52, 178 –188 (1999)
Copyright © 1999 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
The Anti-Estrogenicity of Ah Receptor Agonists in Carp (Cyprinus
carpio) Hepatocytes
Jean M. W. Smeets,* Ineke van Holsteijn,* John P. Giesy,† and Martin van den Berg* ,1
*Research Institute of Toxicology, Utrecht University, Utrecht, The Netherlands; †Department of Zoology, National Food Safety and Toxicology Center,
Institute of Environmental Toxicology, Michigan State University, East Lansing, Michigan
Received February 26, 1999; accepted July 16, 1999
Cultured hepatocytes of female carp (Cyprinus carpio) were
coexposed for 4 days to 200 nM 17b-estradiol (E2), and concentration ranges of nine known Ah receptor (AhR) agonists: 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), 3,3*4,4*5-pentachlorobiphenyl (PCB 126), 2,3*4,4*5-pentachlorobiphenyl (PCB 118),
b-naphthoflavone (BNF), benzo(a)pyrene (BaP), benzo(a)anthracene (BaA), diindolylmethane (DIM), 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) and hexachlorobenzene (HCB). TCDD
caused a greater than 100-fold induction of cytochrome P4501A
(CYP1A) activity, measured as ethoxyresorufin O-deethylase
(EROD), with an EC 50 of 6 pM. Based on EC 50 values, the order of
potency as CYP1A inducers was TCDD > PCB 126 > BNF >
BaP > BaA > PCB 118. DIM and MCDF caused a lower maximum CYP1A induction (< 9-fold), whereas HCB caused no
EROD induction at concentrations up to 6 mM. TCDD, PCB 126,
BNF, BaP, and DIM also caused a concentration-dependent suppression of the secretion of the yolk protein vitellogenin (Vtg),
relative to E2-treated hepatocytes. Suppression of Vtg secretion
was not directly correlated with EROD activity, and the antiestrogenic effects occurred at higher concentrations than the induction of CYP1A. This indicates that the anti-estrogenicity was
not caused by increased metabolism of E2 due to induction of
CYP1A. Nevertheless, the order of potency of the tested compounds for suppression of Vtg secretion was comparable to the
order of potency for CYP1A induction. This concurrence suggests
that the anti-estrogenicity of these compounds is AhR-mediated,
but does not involve CYP1A. This could be relevant for feral fish
populations, as they are frequently exposed to AhR agonists, to an
extent that AhR-mediated effects are observed.
Key Words: TCDD; Ah receptor; vitellogenin; CYP1A; hepatocytes; in vitro; fish; anti-estrogen.
In recent years there has been increasing attention to the
effects of estrogen-mimicking compounds in the aquatic environment. Among the better-documented effects of xenoestrogens is the induction of the yolk protein precursor vitellogenin
(Vtg) in male fish (Sumpter and Jobling, 1995). However,
anti-estrogenic effects have also been reported in fish. Female
To whom correspondence should be addressed. Fax: 131-30 –2535077.
E-mail: m.vandenberg@ritox.vet.uu.nl.
1
English sole (Parophrys vetulus) from contaminated sites exhibited reduced gonadal recrudescence (Johnson et al., 1988).
In other fish species, aryl hydrocarbon receptor (AhR) agonists
like 3,394,49-tetrachlorobiphenyl (PCB 77), Aroclor 1254, and
benzo(a)pyrene (BaP), inhibited secretion of Vtg or impaired
gonadal development (Chen et al., 1986; Monosson et al.,
1994; Thomas, 1990; Wannemacher et al., 1992). Vtg production in the liver of fish is controlled by 17b-estradiol (E2) and
is crucial for oocyte maturation (Wallace, 1985). Therefore, a
reduction of Vtg synthesis could result in impaired gonadal
development and reduced fertility.
AhR agonists include naturally occurring substances, but
also ubiquitous aquatic contaminants such as polycyclic aromatic hydrocarbons (PAHs), polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs)
(Safe, 1995). Some of the most potent agonists, e.g., PCBs,
PCDDs, and PCDFs, bioaccumulate in aquatic organisms because of their lipophilic attributes and their resistance to metabolism. The presence of the AhR has been established in
several mammalian and fish species (Hahn et al., 1994; Safe,
1995; Stegeman and Hahn, 1994), and activation of this receptor has been related to a diverse spectrum of biochemical and
toxic responses in these animals (Safe, 1990). In rodents,
PCDDs and PCDFs cause inhibition of several E2-induced
responses, suggesting a relationship between AhR activation
and anti-estrogenicity (Safe, 1995). Experiments in human
breast cancer cell lines have confirmed this relationship by
demonstrating parallel structure–activity relationships for AhR
binding and anti-estrogenic effects (Krishnan and Safe, 1993;
Spink et al., 1994).
Effects of AhR agonists on fish suggest that these compounds may be anti-estrogenic in fish, as they are in mammals.
