Journal of Experimental Marine Biology and Ecology 325 (2005) 125 – 133
www.elsevier.com/locate/jembe
Short-term bioaccumulation, circulation and metabolism of
estradiol-17h in the oyster Crassostrea gigas
O. Le Curieux-Belfond a, B. Fievet b, G.E. Séralini c,*, M. Mathieu a
a
Laboratoire de Physiologie et Écophysiologie des Mollusques Marins, Unité Mixte de Recherches Ifremer,
Université de Caen, Esplanade de la Paix, 14032 Caen, France
b
Laboratoire d’Études Radioécologiques de la Façade Atlantique, Institut de Radioprotection et de Sûreté Nucléaire,
rue Max Pol Fouchet, 50130 Cherbourg-Octeville, France
c
Laboratoire de Biochimie et Biologie Moléculaire, EA 2608, IBFA, Université de Caen, Esplanade de la Paix, 14032 Caen, France
Received 23 October 2004; received in revised form 25 February 2005; accepted 18 April 2005
Abstract
Steroids are active signal transmitters in Vertebrates. These roles have also been hypothesized in other Phyla and endocrine
disrupting effects have been reported for different estrogen-like compounds in fishes and some marine invertebrates. As
estradiol-17h has shown some physiological activities in the oyster and as estrogens or estrogen-like molecules can be present
in water, we have investigated the bioaccumulation and metabolism of this estrogen in vivo in the oyster Crassostrea gigas.
When dissolved in seawater, in less than 48 h estradiol-17h concentrated up to 31 times in the soft tissues of the suspensionfeeder mollusc. Injected in the adductor muscle, estradiol-17h circulated from muscle to the gonad, the gills, the mantle, the
labial palps, and to a lesser extent to the digestive gland. After 2 h, estradiol flow increased specifically towards this gland.
Different hypotheses were raised concerning the circulation paths. However, in all cases estradiol metabolism primarily
evidenced an in vivo transformation into estrone in the whole oyster and in its digestive gland. This strong 17h-hydroxysteroid-dehydrogenase activity confirms our previous in vitro results. In conclusion, it is proposed that oyster is able to take in
charge estradiol as a potential contaminant in seawater. Therefore, its bioaccumulation and transformation into estrone could be
studied as potential biomarkers of endocrine disruption. Furthermore, the experimental approach with dissolved steroids in the
seawater combined to an anatomical screening appears as an interesting tool to investigate the bivalve endocrinology.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Bioaccumulation; Bivalve; Endocrine disruption; Estradiol; Steroid
1. Introduction
* Corresponding author. Tel.: +33 23 156 5489; fax: +33 23 156
5320.
E-mail address: [email protected] (G.E. Séralini).
0022-0981/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jembe.2005.04.027
Estradiol-17h is a naturally occurring steroid
hormone produced endogenously by all vertebrate
species. This estrogen plays important and varied
126
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
roles in their differentiation, development and reproduction. In invertebrates, the steroid control of reproduction is unclear. Previous studies have reported
in bivalves (Mytilus edulis) the presence of estradiol-17h in gonads by mass spectrometry (Zhu et al.,
2003). Moreover, some effects of this estrogen, such
as the stimulation of vitellogenin accumulation in
oyster (Crassostrea gigas) oocytes (Li et al., 1998)
have been identified, as well as estradiol metabolism, particularly a 17h-hydroxysteroid dehydrogenase activity converting estradiol into estrone (Le
Curieux-Belfond et al., 2001). Elsewhere, a steroid
competition assay has shown in the reproductive
system of the cephalopod Octopus vulgaris an estradiol-17h binding activity linked to a 70-KDa
estrogen receptor-like protein (Di Cosmo et al.,
2002). Some molecules from or close to the steroid
family may also play a role, for example estradiol
stimulates nitric oxide release in the nervous pedia
ganglia of the mussel M. edulis (Stefano et al.,
2003).
