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Veterinary Physiology and Pharmacology (K.R., F.W., I-C.C, S.S), Texas A&M University, College Station, Texas 77843-4466; Department of Pharmacology (J.D.N., D.P.M.), Duke University Medical School, Durham, North Carolina 27709; Chemical Industry Institute of Toxicology (L.S.L., K.W.G.), Research Triangle Park, North Carolina 27709; and Laboratory of Reproductive and Developmental Toxicology (W.P.B., K.S.K.), National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: Stephen Safe, Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A recent study by Arnold and co-workers (18) reported synergistic interactions of binary mixtures of endosulfan, dieldrin, toxaphene, and chlordane in competitive estrogen receptor (ER)-binding assays and in an estrogen-responsive assay in yeast. Less dramatic synergistic interactions between two weakly estrogenic hydroxy PCB congeners were observed also in the yeast assay and in human endometrial cancer cells. Synergistic interactions between estrogenic chemicals have been reported also in other studies using a single concentration of several compounds and their reconstituted mixtures (6, 10, 19); however, the study by Arnold and coworkers (18) reported dramatic synergistic interactions (up to 1600-fold) by comparing the concentration-dependent effects of individual compounds and their binary mixtures. Based on these data, it was suggested that the estrogenic potency of some environmental chemicals, when tested singly, may be underestimated (18). This study has used two compounds, dieldrin and toxaphene, which gave synergistic responses as a binary mixture in the yeast and ER-binding assays in the study reported by Arnold and co-workers (18). The results of this study show that in the mouse uterus, interactions of dieldrin and toxaphene in ER-binding and functional assays were not synergistic; moreover, similar results were observed in MCF-7 human breast cancer cells and yeast-based reporter gene assays using both the mouse and human ER. Thus, the synergistic interactions of weakly estrogenic environmental chemicals are not universally observed in estrogen-responsive assays, and this should be considered in hazard assessment of these environmental contaminants.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
B6C3F1 female mice were bred in an onsite animal facility and
housed 6 to 9 per cage with ad libitum access to food and
water. Test compounds were dissolved in corn oil with slight warming,
and mice (n = 6–9 per group) received 0.1 ml corn oil (control),
E2 at a dose of 0.0053 µmol/kg·day, toxaphene (2.5, 15,
60, or 275 µmol/kg), dieldrin, or an equimolar toxaphene-dieldrin
mixture. The mixture was derived by combining equal molar combinations
of toxaphene and dieldrin. The solutions (0.1 ml per animal) were
administered ip for 3 consecutive days starting at 21 days of age.
Animals were killed by CO2 asphyxiation 20 h after the
last treatment, and uteri were excised, blotted, and weighed. The uteri
were then placed in ice-cold buffer and bisected, such that each half
contained an entire uterine horn.
Cell proliferation assay
MCF-7 cells (50,000) were seeded in six-well multiwell plates in
media containing 2 ml DME/F12 without phenol red, supplemented with 5%
FBS treated with dextran-coated charcoal (FBS-DCC), 2.2 g/liter sodium
bicarbonate, 10 mg/liter apotransferrin, and 20 µg/ml BSA. After
24 h, the media was changed, and the cells were treated with
10-9 M E2,
10-7–10-5 M concentrations of
toxaphene, dieldrin, or dieldrin plus toxaphene (equimolar
concentrations). The media and chemical treatment were changed every 2
days, and the total duration of the proliferation experiments was 11
days. Cells were then trypsinized, washed with media, resuspended in 1
ml of media, and counted using a Coulter counter, as previously
described (22). All determinations were carried out in triplicate, and
results are expressed as means ± SD.
