Conjugation and deconjugation reactions within the fetoplacental compartment in a sheep model: A key factor determining bisphenol a fetal exposure

Article abstract of DOI:10.1124/dmd.114.061291

The widespread human exposure to bisphenol A (BPA), an endocrine disruptor targeting developmental processes, underlines the need to better understand the mechanisms of fetal exposure. Animal studies have shown that at a late stage of pregnancy BPA is efficiently conjugated by the fetoplacental unit, mainly into BPA-glucuronide (BPA-G), which remains trapped within the fetopla-cental unit. Fetal exposure to BPA-G might in turn contribute to in situ exposure to bioactive BPA, following its deconjugation into parent BPA at the level of fetal sensitive tissues. The objectives of our study were 1) to characterize the BPA glucurono- and sulfoconjugation capabilities of the ovine fetal liver at different developmental stages, 2) to compare hepatic conjugation activities in human and sheep, and 3) to evaluate the extent of BPA conjugation and deconjugation processes in placenta and fetal gonads. At an early stage of pregnancy, and despite functional sulfoconjugation activity, ovine fetuses expressed low hepatic BPA conjugation capabilities, suggesting that this stage of development represents a critical window in terms of BPA exposure. Conversely, the late ovine fetus expressed an efficient detoxification system that metabolized BPA into BPA-G. Hepatic glucuronidation activities were quantitatively similar in adult sheep and humans. In placenta, BPA conjugation and BPA-G deconjugation activities were relatively balanced, whereas BPA-G hydrolysis was systematically higher than BPA conjugation in gonads. The possible reactivation of BPA-G into BPA could contribute to an increased exposure of fetal sensitive tissues to bioactive BPA in situ.

Full text of DOI:10.1124/dmd.114.061291

1
521-009X/43/4/467–476$25.00
http://dx.doi.org/10.1124/dmd.114.061291
DRUG
M
ETABOLISM AND
DISPOSITION
Drug Metab Dispos 43:467–476, April 2015
Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics
Conjugation and Deconjugation Reactions within the Fetoplacental
Compartment in a Sheep Model: A Key Factor Determining Bisphenol
A Fetal Exposure
Tanguy Corbel, Elisabeth Perdu, Véronique Gayrard, Sylvie Puel, Marlène Z. Lacroix,
Catherine Viguié, Pierre-Louis Toutain, Daniel Zalko, and Nicole Picard-Hagen
Institut National de Recherche Agronomique, Unité Mixte de Recherche 1331, Toxalim, Research Centre in Food Toxicology,
F-31027; and Université de Toulouse, Institut National Polytechnique de Toulouse, Ecole Nationale Vétérinaire de Toulouse, Ecole
d’Ingénieur de Purpan, Université Paul Sabatier, F-31076, Toulouse, France
Received September 21, 2014; accepted January 6, 2015
ABSTRACT
The widespread human exposure to bisphenol A (BPA), an en-
conjugation and deconjugation processes in placenta and fetal
docrine disruptor targeting developmental processes, underlines gonads. At an early stage of pregnancy, and despite functional
the need to better understand the mechanisms of fetal exposure. sulfoconjugation activity, ovine fetuses expressed low hepatic BPA
Animal studies have shown that at a late stage of pregnancy BPA is
conjugation capabilities, suggesting that this stage of development
efficiently conjugated by the fetoplacental unit, mainly into BPA- represents a critical window in terms of BPA exposure. Conversely,
glucuronide (BPA-G), which remains trapped within the fetopla- the late ovine fetus expressed an efficient detoxification system
cental unit. Fetal exposure to BPA-G might in turn contribute to in
situ exposure to bioactive BPA, following its deconjugation into
that metabolized BPA into BPA-G. Hepatic glucuronidation activities
were quantitatively similar in adult sheep and humans. In placenta,
parent BPA at the level of fetal sensitive tissues. The objectives BPA conjugation and BPA-G deconjugation activities were relatively
of our study were 1) to characterize the BPA glucurono- and balanced, whereas BPA-G hydrolysis was systematically higher than
sulfoconjugation capabilities of the ovine fetal liver at different BPA conjugation in gonads. The possible reactivation of BPA-G into
developmental stages, 2) to compare hepatic conjugation activities BPA could contribute to an increased exposure of fetal sensitive
in human and sheep, and 3) to evaluate the extent of BPA tissues to bioactive BPA in situ.
Introduction
to a lesser extent into BPA-sulfate (BPA-S) by sulfotranferases (SULT).
These reactions are usually considered as mechanisms of detoxification
because these metabolites are not estrogenic (Nakagawa and Tayama,
Bisphenol A (BPA) is a xenoestrogen widely used as a monomer in
the manufacture of epoxy resins and polycarbonate plastics, which
contribute to the almost ubiquitous human exposure to BPA across
a life span (Vandenberg et al., 2010). Much of the concern regarding
BPA safety has arisen from reported adverse effects of BPA, such as
the impairment of reproductive development, as well as effects on
energy metabolism and cognitive development even at low doses when
exposure occurs during the perinatal period (Vandenberg et al., 2013).
In adult humans, BPA is almost completely eliminated in urine
2000; Matthews et al., 2001). In humans, in the fetoplacental compart-
ment, highly variable BPA and BPA-G levels have been reported in
cord blood, placenta, and amniotic fluid (Vandenberg et al., 2010;
Gerona et al., 2013). However, these biomonitoring data remained limited
and were therefore unlikely to reflect fetal exposure throughout
pregnancy. Thus, many uncertainties remain regarding the actual fetal
internal exposure to BPA in humans during this critical period of
pregnancy, which consequently prevents a sound evaluation of human
health risks, based on the effects observed in animal studies and on the
determination of BPA fetal internal exposure in animal models.
Previous BPA toxicokinetic investigations have shown the pre-
ponderance of the glucuroconjugated form of BPA in the plasma of
fetal monkeys (Patterson et al., 2013) and sheep (Corbel et al., 2013;
Viguie et al., 2013) after maternal exposure to BPA. It has been
established from both the pregnant ewe (Corbel et al., 2013) and the
isolated perfused human placenta (Corbel et al., 2014) models that
placental permeability toward BPA-G is very limited. Such observa-
tions suggest that toward the end of gestation, the BPA-G present in
fetal blood may originate from fetal phase II metabolism rather than
from a maternal supply.
(
Volkel et al., 2002) after an extensive hepatic first-pass conjugation into
BPA-glucuronide (BPA-G) by UDP-glucuronosyltransferases (UGT) and
This work was supported by the French Région Midi-Pyrénées [APRTCN
1
00551322] and by the French National Research Agency [ANR-13-CESA0007-1].
