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Author's personal copy
Chemosphere 86 (2012) 65–69
Contents lists available at SciVerse ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Enantioselective effects of alpha-hexachlorocyclohexane (HCH) isomers
on androgen receptor activity in vitro
Nela Pavlíková a, Lucie Bláhová a, Petr Klán b, Sreenivas Reddy Bathula c, Vladimír Sklenář c,
John P. Giesy d,e,f, Ludek Bláha a,⇑
a
Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 3, CZ625 00 Brno, Czech Republic
Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
d
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon, Canada
e
Department of Zoology and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA
f
State Key Laboratory and Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong, SAR, China
b
c
a r t i c l e
i n f o
Article history:
Received 14 July 2011
Accepted 24 August 2011
Available online 2 October 2011
Keywords:
Anti-androgenicity
Optical isomers
Pesticide
Hexachlorocyclohexane
a b s t r a c t
Alpha-hexachlorocyclohexane (alpha-HCH), a part of the HCH pesticide mixture, is one of the most widespread persistent organic pollutants. Interestingly, only limited number of studies addressed the toxicity
of alpha-HCH and the effects of its individual optical isomers have not been investigated in detail. In the
present study we separated two alpha-HCH enantiomers by preparative HPLC and studied their activities
towards androgen receptor (AR) using the MDA-kb2 cell line stably transfected with the luciferase reporter gene under the control of AR. There was no direct effect of alpha-HCH on AR but both isomers significantly suppressed the activity of AR in co-exposure with the natural ligand dihydrotestosterone in a
concentration-dependent manner. One of the enantiomers appeared to be more active at lower concentration, which was also supported by the molecular modeling calculations with AR that showed a slight
difference in estimated free energy of binding and inhibition constant between two enantiomers.
Although studies with other pesticides demonstrated strong enantioselective differences in toxicity,
the present research shows rather minor differences in modulations of AR by both alpha-HCH enantiomers. For the first time, enantioselective effects of alpha-HCH were demonstrated and the results suggest
interaction with multiple regulatory events controlling the AR activity. Full elucidation of the toxicity
mechanism will require further research.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Various pesticides are used in the agriculture around the world
along with increasing demands of food. The public and government
regulators continue to be concerned about the potential hazards
posed to the health of wildlife and humans. Some of the most toxic
and persistent organochlorine pesticides (OCPs) are no longer used
in most developed countries following the ratification of the Stockholm Convention of the United Nations. However, these compounds
are still used in some countries especially for the control of malaria
and other endemic diseases (Maffei et al., 2009; Mansour, 2009).
OCPs are persistent in the environment with half-lives ranging from
months to years and may accumulate to the levels causing adverse
effects in animals (Mingelgrin and Nasser, 2006).
⇑ Corresponding author. Tel.: +420 549493194, mobile: +420 605510953;
fax: +420 549492840.
E-mail address: blaha@recetox.muni.cz (L. Bláha).
0045-6535/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2011.08.052
Chirality is an attribute of natural as well as anthropogenic
compounds including pesticides (Muller and Kohler, 2004; Liu
et al., 2005). Synthesis of pesticides containing stereogenic center
usually results in a mixture of stereoisomers. Once they are
released into the environment, the enantiomeric ratio may be
changed by the enantiospecific or –selective breakdown (Muller
and Kohler, 2004; Smith, 2009). Although the different effects of
enantiomers are known at some pesticides, there is still a lack of
information about their toxicity and fate in the environment
(Muller and Kohler, 2004; Smith, 2009).
Hexachlorocyclohexane (HCH) has been one of the most frequently used organochlorine pesticides after the World War II.
