67, 75– 80 (2002)
Copyright © 2002 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Effects of Primary Exposure to Environmental and Natural Estrogens
on Vitellogenin Production in Carp (Cyprinus carpio) Hepatocytes
T. Rouhani Rankouhi,* ,1 I. van Holsteijn,* R. Letcher,† J. P. Giesy,‡ and M. van den Berg*
*Institute for Risk Assessment Sciences (IRAS), Utrecht University, P. O. Box 80176, 3508 TD, Utrecht, The Netherlands; †Great Lakes Institute
for Environmental Research (GLIER), University of Windsor, Windsor, Ontario, Canada; and ‡Department of Zoology, National Food Safety
and Toxicology Center, Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan 48824
Received July 24, 2001; accepted November 6, 2001
Vitellogenin (vtg) has been used as a biomarker for exposure
to xenobiotics with estrogenic properties in several in vivo and
in vitro studies. Vtg is a precursor of the yolk proteins lipovitelline and phosvitin and is synthesized in the liver of female
oviparous vertebrates as a consequence of estrogen-dependent
gene expression. Estrogen receptor (ER) studies indicate that
the ER is upregulated by (primary) exposure to xenoestrogens
(Lazier and MacKay, 1993; Nimrod and Benson, 1997). Furthermore, in vivo experiments with rainbow trout indicate that
pretreatment of animals with estrone (E1) increases the vitellogenic response of hepatocytes after a secondary exposure to
estradiol (E2; van Bohemen et al., 1982). An enhanced inducibility of the vitellogenin gene via receptor induction is probably responsible for this increase in vitellogenesis. A persistent
induction of the ER might be also responsible for the decrease
of the lag time in E2 stimulated vitellogenesis in estrogen
pretreated animals (“memory effect”), at least in some species
as e.g., Xenopus (Lazier and MacKay, 1993). The vitellogenin
gene is also present in male oviparous vertebrates but under
natural circumstances endogenous estrogen levels are too low
to induce vitellogenesis. However, if male fish are exposed to
xenoestrogens, expression of the vitellogenin gene and vtg
synthesis can occur (Callard and Ho, 1987).
There is serious concern that some persistent manmade
chemicals found in the aquatic environment can disrupt endocrine development in wildlife and laboratory animals (Colborn
et al., 1993; Guillette and Crain, 1995; Sonnenschein and Soto,
1998). Elevated vtg concentrations in the plasma of male trout,
carp, and roach as well as an increased incidence of intersexuality in wild fish populations of roach have been associated
with discharges from sewage treatment plants containing biologically active estrogenic compounds (Folmar et al., 1996;
Jobling et al., 1998; Jobling and Sumpter, 1993; Purdom et al.,
1994; Routledge et al., 1998; Rodgers-Gray et al., 2000).
In order to investigate the possible interactive effects of
coexposure in the environment we measured the effects of
primary exposure of carp hepatocytes to xenoestrogens on vtg
synthesis in response to secondary xenoestrogen exposure in
the CARP-HEP assay (Smeets et al., 1999a).
Vitellogenin (vtg) is a precursor of the yolk proteins lipovitelline
and phosvitin and is synthesized as a consequence of estrogen-dependent gene expression in female and male hepatocytes of egg-laying
vertebrates. Freshly isolated carp hepatocytes of a genetically uniform strain of adult male carp (Cyprinus carpio) were used to investigate the effects of primary exposure to estrogenic compounds on the
vitellogenic response to xenoestrogens. Isolated carp hepatocytes
were first exposed (primary exposure) to 50 or 100 ␮M of either
methoxychlor (MXCL) or bisphenol A (BPA), different concentrations of estrone (E1; 1 or 10 nM) or 17␤-estradiol (E2; 0.1 or 1 nM) for
2 days. Hepatocytes were exposed to xenoestrogens (secondary exposure) at both 2 and 5 days after isolation. Hepatocytes were cultured
for a total period of 8 days. A competitive indirect ELISA was used
to determine the level of vtg after 8 days. The concentration of
chemicals used for the primary exposure induced vtg to a level that
was less than 10% of the response elicited by E2 (1000 nM). A
cytotoxic response, measured by MTT, was not observed after primary exposure to any of the xenoestrogens. After primary exposure to
MXCL, vtg production in response to E2 was increased by 4-fold, and
vitellogenesis in response to E1 treatment was doubled compared
with vitellogenesis without pretreatment. No significant differences
were observed between primary exposure to 50 and 100 ␮M MXCL.
