Experimental and Theoretical Evidence for Diastereomer- and

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Experimental and theoretical evidence for diastereomer- and enantiomerspecific accumulation and biotransformation of HBCD in maize roots Honglin Huang, Shuzhen Zhang, Jitao Lv, Bei Wen, Sen Wang, and Tong Wu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03223 • Publication Date (Web): 14 Oct 2016 Downloaded from http://pubs.acs.org on October 15, 2016

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Environmental Science & Technology

1

Experimental and theoretical evidence for diastereomer- and enantiomer-specific

2

accumulation and biotransformation of HBCD in maize roots

3 4

Honglin Huang †, Shuzhen Zhang †, ‡ *, Jitao Lv†, Bei Wen †,

5

Sen Wang†, § , and Tong Wu†,ǁ

6 7

†

8

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

9

2871, Beijing 100085, China

State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research

10

‡

University of Chinese Academy of Sciences, Beijing 100049, China

11

§

Department of Environmental Sciences, College of Urban and Environmental

12

Sciences, Northwest University, Xi’an 710027, China

13

ǁ

14

Technology, Hebei, 050018, China

School of Environmental Science and Engineering, Hebei University of Science and

1

ACS Paragon Plus Environment


15

ABSTRACT:

16

Diastereomer- and enantiomer-specific accumulation and biotransformation of

17

hexabromocyclododecane (HBCD) in maize (Zea mays L.) were investigated.

18

Molecular interactions of HBCD with plant enzymes were further characterized by

19

homology modeling combined with molecular docking. The (−)α-, (−)β- and

20

(+)γ-HBCD enantiomers accumulated to significantly higher levels in maize than their

21

corresponding enantiomers. Bioisomerization from (+)/(−)-β- and γ-HBCDs to

22

(−)α-HBCD was frequently observed, and (−)γ-HBCD was most easily converted,

23

with bioisomerization efficiency of 90.5 ± 8.2%. Mono- and di-hydroxyl HBCDs,

24

debrominated metabolites including pentabromocyclododecene (PBCDe) and

25

tetrabromocyclododecene (TBCDe), and HBCD-GSH adducts were detected in maize

26

roots. Patterns of hydroxylated and debrominated metabolites were significantly

27

different among HBCD diastereomers and enantiomers. Three pairs of HBCD

28

enantiomers were selectively bound into the active sites and interacted with specific

29

residues of maize enzymes of CYP71C3v2 and GST31. (+)α-, (−)β- and (−)γ-HBCDs

30

preferentially bound to CYP71C3v2, whereas (−)α-, (−)β- and (+)γ-HBCD had strong

31

affinities to GST31, consistent with experimental observations that (+)α-, (−)β- and

32

(−)γ-HBCDs were more easily hydroxylated , and (−)α-, (−)β- and (+)γ-HBCDs were

33

more easily isomerized and debrominated in maize compared to their corresponding

34

enantiomers. This study for the first time provided both experimental and theoretical

35

evidence for stereo-specific behaviors of HBCD in plants.

36 37

KEYWORDS: HBCDs; maize; diastereomer- and enantiomer-specific; accumulation;

38

biotransformation; enzymes

2


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39

INTRODUCTION

40 41

Hexabromocyclododecane (HBCD) is a brominated flame retardant additive mainly

42

used in polystyrene insulation foam and textile coatings and, to a lesser extent, in

43

high-impact polystyrene for electric and electronic devices.1 As a result of its

44

widespread use and physical and chemical properties, HBCD has been found

45

ubiquitously in the environment.1–3 HBCD has a complex stereochemistry, and the

46

commercial formulations consist mainly of α-, β- and γ-diastereomers,4 with each of

47

them having a pair of enantiomers. Enantiomers of a chiral compound have similar

48

physicochemical properties, but can have significantly different behavior in biological

49

systems.5 Therefore, the diastereomer- and enantiomer-specific behaviors of HBCD in

50

organisms, such as uptake and transformation, will largely determine their fates in the

51

environment and have drawn increasing research attention in recent years.

52

Soil is the main receptor for organic contaminants. HBCD has been frequently

53

detected in soils, and high concentrations were found in soils at sites near

54

contamination sources. For example, HBCD has been detected at concentration of

55

140–1300 and 111−23200 ng kg-1 dry weight in soils in Sweden and Belgium,

56

respectively, near HBCD-processing factories.6,7. Heavy discharges of HBCD to soils

57

have also been reported from industrial waste disposal sites and electronic waste

58

recycling areas in the United Kingdom and China.8−11 Therefore, plant uptake and

59

accumulation of HBCD are important processes determining their fates in the

60

terrestrial environment. Nevertheless, the previous studies on bioaccumulation of

61

HBCD have mainly focused on animal species.12−14 Plant uptake and accumulation of

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HBCD have only been demonstrated in one field study and several pot

63

experiments.15−19 The concentration of HBCD was reported to be 3−28 ng/g dry 3



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64

weight in radish, wheat and reed from a natural wetland conservation area near

65

Tianjin, China.18 Diastereomer-specific uptake and accumulation were evidenced in

66

maize by a hydroponic experiment in our previous work.15 But so far the enantiomer

67

selectivity of HBCD in plant uptake and accumulation remains unknown.

