Академический Документы
Профессиональный Документы
Культура Документы
, BMG Labtech-
nologies GmBH, Offenburg, Germany). The amount of estradiol
could be read against a standard curve constructed with the hor-
mone provided in the kits.
2.8. Statistical analysis
Data obtained were analyzed by the software package Prizm5
(GraphPad Software, Inc., San Diego, CA). One Way ANOVA with
Dunnetts post hoc test was used for comparison.
3. Results
3.1. CYP19 expressions in different brain cell lines
CYP19 expressions in the brain cell lines LAN-1, T98G, DBTRG-
05MG (05MG), U-87 MG (U87) and U251 were determined and
compared. Expression in T98G cells was signicantly (p < 0.05)
higher than the other neuroblastoma or glioblastoma cell lines
(Fig. 1). Hence, we chose T98G cell line as the model in our study.
3.2. CYP19 expression was induced by TCDD
In addition to T98G cells, LAN-1 cells were arbitrarily selected
for testing the responses to TCDD exposure. 10 nM TCDD increased
the expression by 14-fold in LAN-1 cells (Fig. 2B), whereas the
same concentration induced 2-fold increase in T98G cells
(Fig. 2A). Doseresponse relationship was demonstrated in both
cell lines. However, aromatase protein and enzyme activity in
LAN-1 cells were not detectable in subsequent experiments.
T98G cells were selected for further study.
3.3. TCDD induced aromatase activity in T98G cells
As the mRNA expression of CYP19 increased, the enzyme activ-
ity should also be induced. We veried that the aromatase activity
in T98G cells was induced by TCDD after 48 h incubation (Fig. 3A)
in a dose-dependent manner. The activity was signicantly
(p < 0.05) increased starting from0.001 nM TCDD, and a vefold in-
crease was observed in cultures treated with 0.01 nM TCDD and
above. Increased estradiol production was also observed in the
presence of testosterone (Fig. 3B), since aromatase catalyzes the
conversion of androgen into estrogen.
3.4. Aromatase protein was increased by TCDD
Increased aromatase protein was also observed in T98G cells
(Fig. 4A). The normalized optical density of the CYP19 was esti-
mated and shown in Fig. 4B. The protein was increased by about
2.7-fold. The result veried that the induced aromatase activity
was regulated at the expression level.
3.5. Specic promoter usage and regulation
By using the Exon-specic probes, we observed that transcripts
from Promoters Ia, I.f, I.2, I.3 and II on Exon 1 were detectable in
L
A
N
-
1
T
9
8
G
0
5
M
G
U
8
7
U
2
5
1
0
1
2
3
30
40
50
60
*
R
e
l
a
t
i
v
e
C
Y
P
1
9
m
R
N
A
E
x
p
r
e
s
s
i
o
n
Fig. 1. Basal CYP19 mRNA expression in various brain cell lines. Brain cells LAN-1,
T98G, DBTRG-05MG (05MG), U-87 MG (U87) and U251 were seeded in 6-well
plates and maintained in culture medium as described in Methods. The amount of
CYP19 mRNA was determined by real time PCR and was normalized by GAPDH
mRNA. Values are means SEM, n = 3. Means labeled with () were signicantly
(p < 0.05) higher than means of the other cell lines.
108 W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112
this cell line (Fig. 5). Since Promoter I.f is reported as the major reg-
ulatory promoter for CYP19 expression in the brain under normal
condition (Yague et al., 2006), we specically looked into the Exon
I.f expression response. RT-PCR assay result showed that relative
Exon I.f-spliced mRNA was raised by TCDD in a dose dependent
manner with signicant deviation (P < 0.05) occurred beyond 0.1
nM (Fig. 6).