Primary exposure to 50 and 100 ␮M BPA increased the maximum
vtg production in response to secondary E2 exposure by about 5- and
7-fold, respectively. Primary exposure to BPA (50 and 100␮M) followed by secondary exposure to E1 showed a 4- and 5-fold increase
of the vtg synthesis in comparison to E1 exposure alone. Primary
exposure to the endogenous estrogens had no significant influence on
the vtg synthesis in response to secondary exposure to E1 or E2.
Compared to hepatocytes exposed only to MXCL or BPA, primary
exposure to E2 increased the vtg synthesis in hepatocytes induced by
MXCL or BPA by almost a factor of 2. Primary exposure to E1
increased vitellogenesis after secondary exposure to MXCL only marginally. The present results indicate that weakly estrogenic environmental chemicals such as MXCL and BPA can increase the sensitivity of carp hepatocytes towards endogenous estrogens with respect to
VTG synthesis.
Key Words: carp hepatocytes; vitellogenin; in vitro; primary
exposure; methoxychlor; bisphenol A.
To whom correspondence should be addressed. Fax: ⫹31-30-2535077.
E-mail: t.rouhani@iras.uu.nl.
1
75
76
RANKOUHI ET AL.
The environmental contaminants methoxychlor (MXCL)
and bisphenol A (BPA) were selected for this study because
they can interact with the estrogen receptor but at concentrations that greatly exceed E2 or E1 concentrations (Eroschenko
et al., 1996; Nimrod and Benson, 1997; Safe et al., 1998;
Schlenk et al., 1998; Sheeler et al., 2000). MXCL and BPA
have an estrogenic potency relative to E2 of respectively 1 ⫻
10 –3 and 1 ⫻ 10 – 4 in carp hepatocytes (Smeets et al., 1999b).
The estrogenicity of these compounds has also been established in several other in vivo and in vitro studies using
different methods, such as the E-screen and the Yeast Estrogen
Screen, vitellogenin synthesis assays and competitive ER binding studies (Arnold et al., 1996; Christiansen et al., 2000; Chun
and Gorski, 2000; Gray et al., 1989; Mehmood et al., 2000;
Shelby et al., 1996; Sonnenschein and Soto, 1998).
MXCL is an organochlorine pesticide used as a substitute for
DDT. In both fish and mammals MXCL has been classified as
a proestrogen as biotransformation generates demethylated
MXCL metabolites that elicit more estrogenic activity than the
parent compound (Bulger et al., 1978). BPA is used in the
manufacture of polycarbonate and epoxy resins. These resins
can be found in various consumer products such as internal
coatings for food cans, plastics, optical lenses, and restorative
materials used in dentistry (Olea et al., 1996; Staples et al.,
2000).
MATERIALS AND METHODS
Experimental animals. The genetically uniform strain of adult male carp
(Cyprinus carpio) used in these experiments was obtained from the hatchery of
the Fish Culture and Fisheries research group at Wagering Agricultural University. The fish were kept in Utrecht municipal tap water at a constant
temperature of 24°C.
Hepatocyte isolation and cell exposure. Carp hepatocytes were isolated
using a collagenase perfusion technique and cultured in phenol red-free
DMEM/F12 medium (D2906, Sigma, St. Louis, MO), as described earlier
(Smeets et al., 1999a). All in vitro experiments were performed twice, using
isolated hepatocytes of 1 fish for each set of experiments. The concentrations
of chemicals used for primary exposure (MXCL: 50 or 100 ␮M; BPA: 50 or
100 ␮M; E1: 1 or 10 nM; or E2: 0.1 or 1 nM) were chosen based on the
criterion that these should induce vitellogenesis less than 10% of the response
elicited by E2 (1000 nM). The concentration-response relationship for vtg
induction by E2 in the CARP-HEP assay was determined earlier (Smeets et al.,
1999a). All chemicals were dissolved in DMSO (final concentration 0.1 %).
