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Agricultural and Environmental Chemistry

Plant Uptake and Metabolism of 2,4-dibromophenol in Carrot: In Vitro Enzymatic Direct Conjugation Jianqiang Sun, Qiong Chen, Zhuxiu Qian, Yan Zheng, Shuai Yu, and Anping Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00543 • Publication Date (Web): 15 Apr 2018 Downloaded from http://pubs.acs.org on April 15, 2018

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Journal of Agricultural and Food Chemistry

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Plant Uptake and Metabolism of 2,4-dibromophenol in Carrot: In Vitro

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Enzymatic Direct Conjugation

3

Jianqiang Sun,† Qiong Chen,† Zhuxiu Qian,† Yan Zheng,† Shuai Yu,† Anping Zhang*,†

4 5

6

†

7

Zhejiang University of Technology, Hangzhou 310014, China

International Joint Research Center for Persistent Toxic Substances, College of Environment,

*

Corresponding author (Tel: 86-571-88320534; Fax: 86-517-88871576; E-mail:

[email protected])

ACS Paragon Plus Environment


8 9

Abstract Plants can extensively uptake organic contaminants from soil and subsequently transform

10

them into various products. Those compounds containing hydroxyl may undergo direct

11

conjugation with endogenous biomolecules in plants, and potentially be preserved as conjugates,

12

thus enabling overlooked risk via consumptions of food crops. In this study, we evaluated the

13

uptake and metabolism of 2,4-dibromophenol (DBP) by both carrot cells and whole plant. DBP

14

was completely removed from cell cultures with a half-life of 10.8 h. Four saccharide conjugates,

15

three amino acid conjugates and one phase I metabolite were identified via UPLC-QTOF-MS

16

analysis. The dibromophenol glucopyranoside (glucose conjugate) was quantitated by

17

synthesized standard and accounted for 9.3% of the initial spiked DBP at the end of incubation.

18

The activity of glycosyltransferase was positively related to the production of

19

DBP-glucopyranoside (p=0.02, R2=0.86), implying the role of enzymatic catalysis involved in

20

phase II metabolism.

21 22

Keywords: brominated phenols, direct conjugation, glycosyltransferase, plant uptake, metabolism, cells


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Introduction

24

Brominated phenols are a group of compounds widely used as biocides, disinfectants, solvents,

25

and especially flame retardants, or intermediates for the synthesis of flame retardants.1, 2 Of these,

26

tetrabromobisphenol A has the largest production volume and is the most extensively consumed.3

27

The approximate worldwide annual production of tetrabromobisphenol A reached 170,000 tons

28

about a decade ago and still grows with continued market demand.4 As another example of

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simple bromophenols, 2,4,6-tribromophenol is also used as a flame retardant and antifungal

30

agent (e.g., wood treatment).1 Such bromophenols may play a role in the chemical defense and

31

ecological deterrence, and they have been suspected to exhibit potential toxicity, for instance,

32

nerve, reproduction and endocrine disruption.1, 3, 5 Indeed, brominated phenols have frequently

33

been detected in soil, river, sewage sludge, and even in biological and human samples.3 For

34

example, as much as 450 µg/g of tetrabromobisphenol A in soil has been reported in

35

contaminated sites.6 Concentrations of 2,4-dibromophenol, 2,6-dibromophenol, and

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2,4,6-tribromophenol in the surface water have been reported at 40,000, 3000 and 300 ng/L,

37

respectively, while 2,4,6-tribromophenol reached 3690 ng/g in sediment samples from the Rhone

38

estuary, France.5

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Given the continuous occurrence of brominated phenols in soils, especially in agricultural

40

soils, there is heightened concern regarding their environmental fate and food safety. Studies

41

have shown that xenobiotics in plants may undergo phase I, II, and III metabolisms.7 In phase I

42

metabolism, molecular usually involves some activation processes such as hydroxylation,

43

oxidation or hydrolysis .8, 9 In phase II, conjugation of the parent compound or intermediate

44

products with polar biomolecules such as amino acids, carbohydrates, or glutathione occurs in



45

plants with increased water solubility.8-10 For example, in vitro and/or in vivo research models in

46

various plants have shown that 4-nonylphenol11, polycyclic aromatic hydrocarbons12,

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strobilurins13, phthalates9, and triclosan14 could be transformed to their hydroxylated or

48

hydrolyzed formations through phase I metabolism. Thus, when considering the exposure of

49

humans to contaminants via vegetables, such formed conjugates could undergo back conversion

50

through cleavage in the human gut, subsequently release the parent molecule with potential

51

adverse effects.15 Nonetheless, information about the phase II metabolism of organic

52

contaminants in plants is still relatively limited.15-17 Furthermore, xenobiotics in plants are

53

transformed under the effect of enzymes, including cytochrome P450 monooxygenase (CYP450),

54

glutathione-S transferase (GST), and glycosyltransferase (GT).18 In a recent study, CYP450

55

enzymatic phase I metabolism of ibuprofen by the plant Phragmites australis was reported.19

56

However, there is no further investigation combining the enzyme-involved phase II metabolism

57

and the transformation process of organic pollutant in the plant.

