one obtained by Dettenmaier et al.,46

321

when they investigated the plant uptake of 25 organic chemicals with logKow ranging


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in -0.8 to 5, similar to logKow values of the target OPEs in the present study.

323

Biotransformation of OPEs in Wheat Seedlings. Seven di-esters and two

324

mono-esters were detected and quantified in wheat roots and shoots, with their

325

concentrations higher in roots than in shoots (Figure 1); but none of them were found

326

in the untreated and unplanted controls (Table S5). All the metabolites were detected

327

in wheat tissues after exposure for only 12 h, indicating metabolism occurred very

328

rapidly. Concentrations of BDCPP, BBEP, DiBP, DnBP, and DPhP in wheat roots and

329

shoots increased first and then decreased noticeably, while concentrations of BCEP

330

and BCPP kept increasing. This suggests that BCEP and BCPP were relatively stable,

331

whereas the other metabolites might be further metabolized inside plants.

332

Concentrations of MnBP and MPhP in both roots and shoots kept increasing during

333

the exposure time, being most likely attributed to the further continuous

334

transformation of di-esters to mono-esters. Metabolites in wheat should derive from a

335

combination of root uptake following metabolism of the parent OPE compounds in

336

the exposure solutions and metabolism occurring inside plants. Although we paid

337

particular attention to avoiding metabolism of OPEs in the exposure solutions caused

338

by microorganisms by sterilizing the culture solution and sealing the exposure

339

reactors, metabolites were detected in the culture solutions during the exposure time

340

(Figure S3). This could have occurred because root-associated microbes can be

341

inoculated in the solution during plant cultivation,47 which would result in metabolism

342

of OPEs in the solutions to some extent. This was confirmed by our observation on

343

the metabolism of OPEs in the fresh root-exudate solutions (see results in Table S7).

344

Further, the metabolites are generally more polar than their parent compounds.

345

Therefore, OPE metabolites should have lower logKow and therefore lower log

346

root-to-solution values than the parent OPEs if only uptake occurred. However, the



347

log root-to-solution values of the metabolites were found to be much higher than

348

those of the corresponding parent compounds (Table S8). This suggested the

349

occurrence of metabolism of OPEs inside plants besides root uptake from exposure

350

solutions.

351

In addition to the metabolites quantified above, we observed some unidentified

352

peaks in the chromatograph (Figure S4), likely belonging to some other unidentified

353

metabolites. The mass balance was tested for each exposure time by calculating the

354

percentages of the total mass of each OPE and its metabolites in the whole plant and

355

the exposure solution to the initial amount added (Table S9). It was found that the

356

recoveries of OPEs decreased with increasing plant exposure time, and 44.3% to

357

83.5% of the OPEs were recovered at the end of the exposure. These results might

358

also suggest the existence of some unidentified metabolites. Unfortunately,

359

commercial standards of the potential metabolites are unavailable, precluding accurate

360

quantification of their concentrations. Therefore, the putative metabolites were

361

identified by UPLC-ESI-Q-TOF-MS, and the mass spectra and chromatograms were

362

collected (Figure 3). The products observed were assigned according to their [M-H]-

363

m/z and retention time, and the chemical information for the metabolites identified is

364

provided in Table S10. A total of 34 metabolites belonging to 8 OPEs respectively

365

were identified in wheat tissues (Figure S5). The levels of the products were higher in

366

roots than in shoots. TCEP exhibited the fewest kinds of metabolites while TBEP had

367

the most. Oxidatively dechlorinated, hydroxylated, and glutathione (GSH)-conjugated

368

products were found to be derived from the halogenated OPEs, whereas the

369

metabolites detected for non-halogenated OPEs included oxidative-dealkylated,

370

hydroxylated, methoxylated and glucuronide-conjugated products (see Figure S6 for

371

details). Similar oxidative-dealkylation, GSH- and glucuronide-conjugated products


Page 16 of 34

Page 17 of 34


372

have been reported for organophosphorus pesticides in previous works.48,49 The

373

metabolites containing hydroxyl, especially the hydroxylated tri-esters, accounted for

374

the largest amounts of the total products in both roots and shoots for all the OPEs. Su

375

et al.24,25 also detected mono- and di- hydroxylated TPhP isomers in a test using

376

chicken embryonic hepatocyte screening, with their levels increasing over the

377

exposure time, and based on this observation they highlighted the importance of the

378

hydroxylation pathway. Van den Eede et al.22 also identified a considerable

379

contribution of hydroxylated products of TCPP, TBEP, and TPhP to metabolism of

380

OPEs in human liver cells. To our knowledge, this is the first time the existence of

381

hydroxylated OPEs in plants has been reported, to which we should pay particular

382

attention since hydroxylated metabolites are sometimes more toxic than their parent

383

compounds.50-54 Patterns of metabolites from diverse OPE congeners were distinct.