Isolated hepatocytes from 1 fish (1 ⫻ 10 6 cells/ml medium) were put into
single containers where either the stock solutions of the chemicals for primary
exposure or only DMSO (0.1 %; controls) were added. Then hepatocytes were
seeded in 96-well tissue culture plates (180 ␮l/well; Greiner, Alphen a/d Rijn,
The Netherlands). Each concentration of a compound was tested in 6 wells on
at least 1 plate. Hepatocytes were pretreated with the xenoestrogens for a
period of 48 h (Fig. 1). Cells were subsequently exposed (secondary exposure)
to E1 (Sigma, St. Louis, MO), E2 (1 to 1000 nM; (Sigma, St. Louis, MO),
MXCL (Riedel de Hahn Ag, Seelze, Germany), or BPA (1 to 100 ␮M; Sigma,
St. Louis, MO), or to DMSO alone, while refreshing the culture medium on the
second and fifth day after isolation. Culture medium (containing the different
concentrations of chemicals used for secondary exposure) was refreshed by
removing 90% of the medium and replacing it carefully using a multichannel
pipette. Hepatocytes were cultured for a total period of 8 days. At the end of
the exposure period, culture medium was transferred to 96-well plates and kept
FIG. 1. Dosing scheme of carp hepatocytes: primary exposure of hepatocytes (for a period of 48 h) to different compounds prior to seeding; secondary
exposure to chemicals or to DMSO alone while refreshing culture medium on
days 2 and 5 (period of secondary exposure, 2 ⫻ 72 h); removal of culture
medium for vtg measurement on day 8.
at –70°C prior to vitellogenin analysis. The remaining cell monolayers were
assessed for cell viability via the MTT assay.
Cell viability and quantification of vitellogenin. The viability of cells
after secondary exposure (on day 8) was determined by measuring the mitochondrial dehydrogenase activity using 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St. Louis, MO) as substrate (Denizot and Lang, 1986). Cell viability was also assessed by microscopic
examination of the cell morphology and the state of cell-to-cell contacts on
days 1, 2, and 5 of the assay. A competitive indirect ELISA was used to
determine the level of vtg in the culture medium on day 8 as described earlier
(Smeets et al., 1999a). Plasma of an E2 treated female carp, containing
approximately 45 ng/ml vtg was used for the standard curve dilution. The
detection limit of the ELISA was defined as the vtg level corresponding to the
lower limit of the linear part of the standard curve. Depending on the sensitivity of the hepatocytes the detection limit of the ELISA corresponded with an
exposure of the cells to between 2 and 5 nM E2 without pretreatment.
Statistical analysis. Statistical calculations were performed using the computer program SPSS. Statistical significance (p ⬍ 0.01) among the treatments
was calculated using a one-way ANOVA and a post hoc test (least squares
difference) with Bonferroni correction.
RESULTS
Cell Viability
Cell viability was determined by comparing mitochondrial
MTT of hepatocytes exposed to medium containing DMSO
(controls) or to medium containing the chemicals (dissolved in
DMSO). Compared to the controls up to 70% greater MTT
activity was observed after primary exposure of hepatocytes to
BPA (50 or 100 ␮M) and secondary exposure to 1 or 10 nM
E2. Secondary exposure of hepatocytes to 50 or 100 ␮M
MXCL resulted in MTT activity reduction that was approximately 30% less than the mitochondrial activity of the controls.
Microscopic observation revealed that during the first day
after seeding carp hepatocytes were similar in shape to freshly
isolated cells, but with a limited degree of intercellular contact.
Two days after seeding the hepatocytes formed clusters with a
high degree of intercellular contact. After 5 days in culture the
degree of cluster formation was similar to the monolayer
hepatocyte cultures of rainbow trout (Flouriot et al., 1993). All
cell layers remained attached to the bottom of the plates
throughout the experiment.
