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(long) Promotion of Endometriosis in Mice by PolychlorinatedDibenzo-p-Dioxins, Dibenzofurans, and Biphenyls-- July EHP
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[Environmental Health Perspectives Volume 105, Number 7, July 1997 ]
Promotion of Endometriosis in Mice by Polychlorinated Dibenzo-p-Dioxins,
Dibenzofurans, and Biphenyls
Krista L. Johnson,1 Audrey M. Cummings,2 and Linda S. Birnbaum2
1Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC
27599-7270; 2U.S. Environmental Protection Agency, National Health and
Environmental Effects Research Laboratory, Environmental Toxicology
Division, Research Triangle Park, NC 27711
* Materials and Methods
Previous studies showed exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin
(2,3,7,8-TCDD) enhances the development of endometriotic lesions. In this
study we examined the effects of other polyhalogenated aromatic hydrocarbons
on endometriotic proliferation. B6C3F1 female mice were treated via oral
gavage a total of five times, with 3 weeks between each dosing, with 0, 1,
3, or 10 Âµg 2,3,7,8-TCDD/kg body weight (bw); 3 or 30 mg
2,2Â«,4,4Â«,5,5Â«-hexachlorobiphenyl (PCB 153)/kg bw; 100, 300, or 1000 Âµg
3,3Â«,4,4Â«,5-pentachlorobiphenyl (PCB 126)/kg bw; 10, 30, or 100 Âµg
2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF)/kg bw; or 2 or 20 mg
1,3,6,8-TCDD/kg at 10 ml/kg bw. Endometriosis was surgically induced during
the week of the second dosing. Three weeks following the final dose, the
mice were euthanized and endometriotic lesions, whole body, liver, ovaries,
uterine horn, and thymus were weighed, and lesion diameters were measured.
Lesions, uterine horns, and ovaries were fixed for histopathology and livers
were processed for measurement of ethoxyresorufin O-deethylase (EROD)
activity. Both 2,3,7,8-TCDD (1 and 3 Âµg/kg bw) and 4-PeCDF (100 Âµg/kg bw)
significantly enhanced the growth of endometrial lesions. No statistically
significant increase in endometriotic lesion size was detected in animals
treated with either PCB 126 or with the highest dose of 2,3,7,8-TCDD,
possibly due to the effects of histologically observed ovarian toxicity. The
nondioxin-like compounds, PCB 153 and 1,3,6,8-TCDD, produced no observable
effects on endometriosis. Hepatic EROD activity was significantly induced by
2,3,7,8-TCDD, 4-PeCDF, and PCB 126, but not by PCB 153 or 1,3,6,8-TCDD. The
results of this study provide preliminary support for the hypothesis that
halogenated aromatic hydrocarbon-promoted endometriosis may be Ah receptor
mediated. Key words: endometriosis, halogenated aromatic hydrocarbon,
polychlorinated biphenyls, polychlorinated dibenzofurans, polychlorinated
dibenzo-p-dioxins, structureÃ�activity relationships, TCDD. Environ Health
Perspect 105:750Ã�755 (1997)
Address correspondence to L.S. Birnbaum, U.S. Environmental
Protection Agency, National Health and Environmental Effects
Research Laboratory, Environmental Toxicology Division, MD-66,
Room L-318, Research Triangle Park, NC 27711 USA.
The research described in this article has been funded in part by
the U.S. Environmental Protection Agency with the University of
North Carolina, Chapel Hill. The manuscript has been reviewed in
accordance with EPA policy and approved for publication; however,
it does not necessarily reflect the views of the agency. Mention
of trade names or commercial products does not constitute
endorsement or use recommendation.
The authors are grateful for the technical assistance of Michael
DeVito, Janet Diliberto, Joan Metcalf, David Ross, Vicki
Richardson, Joe Jackson, Frances McQuaid, Dennis House, Chris
Hurst, and Michael Santostefano. In addition, the authors would
like to thank Michael Santostefano, Barbara Abbott, and Michael
DeVito for reviewing this manuscript.
Received 16 September 1996; accepted 2 April 1997.
Polyhalogenated aromatic hydrocarbons (PHAHs) are a group of environmental
contaminants that include the polychlorinated dibenzo-p-dioxins (PCDDs),
dibenzofurans (PCDFs), diphenylethers (PCDEs), biphenyls (PCBs), and
naphthalenes (PCNs) (1,2). PHAHs evoke a broad range of toxic and
biochemical responses (3,4). One common adverse effect is reproductive
toxicity, including reduced fertility, decreased litter size, diminished
uterine weight, and altered ovarian functioning in several species (5Ã�7).
Recent studies showed an effect on endometriosis in several species
following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD)
(8,9). Endometriosis is the growth of endometrial tissue outside the uterus,
often causing infertility and pain (10). A major characteristic of
endometriosis is the presence of lesions and hemorrhagic cysts in the
peritoneum (11). Proliferation of endometrial lesions is estrogen dependent
(12) and often associated with immune dysfunction (13) and it is possibly
caused by compounds such as the most potent PHAH, 2,3,7,8-TCDD (14).