In the present study, anti-estrogenicity was investigated using
cultured hepatocytes of female carp (Cyprinus carpio), which
can be induced in vitro to synthesize and secrete Vtg (CARPHEP assay) (Smeets et al., 1999a). Carp hepatocytes were
coexposed to E2 and AhR agonists from different compound
classes and of varying potency. These compounds include
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the planar PCB
3,394,495-PCB (PCB 126), the mono-ortho PCB 2,394,495-
178
179
ANTI-ESTROGENICITY IN CARP HEPATOCYTES
PCB (PCB 118), and 6-methyl-1,3,8-trichlorodibenzofuran
(MCDF) (Safe, 1990; Safe, 1995). MCDF has been characterized in mammalian systems as an AhR-binding compound that
is relatively more potent as an anti-estrogen than as inducer of
cytochrome P4501A1 (Safe, 1995). In addition, b-naphthoflavone (BNF), hexachlorobenzene (HCB), the PAHs BaP and
benzo(a)anthracene (BaA), and diindolylmethane (DIM) were
tested. DIM is the more potent gastric self-condensation product of indole-3-carbinol, a constituent of cruciferous vegetables
possessing anti-estrogenic as well as AhR-activating potency
in mammalian systems (Bjeldanes et al., 1991; Bradlow et al.,
1991). The effects of these compounds on Vtg secretion by
carp hepatocytes were compared to their potencies as inducers
of the monooxygenase cytochrome P4501A (CYP1A), which
is one of the most sensitive AhR-mediated responses in many
species (Safe, 1990).
MATERIALS AND METHODS
Animals. Fish used in these experiments were genetically uniform all
female, F1-hybrid progenies of common carp ( Bongers et al., 1994; Komen et
al., 1991). All fish were raised at 25°C in the hatchery of the research group
Fish Culture and Fisheries (Wageningen Agricultural University) and transported to Utrecht 1 to 2 months prior to their use. Subsequently, fish were kept
in Utrecht municipal tapwater at a constant temperature of 24°C. Female carp
(22–30 cm) were approximately 1 year old and possessed gonads with oocytes
in various stages of vitellogenesis.
Cell culture and exposure. Hepatocytes were isolated, cultured and exposed as described earlier (Smeets et al., 1999a). Briefly, a two-step perfusion
technique was used in which a portion of the liver was perfused, first with a
Ca 21- and Mg 21-free medium containing EDTA (0.145 M NaCl; 5.4 mM KCl;
5 mM EDTA; 1.1 mM KH 2PO 4; 12 mM NaHCO 3; 3 mM NaH 2PO 4; 100 mM
HEPES; pH 7.5), and subsequently with medium containing 0.26 mg/ml of
collagenase D (Boehringer, Mannheim, Germany). The perfused liver sections
were then removed, minced, and sieved through nylon mesh. Subsequently, the
hepatocytes were washed three times and resuspended in culture medium The
yield was 1.5 to 4 310 8 cells. Viability was . 90% as assessed with trypan
blue exclusion. The percentage of erythrocytes present was variable but never
exceeded 10%.
The hepatocytes were cultured in phenol red free DMEM/F12 medium
(D2906, Sigma, St. Louis, MO, USA), supplemented with 14.3 mM NaHCO 3,
HEPES (final concentration 20 mM), 50 mg/l gentamycin, 1 mM insulin, 10
mM hydrocortisone, 2% v/v Ultroser-SF (steroid-free) serum (Jones Chromatography, Mid Glamorgan, UK) and 2 mg/l of the protease inhibitor aprotinin
(Fluka, Buchs Switzerland) at pH: 7.4. Cells were seeded in 96-well tissue
culture plates (Greiner, Alphen a/d Rijn, the Netherlands) at 1 3 10 6 cells/ml,
0.18 ml/well, and maintained in air at 24°C. Each concentration of a compound
was tested in six wells on one plate, unless stated otherwise. After 1 day of
acclimatization, the cells were exposed for 4 days to E2 and the test compounds. Toxicant-containing medium was renewed after 2 days9 exposure.
Compounds were dissolved in DMSO (final concentration 0.2% v/v) except
E2 (Sigma Chemical Co., St. Louis, MO, USA), which was dissolved in
ethanol. BaP was obtained from Sigma Chemical Co. (St. Louis, MO, USA)
and a- and b-naphthoflavone (ANF and BNF) (99.5%) from Janssen Chimica
(Geel, Belgium). TCDD originated from Dow Chemical (Midland, USA) and
PCB 126 from Schmidt BV (Amsterdam, the Netherlands). BaA and HCB
were obtained from Riedel de Haën AG (Seelze, Germany) and PCB 118 from
Cambridge Isotope Laboratories Inc. (Woburn, MA, USA). DIM was a gift
from Ir. J. Vuik (TNO Toxicology and Nutrition Institute Zeist, the Netherlands) and MCDF was a gift from Prof. S. Safe (Texas A&M Univ., Dept. of
Veterinary Physiology and Pharmacology). After 4 days9 exposure, culture
medium was transferred into 96-well plates, frozen, and kept at –70°C prior to
analysis of Vtg content. Remaining cell monolayers were either used immediately for determination of cell viability, or frozen and kept at –70°C prior to
EROD determinations.