Primary Exposure to Xenoestrogens
Hepatocytes that were pretreated on day 1 with several
xenoestrogens showed increased sensitivity for vtg induction
77
VITELLOGENIN PRODUCTION IN HEPATOCYTES
TABLE 1
Summary of the Results of the Experiments with and without
Primary Exposure to Xenoestrogens in Carp Hepatocytes
Effect on vitellogenesis
Primary exposure
Secondary
exposure
Without primary
exposure (%)
With primary
exposure (%)
Estradiol (1 nM)
Estradiol (1 nM)
Estrone (10 nM)
Estrone (10 nM)
Estradiol (0.1 nM)
Estradiol (1 nM)
Estrone (1 nM)
Estrone (10 nM)
Estradiol (0.1 nM)
Estradiol (1 nM)
Estrone (1 nM)
Estrone (10 nM)
MXCL (50 ␮M)
MXCL (100 ␮M)
MXCL (50 ␮M)
MXCL (100 ␮M)
BPA (50 ␮M)
BPA (100 ␮M)
BPA (50 ␮M)
BPA (100 ␮M)
Estradiol (1000 nM)
Estrone (1000 nM)
Estradiol (1000 nM)
Estrone (1000 nM)
MXCL (100␮M)
MXCL (100␮M)
MXCL (100␮M)
MXCL (100␮M)
BPA (100␮M)
BPA (100␮M)
BPA (100␮M)
BPA (100␮M)
Estrone (1000 nM)
Estrone (1000 nM)
Estradiol (1000 nM)
Estradiol (1000 nM)
Estrone (1000 nM)
Estrone (1000 nM)
Estradiol (1000 nM)
Estradiol (1000 nM)
100
81
100
81
3
3
3
3
3
3
3
3
81
81
100
100
81
81
100
100
92
72
95
80
4
7*
3
5*
3
6*
3
3
188*
176*
470*
427*
351*
437*
555*
737*
Note. Vtg is expressed as a percentage of the induction by 1000 nM estradiol
(without pretreatment).
*Values significantly different from their controls (without primary exposure; p ⬍ 0.01).
by secondary exposure to xenoestrogens (Table 1). Compared
to cells exposed only to E2 or E1, primary exposure to MXCL
(50 or 100 ␮M) increased the vtg production in response to
1000 nM E2 or E1 by 4- and 2-fold, respectively (Figs. 2 and
3). The enhanced response to E1/E2 exposure was similar
whether 50 or 100 ␮M MXCL was used for primary exposure.
Compared to cells without pretreatment primary exposure to 50
or 100 ␮M BPA, with subsequent exposure to E2 (1000 nM),
increased the production of vtg by approximately 5- to 7-fold
(Fig. 2). In the case of BPA pretreatment (50 or 100 ␮M),
secondary exposure to 1000 nM E1 resulted in 4 and 5 times
greater vtg induction than without primary exposure (Fig. 3).
Compared with cells not pretreated, primary exposure to E1
(1 or 10 nM) or E2 (0.1 or 1 nM) followed by secondary
exposure to 1000 nM E1 or E2 did not affect vitellogenesis
(data not shown). As a consequence of pretreatment with 1 nM
E2 or 10 nM E1, vtg synthesis in hepatocytes induced by 100
␮M MXCL increased by a factor of 2.3 and 1.6, respectively
(Fig. 4). Vitellogenesis doubled after primary exposure of the
hepatocytes to 1 nM E2 and secondary exposure to BPA 100
␮M (Fig. 5). No influence on vitellogenesis was observed
comparing cells exposed to 100 ␮M BPA with cells pretreated
with 1 or 10 nM E1 and subsequent exposure to BPA (100␮M;
Fig. 5).
FIG. 2. Effects of BPA or MXCL pretreatment on vitellogenesis after
secondary exposure to estradiol in carp hepatocytes. Error bars represent SD of
12 determinations. *Values significantly different from their controls (without
primary exposure; p ⬍ 0.01). Controls were exposed to an equivalent concentration of DMSO only.
DISCUSSION
Changes in the synthesis of vtg were observed in the hepatocytes of individual carps after exposure to xenoestrogens,
rather caused by a compound- and concentration-dependent
effect than by cytotoxic effect. MTT activity remained unchanged for most of the compounds tested. Only exposure to
50 or 100 ␮M MXCL reduced MTT activity. However, exposure to both concentrations of MXCL still increased vitello-
FIG. 3. Effects of BPA or MXCL pretreatment on vitellogenesis after
secondary exposure to estrone in carp hepatocytes. Error bars represent SD of
12 determinations. *Values significantly different from their controls (without
primary exposure; p ⬍ 0.01). Controls were exposed to an equivalent concentration of DMSO only.