68

Biotransformation is an important process for organic contaminants with different

69

diastereomer or enantiomer structures. To date only a small number of studies exist on

70

the transformation of HBCD in organisms.20–24 A diastereomeric shift toward

71

α-HBCD by liver microsomes of seals has been reported by Zegers et al.20 The

72

degradation

73

tetrabromocyclododecenes (TBCDe) from HBCD have been detected in chicken eggs,

74

whale and whitefish (Coregonuslavaretus).23 Hydroxylated metabolites including

75

OH-HBCDs,

76

tetrabromocyclododecanols (OH-TBCDs) have been confirmed in in vitro assays with

77

rat and seal liver microsomes .20,24 However, the biotransformation pathway of HBCD,

78

particularly at the enantiomer level, is still unclear. To the best of our

79

knowledge, there have no reported studies on biotransformation of HBCD inside

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plants, although the total biomass of plants is far greater than that of animals on earth,

81

and the “green-liver” concept of plant metabolism in the transformation and

82

degradation of organic contaminants is well known.25

products

of

pentabromocyclododecenes

pentabromocyclododecanols

(PBCDe)

(OH-PBCDs)

and

and

83

Moreover, there is much lacking in the understanding the molecular mechanisms

84

for the selective accumulation and transformation of HBCD diastereomers and

85

enantiomers in organisms. The individual enantiomers of chiral compounds can

86

interact enantio-selectively with enzymes and biological receptors in organisms.5,26

87

For example, Kania-Korwel et al.27 found a greater binding of (+)-PCB 136 to P450

88

isozymes of CYP2B and CYP3A than (−)-PCB 136 in mouse and rat in vivo. The 4


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(−)-o,p,-DDT enantiomer was found to be an active estrogen mimic at the human

90

estrogen receptor, whereas (+)-o,p,-DDT had negligible activity.28 Whether HBCD

91

diastereomers and enantiomers interact selectively with biomacromolecules, and if so

92

how the selective interactions of HBCD with biomacromolecules lead to their

93

diastereo- and enantio-specific bioaccumulation and biotransformation in organisms,

94

are not known. Homology modeling has been successfully applied to construct

95

three-dimensional (3D) models of enzymes in organisms, which have been combined

96

with molecular docking to characterize the interactions of stereo-isomers of PCBs and

97

pesticides with isozymes.29–33 Using homology modeling combined with molecular

98

docking, the function of enzymes can be predicted by their 3-D structures, and the

99

active sites of enzymes involved in stereoisomer recognition as well as the binding

100

modes and binding affinities of stereoisomers to enzymes can be characterized. This

101

information is helpful in elucidating the molecular mechanisms of accumulation and

102

transformation in organisms of contaminants having various stereoisomers, such as

103

HBCD. Therefore,

104

diastereomer-

and

enantiomer-specific

accumulation

and

105

biotransformation of HBCDs in maize were studied in the present study by exposing

106

plants to (−)α-, (−)β- and (+)γ-HBCD enantiomers individually in a hydroponic

107

experiment. Attempts were made for the first time to clarify the molecular interactions

108

between HBCD and plant cytochrome P450 (CYP) and glutathione S-transferase

109

(GST) enzymes by using homology modeling and molecular docking. The theoretical

110

results

111

enantiomer-selective behaviors of HBCD in maize in vivo.

obtained

were

further

used

to

explain

112 113

MATERIALS AND METHODS 5


the

diastereomer-

and


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114 115

Chemicals. Three native HBCD standards (α-, β- and γ-HBCD stereoisomers in

116

toluene, chemical purity > 98%) were obtained from Accu-Standard (New Haven, CT,

117

USA). The

118

Cambridge

119

Racemic-(1,5R,6S,9S,10R)-pentabromocyclododecene (PBCD) was provided by

120

Wellington Laboratories, Inc. (Ontario, Canada). All solvents used were of HPLC

121

grade and purchased from J. T. Baker (Deventer, Netherlands). High-purity water was

122

prepared with a Milli-Q system (Millipore, Bedford, MA). Neutral silica gel

123

(100−200 mesh) and alumina (60−100 mesh) were purchased from Fisher Scientific

124

Inc. (Fair Lawn, NJ).

13

C-labeled α-, β- and γ-HBCDs (purity ≥ 99%) were purchased from Isotope

Laboratories,

Inc.

(Andover,

Massachusetts,

USA).

125

Preparation of Individual HBCD Enantiomers. Enantiomerically pure α-, β-

126

and γ-HBCDs were prepared from standards of the racemic α-, β- and γ-HBCDs by an

127

Agilent HPLC-system (Agilent 1100, USA) with a diode array detector (DAD). The

128

enantiomeric separation was conducted on a chiral Nucleodex β-PM column (5 µm,

129

200 mm × 4 mm i.d.) from Macherey-Nagel GmbH & Co. KG (Düren, Germany)

130

using methanol/water as mobile phase at a flow rate of 0.35 mL min-1. Preparation

131

procedures are described in detail in the Supporting Information (SI). Six individual

132

HBCD enantiomers were successfully prepared and the baseline enantiomeric

133

separation achieved for the mixtures of the three pairs of (+)/(−)- α-, β- and γ-HBCD

134

enantiomers by UPLC-MS/MS is shown in Figure S1 in SI. According to the method

135

described by Heeb et al.4 (−)α-, (−)β- and (+)γ-HBCDs were eluted prior to their

136

opposite enantiomers. The purities of the prepared HBCD enantiomers exceed 98%,

137

and they were found to be stable over half a year when stored at −20 °C.

