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Interdisciplinary Studies on Environmental Chemistry — Environmental Research in Asia,
Eds., Y. Obayashi, T. Isobe, A. Subramanian, S. Suzuki and S. Tanabe, pp. 203–210.
© by TERRAPUB, 2009.
Characterization of the Organohalogen Compounds
Which Affect Gene Expressions Mediated by
Thyroid Hormone Receptors
Haruya S AKAI1, Ayato KAWASHIMA2,
Yuji KASHIMA3 and Toshiko YAMADA-OKABE4
1
Department of Human genetics, Yokohama City University School of Medicine,
Kanagawa, Japan
2
Department of Agriculture, Ehime University, Ehime, Japan
3
Japan Environmental Sanitation Center, Kanagawa, Japan
4
Division of Food Safety, Graduate School of Nutritional Sciences,
Sagami Women’s University, Kanagawa, Japan
(Received 3 February 2009; accepted 1 April 2009)
Abstract—Some organohalogen compounds could augment the gene expression
by thyroid hormone, T3, through the thyroid hormone receptor. But it is not the
case in 4-hydroxy-2′,3,4′,5,6′-pentachlorobiphenyl (PCB–OH). The addition
of this compound to the culture media suppressed the T3-induced reporter gene
expression. 3,3′,5,5′-Tetrabromobisphenol A (TBBPA), which had been reported
to modulate thyroid hormone systems like PCB–OH, was also shown to
suppress the T3-induced gene expression and 4-hydroxylated and 3,5-brominated
phenyl residues of TBBPA were necessary to repress the gene expression
induced by T3. In contrast, 3,3′,5,5′-tetrabromobisphenol S (TBBPS) which
has the same structure of TBBPA such as 4-hydroxylated and 3,5-brominated
phenyl residue, neither enhanced nor suppressed the reporter gene expression
activated by T3. To explain this discrepancy, here we speculated the three
dimensional structure of these compounds by calculation of the geometry
optimization of TBBPA, TBBPS and T3. The optimized geometry showed that
the configurations of two benzene rings of T3 were perpendicular to each other
and that of TBBPA was a twisted one. However the configuration of TBBPS
was face to face. Furthermore, nitrofen (NIP), which we had reported in the TRmediated gene expression has the same geometry as T3, even though it does not
have 4-hydroxy-3,5-dihalogen structure. These results indicate that twist angle
of the three dimensional structure is one of the most important characters for
modulation of TR-mediated gene expression.
Keywords: optimized geometries, thyroid hormone, thyroid hormone receptor,
tetrabromobisphenol A (TBBPA), tetrabromobispenol S (TBBPS), nitrofen
(NIP)
BACKGROUND
Some organohalogen compounds (OHCs) and phenol derivatives were suspected
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H. SAKAI et al.
Fig. 1. Effects of TBBPA and TBBPS on the luciferase activity mediated by TR. (A) HeLaTR-DR4luc cells were cultured in the presence of TBBPA with 7.7 nM T3 (left) or 61.6 nM T3 (right).
The luciferase activity and protein concentrations in the cell extracts were determined. Luciferase
activity was normalized to protein concentration. Mean of tripricate experiments are shown
along with standard deviations. Activity was expressed relative to the control without OHCs.
The statistical difference between the activities in the cells exposed to TBBPA and not exposed
to TBBPA is indicated by asterisks: *p < 0.05; **p < 0.01 (t-test). (B) The cells were cultured
in the presence of TBBPS with 30.8 nM T3 (left) or 61.6 nM T3 (right).
to have harmful effects on wildlife and human by disrupting endocrine systems
(Kupfer and Bulger, 1987; Kelce et al., 1995; Chen et al., 1997; Connor et al.,
1997; Andersen et al., 1999), including thyroid hormone systems (Brouwer et al.,
1998; Brucker-Davis, 1998). Thyroid hormone plays important roles in
metabolism, growth, and development in a wide variety of organisms
(Spangenberg, 1971; Shi and Ishizuya-Oka, 2001). Biological functions of
thyroid hormones, T3, are mediated by thyroid hormone receptors (TRs) that
belong to nuclear hormone receptor families. After binding T3 to the ligandbinding domain of TRs, TRs become activated as transcription factors. The
interaction between OHCs and TRs were reported in several studies. Kitamura et
al. (2002) suggested that 4-hydroxyl residue and two adjacent 3,5-halogen
residues in the phenol ring are essential to disrupt thyroid hormone activity. 3and/or 5-substituents of thyronine are also important for either agonist or
antagonist against TR (Baxter et al., 2002). From these studies, 4,4′-diOH3,3′,5,5′-tetrahalogenated structure is thought to be one of most important
structures to interact with TR.