2,3,7,8-TCDD induced an increase in the prevalence and severity of
endometriosis in rhesus monkeys (8) and provoked an increase in the growth
of surgically induced endometriotic lesions in both rats and mice (9)
following subchronic exposure. Because humans are exposed to a broad range
of PHAHs, identification of the effects of additional PHAHs on endometriosis
Endometriosis is difficult to diagnosis, thus estimates of prevalence vary
widely. Many researchers suggest that the prevalence of endometriosis is
increasing in the general population (15). While a 10% prevalence rate in
the general population has often been accepted (10), estimates are as high
as 60Ã�80% among women with infertility or pain in Belgium (16). Researchers
suggest that the high incidence rate of endometriosis in Belgian women
coincides with elevated concentrations of persistent organochlorine
environmental contaminants in this country (17). For example, elevated blood
levels of PCBs were discovered in Belgian women who suffer from
endometriosis (17). A recent study in Israel has also demonstrated an
association between endometriosis and elevated 2,3,7,8-TCDD levels (18).
The mechanism of action by which dioxin induces increased proliferation of
endometriosis in recent animal studies (8,9) has not yet been determined.
Objectives of this investigation were to determine if PHAH-promotion of
endometriosis may be aryl hydrocarbon (Ah) receptor mediated by evaluating
the structural relevance of PHAHs to the proliferation of endometriosis.
Therefore, a selection of PHAHs with varying degrees of affinity for the Ah
receptor were selected. In addition to 2,3,7,8-TCDD, four other PHAHs were
administered: 3,3Â«,4,4Â«,5,-pentachlorobiphenyl (PCB 126),
2,2Â«,4,4Â«,5,5Â«-hexachlorobiphenyl (PCB 153),
1,3,6,8-tetrachlorodibenzo-p-dioxin (1,3,6,8-TCDD), and
2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF). The hypothesis of this study is
that induction of endometriosis by PHAHs may be Ah receptor-mediated and
will be influenced by structural differences among these chemicals.
2,3,7,8-TCDD and the two additional dioxin-like compounds (PCB 126 and
4-PeCDF) (19) should evoke increased proliferation of endometriotic lesions,
while the nondioxin-like chemicals (PCB 153 and 1,3,6,8-TCDD) (19) should
not induce increased endometriotic growth.
Materials and Methods
Chemicals. Both dioxin-like and nondioxin-like chemicals were used in
addition to 2,3,7,8-TCDD (Radian Corp., Austin, TX) (19). Dioxin-like
chemicals used were PCB 126 (Ultra Scientific Chemical Co., North Kingstown,
RI) and 4-PeCDF (Accustandard, New Haven, CT), and nondioxin-like chemicals
used were PCB 153 (Accustandard) and 1,3,6,8-TCDD (Accustandard.) All
chemicals had purities greater than 98%. No 2,3,7,8-TCDD-like contaminants
were present in the other chemicals.
Animals. Previous studies using rodent models focused on the effects of
2,3,7,8-TCDD exposure on the promotion of endometriosis in rats and mice
(9). Because mice showed a greater degree of endometriotic severity (9),
appeared to lack overt endocrine disruption at the doses studied (9), and
demonstrated greater immunosensitivity (20) following exposure to
2,3,7,8-TCDD than did rats, the mouse model was chosen for this study.
Female B6C3F1 mice were obtained from Charles River Breeding Laboratories
(Raleigh, NC) at 70 days of age, randomly assigned to a treatment group, and
acclimated for 1 week prior to dosing. Mice were maintained at the National
Health and Environmental Effects Research Laboratory of the U.S.
Environmental Protection Agency (EPA). Animal care and treatment were
conducted according to established guidelines. All animals were housed in an
environment with controlled humidity (40Ã�50%), 12:12-hr light:dark cycle,
and constant temperature (20Ã�24Â¡C). Animals received food (Prolab rat,
mouse, hamster 3000, Agway, Syracuse, NY) and water ad libitum.
Treatment. Chemical doses were assigned based on published toxic equivalency
factor (TEF) values (3) for all chemicals and solubility considerations for
PCB 153 and 1,3,6,8-TCDD. Experiment 1 included doses of 0 (corn oil; Sigma
Chemical Co., St. Louis, MO), 1, 3, and 10 Âµg 2,3,7,8-TCDD/kg bw with 10
animals per group. Experiment 2 included doses of 0, 3, and 30 mg PCB 153/kg
bw and 100, 300, and 1000 Âµg PCB 126/kg bw with 10Ã�12 animals per group.