138

Plant Exposure. Plant exposure was carried out according to the methods 6


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139

described in our previous research,34 and the details are provided in SI. In order to

140

minimize metabolism of HBCD caused by anaerobic microorganisms, autoclaved

141

deionized water was used to prepare Hoagland nutrient solution, which was further

142

saturated with oxygen. Exposed solutions were obtained by first dissolving each of

143

the prepared pure (+)/(−)-α-, β- and γ-HBCD enantiomers in methanol individually,

144

and then gradually diluting with the sterile 1/4 strength Hoagland nutrient solution.

145

The concentrations of (+)/(−)-α-, β- and γ-HBCD enantiomers in the initial exposure

146

solution were set and determined at 25.0, 17.5 and 2.0 ng L-1, respectively, lower than

147

or equal to their water solubility.

148

Maize (Zea mays L.) seeds were obtained from the Chinese Academy of

149

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

150

selected and surface-sterilized in 3% (v/v) H2O2 for 10 min, followed by thoroughly

151

washing with distilled water, and subsequently germinated on moist filter paper for 4

152

days. After germination, the maize seedlings were transferred to containers containing

153

sterile 1/4 strength Hoagland nutrient solution for cultivation in a controlled

154

environment growth chamber. After 7 days, a batch of five uniformly sized maize

155

seedlings with three leaves each was transferred to an autoclaved 200 mL brown

156

glass-stoppered flask, which was used as the exposure container. Then 150 mL of

157

each of the exposure solutions of (+)/(−)-α-, β- and γ-HBCD enantiomers were added

158

to the exposure container. Unplanted controls with 150 mL solution of each individual

159

HBCD enantiomers but without seedlings, and blank controls with seedlings but

160

without HBCD, were set up simultaneously. All treatments were set up in triplicate

161

with separate containers. Maize plants were harvested after 7 d of exposure. The

162

maize seedlings were freeze-dried at −50 °C for 48 h in a lyophilizer (FD-1, Beijing

163

Boyikang Instrument Ltd, China), separated into roots and shoots, weighed and finely 7



164

chopped. The roots and shoots of the five seedlings in each container were combined

165

as one root or shoot sample. Nutrient solutions after plant exposure were filtered with

166

0.45 µm filter membranes. All the samples were stored in glass containers at −20 °C

167

before chemical analysis.

168

HBCD Extraction, Clean-up and Analysis. 0.5–1.0 g (dry weight) of plant

169

samples or 5−10 mL of exposed solutions were spiked with 10 ng of surrogates of

170

13

171

HBCDs are based on the method of Wu et al.15 with some modifications. HBCD

172

enantiomers were analyzed by an Acquity UPLC system coupled to a

173

triple-quadrupole mass spectrometer (Waters, Milford, MA, USA). Separation of

174

HBCD enantiomers was carried out on a Nucleodex β-PM column (5 µm, 200 mm × 4

175

mm i.d.). A mobile phase of A (70 : 30 methanol : acetonitrile, v : v) and B (H2O with

176

10 mM ammonium acetate) at a flow rate of 0.4 mL min-1 was applied for elution of

177

the target compounds. The MS system was operated in the electrospray (ESI) negative

178

ionization and multiple reaction monitoring (MRM) mode. The conditions for the MS

179

system were optimized as follows: cone voltage, 20 V; capillary voltage, 3.5 kV;

180

desolvation temperature, 160 °C; source temperature, 110 °C; nebulizing gas flow,

181

400 L h−1; cone gas flow, 50 L h−1; and collision energy, 15 eV. The MRM transitions

182

monitored for HBCD enantiomers were 640.6→79.0 and 652.6→79.0 for the

183

13

184

are provided in the SI.

185

C-α-HBCD and 13C-γ-HBCD. The extraction, clean-up and analysis procedures for

C-labeled HBCDs, respectively. Details of HBCD extraction, clean-up and analysis

Quality Assurance and Quality Control. Matrix effects on signal intensity of 13

UPLC-MS/MS were minimized using

187

standards and

188

solvent blank was added to ensure that the samples were free of contamination.

13

C-α- and

13

186

C-γ-HBCDs as the surrogate

C-β-HBCD as internal standard. For every batch of six samples, a

8


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189

The average recoveries of α-, β- and γ-HBCD were 82.6 ± 9.50%, 90.2 ± 8.90%, and

190

86.5 ± 9.70% in the spiked matrices (n = 3), respectively. Recoveries of surrogate

191

standard were as follows: 85.7 ± 9.20% for

192

13

193

(MDLs) for α-, β- and γ-HBCD were 0.78, 0.05 and 0.14 ng/g, respectively. Details of

194

quality assurance and quality control are provided in the SI.