78
RANKOUHI ET AL.
FIG. 4. Effects of E1 or E2 pretreatment on vitellogenesis after secondary
exposure to MXCL in carp hepatocytes. Error bars represent SD of 6 determinations, except for controls (n ⫽ 12). *Values significantly different from
their controls (without primary exposure; p ⬍ 0.01). Controls were exposed to
an equivalent concentration of DMSO only.
genesis, which would not be expected in the case of cytotoxicity of MXCL. The reduction of MTT activity of cells exposed
to 50 or 100 ␮M MXCL may be a function of a shift in energy
balance within the cells. Future experiments are necessary to
substantiate this suggestion. A decrease of MTT activity in
combination with an increase of vitellogenesis has been described earlier in the CARP-HEP assay by Smeets et al.
(1999a). Further investigation will also be necessary to explain
the mechanisms behind the increase of MTT activity after
pretreatment of hepatocytes with BPA (50 or 100 ␮M) and
secondary exposure to 1 or 10 nM E2. A comprehensive
microscopic examination of the hepatocytes, which was used
as an additional parameter for cell viability, did not reveal any
signs of cell decay, and the cell layers remained attached to the
bottom of the wells throughout the experiment (which is also a
sign of viability of the cells). Therefore, it can be concluded
that the added compounds did not influence the viability of the
cells.
From a quantitative point of view, changes in the synthesis
of vtg were observed in the hepatocytes of individual carps
after exposure to xenoestrogens. These quantitative differences
in vitellogenesis might be due, in part, to individual differences
in sensitivity to estrogens. Also the age of the fish and seasonal
variations in sensitivity to estrogens might play a role. Thus we
normalized the results of each experiment to the level of vtg
induced by 1000 nM E2 (without primary exposure) and expressed vtg synthesis as a percentage of this level for comparison purposes. However, when normalizing the results of the
different sets of experiments, we obtained comparable (qualitative) results in the dose response relationships exposing the
hepatocytes to xenoestrogens.
In spite of the fact that they have only little estrogenic
potency, MXCL and BPA are able to increase vitellogenin
synthesis significantly when combined with a subsequent exposure to E2 or E1. Vitellogenesis was doubled after primary
exposure to E2 followed by exposure to 100 ␮M MXCL or
BPA. Primary exposure to E1 almost doubled the synthesis of
vitellogenin after secondary exposure to MXCL (100␮M).
However, no enhancement of vitellogenesis by secondary exposure to BPA was observed after primary exposure to E1. A
possible explanation for the observed enhancement of vitellogenesis could be that pretreatment with the xenoestrogens at a
low dose level could induce the ER population (priming) at a
concentration below the threshold for vitellogenin synthesis in
males. Consequently, subsequent exposure to another xenoestrogen would induce vitellogenesis to a greater level than
without primary exposure, due to upregulation of the ER. This
hypothesis is supported by the findings from in vivo studies that
primary exposure to estradiol or to the xenoestrogen nonylphenol increased the number of estrogen binding sites. For example, repeated exposure of catfish (on days 1, 4, and 7) to 0.6
mg/kg E2 or to 60.5 mg/kg nonylphenol increased the number
of ER in the cytosolic fraction compared to control males
(Nimrod and Benson, 1997). Another example for the upregulation of the ER is the increased capacity of the uterus of
ovariectomized rats to bind E2 after E2 pretreatment for 3 days
(2 ␮g/100g) and subsequent exposure to E2 (2 ␮g/100g; Eisenfeld and Axelrod, 1966).
Since primary exposure to the endogenous estrogens followed by exposure to E1 or E2 failed to increase vitellogenesis,
factors other than upregulation of ER may affect vitellogenesis
if only endogenous estrogens are involved. There may be
different E2 receptor populations involved (with higher and
lower affinity for endogenous estrogens). It has been reported
that an ER population with a lower affinity for endogenous
estrogens increased when channel catfish were pretreated with
E2 (Nimrod and Benson, 1997). Changes in affinity for endogenous estrogens have also been reported for hepatic ER populations of brown and rainbow trout (Campbell et al., 1994). It
would appear that an increase in the number of hepatic ER in
FIG. 5. Effects of E1 or E2 pretreatment on vitellogenesis after secondary
exposure to BPA in carp hepatocytes. Error bars represent SD of 6 determinations, except for controls (n ⫽ 12). *Values significantly different from their
controls (without primary exposure; p ⬍ 0.01). Controls were exposed to an
equivalent concentration of DMSO only.