Control 0.001 0.01 0.1 1 10
0
5
10
15
*
R
e
l
a
t
i
v
e
C
Y
P
1
9
m
R
N
A
E
x
p
r
e
s
s
i
o
n
i
n
L
A
N
-
1
c
e
l
l
s
TCDD Conc. (nM)
Control 0.001 0.01 0.1 1 10
0
1
2
* *
R
e
l
a
t
i
v
e
C
Y
P
1
9
m
R
N
A
E
x
p
r
e
s
s
i
o
n
i
n
T
9
8
G
c
e
l
l
s
TCDD Conc. (nM)
A B
Fig. 2. TCDD increased CYP19 mRNA expression in brain cells. Brain cells were seeded in 6-well plates and maintained in phenol-red free RPMI 1640 medium supplemented
with 10% charcoal dextran-treated FBS. Cells were treated with TCDD for 24 h. The amount of CYP19 mRNA was determined by real time PCR and was normalized by GAPDH
mRNA in T98G (A) and LAN-1 (B) cells. Values are means SEM, n = 8. Means labeled with various letters were signicantly (p < 0.05) different.
0 0.001 0.01 0.1 1 10
0
100
200
300
*
*
A
r
o
m
a
t
a
s
e
a
c
t
i
v
i
t
y
(
c
p
m
/
m
g
p
r
o
t
e
i
n
/
h
r
)
TCDD Conc (nM)
0 0.001 0.01 0.1 1 10 0 10
0
5
10
15
20
E
s
t
r
a
d
i
o
l
i
n
c
u
l
t
u
r
e
m
e
d
i
u
m
(
p
g
/
m
l
)
*
*
* *
*
TCDD (nM)
10 nM testosterone
* * *
+ + + + + + - -
b
a
A B
Fig. 3. Aromatase activity was induced by TCDD in T98G cells. T98G cells were seeded in six-well plates and maintained in phenol red-free RPMI medium supplemented with
10% charcoal dextrantreated serum. Aromatase activities were determined in cultures treated with TCDD (A). Estradiol concentrations in culture medium were assayed by
ELISA (B). The data represents the means SEM of 3 samples isolated from independent cultures. () mean values were signicantly (p < 0.05) different from that of the
control cultures.
Control 0.001 0.01 0.1 1 10
0
1
2
3
R
e
l
a
t
i
v
e
O
p
t
i
c
a
l
D
e
n
s
i
t
y
TCDD Conc. (nM)
A
B
Fig. 4. TCDD increased aromatase protein in T98G cells. T98G cells were seeded in
6-well culture dishes and treated with TCDD for 24 h. Amounts of CYP19 in protein
extracts were determined by western blot analysis. The image shown in Fig. 4A is a
representation of three independent experiments, and Fig. 4B display the optical
density readings of the proteins. Values are means SEM, n = 3. Means labeled with
() were signicantly (p < 0.05) different.
G
A
P
D
H I
a
I
.
4
I
.
5
I
.
f
I
.
2
I
.
3 I
I
0.0
0.1
0.2
0.3
0.4
0.95
1.00
R
e
l
a
t
i
v
e
A
l
t
e
r
n
a
t
e
S
p
l
i
c
e
d
m
R
N
A
e
x
p
r
e
s
s
i
o
n
Fig. 5. Exon-specic CYP19 expression in T98G cells. T98G cells were seeded in 6-
well plates and maintained in culture medium as described in Methods. The
amounts of exon-specic spliced mRNA species were determined by real time PCR
and were normalized by GAPDH mRNA. Values are means SEM, n = 3. Means
labeled with () were signicantly (p < 0.05) higher than means of the other cell
lines.
W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112 109
3.6. Effects of TCDD on C/EBP binding in Exon I.f
The result of Exon I.f-spliced mRNA assay illustrated that TCDD
might activate the transcriptional activity through the correspond-
ing promoter. Subsequently, we scanned the sequence (500/0) of
ExonI.f of CYP19 using TRANSFAC(version8.3), and one C/EBP bind-
ing site was located in the segment (397/391). EMSA assay was
performedtoverifythe functionalityof the binding. Nuclear extracts
isolated from cultures treated with TCDD displayed an increasing
trend of C/EBP binding from 0.001 to 10 nM (Fig. 7). The band spec-
icity was further demonstrated by the weakening of intensity
when co-incubating with 7 C/EBP unlabeled oligonucleotide (SC)
or the antibody of C/EBP (Santa Cruz Biotechnology) (SS).
