[Date Prev][Date Next][Thread Prev][Thread Next][Date Index][Thread Index]

(long) Promotion of Endometriosis in Mice by PolychlorinatedDibenzo-p-Dioxins, Dibenzofurans, and Biphenyls-- July EHP

  Hope this works on everyone's e-mail software -- sometimes the Greek
  symbols don't come through, but here goes.
  [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
     * Introduction
     * Materials and Methods
     * Results
     * Discussion
  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 µg 2,3,7,8-TCDD/kg body weight (bw); 3 or 30 mg
  2,2«,4,4«,5,5«-hexachlorobiphenyl (PCB 153)/kg bw; 100, 300, or 1000 µg
  3,3«,4,4«,5-pentachlorobiphenyl (PCB 126)/kg bw; 10, 30, or 100 µg
  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 µg/kg bw) and 4-PeCDF (100 µg/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, structure�activity relationships, TCDD. Environ Health
  Perspect 105:750�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 (5�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
  is warranted.
  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 60�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: 3,3«,4,4«,5,-pentachlorobiphenyl (PCB 126),
  2,2«,4,4«,5,5«-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 (40�50%), 12:12-hr light:dark cycle,
  and constant temperature (20�24¡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 µg 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 µg PCB 126/kg bw with 10�12 animals per group.
  Experiment 3 included doses of 0, 10, 30, and 100 µg 4-PeCDF/kg bw and 2 and
  20 mg 1,3,6,8-TCDD/kg bw with 10�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 -70¡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 µl) to provide reaction
  linearity, added to 0.1 M KPO4 buffer containing 1.5 nM ethoxyresorufin, and
  preincubated for 2 min at 37¡C. The production of resorufin was started by
  the addition of 100 µl of §-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 µg 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 µg 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 µg 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 µg 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 µg 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 µg PCB 126/kg bw. Lesion
  weight values for the highest dose of PCB 126 (1000 µg/kg bw) decreased
  nonsignificantly from 300 µg/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
                                   [table 1]
  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 µg 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 µg
  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 µg 2,3,7,8-TCDD (in two of three animals
  examined), 100 µg PCB 126 (one of three animals), and 1000 µg 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 µg 2,3,7,8-TCDD
  (53.3%), 100 µg PCB 126 (60%), or 1000 µg 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.
                                   [table 2]
  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 µg 2,3,7,8-TCDD/kg bw, which was followed by an
  apparent decrease in uterine weights in the highest dose group, 10 µg
  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 µg 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 structure�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 µg
  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 µg 2,3,7,8-TCDD, or 100 or 1000 µg
  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
  structure�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 µg PCB 126/kg bw should be comparable to
  lesions in animals treated with 10 µg 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 µg 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 structure�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 21:51�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 105:391�401 (1995).
  4. Birnbaum LS. The mechanism of dioxin toxicity: relationship to risk
  assessment. Environ Health Perspect 102(suppl 9):157�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 35:553�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 211:463�469 (1979).
  7. Umbreit TH, Hesse EJ, Macdonald GJ, Gallo MA. Effects of TCDD�estradiol
  interactions in three strains of mice. Toxicol Lett 40:1�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 21:433�441 (1993).
  9. Cummings AM, Metcalf JL, Birnbaum L. Promotion of endometriosis by
  2,3,7,8-tetrachlorodibenzo-p-dioxin in rats and mice: time�dose dependence
  and species comparison. Toxicol Appl Pharmacol 138:131�139 (1996).
  10. Olive DL, Schwartz LB. Endometriosis. New Engl J Med 328:1759�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;9�17.
  12. Cummings AM, Metcalf JL. Induction of endometriosis in mice: a new model
  sensitive to estrogen. Reprod Toxicol 9:233�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
  Academic, 1991;97�111.
  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 14:699�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;3�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
  55:759�765 (1991).
  17. Koninckx PR, Braet P, Kennedy SH, Barlow DH. Dioxin pollution and
  endometriosis in Belgium. Hum Reprod 9:1001�1002 (1994).
  18. Mayani A, Barel S, Soback S, Almagor M. Dioxin levels in women with
  endometriosis. Hum Reprod 12:373�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 24:145�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 88:213�224 (1994).
  21. Vernon MW, Wilson EA. Studies on the surgical induction of endometriosis
  in the rat. Fertil Steril 44:684�694 (1985).
  22. Diliberto JJ, Akubue PI, Luebke RW, Birnbaum LS. Dose�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
  130:197�208 (1995).
  23. Bradford MM. A rapid and sensitive method for quantitation of microgram
  quantities of protein utilizing the principle of protein-dye binding. Anal
  Biochem 72:248�254 (1976).
  24. Okey AB, Riddick DS, Harper PA. Molecular biology of the aromatic
  hydrocarbon (dioxin) receptor. TIPS 15:226�232 (1994).
  25. Safe S. Comparative toxicology and mechanism of action of
  polychlorinated dibenzo-p-dioxins and dibenzofurans. Ann Rev Pharmacol
  Toxicol 26:371�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
  structure�activity relationships. Toxicology 41:21�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 32:3803�3813 (1983).
  28. Kafafi SA, Afeefy HY, Said HK, Hakimi JM. A new structure�activity model
  for Ah receptor binding. Polychlorinated dibenzo-p-dioxins and
  dibenzofurans. Chem Res Toxicol 5:856�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 90:243�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
  133:321�327 (1995).
  31. Cummings AM, Metcalf JL. Effects of estrogen, progesterone, and
  methoxychlor on surgically induced endometriosis in rats. Fundam Appl
  Toxicol 27:287�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;139�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 113:1�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 46:483�549 (1994).
  35. Whitlock JPJ. Genetic and molecular aspects of
  2,3,7,8-tetrachlorodibenzo-p-dioxin action. Ann Rev Pharmacol Toxicol
  30:251�277 (1990).
  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 10:349�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 44:3768�3771 (1984).
  Jackie Hunt Christensen
  Food Safety Project Director
  Institute for Agriculture and Trade Policy
  2105 1st Avenue South
  Minneapolis,  MN 55404
  612-870-3424 (direct line)
  612-870-4846 (fax)
  e-mail: <jchristensen@igc.apc.org>
  IATP's Endocrine Disrupter Resource Center: http://www.sustain.org/edrc