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This study aimed to assess the uptake and metabolism of brominated phenols by edible plants

59

concerning their potential risks from contaminated soil to food consumption. The compound

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2,4-dibromophenol was chosen for the potential direct conjugation of its hydroxyl group with

61

biomolecules. The plant uptake, translocation, and metabolism of dibromophenol were

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investigated in both carrot cell culture and whole plant at different times. Carrots were selected

63

because of its high consumption of raw root and high uptake ability of roots to the organic

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compound.14 The activity of the specific enzyme along with the response of the metabolites was

65

measured to provide perspective into the role of the enzyme in the transformation process in the


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plant. If such compounds were masked in the form of conjugated metabolites, which could be

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cleaved to the active parent through enzymatic hydrolysis, their biological activity might have

68

been underestimated.15, 20

69

Materials and Methods

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Chemicals. Standards of DBP (>99.0%) and DBP-d3 (>99.0%) were purchased from

71

Sigma-Aldrich (Shanghai, China). Standard of 4-bromocatechol (>98%) was purchased from

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Aladdin (Beijing, China). The stock solution of DBP and DBP-d3 were prepared in methanol and

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stored in amber glass vials. All organic solvents used were of HPLC grade (J. T. Baker, Shanghai,

74

China). Deionized water was prepared using Milli-Q plus water purification system (Millipore

75

Corporation, Shanghai, China).

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Experimental Design. The carrot cell suspension was grown from a carrot callus which was

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initiated from germinated seeds of little finger carrots (Daucus carota var. sativus) under sterile

78

conditions and exclusion of illumination in Narayan culture medium at 26 °C and 130 rpm.21 The

79

metabolism experiment was performed at the beginning of the subcultivation interval (120 h),

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applying to 10 mL Murashige and Skoog liquid media inoculated with 3 g (wet weight) fresh

81

carrot callus tissue. Same amounts of nutrient solutions were added to the treated groups and the

82

control groups. The initial concentration of DBP in the culture medium was 100 µg/L. Three

83

control treatments including a medium control with DBP but without cells, a blank cell culture

84

control with cells only, and a negative control with DBP and nonviable cells autoclaved at

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121 °C for 20 min were conducted for accurate quality control. All treatments and controls were

86

repeated in triplicates.



87

Three vials were harvested at 0, 2, 8, 24, 48, 72 and 120 h of incubation. Cell culture and cell

88

materials were immediately separated by filtration through cellulose filters. The cell materials

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were washed twice with 5.0 mL deionized water combined with the respective medium, and

90

wrapped in aluminum foil, immediately frozen and stored at -80 °C for further processing. The

91

media were extracted three times with 10 mL dichloromethane to determine the targeted

92

compounds. The extractions were condensed using rotary evaporation and cleaned with 12 mL

93

methanol. Targeted compounds were eluted. The eluent was then concentrated under nitrogen gas

94

and finally reconstituted in 1.0 mL of methanol. To analyze the cell matter, the frozen cell

95

materials were homogenized in liquid nitrogen using a mortar and pestle, transferred into a glass

96

centrifuge tube, supplemented with 5.0 mL water/methanol (1:1 ,V/V), then sonicated for 30 min

97

in a sonication water bath (Fisher Scientific, Pittsburgh, PA). After each extraction, the glass

98

centrifuge tube was centrifuged for 10 min at 6500 rpm at 4 °C, and the supernatants were

99

collected. The same extraction step was repeated for three consecutive times. The supernatants

100

were combined and concentrated using a gentle nitrogen stream, finally adjusted to a total

101

volume 1.0 mL.14-16

102

As for the whole plant, the extraction and analysis methods for DBP were the same as those

103

for the carrot cell materials but without the filtration step. Hoagland’s nutrient solution was used

104

in the cultivation of the whole plant. Negative control (with nonviable plants) was not used for

105

quality control.

106 107

Compound Synthesis. In addition to the parent compound, DBP-glucopyranoside was synthesized for structural confirmation and accurate quantification.22 NMR and IR analyses were


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used to verify the purity and structure of the synthesized standards. Both of retention time and

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accurate mass MS/MS fragmentation pattern were used for unambiguous identification.