3.7. Protein kinase signaling pathways
Since the transcriptional factors C/EBP could be activated by
phosphorylation, we examined the activation states of the related
signaling protein kinases by western blot analysis. TCDD appeared
to increase the phosphor- ERK-1 and -2 in the nuclear protein,
while no effect on JNK or p38MAPK was observed (Fig. 8A).
3.8. Effect of the ERK inhibitor U0126 on TCDD-induced mRNA
expression
Since ERK-1/2 was increased by TCDD treatment, effect of ERK
inhibitor on the RNA expression was investigated in a subsequent
experiment. Cultures pre-treated with U0126 (I + TCDD) displayed
a decreased expression of Exon I.f-specic CYP19 mRNA as com-
pared to cells treated with TCDD alone (Fig. 8B). This result illus-
trated that ERK activation was partially responsible for the
induction of CYP19 expression.
4. Discussion
In the present study, an endocrine system of the brain per-
turbed by TCDD was depicted. The toxicant increased CYP19 mRNA
expression in cultured cells and it might induce the Exon I.f trans-
activity through ERK activation.
TCDD is reported to reduce aromatase protein or mRNA in JEG-3
cells (Drenth et al., 1998) and granulosa cells (Moran et al., 2000;
Dasmahapatra et al., 2000). In contrast, other investigators have
shown that the toxicant induces aromatase in human placental
cells (Augustowska et al., 2003), breast cells (Chan et al., 2010)
and primary Sertoli cells (Lai et al., 2005). The conicting results
could be attributed to the species and tissue differences. In the
present study, TCDD consistently increased the expression of
CYP19 in various human brain cells.
ERK appears to be an effector signaling molecule of TCDD. The
toxicant may upregulate or downregulate gene expression through
ERK activation. Studies have shown that the contaminant induces
TNFa and IL-6 expression in synoviocytes (Kobayashi et al., 2008;
Cheon et al., 2007) and THP-1 cells (Cheon et al., 2007), respec-
tively. On the other hand, TCDD suppresses PPAR-c1 expression
in C3H10T1/2 cells (Hanlon et al., 2005). ERK activated by TCDD
also takes part in other physiological pathways, for instance,
Control 0.001 0.01 0.1 1 10
0
1
2
3
R
e
l
a
t
i
v
e
E
x
o
n
I
.
f
-
s
p
l
i
c
e
d
m
R
N
A
e
x
p
r
e
s
s
i
o
n
TCDD Conc. (nM)
Fig. 6. TCDD increased Exon I.f specic CYP19 mRNA expression in T98G cells. T98G
cells were seeded in 6-well plates and maintained in phenol-red free RPMI 1640
medium supplemented with 10% charcoal dextran-treated FBS. Cells were treated
with TCDD for 24 h. The amount of Exon I.f-spliced CYP19 mRNA was determined
by real time PCR and was normalized by GAPDH mRNA. Values are means SEM,
n = 3. Means labeled with various letters were signicantly (p < 0.05) different.
Fig. 7. Effect of TCDD on the binding of C/EBP sequence within promoter I.f. Nuclear extract samples were prepared from TCDD-treated T98G cells and EMSA was performed.
In this gure, lanes labeled with 0, 0.001, 0.01, 0.1, 1 and 10 are samples treated with the respective TCDD concentration (nM); 10-SC and 10-SS are the samples extracted
from 10 nM TCDD and incubated with 7 C/EBP consensus sequence and C/EBP antibody, respectively. Data represent one of two independent experiments with comparable
results. Non-specic binding bands are seen below the C/EBP bands.