Transient transfection assays
MCF-7 cells for proliferation and transient transfection studies
were maintained in MEM with phenol red and supplemented with 10% FCS
plus antibiotic/antimycotic solution, 0.035% sodium bicarbonate,
0.011% sodium pyruvate, 0.1% glucose, 0.238% HEPES, and 6 x
10-7% insulin. MCF-7 cells were then trypsinized,
reseeded in 100-mm petri dishes with 10 ml of phenol red-free DME/F-12
medium plus 5% charcoal-stripped FBS, and grown until 50–60%
confluent. About 3 h before transfection, medium was replaced with
5 ml charcoal-stripped DME/F-12 medium. Cells in each petri dish were
transfected with 1 ml transfection cocktail containing either 10 µg
CKB-CAT plus 5 µg hER or 5 µg CATH-CAT plus 4 µg hER, 50 µl of
2.5 M CaCl2, and 500 µl HBS (pH 7.05). After
incubation for 14 to 16 h at 37 EC, cells were washed once with 5
ml PBS and treated with test chemicals in 10 ml DCC-stripped DME/F-12
medium. After 48 h, cells were washed once with 5 ml PBS and
harvested by scrapping. Cells were lysed in 200 µl of 0.25
M Tris-Cl (pH 7.6) by three cycles of freezing in liquid
nitrogen for 2 min, thawing at 37 EC for 2 min, sonication for 3 min,
and vortexing for 1 min. Cell debris was pelleted, and the protein
concentration in the supernatant was determined (23) using BSA as
standard. An aliquot of cell lysate equal to 80 µg protein was
diluted to 120 µl with 0.25 M Tris-Cl (pH 7.6) and
incubated with 1 µl [14C]chloramphenicol (53 mCi/mmol)
and 42 µl of 4 mM acetyl CoA for 6 h at 37 EC. The
reaction was stopped by vortexing with 700 µl ethyl acetate. The
extract (600 µl) was dried, redissolved in 20 µl ethyl acetate, and
the acetylated products resolved by TLC (Whatman Lab Sales, Hillsboro,
OR) using a 95:5 chloroform:methanol solvent mixture. The percent
protein conversion into acetylated chloramphenicol was quantitated
using the counts/minute obtained from the Betagen Betascope 603 blot
analyzer (Intelligenetics, Inc., Mountain View, CA). The TLC plates
were subjected to autoradiography using a Kodak X-Omat film (Eastman
Kodak Co., Rochester, NY) for about 20 h.
Progesterone (PR) and ER-binding assays
The uterine bisections of each treatment group were pooled in an
ice-cold TESHMo (10 mM Tris-Cl, pH 7.4, 1.5 mM
EDTA, 15 mM thioglycerol, 10 mM sodium
molybdate) buffer, 1 ml/50 mg tissue. Uteri were homogenized with
3 x 8-sec bursts using a Brinkman/Polytron tissue grinder
(Brinkman Instruments Co., Westbury, NY). Samples were then centrifuged
for 45 min at 105,000 x g, and the clear supernatant,
constituting the cytosol for this experiment, was carefully decanted
and immediately used for competitive binding assays. Cytosolic
fractions described above were incubated with 20 nM
[3H]R5020 in the presence or absence of 2 µM unlabeled
R5020 at 4 EC. After an 8-h incubation, samples were placed on ice and
treated with 0.1 vol DCC suspension (0.5% dextran:5% charcoal, wt/vol
in TESHMo) for 10 min. Samples were then centrifuged at 5,000 x
g for 10 min, and the supernatant was measured by liquid
scintillation counting. PR levels were calculated assuming a 1:1
binding between PR and [3H]R5020. Levels are reported in
femtomoles per uterus. [3H]E2
(10-8 M) was used as a radioligand for
determining the competitive displacement of
[3H]E2 by different concentrations of the
test compounds. Uterine cytosol for ER binding was obtained from
untreated animals.