Experiment 3 included doses of 0, 10, 30, and 100 Âµg 4-PeCDF/kg bw and 2 and
20 mg 1,3,6,8-TCDD/kg bw with 10Ã�12 animals per group. Dosing was
administered via oral gavage a total of five times, with 3-week intervals
between each dosing as described (9). All animals were dosed with a corn oil
dosing vehicle at 10 ml/kg bw.
Surgical technique. Because mice have a closed reproductive tract with a
bursa-enclosed ovary and an estrous cycle instead of a menstrual cycle, they
do not develop endometriosis naturally (12). Endometriosis must be induced
in these animals through surgical methods performed during the week of the
second dosing. The two major steps of the surgical process as described by
Vernon and Wilson (21) in a rat model and extended to mice by Cummings and
Metcalf (12) were 1) ablation of the left uterine horn with longitudinal
bisection to expose the epithelial cells and 2) suturing of uterine segments
onto alternating mesenteric blood vessels in the peritoneal cavity.
Necropsy. At the conclusion of 16 weeks, the animals were euthanized by
carbon dioxide asphyxiation followed by exanguination via cardiac puncture.
Necropsies were performed in a random order to prevent bias during the
measuring of lesion diameters. Lesions, ovaries, uterine horn, liver, and
thymus were extracted and weighed. Lesions, ovaries, and uterine horns were
fixed for histology, while liver and thymus were frozen on dry ice and
stored at -70Â¡C.
EROD assay. Ethoxyresorufin and resorufin were purchased from Molecular
Probes (Eugene, OR). Microsomal proteins were prepared (22) and quantified
(23) using bovine serum albumin (BSA) as a standard. The reaction buffer
contained 0.1 M KPO4, 5 mM Mg2SO4, and 2 mg BSA/ml at pH 7.5. Liver
microsomes were diluted in 0.1 M KPO4 (100 Âµl) to provide reaction
linearity, added to 0.1 M KPO4 buffer containing 1.5 nM ethoxyresorufin, and
preincubated for 2 min at 37Â¡C. The production of resorufin was started by
the addition of 100 Âµl of Â§-NADPH (5 mg/ml) and monitored
spectrofluorimetrically as described (22). A log10 transformation was
utilized to normalize ethoxyresorufin O-deethylase (EROD) data.
Histopathology. Microscopic examination of endometriotic lesions and ovaries
was performed to characterize histological changes associated with exposure
to halogenated aromatic hydrocarbons (HAHs). Animals were selected for
histopathology randomly to obtain an unbiased representative sample for
review. Histology was prepared by Experimental Pathology Laboratories Inc.
(Research Triangle Park, NC) and pathology was performed by John Seely at
PATHCO (Research Triangle Park, NC). Endometriotic lesions were examined for
the presence of inflammation and luminal exudate or transudate, while
ovaries were examined for both the presence of primary, growing, and antral
follicles and the presence of corpora lutea (both active and regressing).
Active corpora lutea were defined as newly formed corpora lutea, as well as
those that became increasingly eosinophilic and those that appeared foamy.
Regressive corpora lutea were characterized by degeneration, necrosis, and
fibrous tissue proliferation.
Statistical analysis. Primary statistical analysis of endometriotic lesion
diameters and secondary analyses of lesion, ovarian, uterine, and thymus
weights from all chemical treatment groups were performed using the
Dunnett's test and a level of probability of statistical significance of
p<0.05. Means with standard deviations (SD) were determined for all dose
groups for body, liver, ovarian, uterine, and lesion weights and lesion
diameters. For EROD activity, the statistical intergroup comparisons were
determined using a one-way analysis of variance (ANOVA) followed by Fisher's
protected least significant difference (PLSD). The levels of probability of
statistical significance for EROD data are p<0.01.
EROD activity is a marker for CYP1A1-dependent enzyme induction by
2,3,7,8-TCDD and related compounds (19). In this study, constitutive EROD
activity in control animals for three separate experiments were similar
(Table 1). A statistically significant (p<0.01) dose-dependent increase in
EROD activity in mice treated with increasing doses of 2,3,7,8-TCDD, PCB
126, or 4-PeCDF was observed; in contrast, mice treated with 1,3,6,8-TCDD or
PCB 153 had EROD activities similar to control groups (Table 1).
Previous studies in a rodent model for 2,3,7,8-TCDD-promoted endometriosis
focused on lesion diameter as an indicator of 2,3,7,8-TCDD-induced responses
(9). Examination of lesion diameter in three separate control groups in this
study indicated similar values with no statistical differences (Table 1).
Treatment of animals with 1 or 3 Âµg 2,3,7,8-TCDD/kg bw resulted in a
statistically significant increase in lesion diameter (p<0.05). Although the
diameter was increased relative to controls at 10 Âµg 2,3,7,8-TCDD/kg bw, it
was not statistically significant (Table 1). An increase in lesion diameter
with dose was observed in animals treated with 4-PeCDF, although a
statistically significant increase was only observed in animals treated with
100 Âµg 4-PeCDF/kg bw (Table 1). Animals treated with PCB 126 resulted in an
apparent increase in lesion diameter; however, this increase was not
statistically significant compared to control animals (Table 1). Analysis of
lesion diameter values for animals treated with PCB 153 or 1,3,6,8-TCDD
resulted in lesion diameter values similar to control animals in all dose
groups (Table 1).
