Molecular docking of bisphenol A and its nitrated and chlorinated metabolites onto human estrogen-related receptor-gamma

Article abstract of DOI:10.1016/j.bbrc.2012.08.065

A xenoestrogen and known endocrine disruptor, bisphenol A (BPA) binds the human estrogen-related receptor-gamma (ERRγ) with high affinity (Kd≈5.5nM). It is likely that BPA undergoes oxidative biotransformation by hypochlorite/hypochlorous acid (^-OCl/HOCl) and peroxynitrite (PN) and the products formed in these reactions may serve as secondary estrogens and contribute to the toxicodynamics of BPA. Therefore, in the present study we have examined the formation of chlorinated and nitrated BPA in reactions of BPA with ^-OCl/HOCl and PN(+CO₂) performed around the neutral pH. We have identified four major products in these reactions and they include 3-chloro-BPA (CBPA), 3,3'-dichloro-BPA (DCBPA), 3-nitro-BPA (NBPA) and 3,3'-dinitro-BPA (DNBPA). Towards understanding the toxicodynamics and estrogenic activity of BPA in biological systems, we have performed molecular docking of BPA, CBPA, DCBPA, DNBPA and NBPA onto the ERRγ using AutoDock 4.2 software and compared the binding energies with those of estradiol, the natural ligand. Based on the genetic algorithm, the three best conformations were selected and averaged for each ligand and a detailed analysis of molecular interactions based on free energies of binding (kcal/mol) was computed. The results indicate the following rank order of binding to ERRγ: BPA (-8.78±0.06)>CBPA (-8.53±0.41)>NBPA (-7.36±0.74)>DCBPA (-5.24±0.17)>DNBPA (-4.95±0.78)>estradiol (-4.94±1.04). The docking studies revealed that the OH group of one of the phenyl rings forms a hydrogen bond with Glu275/Arg316, while the OH group of other phenyl ring was bound to Asp346. These results suggest that both BPA and its putative chlorinated and nitrated metabolites have strong binding affinity compared to estradiol.

Full text of DOI:10.1016/j.bbrc.2012.08.065

Biochemical and Biophysical Research Communications 426 (2012) 215–220
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Molecular docking of bisphenol A and its nitrated and chlorinated metabolites
onto human estrogen-related receptor-gamma
Sainath Babu a, Nadeem A. Vellore b, Agasthya V. Kasibotla a, Harlan J. Dwayne c, Michael A. Stubblefield c,
Rao M. Uppu a,
⇑
a Department of Environmental Toxicology, Southern University and A&M College, Baton Rouge, LA, United States
b Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT, United States
c Department of Mechanical Engineering, Southern University and A&M College, Baton Rouge, LA, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 August 2012
Available online 23 August 2012
A xenoestrogen and known endocrine disruptor, bisphenol A (BPA) binds the human estrogen-related
receptor-gamma (ERR
c
) with high affinity (Kd ꢀ 5.5 nM). It is likely that BPA undergoes oxidative bio-
transformation by hypochlorite/hypochlorous acid (ꢁOCl/HOCl) and peroxynitrite (PN) and the products
formed in these reactions may serve as secondary estrogens and contribute to the toxicodynamics of
BPA. Therefore, in the present study we have examined the formation of chlorinated and nitrated BPA
in reactions of BPA with ꢁOCl/HOCl and PN(+CO2) performed around the neutral pH. We have identified
four major products in these reactions and they include 3-chloro-BPA (CBPA), 3,30-dichloro-BPA (DCBPA),
3-nitro-BPA (NBPA) and 3,30-dinitro-BPA (DNBPA). Towards understanding the toxicodynamics and estro-
genic activity of BPA in biological systems, we have performed molecular docking of BPA, CBPA, DCBPA,
Keywords:
Bisphenol A
Metabolites of bisphenol A
Xenoestrogens
Estrogen receptors
Human estrogen-related receptor
AutoDock 4.2 software
DNBPA and NBPA onto the ERRc using AutoDock 4.2 software and compared the binding energies with
those of estradiol, the natural ligand. Based on the genetic algorithm, the three best conformations were