384

For instance, mono-esters were found for all non-halogenated OPEs but none was

385

identified for the halogenated ones, and the GSH-conjugated products were detected

386

as

387

glucuronide-conjugated ones instead of the GSH-adducts were found for

388

non-halogenated OPEs. In addition, among the three halogenated OPEs, the direct

389

hydroxylation metabolite without removing the Cl atom only appeared for TCPP, and

390

the products of further methoxylation after hydroxylation were found for aryl

391

phosphates (Figure S6), which had not been reported in previous research on the

392

metabolism of TPhP. The hydroxylated and methoxylated di-esters in wheat might be

393

formed through hydroxylation and methoxylation of their corresponding di-esters or

394

through dealkylation of their hydroxylated and methoxylated tri-esters; which of these

395

is the main transformation pathway needs further investigation. Mass balance was

396

then further estimated by including both the quantified and semi-quantified

the

phase

II

metabolites

for

halogenated


OPEs,

whereas

the


397

metabolites (Table S11). 71.1% to 94.6% of the initial OPEs were recovered at the

398

end of exposure, which were improved approximately 8.5% to 30.2% compared to the

399

data acquired with only the quantified metabolites included. Improvement was

400

particularly achieved for TCPP, TBEP and TPhP, which had relatively more

401

metabolites detected.

402

Molecular Interactions between OPEs and Plant Biomacromolecules. The results

403

above showed the diverse accumulation and transformation of different OPEs in

404

wheat tissues. To obtain further insight into the mechanisms involved in these

405

processes, molecular interactions between OPEs and plant biomacromolecules were

406

characterized using homology modeling combined with molecular docking. Dynamic

407

changes of GST and CYP450 activities and gene expressions in wheat roots were first

408

investigated in order to determine their roles in the metabolization of OPEs in wheat

409

plants. Compared with those of blank controls, the significantly increasing activities

410

of the two enzymes (P < 0.05, Figure S7) and the up-regulation in gene expressions of

411

TaGST4-4 and CYP71C6v1 (Fold change ≥ 2, Figure S7) were observed during the

412

exposure time, demonstrating that they were involved in the metabolism of OPEs in

413

wheat. The results provided an experimental basis for molecular docking and for the

414

analysis of interactions between OPEs and the plant biomacromolecules. It was found

415

that all the OPEs could be docked into the active cavities of wheat biomacromolecules

416

TaLTP1.1, TaGST4-4 and CYP71C6v1 (Figure 4 and Figure S8). The main residues

417

in the active sites including Leu34, Ala38, Ala47, Leu51, Leu77, Tyr79, and Ile81

418

belong to TaLTP1.1, Pro14, Ser15, Phe17, Arg114, Arg118, Trp171, and Arg222 to

419

TaGST4-4, and Ser78, Phe79, Leu187, Ala322, Glu325, and Met506 to CYP71C6v1,

420

respectively, were found to be located within a distance of 3 Å to the OPE congeners

421

(Table S12, Figure 4 and Figure S8), implying their important roles in binding to the


Page 18 of 34

Page 19 of 34


422

OPE congeners. All these amino acid residues were hydrophobic and were probably

423

bound with OPEs mainly via hydrophobic interactions. Furthermore, several OPEs

424

formed hydrogen bonds with the residues, such as TBEP with the residue Tyr79 of

425

TaLTP1.1; TCEP with Ser15, TDCPP with Arg114, and TPhP with Arg222 of

426

TaGST4-4; and TiBP with Ser78, and TPhP with Glu325 of CYP71C6v1, respectively

427

(Figure 4 and Figure S8). The interaction energies of TCEP, TCPP, TDCPP, TBEP,

428

TnBP, TiBP, TPhP and EHDPP were from −5.38 to −8.10 kcal/mol for TaLTP1.1,

429

from −4.78 to −6.33 kcal/mol for TaGST4-4, and from −3.02 to −6.82 kcal/mol for

430

CYP71C6v1, respectively (Table S13). Compared with the binding affinity values of

431

the van der Waals force, the effect of the electrostatic energy was negligible (Table

432

S13), suggesting that OPEs were bound to the active sites of plant biomacromolecules

433

mainly via van der Waals forces. The binding affinities varied markedly among

434

different OPEs. The docking energies for OPEs to TaLTP1.1 were found to have a

435

significantly negative correlation to the logKow values of OPEs (Figure S9, P < 0.005),

436

that is to say, OPEs with higher hydrophobicity were more liable to bind with

437

TaLTP1.1, similar to the uptake abilities of OPEs quantified and presented as log

438

root-to-solution values. Therefore, a consistent conclusion was achieved from the

439

experimental and molecular docking results that hydrophobic partitioning into plant

440

biomacromolecules was the major pathway for the uptake of OPEs by wheat.