110 W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112
insulin signaling in MCF-10A cells (Park et al., 2004), and apoptosis
in RAW264.7 (Park et al., 2005) and Jurkat (Kwon et al., 2003) cells.
Our laboratory has also demonstrated that TCDD could increase
the stability of CYP19 transcript in cultured MCF-7 cells through
ERK activation (Chan et al., 2010). Contrasting to the present study,
TCDD appeared to increase the CYP19 transcription in brain cells
through a different downstream pathway of ERK.
C/EBPs are a family of transcriptional factors that could be acti-
vated by ERK (Li et al., 2007). The beta isoform of C/EBP is impor-
tant in consolidating long term memory (Carew and Sutton, 2001).
The isoform may also participate in promoting post-ischemic
inammation and cause brain damage (Yi et al., 2007). The delta
isoform of C/EBP, on the other hand, can bind to PI.3/II of CYP19
and initiates the expression in breast cancer cells (Kijima et al.,
2008). In the present study, the C/EBP binding activity within Exon
I.f segment was enhanced upon TCDD treatment. This could be the
underlying mechanism for the induced CYP19 expression.
Other physiological actions of TCDD on the brain have also
been documented, such as inducing apoptosis in cerebellar
granule cells (Sanchez-Martin et al., 2011), changing the neurode-
velopment in fetal mouse brain (Mitsui et al., 2011), and
stimulating the efux transporter at the blood brain barrier (Wang
et al., 2011). However, change in cell number under TCDD treat-
ment as determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay was not observed in the present
study (data not shown).
In summary, this study illustrated that TCDD could induce the
transcriptional activity of aromatase in glioblastoma cells. Although
the physiological signicance is not known, the upregulation of
aromatase might perturb the hormonal balance in the brain or exac-
erbate the cellular response after injury.
Acknowledgement
This study was supported by The Chinese University of Hong
Kong.
References
Augustowska, K., Gregoraszczuk, E.L., Milewicz, T., Krzysiek, J., Grochowalski, A.,
Chrzaszcz, R., 2003. Effects of dioxin (2,3,7,8-TCDD) and PCDDs/PCDFs
congeners mixture on steroidogenesis in human placenta tissue culture.
Endocr. Regul. 37, 1119.
Bingham, D., Macrae, I., Carswell, H., 2005. Detrimental effects of 17 beta-oestradiol
after permanent middle cerebral artery occlusion. J. Cerebral Blood Flow Metab.
25, 414420.
Birnbaum, L.S., Tuomisto, J., 2000. Non-carcinogenic effects of TCDD in animals.
Food Addit. Contam. 17, 275288.
Cao, J., Patisaul, H.B., Petersen, S.L., 2011. Aryl hydrocarbon receptor activation in
lactotropes and gonadotropes interferes with estradiol-dependent and -
independent preprolactin, glycoprotein alpha and luteinizing hormone beta
gene expression. Mol. Cell. Endocrinol. 333, 151159.
Carew, T.J., Sutton, M.A., 2001. Molecular stepping stones in memory consolidation.
Nat. Neurosci. 4, 769771.
Chan, M.Y., Huang, H., Leung, L.K., 2010. 2,3,7,8-Tetrachlorodibenzo-para-dioxin
increases aromatase (CYP19) mRNA stability in MCF-7 cells. Mol. Cell.
Endocrinol. 317, 813.
Cheon, H., Woo, Y., Lee, J., Kim, H., Kim, H., Cho, S., Won, N., Sohn, J., 2007. Signaling
pathway for 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced TNF-alpha
production in differentiated THP-1 human macrophages. Exper. Mol. Med. 39,
524534.