Originally, HCH was used as a technical mixture of six isomers,
containing approximately 10% of the effective pesticide c-HCH
(lindane). The mixture was composed primarily of a-HCH and
b-HCH, both of which are more persistent than lindane and inert
as insecticides. After replacement of technical HCH with pure lindane during the 1960s, concentrations of a-HCH were expected
to decrease but experimental studies demonstrated higher stability
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of the alpha-isomer compared to c-HCH, (Shen et al., 2004). But
even the long half-life of the a-isomer does not provide sufficient
explanation for its current high environmental concentrations,
and existence of secondary sources of a-HCH has been proposed
(Malaiyandi and Shah, 1984; Iwata et al., 1994).
From the toxicological point of view, a-HCH is the least
explored isomer from all HCH isomers (Willett et al., 1998). It is
considered to affect the central nervous system (Willett et al.,
1998) but unlike lindane it has no or little effect on the gammaaminobutyric acid GABA receptor (Nagata and Narahashi, 1995).
a-HCH was also reported to cause liver cancer in mice and rats
(Ito et al., 1975). Concerning the endocrine disruption, in vitro
inhibitive effect of a-HCH on activated androgen receptor was
reported (Schrader and Cooke, 2000). Specifically, a-HCH was
shown to antagonize the androgen receptor (AR)-mediated effects
of the natural ligand dihydrotestosterone, DHT (Roy et al., 2004).
During the synthesis, two a-HCH enantiomers are formed as a
racemic mixture (Willett et al., 1998) but to our knowledge all previous studies addressed the toxicity of a-HCH as a racemate, and
toxicity of individual enantiomers has not been explored.
The present research aimed to study possible effects of isolated
a-HCH enantiomers towards androgen receptor. The enantiomers
were separated and concentrated using the semi-preparative HPLC
and the relative potencies to interact with AR were determined (i)
experimentally using the in vitro MDA-kb2 reporter gene assay,
and by (ii) molecular modeling of interactions between HCH and
AR.
2. Materials and methods
2.1. Chemicals
a-HCH (99% purity) was provided by Zbynek Prokop and Jiri
Damborsky (Loschmidt laboratories, Faculty of Science, Masaryk
University, Brno, Czech Republic), 5a-Androstan-17b-ol-3one (synonym 4,5a-dihydrotestosterone, DHT; CAS number 52118-6, purity P99.0%) was purchased from Sigma–Aldrich. Other
chemicals, solvents (the highest possible purity) and the components of the cell culture media were purchased from Sigma–Aldrich
unless stated otherwise.
2.2. HPLC separation of the a-HCH enantiomers
Separation of the a-HCH enantiomers was performed using an
Agilent 1100 series Chromatograph equipped with a UV–VIS diode
array detector. Several chiral HPLC columns were tested and the
best separation was achieved by using a CHIRALCEL OD-H column
(cellulose tris-3,5-dimethylphenyl carbamate, 150 2.1 mm;
Chiral Technologies Europe, 67404 Illkirch – Cedex, FRANCE). Pure
hexane (Pestanal, Fluka; for residual analysis) was used as a mobile
phase (flow rate of 250 lL min–1). Temperature of the column and
the collector was kept at 25 °C; the analytes were detected at
k = 210 and 450 nm. After separation of the enantiomers, the solvent was evaporated, and individual enantiomers were dissolved
in hexane. Concentrations were confirmed by external calibration
curves. For toxicity testing, the enantiomers were dissolved in
dimethylsulfoxide (DMSO; 10 mM stock solutions), a nontoxic solvent often used in biological studies.
2.3. Cell culture
The effects towards AR were tested using the human breast carcinoma cell line MDA-kb2 stably transfected with the luciferase
gene under the control of AR (Wilson et al., 2002).The cell line
was routinely cultured in L-15 Leibovitz medium supplemented
with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator under atmospheric conditions (no external addition of CO2).