110

Chemical Analysis. Before instrumental analysis, the final extracts from the cells/plant and

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aqueous medium were mixed by a rotator for 5 min and further filtered through 0.22 µm

112

polytetrafluoroethylene (PTFE) membrane into a 2-mL glass vial.

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Quantitative analysis via UPLC-MS/MS. A system of Waters ACQUITY ultra-performance

114

liquid chromatography (UPLC) in tandem with two-stage mass spectrometers (Waters

115

Corporation, Milford, MA), and a column (50 mm × 2.1 mm i.d., 1.7µm, ACQUITY UPLC BEH

116

C-18 (Waters)), applying electrospray ionization (ESI) were used for quantitating analysis. The

117

column temperature is at 30 °C. The mobile phases were water (solvent A) and methanol (solvent

118

B). At a constant flow of 0.2 mL/min, the gradient was as follows: 0-0.5 min, 95% B; 0.5-3.5

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min, 95-70% B, held for 0.5 min; 4-5 min, 70-95% B. The injection volume of each sample was

120

10 µL, and equilibration time between each sample run was set for 1 min. In the ESI negative

121

mode, the mass data of the targeted compound were acquired in multiple reactions monitoring

122

(MRM) mode and product ion scan. The instrument control and data acquisition were processed

123

using the MassLynx Workstation software, ver. 4.1 (Waters).

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Untargeted analysis via UPLC-QTOF-MS. The cell extracts were further analyzed on an

125

AQUITY UPLC (waters) in tandem with a micrOTOF-QII mass spectrometer (Bruker Daltonics,

126

Bremen, Germany) with an electrospray ionization (ESI) interface. The column and mobile

127

phases applied for untargeted analysis were as described above. The mobile phase gradient

128

program was as follows: 0-0.5 min, 5% B; 0.5-3.5 min, 5%-50% B; 3.5-6.5 min, 50%-100% B;



129

6.5-7 min, 0% B; and 7-10 min, 100%-5% B. The conversion of raw data files to mzXML files

130

and analysis of converted files were processed using Hystar and MZmine 2.9, respectively.23 The

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exact m/z, retention times of peak, peak abundant, and presence in different treatments and

132

controls were compared to obtain the proposed candidate metabolites. These masses of primary

133

candidates obtained by Q-TOF were further used for the semi-quantitatively determination on

134

UPLC-QqQ-MS/MS.

135

Enzyme Extraction and Activity Determination. The extraction of glycosyltransferase was

136

conducted according to the methods described previously.24, 25 The buffers were precooled in a

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refrigerator before extraction, and the cell samples (3 g) were kept on ice during processing to

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maintain the integrity of the enzyme. The frozen cells were homogenized under liquid nitrogen

139

with a mortar and pestle to a fine powder, and then, the homogenized cells were transferred into

140

a glass centrifuge tube and mixed with 10 mL 0.1 M of sodium phosphate buffer (pH 7.4) that

141

contained 250 mM of sucrose and 1 mM of ethylenediaminetetraacetic acid. The mixture was

142

centrifuged at 10,000 g for 15 min at 4 °C. The same extraction step was repeated three times.

143

The supernatant was collected and then precipitated twice by adding ammonia sulfate. The

144

suspension was centrifuged at 18,500 g for 30 min at 4 °C. In the final step, the precipitate was

145

redissolved in 2.5 mL of 200 mM Tris / HCl buffer (pH 7.3) and subsequently stored at -20 °C

146

for further analysis.19 GT activity was determined by Plant GT ELISA Kit (Shanghai Hengyuan

147

Co., Ltd, Shanghai, China) and analyzed by a microplate reader.

148 149

Data processing. All the reported values are mean and standard deviation of three replicates. The statistical treatment was carried out by a one-way analysis of variance (ANOVA) at the


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significance level of 0.05 using Origin Pro, ver. 8.0 (OriginLab, Northampton, MA).

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Results and Discussion

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Uptake, Accumulation, and Translocation of DBP in Whole Plants. No DBP was detected

153

in the medium or plant blanks which were not spiked DBP. However, approximately 20% of

154

DBP was lost in the abiotic control over the 120 h of incubation, likely due to the slight

155

degradation (e.g., photolysis).3 There was no significant difference in plant biomasses between

156

the spiked and nonspiked groups, indicating that the treatment of DBP did not affect the growth

157

of the plants. The dissipation of DBP in the solution was significantly accelerated than those in

158

the abiotic control, with its average amounts decreasing from an initial amount of 996 ± 94.4 ng

159

to 282.7 ± 74.4 ng after 2 h exposure and to 4.0 ± 3.3 ng after 120 h exposure, respectively

160

(Figure 1A). DBP in the hydroponic medium appeared to be rapidly taken up, providing direct

161

evidence for the uptake ability of carrot.