Steroids and steroid-like compounds may be present in the environment and perturb the bivalves. For
example, when the oyster Saccostrea commercialis is
deployed at sewage disturbed marine locations, the
lipid fraction of its gills shows a significant rise in hsitosterol level, a plant sterol derived from domestic
sewage and marine algae (Avery et al., 1998). Vertebrates naturally produce estrogens that pollute the
environment. For example, field-applied poultry litter
containing up to 1.28 Ag/l of estradiol-17h contributes
to the runoff of this hormone, which then persists at
least 7 days under field conditions (Nichols et al.,
1997). The presence of estradiol-17h is also regularly
recorded in sludge effluents (Croley et al., 2000) and
causes the feminization of the males (Metcalfe et al.,
2001) leading to a decrease of their reproductive
success (Matthiessen et al., 2002). Some physiological perturbations in bivalves could also be linked to
hormone-like contaminants as estrogenic or anti-estrogenic compounds (Gauthier-Clerc et al., 2002). All
the more since the induction by ethinylestradiol of the
reproduction response and the effects of estrogenic
effluent on embryo production were shown comparable in a gastropod mollusc and in fishes (Jobling et al.,
2003).
In order to elucidate the factors that could modulate the oyster biological cycle, we raised the
question as to whether estradiol dissolved in the seawater is accumulated and metabolized in the bivalve
C. gigas.
2. Materials and methods
2.1. Biological samples
Three-year-old Pacific oysters C. gigas were
obtained from a farm in Saint-Vaast-La-Hougue (Normandy, France) in April, a period during which
gonad maturation, started in winter, is in progress
until spawning in summer. Before the experiment,
oysters were kept during 48 h at seasonal temperature (12–14 8C) in sand bed filtered seawater oxygenated by airflow, in order to acclimatize them.
Total lipid content of the different organs of 12
oysters was extracted by the Bligh and Dyer’s method
(1959) and measured according to the method of
Marsh and Weinstein (1966) using palmitic acid as
standard. The water fraction of each organ was evaluated on 12 oysters by the subtraction of the lyophilized weight from the wet weight. The lipophilic
index, corresponding to the ratio of lipid to water,
was used to classify the organs.
Hemolymphatic courses between the adductor
muscle and other organs were estimated anatomically
on six oysters to define the bproximity indexQ and thus
to classify the organs according to their distance from
the injection point.
2.2. Chemicals
[4-14C]-estradiol (1,3,5(10)-estratriene-3,17h-diol
52 mCi/mmol) and [1,3,5,6-3H] estrone were obtained
from New England Nuclear Corp. (Zaventem, Belgium). The unlabelled steroids were obtained from
Sigma (St Quentin Fallavier, France). Solvents of
analytical grade or of HPLC-grade were purchased
from Prolabo (Fontenay-Sous-Bois, France). The
scintillation cocktails Ultima Gold XR and Ultima
Flo M (Packard, France) were used respectively for
the radioactivity measurements in samples (sample/LS
ratio: 1.5–16, v/v) and the HPLC monitoring (LS/
HPLC ratio: 1–3, v/v). The tissue solubilizer Solvable
was supplied by Packard Bioscience (Groningen, The
Netherlands).
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
2.3. Bioaccumulation study
Oysters were kept 0–48 h in tanks (2 oysters per
tank) containing 10 l of sand bed filtered seawater,
at the seasonal temperature (12–14 8C), and oxygenated by airflow. [4-14C]-estradiol was dissolved
in seawater to a final concentration of 0.046 AM
(2.4 ACi/l). The stability of this estrogen in seawater was verified by HPLC analysis at 48 h (chromatogram shows a single peak that displays a
retention time similar to standard estradiol). After
incubation, oysters were removed from their shells,
rinsed abundantly, and finally whole oyster soft
tissues were used for measurement of the bioaccumulated radioactivity.
The bioaccumulation index that we calculated
corresponds to the ratio of the radioactivity present
in 1 g of soft tissues on the radioactivity in 1 ml of
seawater. Alkaline lyses of the homogenized tissues
of whole oysters (pooled by 2) were performed
during 3 h at 50 8C with five volumes of Solvable
solution (a mixture of 3% sodium hydroxide and
4% alcohol). The 14C measurements in seawater
and lysed tissue samples were performed with a
Tri-Carb 1600 (Packard) liquid scintillation counter,
using a 15-min counting time and a tSIE protocol
for quenching correction. Quenching was found to
be linear and similar in both seawater and tissue
samples in the 0–50 nM steroid concentration
range.