Part of this work was presented as follows: Corbel T, Perdu E, Lacroix MZ, Puel S,
Gayrard V, Viguié C, Toutain PL, Zalko D, and Picard-Hagen N (2012) The free/
conjugated bisphenol A ratio in fetoplacental compartment: a key parameter
determining bisphenol A prenatal exposure. Connaissances récentes sur les effets
des perturbateurs endocriniens sur l’environnement et la santé; 2012 Dec 10–11;
Paris, France. Ministère de l’Écologie et du Développement Durable et de l’Énergie/
National Endocrine Disrupter Research Programme (PNRPE), Paris, France.
dx.doi.org/10.1124/dmd.114.061291.
ABBREVIATIONS: BPA, bisphenol A; BPA-G, bisphenol A-glucuronide; BPA-S, bisphenol A-sulfate; CL, clearance; DMEM, Dulbecco’s modified Eagle’s
medium; HPLC, high-performance liquid chromatography; LOQ, limit of quantification; MS, mass spectrometry; PAPS, 39-phosphoadenosine
5
9-phosphosulfate; SULT, sulfotransferase; UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase.
467

4
68
Corbel et al.
It is noteworthy that the results obtained in near-term fetuses are not fractions were purchased from TEBU (pools of 10 female donors), Life
Technologies and Sigma-Aldrich (pools of 10 mixed gender donors).
necessarily representative of BPA fetal exposure at earlier stages of
pregnancy, including during the most critical period of development,
which are characterized by a weak expression of drug-metabolizing
systems (Domoradzki et al., 2004). Moreover, conjugated metabolites
can be regenerated back to the corresponding parent compounds by
b-glucuronidases and sulfatases, which are expressed in many tissues
In Vitro Incubations. The protein content of the subcellular fractions was
determined with bovine serum albumin as a standard (Bradford, 1976). The
linearity of the UGT reaction with time was investigated using incubation times
of 10, 20, and 30 minutes, with a liver microsomal protein concentration of
1
mg/ml; the linearity of the SULT reaction with time was investigated using
incubation times of 10, 20, 30, and 60 minutes, with a liver cytosolic protein
concentration of 0.4 mg/ml. All assays were optimized with respect to substrate
concentration range. All preincubations and incubations were performed in
(
Warren and French, 1965; Sperker et al., 1997). Accordingly, it has
been shown that 4.4% of the BPA-G dose passed through the placental
membrane is converted back to parent BPA in the rat fetal compartment glass vials containing 0.5 M Tris-Maleate buffer (pH 7.4) and 10 mM MgCl at
2
(Nishikawa et al., 2010). Thus, high levels of BPA metabolites in the 37ꢀC under shaking. BPA (purity $99%) and BPA-G and BPA-S (purity $95%)
fetal unit raise the question of the deleterious consequences of in situ stock solutions were dissolved in ethanol and were diluted extemporaneously in
the incubation buffer. The final concentration of ethanol in the incubation media
was 0.5% for BPA, 0.8% for BPA-G, and 0.6% for BPA-S.
Glucuronidation Assays. In vitro glucuronidation of BPA was evaluated by
incubating 0.5 mg of hepatic microsomal proteins for 20 minutes with BPA at
reactivation of BPA metabolites through deconjugation at the level of
fetal tissues. Together, these data highlight the urgent need for models
relevant to the human situation for investigating of the dynamics of the
balance between BPA conjugation and back deconjugation throughout
different concentrations (2.5, 5, 10, 50, 100, and 200 mM), in a volume of
the whole gestation period.
0
.5 ml buffer containing 1 mM UDP-glucuronic acid (UDPGA). The reaction
In this context, the present study 1) characterizes in vitro fetal hepatic
BPA conjugation activities at different pregnancy stages in sheep, 2)
compares in vitro hepatic conjugation activities between adult humans
and sheep to ascertain the validity of the sheep model, and 3) evaluates
was stopped by adding 1 ml of ice-cold acetonitrile and the samples were stored
at 220ꢀC until assayed. Blank samples without UDPGA or without microsomal
proteins were used as controls.
Sulfoconjugation Assays. In vitro sulfoconjugation activity was evaluated
ex vivo the BPA conjugation and back deconjugation in the placenta using cytosolic fractions. For each assay, 0.2 mg of cytosolic protein was
and fetal gonads, which are target organs susceptible to estrogenic preincubated for 30 minutes with 5 mM ATP and 2 mM Na SO in a volume
of 0.5 ml of incubation buffer. The assay conditions for the generation of
9-phosphoadenosine 59-phosphosulfate (PAPS) in vitro by preincubation of
2
4
interferences, at different developmental stages.
3
cytosolic proteins at 37ꢀC for 30 minutes with inorganic sulfate (2 mM) and
ATP (5 mM) in presence of Mg (10 mM) were adapted from the method
Materials and Methods
2+
Animals and Tissue Collection
optimized by Nemoto et al. (1978), Wong et al. (1991), and Wong and Wong
(
1996). In a preliminary experiment, we showed that the rate of BPA-S
All animal procedures were performed in accordance with accepted standards
of humane animal care under agreement number 31-1155545 from the French
Ministry of Agriculture and were approved by the Midi-Pyrénées ethics committee
formation was similar in presence of a PAPS generation system or of 240 mM
PAPS, indicating that this preincubation would generate a sufficient amount of
PAPS to optimize BPA sulfoconjugation. BPA at different concentrations (2.5,
(ref: MP/01/15/03/12).
5
, 10, 50, 100, 200, 500, and 1000 mM) was then added to the incubation
medium. The reaction was stopped after 20 minutes of incubation by adding
ml of ice-cold acetonitrile and the samples were stored at 220ꢀC until assayed.
Three nonpregnant and 12 pregnant Lacaune ewes (54–57 days of gestation,
corresponding to early-stage fetus, n = 8; and 120–135 days of gestation
corresponding to near-term fetus, n = 4) were used. Ewes were sacrificed by
electrical stunning immediately followed by exsanguination. Fetuses were rapidly
extracted from the uterus (six males and eight females, and three males and three
females, for the stages of 54–57 days and 120–135 days of pregnancy, respectively).
Fetal gonads and placental cotyledons were immediately collected and chilled
in saline solution at 4ꢀC and were incubated within 1 hour after collection.
Ovaries were also collected from the three nonpregnant ewes and from three
adult ewes at 120–135 days of pregnancy.
For early-stage fetuses, fetal livers were collected, immediately frozen in
liquid nitrogen, and stored at 280ꢀC. The livers from six near-term fetuses and
from three nonpregnant ewes were immediately perfused for 5 minutes after
their collection using a saline solution at 4ꢀC. A fragment of about 10 g of the
right lobe was frozen in liquid nitrogen and stored at 280ꢀC.
1
Blank samples without ATP and Na
used as controls.