Uterine peroxidase (UPO) assay
Uterine bisections were pooled into treatment groups and
homogenized as described above. Homogenates were centrifuged at
39,000 x g at 2 EC for 45 min, and the resulting
pellet was washed and resuspended in 10 mM Tris-Cl buffer
containing 0.5 M CaCl2. The extract was
clarified by centrifugation for 45 min at 39,000 x g
at 2 EC. UPO activity of the supernatant was determined as described
(24). The assay mixture (3.0 ml total) contained 13 mM
guaiacol and 0.3 mM H2O2 in the
extraction buffer. The reaction was started by addition of 1.0 ml of
the CaCl2 extract. The initial rate (1 min) of guaiacol
oxidation was monitored at 
= 470 nm on a Beckman
spectrophotometer (Beckman Instruments Inc., Fullerton, CA). An enzyme
unit was defined as the amount of enzyme required to produce an
increase of 1 absorbance U per min under the assay conditions
described. Enzyme activity is expressed per milligram of extract
protein, measured by the method of Bradford (23).
ER binding in MCF-7 cells
A suspension of MCF-7 cells in 10 ml serum-free media was
incubated with 10-9 M
[3H]E2 alone or in the presence of 2 x
10-7 M unlabeled E2,
10-5 M toxaphene, 10-5
M dieldrin, or 10-5 M
dieldrin/toxaphene mixture for 1 h at 37 EC. Cells were harvested
by centrifugation and washed with PBS (20 ml x2). Cells were pelleted
by centrifugation, resuspended in 2 ml HED buffer (25 mM
HEPES, 1.5 mM EDTA, 1 mM dithiothreitol, pH
7.6), incubated on ice for 10 min, and vigorously homogenized using a
teflon pestle/drill apparatus. The homogenate was transferred to a
centrifuge tube, rinsed with 2 ml HED buffer, and centrifuged at
1500 x g at 4 EC for 15 min, and the supernatant was
collected and saved. The pellet was resuspended in 1 ml HEGDK (25
mM HEPES, 1.5 mM EDTA, 10% glycerol, 1
mM dithiothreitol, 0.5 M KCl; pH 7.6) and
incubated on ice for 1 h with gentle shaking. After centrifugation
at 105,000 x g for 1 h at 4 EC, the supernatant
was collected and combined with the supernatant noted above. Samples
were then placed on ice and treated with an 0.1 vol DCC suspension
(0.5% dextran, 5% charcoal, wt/vol in TESHMo) for 10 min. Samples
were then centrifuged at 5,000 x g for 10 min, and the
entire supernatant was measured for radioactivity by liquid
scintillation counting. The results are expressed as means ±
SE for three separate determinations.
Yeast expression/reporter system using the mouse and human ER
The yeast strain BJ2168 (MAT a, prc 1–407, prb 1–1122, pep
4–3, leu 2, trip-1, ura 3–52) was transformed with a yeast expression
plasmid containing the wild-type mER cDNA, and a reporter plasmid
containing a single vitellogenin estrogen responsive element (ERE)
linked to the LacZ gene (25). The transformations were
performed using the lithium acetate technique, and the yeast
transformants were selected by uracil and/or tryptophan auxotrophy. A
yeast culture was grown overnight at 30 EC in complete minimal dropout
medium lacking uracil and tryptophan. The yeast culture was diluted to
OD600 = 0.3 and grown for 2 h before the addition of
the environmental compounds alone or in combination (see Fig. 3
). The compounds tested in this system were dissolved
in dimethyl sulfoxide (DMSO) and 10 µl of an individual compound, or
5 µl each of two compounds were added to 1 ml of yeast culture such
that the concentration of DMSO did not exceed 1% by vol. The yeast
cultures were grown for an additional 2.5 h before harvesting for
measurement of ß-galactosidase (ß-gal) activity.