selected and averaged for each ligand and a detailed analysis of molecular interactions based on free ener-
gies of binding (kcal/mol) was computed. The results indicate the following rank order of binding to ERRc:
BPA (ꢁ8.78 0.06) > CBPA (ꢁ8.53 0.41) > NBPA (ꢁ7.36 0.74) > DCBPA (ꢁ5.24 0.17) > DNBPA
(ꢁ4.95 0.78) > estradiol (ꢁ4.94 1.04). The docking studies revealed that the OH group of one of the
phenyl rings forms a hydrogen bond with Glu275/Arg316, while the OH group of other phenyl ring was
bound to Asp346. These results suggest that both BPA and its putative chlorinated and nitrated metabo-
lites have strong binding affinity compared to estradiol.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
and, possibly, neutrophil and macrophage derived oxidants, viz.,
hypochlorite/hypochlorous acid (ꢁOCl/HOCl) and peroxynitrite
Bisphenol A (BPA; Fig. 1), a xenoestrogen and semi-persistent
organic pollutant in urban environments, adversely affects the
endocrine system even at low doses in experimental animals and
humans [1–3]. Recent studies show that BPA binds strongly to
(PN). The direct reaction of BPA with ꢁOCl/HOCl has been studied
in the context of bioremediation and treatment of waste water
and industrial effluents [10,11]. These reactions mainly produce
chlorinated BPA. Interestingly, the reaction of BPA with PN has
never been studied; however, an analogy of reactions of PN
( CO2) with most phenolic compounds [12–14] suggests nitrated
BPA could be the major products. Studies have shown that chlori-
nated and nitrated BPA exhibit greater toxicity than native BPA it-
self and elaborates mutagenic and/or genotoxic effects [15–17].
Therefore, it is likely that these chlorinated and nitrated BPA if
formed in biological systems can serve as secondary estrogens
and relay, at least, in part, the toxic effects of BPA.
In order to understand the molecular targets for BPA and its
putative chlorinated and nitrated products and the likely disrup-
tion of endocrine function, in the current study, we carried out
the reactions of ꢁOCl/HOCl and PN (+CO2) with BPA around the
neutral pH. We identified the major products formed in these
the human estrogen-related receptor-gamma (ERR
as compared to the estrogen receptor (ER) itself [4,5]. ERR
to the group orphan nuclear receptors, and these are closely related
to ER. As on date, three different ERR proteins ( , b, ) have been
identified [5]. The function of ERR is not well known, however,
c
; Kd ꢀ 5.5 nM)
c
belongs
a
c
c
high levels of this receptor are expressed in the fetal brain and in
other tissues of adult rodents and humans [6,7].
Approximately 20–25% of BPA is known to undergo metabolic
transformation by enzymes of cytochrome P450 system [8,9]
⇑
Corresponding author. Fax: +1 225 771 5350.
E-mail address: rao_uppu@subr.edu (R.M. Uppu).
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2012.08.065

216
S. Babu et al. / Biochemical and Biophysical Research Communications 426 (2012) 215–220
mobile phase consisting of 0.1% TFA and 50% acetonitrile in water.
The flow of the mobile phase was set at 1 mL/min and the eluent
was monitored at 292 nm. A Thermo Scientific Spectra System
SCM 1000 equipped with a Spectra System P4000 (quaternary
pump) and a Spectra System UV2000 (dual wavelength detector)
was used for the purpose of sample analysis.
2.4. Synthesis and characterization of NBPA
Nitration of BPA was performed using a combination of sodium
nitrate and an acidic ionic liꢁquid, [MIM]+ HSO4 [23]. BPA (3.32 g;
ꢁ
20 mmol) and [MIM]+ HSO4 (5.72 g; 20 mmol) were dissolved in
80 mL of acetonitrile. To this mixture, 1.7 g (20 mmol) of NaNO3
was added and stirred overnight at 25 1 °C. At the end, the mix-
ture was filtered and the filtrate was evaporated under reduced
pressure at ca. 50 °C. The thick brown liquid-like residue was ex-
tracted with hot hexane, which was then evaporated under re-
duced pressure.