Deconjugation Assays. Hepatic deconjugation activity was evaluated on
ovine microsomal liver fractions from one pool of early-stage fetuses, one near-
term fetus, and one adult ewe. We incubated 0.5 mg of microsomal protein for
2 4
SO or without cytosolic proteins were
2
0 minutes with BPA-G or BPA-S at three different concentrations (2.5, 50,
and 100 mM) without cofactors under the same experimental conditions as
those of the conjugation assay. The reaction was stopped by adding 1 ml of ice-
cold acetonitrile and the samples were stored at 220ꢀC until assayed.
Analytic Procedure. BPA and its metabolites concentrations were determined
after extraction with acetonitrile using a validated ultra-performance liquid
chromatography with tandem mass spectrometry (UPLC-MS/MS) system,
adapted for incubation media, from a method previously described elsewhere
(
2
Lacroix et al., 2011).The calibration curves ranged from 4.4 nM to 219 mM,
4.8 nM to 248 mM, and 32.5 nM to 325 mM, for BPA, BPA-G, and BPA-S,
Characterization of BPA Conjugation/Deconjugation Activities in Hepatic
Subcellular Fractions
respectively. The mean intraday and interday coefficients of variation for three
concentrations of BPA, BPA-G, and BPA-S were lower than 15%, and the limits
of quantification (LOQ) were validated at 4.4 nM, 24.8 nM, and 32.5 nM,
respectively.
Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich
(Saint-Quentin Fallavier, France).
Preparation of Hepatic Subcellular Fractions. Because of the small size
of the organs (around 2g; Bazer et al., 2012), the livers from six male and six
female early-stage fetuses were pooled as follows: three pools of two males and
three pools of two females. Livers from the six near-term fetuses (three males,
three females) and from the three nonpregnant ewes were processed individually.
Hepatic subcellular fractions were prepared by differential centrifugation to
obtain microsomal and cytosolic fractions as previously described elsewhere
Determination of Michaelis-Menten Parameters. All data were computed
using WinNonlin software (version 5.3; Pharsight Corporation, Mountain View,
CA) and the Michaelis-Menten parameters Vmax and K
m
were determined by
nonlinear regression analysis using the following model:
Vmax ꢀ ½Sꢁ
V ¼
ð1Þ
(Zalko et al., 2006) within 1 month after collection. All fractions were then stored
Km þ ½Sꢁ
in sodium phosphate buffer (0.1 M), pH 7.4, containing 20% glycerol at 280ꢀC
until the assays.
Human microsomal fractions were obtained from pools of 10 female donors
and purchased from TEBU (Le Perray-en-Yvelines, France), BD Gentest
where Vmax was the maximum reaction velocity expressed in pmol per mg of
microsomal or cytosolic protein and per min, K was the Michaelis constant
which corresponds to the BPA micromolar concentration at half Vmax, and [S]
m
(Erembodegem, Belgium) and Life Technologies (Saint-Aubin, France). Cytosolic was the micromolar substrate concentration.

Fetoplacental BPA Conjugation/Deconjugation Reactions
469
For the incubations of hepatic subcellular pool fractions of early-stage (Csanady et al., 2002), respectively], and B was the ratio between the liver
fetuses, several concentrations of BPA-G were below the LOQ. Consequently, weight and the body weight [12 g/kg in sheep (Boxenbaum, 1980) and 26 g/kg
the sparse data option of WinNonlin was used for each sex group, allowing a
computation of the different standard errors (S.E.) associated with the estimated
parameters.
in humans (Arms and Travis, 1988)].
BPA Conjugation/Deconjugation Reactions in Target Tissues
Hepatic Intrinsic Clearance. From the Michaelis-Menten parameters de-
termined both for the glucurono- and sulfoconjugation reactions, the hepatic
intrinsic clearance (CLint) was calculated with the following equation, expressed
in ml/min per g of liver:
3
3
Biochemical Synthesis of [ H]-BPA-Glucuronide and [ H]-BPA-Sulfate.
Tritiated metabolites of BPA, not available commercially, were synthesized
from [ H]-BPA (radiopurity: .97%, specific activity: 185 GBq/mmol; Moravek
Biochemicals, Brea, CA) as previously described by Bursztyka et al. (2008).
[ H]-BPA-glucuronide ([ H]-BPA-G) was produced by incubating 2 mg of
guinea pig liver microsomal protein with [ H]-BPA (1133 kBq) fortified with
0 nmol BPA and 2 mM UDPGA in 1 ml 0.5 M Tris buffer (pH 7.4) containing
3
3
3
Vmax
CLint ¼
ꢀ ꢀ A
ð2Þ
3
Km
4
where A was the amount of protein (microsomal or cytosolic for glucuronidation 10 mM MgCl and 0.1% Triton X-100 for 2 hours at 37ꢀC. After the addition
2
and sulfoconjugation, respectively) per gram of liver. In sheep, the amount of of 3 volumes of methanol and a 10-minute centrifugation at 8000g, the superna-
3
protein per gram of liver was determined by the Bradford method (Bradford, tant was collected, and [ H]-BPA-G synthesis was checked using the radio-high-
3
1
976). For human subcellular fractions, the values of the amounts of microsomal pressure liquid chromatography (HPLC) system described later. [ H]-BPA-G was
protein and cytosolic protein per gram of liver were 45 mg/g (Riley et al., 2005) collected using the same HPLC system coupled to a Gilson model 201/202 fraction
3
and 80.7 mg/g (Cubitt et al., 2011), respectively.
collector. [ H]-BPA-G was purified using a 0.5 g Chromabond C18 glass cartridge
Macherey Nagel, Hoerdt, France) as described previously elsewhere (Cabaton et al.,
2
Correction for Nonspecific Protein Binding. Glucuronidation and sulfo-
conjugation BPA intrinsic clearances were corrected for nonspecific protein
binding in the microsomal or cytosolic incubation medium (fuinc) using the
following equation:
(
3
008). The [ H]-BPA-G structure, with a specific activity of 28.3 GBq/mmol, was
confirmed by mass spectrometry.
H]-BPA-sulfate ([ H]-BPA-S) was produced by incubating 2 mg of
guinea pig liver cytosolic protein with 40 nmol [ H]-BPA (1133 kBq) and
3
3
[
3
Clint
CLint;u ¼
fuinc
ð3Þ 2 mM PAPS in a final volume of 1 ml 0.5 M Tris buffer (pH 7.4) containing
1
2
0 mM MgCl for 2 hours at 37ꢀC. After protein precipitation using 3 volumes
3
of methanol and centrifugation, the supernatant was evaporated. [ H]-BPA-S
where CLint,u was the unbound intrinsic clearance and fuinc the percentage of
BPA unbound from protein in the incubation.
The free fraction of BPA in the microsomal incubation medium (fuinc) was
estimated with the following equation (Austin et al., 2002):
3
production was checked by radio-HPLC. [ H]-BPA-S was collected and purified
using a 0.5 g Chromabond C18 glass cartridge (Macherey Nagel) as described
elsewhere (Cabaton et al., 2008). The [ H]-BPA-S structure, with a specific
activity of 28.3 GBq/mmol, was confirmed by mass spectrometry.