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The yeast strain BJ2407 (a/
prb1–1122/prb1–1122
prc1–407/prc1–407 pep4–3/pep4/3 leu2/leu2 trp1/trp1
ura3–52/ura3–52) was transformed with the ER expression plasmid
YePE10, and a reporter vector YRPE2 containing two copies of the
vitellogenin ERE was inserted into the promoter of an enhancerless
CYC-1-ß-gal fusion (26, 27). Individual transformants were grown
overnight to a density (OD600) of approximately 0.8 and
then combined with an equal volume of minimal medium containing copper
sulfate (to induce receptor synthesis from the CUP1 promoter),
E2, or the compound to be tested. The final concentration
of copper sulfate was 50 µM. Increasing concentrations of ligands
dissolved in DMSO were tested as indicated in the figure legends. Cells
were induced for 24 h in 96-well plates and then lysed, and
ß-galactosidase activity was measured. Relative ß-gal units were
calculated using the following formula: u = 1000 ·
[OD420 - (1.75 · OD550)]/(0.1 ·
OD600 · min).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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show that neither
dieldrin nor toxaphene (10-8–10-5
M) significantly bound to the mouse uterine ER in a
competitive binding assay using [3H]E2 as the
radioligand. In addition, no significant binding was observed using an
equimolar concentration of a dieldrin/toxaphene mixture (Fig. 1), and
these results contrast with previous studies that reported synergistic
binding of this mixture to recombinant baculovirus expressed hER (18).
The estrogenic effects of toxaphene, dieldrin, toxaphene plus dieldrin,
and E2 were determined in the immature B6C3F1 mouse uterus
(Table 1). Treatment with E2 (0.0053
µmol/kg·day) resulted in a 3.1-, 4.8-, and 7.8-fold increase in
uterine wet weight, peroxidase activity, and PR binding, respectively.
At doses of 2.5, 15, and 60 µmol/kg·day (x3), dieldrin did not
significantly induce uterine wet weight or PR binding; however, a
slight increase in UPO activity was observed only at the intermediate
dose of dieldrin (15 µmol/kg). The highest dose of dieldrin used in
this study (275 µmol/kg) was lethal to the animals. Similar results
were obtained with the dieldrin/toxaphene mixture (275 µmol/kg). The
dose-dependent estrogenic effects of toxaphene also were investigated
in the immature mouse uterus (Table 1). No significant increases were
observed in uterine wet weight or PR binding at any of the doses (2.5,
15, 60, and 275 µmol/kg·day). UPO activity was increased slightly
at the three lower doses, whereas all the uterine responses were
decreased at the 275-µmol/kg dose, and this was caused by some
toxicity observed in this treatment group. Immature mice also were
cotreated with equimolar doses of toxaphene and dieldrin combined (2.5,
15, and 60 µmol/kg·day x3) to investigate possible synergistic
interactions between the two weakly estrogenic pesticides, as
previously described (18). The results (Table 1) showed that treatment
with the binary mixture did not cause a dose-dependent increase in
uterine wet weight, peroxidase activity, or PR binding; however, PR
levels at the 2.5- and 60-µmol/kg·day dose were significantly
induced, whereas no significant induction was observed with the
compounds alone.
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indicate that at concentrations
ranging from 10-8–10-5 M,
neither toxaphene nor dieldrin induced CAT activity in MCF-7 cells
transiently transfected with either the CKB-CAT or CATH-CAT constructs.
In contrast, 10-9 M E2 induced a
24-fold increase in CAT activity in MCF-7 cells transiently transfected
with CKB-CAT and a 3.8-fold increase in cells transiently transfected
with the CATH-CAT construct. Combined treatment of MCF-7 cells with an
equimolar mixture of dieldrin plus toxaphene
(10-8–10-5 M) resulted in no
induced response and was similar to that observed for the individual
compounds. The results in Fig. 2 summarize the
concentration-dependent effects of toxaphene, dieldrin, and toxaphene
plus dieldrin (equimolar concentrations) on proliferation of MCF-7
cells. There was not a concentration-dependent increase in cell
proliferation by either compound alone or the mixture
(10-7–10-5 M); however, small
but significant increases in cell proliferation were observed for
toxaphene and dieldrin plus toxaphene at the 10-7
M concentration. In contrast to both organochlorine
pesticides, E2 induced a 6-fold increase in MCF-7 cell
proliferation.
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demonstrate that binding of [3H]E2 to the ER
in MCF-7 cells was not competitively decreased by cotreatment with
10-5 M toxaphene, dieldrin, or the
toxaphene/dieldrin mixture, whereas a 200-fold excess of unlabeled
E2 caused a 94% decrease in ER binding (the
specifically-bound ER was the difference between the total binding
minus the binding after competition with unlabeled E2).
