steroids 73 (2008) 1242–1251
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Effects of endocrine disruptors on genes associated with
17-estradiol metabolism and excretion
Nathalie Hanet
a,1
, Allan Lancon
b,2,3
, Dominique Delmas
b,2,3
, Brigitte Jannin
b,2,3
,
Marie-C
Yves A
a
Unité Mix
Faculté de
b
Laboratoi
21000 Dijo
artic
Article histo
Received 7
Received in
Accepted 1
Published
Keywords:
Estradiol m
Endocrine
ATP-bindin
UDP-glucu
Sulfotrans
∗
Corresp
fax: +33 38
E-mail
1
Presen
Faculté de
2
Presen
3
Presen
(LBMN), Di
0039-128X
doi:10.1016hristine Chagnon
a,1
, Moustapha Cherkaoui-Malki
b,2,3
, Norbert Latruffe
b,2,3
,
rtur
a,1
, Jean-Marie Heydel
a,∗,1
te de Recherche 1234 Toxicologie Alimentaire, Institut National de la Recherche Agronomique- Université de Bourgogne,
Pharmacie, 7 bd Jeanne d’Arc, 21000 Dijon, France
re de Biologie Molé et Cellulaire (E.A.2978), Université de Bourgogne, Faculté des Sciences, 6 boulevard Gabriel,
n, France
le info
ry:
April 2008
revised form 7 May 2008
1 June 2008
on line 26 June 2008
etabolism
disruptors
g cassette transporters
ronosyltransferase
ferase
abstract
In order to provide a global analysis of the effects of endocrine disruptors on the hor-
mone cellular bioavailability, we combined 17-estradiol (E2) cellular flow studies with
real-time PCR and Western blot expression measurements of genes involved in the hor-
mone metabolism and excretion. Three endocrine disruptors commonly found in food
were chosen for this study, which was conducted in the estrogen receptor (ER) nega-
tive hepatoblastoma HepG2 cell line: bisphenol A (BPA), genistein (GEN) and resveratrol
(RES). We showed that 24 h after a single dose treatment with genistein, resveratrol or
bisphenol A, the expression of ATP-binding cassette transporters (the multidrug resistance
or MDR, and the multidrug resistance associated proteins or MRP) uridine diphosphate-
glucuronosyltransferases (UGT) and/or sulfotransferases (ST) involved in 17-estradiol
elimination process were significantly modulated and that 17-estradiol cellular flow was
modified. Resveratrol induced MDR1 and MRP3 expressions, bisphenol A induced MRP2 and
MRP3 expressions, and both enhanced 17-estradiol efflux. Genistein, on the other hand,
inhibited ST1E1 and UGT1A1 expressions, and led to 17-estradiol cellular retention.
Thus, we demonstrate that bisphenol A, genistein and resveratrol modulate 17-estradiol
cellular bioavailability in HepG2 and that these modulations most probably involve regula-
tions of 17-estradiol phase II and III metabolism proteins. Up to now, the estrogenicity of
environmental estrogenic pollutants has been based on the property of these compounds
to bind to ERs. Our results obtained with ER negative cells provide strong evidence for the
existence of ER-independent pathways leading to endocrine disruption.
© 2008 Elsevier Inc. All rights reserved.
onding author at: UMR 1129 FLAVIC, Faculté de Pharmacie, 7 bd Jeanne d’Arc, 21000 Dijon, France. Tel.: +33 380 39 32 17;
0 39 32 18.
address: jean-marie.heydel@u-bourgogne.fr (J.-M. Heydel).
t address: Unité Mixte de Recherche 1129 FLAVIC, Institut National de la Recherche Agronomique- Université de Bourgogne,
Pharmacie, 7 bd Jeanne d’Arc, 21000 Dijon, France.
t address: Inserm U866, Dijon F-21000, France.
t address: Université de Bourgogne, Faculté des Sciences Gabriel, Centre de Recherche-Biochimie Métabolique et Nutritionnelle
jon F-21000, France.
/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
/j.steroids.2008.06.005

steroids 73 (2008) 1242–1251 1243
1. Introduction
Endocrine
the potenti
of humans
tioning of
manners: b
hormone, b
the recepto
fore preven
the synthe
ifying the s
hormones.
tal estrogen
EDCs), that
Various na
been ident
ing pharma
metals and
ity of e-ED
compound
sequently e
estrogen re
get genes.
ERs could
EDCs.
Hormon
pally by the
comprises i
subsequen
proteins in
There a
biological e
rupting the
while most
phase II en
sured gene
none has i
excretion, n
expression
This pa
monly foun
elimination
line was us
retained m
most of the
of the hum
receptor at
induce tran
gene in tran
gation of an
The e-ED
like respon
bisphenol A
bonate flas
and dental
the stilben
estrogenic
natural com
In human hepatocytes, E2 glucuronides are formed by
UDP (uridine diphosphate)-glucuronosyltransferases (UGTs)
7 an
11,12
sser
A1) c
tran
re AT
conj
rotei
nd t
RP3
BPA,
wer
f gen
y E2 c
reme
hob
and
s of
he e
l, suc
hole
pres
at e
ilabi
ation
g to e
Ex
Ch
tradi
ide (
sed
ein
don,
84 TB
erkin
cals
n k
–Aldr
Cel
cultu
oaci
ated
gen
blas
ean
ring
yco
ere
l-red
men
oaci
O
2
adisrupting compounds (EDCs) are chemicals with
al to elicit negative effects on endocrine systems
and other animals. They interfere with the func-
the endocrine system in at least three possible
y mimicking the action of a naturally produced
inding to their hormone receptors; by blocking
rs in target cells for these hormones and there-
ting the action of natural hormones; or by altering
sis and function of hormone receptors and mod-
ynthesis, transport, metabolism and excretion of
The most studied of all EDCs are environmen-
s which we will designate as e-EDCs (estrogenic
mimic the gonadal hormone 17-estradiol (E2).