Ex Vivo Incubations of [ H]-BPA, [ H]-BPA-G, and [ H]-BPA-S with
Target Organs from Adult Sheep and Fetuses. Approximately 5 mg of fetal
ovaries or fetal testes at an early stage of pregnancy (54–57 days, n = 3) and
approximately 50 mg of fetal testes and fetal ovaries from 120- to 135-day-old
fetuses (n = 3), placental cotyledons, and maternal ovaries (n = 3) were
incubated in 12-well plastic culture plates containing 100 pmol [ H]-BPA
2.83 kBq) in 1 ml Dulbecco’s modified Eagle’s medium (DMEM), or 100 pmol
H]-BPA-G (2.83 kBq) in 1 ml sodium chloride 0.9%, or in 1 ml sodium acetate
buffer 0.2 M, pH 4.5, or 100 pmol [ H]-BPA-S (2.83 kBq) in 1 ml sodium
chloride 0.9% .
3
3
3
3
1
fuinc ¼
ð4Þ
C ꢀ 100:56ꢀlogꢀ P=D 21:41ꢀ þ 1
where C was the microsomal protein concentration in the incubation medium
mg/ml) and logP/D, corresponding to logKow for a basic compound, was 3.4
(
for BPA (Austin et al., 2002; Mazur et al., 2010).
To verify the predicted fuinc determined by eq. 4 in our experimental conditions,
fuinc was determined experimentally in duplicate by equilibrium dialysis over
3
(
[
3
20 minutes at 37ꢀC, with a cellulose membrane with a molecular mass cutoff of
3
5
kDa, as described previously elsewhere (Corbel et al., 2013). The binding of BPA
to microsomal proteins was determined at protein concentrations of 1 mg/ml for
only one adult sheep and for three BPA concentrations (1, 10, and 100 mM).
Because no equation was described to estimate the free fraction of BPA in
the cytosolic incubation medium, the binding of BPA to cytosolic proteins was
determined for hepatic cytosolic fractions at a protein concentration of 0.4 mg/ml
from one pool of early-stage fetuses, one near-term fetus, one adult sheep, and
one human pool and for two concentrations of BPA (10 and 200 mM).
BPA concentrations in the incubation medium and dialyzed subcellular
fraction sample compartments were measured by ultra-performance liquid
chromatography with tandem mass spectrometry (UPLC-MS/MS), as described
earlier. The free fraction of BPA (fuinc) was determined as the ratio of the
BPA concentration measured in the incubation medium compartment to that
in the dialyzed compartment containing the microsomal or the cytosolic
fractions.
In Vitro–In Vivo Extrapolation for Hepatic Clearance. To understand
better the species- and development stage-dependent variation in BPA hepatic
pharmacokinetics, the unbound intrinsic clearance (CLint,u) was incorporated
into the well-stirred model (Ito and Houston, 2004) to calculate the hepatic
H
clearance (CL ) for ovine near-term fetuses and for adult sheep and humans, as
shown in the following equation derived from Miners et al. (2006):
For the three molecules, control incubations were performed in the same
conditions with 100 pmol [ H]-BPA, [ H]-BPA-G, or [ H]-BPA-S (2.83 kBq)
without tissues. All incubations were maintained for 8 hours at 37ꢀC under 90 rpm
2 2
shaking and an O /CO 95%/5% atmosphere. At the end of the incubations, media
and tissues were collected separately in glass vials. Tissues were extracted in 1 ml of
ethanol. All samples were kept at 220ꢀC until analysis.
Analytic Procedure. Radioactivity recovery and BPA and metabolite
quantifications were performed with the analytic procedures previously described
by Bursztyka et al. (2008). The radioactivity in each incubation medium and
tissue was measured from aliquots (10 ml) by liquid scintillation counting in
a Packard Tricarb 4430 counter with Ultima Gold (Packard Instruments, Meriden,
CT) as the scintillation cocktail. Aliquots of each incubation medium were
analyzed directly by radio-HPLC. Ethanolic extracts from tissues were evaporated
before radio-HPLC analysis.
3
3
3
The HPLC system used to analyze BPA metabolites consisted of a Spectra
Physics system (P1000) coupled to Flo-One A500 detector, with Flo-scint II as
scintillation cocktail (Packard Instruments) for online radioactivity detection.
The column was a Zorbax C18 column (250 ꢀ 4.6 mm, 5 mm) (Interchim,
Montluçon, France) coupled to a Kromasil C18 guard column (18 ꢀ 4.6 mm,
5
mm). The mobile phases consisted of ammonium acetate buffer (20 mM,
pH 3.5) and acetonitrile 95:5 v/v and 10:90 v/v in A and B, respectively. The
flow rate and temperature were set at 1 ml/min and 35ꢀC. The gradient used
was 0–4 minutes 100% A; 4–6 minutes of linear gradient from 100% A to A:B
85:15 v/v; 6–16 minutes A:B 85:15 v/v; 16–18 minutes linear gradient from
QH ꢀ fu ꢀ ðB ꢀ CLint;uÞ
CLH ¼
ð5Þ
QH þ fu ꢀ ðB ꢀ CLint;uÞ
H
where Q was the liver blood flow [75, 50, and 23 ml/kg per min in near-term
fetuses (Rudolph, 1985), adult ewes (Boxenbaum, 1980), and humans (Arms 15% B to 25% B; 18–28 minutes A:B 75:25 v/v; 28–30 minutes linear gradient
and Travis, 1988), respectively], fu was the free fraction of BPA in blood [11%,
%, and 5% in near-term fetuses, adult ewes (Corbel et al., 2013) and humans
from 25% B to 30% B; 30–37 minutes A:B 70:30 v/v; 37–39 minutes linear
gradient from 30% B to 70% B; 39–45 minutes A:B 30:70 v/v.
7

4
70
Corbel et al.
BPA and metabolites were identified by comparison of radio-HPLC retention to 1 (mean 0.9760.10; range: 0.84–1.07) under all conditions. Thus,
times with those of authentic standards before and after enzymatic hydrolysis.
Metabolites were quantified by integrating the area under the detected radioactive
peaks.
the nonspecific protein binding correction in the cytosol was disregarded
to calculate CLint,u
.
Glucuronidation of BPA. The formation of BPA-G by hepatic
microsomal fractions of human, adult ewes, and early- and late-ovine
fetuses, expressed as a function of BPA concentration, followed
Michaelis-Menten kinetics (Fig. 1). BPA-G formation showed enzyme
saturation for BPA concentrations around 10 mM and 50 mM, in ovine
fetuses at early and late stages of pregnancy, respectively, and around
Statistical Analysis
Data were expressed as mean 6 S.D., unless otherwise specified. Statistical
analyses were performed using Systat 12.0 software (SPSS/IBM, Chicago, IL).