2.4. Circulation study
For each oyster, a little piece of the posterior edge
of the shell was removed with a small circular saw,
without hurting the coat. Injections in the oysters
were performed with a 10-min interval that was
necessary for excision of the organs of each oyster
at the end of the incubation. An ethanol solution of
estradiol was injected in the adductor muscle (needle
length 40 mm, diameter 0.8 mm). The injected volume was adapted to the soft tissues weight of each
oyster, in order to correspond to a final concentration
in the total soft tissues of 4 AM. A preliminary
experiment on 12 oysters of the same group gave
the coefficient total mass/soft tissues mass (4.83)
which allowed an estimation of the soft tissues
mass of each oyster.
127
The organs were precisely dissected in the following order: gills, labial palps, visceral mass, body
mantle and adductor muscle. The visceral mass was
separated into two parts: the digestive gland associated with the gonad, and the rest of the visceral
organs including the digestive tract and the heart.
Then, these tissues were partially frozen to help
gonad separation from the digestive gland (digitations of the gonad penetrate into the digestive gland).
The separated organs of seven oysters were pooled,
homogenized at 4 8C without any buffer, and then
deep-frozen in liquid nitrogen before stocking at
20 8C. Radioactivity was counted in the organs 2
and 48 h after injection. The results were expressed
as flows by difference of radioactivity between time
0 and the second hour after injection, and between 2
and 48 h after injection. A Student’s t-test was
applied.
2.5. Metabolism study
The samples used in these analyses derived from
preparations described in Section 2.4. The samples
collected at time zero, immediately after injection,
were used as negative controls to study metabolism
after 2 or 48 h of incubation. To protect the steroids
and enhance their extraction, non-radiolabelled estrone, estradiol, androstenedione and testosterone
(0.1 AM each; dissolved in an ethanol solution of
ascorbic acid 0.2%) were added. The precursor and
its metabolites in 20 g of homogenate were extracted
three times with three volumes of diethylether. The
lipids were partially removed from the residues dissolved in methanol/water mixture (7:3, v/v) and frozen at 20 8C for 48 h by discarding the pellet of an
800 g centrifugation. The methanol/water fraction,
after a partial evaporation, was extracted three times
with two volumes of diethylether. After a complete
evaporation under a nitrogen stream at 34.5 8C, the
residue dissolved in 50 Al of ethanol was purified by
thin layer chromatography on silica gel plates (Kieselgel 60 F254, 20 20 cm, 0.25 mm, Merck) by two
successive runs in a cyclohexane/ethylacetate (1:1, v/
v) solvent system. The steroids area was scraped off,
transferred into a glass pipette fitted with glass-wool
cork and finally eluted with ascorbic ethanol. The
small particles of silica were removed by a smooth
centrifugation of 800 g during 5 min. After evapo-
128
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
Bioaccumulation Index
3. Results
30
3.1. Bioaccumulation
The bioaccumulation index of estradiol in oysters
increased during the 48 h of the experiment. The
process is rapid within 2 hours, and then slows
down. After 48 h, the radioactivity, corresponding to
estradiol but also to its metabolites, was 31 times
more concentrated in 1 g of oyster tissue than in 1
ml of seawater (Fig. 1).
20
10
0
0
12
24
36
48
hours
Fig. 1. Bioaccumulation of estradiol in the oyster. After immersion
of the oysters in seawater containing 45 nM [4-14C]-estradiol for 1
min to 2 days, the bioaccumulation index of the steroid in the oyster
organism was estimated by the ratio of radioactivity in 1 g of soft
tissue on 1 ml of seawater. For each point, the radioactivity was
measured in two oysters. y = 0.49 ln(x) + 13.23. R 2 = 0.989. Student’s
t-test: P b 0.01.
ration, the dried residue was purified on a Sep-Pak
C18 column (360 mg particles of 55–100 Am, Millipore), washed with ethanol, flushed with ultra-pure
water and eluted with ascorbic ethanol. The evaporated residue was dissolved in 100 Al ethanol and frozen
until HPLC analysis. The extraction and purification
global yield for estradiol was 54.4 F 9%.
The High Performance Liquid Chromatography
(HPLC Spectra System P1000XR, Thermo-Separation-Products) was performed on a 250 mm 4.6
mm 5 Am RP18 Supelcosil LC18 column (Supelco)
fitted with a 25 mm 4 mm 4 Am RP18 Lichrosorb pre-column (Merck). The sample was eluted
with a gradient of methanol (5–95% in 30 min) in a
water/acetonitrile mixture (60:34, v/v) at a flow rate
of 1 ml/min. The effluent was monitored with a
radioactive flow detector (Flow One beta A-500,
Packard). The metabolites were compared in separate analysis with tritiated internal standards. The
proportions between the different steroids present in
one chromatogram were estimated by calculation of
the corresponding areas and the ratio of these values
to the total radioactivity recorded during the run.