C12-β-HBCD, and 87.5 ± 8.40% for

13

C12-α-HBCD, 89.6 ± 10.3% for

13

C12-γ-HBCD. The method detection limits

195

Metabolite Identification. An Agilent 6545 Q-TOF LC-MS (USA) was used to

196

identify the possible metabolites of HBCD enantiomers. Q-TOF MS was operated

197

with an electrospray source in negative ion mode. The main instrument setting for the

198

Q-TOF was as follows: drying gas temperature 150 °C, drying gas flow 10 L/min,

199

capillary voltage 3,500 V, fragmentor voltage 100 V. The Q-TOF chromatograms

200

were acquired in a scan range from 50 to 1050 m/z. The [M−H]− brominated clusters

201

monitored for the putative metabolites of HBCD in maize are listed in Table S1 in SI.

202

It was assumed that the response factors for the isomers of metabolites were equal to

203

each other based on the report by Abdallah et al.,35 and then the semi-quantitative

204

concentrations of the metabolites were determined by using peak area.

205

Homology Modeling and Molecular Docking. Previous studies have confirmed

206

that CYP450 and GST enzymes play the key roles in the hydroxylation and reduction

207

of xenobiotics such as PCBs and triazine, and CYP71C3v2 and GST31 are the most

208

important CYP450 and GST isozymes in maize, respectively.36,37 Therefore, the 3-D

209

structures of CYP71C3v2 and GST31 isozymes were used for homology modeling.

210

The primary sequences of CYP71C3v2 and GST31 are available at the National

211

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

212

The

213

(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The human CYP450 1A1 (PDB ID: 4I8V,

template

proteins

were

identified

by

the

9


BLAST

program


214

resolution: 2.60 Å) and wheat GST TaGST4-4 (PDB ID: 1GWC, resolution: 2.20 Å)

215

were employed as the templates for CYP71C3v2 and GST31, respectively (Figure S2,

216

SI). The sequence identity was 31% for CYP71C3v2 to CYP1A1, and 46% for

217

GST31 to TaGST4-4. CYP71C3v2 contained four α helices and two random coils,

218

and GST31 contained six α helices and four β sheets (Figure S3A, SI). The initial 3-D

219

structural models were energy-minimized to release the bad atomic contacts and tune

220

unreasonable local structural conformations. After that, the final models were

221

assessed by the Procheck program to check the stereochemical quality of a protein

222

structure. Concerning the Procheck assessment, the percentage of the residues in the

223

core Ramachandran region were 87.3% for CYP71C3v2 and 93.5% for GST31

224

(Figure S3B, SI), respectively. Residues located in the unfavorable regions were far

225

from the substrate-binding domain and did not affect the ligand-protein binding

226

simulations, suggesting that the models of the two plant enzymes are of reasonable

227

quality compared with the crystal structures of the templates.

228

Molecular interactions of HBCD enantiomers with plant enzymes were

229

characterized by using molecular docking. The structural models of the three pairs of

230

HBCD enantiomers, whose energies were minimized using the molecular mechanics

231

method (MM2), were generated by ChemDraw (CambridgeSoft, Waltham, MA, USA).

232

The docking on the monomer model of plant enzymes with HBCD was performed

233

using Autodock 4.0. Generally, polar hydrogen atoms were added and Kollman

234

charges were assigned to all atoms of the proteins. The docking area was defined

235

using AutoGrid 4. A 40 × 40 × 40 3-D affinity grid centered around the antifolate

236

binding site was calculated. The spacing between grid points was set as 0.375 Å. For

237

the ligand, Gasteiger charges were added, and nonpolar hydrogen atoms were merged.

238

For docking simulations, the Lamarckian genetic algorithm (LGA) was selected for 10


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239

ligand conformation and all bond rotations for ligands were ignored. In the docking

240

protocol the number of individuals in each population was set to 150 and the

241

maximum number of energy evaluations was set to 25,000,000, whereas the

242

maximum number of generations was set to 1000. The maximum number of top

243

individuals, the rate of gene mutation and crossover were set as default values.

244

Energetic evaluations were calculated using the MOLDOCK score algorithm. The

245

best orientation with the highest docking score was finally selected. The lowest

246

energy docking models were selected from the fine grid docking using several scoring

247

functions for configuration analysis.

248

Statistical Analysis. Data analysis was performed using SPSS 16.0 (SPSS Inc.,

249

Chicago IL, USA). One-way analysis of variance (ANOVA) accompanied by Tukey’s

250

test was used to determine the differences in the accumulation and biotransformation

251

of HBCD diastereomers and enantiomers in maize roots. The level of significance

252

was set at P = 0.05.

253 254

RESULTS AND DISCUSSION

255 256

Diastereomer- and Enantiomer-specific Accumulation of HBCD in Maize. All

257

the three pairs of HBCD enantiomers were detected in maize roots (Figure 1A), while

258

none of them was found in the blank controls, indicative of their uptake by roots.

259

HBCD enantiomers were also detected in shoots but at much lower concentrations

260

(Table S2) compared with root concentrations, with root-to-shoot translocation ratios

261

in the range of 0.02−0.06 (Table S3, SI). Therefore, only diastereomer- and

262

enantiomer-specific accumulation of HBCD in roots will be discussed in the

263

following. Accumulation of HBCD in maize roots was diastereomer- and 11



264

enantiomer-specific. (−)α-, (−)β- and (+)γ-HBCDs accumulated in maize roots were at

265

the concentrations of 2498, 3489 and 2238 ng g-1 based on dry weight, respectively,

266

which were consistently higher than those of their corresponding enantiomers (665,

267

2230 and 1876 ng g-1 for (+)α-, (+)β- and (−)γ-HBCDs, respectively). Our previous

268

study confirmed the diastereomer-specific uptake and translocation of HBCD by

269

maize,15 but no studies have been performed to date regarding its enantiomer-specific

270

uptake by plants. Root concentration factors (RCFs) of the three pairs of HBCD

271

enantiomers were further calculated by dividing the concentration of each enantiomer

272

in maize roots by the equalized concentration in the exposure solution (Figure 1B).