Endometriotic lesion weight was used as an additional marker to examine
HAH-promoted endometriosis. Examination of lesion weights in three separate
control groups revealed high variability. Lesion weights for control animals
in experiment 1 were significantly different from control animals in
experiments 2 and 3, which may contribute to the variability in results
among dose and chemical groups (Table 1). Treatment of animals with 1, 3, or
10 Âµg 2,3,7,8-TCDD/kg bw resulted in a dose-dependent decrease in
endometriotic lesion weight. However, endometriotic lesion weights in all
dose groups of 2,3,7,8-TCDD-treated mice were significantly elevated
compared to control animals in experiment 1 (Table 1). Lesion weights of
animals treated with 1 or 3 Âµg 2,3,7,8-TCDD/kg bw also appear elevated when
compared to control animals of experiments 2 or 3. Elevated endometriotic
lesion weights were not observed in the lowest treatment groups for 4-PeCDF
or PCB 126 when compared to controls, although an apparent, but
nonsignificant dose-dependent increase in lesion weight was observed in
animals treated with 4-PeCDF/kg bw or 100 or 300 Âµg PCB 126/kg bw. Lesion
weight values for the highest dose of PCB 126 (1000 Âµg/kg bw) decreased
nonsignificantly from 300 Âµg/kg bw. High variability was present in all
treatment groups, especially in lesion weight data of animals treated with
PCB 126 or 4-PeCDF. Analysis of lesion weights in mice treated with PCB 153
or 1,3,6,8-TCDD resulted in endometriotic lesion weights similar to control
Another tissue examined in this study to assess the effects of HAH exposure
on endometriosis was the ovary. Because body weight values did not vary
significantly across chemical classes or doses, similar results were
observed between crude ovarian weights (Table 1) and ovarian weight/bw
ratios (data not shown). Ovarian weights from ablated uterine horns appeared
slightly lower than the ovarian weights from intact uterine horns. However,
trends in both sets of ovarian weights are consistent within most dose
groups. Therefore, the crude ovarian weights from intact uterine horns
(Table 1) were used as markers to describe the dose-dependent effects of
administered compounds on ovarian weights. Although no significant
differences in ovarian weight were found in comparisons of treated to
control animals, possibly due to high data variability, examination of
ovarian weights from animals treated with 1 or 3 Âµg 2,3,7,8-TCDD/kg bw
revealed a trend toward an increase in ovarian weights, which was followed
by an apparent decrease in ovarian weight in animals treated with 10 Âµg
2,3,7,8-TCDD/kg bw (Table 1). Animals treated with PCB 153 exhibited an
apparent but nonsignificant increase in ovarian weight with increasing dose
(Table 1). In contrast, mice treated with PCB 126 or 4-PeCDF showed an
apparent decrease in ovarian weight with increasing dose (Table 1).
Furthermore, analysis of ovarian weights from mice treated with 1,3,6,8-TCDD
showed ovarian weights similar to control animals at all dose levels (Table
1). A microscopic examination of endometriotic lesions and ovarian tissue
was used to characterize histological changes associated with exposure. In
all endometrial lesions examined, endometrial epithelium, endometrial glands
with stroma, and the myometrium were present, but these structures varied in
thickness and prominence, without relation to treatment. The presence of
luminal exudate or transudate was more severe in lesions characterized as
nonstandard than in those characterized as standard, but no significant
dose-dependent distribution of severity was noticed because incidence rates
of nonstandard lesions, such as discolored or hardened lesions, were
consistent across dose groups and chemical classes. Inflammation of the
endometriotic uterine segments was consistent across all animals and, in
most instances, appeared acute (associated with polymorphonuclear cells.)
Examination of ovarian tissue revealed the absence of active corpora lutea
in animals only from the 10 Âµg 2,3,7,8-TCDD (in two of three animals
examined), 100 Âµg PCB 126 (one of three animals), and 1000 Âµg PCB 126/kg bw
(two of three animals) treatment groups. In addition, animals with the
highest number of regressive corpora lutea compared to total corpora lutea
(both active and regressive) were animals treated with 10 Âµg 2,3,7,8-TCDD
(53.3%), 100 Âµg PCB 126 (60%), or 1000 Âµg PCB 126/kg bw (75%). In contrast,
only 26.7% of the total corpora lutea in control animals, was characterized
as regressive. The absence of active corpora lutea and the high percentages
of regressive corpora lutea in these dose groups indicate either a direct or
indirect chemically induced atrophic effect on the ovaries in these animals.