Cell viability, CYP1A activity, and protein. The viability of cells, exposed
for 4 days to the various compounds, was assessed by measuring mitochondrial
dehydrogenase activity, using MTT (3-(4,5-dimethyl-thiazol-2yl]-2,5-diphenyltetrazolium bromide) as a substrate (Denizot and Lang, 1986). In addition,
leakage of lactate dehydrogenase (LDH) was measured, which is indicative of
membrane integrity (Bergmeyer et al., 1965). CYP1A activity was measured as
ethoxyresorufin O-deethylase activity (EROD) (Burke and Mayer, 1974). The
determination of viability parameters and EROD activity were performed as
described earlier (Smeets et al., 1999a). Protein was measured according to the
method of Bradford (Bradford, 1976).
Vtg determination. The secretion of Vtg into the culture medium was
quantified by means of an indirect competitive ELISA as described earlier
(Smeets et al., 1999a). Ninety-six-well EIA/RIA plates were coated with
diluted blood plasma of an E2-treated female carp, containing approximately
45 mg/ml of Vtg as the dominant protein. The same plasma was used in a
duplicate standard dilution curve on every plate (28,125- to 7,200,00- fold
dilutions). Medium samples were diluted 100- to 2000-fold, in order to obtain
Vtg concentrations that were within the linear part of the log-transformed
standard curve. Every sample was measured in duplicate at two dilutions
differing by a factor of 2. The primary antibody used was a polyclonal rabbit
antibody against goldfish (Carassius auratus) Vtg that cross-reacts with Vtg
from other cyprinid species such as carp (Nichols et al., 1999). The secondary
antibody was an alkaline phosphatase conjugated monoclonal mouse antirabbit IgG (clone RG-96, Sigma, St. Louis, MO, USA). 4-Methylumbelliferylphosphate was used as substrate and quantified with a fluorescence plate
reader (excitation, 360 nm; emission, 460 nm).
Calculations and statistics. Dose–response curves were fitted using the
dose–response or sigmoidal curve fit option of the graphics computer program
Slide Write Plus for Windows version 4.0 (Advanced Graphics Software Inc.,
Carlsbad, Ca, USA). Curve fits of the EROD dose–response relationships were
calculated using only the rising part of the curve in order to make a sigmoidal
fit possible. Statistical significance (p , 0.05) was calculated using a two-way
ANOVA.
RESULTS
CYP1A Induction in Carp Hepatocytes
All compounds induced EROD activity, in a concentrationdependent manner (Figs. 1– 4), with the exception of HCB,
which did not cause significant induction of EROD activity
over a concentration range of 6 nM to 6 mM (data not shown).
In order of increasing EC 50 values, the most potent EROD
inducers were: TCDD, PCB 126, BNF, BaP and BaA (Table
1). These compounds induced EROD activity to a maximum
value between 100 and 200 pmol/mg cellular protein/min,
whereas control activity was below the detection limit of 1.7
pmol/mg/min. PCB 118, MCDF, and DIM were less potent and
caused maximum EROD activities between 10 and 25 pmol/
mg/min. The EROD induction levels and EC 50 values of these
compounds also depended on the exposure time (Figs. 1– 4,
Table 1). TCDD, PCB 126, and BNF had a lower EC 50 value
after 96-h exposure than after 18 h (Table 1). In contrast, BaP,
BaA, MCDF, and DIM had lower EC 50 values and lowest
observed effect concentrations (LOECs) after 18-h exposure
180
SMEETS ET AL.
FIG. 1. EROD induction, Vtg production, and cytotoxicity in carp hepatocytes coexposed to 200 nM E2 and various concentrations of TCDD or PCB 126.
MTT activity (■) is expressed as percentage of the activity in the solvent control. LDH leakage (h) is expressed as percentage LDH activity in the culture
medium relative to the total activity. Vtg production (●) is expressed as percentage of the Vtg production by 200 nM E2. EROD induction was measured after
18-h exposure (}), or 96-h exposure ({). Error bars represent SD of six or four (LDH, EROD 18 h) measurements.
than after 96 h (Figs. 2– 4). To investigate the effect of E2 on
TCDD-induced CYP1A induction, hepatocytes were coexposed to 6 nM TCDD and a concentration range of E2 (20 to
600 nM). No significant effect of E2 was observed on EROD
induction by TCDD (data not shown).