Dasmahapatra, A.K., Wimpee, B.A., Trewin, A.L., Wimpee, C.F., Ghorai, J.K., Hutz, R.J.,
2000. Demonstration of 2,3,7,8-tetrachlorodibenzo-p-dioxin attenuation of
P450 steroidogenic enzyme mRNAs in rat granulosa cell in vitro by
competitive reverse transcriptase-polymerase chain reaction assay. Mol. Cell.
Endocrinol. 164, 518.
Drenth, H.J., Bouwman, C.A., Seinen, W., Van den Berg, M., 1998. Effects of some
persistent halogenated environmental contaminants on aromatase (CYP19)
activity in the human choriocarcinoma cell line JEG-3. Toxicol. Appl. Pharmacol.
148, 5055.
Endo, T., Kakeyama, M., Uemura, Y., Haijima, A., Okuno, H., Bito, H., Tohyama, C.,
2012. Executive function decits and social-behavioral abnormality in mice
exposed to a low dose of dioxin in utero and via lactation. PLoS One 7, e50741.
Fernandez, M., Paradisi, M., DIntino, G., Del Vecchio, G., Sivilia, S., Giardino, L., Calza,
L., 2010. A single prenatal exposure to the endocrine disruptor 2,3,7,8-
tetrachlorodibenzo-p-dioxin alters developmental myelination and
remyelination potential in the rat brain. J. Neurochem. 115, 897909.
Garcia-Segura, L., Naftolin, F., Hutchison, J., Azcoitia, I., Chowen, J., 1999. Role of
astroglia in estrogen regulation of synaptic plasticity and brain repair. J.
Neurobiol. 40, 574584.
Garcia-Segura, L.M., Wozniak, A., Azcoitia, I., Rodriguez, J.R., Hutchison, R.E.,
Hutchison, J.B., 1999. Aromatase expression by astrocytes after brain injury:
implications for local estrogen formation in brain repair. Neuroscience 89, 567
578.
Haijima, A., Endo, T., Zhang, Y., Miyazaki, W., Kakeyama, M., Tohyama, C., 2010. In
utero and lactational exposure to low doses of chlorinated and brominated
dioxins induces decits in the fear memory of male mice. Neurotoxicology 31,
385390.
Hanlon, P.R., Cimafranca, M.A., Liu, X., Cho, Y.C., Jefcoate, C.R., 2005. Microarray
analysis of early adipogenesis in C3H10T1/2 cells: cooperative inhibitory effects
of growth factors and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl.
Pharmacol. 207, 3958.
Kijima, I., Ye, J., Glackin, C., Chen, S., 2008. CCAAT/enhancer binding protein delta
up-regulates aromatase promoters I.3/II in breast cancer epithelial cells. Cancer
Res. 68, 44554464.
Kobayashi, S., Okamoto, H., Iwamoto, T., Toyama, Y., Tomatsu, T., Yamanaka, H.,
Momohara, S., 2008. A role for the aryl hydrocarbon receptor and the dioxin
TCDD in rheumatoid arthritis. Rheumatology 47, 13171322.
Kwon, M.J., Jeong, K.S., Choi, E.J., Lee, B.H., 2003. 2,3,7,8-Tetrachlorodibenzo-p-
dioxin (TCDD)-induced activation of mitogen-activated protein kinase signaling
pathway in Jurkat T cells. Pharm. Toxicol. 93, 186190.
Lai, K.P., Wong, M.H., Wong, C.K., 2005. Effects of TCDD in modulating the
expression of Sertoli cell secretory products and markers for cell-cell
interaction. Toxicology 206, 111123.
Latchney, S.E., Lioy, D.T., Henry, E.C., Gasiewicz, T.A., Strathmann, F.G., Mayer-
Proschel, M., Opanashuk, L.A., 2011. Neural precursor cell proliferation is
disrupted through activation of the aryl hydrocarbon receptor by 2,3,7,8-
tetrachlorodibenzo-p-dioxin. Stem. Cells Dev. 20, 313326.