Between two consecutive analyses, the column was
washed with methanol during 15 min, equilibrated
with the elution mixture and tested with a blank
chromatogram.
3.2. Circulation
Circulation was studied by injection of estradiol in
the adductor muscle. The radioactivity released by the
oysters in seawater was compared to the total radioactivity injected (Fig. 2). Nearly one-half of injected
radioactivity stayed in the oyster, whereas the nonclosed hemolymphatic circulating system lost the
other half within 10 min. The adductor muscle retains,
in its hemolymphatic sinusal lacuna, around 50% of
the total radioactivity present in the oyster within the
first 2 h, and 30% after 48 h.
The distribution of the radioactive steroid and its
metabolites in oysters was measured in various organs
µCi 50
40
30
20
10
0
0
1
2
3
hours
Fig. 2. Loss of radioactivity in seawater after injection of [4-14C]estradiol in the adductor muscle of oysters. Ten minutes separate
two consecutive injections in seven oysters. The cumulated radioactivity injected in oysters (dotted line) is compared over time to the
seawater radioactivity (black curve). One half of the injected estradiol appears to be held by oysters, whereas the other half is released
within 10 min after injection. Values are the averages of three
analyses F SE.
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
129
from 0.17 to 2.36 nmol (estradiol-equivalent)/g of wet
weight tissues, in the following order: gonad N gills N
mantle N labial palps N digestive gland. However, during the second period from the 2nd to the 48th hour,
the adductor muscle outcome decreased to 5.88 nmol
estradiol-equivalent per gram adductor muscle (or
24.5 nmol per whole adductor muscle) and the
flows also changed dramatically in the other organs.
The incoming flows diminished in the labial palps and
the gills 4 and 15 times, respectively, and even reversed in the gonad and the mantle, coming out of
these organs. Synchronously, the flow coming into the
digestive gland increased 2.3 times.
The transfers in a given organ expressed as flows
per whole organ of oyster exhibited some small differences when compared to the results in terms of
flows per gram of organ (Fig. 3B). More especially,
during the first 2 h, the mantle, weighing nearly 29%
of the total soft tissues, appeared to have the most
important incoming flow, while the labial palps
weighing nearly 5% have a light incoming flow comparable to the digestive gland.
Fig. 3. Circulation of estradiol and its metabolites in the oyster.
After injection of [4-14C]-estradiol in the adductor muscle, radioactivity was measured in different organs. Steroid flows from 0 to
2 h (light grey histograms in A and B) and from 2 to 48 h (dark
histograms in A and B) are expressed in nmol estradiol-equivalent
per gram soft tissues (A) or per organ (B). An incoming flow is
noted positive on the Y scale, whereas an outcoming flow is noted
negative. During the first 2 h, the adductor muscle outgoing flow is
8.96 nmol/g adductor muscle or 37.3 nmol per whole muscle. From
the 2nd to the 48th hour, the adductor muscle outcoming flow is
5.88 nmol/g adductor muscle or 24.5 nmol per whole adductor
muscle. These results are dependent on the lipophilic index (dotted
line) and the proximity index of the organs derived from anatomical
data (dark line) (C). Values are the averages of three analyses F SE.
All differences between the two periods are significant ( P b 0.05).
2 and 48 h after the injection of [14C]-estradiol in the
adductor muscle. The results were expressed as flows
of radioactivity coming in or out of the organs (Fig.
3A). At time zero, 104.9 F 5.2 nmol estradiol (25.2
nmol/g) were present in the adductor muscle. During
the first 2 h, 8.96 nmol/g adductor muscle (or 37.3
nmol per whole adductor muscle) went to the seawater
and to the other organs. The incoming flows ranged
LP
DG
Go
Gi
He
AM
Ma
Fig. 4. Two possible circulation paths of estradiol and its metabolites. Circulation through the hemolymphatic system (dark line
arrows): estradiol injected (double line arrow) in the adductor
muscle (AM) circulates through the gills (Gi) to the heart (He),
and is then distributed to the other organs, including the gonad (Go),
the digestive gland (DG) and the labial palps (LP). Circulation of
the fraction lost in the seawater (dotted line arrows): absorbed by the
filtration and digestive systems, the steroids can also diffuse directly
through the membranes, especially in the mantle and gonad tissues.