For the early-stage fetuses (only for sulfoconjugation) and the 120- to 135-day-
old fetuses, the formation rate of BPA metabolites by subcellular hepatic
fractions was analyzed by a two-way analysis of variance (ANOVA) with
substrate concentration, sex, and their interaction as the fixed-effect factors;
individuals were nested within the sex group as a random-effect factor. As no
gender effect was observed, male and female fetuses were grouped together.
1
00 mM in adult ewes and humans (Fig. 1). In near-term ovine fetuses,
the kinetics of BPA-G formation did not differ according to gender
(ANOVA, N.S.).
Table 1 presents the mean 6 S.D. values for the Michaelis-Menten
m
The effect of the developmental stage (early-stage versus near-term fetuses only parameters (Vmax and K ) and the hepatic unbound intrinsic clearance
for sulfoconjugation and near-term fetus versus adult ewes on both glucurono- (CLint,u) of BPA glucuronidation in humans, in ovine adults and in
and sulfoconjugation) and the effect of species (human versus ovine) on the early- and late-stage ovine fetuses. The mean Vmax of glucuronidation
m
liver metabolic parameters (Vmax, K , and intrinsic clearance) were evaluated
by Student’s t test or by Aspin-Welch test in the case of unequal variance. For
each analysis, P values were corrected by the number of comparisons.
increased throughout the development: it was 512-fold lower in early-
stage fetuses and 13-fold lower in near-term fetuses (P = 0.012) than
in adult sheep. The mean Vmax of BPA glucuronidation was significantly
higher for incubations performed with adult ovine hepatic microsomal
fractions, compared with human hepatic microsomes (6086 6 862 versus
Results
Characterization of BPA Conjugation/Deconjugation Activities in
Hepatic Microsomes
2770 6 250 pmol/min per mg, P = 0.006).
The Michaelis-Menten constant (K ) of BPA glucuronidation in-
m
The mean (6 S.D.) amounts of microsomal proteins were 5.15 6 creased with the developmental stage in sheep: it was about 20-fold and
.24 and 6.80 6 1.95 mg/g in early-stage and near-term fetal livers, 10-fold lower (P = 0.03) for early (4.06 mM) and late (9.59 mM) stages of
1
respectively, and were about 2-fold higher in adult livers (12.65 6 pregnancy, respectively, compared with adults (82.0 mM). In adults, the
.12 mg/g, t test P , 0.05). The mean (6 S.D.) amounts of cytosolic
for glucuronidation was not significantly different between ewes and
proteins were 10.78 6 1.49, 12.45 6 4.37, and 16.28 6 1.65 mg/g in humans (59.6 mM, N.S.).
2
K
m
early-stage, near-term fetal livers and in adult livers, respectively. The
In sheep, the hepatic unbound intrinsic clearance of BPA glucuroni-
average body weight of the near-term fetuses was 2.93 6 0.86 kg, and dation was about 65-fold and 2-fold lower for early (0.060 ml/min per g
their liver weight was 89.2 6 23.8 g.
of liver) and late (1.73 ml/min per g of liver, P = 0.04) stages of
Correction for Nonspecific Protein Binding. The experimental pregnancy, respectively, compared with adults (3.93 ml/min per g of
BPA free fraction (fuinc) in the microsomal incubation medium under liver). The human unbound intrinsic clearance (9.00 ml·min per g of liver)
all conditions was similar to the predicted value (eq. 4) (mean 6 S.D.: was in the same range as observed for adult sheep, although it tended to
0
.26 6 0.04 versus 0.24). The experimental fuinc in cytosol was close be higher (P = 0.06).
Fig. 1. Kinetics of bisphenol A glucuronidation by liver microsomes. BPA-G formation rate (pmol/mg microsomal protein per min) as a function of BPA concentrations by
liver microsomes from ovine fetus at early and late stages of pregnancy and from adult ewes and humans, incubated for 20 minutes. Symbols indicate mean (6 S.D.) values
of three determinations, and lines indicate fitted values of BPA-G formation.

Fetoplacental BPA Conjugation/Deconjugation Reactions
471
TABLE 1
Bisphenol A glucuronidation by hepatic microsomes
Michaelis-Menten apparent kinetic constants (Vmax and K
m
) for BPA glucuronidation by hepatic microsomal fractions of early-stage
(54- to 57-day-old) and near-term (120- to 135-day-old) ovine fetuses and of adult sheep and humans after incubation with BPA for
20 minutes. Hepatic glucuronidation unbound intrinsic clearance (CLint,u) and extrapolated hepatic clearance (CL
H
) (mean 6 S.D.).
Subjects
V
max
K
m
CLint,u
CL
H
pmol/mg microsomal protein per min
mM
ml/min per g of liver
ml/kg per min
Ovine fetusesa
Early stage
b
Female (n = 3)
Male (n = 3)
15.3 (S.E. 2.26)
8.50 (S.E. 1.70)
4.71 (S.E. 2.95)
3.41 (S.E. 2.93)
0.075
0.044
NA
NA
b
Near-term
Female (n = 3)
Male (n = 3)
519 6 258
446 6 316
10.8 6 5.94
8.38 6 7.05
1.53 6 1.16
1.93 6 1.21
4.76 6 3.68
6.40 6 4.28
Adult
Ewes (n = 3)
Humans (n = 3)
6086 6 862
2770 6 250
82.0 6 18.1
59.6 6 15.6
3.93 6 0.85
9.00 6 2.47
3.14 6 0.43
7.67 6 1.35
NA, not applicable.
a
At 54–57 days of pregnancy, several concentrations of BPA-G were below the LOQ (25 nM), so the data were computed together for
each sex group to estimate Michaelis-Menten parameters and the associated standard errors (S.E.).
b
Three pools of two fetuses.
Sulfoconjugation of BPA. The formation of BPA-S by hepatic unbound intrinsic clearance of BPA sulfoconjugation. The overall
cytosolic fractions of humans adult ewes and early- and late-ovine fetuses, mean Vmax of hepatic sulfoconjugation for early-stage and late-stage
expressed as a function of BPA concentration, followed Michaelis- fetuses, respectively, was similar (78.3 6 1.32 versus 104 6 16.2 pmol/min
Menten kinetics (Fig. 2). BPA-S formation showed enzyme saturation for per mg of cytosolic proteins, N.S.) but was lower than that calculated
a BPA concentration around 50 mM for ovine fetuses, regardless of the for ewes (655 6 309 pmol/min per mg of cytosolic proteins). Com-
stage of pregnancy, and for 100 and 500 mM, for humans and adult ewes, pared with adult sheep, the mean human sulfoconjugation Vmax (7.03 6
respectively (the latter concentration is not shown in Fig. 2). In ovine, 4.37 pmol/min per mg of cytosolic proteins) was very low.
hepatic sulfoconjugation plots clearly suggest similar sulfoconjugation
m
The overall mean K for sulfoconjugation was similar between early-
activities, whatever the stage of pregnancy. By contrast, the sulfoconju- and late-stage fetuses (15.67 6 8.11 versus 7.51 6 1.48 mM, N.S.) and
gation rate calculated for the 3 human cytosolic fraction pools showed was 37- and 77-fold lower than in the adult sheep (575 6 267 mM),
considerable variation, and was lower than observed for adult sheep. For respectively. In the adult, the K
the early- and late-stage ovine fetuses, the kinetics of BPA-S formation when using human cytosols (40.2 6 6.16 mM) than when using adult
for sulfoconjugation was 14-fold lower
m
did not differ according to the sex (ANOVA, N.S.).
sheep cytosols.