Latchney, S.E., Hein, A.M., OBanion, M.K., Dicicco-Bloom, E., Opanashuk, L.A., 2012.
Deletion or activation of the aryl hydrocarbon receptor alters adult
hippocampal neurogenesis and contextual fear memory. J. Neurochem.
DMSO TCDD I+TCDD
0
1
2
3
R
e
l
a
t
i
v
e
C
Y
P
1
9
m
R
N
A
E
x
p
r
e
s
s
i
o
n
A
B
Fig. 8. Immunoblot of p-P38, p-ERK and p-JNK in T98G cells treated with TCDD.
T98G cells were seeded in 6-well culture dishes and treated with TCDD for 24 h.
Amounts of p-P38, p-ERK and p-JNK in nuclear extracts were determined by
western blot analysis. The image shown in Fig. 8A is a representation of two
independent experiments and the Histone 4-normalized optical density readings
are shown under the band image. In Fig. 8B, cells were seeded in 6-well plates and
were cotreated with TCDD and ERK inhibitor (U0126) for 24 h. The amount of
CYP19 mRNA was determined by real time PCR and was normalized by GAPDH
mRNA. Values are means SEM, n = 4. Means labeled with () were signicantly
(p < 0.05) different than the other two.
W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112 111
Li, H., Gade, P., Xiao, W., Kalvakolanu, D.V., 2007. The interferon signaling network
and transcription factor C/EBP-beta. Cell. Mol. Immunol. 4, 407418.
Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25,
402408.
Means, G.D., Mahendroo, M.S., Corbin, C.J., Mathis, J.M., Powell, F.E., Mendelson, C.R.,
Simpson, E.R., 1989. Structural analysis of the gene encoding human aromatase
cytochrome P-450, the enzyme responsible for estrogen biosynthesis. J. Biol.
Chem. 264, 1938519391.
Mitsui, T., Taniguchi, N., Kawasaki, N., Kagami, Y., Arita, J., 2011. Fetal exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin induces expression of the chemokine genes
Cxcl4 and Cxcl7 in the perinatal mouse brain. J. Appl. Toxicol. 31, 279
284.
Moon, B.H., Hong, C.G., Kim, S.Y., Kim, H.J., Shin, S.K., Kang, S., Lee, K.J., Kim, Y.K., Lee,
M.S., Shin, K.H., 2008. A single administration of 2,3,7,8-tetrachlorodibenzo-p-
dioxin that produces reduced food and water intake induces long-lasting
expression of corticotropin-releasing factor, arginine vasopressin, and
proopiomelanocortin in rat brain. Toxicol. Appl. Pharmacol. 233, 314322.
Moran, F.M., Conley, A.J., Corbin, C.J., Enan, E., VandeVoort, C., Overstreet, J.W.,
Lasley, B.L., 2000. 2,3,7,8-Tetrachlorodibenzo-p-dioxin decreases estradiol
production without altering the enzyme activity of cytochrome P450
aromatase of human luteinized granulosa cells in vitro. Biol. Reprod. 62,
11021108.
Nguyen, A.T., Nishijo, M., Hori, E., Nguyen, N.M., Pham, T.T., Fukunaga, K., Nakagawa,
H., Tran, A.H., Nishijo, H., 2013. Inuence of maternal exposure to 2,3,7,8-
tetrachlorodibenzo-p-dioxin on socioemotional behaviors in offspring rats.
Environ. Health Insights 7, 114.
Nishijo, M., Kuriwaki, J., Hori, E., Tawara, K., Nakagawa, H., Nishijo, H., 2007. Effects
of maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on fetal brain
growth and motor and behavioral development in offspring rats. Toxicol. Lett.
173, 4147.
Ohtake, F., Takeyama, K., Matsumoto, T., Kitagawa, H., Yamamoto, Y., Nohara, K.,
Tohyama, C., Krust, A., Mimura, J., Chambon, P., Yanagisawa, J., Fujii-Kuriyama,
Y., Kato, S., 2003. Modulation of oestrogen receptor signalling by association
with the activated dioxin receptor. Nature 423, 545550.