The final parameters measured in the study were uterine, thymus, and liver
weights. Uterine weights of all treated animals were not statistically
different from values of control animals, and analysis of uterine weights
for three separate control experiments revealed no statistical differences
(Table 2). There was an apparent nonsignificant increase in uterine weights
of mice treated with 3 Âµg 2,3,7,8-TCDD/kg bw, which was followed by an
apparent decrease in uterine weights in the highest dose group, 10 Âµg
2,3,7,8-TCDD/kg bw. These effects on uterine weights were similar to those
observed in animals treated with PCB 126. A trend toward a slight decrease
in uterine weights with increasing dose of 4-PeCDF was observed. Treatment
of animals with all doses of PCB 153 resulted in a slight, nonsignificant
dose-dependent increase in uterine weights as compared to controls. However,
all animals treated with 1,3,6,8-TCDD had uterine weights similar to control
animals. In contrast, a dose-dependent decrease in thymus weights with
increasing dose of 4-PeCDF or PCB 126 was observed (Table 2). Thymus weights
of animals treated with 30 or 100 Âµg 4-PeCDF/kg bw were significantly less
than control thymus weights (p<0.05), while the suggested decrease in thymus
weights in animals treated with PCB 126 was nonsignificant. Finally,
treatment of animals with 2,3,7,8-TCDD, PCB 126, or 4-PeCDF resulted in an
apparent increase in liver weight with increasing dose, although this was
only significant in animals treated with the middle and high doses of
4-PeCDF (Table 2). In contrast, exposure of animals to PCB 153 or
1,3,6,8-TCDD resulted in liver weights in all dose groups similar to
The promotion of endometriosis only by PHAHs with high binding affinity to
the Ah receptor is consistent with the hypothesis that PHAH promotion of
endometriosis is Ah receptor mediated. Due to variability in these results
from study limitations such as individual animal variability, bioassay
insensitivity, and gross invasiveness of the surgical procedures,
conclusions about the mechanism of action by which HAHs promote
endometriosis are only preliminary. Follow-up studies should attempt to
refine the surgical model to more accurately assess the effects of HAHs on
As in previous studies that focused on lesion diameter as the primary
endpoint to assess the influence of 2,3,7,8-TCDD on the promotion of
surgically induced endometriosis (9), this study evaluated the effects of
2,3,7,8-TCDD, PCB 126, PCB 153, 1,3,6,8-TCDD, and 4-PeCDF on endometriotic
lesion diameter (Table 1). The mechanism by which PHAHs such as
2,3,7,8-TCDD, PCB 126, or 4-PeCDF increase lesion diameter is unknown, but
it appears to be Ah receptor mediated, as is true for all other
well-characterized 2,3,7,8-TCDD-induced responses (24).
Structure binding relationships (SBRs), based on affinity of ligand binding
for the Ah receptor, are often indirectly assessed by induction of
arylhydrocarbon hydroxylase (AHH), EROD, and other enzyme activities, and
are described by structureÃ�activity relationships (SARs). Previous studies
showed a correlation between endpoints, such as CYP1A1 induction, and
affinity for the Ah receptor expressed through SARs (25). For example, in
this study 2,3,7,8-TCDD, PCB 126, and 4-PeCDF, the congeners that increased
endometriotic lesion diameters (statistically significant only in animals
treated with 2,3,7,8-TCDD or 4-PeCDF), have the strongest binding affinities
for the Ah receptor (1,26,27). In contrast, compounds such as 1,3,6,8-TCDD
and PCB 153, which did not induce any increases in endometriotic lesion
diameter, have weak affinities towards the Ah receptor (27,28).
Endometriotic lesion weights were also measured for secondary analysis of
changes in endometriotic lesion sizes due to HAH exposure (Table 1).
Increases in lesion weight, like increases in lesion diameter, occured in
animals exposed to chemicals with the highest affinities for the Ah receptor
(statistically significant increases induced by 2,3,7,8-TCDD and
nonsignificant increases induced by PCB 126 and 4-PeCDF) and not in animals
exposed to chemicals with significantly lower binding affinities (PCB 153
and 1,3,6,8-TCDD). This supports the hypothesis that promotion of
endometriosis by HAHs may be Ah receptor mediated.
This study also revealed changes in ovarian weight and differences in
ovarian histopathological evaluation based on chemical and dose (Table 1).