Table 2 gives the mean (6 S.D.) values of Michaelis-Menten
In sheep, the hepatic unbound intrinsic clearance of BPA sulfoconju-
m
parameters (Vmax and K ) for BPA-S formation and for the hepatic gation was 3-fold lower for early-stage fetuses than it was for near-term
Fig. 2. Kinetics of bisphenol A sulfoconjugation by liver cytosols. BPA-S formation rate (pmol/mg cytosolic protein per min) as a function of BPA concentrations using
liver cytosol from ovine fetuses at early and late stages of pregnancy and from adult ewes and humans, incubated for 20 minutes. Symbols indicate mean (6 S.D.) values of
three determinations, and lines indicate fitted values of BPA-S formation.

4
72
Corbel et al.
TABLE 2
Bisphenol A sulfoconjugation by hepatic cytosols
Michaelis-Menten apparent kinetic constants (Vmax and K
m
) for BPA sulfoconjugation by hepatic cytosolic fractions of 54- to 57-day-
old (early-stage) and near-term ovine fetuses and of adult sheep and humans. Hepatic sulfoconjugation unbound intrinsic clearance (CLint,u
)
H
and extrapolated hepatic clearance (CL ) (mean values 6 S.D.)
Subjects
V
max
K
m
CLint,u
CL
H
pmol/mg cytolic protein per min
mM
ml/min per g of liver
ml/kg per min
Ovine fetuses
Early stage
a
Female (n = 3)
Male (n = 3)
79.2 6 5.74
77.3 6 18.3
12.3 6 0.84
19.0 6 11.4
0.279 6 0.020
0.203 6 0.076
NA
NA
a
Near term
Female (n = 3)
Male (n = 3)
116 6 27
93.0 6 24.0
7.96 6 0.44
7.05 6 2.16
0.660 6 0.370
0.716 6 0.070
2.18 6 1.36
2.50 6 0.64
Adults
Ewes (n = 3)
Humans (n = 3)
655 6 309
7.03 6 4.37
575 6 267
40.2 6 6.16
0.081 6 0.034
0.015 6 0.012
0.070 6 0.034
0.020 6 0.015
NA, not applicable.
a
Three pools of two fetuses.
fetuses (0.241 6 0.065 versus 0.688 6 0.240 ml/min per g of liver, P = BPA Conjugation/Deconjugation Reactions in Target Tissues
0
.015). The adult ewes hepatic unbound intrinsic clearance of BPA
Overall radioactivity recovery for incubation media and tissues
fetal gonads, adult ovaries, and placenta), after 8 hours of incubation
sulfoconjugation was 8-fold lower than that of fetuses at a late stage
of pregnancy (P = 0.007) (Table 2). The hepatic unbound intrinsic
clearance of sulfoconjugation was in the same range in humans and in
ewes (0.015 6 0.012 versus 0.081 6 0.034 ml/min per g of liver, N.S.).
In sheep, the ratio between the unbound intrinsic clearance of BPA
glucuronidation and BPA sulfoconjugation increased with the stage of
pregnancy from 0.24 (early stage) to 2.5 (late stage), and reached 48 in
adult ewes. In adult humans, BPA hepatic glucuronidation was highly
predominant, with a ratio of about 600.
Prediction of In Vivo Hepatic Clearance from the In Vitro Data
on BPA-G and BPA-S Formation by Hepatic Subcellular Fractions.
The glucuronidation hepatic clearance CLH (Table 1) was of the same
order of magnitude in ovine near-term fetuses and in adult sheep (5.58 6
(
3
3
3
with 100 pmol [ H]-BPA, [ H]-BPA-G, or [ H]-BPA-S (2.83 kBq) was
05 6 15%. The fraction of the radioactivity recovered from incubation
1
3
media was 65% 6 18% for [ H]-BPA (DMEM), 57% 6 23% for
3
3
[
H]-BPA-G (sodium acetate buffer), 80% 6 15% for [ H]-BPA-G (saline
3
solution) and 74% 6 16% for [ H]-BPA-S (saline solution). The respective
percentages of radioactivity recovered for the same assays, from tissue
extracts, were 57% 6 12%, 45% 6 21%, 20% 6 13%, and 21% 6 8%.
Representative radio-HPLC profiles for these assays are displayed
3
in Fig. 3 for 100 pmol [ H]-BPA incubated in 1 ml of DMEM, and
3
1
00 pmol [ H]-BPA-G incubated in saline solution without tissue
(control) or incubated with placenta obtained at 54–57 days of pregnancy.
Blank incubations of radiolabeled substrates without tissue did not reveal
any degradation of BPA or of BPA-G. Under our HPLC conditions, the
3.68 versus 3.14 6 0.43 ml/kg per min, N.S.). However, it was 2-fold
3
3
3
higher in humans compared with adult sheep (7.67 6 1.35 versus 3.14 6
retention times of [ H]-BPA, [ H]-BPA-G, and [ H]-BPA-S were 43.8,
9.0, and 42.5 minutes, respectively. After incubation of BPA with
0.43 ml/kg per min; P = 0.01).
2
The sulfoconjugation hepatic clearance (Table 2) was very low in
placenta, the only metabolite produced was BPA-S. Incubation of
BPA-G with placenta tissue in saline solution showed a hydrolysis of
BPA-G into BPA. Integrating the area under the radioactive peaks
permitted quantification of BPA and metabolites in pmoles and comparison
of BPA conjugation with BPA-G hydrolysis.
adult sheep and humans (0.070 6 0.034 and 0.020 6 0.015 ml/kg per
min) and accounted for only 2.2% and 0.26% of the overall conjugation
hepatic clearance, respectively. The near-term fetal sulfoconjugation
hepatic clearance was 33-fold higher than that in adult sheep (2.34 6
0
.97 ml/kg per min; P = 0.001) and was as high as 30% of the
Figure 4 shows for each tissue (fetal and adult gonads, placenta) the
3
3
conjugation hepatic clearance.
amount of [ H]-BPA that was conjugated and the amount of [ H]-BPA-G
that was hydrolyzed after 8 hours of incubation of 100 pmol of
radiolabeled BPA (in DMEM) or 100 pmol of radiolabeled BPA-G (in
Deconjugation of BPA Metabolites. The deconjugation assays
(
glucuronidase and sulfatase activities) performed with ovine hepatic
microsomal fractions, under the same conditions of incubation as the saline solution). In placenta (Fig. 4A), at early as well as at late stages of
conjugation assays but without cofactors, showed a very low percentage pregnancy, BPA conjugation (range: 4.89–29.64 pmol/8 h and 6.67–
of hydrolysis of BPA-G and BPA-S into BPA: below 1.5% and 2.5% 47.7 pmol/8 h, respectively) was highly variable and was mainly associated
for the sulfatase and the glucuronidase activities, respectively, whatever with the production of BPA-S.
the concentration of the substrate and the stage of development.