The yellow liquid residue, dissolved in dichloromethane, was
analyzed by GC/MS/EI using an Agilent Technologies 7890A GC
equipped with the Agilent Technologies 5975 C V L triple-axis
MSD and a HP-5MS capillary column (length: 30 m; internal diam-
Fig. 1. Structures of estradiol, BPA, nitrated BPA (NBPA and DNBPA), and
chlorinated BPA (CBPA and DCBPA).
reactions, and investigated their estrogenic activities along with
BPA based on molecular docking onto ERRc.
eter: 0.25 mm; and film thickness: 0.25 lm). Helium was used as
the carrier gas (total flow: 3 mL/min; split ratio: 1:50) with tem-
perature programming as follows: 40 °C for 2 min (isothermal);
20 °C/min up to 150 °C (ramp 1), 150 °C for 3 min (isothermal);
20 °C/min up to 300 °C (ramp 2), and 300 °C for 2 min (isothermal)
(total run time: 20 min; temperature of the inlet port: 270 °C).
When analyzed by GC/MS/EI, the nitro product resolved as a
single peak at 17.34 min. The ion chromatogram of this product
showed a molecular ion (MÅ+) at m/z 273 and a daughter ion at
m/z 258 (100%; base peak; [Mꢁ15]+), confirming that the chemical
identity of the product is 3-nitro-BPA.
2. Materials and methods
2.1. Materials
Chemicals and reagents were obtained as follows: bisphenol A,
H2O2 (30%), isoamyl nitrite (96%), dichloromethane (DCM), DMSO-
d6, MnO2 (granular), 1-methylimidazolium hydrogen sulfate
([MIM]+ HSO4ꢁ), NaHCO3, NaNO3, potassium phosphate monoba-
sic, sodium phosphate dibasic, and sodium hypochlorite (chlorine
content: ca. 5%) from Sigma (St. Louis, MO); diethylenetriamine-
pentaacetic acid (DTPA) from ACROS Organics (New Jersey, NJ);
HNO3 (70%) and trifluroacetic acid (TFA) from Fisher Scientific (Fair
Lawn, NJ); and acetonitrile and acetone from Mallinckrodt (Phil-
lipsburg, NJ). Peroxynitrite (PN) and 3,30-dinitro-BPA (DNBPA)
were synthesized as described in our previous publications
[18,19]. Water used in all experiments was deionized to a final
2.5. Synthesis and characterization of CBPA, DCBPA and TCBPA
BPA (1.37 g; 6 mmol) was allowed to react with excess ꢁOCl/
HOCl (ca. 25 mmol) in 20 mL of 50% methanol in water, as de-
scribed by Fukazawa et al. [10]. Following an overnight stirring,
aliquots (200
lL each) were subjected to RP-HPLC analysis using
resistance of 18.0 M
The concentration of ꢁOCl and H2O2 in stock solutions was
determined using
= 350 Mꢁ1 cmꢁ1 at 292 nm [20] and = 41 Mꢁ1-
X/cm or higher.
an Econosphere C8-column (250 ꢂ 10 mm; particle size: 10
l)
and a mobile consisting of 50% acetonitrile in water that also
contained 0.1% TFA (flow: 3 mL/min). The eluent was monitored
at 292 nm. A Lab Alliance series II/III liquid chromatograph
equipped with a Lab Alliance model 500 UV–Vis detector was
used. Peaks with significant absorbance were collected and pooled
individually from several HPLC runs and concentrated by flash
evaporation.
e
e
cmꢁ1 at 240 nm [21], respectively. Working standards of PN and ꢁ-
OCl/HOCl (2 or 5 mM) were prepared by dilution of the respective
stock solutions with water and used immediately (usually within
5 min). Stock solutions of BPA (10 mM) were prepared in 0.01 M
NaOH and stored in aliquots of 1 mL each at ꢁ20 °C until use.
GC/MS/EI analysis of chlorinated BPA was performed using a
Hewlett Packard-6890 GC equipped with HP-5973 MS and a
RTX-5MS capillary column (length, 30 m; internal diameter,
2.2. Reactions of BPA with PN and ꢁOCl/HOCl
BPA (200 lM) was allowed to react with 0 to 500 lM of PN (or
0.25 mm; and film thickness, 0.25 lm). Helium was used as the
ꢁOCl/HOCl) in 2 mL of 0.08 M phosphate buffer, pH 7.0 that also
contained 0.1 mM DTPA. In reactions performed with PN, NaHCO3
was included in the assay at a final concentration of 10 mM [22].