As with other endpoints, chemicals with greater binding affinities for the
Ah receptor evoked greater toxic responses such as decreases in ovarian
weights. Ovarian histological examination of animals treated with 1 or 3 Âµg
2,3,7,8-TCDD/kg bw, PCB 153, 1,3,6,8-TCDD, or 4-PeCDF was consistent with
ovarian weights for these animals and indicated no presence of ovarian
atrophy. The absence of corpora lutea and the high percentages of regressive
corpora lutea in animals treated with 10 Âµg 2,3,7,8-TCDD, or 100 or 1000 Âµg
PCB 126/kg bw supports the hypothesis that ovarian atrophy may have occurred
in animals treated with high doses of chemicals with strong binding
affinities for the Ah receptor. The lack of ovarian atrophy observed in the
4-PeCDF-treated animals may be due to the low concentration of 4-PeCDF
available to extrahepatic tissues due to its sequestration in the liver
(29). Still, the results of the histological evaluations are preliminary
because only a small, randomly selected representative sample from each dose
group was examined.
Ovarian atrophy at high doses of 2,3,7,8-TCDD and PCB 126 is consistent with
the results of decreased lesion diameters and weights observed in animals
administered high doses of these compounds compared to the resulting lesion
diameters and weights in animals administered lower doses. High doses of
PHAHs may cause antiestrogenic effects, leading to either indirect or direct
ovarian toxicity or atrophy (30). The resulting decrease in the promotion of
endometriosis (observable in the variability in lesion diameter and lesion
weight values at the high dose levels of 2,3,7,8-TCDD and PCB 126) is
consistent with the requirement for estrogen to promote lesion growth (31).
Thus, Ah receptor binding may correlate with ovarian atrophy, as well as
lesion diameter and lesion weight. Future studies examining endometrial
promotion should use lower dose levels of 2,3,7,8-TCDD and PCB 126 to avoid
adverse effects on the ovary.
Several additional endpoints were measured in this study to assess the
adverse effects of PHAH exposure. Resulting antiestrogenic effects from HAH
exposure could also be mediated by an indirect route via a decrease in the
concentration of circulating estrogens (32). This can also lead to a
decrease in uterine weights (33). Analysis of uterine weights in this study
revealed a correlation between changes in uterine weights and binding
affinities for the Ah receptor. Chemicals that bind strongly to the Ah
receptor (2,3,7,8-TCDD, PCB 126, and 4-PeCDF) caused decreases in uterine
weights at high doses, while chemicals that bind weakly to the Ah receptor
(PCB 153 and 1,3,6,8-TCDD) evoked no apparent decreases in uterine weights
when compared to controls. Also, even though changes in uterine weights were
not statistically significant, they appeared to correlate with changes in
ovarian weights and ovarian histopathological evaluations, supporting the
theory of ovarian atrophy in animals treated with the highest doses of
2,3,7,8-TCDD or PCB 126. Therefore, a relationship appears to exist between
structureÃ�activity and binding relationships and antiestrogenic effects,
such as decreases in uterine weights.
Another significant effect often associated with 2,3,7,8-TCDD exposure is
thymic atrophy, shown by previous studies to be Ah receptor mediated (25).
Originally, this study design was for levels of chemical exposure to produce
no overt toxicity. However, analysis of thymic atrophy in this study
revealed decreases in thymus weights only at high doses of chemicals with
high binding affinities for the Ah receptor (2,3,7,8-TCDD, PCB 126, and
4-PeCDF); this was only statistically significant in animals treated with
either of the two highest doses of 4-PeCDF. Therefore, the mechanism of
thymic atrophy correlates with the structural relationships of PHAHs
observed in mice with endometriosis.
Hepatotoxicity is a common response in mice following exposure to PHAHs (32)
and often results in increased liver weights (34). In this study,
nonsignificant increased liver weights with increasing dose were apparent
only in animals treated with 2,3,7,8-TCDD or PCB 126, while statistically
significant increases were apparent in animals treated with 4-PeCDF. A
greater hepatotoxic response may have been observed in animals treated with
4-PeCDF than in animals treated with 2,3,7,8-TCDD or PCB 126 because 4-PeCDF
is sequestered in the liver (29). Because these chemicals bind with great
affinity to the Ah receptor, increases in liver weight and hepatotoxicity
appear to correlate with binding affinities for the Ah receptor.
In addition to induction of toxic responses such as thymic atrophy and
hepatotoxicity, PHAHs have been identified as microsomal monooxygenase
inducers (35), frequently inducing cytochrome P450 isozymes (36). CYP4501A1
enzyme induction is often measured by AHH or EROD activity. Increased enzyme
activity coincides with increased gene expression and PHAH exposure (37).
Therefore, EROD activity was measured in this study as an indicator of
PHAH-induced effects and to interpret information about endometrial growth.
Based solely on analysis of EROD activities (Table 1) for animals treated
with PCB 126 versus animals treated with 2,3,7,8-TCDD, lesion size of
animals treated with the 100 Âµg PCB 126/kg bw should be comparable to
lesions in animals treated with 10 Âµg 2,3,7,8-TCDD/kg bw. Thus, lesion
diameters of animals treated with PCB 126 should have decreased with
increasing dose as did lesion diameters in animals treated with the highest
dose of 2,3,7,8-TCDD when compared to lower doses. This effect is most
probably the result of ovarian atrophy as seen histologically. Even though
the EROD values of animals treated with the highest dose of 4-PeCDF were
comparable to EROD values in animals treated with 300 Âµg PCB 126/kg bw, no
ovarian atrophy was observed in these animals, possibly because of the lack
of availability of this chemical to extrahepatic tissue due to its extensive
sequestration in the liver (29). Thus, lesion diameters continued to
increase because no ovarian atrophy occurred and circulating estrogen levels
were probably normal.