tural and synthetic chemical compounds have
ified that induce estrogen-like responses includ-
ceuticals, pesticides, industrial chemicals, heavy
phytoestrogens [1]. Up to now, the estrogenic-
Cs has been based on the property of these
s to bind to estrogen receptors (ERs), which sub-
xert transcriptional effects when binding to the
sponse element (ERE) on the promoter of tar-
However, several nuclear receptors other than the
be involved in E2 target genes regulation by e-
al estrogens are eliminated from the body princi-
liver, where the major pathway of E2 elimination
ts conjugation by phase II detoxifying enzymes and
t excretion of the conjugate by phase III transporter
to the bile.
re evidences that e-EDCs might influence the
ffects of E2 by modulating its metabolism and dis-
balanced generation of metabolites [2–5]. However,
of these studies investigated the modulation of E2
zymes activities, fewer studies have actually mea-
s expression (always in ER positive models), and
nvestigated the effects of e-EDCs on phase III E2
either on transport activities nor on transporters
.
per focuses on the effects of three e-EDCs com-
d in human diet on the regulation of E2 cellular
in the human hepatoblastoma HepG2. This cell
ed as a model because it is well differentiated, it has
any hepatocyte-specific functions and it expresses
proteins involved in E2 phase II and III metabolism
an liver. Conversely, HepG2 cells expresses ER
very low levels which are unsufficient for ligand to
sactivation of an ERE-containing synthetic target
sient transfection [6,7], thus enabling the investi-
ER-independent effect of these e-EDCs.
Cs chosen for this study mimic or induce estrogen-
se with varying degrees of potency. The chemical
(BPA) has been shown to be released from polycar-
ks during sterilization, inner coating of food cans
sealants [8]. The isoflavone genistein (GEN) and
e resveratrol (RES) were chosen as representative
polyphenols to which we are exposed, as they are
pounds of food and wine [9,10].
1A1, 2B
2B15 [
to a le
(SULT1
Major
cytes a
for E2
glycop
gene a
[17], M
E2,
HepG2
sions o
then b
measu
hydrop
other h
porter
Thus t
the cel
as a w
The
esis th
bioava
elimin
leadin
2.
2.1.
17-es
sulfox
purcha
Genist
(Abing
ity: 1.
from P
Chemi
poratio
Sigma
2.2.
Cell
l-amin
inactiv
Invitro
hepato
(Europ
monito
free (M
cells w
pheno
supple
l-amin
of 5% Cd with a minor contribution by UGT1A3, 1A4 and
]. The estrogen sulfotransferase (SULT1E1), and
extent the thermostable phenolsulfotransferase
atalyze the formation of E2 sulfoconjugates [13,14].
sporters responsible for E2 excretion by hepato-
P-binding cassette proteins with varying affinities
ugates. These include, for the most important, P-
n, encoded by MDR1 [15] (multidrug resistance)
he multidrug resistance proteins MRP1 [16], MRP2
[18] and MRP4 [19].
RES and GEN effects on E2 cellular metabolism in
e first investigated by measurements of the expres-
es involved in E2 phase II and III metabolism, and
ellular flow studies. E2 flow studies are a combined
nt of E2 influx and efflux from the cells. Due to E2
icity, E2 influx can be considered as passive. On the
, E2 efflux results from active excretion by trans-
conjugates issued from E2 phase II metabolism.
fflux represents the transport activity of E2 across
h that the E2 elimination process can be analyzed
by this method.
ent experiments were designed to test the hypoth-
ndocrine disrupting chemicals can modulate E2
lity through a modification of E2 metabolism and
. The tests examined ER-independent pathways
ndocrine disruption.
perimental
emicals
ol (E2), bisphenol A, trans-resveratrol, dimethyl-
DMSO) and 30% acrylamide/bis-acrylamide were
from Sigma–Aldrich (St. Quentin Fallavier, France).
(GEN) was obtained from AApin Chemicals
UK). [
3
H]-17-estradiol ([
3
H]-E2; specific activ-
q/mmol; labeled in 6 and 7) was purchased
Elmer Life and Analytical Sciences (Boston, US).
used for Western blotting and cellular incor-
inetic, unless specified, were purchased from
ich.
l and culture media
re media, l-glutamine and non-essential
ds were purchased from Sigma–Aldrich. Heat-
fetal bovine serum (FBS) was obtained from
(Cergy-Pontoise, France). The HepG2 human
toma cell line was obtained from the ECACC
Collection of Cell Culture, Salisbury, UK). Routine
has shown the HepG2 cells to be mycoplasma
alert Kit from Cambrex, Verviers, Belgium). The
grown in monolayer culture and maintained in
Dulbecco’s Modified Eagle’s Medium (DMEM)
ted with 2 mM l-glutamine, 1% non-essential
ds, and 10% FBS (v/v) in an humidified atmosphere
nd at 37
◦
C. All the experiments were carried out