130
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
The lipophilic index and the proximity of injection (Fig. 3C) both seemed to contribute to the
distribution of estradiol and its metabolites within
the organs (Fig. 4).
1500
dpm
A
1250
E1
1000
750
3.3. Metabolism
E2
500
Metabolism of estradiol was observed in the whole
oyster (Fig. 5). After 2 h, HPLC analyses showed that
57% of estradiol was converted into estrone, while the
rest of the radioactivity corresponded essentially to the
non-metabolized precursor (Fig. 5A). After 48 h the
estrone fraction reached 72% of the total radioactivity
found in the oyster, and two minor unidentified meta-
m1
250
0
1500
dpm
B
1250
1000
500
E1
dpm
A
400
750
500
250
300
0
E2
200
0
4
8
12
16
20
24
28
Min.
100
Fig. 6. Detection of estradiol and its metabolites in the digestive gland
of the oyster by HPLC. See legend of Fig. 5. Analysis of estradiol and
its metabolites (radiodetection window = 18.5–156 keV) (A) was
followed by an identical analysis with [2,4,6,7-3H]-estrone as internal
standard (radiodetection window = 0–18.5 keV) (B).
0
500
dpm
B
E1
400
300
200
m2 m1
100
E2
0
0
4
8
12
16
20
24
28
bolites, m1 and m2, also appeared representing respectively 7% and 4% of the total radioactivity (Fig. 5B).
Two hours after the estradiol injection in the adductor muscle, the digestive gland showed a pathway
similar with that of the whole oyster, metabolizing
58% of estradiol into estrone (Fig. 6A). The identity
of estrone was confirmed by its co-elution with the
tritiated internal standard (Fig. 6B). The metabolite
m1 was also detected in the digestive gland as from
these first 2 h in similar or higher proportion (12%).
Min.
Fig. 5. Detection of estradiol and its metabolites in the oyster after
2 h (A) or 48 h (B), by HPLC and radiodetection. A peak of
estradiol-17h was observed at time zero. The metabolism of the
bioaccumulated [4-14C]-estradiol (E 2 = 13.5 F 0.2 min) in the oyster
was evidenced in estrone (E 1 = 16.0 F 0.2 min), and at least in two
other minor, more polar metabolites (m2 = 3.4 F 0.2 min,
m1 = 5.3 F 0.2 min). Analyses were replicated twice.
4. Discussion
4.1. Bioaccumulation of estradiol in the oyster
Since in bivalves the uptake of contaminants from
particles seems to be slower than directly from the
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
water (Bruner et al., 1994), the short-term study
using filtered seawater should reflect in situ oyster
physiology.
Natural and synthetic estrogens represent a part of
the pollutants found in river, coastal and estuarial
waters, especially near sewage plants. For example,
estrone and estradiol range, in seawater samples of the
coastal area of Tokyo Bay, more than 30 and 1.3 ng/l,
respectively (Kawai et al., 2002). Numerous urbanderived estrogenic contaminants affect the benthic
macrofauna of the coastal areas, especially of the
estuaries (Morrissey et al., 2003; Korner et al.,
1999). Even concentrations of estrogens in the ng/l
range are able to induce intersex in fishes and to alter
the sex ratio of their population in favour of females
(Metcalfe et al., 2001). Furthermore, synergistic interactions of estrogens and xenoestrogens in mixture
may amplify the estrogenic effects of wastewaters
(Arnold et al., 1996). Therefore, estrogens that are
discharged in the environment at low concentrations
or discontinuously but in higher concentrations are
likely to cause physiological dysfunction in the oyster.
For example, in vivo estradiol is able to reduce the
lysosomal stability in the digestive gland cells of the
mussel M. edulis (Moore et al., 1978). In certain
summers, gonad maturation of the oyster appears to
be more intensive and prolonged. Synchronously,
higher mortality rates were recorded, sometimes
attaining more than 50% (Bricelj et al., 1992).
Among the multiple factors suspected, pollutants
could play a role, notably estrogenic ones (Cheney
et al., 1997).