273

The values followed the order (+)γ- > (−)γ- > (−)β- > (+)β- > (−)α- > (+)α-HBCD.

274

RCF values were higher for (−)α-, (−)β- and (+)γ-HBCDs than their corresponding

275

enantiomers, further confirming that (−)α-, (−)β- and (+)γ-HBCDs were the

276

preferential configurations accumulated in maize. A significant positive correlation

277

was found between the values of logRCF for HBCD enantiomers and logKow (R2 =

278

0.68, P < 0.05) (Figure S4), indicating the contribution of hydrophobicity to plant

279

uptake of HBCD enantiomers. Diastereomer- and enantiomer-selective accumulation

280

of HBCD has also been found in organisms. However, the results were inconsistent

281

for different organisms, with some similar to14,38 and some different from39,40 the

282

results we obtained for maize.

283

Bioisomerization of HBCD Diastereomers and Enantiomers in Maize. Very

284

limited bioisomerization products (less than 3.1% of the initial amount) were found in

285

the solution of the unplanted control (Table S4, SI). The chromatograms of HBCD

286

bioisomerization products in maize roots are shown in Figure 2. Isomerization

287

occurred for all the tested HBCD stereoisomers with the sole exception of

288

(+)α-HBCD. Taking (+)γ-HBCD as an example, it converted mainly into (−)α-HBCD, 12


Page 12 of 31

Page 13 of 31


289

followed by (−)γ-HBCD and then (−)β-HBCD. (−)α-HBCD was frequently detected

290

in maize roots after they were exposed to the individual (+)/(−)-β- or γ-HBCD

291

stereoisomers, suggesting the capability of maize roots to bioisomerize (+)/(−)-β- or

292

(+)/(−)-γ-HBCD to (−)α-HBCD. The isomerization rate was further calculated (Table

293

S5, SI), and the results indicated that bioisomerization of HBCD in maize roots was

294

enantiomer-specific, with the rates for (−)α-, (−)β- and (+)γ-HBCDs higher than those

295

of their corresponding enantiomers. Among the three pairs of HBCD enantiomers,

296

(+)/(−)-γ-HBCDs were more prone to isomerization, with isomerization efficiency of

297

90.5 ± 8.24% and 88.5 ± 7.98%, respectively. The inter-conversion pathway of HBCD

298

is given in Figure S5A in the SI by taking the (+)/(−)-γ-HBCD enantiomers as an

299

example. Conversions of (−)γ-HBCD to (−)α-HBCD and (+)γ-HBCD to (+)α-HBCD

300

indicated the occurrence of regio- and stereo-selective isomerization of HBCD

301

enantiomers in maize roots. In addition, (+)/(−)-β- and γ-HBCDs were

302

stereoselectively rearranged, with (−)β-HBCD converting into (+)β-HBCD,

303

(−)γ-HBCD into (+)γ-HBCD and vice versa. The diastereomeric shift of β- and

304

γ-HBCDs towards α-HBCD in maize was similar to the bioisomerization typically

305

occurring in other organisms such as juvenile rainbow trout and mice,41,42 which can

306

be one of the reasons for the dominance of α-HBCD in the majority of organisms.43

307

More interestingly, bioisomerization of (−)α-HBCD towards (+)α-, (−)β-, and

308

(+)/(−)-γ-HBCD were observed in maize roots, but these isomerization reactions have

309

never been reported for animals.

310

Metabolization of HBCD in Maize by Debromination and Hydroxylation. The

311

metabolites derived from each HBCD enantiomer in maize roots were identified

312

(Figure 3), but none of them was detected in shoots. In order to exclude the possibility

313

for maize roots to take up metabolites directly from nutrition solutions, the solutions 13



Page 14 of 31

314

of unplanted controls were analyzed and no metabolite product was detected. The

315

mass balance of HBCD and metabolites was further calculated and the results (Table

316

S4) showed that the products of isomerization, debromination and hydroxylation

317

metabolites in solution after plant exposure were 0.08−0.5, 0.009−0.07; 0.002−0.02

318

ng/mL (Table S2), accounting for 0.04−3.84%, 0.05−0.27% and 0.03−0.09% of their

319

total mass (Table S4), respectively. This suggests that the contribution of microbial

320

biotransformation of HBCD in solution to root accumulation of metabolites can be

321

neglected.