162

On the other hand, the amounts of DBP in plants increased quickly to a maximum of 206.8 ±

163

27.4 ng after 8 h of exposure, and then, the amounts decreased slowly to a final content of 60.2 ±

164

14.5 ng after 120 h exposure, indicating the process of accumulation of DBP from the medium

165

into the root and, subsequently, transformation within the plants. At the end of incubation,

166

approximately 7.5% and 0.5% of DBP was distributed in the plants and mediums, respectively.

167

The rapid uptake and transformation of DBP by plants are comparable to some other organic

168

compounds, such as triclosan by carrot and 2-mercaptobenzothiazole by Arabidopsis but is much

169

faster than biodegradation by bacteria.14, 16

170

Accumulation of chemicals occurs after the first step of the uptake through plant roots. In this



171

study, the bioconcentration factor (BCF, the extent of the accumulation in plant tissue) and

172

translocation factor (TF, the in-plant transport of contaminant from roots to stems and leaves)

173

were assessed at the time of 24 h. The root average BCF of DBP was 0.66 ± 0.12 L g-1, the stem

174

and leaf part average BCF was 0.52 ± 0.17 L g-1, and the average TF was 0.79 ± 0.05. The

175

average BCF value was consistently less than 1, which was comparable to other hydrophobic

176

chemicals.9, 26 The average TF value was less than 1, suggesting that DBP was relatively difficult

177

to transport from the roots to stems or leaves. Hydrophobic organic contaminants (log Kow>3.0)

178

are easier to absorb by roots and difficult to transport to stems or leaves; thus, the transmission

179

capacity was relevant to the log Kow.27 The DBP with a log Kow value at 3.48 is too hydrophobic

180

to translocate to a greater extent.

181

Dissipation of Parent DBP in Carrot Cell Cultures. Plant cell cultures have been served

182

as a model system to explore the metabolism potentials of a variety of xenobiotics because cells

183

contain simpler matrices to exclude matrices interference and are more easily and rapidly

184

manipulated than whole plants.11-14 The time-dependence of DBP in carrot cell cultures was

185

comparable to the trend in intact plants. Amounts of DBP in the culture medium reduced rapidly,

186

from an initial 987 ± 64 ng to 262.5 ± 42.1 ng after 2 h exposure and to 3.8 ± 2.3 ng after 120 h

187

exposure, respectively (Figure 1B). For the carrot callus, the content of DBP rapidly increased

188

and reached the maximum at 2 h with the value of 134.1 ± 15.7 ng, likely indicating the

189

absorption of DBP from the medium. However, the amounts of DBP in cells were steadily

190

reduced to a final of 3.0 ± 0.7 ng after 120 h incubation. Thus, the dissipation of DBP from the

191

medium with carrot cells may be ascribed to the uptake and the subsequent intracellular


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metabolism. Statistical analysis of the DBP amounts showed that the dissipation of DBP detected in the

194

system followed a second order reaction, for the curve of 1/ [At, DBP] versus time was linear

195

(r2=0.94). The reaction rate constant k and a half-life (t1/2) were calculated to be 0.0221 ng-1 h-1

196

and 10.8 h, respectively. The dissipation rate of DBP in this study was comparable to those

197

reported results in similar type cell cultures with the half-life value of 9 h for triclosan and

198

exponentially faster than those of 112 h for di-n-butyl phthalate and 5000 h for di(2-ethylhexyl)

199

phthalate.9, 14 Similar to other organic contaminants, the reduction of DBP in this study could

200

clearly be attributed to the uptake and metabolism by plants.14

201

Occurrence and Identification of DBP Metabolites. The cell extracts of the samples at 120

202

h incubation were used for UPLC-QTOF-MS analysis to detect and identify metabolites of DBP.

203

Eight metabolites absent from all the controls were proposed upon the data (Figure S1, Table 1).

204

Phase I Conjugate (Group 1). Compound 1 was identified as 4-bromocatechol by comparing

205

the retention time and mass spectrometric data of its authentic standard, showing neither DBP

206

fragmentation nor neutral loss of DBP, and it readily produced fragment ions due to the loss of

207

bromine and hydroxyl. The ability of the carrot cell culture to form phase I transformation

208

products was previously observed and can be ascribed to the enzymatic reaction of P450

209

monooxygenase in plant cells.19 No further phase I metabolite of DBP was detected, such as

210

oxidation and hydroxylation products.