Anti-estrogenicity of AhR Agonists
Anti-estrogenicity was measured as reduced secretion of Vtg
by carp hepatocytes coexposed to the test compounds and 200
nM E2. BaA, PCB 118, HCB (not shown), and MCDF did not
cause a significant reduction in vitellogenesis at the concentrations tested (Figs. 3, 4; Table 1). Only at the highest concentrations of BaA and PCB 118, was a slight downward trend
in Vtg secretion observed. In contrast, the highest tested concentrations of TCDD, PCB 126, BNF, BaP, and DIM all
caused a significant reduction of Vtg secretion to less than 50%
of the Vtg secretion caused by 200 nM E2 (Figs. 1–3; Table 1).
The anti-estrogenic potency of these compounds is expressed
as the IC 50 for Vtg secretion, which is the concentration that
caused 50% reduction in Vtg relative to 200 nM E2 (Table 1).
To investigate whether the anti-estrogenic effects of TCDD and
BNF are additive, a single concentration of TCDD (60 pM) was
combined with three concentrations of BNF (0.6, 2, and 6 mM)
(Fig. 5). TCDD alone caused a 49% reduction of Vtg production
relative to 200 nM E2. The three tested BNF concentrations alone
caused 52 to 81% reduction of Vtg. The combination of 60 pM
TCDD and these three BNF concentrations caused a significantly
greater decrease in Vtg secretion than the single compounds (76 to
85%) (Fig. 5). Coaddition of BNF had no significant ( p, 0.05)
effect on EROD induction by TCDD, as EROD was already
maximally induced by 60 pM TCDD (data not shown).
Four days9 exposure to TCDD, PCB 126, BaP, or BaA at the
concentrations that were also used in the anti-estrogenicity
experiments did not significantly ( p, 0.05) induce Vtg secretion in carp hepatocytes (not shown). This indicates that these
compounds do not have estrogenic activity.
ANTI-ESTROGENICITY IN CARP HEPATOCYTES
181
FIG. 2. EROD induction, Vtg production, and cytotoxicity in carp hepatocytes coexposed to 200 nM E2 and various concentrations of BNF or DIM. MTT
activity (■) is expressed as percentage of the activity in the solvent control. LDH leakage (h) is expressed as percentage LDH activity in the culture medium
relative to the total activity. Vtg production (●) is expressed as percentage of Vtg production by 200 nM E2. EROD induction was measured after 18-h exposure
(}), or 96-h exposure ({). Error bars represent SD of six or four (LDH, EROD 18 h) measurements.
Effects on Cell Viability
Viability of carp hepatocytes was determined by measurement of MTT activity and LDH leakage. LDH leakage was not
significantly increased by exposure to E2, TCDD, PCB 126, or
BNF, at any concentration. In contrast, MTT activity was
significantly reduced by 21 to 29% in cells exposed to 200 nM
E2, relative to solvent-treated hepatocytes (Figs. 1– 4). The
most potent CYP1A-inducing compounds, TCDD, PCB 126,
and BNF, caused an additional significant decrease in MTT
activity of maximally 33 to 43% relative to E2-treated cells
(Figs. 1 and 2). The decrease of MTT activity occurred at
concentrations where EROD induction was maximal. However, at the highest concentrations of TCDD, PCB 126, and
BNF, MTT activity was less suppressed. BaP, BaA, and PCB
118 also caused a significant 17 to 26% decrease of MTT
activity relative to E2-treated cells, but only at the highest
tested concentrations of these compounds (Figs. 3 and 4).
MCDF and HCB (not shown) had no significant effect on MTT
activity (Fig. 4), whereas 50 mM DIM caused a significant
increase in MTT activity to 101 6 13% of the activity in
hepatocytes exposed only to DMSO.
The Effects of Coexposure to TCDD and ANF
Because ANF acts as an AhR antagonist in some experimental models, it was investigated whether this compound can
inhibit the anti-estrogenicity and CYP1A induction of TCDD.
Therefore, carp hepatocytes were exposed to TCDD (0.06 and
6 nM) and ANF (1, 5, 25 mM) alone and in combination. ANF
(25 mM) caused a $ 9-fold induction of EROD activity (Fig.
6). EROD induction was more pronounced after an 18-h exposure period then after 96-h exposure (Fig. 6). Twenty-five
micromolar ANF also caused a significant 39% decrease in Vtg
secretion relative to E2-treated hepatocytes (Fig. 7). MTT
activity was not significantly influenced by exposure to ANF at
these concentrations (not shown).
Coexposure of hepatocytes to ANF and TCDD enhanced the
182
SMEETS ET AL.
FIG. 3. EROD induction, Vtg production, and cytotoxicity in carp hepatocytes coexposed to 200 nM E2 and various concentrations of BaP or BaA.