322

The hydroxylated metabolites detected in roots included mono- and di-

323

OH-HBCDs and mono- and di- OH-pentabromocyclododecanols (OH-PBCDs),

324

mono- glutathione (GSH)-HBCD adducts, and the debrominated metabolites included

325

pentabromocyclododecenes (PBCDe) and tetrabromocyclododecenes (TBCDe)

326

(Figure 3A). Higher polarity di- and mono- hydroxyl metabolites eluted earlier than

327

the debrominated metabolites, while the latter eluted earlier than their respective

328

HBCD isomers (Figure 3B), consistent with the observations obtained for three

329

wildlife species (tern egg, seal and flounder) and Wistar rats.24 The results suggested

330

that debromination and hydroxylation were the two metabolic pathways for HBCDs

331

in maize (Figures S5B, 5C in SI). Debrominated and hydroxylated metabolites have

332

also been detected in both abiotic and biotic samples;21,23,44−46 however there has been

333

no

334

biotransformation in organisms at the individual HBCD enantiomer level. Patterns of

335

debrominated and hydroxylated derivatives from each HBCD enantiomers in maize

336

roots were distinct (Table S1 in SI). For example, two mono- and two di- OH-PBCDs

337

and two OH-HBCDs were obtained from (−)β-HBCD, while no mono-OH-HBCD or

338

di-OH-PBCD were formed from β(+)-HBCD, and HBCD-GSH conjugates only

research

involving

the

selective

debromination

14


and

hydroxylation

Page 15 of 31


339

appeared for the transformation of (+)/(−)-β-, (−)α- and (+)γ-HBCDs. Unfortunately,

340

native or isotopically labeled commercial standards for these isolated metabolites are

341

unavailable, precluding accurate quantification of their concentrations. Therefore, a

342

semi-quantitative analysis was performed using peak areas. It was interestingly found

343

that more debrominated products were detected in maize roots after being exposed to

344

(−)α-, (−)β- and (+)γ-HBCDs, while more hydroxylated products were obtained for

345

the exposures to (+)α-, (−)β- and (−)γ-HBCDs compared with their corresponding

346

enantiomers (Figure S6), which further confirmed that metabolization of HBCD by

347

debromination and hydroxylation in maize was enantio-specific and selective.

348

Molecular Interactions between HBCD and Plant Enzymes. The above results

349

provided

experimental

evidence

of

diastereomer-

and

350

accumulation and biotransformation of the individual HBCD enantiomers in maize

351

roots. It is necessary to gain further insight into the molecular mechanisms involved in

352

the selective interactions between HBCD enantiomers and maize roots. Selective

353

molecular interactions of HBCD enantiomers with maize enzymes of CYP71C3v2

354

and GST31 were characterized by homology modeling and molecular docking

355

methods. All three pairs of HBCD enantiomers could be docked into the active

356

cavities of maize enzymes of CYP71C3v2 and GST31 (Figure S7 in SI), and the

357

locations and conformations were diastereo- and enantio-distinct (Figure 4 and Figure

358

S8 in SI). When HBCD enantiomers were docked into CYP71C3v2 and GST31, eight

359

residues including Pro476, Arg126, Leu401, Leu400, Asn326, Phe468, Cys475 and

360

Thr334 in the active site of CYP71C3v2, and six main residues including Leu11,

361

Ser14, Pro13, Trp110, Trp168 and Phe222 in the active site of GST31 were found to

362

be located within a distance of 5 Å to the HBCD enantiomer, and suggested their

363

important roles in binding to the HBCD enantiomers (Table 1 and Figure S9 in SI). 15


enantiomer-selective


364

All these amino acid residues were hydrophobic and would bind with hydrophobic

365

compounds such as HBCD mainly via hydrophobic effects.47 Furthermore, HBCD

366

enantiomers also showed distinctly different binding modes with the residues in the

367

active sites of maize enzymes. Taking the interaction of (+)/(−)-α-HBCD with maize

368

enzymes as an example, (−)α-HBCD formed one halogen bond with the residues of

369

Arg473 and Cys476 of CYP71C3v2, respectively; whereas (+)α formed one halogen

370

bond with the residue of Asn326 (Figure S9a in SI). (+)α-HBCD did not form halogen

371

bonds with GST31 residues, while (−)α-HBCD formed halogen bonds with the

372

residue Ser14 of GST31 (Figure S9b in SI). The binding affinity values of HBCD

373

enantiomers were within the range of −7.92~−6.72 kcal/mol for CYP71C3v2 and

374

−8.42~−7.15 kcal/mol for GST31 (Table 1), respectively. Compared with the der

375

Waals force, the binding energy through electrostatic interaction was negligible (Table

376

S6), suggesting that HBCD enantiomers were bound to the active sites of

377

CYP71C3v2 and GST31 mainly via van der Waals forces. The binding affinity of

378

HBCD with plant enzymes exhibited both enantiomer-specific differences and

379

enzyme selectivity. (−)α-, (−)β- and (+)γ-HBCDs, with higher binding affinities than

380

their corresponding enantiomers, were found to fit better into the active sites of

381

GST31 (Table 1), which was consistent with the experimental results that more (−)α-,

382

(−)β- and (+)γ-HBCDs were isomerized and debrominated in maize. It has been

383

reported that GST enzymes exhibit catalytic activities in the reduction and

384

isomerization reactions of xenobiotics in plants,48 whereas, (+)α-, (−)β- and

385

(−)γ-HBCDs were the preferentially binding conformations for CYP71C3v2,

386

suggesting that these three isomers were more easily metabolized by CYP71C3v2.