The bioaccumulation of estradiol in oysters indicates that seawater could carry steroidal messengers to
or through an oyster population. Therefore, estradiol,
or similar molecules and even sterols, could influence
the gonad maturation processes through this seawater
pathway. After the first hours, the bioaccumulation
increases only slowly (Fig. 1), probably due to intense
metabolism and excretion, and to losses through the
open circulating system.
4.2. Circulation of estradiol and its metabolites in the
oyster
From the adductor muscle vessels and sinus, the
hemolymph is collected in the afferent veins and lines
the gills. There, the oxygenated hemolymph is pumped
131
to the heart and distributed to the other organs. This
circulatory system is not completely enclosed and
losses occur.
The flows of estradiol and its metabolites appeared
to have taken at least two ways of circulation (Fig. 4).
One possible way was through the hemolymphatic
system: estradiol went to the gills, from the gills to
the heart, and was then distributed to the other organs,
among which the mantle, the gonad tissues (within the
mantle), the digestive gland and the labial palps. At
the level of these organs, the hemolymphatic system
becomes open and the circulation back to the sinusal
system of the adductor muscle was less efficient. This
may explain in part the steroid loss in the seawater.
For the other way, estradiol and its metabolites lost in
the water may be captured back by the gills and the
labial palps. These steroids dissolved in the oyster
environment may also diffuse through the teguments,
notably the teguments of the mantle and the gonad
that possess large surfaces in contact with the water.
The lipophilic index and the proximity of injection
(Fig. 3C) both seemed to contribute to the distribution
of estradiol and its metabolites within the organs, but
other active processes also influenced the distribution,
like the intensity of steroid metabolism in the different
organs and the unclosed circulating system. Therefore, the gills, the mantle and the gonad were the
first organs distributed by the hemolymphatic system,
and in the first period had the highest incoming flows.
Whereas the digestive gland and the labial palps,
situated at a greater distance, accumulated to a lesser
extent during the first period. Although the gonad was
farther from the injection point than the gills, it accumulated a little more, probably because of its higher
lipid/water ratio. Moreover, the mantle and the gonad,
which anatomically belong to the mantle, have similar
incoming flows although they do not have the same
lipid/water ratio, which is consistent with the assumption that the hemolymphatic system drives the distribution of the radioactivity during the first 2 h.
Although the labial palps had a lipid/water ratio similar to that of the gonad, the incoming flow was
continuous and even increasing in the labial palps,
whereas it was reversing in the gonad during the
second period after 2 h. It can also be observed that
the digestive gland, rather hydrophobic but more distant to the injection point, accumulates poorly during
this first 2 h.
132
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
The gonad appears to eliminate the steroid and its
metabolites afterwards, as it was secreting this family
of substances, while the digestive gland benefits from
the bioaccumulation in contrast to all other organs.
Both the high lipid/water ratio of the digestive gland
and the excretion of steroids from the other organs
may explain this result. This new distribution from 2
to 48 h depends probably on active processes, since
the distribution of radioactivity 48 h after injection of
estradiol in the adductor muscle (adductor muscle N
gills N gonad and mantle N digestive gland and palps)
does not match the natural distribution of sterols in the
oyster recorded elsewhere (labial palps N visceral
mass N mantle and gills) (Gordon and Collins, 1982).
The bioaccumulation of the steroid within the oyster
feed may not be excluded. Moreover, gills generate
water currents and are well drained by hemolymph;
these characteristics favor steroids uptake from water
and this probably compensates losses during the second period.
4.3. Metabolism of estradiol in the whole oyster and
in its digestive gland
During the first 2 h after injection, in the whole
oyster estradiol-17h has been metabolized essentially
or almost exclusively into estrone. This reveals an
intensive 17h-hydroxysteroid dehydrogenase-like activity that was also evidenced previously in vitro (Le
Curieux-Belfond et al., 2001). After 48 h, two other
minor metabolites m1 and m2 are detected. Regarding
the HPLC conditions and in comparison to mammals,
they could correspond to more polar estrogens, such
as catechol estrogens (Cheng et al., 2001a,b). Their
identification could only be possible by collecting
larger quantities with a non-radiolabelled precursor.
The digestive gland concentrated the same metabolites. Estrone was in high quantities, whereas m1
was also detected in this organ. Therefore, in further
studies, not only estradiol, but also estrone, m1 and
m2 should be considered as potential regulators or
disrupters of oyster physiology.