441

Considering the interaction of OPEs with the wheat enzymes, the non-halogenated

442

OPEs were found to have more negative binding energies and therefore were more

443

liable to fit into the active sites of CYP71C6v1 and TaGST4-4 compared with the

444

halogenated OPEs, supporting the experimental observation that non-halogenated

445

OPEs such as TBEP, TiBP and EHDPP were more liable to be metabolized. The

446

binding energies of halogenated OPEs for TaGST4-4 were more negative than those



447

for CYP71C6v1, indicating that halogenated OPEs were more prone to bind with

448

wheat GST enzyme. This is in accordance with the experimental results that GSH

449

conjugate metabolites were found for halogenated OPEs. By contrast, the

450

non-halogenated OPEs were preferentially bound with CYP71C6v1 rather than

451

TaGST4-4, in line with the experimental observation of the existence of the

452

mono-esters and the di-hydroxylated products, which have been evidenced to be

453

mainly produced by the CYP isozymes.55,56 Moreover, it was worth noting that TBEP

454

had the most negative binding energies for both TaGST4-4 and CYP71C6v1, and

455

therefore possessed the strongest interactions with wheat enzymes, which was in

456

agreement with the experimental evidence that TBEP was the most readily

457

metabolized and generated the most metabolites in wheat. Therefore, the conclusion

458

can be made that the diverse accumulation and transformation of OPE congeners may

459

derive from their different binding modes and affinities to plant biomacromolecules.

460

Environmental Implications. This study provides both experimental and theoretical

461

evidence for the uptake, accumulation and biotransformation of OPEs in wheat plants.

462

Uptake by roots and translocation to shoots indicate the potential for OPEs to transfer

463

into the food chain. Transformation of OPEs in plants was evidenced with a total of

464

43 metabolites detected, which may cause adverse effects on the environment and

465

human health. Molecular interaction between OPEs and the wheat biomacromolecules

466

nsLTP, GST, and P450 explained the diverse accumulation and transformation of

467

OPEs in wheat tissues observed in the experiments. Results obtained by this study are

468

useful to gain further understanding of the fates of OPEs in the environment. Further

469

research is necessary on the generation and behaviors of OPE metabolites in the

470

soil-plant system, which is critical for evaluating the fate and potential toxicity of

471

OPEs in the environment.


Page 20 of 34

Page 21 of 34


472

ASSOCIATED CONTENT

473

Supporting Information. Additional figures and experimental details regarding

474

extraction and analytical methods of OPE congeners and metabolites, homology

475

modeling and molecular docking methods, OPE metabolite identification and

476

distribution, mass balance of OPEs, 3-D structures of plant biomacromolecules, and

477

OPEs binding with plant biomacromolecules. This material is available free of charge

478

via the Internet at http://pubs.acs.org.

479 480

AUTHOR INFORMATION

481

Corresponding author

482

*phone: +86-10-62849683; fax: +86-10-62923563; e-mail: [email protected].

483

Notes

484

The authors declare no competing financial interest.

485 486

ACKNOWLEDGMENTS. This work was funded by the National Natural Science

487

Foundation of China (Projects 21537005 and 21621064) and the Strategic Priority

488

Research Program of the Chinese Academy of Sciences (Grant XDB14020202).

489 490

References

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Figure captions

663

Fig. 1 Time-dependent concentrations of OPEs and quantified metabolites in wheat

664 665 666

roots and shoots. Error bars represent standard deviation values (n = 3). Fig. 2 Relationships between the mean log root-to-solution values (A), the mean TSCF values (B) of different exposure time and logKow values of OPEs.