387

This is in accordance with the experimental results obtained, where hydroxylation

388

reactions were easier for these three conformations than their corresponding 16


Page 16 of 31

Page 17 of 31


389

enantiomers (Figure S6). Therefore, enantiomer-selective accumulation and

390

biotransformation of HBCD enantiomers may derive from their different binding

391

affinities and binding modes to maize enzymes.

392

Environmental Implications. This study demonstrated that accumulation of

393

HBCDs in maize roots was diastereomer- and enantiomer-specific. Patterns of

394

isomerization, debromination and hydroxylation metabolites for each HBCD

395

enantiomer were significantly different, which comprehensively confirmed the

396

enantio-selective biotransformation of HBCDs in maize roots. Molecular interaction

397

between the individual HBCD enantiomers and maize enzymes of CYP71C3v2 and

398

GST31 well explained the diastereomer- and enantiomer-selective accumulation and

399

in vivo biotransformation of HBCDs in maize roots. This study provided both

400

experimental and theoretical evidence for steroisomer-specific biotic processes of

401

HBCD in maize. In order to clarify diastereomer- and enantiomer-selective metabolic

402

pathways of HBCDs in maize, this study was conducted under hydroponic conditions.

403

However due to its hydrophobic character, HBCD is strongly bound to soil particles

404

particularly to soil organic matter, which substantially decrease the its uptake by

405

plants. Therefore, there is a gap between the results obtained for HBCD in this study

406

and its behavior in the real environment, and further investigation of the diastereomer-

407

and enantiomer-selective behavior of HBCD in the soil-plant system is necessary.

408 409

ASSOCIATED CONTENT

410

Supporting Information

411

Detailed extraction and analytical methods of HBCD and chiral separation of

412

HBCD enantiomers by using UPLC-MS/MS, detection frequency of debrominated

413

and hydroxylated products in maize, mass balance of HBCD, 3-D structures of 17



Page 18 of 31

414

CYP71C3v2 and GST31 and HBCD diastereomer- and enantiomer-specific binding

415

with plant enzymes, including nine figures and six tables.

416 417

418

Corresponding Author

419

∗

420

Notes

421

The authors declare no competing financial interest.

422

AUTHOR

INFORMATION

Phone: +86 10 62849683; Fax: +86 10 62923563; E-mail: [email protected]

ACKNOWLEDGMENTS

423

This work was supported by the National Natural Science Foundation of China

424

(Projects 21537005, 21577155 and 21321004), and the Strategic Priority Research

425

Program of the Chinese Academy of Sciences (XDB14020202).

426 427

428

(1) Covaci, A.; Gerecke, A. C.; Law, R. J.; Voorspoels, S.; Kohler, M.; Heeb, N. V.;

429

Leslie, H.; Allchin, C. R.; de Boer, J. Hexabromocyclododecanes (HBCDs) in the

430

environment and humans: a review. Environ. Sci. Technol. 2006, 40 (12), 3679–3688.

431

(2) UNEP. United Nations Environment Programme. Report of the conference of the

432

parties of the Stockholm convention on persistent organic pollutants on the work of its

433

sixth meeting. 2013, UNEP/POPS/COP6. June 2013.

434

(3) Koch,

435

hexabromocyclododecane (HBCD) with a focus on legislation and recent publications

436

concerning toxicokinetics and -dynamics. Environ. Pollut. 2015, 199, 26–34.

437

(4) Heeb, N. V.; Schweizer, W. B.; Kohler, M.; Gerecke, A. C. Structure elucidation

438

of hexabromocyclododecanes – a class of compounds with a complex stereochemistry.

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enzyme of Sphingobium indicum B90A. Chemosphere 2015, 122, 70–78.

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586

24


Page 24 of 31

Page 25 of 31


587

FIGURE LEGENDS

588

Figure 1. Accumulation (A) and the root concentration factors (RCFs) (B) of HBCD

589

enantiomers in maize roots exposed to individual (+)/(−)α-, β- and γ-HBCD

590

enantiomers, with their concentrations at 25.0, 17.5 and 2.00 ng L-1, respectively.

591

Error bars represent one standard deviation. Means (n = 3) with the same letter

592

are not significantly different at the 5 % level.

593 594

Figure 2. Chromatogram profiles of bioisomerization products when maize was exposed to individual (+)/(−)- α-, β- and γ-HBCD enantiomers.

595

Figure 3. [M-H]− brominated clusters for the hydroxylated and debrominated

596

metabolites of HBCD enantiomers in maize roots monitored by Q-TOF LC-MS

597

(A), and the chromatogram profile of metabolites for HBCD enantiomers, taking

598

maize exposed to (−)β-HBCD as an example (B).

599

Figure 4. Enantiomer-specific binding modes of HBCDs bound to CYP71C3v2 and

600

GST31. The ligands are represented by stick drawings, and proteins are

601

presented in cartoons.