In incubations performed with adult ovaries, the BPA conjugation
Because glucuronidation of BPA and hydrolysis of BPA-G were rates (median and range) were 21.8 pmol/8 h (11.6–35.3) for ovaries
measured under the same incubation conditions, resulting in the maximal obtained from pregnant ewes and 18.2 pmol/8 h (1.08–24.9) for ovaries
glucuronidation and hydrolysis rates, the fraction of BPA-G formed in of nonpregnant ewes (Fig. 4B).
presence of 100 mM of BPA (with UDPGA) and the fraction of BPA-G
100 mM) hydrolyzed were compared. In early-stage fetuses, the per- undetectable, whether obtained from early- or late-stage pregnancy
centage of BPA-G hydrolyzed (0.72%) was very low, even if it was fetuses, and ranged from 0 to 2.29 pmol/8 h (median: 0 pmol/8 h) in
-fold higher than the rate of BPA-G formed (0.24%). In ovine near-term fetal ovaries (Fig. 4C) and from 0 to 8.48 pmol/8 h (median:
fetuses and in adult sheep, the rate of hydrolysis of BPA-G was low, even 4.15 pmol/8 h) in fetal testes (Fig. 4D).
negligible, compared with the percentage of glucuronidation (2.38% versus
In optimal conditions for glucuronidase activity (incubation in
2.7% in near-term fetuses and 1.15% versus 72.8% in adult sheep). sodium acetate buffer, pH 4.5), the overall mean hydrolysis percentage
In fetal gonads, the conjugation of BPA was either very low or
(
3
1

Fetoplacental BPA Conjugation/Deconjugation Reactions
473
Fig. 3. Representative radiochromatograms show-
ing BPA conjugation and BPA-G deconjugation
by placenta tissue. Representative radiochromato-
graphic profiles obtained after incubation of
3
1
00 pmol [ H]-BPA for 8 hours in DMEM
medium without tissue (A) or with early-stage
pregnancy placenta (B) and from incubation of
3
1
00 pmol [ H]-BPA-G for 8 hours in saline
solution without tissue (C) or with early-stage
pregnancy placenta (D).
of radiolabeled BPA-G was high, 91% 6 15%, whatever the tissue
and the stage of development, indicating the presence and function- showed that the ovine fetus at the first third of pregnancy, expressed
ality of this enzyme in our ex vivo conditions. limited activities of BPA glucuronidation, with an intrinsic clearance
Hepatic BPA Conjugation/Deconjugation Activities. Our study
In saline solution, for fetal gonads, BPA-G hydrolysis rates were about 30-fold lower than in the last third of pregnancy. By contrast,
relatively high both for early- and late-stage pregnancy [median the capability of the liver to glucurono-conjugate BPA in near-term
(
range): 6.99 pmol/8 h (4.13–15.9) for fetal ovaries (Fig. 4C) and 27.5 lambs was nearly as high as in adults. These results are consistent with
pmol/8 h (16.8–59.3) for fetal testes (Fig. 4D)]. For adult ovaries (Fig. the development of the expression of isoforms of UGT in the ovine
B), BPA-G hydrolysis rates were similar between pregnant and near-term fetus and newborn (Dvorchik et al., 1986; Pretheeban et al.,
nonpregnant ewes [median (range): 49.5 pmol/8 h (49.1–58.4) versus 2011). The two parameters Vmax and K increased throughout pregnancy
4
m
4
3.1 pmol/8 h (26.0–54.8)]. In our experimental conditions, for fetal and in adulthood, indicating both an increase in UGT enzyme expression
gonads (at all stages of pregnancy) and adult ovaries, the BPA-G and a decrease in their affinity, depending on the UGT isoforms. How-
hydrolysis rate was systematically higher than the BPA conjugation ever, as the in vitro BPA glucuronidation was evaluated under native
rate. For placenta, the BPA-G hydrolysis rate was lower at an early incubation conditions (without activation), we cannot rule out the
stage than at a late stage of pregnancy [median (range): 3.42 pmol/8 h hypothesis that the UDPGA cofactor transport could be a rate-limiting
(
3.13–10.1) and 24.5 pmol/8 h (18.3–28.6), respectively] (Fig. 4A).
step for BPA glucuronidation in ovine fetuses.
In human liver microsomes, BPA is mainly glucuronidated by the
3
[
H]-BPA-S hydrolysis rates (data not shown) were relatively low
(
median: 1.44 pmol/8 h) and ranged from 0 to 5.20 pmol/8 h for the UGT2B15 and 2B7 isoforms (Hanioka et al., 2008; Mazur et al.,
fetal gonads (all stages of pregnancy) and adult ovaries. For placenta, 2010). The expression of these two isoforms is detectable in human
BPA-S hydrolysis rates ranged from 0 to 7.58 pmol/8 h and from 3.92 fetal livers during the second trimester of pregnancy, and has been
to 14.0 pmol/8 h at early and late stages of pregnancy, respectively, stated to account for 18% (Divakaran et al., 2014) or 10%–20% of the
and were highly variable, precluding any comparison between conjuga- values calculated in adults.
tion and BPA-S deconjugation activities throughout gestation.
We showed that for BPA the glucuronidation unbound intrinsic
clearance of hepatic microsomal fractions in humans was close to that
calculated for sheep. This suggests that sheep, unlike immature rats
Discussion
(Matsumoto et al., 2002), represent a valuable model to evaluate metabolic
Pregnancy is a critical window of exposure during which developmental processes toward BPA.
processes are extremely sensitive to estrogenic interferences. BPA is a xeno-
The estimated value of hepatic glucuronidation clearance for adult
estrogen of major concern for fetal development. The degree of human fetal humans (7.7 ml/kg per min) was consistent with the values determined
exposure to BPA and its metabolites is still unclear. Addressing the in in vitro studies using liver microsomal fractions [13.7 ml/kg per min
metabolism of the fetoplacental unit in animal models is a key issue for (Elsby et al., 2001); 15.5 ml/kg per min (Mazur et al., 2010)] or
the understanding of fetal exposure to bioactive (parent) BPA. In the cryopreserved hepatocytes [4.4 ml/kg per min (Kurebayashi et al.,
present study, we used human and ovine subcellular liver fractions as 2010); 6 ml/kg per min (Kuester and Sipes, 2007)].
well as sheep target tissues to evaluate the ontogeny of metabolic activities
involved in BPA conjugation/deconjugation reactions.