The reaction in all cases was initiated by the addition of PN
(or ꢁOCl/HOCl). Throughout the course of addition of PN
(or ꢁOCl/HOCl), which typically took 5 s, the contents were con-
stantly stirred and stirring continued for an addition 10 s. Before
further analysis by reversed phase HPLC (see below), the reaction
mixtures were left at room temperature (25 1 °C) for 5–15 min.
carrier gas (total flow: 1 mL/min; splitless) with temperature pro-
gramming as follows, 150 °C for 2 min (isothermal); 30 °C/min up
to 270 °C (ramp), and held at 270 °C for 6 min (isothermal) (total
run time: 12 min; temperature of the inlet port was 260 °C). All
three products resolved as single peaks. Each of them showed
M+Å corresponding to the predicted molecular weight of 262 for
NBPA, 296 for DCBPA, and 330 for TCBPA. In each case, the daugh-
ter fragment corresponded to [Mꢁ15]+.
2.6. Preparation of receptor structure
2.3. RP-HPLC analysis of BPA and its oxidation products
The structure of ligand binding domain of ERRc was obtained
from Research Collaboratory for Structural Bioinformatics (RCSB)-
The reaction mixtures of PN-BPA and ꢁOCl/HOCl-BPA were ana-
lyzed using a Hypersil Gold LC18 column (150 ꢂ 3.6 mm) and a
Protein Data Bank (PDB). The PDB entry 2E2R was selected for

S. Babu et al. / Biochemical and Biophysical Research Communications 426 (2012) 215–220
217
structural analysis based on its high resolution (1.6 Å) and specific-
ity of binding to bisphenol Z [5]. The ligand binding domain com-
prises of 232–458 amino acid range (chain length of 226 amino
acids) was co-crystallized with BPA compound as the ligand. For
docking studies, the pdb file was refined by removing the ligand
and water molecules from the protein X-ray structure. The initial
refinement was done by utilizing various tools present in Discov-
ery Studio ViewerPro program. The final refinement was made
with AutoDock Tools 4.2 [24; The Scripps Research Institute, La Jol-
la, CA] by adding missing hydrogens and adding partial charges on
all the atoms based on Kollman charges. The active site was de-
fined by a grid box of 52 ꢂ 52 ꢂ 52 points and spacing of 0.375 Å
with the ligand binding site as the center. The final structure was
then saved in .pdbqt format.
2.7. Ligand structure
Fig. 2. Typical RP-HPLC chromatograms of the reaction mixtures in which BPA
BPA and its putative metabolites (viz., CBPA, DCBPA, NBPA and
DNBPA) were selected for the docking studies against ERRc.
(initial concentration: 200
lM) was allowed to react with (A) 0, (B) 200, or (C)
500 peroxynitrite in 2.0 mL of 0.08 M phosphate buffer, pH 7.0 that also
l
M
contained 0.1 mM DTPA and 0.01 M NaHCO3. Aliquots (20
lL each) of the reaction
Estradiol, the natural ligand, was also used to compare the docking
and binding efficiency. The .pdb format of DNBPA was obtained
using the crystal structure reported earlier (.cif format) from our
laboratory [19]. The structural coordinates of BPA, NBPA, CBPA,
DCBPA and estradiol were obtained from ChemDraw and con-
verted to .pdb format by Mercury software. The 3D conformations
of all the ligands were generated by DS viewerpro program and
minimized using 500 steps of steepest descent algorithm. These
.pdb files were imported to AutoDock 4.2 program and minimized
further by adding kollman charges and setting the charged resi-
dues on the active site to be flexible. The multiconformation library
of all the four ligands was generated by exploring the torsional
space of the ligands using AutoDock program. The final ligand
structures were saved in .pdbqt format.
mixture were analyzed as described in Section 2.
the extent of BPA oxidation was calculated as 20 ( 0.5) mol% (i.e.,
relative to the initial concentration of PN employed in the reac-
tion). Similar lower yields of oxidation have been reported to be
typical of PN (+CO2)-mediated reactions of phenols [12–
14,18,22], which typically follow zero-order kinetics. Thus, it ap-
peared that the PN ( CO2) reaction with BPA was mediated mainly
Å
Åꢁ
by the free radical oxidants including NO2 and CO3 rather than
PN itself.