These results indicate minimal competition by toxaphene, dieldrin, or
dieldrin plus toxaphene for the ER in MCF-7 cells using this whole
cell-binding assay.
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summarize the effects of
toxaphene, dieldrin, and other pesticides on ß-gal activity in a
yeast system using the mER and a reporter plasmid containing a single
ERE linked to the LacZ gene (25). At a concentration of
2.5 x 10-7 M, the pesticides did not
induce ß-gal activity (Fig. 3A), as previously reported using a yeast
assay system expressing the hER (18). However, 2.5 x
10-5 M of any compound individually was
capable of inducing activity from 1.5-fold (dieldrin) to 4-fold
(endosulfan). This induction was approximately 7–18% of the activity
induced by 1 x 10-8 M Diethylstilbestrol
(DES), which was 22-fold. No synergistic induction by the mER was
demonstrated with any of the chemical combinations ranging from
1–3 x 10-7 M. Furthermore, the chemical
mixtures did not induce activity above background, as in the case of
each compound individually at 2.5 x 10-7
M. Synergistic transactivation also was absent when any two
compounds were mixed at the higher concentration (2.5 x
10-5 M) (Fig. 3A). The maximum level of
activity attained by any chemical alone or in combination at 2.5
x 10-5 M was never above 18% of the level
obtained with 1 x 10-8 M DES. This
observation directly contrasts with the hER system, which showed that
binary mixtures of these compounds synergistically induced up to 65%
of the activity observed for 10-8 M
E2 (18). A further comparison of mER with the hER (18)
yeast-based systems was determined using a longer (16 h) incubation
time (Fig. 3B). Induction by 10-8 M DES was
increased to 71-fold; however, the organochlorine pesticides alone
(2.5 x 10-7 M) or in combination (1 to
3 x 10-7 M) were inactive. At higher
concentrations (2.5 x 10-5 M), only
dieldrin and endosulfan were weakly estrogenic, and synergism was not
observed for the binary mixtures.
The estrogenic activity of dieldrin, toxaphene, other organochlorine
pesticides, and their binary mixtures also were investigated using
yeast transformants containing the human ER plus a reporter gene
containing two vitellogenin EREs upstream of the ß-gal gene (26, 27).
The cells were treated with increasing concentrations of either
E2 as a positive control or endosulfan, chlordane,
toxaphene, and dieldrin (singly or in combination) (Fig. 4). E2 (10-8 M)
induced a 5000-fold increase in ß-gal activity. Of the other
chemicals tested, only endosulfan (10-4 M)
induced ß-gal activity above background. Equimolar concentrations of
combinations of endosulfan, chlordane, toxaphene, and dieldrin did not
result in synergistic induction of ß-gal activity. The failure to
observe synergistic interactions for dieldrin plus toxaphene and other
binary mixtures in the two yeast-based assays (Figs. 3 and 4) was
consistent with results observed in the mouse uterus (Fig. 1 and Table 1) and MCF-7 human breast cancer cells (Fig. 2 and Table 2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arnold and co-workers (18) recently reported that interactions between weakly estrogenic pesticides such as dieldrin, toxaphene, and endosulfan resulted in enhanced binding to baculovirus-expressed hER and synergistic induction of estrogen-responsive reporter gene activity in a yeast bioassay. It was surprising that toxaphene alone was only weakly estrogenic because toxaphene is a mixture of C10H10Cl8 isomers that undoubtedly exhibit estrogenic and nonestrogenic activities. Interestingly, chlordane also synergistically interacted with the estrogenic pesticides in the same assay system, even though chlordane does not seem to be estrogenic (6, 18). These results have significant mechanistic implications regarding ER-mediated gene expression; moreover, the apparent synergistic effects observed for the weakly estrogenic and nonestrogenic organochlorine pesticides have heightened concern regarding potential environmental and human health implications of exposure to mixtures of environmental contaminants (31, 32). This study was designed to further investigate the estrogenic activity and interactions of toxaphene and dieldrin using three well-characterized models, namely the immature mouse uterus, MCF-7 human breast cancer cells, and the yeast-based ER assay.