Before the experiment, cells were trypsinized, mixed with L-15
Leibovitz medium supplemented with 10% dialyzed FBS (serum
steroids removed) and seeded into 96-well plates at a density of
10 000 cells well1. After the 24-h pre-incubation, cells were
exposed in three replicates to the solvent DMSO, 1 nM DHT
(positive control) or a range of HCH concentrations (either without
or in the presence of DHT). Maximum DMSO concentration in the
test system was 1% v/v and it had no effect on the cell viability
or reporter luciferase expression. After 24-h exposure, the medium
was removed, the cells were washed with the phosphate-buffered
saline (PBS), lysed for 30 min at room temperature by addition of
25 lL of lysing buffer per well (Promega E1531). Luminescence
(activity of the reporter luciferase) was measured using the flash
mode with a multiwell plate reader (Luminoscan Ascent, Thermo
Fisher Scientific Inc., Waltham, MA, USA) by use of a luciferase
assay reagent injected by a dispenser to each well just before
luminescene measurement. Luciferase assay reagent consisted of
20 mM Tricine, 1.07 mM Mg(CO3)Mg(OH)2, 2.67 mM MgSO47H2O,
0.1 mM EDTA disodium salt, 33.3 mM dithiothreitol, 270 lM of
coenzyme A, 470 lM luciferin, and 530 lM of ATP in redistilled
water, pH = 7.8. Viability of cells was determined using a neutral
red method (Freyberger and Schmuck, 2005; Benisek et al.,
2008). Neutral red (0.5 mg mL1) was added to each well and the
microplate was incubated at 37 °C for 1 h. Medium was then
removed, cells lysed with 1% acetic acid in 50% ethanol and absorbance at 570 nm was measured (only viable cells accumulated
neutral red). Effects of both enantiomers and the racemic HCH
mixture were tested in three independent experiments and each
exposure variant was tested in three replicated wells. Results are
presented as means ± SEM of N = 3 independent experiments.
2.4. Molecular modeling of the HCH binding to AR
The binding affinity of HCH to AR was studied by molecular
modeling using an AutoDock software – file PDB ID: 2Q7I. This file
contains ligand binding domain (LBD) of the AR, activation function 2 (AF2) and testosterone. Various isomers of HCH were docked
into 2Q7I, and binding affinities were calculated either in the presence or the absence of AF2 and testosterone. Inside docking calculations, MMFF94 force field (Halgren, 1996) was used for energy
minimization of ligand molecules (HCH). Gasteiger partial charges
were added to the ligand atoms. Non-polar hydrogen atoms were
merged, and rotatable bonds were defined. This type of docking
is referred to as a rigid body docking. Essential hydrogen atoms,
Kollman united atom type charges, and solvation parameters were
added. Affinity (grid) maps of 70 70 70 Å grid points and
0.375 Å spacing were generated using the Autogrid program
(Morris et al., 1998). Docking simulations were performed using
the Lamarckian genetic algorithm (LGA) and the Solis & Wets local
search method (Solis and Wets, 1981). Each docking experiment
was derived from 10 different runs that were set to terminate after
a maximum of 250 000 energy evaluations. The population size
was set to 150. During the search, a translational step of 0.2 Å,
and quarternion and torsion steps of 5 were applied.
2.5. Statistics
Responses of treatments and controls were compared using the
one-way analysis of variance (ANOVA) followed by the Dunnet’s
multiple range test. Differences between the effects of individual
enantiomers used at the same concentrations were analyzed by
Student’s t-test. For all statistics, p-values less than 0.05 were considered statistically significant. Calculations were performed in
Statistica 8.0 (StatSoft Inc., Tulsa, OK, USA).
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N. Pavlíková et al. / Chemosphere 86 (2012) 65–69
70
Norm.
60
50
40
30
E2 : 7,877
20
E1 : 6,809
10
0
2
4
6
8
10
12
14
min
Fig. 1. Chromatogram of a-HCH enantiomers separated on a CHIRALCEL OD-H column.