211

DBP Saccharide Conjugates (Group 2). All detected saccharide metabolites produced a

212

fragment at 250.8954, corresponding to the molecular anion of DBP (C6H4Br2O), in the ESI



213

negative mode. This fragment ion indicated that these saccharide conjugates were formed from

214

DBP and the core structure of DBP was not changed in the formed molecules.

215

DBP-glucopyranoside (2) was verified as Level 1 confidence by comparing with the retention

216

time and fragmentation data of the synthesized standard.28 Compound 2 produced the fragments

217

at m/z 250.8954 and 163.1485, which corresponds to DBP and glucose (C6H11O5), suggesting the

218

direct conjugation occurred at the phenol moiety of DBP. However, it was difficult to quantitate

219

these conjugates in the plant extracts due to the lack of the commercial standard for other

220

metabolites. Glucopyranosyl-(malonyl)-glucopyranoside (3) produced fragments at m/z

221

250.8954 and 410.3274, corresponding to the molecular anion of DBP and a hydroxyl

222

glucopyranosyl-(malonyl)-pentofuranoside (C15H23O13), respectively. Both

223

DBP-glucopyranosyl-Pentofuranoside and DBP-glucopyranosyl-Desoxyhexopyranoside (4 and 5)

224

were identified as the disaccharide conjugates. Both compounds 4 and 5 produced a fragment at

225

m/z 250.8954 via the loss of DBP. In negative mode, the fragmentation patterns of the glycosidic

226

bond of disaccharide conjugates did not occur.14

227

DBP Amino Acid Conjugates (Group 3). The untargeted search also included amino acid

228

conjugated DBP metabolites. Amino acid conjugation has been observed infrequently in plants

229

and is thought to be a side reaction rather than a main route of detoxification.29 In the positive

230

ionization mode, a signal at m/z 339.9938 was detected. DBP-alanine (6) was detected at a

231

retention time of 5.2 min with a fragment of m /z 89.0932, corresponding to amino acid

232

(C3H6NO2). In negative ionization, Compounds 7 and 8 produced fragment ions at m/z 129.1140

233

and 173.1235 via the loss of acetyl-alanine (C5H8NO3) and aspartic acid (C6H8NO5), respectively.


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The precursor ion m/z 250.8954, corresponding to DBP, was also observed as product ion for

235

compounds 7 and 8, indicating that they were likely DBP acetyl-alanine and DBP acetyl-aspartic

236

acid, respectively.

237

Possible Metabolic Pathways. Based on the eight metabolites measured in this study, there

238

seem to be three main metabolic pathways for DBP in carrot cells: debromination/hydroxylation,

239

glycosylation, and conjugation with amino acids (Figure 2). Debromination/hydroxylation as the

240

first pathway occurred by removing a bromine atom and hydroxylation in the ortho position of

241

DBP, and produced the only phase I metabolite, 4-bromocatechol. This metabolic pathway has

242

also been observed in other studies, for example, BDE-47 can undergo

243

debromination/hydroxylation by enzymatic degradation in animals, producing two hydroxylated

244

tri-BDEs.30 Other possible congeners, such as bromohydroquinone and 4-bromoresorcinol, were

245

not detected, probably because the ortho-positioned bromine atoms may be more easily replaced

246

than the para-positioned.31 Additionally, direct hydroxylation, debromination, and Br-shift

247

during plant metabolism of organic contaminants may not have occurred in this study. Moreover,

248

none of the theoretical phase II metabolite of 4-bromocatechol was detected.

249

Glycosylation is a biological plant detoxification strategy by the addition of sugar monomers

250

to reduced toxicity, increase polarity, sequentially help to export to tissue or subcellular

251

compartments.16, 32 When slower phase I activation is not necessary on the aromatic structures,

252

glycosylation occurs rapidly in plants by catalysis of the glycosyltransferase.8 Compound 2 was

253

identified as a β-D-glucopyranose moiety because the glycosides were mainly in the form of

254

β-glycoside conjugates, forming an O(1) glucosidic bond binding with the hydroxy of DBP.33 For



255

hydroxyl-containing contaminants, such as triclosan and chlorophenol, the same direct binding in

256

plants has been observed.14, 34 Compound 2 could then be formed into the disaccharide

257

conjugates (3, 4 and 5) by adding pentose or hexose , respectively. It is common in plants

258

conjugation with monosaccharide β-D-glucose occurs, unlike conjugation with disaccharide.35, 36

259

The second molecule in compound 3 is most similar to the second glucopyranoside that forms an

260

O-glucosidic bond.37 The participation of a pentose, such as compound 4, has rarely been

261

detected so far.36 Compound 5 contains a disaccharide consisting of deoxyhexose and

262

glucopyranose, which is most likely rhamnose.38, 39

263

Amino acids have been found to participate in multifunctional secondary metabolism in plant