79
VITELLOGENIN PRODUCTION IN HEPATOCYTES
response to primary exposure to E1 and E2 is compensated by
a decrease in affinity to those estrogens. This could partly
explain the lack of enhancement of vitellogenesis observed by
primary and secondary exposure to endogenous estrogens in
the carp hepatocytes. Further studies, including receptor binding kinetics and affinities in these carp hepatocytes might
elucidate the mechanisms behind these effects.
The present and previous results obtained using the CARPHEP assay (Letcher et al., submitted; Smeets et al., 1999b)
demonstrate that BPA has a lower estrogenic potency than
MXCL. However primary exposure to BPA is more effective
in enhancing the vitellogenin production in response to E2 or
E1 than pretreatment with MXCL. A possible explanation for
these differences could be that while exposing hepatocytes for
period of 7 days to MXCL, not only the parent compound, but
also its bioactivated metabolites, bind to the ER. It has been
reported that the o-demethylated metabolites of MXCL have a
greater binding affinity to the hepatic ER of channel catfish and
the rat uterine estrogen receptor than their parent compound
(Nimrod and Benson, 1997; Ousterhout et al., 1981; Schlenk et
al., 1998). Therefore the bioactivated MXCL compounds probably induce vitellogenesis more than the parent compound
does. Probably the pretreatment period of 48 h is not long
enough to bioactivate MXCL as effectively as after 6 days of
secondary exposure.
In vivo experiments with rainbow trout showed that repeated
pretreatment of the animals with E1 increased the vitellogenic
response to estradiol treatment (van Bohemen et al., 1982).
This is in contrast to our in vitro results obtained with carp
hepatocytes. A possible reason for this difference may be that
the in vivo effects we described on vitellogenesis in the trout
experiments are more evident due to the greater frequency of
exposure. Both the pretreatment with E1 and the secondary
exposure to E2 were repeated daily for 7 days.
These results indicate that relatively high concentrations of
environmental pollutants with a weak estrogenic potency, such
as MXCL and BPA, can increase the sensitivity of carp hepatocytes in vitro to endogenous estrogens, using vitellogenesis
as endpoint. Compared to cells exposed only to E1 or E2,
primary exposure of carp hepatocytes to 100 ␮M MXCL or
BPA enhanced the vitellogenin induction in response to secondary E1 or E2 exposure with a factor 2 to 5 and 4 to 7,
respectively. Because of the interactive effects between xenoestrogens and endogenous estrogens, physiological effects
of xenoestrogens could be underestimated by basing an assessment solely on an in vitro estrogen-dependent response. It
should be noted that for MXCL the maximum contaminant
level in drinking water according to the EPA standards is 40
␮g/l (0.1 ␮M), and BPA in surface water samples did not
exceed a concentration range from 2 to 8 ␮g/l (predicted no
effect concentration is 64 ␮g/l [0.28 ␮M]; Staples et al., 2000).
Therefore the interactive effects with endogenous estrogens
that have been observed at rather high concentrations (50 or
100 ␮M) of MXCL and BPA could indicate that the observed
interactions, if occurring in environmental situations, might be
most relevant for those xenoestrogens that have a bioaccumulative potential.
REFERENCES
Arnold, S. F., Robinson, M. K., Notides, A. C., Guillette, L. J., Jr., and
McLachlan, J. A. (1996). A yeast estrogen screen for examining the relative
exposure of cells to natural and xenoestrogens. Environ. Health Perspect.
104, 544 –548.
Bulger, W. H., Muccitelli, R. M., and Kupfer, D. (1978). Studies on the in vivo
and in vitro estrogenic activities of methoxychlor and its metabolites. Role
of hepatic mono-oxygenase in methoxychlor activations. Biochem. Pharmacol. 27, 2417–2423.