3. Results and discussion
The separation of a-HCH enantiomers performed in this study
was inspired by a previously suggested protocol (Champion et al.,
2004), in which a chiral column CHIRALCEL OJ was used. For separation we used a chiral column (Vetter et al., 1997; Jantunen and
Bidleman, 1998) rather than enantioselective capillaries (Wiberg
et al., 1998). Several chiral columns were tested in our experiments
for their ability to separate enantiomers and the optimal separation was obtained with the CHIRALCEL OD-H column (Fig. 1) using
experimental conditions described in Section 2. Since we were not
able to fully determine the absolute configuration of the separated
enantiomers, they are further labeled En1 and En2 (enantiomers 1
and 2) considering their retention time from the column.
Environmental levels, degradation and changes in the enantiomeric ratio of HCH enantiomers has been subject of several research papers (Klobes et al., 1998; Covaci et al., 2004; Muller and
Kohler, 2004) but to our knowledge, this is the first study, which
attempted to investigate biological effects of isolated a-HCH enantiomers. First, the activity of the racemic a-HCH towards the AR
was tested using the MDA-kb2 cellular reporter gene assay. No direct activation of the AR by a-HCH was found up to 50 lM (data
not shown), which corresponded to the previously reported results
(Roy et al., 2004). Interaction of a-HCH with DHT, the natural ligand of AR, was then studied. Selected 1 nM DHT concentration
caused approximately 50% luciferase induction (Fig. 2a), which allowed assessment of both stimulatory and inhibitory effects of aHCH. As shown in Fig. 2b, racemic a-HCH significantly suppressed
the AR activation by DHT in a concentration-dependent manner
and two higher a-HCH concentrations (10 lM and 50 lM) caused
complete inhibition of reporter luciferase. No significant cytotoxicity was detected after exposures to a-HCH alone or in combination
with DHT (Fig. 2b; circle symbols).
Pronounced anti-androgenic effects of individual a-HCH enantiomers En1 and En2 on the DHT-induced AR activation are shown in
Fig. 3. While En1 suppressed the AR activity within the same concentrations as racemate (10–50 lM), En2 appeared to be more
effective at lower 2 lM concentration. On the other hand variable
responses were observed at higher concentrations with partial
recovery at 50 lM, which was confirmed by repeated experiments
(Fig. 3).
The original hypothesis tested in our experiments considered
possible interference of a-HCH and DHT with the binding site at
AR. Molecular modeling results (Table 1) suggested that most
HCH isomers may directly interact with the AR binding site.
Fig. 2. The effect of dihydrotestosterone (DHT) and a-HCH racemate on MDA-kb2
cells. (a) calibration curve of DHT, (b) effect of a-HCH racemate on luciferase
expression in the cells stimulated by 1 nM DHT (bars) and on the cell viability
(circles with dashed line). Mean values (±SEM) of three independent experiments
are presented. Asterisks (⁄) indicate values significantly different from the control,
analyzed by one way ANOVA followed by Dunnet’s test.
However, the frequency of successful docking for a-HCH enantiomers appeared to be greater (50–60%) than for other HCH
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N. Pavlíková et al. / Chemosphere 86 (2012) 65–69
Table 1
Binding energy of hexachlorocyclohexane (HCH) isomers to androgen receptor in the presence of AF2 and testosterone (file PDB ID: 2Q7I).
a
Molecule
Est. free energy of binding
(kcal mol–1)
Est. inhibition
constant, Ki (lM)
vdW + Hbond + desolv
Energy (kcal mol–1)
Electrostatic energy
(kcal mol–1)
Total intermolec. energy
(kcal mol–1)
Frequency
(%)
(+) a-HCH
() a-HCH
b-HCH
c-HCH
D-HCH
E-HCH
TES(control)a
5.86
5.95
5.40
5.91
6.01
5.59
9.91
51.06
43.41
109.82
46.39
39.60
80.40
54.46
5.70
5.78
5.36
5.85
5.88
5.50
10.14
0.16
0.17
0.05
0.06
0.13
0.09
0.12
5.86
5.95
5.40
5.91
6.01
5.59
10.26
60
50
20
20
30
20
10
Testosterone (TES) binding energy to androgen receptor in the presence of AF2.