MTT-activity (■) is expressed as percentage of the activity in the solvent control. Vtg production (●) is expressed as percentage of Vtg production by 200 nM
E2. EROD induction was measured after 18-h exposure (}), or 96-h exposure ({). Error bars represent SD of six or four (EROD 18 h) measurements.
anti-estrogenicity of TCDD, rather than cause inhibition (Fig.
7). Coexposure to ANF also caused a significant decrease of
TCDD-induced EROD activity, but only at the highest concentration of ANF (25 mM) (Fig. 6). To investigate whether
ANF decreased EROD activity by direct catalytic inhibition of
the CYP1A enzyme, 1, 5, or 25 mM ANF were added during
the EROD incubations of TCDD-induced hepatocytes. These
concentrations of ANF caused significant reductions of EROD
activity by 58, 89, and 96%, respectively, relative to EROD
incubations without ANF (data not shown). This indicates a
direct catalytic inhibition of CYP1A by ANF.
DISCUSSION
CYP1A Induction in Carp Hepatocytes
CYP1A induction is one of the most sensitive markers for
activation of the AhR. In the present study, all compounds
except HCB caused induction of CYP1A activity. Therefore
these substances have AhR agonist properties in carp. In the
case of TCDD, PCB 126, BNF, BaP, and DIM, EROD induction decreased with increasing concentration after reaching a
maximum. This is not in agreement with the general theory of
receptor-mediated enzyme induction, which predicts a sigmoidal dose–response relationship. Decreases of EROD induction
at high concentrations of inducer have been previously observed, both in vitro and in vivo (Haasch et al., 1993; Smeets
et al., 1999b). A decline of EROD activity often does not
coincide with a decrease in CYP1A protein or mRNA. The
effect is thought to be caused by inhibition of CYP1A due to
(competitive) binding of residual compound to the catalytic site
of the enzyme (Hahn et al., 1993). This phenomenon can lead
to underestimation of the maximum EROD induction and EC 50,
and consequently to overestimation of the AhR-activating potency of a compound. Biotransformation can also influence the
dose–response relationships of AhR agonists, depending on the
exposure period. The LOECs and EC 50 values of DIM, MCDF,
BaP, and BaA were higher after 96-h exposure than after 18 h.
Biotransformation may have lowered the effective concentra-
ANTI-ESTROGENICITY IN CARP HEPATOCYTES
183
FIG. 4. EROD induction, Vtg production, and cytotoxicity in carp hepatocytes coexposed to 200 nM E2 and various concentrations of PCB 118 or MCDF.
MTT activity (■) is expressed as percentage of the activity in the solvent control. Vtg production (●) is expressed as percentage of Vtg production by 200 nM
E2. EROD induction was measured after 18-h exposure (}), or 96-h exposure ({). Error bars represent SD of six or four (EROD 18 h) measurements.
tions of these compounds during the 96-h exposure period. In
contrast, TCDD and PCB 126, which have a very slow biotransformation rate in many species (van den Berg et al., 1994),
had lower EC 50 values for EROD induction after the longer
exposure period. Lipophilic compounds such as these partition
into the cells. After a medium change, the effective concentrations of these compounds may therefore be higher than before
the medium change. To minimize the influence of biotransformation in the CARP-HEP assay, it may be more appropriate to
compare EROD-inducing potencies of compounds after an
exposure time of 18 h.
TCDD, PCB 126, BNF, BaP, and BaA all caused maximum
EROD activities between 100 and 200 pmol/mg/min, which
suggests that these compounds were full agonists of the AhR in
carp. Based on EROD EC 50 values, PCB 126 had a relative
potency (REP) of 0.04 to 0.08 compared to TCDD in carp
hepatocytes. This REP is similar to the proposed WHO/IPCS
Toxic Equivalency Factor (TEF) of 0.1 for PCB 126 in mammals, but higher than that proposed for fish (0.005) (van den
Berg et al., 1998). However, the latter TEF value is based on
early life stage mortality, which generally produces lower
REPs than those derived from in vivo or in vitro CYP1A
induction ( Bols et al., 1997; Newsted et al., 1995). BaP was
12,000-fold less potent than TCDD in carp hepatocytes after
18-h exposure. This in vitro potency is in agreement with data
from an in vivo study with carp in which a 40,000-fold greater
concentration of BaP was required to cause an EROD induction that was comparable to that of TCDD (van der Weiden,
1993; van der Weiden et al., 1994).
The low but significant induction of EROD activity by
MCDF and DIM in carp hepatocytes could indicate that these
compounds are partial AhR agonists. Mammalian studies have
shown that these compounds can be both agonists and antagonists of the AhR (Chen et al., 1996; Safe, 1995). In rats,
MCDF has a moderate to high binding affinity for the AhR, but
is . 1.5 3 10 5-fold less potent than TCDD as EROD inducer.
