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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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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
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ABSTRACT:
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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
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INTRODUCTION
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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
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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.
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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
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contamination sources. For example, HBCD has been detected at concentration of
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140–1300 and 111−23200 ng kg-1 dry weight in soils in Sweden and Belgium,
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respectively, near HBCD-processing factories.6,7. Heavy discharges of HBCD to soils
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have also been reported from industrial waste disposal sites and electronic waste
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recycling areas in the United Kingdom and China.8−11 Therefore, plant uptake and
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accumulation of HBCD are important processes determining their fates in the
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terrestrial environment. Nevertheless, the previous studies on bioaccumulation of
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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
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experiments.15−19 The concentration of HBCD was reported to be 3−28 ng/g dry 3
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weight in radish, wheat and reed from a natural wetland conservation area near
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Tianjin, China.18 Diastereomer-specific uptake and accumulation were evidenced in
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maize by a hydroponic experiment in our previous work.15 But so far the enantiomer
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selectivity of HBCD in plant uptake and accumulation remains unknown.
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Biotransformation is an important process for organic contaminants with different
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diastereomer or enantiomer structures. To date only a small number of studies exist on
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the transformation of HBCD in organisms.20–24 A diastereomeric shift toward
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α-HBCD by liver microsomes of seals has been reported by Zegers et al.20 The
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degradation
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tetrabromocyclododecenes (TBCDe) from HBCD have been detected in chicken eggs,
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whale and whitefish (Coregonuslavaretus).23 Hydroxylated metabolites including
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OH-HBCDs,
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tetrabromocyclododecanols (OH-TBCDs) have been confirmed in in vitro assays with
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rat and seal liver microsomes .20,24 However, the biotransformation pathway of HBCD,
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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,
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and the “green-liver” concept of plant metabolism in the transformation and
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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
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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
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estrogen receptor, whereas (+)-o,p,-DDT had negligible activity.28 Whether HBCD
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diastereomers and enantiomers interact selectively with biomacromolecules, and if so
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how the selective interactions of HBCD with biomacromolecules lead to their
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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
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transformation in organisms of contaminants having various stereoisomers, such as
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HBCD. Therefore,
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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
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results
111
enantiomer-selective behaviors of HBCD in maize in vivo.
obtained
were
further
used
to
explain
112 113
MATERIALS AND METHODS 5
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Chemicals. Three native HBCD standards (α-, β- and γ-HBCD stereoisomers in
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toluene, chemical purity > 98%) were obtained from Accu-Standard (New Haven, CT,
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USA). The
118
Cambridge
119
Racemic-(1,5R,6S,9S,10R)-pentabromocyclododecene (PBCD) was provided by
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Wellington Laboratories, Inc. (Ontario, Canada). All solvents used were of HPLC
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grade and purchased from J. T. Baker (Deventer, Netherlands). High-purity water was
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prepared with a Milli-Q system (Millipore, Bedford, MA). Neutral silica gel
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(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).
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Preparation of Individual HBCD Enantiomers. Enantiomerically pure α-, β-
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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
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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
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procedures are described in detail in the Supporting Information (SI). Six individual
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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
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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.
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Plant Exposure. Plant exposure was carried out according to the methods 6
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described in our previous research,34 and the details are provided in SI. In order to
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minimize metabolism of HBCD caused by anaerobic microorganisms, autoclaved
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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
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the prepared pure (+)/(−)-α-, β- and γ-HBCD enantiomers in methanol individually,
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and then gradually diluting with the sterile 1/4 strength Hoagland nutrient solution.
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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.
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Maize (Zea mays L.) seeds were obtained from the Chinese Academy of
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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
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days. After germination, the maize seedlings were transferred to containers containing
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sterile 1/4 strength Hoagland nutrient solution for cultivation in a controlled
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environment growth chamber. After 7 days, a batch of five uniformly sized maize
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seedlings with three leaves each was transferred to an autoclaved 200 mL brown
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glass-stoppered flask, which was used as the exposure container. Then 150 mL of
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each of the exposure solutions of (+)/(−)-α-, β- and γ-HBCD enantiomers were added
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to the exposure container. Unplanted controls with 150 mL solution of each individual
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HBCD enantiomers but without seedlings, and blank controls with seedlings but
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without HBCD, were set up simultaneously. All treatments were set up in triplicate
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with separate containers. Maize plants were harvested after 7 d of exposure. The
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maize seedlings were freeze-dried at −50 °C for 48 h in a lyophilizer (FD-1, Beijing
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Boyikang Instrument Ltd, China), separated into roots and shoots, weighed and finely 7
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chopped. The roots and shoots of the five seedlings in each container were combined
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as one root or shoot sample. Nutrient solutions after plant exposure were filtered with
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0.45 µm filter membranes. All the samples were stored in glass containers at −20 °C
167
before chemical analysis.
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HBCD Extraction, Clean-up and Analysis. 0.5–1.0 g (dry weight) of plant
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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
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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
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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;
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desolvation temperature, 160 °C; source temperature, 110 °C; nebulizing gas flow,
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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.
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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
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C-γ-HBCDs as the surrogate
C-β-HBCD as internal standard. For every batch of six samples, a
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The average recoveries of α-, β- and γ-HBCD were 82.6 ± 9.50%, 90.2 ± 8.90%, and
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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
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Metabolite Identification. An Agilent 6545 Q-TOF LC-MS (USA) was used to
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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,
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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.
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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.
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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).
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The
213
(http://blast.ncbi.nlm.nih.gov/Blast.cgi). The human CYP450 1A1 (PDB ID: 4I8V,
template
proteins
were
identified
by
the
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resolution: 2.60 Å) and wheat GST TaGST4-4 (PDB ID: 1GWC, resolution: 2.20 Å)
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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).
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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.
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For docking simulations, the Lamarckian genetic algorithm (LGA) was selected for 10
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ligand conformation and all bond rotations for ligands were ignored. In the docking
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protocol the number of individuals in each population was set to 150 and the
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maximum number of energy evaluations was set to 25,000,000, whereas the
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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
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enantiomer-specific. (−)α-, (−)β- and (+)γ-HBCDs accumulated in maize roots were at
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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
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followed by (−)γ-HBCD and then (−)β-HBCD. (−)α-HBCD was frequently detected
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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
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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
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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
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Environmental Science & Technology
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
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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
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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
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sixth meeting. 2013, UNEP/POPS/COP6. June 2013.
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(3) Koch,
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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
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of hexabromocyclododecanes – a class of compounds with a complex stereochemistry.
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Chem. Biol, 2011, 5(1), 1–11.
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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
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Environmental Science & Technology
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
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(+)γ (+) γ
Page 27 of 31
Environmental Science & Technology
(−)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
14 16 Retention time (min)
18
10
12 14 Retention time (min)
(+)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
12 14 Retention time (min)
610 611
18
(−)β (−) β
(−)α (−)α (+)γ (+)γ (−)β (−)β (+)β (+)β
16
16
Figure 2.
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(−)γ (−) γ 16
18
Environmental Science & Technology
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
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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.
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GST31
CYP71C3v2
617 618
Figure 4.
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619
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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
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