The results illustrated in Fig. 1 demonstrate that toxaphene, dieldrin,
and a mixture containing equimolar concentrations of both compounds do
not competitively displace [3H]E2 from the
mouse uterine ER. A previous study (33) also showed that dieldrin (3
mg/kg x7) and toxaphene (25 mg/kg x7) and several other
organochlorine pesticides did not induce a uterine wet weight increase
in the immature female rat. In this study, using a standard immature
mouse protocol for estrogenicity, toxaphene and dieldrin alone
exhibited minimal estrogenic activity. The toxaphene/dieldrin mixture
induced a small increase in uterine PR levels (Table 1); however, this
response was not dose dependent. For the remaining responses, no
apparent synergistic interactions were observed for the
toxaphene/dieldrin mixture (Table 1). These results indicate that there
are major differences between the interactive estrogenic effects and ER
binding of toxaphene and dieldrin in the mouse uterus (Table 1; Fig. 1)
compared with results obtained with the hER construct used in the
baculovirus-expressed system or the yeast assay system used by Arnold
and co-workers (18), even though the mouse ER binds with variable
affinities to other environmental chemicals such as hydroxy-PCBs (3)
and alkylphenols (10).
The potential interactions of toxaphene and dieldrin were investigated
further in MCF-7 human breast cancer cells. Soto and co-workers (5)
previously reported that both toxaphene and dieldrin induced
proliferation of MCF-7 cells in their E-screen assay; however, this
response was only induced at the highest concentration
(10-5 M). In the present study,
10-5 M toxaphene, 10-5
M dieldrin, and 10-5 M dieldrin
plus toxaphene did not induce cell proliferation, whereas
10-9 M E2 caused a 6-fold increase
in cell growth (Fig. 2). In contrast, other xenoestrogens previously
identified as weak estrogens in the E-screen, including nonylphenol,
2',4',6'-trichloro-4-biphenylol and bisphenol-A, induced MCF-7 cell
proliferation in our assay system (data not shown). The reason for the
differences between results of this study with toxaphene and dieldrin
vs. previous reports (5, 6) may be caused by several
factors, including cell culture conditions, cell passage number, or
serum (34). The estrogenic activity of toxaphene, dieldrin, and
toxaphene plus dieldrin also was investigated in transient transfection
assays, which used two constructs (CKB-CAT and CATH-CAT) that contain
estrogen-responsive 5'-promoter sequences from the CKB and cathepsin D
genes (20, 21). In transient transfection studies, E2
significantly induced CAT activity in MCF-7 cells, whereas induction
was not observed with toxaphene, dieldrin, or their combination (Table 2). These results complement ER-binding studies, which showed that the
organochlorine pesticides (alone or in combination) did not
competitively bind to the hER in MCF-7 cells (Table 3).
The effects of toxaphene, dieldrin, other organochlorine pesticides,
and binary mixtures of these compounds also were investigated in
yeast-based reporter gene assays (25, 26, 27). The results obtained in
yeast transformed with an expression plasmid that contained the
wild-type mouse ER and a reporter plasmid containing a single ERE
linked to the LacZ gene (Fig. 3) indicated that the
estrogenic activities of dieldrin and toxaphene were minimal, and no
synergistic effects were observed for a binary mixture of these and
other organochlorine pesticides. The potential interactions of
toxaphene and dieldrin, as well as endosulfan and chlordane, also were
investigated in a yeast-based ER assay that used the same yeast strain
and reporter gene construct as reported by Arnold and co-workers (18).