The reaction of PN (+CO2) with BPA (Fig. 2) resulted in the for-
mation of two major products, NBPA (retention time: 4.68 min)
and DNBPA (retention time: 8.30 min). These products were iden-
tified based on a comparison of retention times with authentic
samples. As expected, the yields of NBPA appeared to be higher
2.8. Docking protocol
than that of DNBPA at the concentration of PN of 200
B). This, however, was not the case when higher concentrations
of PN (500 M) was employed in the reaction. There was more of
lM (A and
A grid box was generated using the parameters described in
protein preparation section. The genetic algorithm was used to find
the probable fit for each ligand to receptor. The docking was done
with Lamarckian genetic algorithm with population size of 150.
l
DNBPA formed and the yields of BPA oxidation on a mole-to-mole
basis with PN ( CO2) was somewhat lowered (15 mol%) (A and C).
Even in these reactions, there was significant amount of BPA
(125 lM) that was left unreacted. These results were consistent
with higher yields of DNBPA which required two consecutive nitra-
tions on the same BPA molecule by PN (+CO2). Hence, this observa-
tion suggested that in the biological systems where PN could be
truly a limiting reactant, NBPA would be the most predominant
nitroproduct of BPA.
3. Results and discussion
3.1. Reaction of PN (+CO2) with BPA forms NBPA and DNBPA as major
products
Earlier, it has been shown that nitrated BPA, in particular NBPA
and DNBPA, can be formed in reactions of nitrite with BPA under
simulated acidic conditions of the stomach [16]. We reasoned that
similar nitrated BPA could also be formed predominantly in the
cellular environment as a result of BPA reaction with PN (+CO2).
Both NBPA and DNBPA are strongly mutagenic either with or with-
out a metabolic activation system (S9 mix) and also have been
shown to be clastogenic [15,16]. Given these important properties
of nitrated BPA, in the present study, we explored the oxidative
biotransformation of BPA by PN under biologically reverent condi-
tions of pH and carbonate concentration. Using a combination of
several synthetic and analytical techniques, we identified that both
NBPA and DNBPA in reactions of PN with BPA (see below).
3.2. Reactions of ꢁOCl/HOCl with BPA forms mainly CBPA and DCBPA
with small yields of TCBPA
Often, the chlorinated BPA species have been studied in connec-
tion with bioremediation and advanced oxidations [10,11]. It has
been shown that chlorinated BPA products are frequently found
in waste and runoff water [10] and they display greater affinities
of binding to the estrogen receptor [25]. Thus, it appears that sim-
ilar to nitrated BPA products, the chlorinated BPA species can serve
as potential mediators of toxicity and/or estrogenic activity of BPA.
Our current focus on the ꢁOCl/HOCl-mediated oxidation of BPA
stems from the reasoning that there could be a likelihood of forma-
tion of the chlorinated BPA products under biologically relevant
conditions, mainly at the inflammatory sites due to excessive pro-
duction of ꢁOCl/HOCl by the neutrophils and macrophages. Also, as
mentioned earlier (see Section 1), the ꢁOCl/HOCl-mediated oxida-
tions could partly contribute to the Phase I biotransformation of
BPA.
When BPA (200 lM) was allowed to react with equimolar PN at
pH 7.0 in the presence of 10 mM carbonated species, there was
marked oxidation of BPA (Fig. 2). In this particular assay, the con-
centration of residual and unreacted BPA was analyzed along with
the putative nitration products by utilizing the RP-HPLC. Based on
the decrease in the peak areas corresponding to BPA (see A and B),

218
S. Babu et al. / Biochemical and Biophysical Research Communications 426 (2012) 215–220
Table 1
Binding energies (kcal/mol) of BPA, CBPA, NBPA, DCBPA, DNBPA, and estradiol with
ERR
c
.