264

cells. The production of amino acids conjugations implies consumption of certain amino acids

265

involved in plant development. Previous studies have found that both of benzotriazole and

266

2-mercaptobenzothiazole were substituted for the indole mimic and underwent the tryptophan

267

biosynthesis processing.16, 40 Compounds 6, 7 and 8 likely followed the similar transformation

268

processing, and are structurally identical to the normal physiologically functional plant

269

compounds, suggesting that DBP may also be incorporated into the same transformation pathway,

270

even if the structure of DBP is more different from indole than the structure of benzotriazole.41 It

271

is well-known that many plant amino acid transporters have certain non-characteristic properties;

272

thus, the amino acid conjugates of DBP may occur commonly on other contaminants with similar

273

structure.42

274

Transformation Kinetics of Metabolites in Cell Cultures. Based on the abundance of peak

275

intensities on UPLC-QqQ-MS/MS analysis, two saccharide conjugates (2 and 3) and two amino


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acid conjugates (7 and 8) were chosen as the main metabolites, which accounts for 83.1-98.3%

277

of the total area of eight peaks. The semi-quantitatively determined peak areas were used to

278

describe the kinetics in cells and the release to the mediums (Figure 3, Figure S5). After 120 h

279

incubation, the responses of the four compounds in cell extracts are as follows: compound

280

8>compound 2>compound 3> compound 7. The levels of compounds 2 and 8 sharply increased

281

in the first 48 h and then remained flat in the cell extracts. In contrast, the relative composition of

282

compounds 3 and 7 consistently increased during the incubation of cells. It was clear that the

283

total responses of compounds 2 and 8 accounted for approximately 71.8-88.1% of the four

284

dominant metabolites in cell extracts, of which the percentage of compound 8 was always higher

285

than that of compound 2. Although the parent DBP was completely depleted in 120 h, the

286

response of these four conjugates only accounts for the extractable part of all the dissipation in

287

the system, and the non-extractable part by phase III metabolism was still not counted.

288

To test if the identified conjugates could release back to the hydroponic medium, the medium

289

samples were taken into account in the targeted analysis. Because the direct transformation at the

290

root surface was excluded in the cell experiment, the presence of conjugates in the medium was

291

certainly due to the release. These four metabolites were also successfully detected in the

292

mediums, and their levels obviously increased through time, indicating their excretion from cells.

293

This phenomenon has been observed on the metabolism of triclosan in carrot cells and

294

benzotriazole in Arabidopsis plants .14, 16 However, their peak areas in mediums were

295

exponentially lower than those in cells (Table S2 and S3), showing that disaccharide and amino

296

acid conjugates were not easily released back into the hydroponic mediums. This is because



297

many conjugated pollutant metabolites are generally assumed to be sequestered in vacuoles or

298

cell walls as storage and difficult to pass through a lipid membrane.43

299

The ratios of the responses of each conjugate in mediums to those in cells at 120 h illustrate

300

compound 2 had the most potential to release, followed by compounds 3, 7 and 8. Furthermore,

301

the percentages of compounds 2 and 3 to the sum of the four peaks in mediums were consistently

302

significantly higher than those in cells at different times, and compound 2 continued to account

303

for approximately 59.3-71.0% in the mediums, showing that the excretion of saccharide

304

conjugates was relatively more common than those of amino acid conjugates. This is principally

305

due to the different sites of the different enzymatic reactions within the cell. For instance, the

306

enzymes for amino acid conjugation are resided in mitochondria, while the CYP and

307

glucuronosyltransferase are located in endoplasmic reticulum.44 Because of the impermeability

308

of the inner mitochondrial membrane, the conjugated metabolites cannot readily escape from the

309

mitochondrial matrix.44 Release of conjugates from plants has implications for phytoremediation

310

application because a portion of the parent compound may not be detected but is present in a

311

slightly altered form.

312

Role of Glycosyltransferase Enzyme in Phase II Direct Conjugation. The formation of

313

metabolites in this study could be attributed to the effect of specific enzymes in plants. The

314

debromination and hydroxylation in the process of formation of 4-bromocatechol could be

315

attributed to CYP450.45 Furthermore, it was verified that CYP450 was involved in the

316

debromination of (α-bromo-iso-valeryl)urea to (3-methyl butyryl)urea.46 However, information

317

on the role of enzyme-medicated biotransformation in plants is still scarce. In plants,


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318

glycosylation is catalyzed by a specific family of enzymes called glycosyltransferase that

319

catalyze the transfer of sugar residues from uridine diphosphate to O(OH- and COOH-), N, S and

320

C atoms on an acceptor.47 According to the relatively high abundance of glycosylated products in