% of control (DHT 1nM)
En1
En2
+ DHT 1nM
150
*
(a / b)
100
*
(a / b)
50
*
*
*
(Wang et al., 2007). Our results for the first time demonstrate antiandrogenic action of different a-HCH stereoisomers. Although the
original hypothesis expected pronounced differences between the
two a-HCH isomers, we were able to demonstrate rather minor –
but still significant – differences. Molecular modeling suggested that
a-HCH interaction with AR could be responsible for the effects but
several up-stream regulatory events could also be affected. Further
research should address both in vivo relevance and molecular mechanisms of a-HCH anti-androgenicity with special respect to its high
environmental persistence.
*
0
C
0,08
0,4
2
10
50
HCH enantiomers [ M]
Fig. 3. Inhibitive effect of separated a-HCH enantiomers (En 1, En 2) on MDA-kb2
cells stimulated by 1 nM dihydrotestosterone (DHT). Mean values (±SEM) of three
independent experiments are presented. Asterisks (⁄) indicate values significantly
different from the control, analyzed by one way ANOVA followed by Dunnet’s test.
Letters (a/b) indicate significant differences between effects of individual enantiomers (t-test).
diastereomers, which may partially explain the a-HCH anti-androgenicity observed in the present work and previously (Roy et al.,
2004). Interestingly, molecular modeling also suggested weak but
significant differences between the estimated free energy of binding to the AR of two a-HCH enantiomers (Table 1), which could
explain slight differences between enantiomers observed in vitro
(Fig. 3).
Nevertheless, a-HCH could also affect various upstream signaling events, which control the AR action. Some of the known
upstream AR regulators are for example protein kinase A (Nazareth
and Weigel, 1996), mitogen-activated protein kinases – MAPKs, and
phosphatidylinositol 3-kinase/Akt signaling (Jia et al., 2004).
Recently, the controlling role of other less explored co-repressors/
co-activators has been described (Askew et al., 2009). Modulations
of these regulatory pathways by persistent organic compounds
have been documented at pesticides (Tessier and Matsumura,
2001) or polychlorinated biphenyls (Machala et al., 2003), and these
evidences may indirectly suggest similar action of a-HCH. Also
polycyclic aromatic hydrocarbons modulated other intracellular
receptors RAR/RXR with highly variable concentration–responses,
and significant role of the up-stream regulators has been discussed
(Benisek et al., 2008, 2011). More research efforts are thus needed
to fully understand cellular toxicity mechanisms of organic toxicants including a-HCH.
In summary, various insecticides were shown to have enantioselective effects related to their major targets such as acetylcholinesterase including – for example – isocarbophos (Lin et al., 2008),
fipronil (Wilson et al., 2008), chloramidophos (Zhou et al., 2007) or
acetophenate (Xu et al., 2008). Recently, enantioselective effects towards non-target cellular regulators such as estrogen receptor has
been documented for o,p0 -DDT (Wang et al., 2009) and bifenthrin
Acknowledgements
The authors would like to thank to Prof. Jiri Damborsky and
Dr. Zbynek Prokop (Loschmidt laboratories, Faculty of Science,
Masaryk University) for providing us with a-HCH racemate. The
research was supported by Ministry of Education, C.R. Projects
INCHEMBIOL (MSM0021622412) and ENVISCREEN (NPVII
2B08036). The infrastructure of the RECETOX is supported by the
project CETOCOEN (No. CZ.1.05/2.1.00/01.0001) from the European
Regional Development Fund. Prof. Giesy was supported by the
Canada Research Chair program and an at large Chair Professorship
at the Department of Biology and Chemistry and Research Centre
for Coastal Pollution and Conservation, City University of Hong
Kong. The research was partially supported by a Discovery Grant
from the National Science and Engineering Research Council of
Canada (Project # 326415-07) and a Grant from the Western Economic Diversification Canada (Project # 6578 and 6807).
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