Callard, I. P., and Ho, S.-M. (1987). Structure, biochemistry, and molecular
biology of vitellogenin (yolk). In Fundamentals of Comparative Vertebrate
Endocrinology I. (P. M. Chester-Jones, P. M. Ingleton, and J. G. Phillips,
Eds.), pp. 270 –282. Plenum Press, New York.
Campbell, P. M., Pottinger, T. G., and Sumpter, J. P. (1994). Changes in the
affinity of estrogen and androgen receptors accompany changes in receptor
abundance in brown and rainbow trout. Gen. Comp. Endocrinol. 94, 329 –
340.
Christiansen, L. B., Pedersen, K. L., Pedersen, S. N., Korsgaard, B., and
Bjerregaard, P. (2000). In vivo comparison of xenoestrogens using rainbow
trout vitellogenin induction as a screening system. Environ. Tox. Chem. 19,
1867–1874.
Chun, T.-Y., and Gorski, J. (2000). High concentrations of bisphenol A induce
cell growth and prolactin secretion in an estrogen-responsive pituitary tumor
cell line. Toxicol. Appl. Pharmacol. 162, 161–165.
Colborn, T., vom Saal, F. S., and Soto, A. M. (1993). Developmental effects
of endocrine-disrupting chemicals in wildlife and humans. Environ. Health
Perspect. 101, 378 –384.
Denizot, F., and Lang, R. (1986). Rapid colorimetric assay for cell growth and
survival. Modifications to the tetrazolium dye procedure giving improved
sensitivity and reliablity. J. Immunol. Methods 89, 271–277.
Eisenfeld, A. J., and Axelrod, J. (1966). Effect of steroid hormones, ovariectomy, estrogen pretreatment, sex and immaturity on the distribution of
3H-estradiol. Endocrinology 79, 38 – 42.
Eroschenko, V. P., Rourke, A. W., and Sims, W. F. (1996). Estradiol or
methoxychlor stimulates estrogen receptor (ER) expression in uteri. Reprod.
Toxicol. 10, 265–271.
Flouriot, G., Vaillant, C., Salbert, C., Pelissero, G., Guiraud, J. M., and
Valotaire, Y. (1993). Monolayer and aggregate cultures of rainbow trout
hepatocytes: Long-term and stable liver-specific expression in aggregates.
J. Cell Sci. 105, 407– 416.
Folmar, L. C., Denslow, N. D., Rao, V., Chow, M., Crain, D. A., Enblom, J.,
Marcino, J., and Guillette, L. J., Jr. (1996). Vitellogenin induction and
reduced serum testosterone concentrations in feral male carp (Cyprinus
carpio) captured near a major metropolitan sewage treatment plant. Environ.
Health Perspect. 104, 1096 –1101.
Gray, L. E., Jr., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R.,
Goldman, J., Slott, V., and Laskey, J. (1989). A dose-response analysis of
methoxychlor-induced alterations of reproductive development and function
in the rat. Fundam. Appl. Toxicol. 12, 92–108.
Guillette, L. J., Jr., Crain, D. A., Rooney, A. A., and Pickford, D. B. (1995).
Organization versus activation: The role of endocrine-disrupting contaminants (EDCs) during embryonic development in wildlife. Environ. Health
Perspect. 103(Suppl. 7), 157–164.
Jobling, S., Nolan, M., Tyler, C. R., Brighty, G., and Sumpter, J. P. (1998).
Widespread sexual disruption in wild fish. Environ. Sci. Technol. 32, 2498 –
2506.
80
RANKOUHI ET AL.
Jobling, S., and Sumpter, J. P. (1993). Detergent components in sewage
effluent are weakly oestrogenic to fish: An in vitro study using rainbow trout
(Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 27, 361–372.
Lazier, C. B., and MacKay, M. E. (1993). Vitellogenin gene expression in
teleost fish. In Biochemistry and Molecular Biology of Fishes, Vol. 2.
(P. W. Hochachka and T. P. Mommsen, Eds.), pp. 391– 405. Elsevier
Science, Amsterdam.
Mehmood, Z., Smith, A. G., Tucker, M. J., Chuzel, F., and Carmichael, N.G.