321

DBP metabolites, we measured the activity of glycosyltransferase as a specific metabolic enzyme

322

in cell materials of treated groups and compared it with those of untreated groups to investigate

323

the role that glycosyltransferase played in dibromophenol phytotransformation. To relate the

324

enzyme activities to the formation of conjugates, the absolute amounts of DBP-glucopyranoside

325

(2) in cells were accurately quantitated using the synthesized standard. Figure 4 shows the

326

time-dependent activities of glycosyltransferase and the trend of compound 2 in cells. The

327

amount of compound 2 increased rapidly to 57 ± 5.4 ng after 2 h exposure, continued a growing

328

trend until 48 h, and then the detected compound 2 amounts in cell materials increased slowly to

329

a maximum of 153 ± 11.4 ng.

330

In general, the glycosyltransferase activity in the treated groups was significantly higher than

331

that in untreated groups after 2 h exposure until the end of the incubation, indicating its positive

332

response to the formation of glycosylated conjugates. With the rapid increase of compound 2

333

until 48 h, the difference in the values of glycosyltransferase activities between the treated and

334

controls was kept at relatively high levels. Glycosyltransferase activity that contributed most to

335

the difference values was highest at 8 h and followed by 24 h, accompanied by a faster rate of

336

production than those at other time points. From 48 h, the different value of glycosyltransferase

337

activities continued to decrease with the deceleration of DBP-glucopyranoside production. In

338

summary, our results showed a clear relatedness between the elevation of enzyme activities and



339

the levels of DBP-glycosylated products. Although the amino acid conjugates showed an

340

important role in the metabolites from cell extracts in this study, it still could not verify the

341

correlation with enzyme bioactivities, such as amino transferase and glutathione transferase. The

342

enzymology knowledge of amino acid conjugation is not recognized comprehensively in

343

comparison to that of CYP450 and uridine 5'-diphosphate (UDP)-glucuronosyltransferases.48

344

A multitude of contaminates bearing hydroxyl groups in their molecules are inevitably being

345

introduced into the soil-plant ecosystem by agricultural biosolids, wastewater irrigation, direct

346

application and spatial migration.49 This study demonstrated that DBP as a model was rapidly

347

taken up and metabolized by carrot via enzymatic reaction, predominantly resulting in several

348

phase II saccharide and amino acid conjugates. When the parent DBP was completely depleted at

349

the end of the incubation, there still exist a large number of conjugates. If this direct conjugation

350

was pervasive in more plant species, more types of phenol compounds, and with more kinds of

351

biomolecules, especially in the natural environment, it is critical to take phytotransformation and

352

conjugates into realistic consideration when performing the potential risk assessment of

353

pollutants on environmental fate or human health. For example, previous uptake studies on

354

organic compounds may have underestimated the actual extent of uptake, because only the

355

amount of non-metabolized portion has been counted in.14 In addition, conjugated contaminants

356

are not targeted for analysis. In particular, the extraction and instrumental analysis method may

357

not be suited for those conjugates with the more polar properties, and measurement of the parent

358

compounds alone in plant tissues or other environmental media may underestimate total mass.

359

As the core structure of parent compound is not altered during biotransformation but instead


Page 18 of 31

Page 19 of 31


360

conjugated to form other larger more complex compounds, the direct conjugation of phenol

361

compound may mask its presence of the parent in the environment. Further research is needed to

362

investigate the occurrence and fate of the metabolites of more phenol contaminants via in planta

363

direct conjugation in the environment, and their biological activity and bioavailability in food

364

crops for comprehensive risk assessment.

365

Supporting Information

366

This material is available free of charge via the Internet at http://pubs.acs.org. Additional

367

details on plant cultivation and treatment, instrumental analysis, synthesis of DBP metabolite

368

(DBP-glucopyranoside), metabolite identification on UPLC-QTOF-MS, peak areas and peak

369

proportions of DBP metabolites (2, 3, 7 and 8) on UPLC-MS/MS.

370

Acknowledgments

371

This study was supported by the National Natural Science Foundation of China (21577127,

372

21307111), the Natural Science Foundation of Zhejiang Province (LY17B070006) and the

373

College Student Science and Technology Innovation Program of Zhejiang Province (Xinmiao

374

Talents Program). We appreciate Zili Guo and Shoufeng Dai from the Collaborative Innovation

375

Center of Yangtze River Delta Region Green Pharmaceuticals for UPLC-QTOF-MS analyses.



376

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508

Figure Captions

509

Figure 1 Amounts (ng) of DBP in carrot (A) whole plant experiment and (B) cell cultures versus

510

incubation time (h). Data shown are means ± standard deviation from three replicates.