5. Conclusions
Estradiol accumulates easily and rapidly in the
oyster. This estrogen may thus interact with the oyster
physiology, either as an endocrine signal, or as a
xenobiotic. Nevertheless, this way of administration
should be of interest for studying in vivo the estrogen
effects in oysters.
Distribution of estradiol in the oyster is, in the first
hours of exposition, probably and essentially dependent on the hemolymphatic circulation system. The
specific lipophilicity of each organ influences the
general distribution rather later. Furthermore, it is
thwarted by active processes such as the metabolism.
Thus, gonad uptake is fast and is rapidly followed by
elimination, whereas the digestive gland seems to be
an organ of longer-term accumulation.
Estradiol-17h is essentially converted into estrone
by a 17h-HSD-like activity, maybe in all organs of the
oyster. Nevertheless, the digestive gland also evidences other minor metabolites, which have a polarity
similar to catecholestrogens. These could be endocrine active steroids or excretion forms, and may
participate to endocrine disruption.
Further studies could test steroidogenic enzymes
like the oyster 17h-HSD as biomarkers of seawater
contaminants that exhibit structures close to estrogens.
Acknowledgements
This work was supported by the bConseil Regional
de Basse-NormandieQ (CRAB) and the European
Union (FEDER). [SS]
References
Arnold, S.F., Klotz, D.M., Collins, B.M., Vonier, P.M., Guillette,
L.J., McLachlan, J.A., 1996. Synergistic activation of estrogen
receptor with combinations of environmental chemicals. Science
272 (5267), 1489 – 1492.
Avery, E.L., Dunstan, R.H., Nell, J.A., 1998. The use of lipidic
metabolic profiling to assess the biological impact of marine
sewage pollution. Arch. Environ. Contam. Toxicol. 35,
229 – 235.
Bligh, E.G., Dyer, W.F., 1959. A rapid method of total lipid
extraction and purification. Can. J. Biochem. Physiol. 47,
911 – 917.
Bricelj, M.V., Ford, S.E., Borrero, F.J., Perkins, F.J., Rivara, G.,
Hillman, R.E., Elston, R.A., Chang, J., 1992. Unexplained
mortalities of hatchery-reared, juvenile oysters, Crassostrea
virginica. J. Shellfish Res. 11 (2), 331 – 347.
Bruner, K.A., Fisher, S.W., Landrum, P.F., 1994. The role of the
Zebra Mussel Dreissena polymorpha in contaminant cycling: II.
O. Le Curieux-Belfond et al. / J. Exp. Mar. Biol. Ecol. 325 (2005) 125–133
Zebra mussel contaminant accumulation from algae and suspended particles, and transfer to the benthic invertebrates Gammarus fasciatus. J. Great Lakes Res. 20 (4), 735 – 750.
Cheney, M.A., Fiorillo, R., Criddle, R.S., 1997. Herbicide and
estrogen effects on the metabolic activity of Elliptio complanata
measured by calorespirometry. Comp. Biochem. Physiol., C,
Pharmacol Toxicol. Endocrinol. 118 (2), 159 – 164.
Cheng, Z.N., Huang, S.L., Tan, Z.R., Wang, W., Zhou, H.H., 2001a.
Determination of estradiol metabolites in human liver microsome by high performance liquid chromatography-electrochemistry detector. Acta Pharmacol. Sin. 22 (4), 369 – 374.
Cheng, Z.N., Shu, Z.Q., Liu, Z.Q., Wang, L.S., Ou-Yang, D.S.,
Zhou, H.H., 2001b. Role of cytochrome P450 in estradiol
metabolism in vitro. Acta Pharmacol. Sin. 22 (2), 148 – 154.
Croley, T.R., Hughes, R.J., Metcalfe, C.D., March, D.E., 2000.
Mass spectrometry applied to the analysis of estrogens in the
environment. Rapid Commun. Mass Spectrom. 14 (13),
1087 – 1093.
Di Cosmo, A., Di Cristo, C., Paolucci, M., 2002. An estradiol-17h
receptor in the reproductive system of the female of Octopus
vulgaris: characterization and immunolocalization. Mol. Reprod.
Dev. 61 (3), 367 – 375.
Gauthier-Clerc, S., Pellerin, J., Blaise, C., Gagne, F., 2002. Delayed
gametogenesis of Mya arenaria in the Saguenay fjord (Canada):
a consequence of endocrine disruptors? Comp. Biochem. Physiol., C, Toxicol. Pharmacol. 131 (4), 457 – 467.