Conformation
BPA
CBPA
NBPA
DCBPA
DNBPA
Estradiol
1
2
3
ꢁ8.84
ꢁ8.76
ꢁ8.73
ꢁ8.78
0.06
ꢁ8.98
ꢁ8.43
ꢁ8.19
ꢁ8.53
0.41
ꢁ8.22
ꢁ6.97
ꢁ6.90
ꢁ7.36
0.74
ꢁ5.36
ꢁ5.32
ꢁ5.04
ꢁ5.24
0.17
ꢁ5.84
ꢁ4.64
ꢁ4.38
ꢁ4.95
0.78
ꢁ5.94
ꢁ5.03
ꢁ3.85
ꢁ4.94
1.04
Mean
SD
which three best molecules were selected based on the scoring
method and averaged. Table 1 shows the results of binding affini-
ties of various ligands. The average binding energies were BPA,
CBPA, NBPA, DCBPA, DNBPA and estradiol were ꢁ8.78 ( 0.06),
ꢁ8.53 ( 0.41), ꢁ7.36 ( 0.74) ꢁ5.24 ( 0.17), ꢁ4.95 ( 0.78), and
ꢁ4.94 ( 1.04) kcal/mol, respectively. The highest binding affinity
was exhibited by BPA whereas the lowest affinity was exhibited
by estradiol.
Fig. 3. Typical RP-HPLC chromatograms of the reaction mixtures in which BPA
In general, in all the BPA compounds (Fig. 4), the hydroxyl group
of one of the phenyl rings formed a hydrogen bond with Glu275
and/or Arg316, while the hydroxyl group of other phenyl ring
was bound to Asp346. The lowest binding affinity for estradiol
was evident because of the conjugate rings, which rendered it
more inflexible and gave the undesired restrain energy to dock at
the active site. Compared to the chloro- group, the nitro group
was slightly bulkier, which made the phenol backbone less avail-
able for the hydrophobic interactions. The CBPA and NBPA was
bound tight to the active site due to the electrostatic interaction
provided by the functional group, which then formed hydrogen
bond with Glu275/Arg316 at front end and with Asn346 at rear
end. The hydrophobic region of chloro-ligand formed a T-shaped
(initial concentration: 200
lM) was allowed to react with (A) 0 or (B) 200 lM
hypochlorite in 2.0 mL of 0.08 M phosphate buffer, pH 7.0 that also contained
0.1 mM DTPA. Aliquots (20
described in Section 2.
lL each) of the reaction mixture were analyzed as
The current study also revealed that ꢁOCl/HOCl could mediate
the oxidation of BPA under biologically relevant conditions of pH.
When ꢁOCl/HOCl (200
lM) was allowed to react with equimolar
BPA, three major products of BPA were formed (Fig. 3). These in-
cluded CBPA (retention time: 3.36 min), DCBPA (retention time:
4.66 min), and TCBPA (6.57 min). All these chlorinated BPA prod-
ucts were identified based on a comparison of retention times with
authentic samples and by co-chromatography. Unlike in the case of
PN (+CO2), the ꢁOCl/HOCl gave rise to higher yields of BPA oxida-
tion products (52 mol%; compare the peak area of BPA in A and
B). Among the three chlorinated BPA products, the relative yields
were as follows: CBPA (51%), DCBPA (39%) and TCBPA (10%)
(Fig. 3B).
stacking with Tyr326 and
Leu346, 271, 256. Even though this
p
–
p
interaction with Phe435 and
interaction was seen in
p–p
other ligands, the stereo-chemical properties of chloro-ligand
made it more available to the hydrophobic region. The lower affin-
ity of binding of DNBPA as compared to DCBPA could be due to the
formation of intra-molecular hydrogen bonding between the OH
and NO2 groups which prevented the interaction of phenolic OH
groups with polar amino acid residues.