We found no gender-related differences for the Vmax or K
nor for the unbound intrinsic glucuronidation clearance of BPA in
m
values,

4
74
Corbel et al.
3
3
Fig. 4. BPA conjugation/deconjugation reactions in placenta and in gonads. Conjugation rate of [ H]-BPA and hydrolysis rate of [ H]-BPA-G in fetal and adult ovine tissues
after incubation for 8 hours in DMEM and saline solution, respectively. BPA conjugation and BPA-G hydrolysis activities are represented individually for three fetuses at an
early stage of pregnancy (solid symbols) and three fetuses at a late stage of pregnancy (open symbols): (A) in placenta, (B) in fetal ovaries, (C) in fetal testes, and (D) in adult
ovaries of three pregnant (solid symbols) and three nonpregnant ewes (open symbols).
ovine fetuses. This suggests that UGT’s activities toward BPA are sulfoconjugation was more pronounced in vitro (30%) than previously
similar in male and female fetal livers at the examined stages of reported in vivo, at a steady state in the fetal lamb after intravenous
pregnancy. This result is in agreement with data obtained with human infusion of BPA (4%; Corbel et al., 2013). This difference could be due to
liver microsomes (Elsby et al., 2001; Mazur et al., 2010) and hepatocytes the limitation of in vitro assays (Miners et al., 2006).
(Kuester and Sipes, 2007), as well as with the BPA pharmacokinetic data
Inasmuch as conjugation plays an important role in the detoxification
previously reported for sheep (Corbel et al., 2013). By contrast, a gender of BPA, it is necessary to examine the possible contribution of the liver
difference in the hepatic glucuronidation of BPA was observed in vitro in to the deconjugation of BPA metabolites, which could in turn modulate
adult rats (Mazur et al., 2010).
the elimination of BPA. Our results indicate that b-glucuronidase activity
We also assessed the contribution of the hepatic sulfoconjugation had only a minor influence on the liver BPA glucuronidation activity of
pathway using liver cytosols. The ability of ovine fetal livers to conjugate ovine near-term fetuses and adult sheep. By contrast, in early-stage
BPA through sulfoconjugation was found to be high, even at an early fetuses, as the glucuronidation activity was very low, the balance between
stage of pregnancy. In fact, it was about 3- to 8-fold higher in fetuses than glucuronidation and deconjugation was in favor of hydrolysis, reinforcing
in adults. This precocious expression of phenol liver sulfoconjugation the limited capacity of detoxification by BPA glucuronidation pathway in
activities has already been reported in midgestation human fetuses, with the fetus at an early-stage of pregnancy.
levels slightly below or equal to the activities calculated in adults
Pacifici, 2005; Stanley et al., 2005; Hines, 2008).
BPA Conjugation/Deconjugation Reactions in Target Tissues.
The placenta is considered to play a protective role during gestation
(
The respective contribution of these two conjugative pathways to through the metabolism and clearance of xenobiotics. In our study, the
the hepatic clearance of BPA was then evaluated. In early-stage fetuses, placenta showed wide interindividual variability regarding BPA conju-
the unbound intrinsic sulfoconjugation clearance of BPA was 4-fold gation and hydrolysis activities. These data are in good accordance with
higher than that of glucuronidation. These data are in accordance with the highly variable ratio (from 0.32 to 6.6) of conjugated over unconjugated
the exposure to BPA-S observed in human umbilical cord blood at BPA measured in human placenta (Zhang et al., 2011). Similar high
midgestation (Gerona et al., 2013). Conversely, BPA’s sulfoconjugation interindividual variations in UGT and b-glucuronidase expression and
hepatic clearance was very low in adult sheep and humans, accounting activities have previously been reported for human midterm and late-term
for only 2.2% and 0.26% of the total conjugation hepatic clearance. This placenta (Collier et al., 2002a; Collier et al., 2002b). This variability
result is consistent with the low fraction of BPA-S (1.4% of total BPA) remained unexplained, but was partly attributed to a variability in the
observed at a steady state in ewes after BPA intravenous infusion (Corbel fraction of trophoblast contained in the placental samples. Indeed, UGT
et al., 2013). In adult human hepatocytes incubated with BPA, it was enzymes are localized in the trophoblast layer.
previously reported that the BPA-S fraction was rather high (9% of the
Under our conditions, BPA-S was the main metabolite produced by
total metabolites) but remained low compared with BPA-G (Kurebayashi ovine placental tissues, and the sulfoconjugation activity was already
et al., 2010). In the near-term ovine fetus, the contribution of hepatic BPA observed at an early stage of pregnancy. This result is consistent with

Fetoplacental BPA Conjugation/Deconjugation Reactions
Conducted experiments: Corbel, Perdu, Gayrard, Picard-Hagen, Puel.
475
the fact that the SULT1A family is expressed in human placenta
Stanley et al., 2001), and this isoform is known to provide a relevant
Contributed new reagents or analytic tools: Perdu, Puel, Lacroix.
(
Performed data analysis: Corbel, Perdu, Gayrard, Toutain, Picard-Hagen.
Wrote or contributed to the writing of the manuscript: Corbel, Perdu,
Gayrard, Lacroix, Viguié, Toutain, Zalko, Picard-Hagen.
contribution to the sulfoconjugation of BPA (Nishiyama et al., 2002).
Furthermore, the BPA-G and BPA-S hydrolysis activities were low at
an early stage of pregnancy, when hepatic sulfoconjugation was pre-
dominant. These results suggest that the deconjugation of BPA
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In conclusion, our data suggest that the dynamics of the balance
between the conjugation of BPA and the deconjugation of BPA-G
throughout development in hepatic, placental, and gonadal tissues
could play a role in determining the level of exposure of target tissues
to BPA, providing key information for predicting human fetal BPA
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a high fetal exposure to the BPA conjugates. Furthermore, our data show
that the b-glucuronidase activity in the fetal gonads could lead to a
reactivation of BPA-G to BPA, thereby modifying locally the exposure to
active BPA. The critical importance of this conjugation-deconjugation
cycle must be further investigated, to determine whether the fetal over-
exposure to BPA conjugates, assumed to be inactive in terms of
estrogenicity, has to be taken into account in risk assessment for
human health related to fetal BPA exposure.
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Authorship Contributions
Participated in research design: Corbel, Perdu, Gayrard, Lacroix, Viguié,
Toutain, Zalko, Picard-Hagen.

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Address correspondence to: Nicole Picard-Hagen, Ecole Nationale Vétérinaire
de Toulouse, 23, Chemin des Capelles, 31076 Toulouse CEDEX 3, France. E-mail:
n.hagen-picard@envt.fr