Gordon, D.T., Collins, N., 1982. Anatomical distribution of sterols
in oyster Crassostrea gigas. Lipids 17 (11), 811 – 817.
Jobling, S., Casey, D., Rodgers-Gray, T., Oehlmann, J., ShulteOehlmann, U., Pawlowski, S., Baunbeck, T., Turner, A.P.,
Tyler, C.R., 2003. Comparative responses of molluscs and fish
to environmental estrogens and an estrogenic effluent. Aquat.
Toxicol. 65 (2), 205 – 220.
Kawai, S., Kurokawa, Y., Matsuoka, S., Nakatsukuri, M., Yamada,
H., 2002. Potential estrogenic substances in coastal seawater,
river water and effluents of sewage treatment plant in Japan
using three kinds of in vitro assays. United Nations University—UNESCO International Conference, 8–10 July 2002, Iwate,
Japan.
Korner, W., Hanf, V., Schuller, W., Kempter, C., Metzger, J., Hagenmaier, H., 1999. Development of a sensitive E-screen assay for
quantitative analysis of estrogenic activity in municipal sewage
plant effluents. Sci. Total Environ. 225 (1–2), 33 – 48.
133
Le Curieux-Belfond, O., Moslemi, S., Mathieu, M., Séralini, G.E.,
2001. Androgen metabolism in oyster Crassostrea gigas:
evidence for 17h-HSD activities and characterization of an
aromatase-like activity inhibited by pharmacological compounds and a marine pollutant. J. Steroid Biochem. Mol.
Biol. 78, 359 – 366.
Li, Q., Osada, M., Susuki, T., Mori, K., 1998. Changes in vitellin
during oogenesis and effect of estradiol-17h on vitellogenesis in
the pacific oyster Crassostrea gigas. Invertebr. Reprod. Dev. 33
(1), 87 – 93.
Marsh, J.B., Weinstein, D.B., 1966. Simple charring method for
determination of lipids. J. Lipid Res. 7 (4), 574 – 576.
Matthiessen, P., Allen, Y., Bamber, S., Craft, J., Hurst, M., Hutchinson, T., Feist, S., Katsiadaki, I., Kirby, M., Robinson, C.,
Scott, S., Thain, J., Thomas, K., 2002. The impact of estrogenic
and androgenic contamination on marine organisms in the
United Kingdom—summary of the EDMAR programme. Mar.
Environ. Res. 54 (3–5), 645 – 649.
Metcalfe, C.D., Metcalfe, T.L., Kiparissis, Y., Koenig, B.G.,
Khan, C., Hughes, R.J., Croley, T.R., March, R.E., Potter,
T., 2001. Estrogenic potency of chemicals detected in sewage
treatment plant effluents as determined by in vivo assays with
Japanese medaka Oryzias latipes. Environ. Toxicol. Chem. 20
(2), 297 – 308.
Moore, M.N., Lowe, D.M., Fieth, P.E., 1978. Responses of lysosomes in the digestive cells of the common mussel Mytilus
edulis to sex steroids and cortisol. Cell Tissue Res. 188, 1 – 9.
Morrissey, D.J., Turner, S.J., Mills, G.N., Williamson, R.B., Wise,
B.E., 2003. Factors affecting the distribution of benthic macrofauna in estuaries contaminated by urban runoff. Mar. Environ.
Res. 55 (2), 113 – 136.
Nichols, D.J., Daniel, T.C., Moore, P.A., Edwards, D.R., Pote,
D.H., 1997. Runoff of estrogen hormone 17h-estradiol from
poultry litter applied to pasture. J. Environ. Qual. 26,
1002 – 1006.
Stefano, G.B., Cadet, P., Mantione, K., Cho, J.J., Jones, D., Zhu,
W., 2003. Estrogen signaling at the cell surface coupled to nitric
oxide release Mytilus edulis nervous system. Endocrinology 144
(4), 1234 – 1240.
Zhu, W., Mantione, K., Jones, D., Salomon, E., Cho, J.J., Cadet, P.,
Stefano, G.B., 2003. The presence of 17-beta estradiol in Mytilus edulis gonadal tissues: evidence for estradiol isoforms.
Neuroendocrinol. Lett. 24 (3–4), 137 – 140.