602

25



a

2500

(+)γ (+)γ-HBCD (−)γ (−)γ-HBCD (+)β (+)β-HBCD (−)β (−)β-HBCD (+)α (+)α-HBCD (−)α (−)α-HBCD

3500

2500

3000

2000

2000 2500

1500

1500

2000 1500

1000

1000

1000 500

B

300

200

100

500

b

400

RCF(Croot/C0)

HBCD in plant (ng/g dry weight)

A

c de

d

500

f 0

(−)α (−)α

(+)α (+)α

0 (−)β (−)β

(+)β (+)β

0

0 (−)γ (−)γ

(+)γ (+)γ

(−) α

Plant exposed to HBCD enantiomers

(+) α

(−)β (−) β

(+)β (+) β

(−) γ

Plant exposed to HBCD enantiomers

603 604 605

Page 26 of 31

Figure 1.

606

26


(+)γ (+) γ

Page 27 of 31


(−)α (−)α

(−)β (−)β

(+)α (+)α

(−)β (−)β

(−)β (−)β (+)γ (+)γ (−)γ (−)γ

(-)α

No isomerization

(−)α (−)α (+)β (+)β (+)γ (+)γ

(-)β (−)α (−)α

(+)γ (+)γ (−)γ (−)γ

607 608

10

12 14 Retention time (min)

(+)γ (+)β β(+)γ (+) 16

18

10

12


18

10


(+)β (+) β

(−)β (−) β

(−)α (−) α

(−)γ (−)γ

(+)β (−)β (−) β

(−)β (−) β

(−)α (−) α

(−)α (−)α

609

10

12

14 16 Retention time(min)

18

10

12

(+)γ (+) γ (+)β β (+)

14 Retention time (min)

(−)α (−)α

(+)γ (+)γ

(−)α (−) α (−)γγ (−) (−)β (−) β

(+)γ (+) γ (−)γ (−) γ 18 10


610 611

18

(−)β (−) β

(−)α (−)α (+)γ (+)γ (−)β (−)β (+)β (+)β

16

16

Figure 2.

27


(−)γ (−) γ 16

18


942.9

652.6

A

940.9

636

640

644

944.9

650.6 654.6

Mono-OH-HBCD [C12H18OBr6]

HBCD-GSH [C12H18Br6-C10H15N3O6S]

648.6

648 676.6

652

656

660

930

940

950

670

680

PBCDe [C12H16Br5] 566.3 562.3 690

546

552

558

574.6 578.6

564

572

480.3 TBCDe [C12H14Br4]

572.6 Mono-OH-PBCDe 580.6 576.6 [C12H17OBr5] 478.3 476.4 550

560

570

956

564.3

678.6 Di-OH-HBCD 674.6 [C12H18O2Br6] 680.6 672.6 660

Page 28 of 31

580

590

470

598

484.3

474 478 482 Counts vs. (m/z)

486

586.6 588.6 Di-OH-PBCDe 586.6 [C12H17O2Br5] 592.6 578

584 590 596 Counts vs. (m/z)

602

606

612 613 RT=10.3, 11.8min (-)β-mono-OH-PBCD

B RT=7.2min (-)β-TBCD

1

5

10

15

20

1

5

10

1

5

10

15

15

20

RT=11.2min (-)β-PBCD

RT=7.5min (-)β-di-OH-HBCD

1

20

5

10

15

20

RT=9.6min (-)β-di-OH-PBCD RT=14.1min (-) β-HBCD-GSH

1

5

10

15

20

1

5 10 15 20 Counts vs. Acquisition Time (min)

RT=10.4min (-)β-mono-OH-HBCD

614 615 616

1 10 15 20 5 Counts vs. Acquisition Time (min)

Figure 3.

28


Page 29 of 31


GST31

CYP71C3v2

617 618

Figure 4.

29



619

Page 30 of 31

Table 1. Binding affinities of HBCD enantiomers and the main interaction active site residues with CYP71C3v2 and GST31. Isomer

(−)α-HBCD

(+)α-HBCD

(−)β-HBCD

(+)β-HBCD

(−)γ-HBCD

(+)γ-HBCD

length (Å)

8.3

8.3

8.5

7.6

8.3

9.3

Docking energy (kcal/M)

Active site residues

CYP71C3v2

GST31

CYP71C3v2

GST31

−6.72

−7.57

Trp151, Pro476, Cys475, Ile474,

Leu11, Ser14, Phe16, Trp110,

Arg473, Ala143, Phe468

Ala114, L115, Phe222

Arg126, Leu401, Leu400, Leu519,

Leu11, Pro13, Ser14, Lys54,

Asn326, Ala330, His403

Ile55, Trp168, Phe212, Phe222

Arg136, Arg155, Ala330, Thr334,

Leu11, Pro13, Ser14, Val38,

Phe468, Cys475, Met520

Lys54, Ile55, Trp110, Trp168, Phe222

Arg128, Asla330, Luer 399, Lys475,

Leu11, Pro13, Ser14, Val38, Trp110,

Leu399, Leu519, Met520

Ala111, Trp168, Phe222

Arg126, Ala143, Ala330, Thr334,

Pro13, Ser14, Trp110, Ala111, Trp168,

Phe468, Cys475, Leu519

Phe222, Ser223, Asn227

Pro467, Thr334, Gly397, Ala330,

Leu11, Phe16, Val38, Lys41, Lys53,

Leu400, Leu401, Asn326

Ile55, Trp168, Phe222

−7.82

−7.60

−7.35

−7.92

−7.34

−7.39

−7.29

−7.15

−7.75

−8.42

620

30


Page 31 of 31



Experimental and Theoretical Evidence for Diastereomer- and

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