Park, S., Mazina, O., Kitagawa, A., Wong, P., Matsumura, F., 2004. TCDD causes
suppression of growth and differentiation of MCF10A, human mammary
epithelial cells by interfering with their insulin receptor signaling through c-
Src kinase and ERK activation. J. Biochem. Mol. Toxicol. 18, 322331.
Park, S., Yoon, W., Kim, H., Son, H., Cho, S., Jeong, K., Kim, T., Kim, S., Kim, S., Ryu, S.,
2005. 2,3,7,8-Tetrachlorodibenzo-p-dioxin activates ERK and p38 mitogen-
activated protein kinases in RAW 264.7 cells. Antican. Res. 25, 28312836.
Saldanha, C., Duncan, K., Walters, B., 2009. Neuroprotective actions of brain
aromatase. Front. Neuroendocrinol. 30, 106118.
Sanchez-Martin, F.J., Fernandez-Salguero, P.M., Merino, J.M., 2011. Aryl
hydrocarbon receptor-dependent induction of apoptosis by 2,3,7,8-
tetrachlorodibenzo-p-dioxin in cerebellar granule cells from mouse. J.
Neurochem. 118, 153162.
Simpson, E.R., Michael, M.D., Agarwal, V.R., Hinshelwood, M.M., Bulun, S.E., Zhao, Y.,
1997. Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an
unusual case of alternative promoter usage. FASEB J. 11, 2936.
Steenland, K., Bertazzi, P., Baccarelli, A., Kogevinas, M., 2004. Dioxin revisited:
developments since the 1997 IARC classication of dioxin as a human
carcinogen. Environ. Health Perspect. 112, 12651268.
Toda, K., Terashima, M., Kawamoto, T., Sumimoto, H., Yokoyama, Y., Kuribayashi, I.,
Mitsuuchi, Y., Maeda, T., Yamamoto, Y., Sagara, Y., et al., 1990. Structural and
functional characterization of human aromatase P-450 gene. Eur. J. Biochem.
193, 559565.
Wang, Y., Chan, F.L., Chen, S., Leung, L.K., 2005. The plant polyphenol butein inhibits
testosterone-induced proliferation in breast cancer cells expressing aromatase.
Life Sci. 77, 3951.
Wang, Y., Lee, K.W., Chan, F.L., Chen, S., Leung, L.K., 2006. The red wine polyphenol
resveratrol displays bilevel inhibition on aromatase in breast cancer cells.
Toxicol. Sci. 92, 7177.
Wang, Y., Man Gho, W., Chan, F.L., Chan, S., Leung, L.K., 2008. The red clover
(Trifolium pratense) isoavone biochanin A inhibits aromatase activity and
expression. Br. J. Nutr. 99, 303310.
Wang, X., Hawkins, B.T., Miller, D.S., 2011. Aryl hydrocarbon receptor-mediated up-
regulation of ATP-driven xenobiotic efux transporters at the blood-brain
barrier. FASEB J. 25, 644652.
Yague, J.G., Munoz, A., de Monasterio-Schrader, P., Defelipe, J., Garcia-Segura, L.M.,
Azcoitia, I., 2006. Aromatase expression in the human temporal cortex.
Neuroscience 138, 389401.
Ye, L., Chan, M.Y., Leung, L.K., 2009. The soy isoavone genistein induces estrogen
synthesis in an extragonadal pathway. Mol. Cell. Endocrinol. 302, 7380.
Yi, J.H., Park, S.W., Kapadia, R., Vemuganti, R., 2007. Role of transcription factors in
mediating post-ischemic cerebral inammation and brain damage. Neurochem.
Int. 50, 10141027.
112 W. Tan et al. / Molecular and Cellular Endocrinology 375 (2013) 106112