In addition, MCDF partly inhibits CYP1A1 induction by
TCDD (Astroff and Safe, 1988; Astroff et al., 1988). Similarly,
DIM can cause CYP1A1 induction in rats and bind to the AhR
with an affinity 25,000-fold less than that of TCDD (Jellinck et
184
SMEETS ET AL.
TABLE 1
EROD Induction and Reduction of Vitellogenesis in Carp Hepatocytes Exposed to 200 nM E2 and Concentration Ranges of AhR
Agonists for 18 and 96 H, Respectively
EROD (18 h)
EROD (96 h)
EC 50a
Compound
TCDD
PCB 126
BNF
BaP
BaA
DIM
MCDF
PCB 118
HCB
Vtg (96 h)
EC 50
IC 50c
mM
E maxb
mM
E max
3 3 10 –5
4 3 10 –4
0.09
0.4
0.5
4
0.4
$3
ND
186
134
138
90
66
10
15
$26
ND
6 3 10 –6
1.5 3 10 –4
0.07
0.7
2
10
$2
$3
ND
139
157
180
119
96
4
$5
$26
ND
mM
1310 –4
1310 –3
2
20
ND(.20)
30
ND
ND(. 6)
ND
Note. ND, not detected.
a
Concentration at which EROD induction was 50% of the maximum induction by this compound.
b
Maximum EROD activity induced by this compound in pmol resorufin/mg cellular protein/min.
c
Concentration that caused a 50% decrease of Vtg secretion.
al., 1993). However, in human breast cancer cells, DIM inhibited TCDD-induced CYP1A activity and mRNA (Chen et al.,
1996).
ANF caused a low induction of EROD activity in carp
hepatocytes, indicating that ANF is a partial AhR agonist in
carp, similar to MCDF and DIM. ANF also partly inhibited
TCDD-induced EROD activity (Fig. 6). As we have shown,
ANF can cause direct catalytic inhibition of CYP1A in carp.
However, ANF may also have an inhibiting effect on AhR
activation by TCDD. In rats and human breast cancer cells,
ANF is a (partial) AhR antagonist, as it binds the AhR but does
not cause receptor activation (Gasiewicz and Rucci, 1991;
Merchant et al., 1993)
PCB 118 did not promote a maximum induction of EROD
activity over the dose range tested in this study. Based on
LOECs, PCB 118 had a potency of 10 – 6 relative to TCDD. This
low REP is in agreement with the general observation that
mono-ortho PCBs have lesser AhR potency in fish (TEF , 5 3
10 – 6) than in mammals (TEF 1 3 10 – 4) (van den Berg et al.,
1998). HCB did not cause CYP1A induction in carp hepatocytes. Based on the LOEC of TCDD for EROD induction in
this study (0.6 pM), a possible REP of less than 10 –7 can be
FIG. 5. Vtg production in carp hepatocytes coexposed for 4 days to 200
nM E2 1 BNF or to E2 1 60 pM TCDD 1 BNF. Error bars represent SD of
six measurements. *Significantly different (p , 0.05) from BNF only.
FIG. 6. EROD induction in carp hepatocytes coexposed for 18 or 96 h to
200 nM E2 1 ANF; for 96 h to E2 1 60 pM TCDD 1 ANF; or to E2 1 6 nM
TCDD 1ANF. Error bars represent SD of six (96 h), or four (18 h) measurements. *Significantly different (p , 0.05) from respective control (DMSO).
ANTI-ESTROGENICITY IN CARP HEPATOCYTES
185
CYP1A Induction, E2 Metabolism, and Anti-estrogenicity
FIG. 7. Vtg production in carp hepatocytes coexposed for 4 days to 200
nM E2 1 ANF; to E2 1 60 pM TCDD 1 ANF; or to 200 nM E2 1 6 nM
TCDD 1 ANF. Error bars represent SD of six measurements. Nd, not determined. *Significantly different (p , 0.05) from respective control (DMSO).
calculated for HCB in carp. This is three orders of magnitude
lower than the REP that has been proposed based on data from
rat and chicken (van Birgelen, 1998).
Cell viability, CYP1A Induction, and Vtg Secretion
The most potent AhR agonists, TCDD, PCB 126, and BNF,
were not cytotoxic at the tested concentrations, as evidenced by
the lack of LDH leakage. The observed suppression of Vtg
secretion by these compounds was therefore not caused by
reduced cell viability. The mentioned compounds did cause a
decrease of MTT activity. This secondary viability parameter
was suppressed at concentrations of the test compounds that
caused maximal EROD induction. In addition, exposure to 200
nM E2 caused a pronounced suppression of MTT activity.