In conclusion, this study was an analysis of the influence of the structural
relevance of PHAHs on the proliferation of surgically induced endometriotic
lesions in B6C3F1 female mice. Analysis of all the parameters measured,
especially lesion diameter, suggests that the mechanism of PHAH-promoted
endometriosis may be Ah receptor mediated, with structureÃ�activity and
binding relationships influencing the degree of endometriotic proliferation.
Some endpoints in this study, such as endometriotic lesion diameter, did not
correlate directly with dose level or hepatic EROD induction because of
influences of additional responses such as hormonal interactions and
possible antiestrogenic effects. Chemicals exerting antiestrogenic effects
at high doses can induce ovarian atrophy, which in turn may decrease
circulating estrogen levels and proliferation of endometriosis. In general,
the responses in lesion diameter, lesion weight, ovarian weight, uterine
weight, and thymus weight correlate with structure binding relationships of
the administered PHAHs. Specifically, analysis of the primary endpoint
measured, endometriotic lesion diameter, demonstrates a dose-dependent
increase in size following administration of chemicals with strong binding
affinities for the Ah receptor, when the effects of ovarian atrophy on
lesion diameter are controlled. Therefore, the results of this study provide
preliminary support for the hypothesis that PHAH promotion of endometriosis
may be Ah receptor mediated.
1. Safe S. Polychlorinated biphenyls (PCBs), dibenzo-p-dioxin (PCDDs),
dibenzofurans (PCDFs), and related compounds: environmental and mechanistic
considerations which support the development of toxic equivalency factors
(TEFs). Crit Rev Toxicol 21:51Ã�74 (1990).
2. Safe S, Hutzinger O. PCDDs and PCDFs: sources and environmental impact.
In: Environmental Toxin Series, vol 3. Heidelberg, Germany:Springer Verlag,
3. Birnbaum LS, DeVito MJ. Use of toxic equivalency factors for risk
assessment for dioxins and related compounds. Toxicology 105:391Ã�401 (1995).
4. Birnbaum LS. The mechanism of dioxin toxicity: relationship to risk
assessment. Environ Health Perspect 102(suppl 9):157Ã�167 (1994).
5. Kociba RJ, Keller PA, Park CN, Gehring PJ.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD): results of a 13-week oral
toxicity study in rats. Toxicol Appl Pharmacol 35:553Ã�574 (1976).
6. Barsotti DA, Abrahamson LJ, Allen JR. Hormonal alterations in female
rhesus monkeys fed a diet containing 2,3,7,8-tetrachlorodibenzo-p-dioxin.
Bull Environ Contam Toxicol 211:463Ã�469 (1979).
7. Umbreit TH, Hesse EJ, Macdonald GJ, Gallo MA. Effects of TCDDÃ�estradiol
interactions in three strains of mice. Toxicol Lett 40:1Ã�9 (1987).
8. Rier SE, Martin DC, Bowman RE, Dmowski WP, Becker JL. Endometriosis in
rhesus monkeys (Macaca mulatta) following chronic exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin. Fundam Appl Toxicol 21:433Ã�441 (1993).
9. Cummings AM, Metcalf JL, Birnbaum L. Promotion of endometriosis by
2,3,7,8-tetrachlorodibenzo-p-dioxin in rats and mice: timeÃ�dose dependence
and species comparison. Toxicol Appl Pharmacol 138:131Ã�139 (1996).
10. Olive DL, Schwartz LB. Endometriosis. New Engl J Med 328:1759Ã�1769
11. Brosens I, Vasquez G, Deprest J, Puttemans P. Pathogenesis of
endometriosis. In: Endometriosis: Advanced Management and Surgical
Techniques (Nezhat CR, Berger GS, Nezhat FR, Buttram VCJ, Nezhat CH, eds).
New York:Springer-Verlag, 1995;9Ã�17.
12. Cummings AM, Metcalf JL. Induction of endometriosis in mice: a new model
sensitive to estrogen. Reprod Toxicol 9:233Ã�238 (1995).
13. Dmowski WP, Braun D, Gebel H. The immune system in endometriosis. In:
Modern Approaches to Endometriosis (Thomas EJ, Rock JA, eds). Boston:Kluwer
14. Lundberg K, Dencker L, Grovnik K-O. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD) inhibits the activation of antigen-specific T-cells in mice. Int J
Immunopharmacol 14:699Ã�705 (1992).