511

Figure 2 Proposed metabolic pathway of DBP in carrot cells. Blue arrows and circles represent

512

phase I metabolism reaction, and orange arrows and circles represent phase II metabolism.

513

Figure 3 Time-dependent peak proportions of DBP metabolites detected by UPLC-MS/MS in

514

cells and culture mediums.

515

Figure 4 Glycosyltransferase (GT) activity and quantitated amounts of DBP-glucopyranoside in

516

cell extracts versus incubation time (h). The bar graphs are glycosyltransferase activity, the line

517

graph is amounts of DBP-glucopyranoside. Data are mean activity/amount ± standard deviation

518

(n = 3).



Page 26 of 31

Table 1 Summary of Proposed DBP Metabolites Measured in Carrot Cells Metabolite (No.)

RT (min)

ESI Mode

Obs. m/z

Calc. m/z

Predicated formula

Fragments (m/z)

4-bromocatechol (1)

6.1

-H

187.9952

187.9988

C6H5BrO2

109.1027(-Br) 170.9914(-OH)

DBP-glucopyranoside (2)

4.1

C12H14Br2O6

250.8954(-DBP) 163.1485(-C6H11O5) 207.8938(250-CO2)

C21H26Br2O14

250.8954(-DBP) 410.3274(-C15H23O13)

C17H22Br2O10

250.8954(-DBP) 170.9914(-Br) 528.1433(-OH)

C18H26Br2O10

250.8954(-DBP) 170.9914(-Br)

C9H9Br2NO3

250.8954(-DBP) 87.0773(-C3H6NO2)

DBP glucopyranosyl(malonyl)-glucopyranoside (3)

2.9

DBP-glucopyranosylPentofuranoside (4)

1.6

DBP-glucopyranosylDesoxyhexopyranoside (5)

0.7

DBP-alanine (6)

5.2

DBP-acetyl-alanine (7)

5.5

DBP-acetyl-aspartic acid (8)

5.8

-H

-H

-H

-H

+H

-H

-H

413.0362

661.2216

545.1508

562.2013

339.9938

380.0097

424.0123

413.0360

661.2228

545.1506

562.2010

339.9886

380.0094

424.0189

C11H11Br2NO4

250.8954(-DBP) 129.1140 (-C5H8NO3)

C12H11Br2NO6

250.8954(-DBP) 173.1235(-C6H8NO5)


Confidence level Level 1 Authentic Standard, RT, MS, MS2 Level 1 Synthetic Standard, RT, MS, MS2 Level 2b MS, MS2 Level 2b MS, MS2 Level 2b MS, MS2 Level 2b MS, MS2 Level 2b MS, MS2 Level 2b MS, MS2

Page 27 of 31


Amounts of DBP (ng)

DBP in medium DBP in plant DBP in control

A

1200 1000

1000

800

800

600

600

400

400

200

200

0

0 0

20

40

60

80

100

120

DBP in medium DBP in cell materials DBP in abiotic control DBP in negative control

B

1200

0

20

Incubation time (h)

40

60

80

Incubation time (h) Figure 1


100

120


Page 28 of 31

OH O OH O OH

O O

OH

OH O

O

OH

O

OH Br O OH

Br

Br

DBP-glucopyranosylpentofuranoside, 4

O

O O O

OH

OH

Br

OH

4-Bromocatechol, 1

OH

OH

OH OH O

OH

OH N2H

OH

O OH

Br Br DBP-glucopyranosyl-(malonyl)glucopyranoside, 3

Br

DBP-acetyl-alanine, 7

O O

NH

O

OH

OH

O

OH Br

Br Br DBP-glucopyranoside, 2

Br

2,4-Dibromophenol

Br

Br

DBP-alanine, 6

OH O O O O

OH OH

OH

OH OH

O

OH

O

OH Br

DBP-glucopyranosyldesoxyhexopyranoside, 5

Figure 2


O OH O

Br

Br

NH

Br

DBP-acetyl-aspartic-acid, 8

Page 29 of 31


Peak proportion (%)

compound 8 Cell

compound 7

compound 3 compound 2 Cell culture

100

100

80

80

60

60

40

40

20

20

0

0 2h

8h

24h

48h

72h

120h

2h

Incubation time (h)

8h

24h

48h

Incubation time (h)

Figure 3


72h

120h


0h

2h

8h

24h

48h

Page 30 of 31

72h

120h

1500

treated groups untreated groups

GT activity (IU/L)

1200 120 900 80 600

40

300

0

0 0h

2h

8h

24h

48h

Incubation time Figure 4


72h

120h

Amounts of DBP-glucopyranoside

160

Page 31 of 31


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