However, this E2-induced suppression of MTT activity is not
indicative of reduced cell viability, but possibly a side effect of
large-scale Vtg production in female-derived carp hepatocytes
(Smeets et al., 1999a). In cultured trout hepatocytes, E2 treatment caused a lowering of glucose production relative to CO 2
production (Korsgaard and Mommsen, 1993). It is conceivable
that such a change in the energy balance of the hepatocytes
could result in decreased mitochondrial MTT activity. A similar relationship might exist between CYP1A induction and a
decrease of MTT activity. This would explain the concurrence
of decreased EROD activity and increased MTT activity at the
highest concentrations of TCDD, PCB 126, and BNF. These
hypotheses are also supported by the observation that 50 mM
DIM, which induced only a very low CYP1A activity, caused
a . 50% decrease of Vtg secretion concurrent with a 40%
increase in MTT activity relative to E2-treated cells.
The anti-estrogenic effects of the tested compounds could be
caused by accelerated metabolism of E2 in carp hepatocytes. In
human breast cancer cells (MCF-7), the anti-estrogenic effect
of dioxinlike compounds has been linked to the induction of
CYP1A-mediated E2 hydrolase activities (Spink et al., 1994).
However, there is strong evidence supporting nonmetabolic
pathways of anti-estrogenicity of AhR agonists in MCF-7 cells
(Safe, 1995). In the present study, no evidence was found for
a role of CYP1A in anti-estrogenic effects. The dose–response
curves of the suppression of Vtg secretion were shifted to
higher concentrations compared to the dose–response curves of
EROD induction. Furthermore, coexposure to the CYP1A antagonist ANF did not inhibit the anti-estrogenic effect of
TCDD, but rather enhanced it. These results are consistent with
the observation that CYP1A1 did not catalyze oxidative metabolism of E2 in BNF-treated scup (Stenotomus chrysops)
(Snowberger and Stegeman, 1987).
Another mechanism that could possibly accelerate the metabolism of E2 is AhR-mediated induction of the phase II
enzyme UDP-glucuronyltransferase (GT). Glucuronidation is
an important mechanism in the hepatic elimination of E2 in fish
(Forlin and Haux, 1985). However, studies in plaice have
shown that the GT isoenzymes responsible for 17b-steroid
conjugation are different from the main AhR-inducible GT
isoenzyme (George, 1994). This, and the expected low magnitude of GT induction, make it unlikely that it is the cause of
the observed decrease in Vtg secretion.
AhR-Mediated Anti-estrogenicity
The most potent AhR agonists, TCDD, PCB 126, BNF, and
BaP had the same order of potencies as anti-estrogens and as
CYP1A inducers. This supports the hypothesis that the antiestrogenicity of these compounds is AhR-mediated. Further
support is provided by the observation that the anti-estrogenic
effects of TCDD and BNF were additive, which would be
expected in the case of an AhR-mediated effect. The TEF
concept for AhR agonists applies to two compounds tested in
this study, namely the PHAHs TCDD and PCB 126. The
relative potency (REP) of PCB 126 for Vtg reduction was 0.1
compared to TCDD (based on IC 50s), which is very similar to
the REP for EROD induction after 18-h exposure (0.08). The
similarity of these two REPs suggests that the TEF concept
may also apply to the anti-estrogenic effects of AhR agonists.
This hypothesis is in agreement with previous observations in
rainbow trout (Oncorhynchus mykiss) hepatocytes (Anderson
et al., 1996a).
ANF is a known AhR antagonist in MCF-7 cells, inhibiting
the anti-estrogenic effect of TCDD (Merchant et al., 1993).
However, in carp ANF seems to be a partial AhR agonist
whose anti-estrogenic effect was additive to that of TCDD.
ANF, and also DIM, caused anti-estrogenicity despite the fact
that their maximum EROD induction was relatively low. Safe
186
SMEETS ET AL.
and coworkers (Safe, 1995) have shown that compounds can
be weak (partial) AhR agonists for CYP1A1 induction, and
still be potent anti-estrogens. For example, MCDF was 300 –
570 times less active than TCDD as an anti-estrogen in rat, but
it was more than 10 5-fold less potent as an inducer of CYP1A1
(Astroff and Safe, 1988). In this study, MCDF did not show
anti-estrogenic activity, but it was only tested at concentrations
as high as 4 mM, whereas ANF and DIM caused decreased Vtg
levels at concentrations of 25 and 20 mM, respectively.
AhR-mediated anti-estrogenicity has been extensively investigated in rodents and human breast cancer cells, but the
exact mechanism of the effect is still unclear. A number of
possible mechanisms have been proposed and the mechanism
may be tissue and effect specific (Safe, 1995). One of these
mechanisms is the AhR-mediated induction of phase I and II
enzymes involved in E2 metabolism, which has been discussed
in a previous paragraph. Other possible mechanisms of AhR
mediated reduction of Vtg secretion are the following: a)
binding of the activated AhR to a repressor site in the promotor
region of the Vtg gene, or induced synthesis of a modulatory
protein that binds this site ( Krishnan et al., 1995; White and
Gasiewicz, 1993; Zacharewski et al., 1994). b) Downregulation
of ER levels by activated AhR, resulting in reduced activation
of the Vtg gene (Chaloupka et al., 1992; Harris et al., 1990;
Wang et al., 1993; Zacharewski et al., 1991). This can be
caused by a repressor site in the promotor region of the ER
gene (White and Gasiewicz, 1993) or by inhibition of ER
synthesis at the post-transcriptional level (Wang et al., 1993).