In contrast to the previous study, synergistic activity was not
observed for binary mixtures of toxaphene/dieldrin or other pesticide
combinations. Endosulfan exhibited estrogenic activity in both yeast
assays; however, binary mixtures of endosulfan plus the other
organochlorine compounds did not induce synergistic responses (Figs. 3 and 4). The reasons for the differences between results of this study
and that reported by Arnold and co-workers (18) are unclear but may be
caused by a unique aspect of the yeast strain maintained in either
laboratory or to differences in ER constructs used by either
laboratory. The differences could not be accounted for by levels of ER
expression because varying the level of ER expression did not alter the
results (data not shown). These data (Fig. 4) demonstrate that
synergism between weakly estrogenic chemicals is not universal, even
within the same strain of yeast.
In summary, this study has compared the estrogenic activities of
toxaphene, dieldrin, and an equimolar mixture of the two compounds in
both ER-binding and functional assays in the mouse uterus, MCF-7 human
breast cancer cells, and in estrogen-responsive yeast assays. The
activities of both compounds alone were minimal, and no synergistic
effects were observed with the binary mixture in the mouse uterus and
MCF-7 cells (Tables 1–3; Figs. 1 and 2). Similar results were observed
for toxaphene/dieldrin and other binary mixtures of organochlorine
insecticides in two yeast-based assays (Figs. 3 and 4). These data
contrasted with results reported by Arnold and co-workers (18) in the
ER-binding and yeast assays and other studies which have reported
nonadditive (synergistic) interactions with hydroxy PCBs and other
mixtures of organochlorine pesticides and phenolics (6, 10). It should
be noted also that results of previous studies with mixtures of
estrogenic/nonestrogenic compounds do not always give synergistic
responses. Toxaphene is not a single chemical but a chlorinated
camphene mixture containing several
C10H10Cl8 isomers that undoubtedly
exhibit estrogenic and nonestrogenic activity. However, the toxaphene
mixture is only weakly estrogenic or nonestrogenic in both in
vitro and in vivo assays (6, 18, 33). Similarly,
Aroclor 1221 and nonylphenol also are mixtures of active and inactive
congeners, and the estrogenic activities of both mixtures also are
relatively weak (5, 6, 7, 10, 35). This suggests that synergistic
interactions of weakly estrogenic or nonestrogenic chemicals may be
highly structure dependent. Moreover, the results obtained for a binary
mixture of dieldrin and toxaphene and other organochlorine pesticides
in this study indicate that synergistic interactions are both response-
and assay-specific. Thus, the recent scientific, regulatory, and public
concern regarding the potential adverse environmental and human health
impacts associated with the synergistic effects induced by
organochlorine pesticide mixtures (18) should be tempered by the
results reported in this study, which show that in 10 different assays,
synergistic interactions were not observed (36).
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Footnotes |
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2 A Sid Kyle Professor of Toxicology.
Received October 16, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C. M. Merritt, L. Torrentera, K. M. Winter, C. K. Tornehl, K. Girvin, and S. I. Dodson Dieldrin reduces male production and sex ratio in Daphnia galeata mendotae Toxicology and Industrial Health, February 1, 1999; 15(1-2): 193 - 200. [Abstract] [PDF]
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 K. Ramamoorthy, M. S. Gupta, G. Sun, A. McDougal, and S. H. Safe 3,3',4,4'-Tetrachlorobiphenyl exhibits antiestrogenic and antitumorigenic activity in the rodent uterus and mammary cells and in human breast cancer cells Carcinogenesis, January 1, 1999; 20(1): 115 - 123. [Abstract] [Full Text] [PDF]
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 G. G. J. M. Kuiper, J. G. Lemmen, B. Carlsson, J. C. Corton, S. H. Safe, P. T. van der Saag, B. van der Burg, and J.-A. Gustafsson Interaction of Estrogenic Chemicals and Phytoestrogens with Estrogen Receptor {beta} Endocrinology, October 1, 1998; 139(10): 4252 - 4263. [Abstract] [Full Text] [PDF]
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),
dieldrin (
), and dieldrin/toxaphene (
), and the displacement of
radiolabeled hormone was determined as described in Materials
and Methods. Only unlabeled E2 significantly
decreased binding of [3H]E2 to the uterine
ER.
),
toxaphene (
), or dieldrin/toxaphene (