667

Fig. 3 Identification of the potential metabolites of OPEs in wheat roots by

668

UPLC-Q-TOF-MS taking TPhP as an example. The mass spectra (A) indicate the

669

types of fragment ions and masses (shown as arrows and dotted lines). The

670

chromatograms (B) provide the approximate retention times of the metabolites,

671

which include mono-hydroxylated diphenyl phosphate (M1), mono-methoxylated

672

diphenyl phosphate (M2), mono- glucuronic acid conjugated metabolites after

673

hydroxylation (M3), mono-hydroxylated TPHP (M4), di-hydroxylated TPHP (M5),

674

mono-methoxylated TPHP (M6), and glucuronic acid conjugated metabolites after

675

di-hydroxylation (M7).

676

Fig. 4 Residues in the active sites of TaLTP1.1 interacting with OPE congeners. The

677

hydrogen bonds formed between TBEP and the residue Tyr79 are displayed with

678

green dotted lines.


BCEP-Root BCEP-Shoot

3.0 2.5

3.0 2.0 2.5 2.0

1.5

1.5

1.0

1.0

0.5

0.5 0.0 12

24

36

48

72

96 144 192 240

Concentration of TCPP (µg/g dw)

TCEP-Root TCEP-Shoot

3.5

8

TCPP-Root TCPP-Shoot

7

1.2

5 4

0.8

3 2

0.4

1 0

0.0 12

24

2

1

1

0

0

36

48

72

96 144 192 240

Concentration of TBEP (µg/g dw)

Concentration of TDCPP (µg/g dw)

5

24

20

3

10

2

5 2.0

1

1.5

0.4

1.0 0.2

0.5 0.0

1 0.6

1.0 0.5

0.3

0.0

0.0 36

48

72

96 144 192 240

0.0 12

24

Concentration of TPhP (µg/g dw)

DPhP-Root DPhP-Shoot

10

MPhP-Root MPhP-Shoot 8

6 4 2 0.6 0.4

0.5

0.2 0.0

0.0 12

24

36

48

72

96 144 192 240

Exposure Time (h)

Concentration of DPhP&MPhP (µg/g dw)

TPhP-Root TPhP-Shoot

36

48

72

96 144 192 240

24

TnBP-Root TnBP-Shoot

20 16

DnBP-Root DnBP-Shoot

MnBP-Root MnBP-Shoot

6

4

12 8

2

4 1.5

0.9

1.0

0.6

0.5

0.3 0.0

0.0

12

24

36

48

72

96 144 192 240

Exposure Time (h)

Exposure Time (h) 28 24 20 16 12 8 1.0

Concentration of TnBP (µg/g dw)

2

24

BBEP-Root 5 BBEP-Shoot 4

15

Concentration of EHDPP (µg/g dw)

Concentration of TiBP (µg/g dw)

DiBP-Root 5 DiBP-Shoot 4 3

12

96 144 192 240

Exposure Time (h) Concentration of DiBP (µg/g dw)

TiBP-Root TiBP-Shoot

72

TBEP-Root TBEP-Shoot

25

Exposure Time (h) 18 15 12 9 6 3

48

40

EHDPP-Root EHDPP-Shoot

32

DPhP-Root DPhP-Shoot

MPhP-Root MPhP-Shoot 6

24

4

16

2 0.9

0.6

0.6

0.4 0.3

0.2 0.0

0.0 12

24

36

48

72

96 144 192 240

Exposure Time (h)

Figure 1


Concentration of BBEP (µg/g dw) Concentration of DnBP&MnBP (µg/g dw) Concentration of DPhP&MPhP (µg/g dw)

3

Concentration of BDCPP (µg/g dw)

4

10

12

36

Exposure Time (h)

BDCPP-Root BDCPP-Shoot

TDCPP-Root TDCPP-Shoot

15

BCPP-Root BCPP-Shoot 1.6

6

Exposure Time (h) 20

Page 30 of 34

Concentration of BCPP (µg/g dw)

4.0

Concentration of BCEP (µg/g dw)

Concentration of TCEP (µg/g dw)


Page 31 of 34


0.5 A

3.5

R = 0.929 P < 0.005

0.4

3.0

TSCF

log root-to-solution ratio

4.0

2.5 2.0

R = 0.957 P < 0.001

1.5

0.3 0.2 0.1 B

0.0 1.0 1

2

3

4 logKow

5

6

1

2

3

4 logKow

Figure 2


5

6


A (a)

(b)

(c)

(e)

(f)

(g)

B (a)

(b)

(c)

(e)

(f)

(g)

(d)

(d)

Figure 3


Page 32 of 34

Page 33 of 34


Figure 4




Page 34 of 34

Uptake, Translocation, and Biotransformation of Organophosphorus

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