3.3. Active site in human ERR
c
Primarily, the active site was analyzed in detail to understand
the nature of interaction of BPA and its metabolites with the ERR
3.5. Summary and general implications
c
protein. Based on the ligand–protein interactions, the active site
was mainly composed of hydrophobic core and polar charged ami-
no acids. The ends of the active site contains charged residues lin-
ing outside the active site, providing hydrogen bonding affinity to
the alcohol group of the ligand, while the cleft in the middle re-
gions comprises of aliphatic amino acids interacting with the
non-polar part of the BPA ligands. In general, three pharmaco-
phores were identified,
Bisphenol A is a high volume chemical used in the manufacture
of polycarbonate plastics (PPs), epoxy resins and, to a limited ex-
tent, in thermal and carbonless paper. The extensive use of PPs in
consumer and non-consumer products, and the likelihood that
they contain small amounts of monomeric BPA [26], propelled
studies of metabolic fate and toxicokinetics of BPA in rodents
and humans [27–29]. Studies have shown that more than 75% of
BPA is converted to much less toxic products, namely, BPA-
glucuronide and BPA-sulfate, and are eliminated rapidly [29,30].
This probably is the reason for much less focus on metabolites of
phase I biotransformation (20–25%), particularly those involving
the cellular oxidants, peroxynitrite (+CO2) and ꢁOCl/HOCl. We have
observed for the first time that chlorinated and nitrated BPA could
be formed in reactions of these oxidants under biologically rele-
vant conditions, and these are reported in the present communica-
tion. Further, we observed that these putative products exhibit
(1) Core hydrophobic moiety making van der Waal interactions
provided by Tyr326, Phe435, and Leu256, 271, 346
(2) Terminal charged pocket formed by Arg316, Glu275, and
(3) Terminal Asn346.
In addition to the scores and ranking obtained from AutoDock,
the interaction provided by three pharmacophores was mainly se-
lected as criteria for identifying the best possible fit for protein.
strong binding affinity to ERRc much the same way as described
for the native BPA. The studies of molecular docking described here
can decipher ligand–protein interactions with respect to the
participating functional groups. We anticipate that application of
such studies to BPA isomers [2,2-bis(2-hydrophenyl)propane and
2-(2-hydroxyphenyl), 2-(4-hydroxyphenyl)propane] and other
bisphenols [bisphenol B, bisphenol E, bisphenol F, bisphenol S
3.4. Biding energies reveal a rank order in which estradiol is the least
favorable ligand
A total of 150 independent docking trials were performed using
Lamarckian genetic algorithm for each of the target ligands. Out of

S. Babu et al. / Biochemical and Biophysical Research Communications 426 (2012) 215–220
219
Fig. 4. Binding poses of (A) BPA, (B) CBPA, (C) NBPA, (D) DCBPA, (E) DNBPA, and (F) estradiol with ERRc at its active site.
[6] C.P. Cheung, S. Yu, K.B. Wong, L.W. Chan, et al., Expression and functional study
of estrogen receptor-related receptors in human prostatic cells and tissues, J.
Clini. Endocrinol. Met. 90 (2005) 1830–1844.
and bisphenol Z) and their metabolites could provide valuable
information concerning the possible structure–activity relation-
ships and furthering our understanding of the estrogenic activity.
[7] Y. Takeda, X. Liu, M. Sumiyoshi, A. Matsushima, M. Shimohigashi, Y.
Shimohigashi, Placenta expressing the greatest quantity of bisphenol
receptor ERR among the human reproductive tissues: predominant
expression of type-1 ERR isoform, J. Biochem. 146 (2009) 113–122.
A
c
Acknowledgments
c
[8] S. Nakamura, Y. Tezuka, A. Ushiyama, C. Kawashima, et al., Ipso substitution of
bisphenol A catalyzed by microsomal cytochrome P450 and enhancement of
estrogenic activity, Toxicol. Lett. 203 (2011) 92–95.
This publication was made possible by National Institutes of
Health (NIH) Grant P20 RR16456 (from the BRIN Program of the
National Center for Research Resources), National Science Founda-
tion (NSF) Grants and HRD1043316 (HBCU-UP ACE implementa-
tion program), and the US Department of Education (Title III, Part
B – Strengthening Historically Black Graduate Institutions, HBGI;
Grant number: PO31B040030). The authors acknowledge the year-
long fellowship support from LONI-Institute for Sainath Babu and
the use of high-performance computing (HPC) resources. The con-
tents are solely the responsibility of authors and do not necessarily
represent the official views of the NIH, NSF or the US Department
of Education.
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