15. Berger GS. Epidemiology of endometrosis. In: Endometriosis: Advanced
Management and Surgical Techniques (Nezhat CR, Berger GS, Nezhat FR, Buttram
VCJ, Nezhat CH, eds). New York:Springer-Verlag, 1995;3Ã�7.
16. Koninckx PR, Meuleman C, Demeyere S, Lesaffre E, Cornillie F. Suggestive
evidence that pelvic endometriosis is a progressive disease, whereas deeply
infiltrating endometriosis is associated with pelvic pain. Fertil Steril
17. Koninckx PR, Braet P, Kennedy SH, Barlow DH. Dioxin pollution and
endometriosis in Belgium. Hum Reprod 9:1001Ã�1002 (1994).
18. Mayani A, Barel S, Soback S, Almagor M. Dioxin levels in women with
endometriosis. Hum Reprod 12:373Ã�375 (1997).
19. DeVito MJ, Birnbaum LS. The importance of pharmacokinetics in
determining the relative potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin and
2,3,7,8-tetrachlorodibenzofuran. Fundam Appl Toxicol 24:145Ã�148 (1995).
20. Hanson CD, Smialowicz RJ. Evaluation of the effect of low-level
2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on cell mediated immunity.
Toxicology 88:213Ã�224 (1994).
21. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis
in the rat. Fertil Steril 44:684Ã�694 (1985).
22. Diliberto JJ, Akubue PI, Luebke RW, Birnbaum LS. DoseÃ�response
relationships of tissue distribution and induction of CYP1A1 and CYP1A2
enzymatic activities following acute exposure to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. Toxicol Appl Pharmacol
23. Bradford MM. A rapid and sensitive method for quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal
Biochem 72:248Ã�254 (1976).
24. Okey AB, Riddick DS, Harper PA. Molecular biology of the aromatic
hydrocarbon (dioxin) receptor. TIPS 15:226Ã�232 (1994).
25. Safe S. Comparative toxicology and mechanism of action of
polychlorinated dibenzo-p-dioxins and dibenzofurans. Ann Rev Pharmacol
Toxicol 26:371Ã�379 (1986).
26. Mason G, Farell K, Keys B, Piskorska-Pliszczynska J, Safe L, Safe S.
Polychlorinated dibenzo-p-dioxins: quantitative in vitro and in vivo
structureÃ�activity relationships. Toxicology 41:21Ã�31 (1986).
27. Bandiera S, Sawyer T, Romkes M, Zmudzka B, Safe L, Mason G, Keys B, Safe
S. Competitive binding to the cytosolic 2,3,7,8-TCDD receptor: effects of
structure on the affinities of substituted halogenated biphenyls--a QSAR
approach. Biochem Pharmacol 32:3803Ã�3813 (1983).
28. Kafafi SA, Afeefy HY, Said HK, Hakimi JM. A new structureÃ�activity model
for Ah receptor binding. Polychlorinated dibenzo-p-dioxins and
dibenzofurans. Chem Res Toxicol 5:856Ã�862 (1992).
29. Brewster DW, Birnbaum LS. Disposition and excretion of
2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF) in the rat. Toxicol Appl
Pharmacol 90:243Ã�252 (1987).
30. Li X, Johnson DC, Rozman KK. Reproductive effects of
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in female rats: ovulation,
hormonal regulation and possible mechanisms. Toxicol Appl Pharmacol
31. Cummings AM, Metcalf JL. Effects of estrogen, progesterone, and
methoxychlor on surgically induced endometriosis in rats. Fundam Appl
Toxicol 27:287Ã�290 (1995).
32. DeVito MJ, Birnbaum LS. Toxicology of dioxins and related chemicals. In:
Dioxins and Health (Schecter A, ed). New York:Plenum Press, 1994;139Ã�162.
33. DeVito MJ, Thomas T, Martin E, Umbreit TH, Gallo MA. Antiestrogenic
action of 2,3,7,8-tetrachlorodibenzo-p-dioxin: tissue-specific regulation of
estrogen receptor in CD1 mice. Toxicol Appl Pharmacol 113:1Ã�9 (1992).
34. Pohjanvirta R, Tuomisto J. Short-term toxicity of
2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals: effects,
mechanisms, and animal models. Pharmacol Rev 46:483Ã�549 (1994).
35. Whitlock JPJ. Genetic and molecular aspects of
2,3,7,8-tetrachlorodibenzo-p-dioxin action. Ann Rev Pharmacol Toxicol
36. Poland A, Glover E. Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a
potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene.
Mol Pharmacol 10:349Ã�359 (1974).
37. Goldstein JA, Weacer R, Sundheimer DW. Metabolism of
2-acetylaminofluorene by two 3-methylcholanthrene inducible forms of rat
liver cytochrome P-450. Cancer Res 44:3768Ã�3771 (1984).
Jackie Hunt Christensen
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