c) Inhibited binding of E2 to the ER, either by direct interaction
with activated AhR, or by AhR-mediated induction of a gene
product that inhibits formation of the E2-ER complex. Alternatively, the activated AhR may inhibit binding of the E2-ER
complex to an estrogen-responsive element in the Vtg promotor region. The latter suggestion is supported by results from a
study with MCF-7 cells that found that activated ER and AhR
mutually inhibit the DNA-binding capacity of the receptor
complexes (Kharat and Saatcioglu, 1996). Our studies in carp
hepatocytes have demonstrated that E2 does not influence
CYP1A induction in this system. Therefore, they give no
support for a mechanism of mutual inhibition of the ER and
AhR (Smeets et al., 1999a).
Possible Implications for Feral Fish Populations
Induction of CYP1A is regularly observed in fish from
contaminated areas. It has been used as a biomarker for exposure of fish to PAHs and PHAHs (Goksoyr and Forlin, 1992).
Results presented in this study indicate that CYP1A induction
could coincide with a decrease in Vtg production in female
fish, confirming previous results in rainbow trout hepatocytes
(Anderson et al., 1996a). Few studies have investigated the
effects of AhR-activating substances on vitellogenesis in vivo.
In juvenile rainbow trout, Aroclor 1254 caused a decrease in
E2-induced Vtg levels (Chen et al., 1986), whereas in atlantic
croakers (Micropogonias undulatus), Aroclor 1254 and benzo(a)pyrene (BaP) caused a reduction of Vtg levels during the
vitellogenic period (Thomas, 1990). However, in rainbow trout
exposed simultaneously to E2 and BNF, Vtg levels both increased and decreased, compared to E2-treated fish, depending
on the dose levels (Anderson et al., 1996b). In the latter study,
no dose–response relationship was determined for CYP1A
induction by BNF, therefore, the effects on Vtg levels cannot
be directly compared to CYP1A induction. In carp hepatocytes,
the anti-estrogenic effects of all the tested compounds occurred
only at concentrations at which EROD induction was maximal.
Whether this difference between the dose–response relationships of these two effects will also occur in vivo and in other
fish species needs to be investigated further.
Vtg synthesis is essential for oocyte maturation (Wallace,
1985), hence a reduction of Vtg secretion due to dioxinlike
compounds could impair gonadal development in fish and
consequently reproduction. This adverse effect has been shown
in several in vivo studies. In zebrafish (Brachydanio rerio)
TCDD caused a dose-related reduction of egg numbers and
severely impaired development of previtellogenic and vitellogenic oocytes (Wannemacher et al., 1992). In female white
perch (Morone americana) the planar PCB 3,394,49-tetrachlorobiphenyl (PCB 77) impaired gonadal maturation (Monosson
et al., 1994). Moreover, field studies with white sucker (Catostomus commersoni) and English sole (Parophrys vetulus)
have found that CYP1A induction accompanied impaired gonadal development (Johnson et al., 1988; Munkittrick et al.,
1994). These adverse effects could be explained by AhRmediated inhibition of Vtg secretion by the liver. In addition,
both the ER and AhR are present in various tissues and at
different life stages. Therefore, anti-estrogenic effects of AhRagonists in extrahepatic tissues could be equally important or
more important than a reduction of Vtg secretion by the liver.
In conclusion, the results of this study have demonstrated
that CYP1A-inducing compounds can suppress the E2-induced
secretion of Vtg by carp hepatocytes. This anti-estrogenic
effect was not caused by the induction of CYP1A activity.
Nevertheless, the concurrence between the anti-estrogenicity
and the CYP1A-inducing potency of the compounds suggests
that the suppression of Vtg secretion was AhR mediated. These
results stress the importance of anti-estrogenic effects in the
aquatic environment next to the more frequently investigated
estrogenic effects.
ACKNOWLEDGMENTS
A donation of Shell International Oil Company is highly acknowledged.
Carp used in this study were a kind gift of Dr. Hans Komen of the research
group Fish Culture and Fisheries of Wageningen Agricultural University. The
Vtg antibody was developed by Krista M. Nichols at the Dept. of Fisheries and
Wildlife, Michigan State University (East Lansing, Michigan, USA), supported by grants from the Chlorine Chemistry Council of the Chemical
Manufacturers Association, the National Institutes of Health (grant NIH-ES-
ANTI-ESTROGENICITY IN CARP HEPATOCYTES
04911), and the U. S. Environmental Protection Agency Exploratory Research
Program (Biology Panel grant R825371– 01).
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