(2000). The development of methods for assessing the in vivo oestrogen-like
effects of xenobiotics in CD-1 mice. Food Chem. Toxicol. 38, 493–501.
Nimrod, A. C., and Benson, W. H. (1997). Xenobiotic interaction with and
alteration of channel catfish estrogen receptor. Toxicol. Appl. Pharmacol.
147, 381–390.
Olea, N., Pulgar, R., Pérez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell,
A., Pedraza, V., Soto, A.M., and Sonnenschein, C. (1996). Estrogenicity of
resin-based composites and sealants used in dentistry. Environ. Health
Perspect. 104, 298 –305.
Ousterhout, J., Struck, R. F., and Nelson, J. A. (1981). Estrogenic activities of
methoxychlor metabolites. Biochem. Pharmacol. 30, 2869 –2871.
Purdom, C. E., Hardiman, P. A., Bye, V. J., Eno, N. C., Tyler, C. R., and
Sumpter, J. P. (1994). Estrogenic effects of effluents from sewage treatment
works. Chem. Ecol. 8, 275–285.
Rodgers-Gray, T. P., Jobling, S., Morris, S., Kelley, C., Kirby, S., Janbakahsh, J.,
Harries, J. E., Waldock, M. J., Sumpter, J. P., and Tyler, C.R. (2000). Long-term
temporal changes in the estrogenic composition of treated sewage effluent and
its biological effects on fish. Environ. Sci. Technol. 34, 1521–1528.
Routledge, E. J., Sheahan, D., Desbrow, C., Brighty, G. C., Waldock, M., and
Sumpter, J. P. (1998). Identification of estrogenic chemicals in STW effluent. 2. In vivo responses in trout and roach. Environ. Sci. Technol. 32,
1559 –1565.
Safe, S., Connor, K., and Gaido, K. (1998). Methods for xenoestrogen testing.
Toxicol. Lett. 102–103, 665– 670.
Schlenk, D., Stresser, D. M., Rimoldi, J., Arcand, L., McCants, J., Nimrod,
A. C., and Benson, W. H. (1998). Biotransformation and estrogenic activity
of methoxychlor and its metabolites in channel catfish (Ictalurus punctatus).
Mar. Environ. Res. 46, 159 –162.
Sheeler, C.Q., Dudley, M. W., and Khan, S. A. (2000). Environmental estrogens
induce transcriptionally active estrogen receptor dimers in yeast: Activity potentiated by the coactivator RIP140. Environ. Health Perspect. 108, 97–103.
Shelby, M. D., Newbold, R. R., Tully, D. B., Chae, K., and Davis, V. L. (1996).
Assessing environmental chemicals for estrogenicity using a combination of in
vitro and in vivo assays. Environ. Health Perspect. 104, 1296 –1300.
Smeets, J. M. W., Rankouhi, T. R., Nichols, K. M., Komen, H., Kaminski,
N. E., Giesy, J. P., and van den Berg, M. (1999a). In vitro vitellogenin
production by carp (Cyprinus carpio) hepatocytes as a screening method for
determining (anti) estrogenic activity of xenobiotics. Toxicol. Appl. Pharmacol. 157, 68 –76.
Smeets, J. M. W., van Holsteijn, I., Giesy, J. P., Seinen, W., and van den Berg,
M. (1999b). Estrogenic potencies of several environmental pollutants, as
determined by vitellogenin induction in a carp hepatocyte assay. Toxicol.
Sci. 50, 206 –213.
Sonnenschein, C., and Soto, A. M. (1998). An updated review of environmental estrogen and androgen mimics and antagonists. J. Steroid Biochem. Mol.
Biol. 65, 143–150.
Staples, C. A., Dorn, P. B., Klecka, G. M., O’Block, S. T., Branson, D. R., and
Harris L. R. (2000). Bisphenol A concentrations in receiving waters near US
manufacturing and processing facilities. Chemosphere 40, 521–525.
van Bohemen, C. G., Lambert, J. G., and Van Oordt, P. G. (1982). Vitellogenin
induction by estradiol in estrone-primed rainbow trout, Salmo gairdneri.
Gen. Comp. Endocrinol. 46, 136 –139.