Importance of Twist Angle Structure between Two Benzene Rings
3
4
205
2 1
3
4
2
1
Angle 1
3
4
3 1
2
2 1
4
Angle 2
Fig. 2. Illustration of the principal structure of the two internal single bond axes. The two internal
single bond axes of OHCs could rotate freely and it might show various possible configurations.
Dihedral angles 1 and 2 were calculated using the GAUSSIAN 03 software package.
REPORTER GENE ASSAY
Previously, we established the cell line, HeLaTR, in which human TRα were
stably expressed and thyroid hormone T3 as well as some chemicals could
activate the gene expression through TR (Yamada-Okabe et al., 2003, 2005). We
also made HeLaTR-DR4-luc in which reporter gene was stably transfected in
HeLaTR cells and the TR-mediated gene expression was examined. In HeLaTRDR4-luc cells, at least 7.7 nM T3 induced the luciferase activities about three fold
and higher concentration of T3 induced more luciferase activities (for example
61.4 nM T3 caused 3.5 fold increase) (Sakai et al., 2003). To examine the effect
of 3,3′,5,5′-tetrabromobisphenol A (TBBPA) on TR mediated gene expression,
several concentrations of TBBPA together with 7.7 nM T3 were added to the
medium of HeLaTR-DR4-luc cells followed by incubation for 36 hrs (Fig. 1A
left). Even 5 µM TBBPA decreased 20% luciferase activities induced by 7.7 nM
T3 and this inhibitory effect by TBBPA was dose dependent. 40 µM TBBPA
decreased luciferase activity to less than 30% of control (T3 alone). Higher
concentrations of TBBPA had more potent ability to inhibit T3-induced gene
expression, because luciferase activity induced by 61.4 nM T3 was strongly
suppressed by 50 µM TBBPA (Fig. 1A right). But no decrease was observed when
HeLaTR-DR4-luc cells were treated with less than 10 µM TBBPA, and even 25
µM TBBPA did not have significant effect on TR mediated gene expression.
Furthermore, bisphenol A (BPA) which is a debrominated compound of TBBPA,
showed similar effects as TBBPA. In presence of higher concentration of T3 as
much as 61.4 nM, the luciferase activity induced by T3 was not affected by the
addition of BPA. However, when T3 concentration was reduced to 30.8, 15.4 and
7.7 nM, the addition of 40 µM BPA decreased the luciferase activity to 85.4%,
82% and 73.5% compared to T3 alone, respectively (data not shown). These anti-
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H. SAKAI et al.
I
Br
O
I
NH2
I
HO
OH
HO
COOH
I
Br
CH3
CH3
Br
H
Br
Br
O
N
O
I
O
Br
H
Br
O
H
O
I
Br
T3
TBBPA
Fig. 3. Compounds used in the reporter gene assay and their estimated three dimensional structures
based on computer simulation. The optimized geometry of compounds was calculated using the
GAUSSIAN 03 software package.
thyroid hormone-like effects by TBBPA and BPA were consistent with previous
studies (Kitamura et al., 2002; Moriyama et al., 2002; Iwamuro et al., 2003). On
the contrary, 50 µM 3,3′,5,5′-tetrabromobisphenol S (TBBPS), which was
analogous to TBBPA, did not show any significant inhibitory effect on luciferase
Importance of Twist Angle Structure between Two Benzene Rings
Br
HO
OH
S
Br
Cl
Br
O
O
207
Cl
O
NO2
Br
Cl
Cl
S
O
O
O
Br
O
H
Br
O
Br
H
O
O
N
O
Br
TBBPS
NIP
Fig. 3. (continued).
activity induced by 30.8 nM (Fig. 1B left) or 61.6 nM T3 (Fig. 1B right). The
luciferase activities induced by lower concentration of T3, like 7.7 nM, were not
inhibited even by 40 µM TBBPS (data not shown). This result was unexpected,
because both of TBBPA and TBBPS have structure necessary for binding to TRs,
such as a 4-hydroxyl residue and two adjacent 3,5-halogen residues in the phenol
ring (Kitamura et al., 2002) (Fig. 3).
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H. SAKAI et al.
Table 1. Dihedral angles of T3, TBBPA, TBBPS and NIP.
Angle 1
T3
TBBPA
TBBPS
NIP
2
–52
94
2
Angle 2
90
131
94
89
Fig. 4. Effects of NIP on the luciferase activity mediated by TR. HeLaTR-DR4-luc cells were
cultured in the presence of NIP. The luciferase activity and protein concentrations in the cell
extracts were determined. Luciferase activity was normalized to protein concentration. Mean of
the tripricate experiments are shown along with standard deviations. Activity was expressed
relative to the control without NIP. The statistical difference between the activities in the cells
exposed to NIP and not exposed to NIP is indicated by asterisks: **p < 0.01 (t-test).
THREE-DIMENSIONAL STRUCTURE AND TR MEDIATED GENE EXPRESSION
We hypothesized that the three-dimensional structure of compounds may
affect the TR mediated gene expressions, and the structure of phenyl group may
be one of the important factors determining the binding affinity and/or forming
a ligand and receptor complex. Therefore, we calculated the optimized geometry
of TBBPA, TBBPS and T3 using the GAUSSIAN 03 software package, which is
fully optimized by density functional theory (DFT) at the B3LYP level. 6-31G(d)
basis set was used for the analysis of TBBPA and TBBPS, and 3-21G** basis set
for T3 (Frisch et al., 2003). Dihedral angles (Fig. 2) of TBBPA, TBBPS and T3
are shown in Table 1. As shown in Fig. 3, the difference in the three dimensional
structures between TBBPA and TBBPS is obvious; i.e. two dibromophenol rings
bound perpendicular to each other (twist angle) was observed in TBBPA, but not
in TBBPS. Furthermore, TBBPA seemed to have similar structure as T3, because
the two benzene rings in both compounds showed twisted angle. The dihedral
angle 1 and 2 of T3 calculated were almost the same as TBBPA. It was not the case
for TBBPS. We speculated that this structural similarity might be a main reason
for the suppressive effects by TBBPA. To prove our hypothesis, we chose
nitrofen (NIP), which has twisted angle but not phenol or halogenated phenol
rings. Previously we reported that NIP has an ability to affect T3-mediated gene
expression (Yamada-Okabe et al., 2005). The three dimensional structure and
Importance of Twist Angle Structure between Two Benzene Rings
209
dihedral angles of NIP were calculated. As shown in Table 1 and Fig. 3, two
benzene rings of NIP are bound perpendicular to each other. Although NIP has
neither phenolic ring nor halogenated phenol ring, these characterizations of
three dimensional structures suggest that NIP is analogous to T3. Further, the
calculated dihedral angles of NIP were almost the same as T3 (Table 1). Upon
using HeLaTR-DR4-luc cells, 20 µM NIP was found to have an ability to increase
the luciferase activity significantly (Fig. 4). NIP was reported to bind TR in a
noncompetitive manner to T3 and activate transcription (Brandsma et al., 1994).
From these results, it is evident that the compounds, which showed the
effects on the T3-induced gene expression, examined by reporter gene assay,
have similar three dimensional structures, such as three dimensional twist angle
and 4-hydroxyl residue and two adjacent 3,5-halogen residues in the phenol ring.
The importance of the residues in phenol ring was clear because stronger gene
suppression activity was found by TBBPA than BPA. The importance of the
substituents in the 3′- and /or 5′-position(s) of the thyronine structure was also
mentioned (Baxter et al., 2002). Furthermore, we suggested that the twist angle
structure was essential. This was supported by our findings that TBBPA exhibit
gene suppression but TBBPS did not, although these two compounds have two
3,5-dibromophenol rings. It remains to be determined whether the suppression or
activation effect of TR mediated gene expression by some compounds is based on
three dimensional modeling. Further studies on three dimensional modeling of
TR and OHC complex are necessary to understand TR-mediated functions of
OHCs.
In conclusion, twist angle structure between two benzene rings of OHCs is
one of the most important characters for modulating TR mediated gene expression.
Our model based on computer simulation might be useful for screening other
OHCs for their potential to interfere with TR mediated gene expression. This in
silico screening method might be cheaper and faster and would be suitable as the
first choice before carrying out reporter gene assay or in vivo experiments to
determine the effect on thyroid hormone system by OHCs.
Acknowledgments—This study was supported in part by a grant from the Ministry of
Education, Culture, Sports, Science and Technology, Japan to H.S. and T.Y.-O.
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