Toxicological Sciences 67, 63-74 (2002)
Copyright © 2002 by the Society of Toxicology
ENDOCRINE TOXICOLOGY |
Persistent Abnormalities in the Rat Mammary Gland following Gestational and Lactational Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
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* Division of Reproductive Toxicology and
Division of Experimental Toxicology, Office of Research and Development, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and
Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599
Received September 17, 2001; accepted November 14, 2001
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exposure during gestation has revealed reproductive anomalies in rat offspring, including inconclusive reports of stunted mammary development in females (Brown et al., 1998
, Carcinogenesis 19, 1623–1629; Lewis et al., 2001, Toxicol. Sci. 62, 46–53). The current studies were designed to examine mammary-gland development in female offspring exposed in utero and lactationally to TCDD, and to determine a critical exposure period and cellular source of these effects. Long-Evans rats were exposed to 1 µg TCDD/kg body weight (bw) or vehicle on gestation day (GD) 15. TCDD-exposed females sacrificed on postnatal days (PND) 4, 25, 33, 37, 45, and 68 weighed significantly less than control litter mates, and peripubertal animals exhibited delayed vaginal opening and persistent vaginal threads, yet did not display altered estrous cyclicity. Mammary glands taken from TCDD-exposed animals on PND 4 demonstrated reduced primary branches, decreased epithelial elongation, and significantly fewer alveolar buds and lateral branches. This phenomenon persisted through PND 68 when, unlike fully developed glands of controls, TCDD-exposed rats retained undifferentiated terminal structures. Glands of offspring exposed to TCDD or oil on gestation days 15 and 20 or lactation days 1, 3, 5, and 10 were examined on PND 4 or 25 to discern that GD 15 was a critical period for consistent inhibition of epithelial development. Experiments using mammary epithelial transplantation between control and TCDD-exposed females suggested that the stroma plays a major role in the retarded development of the mammary gland following TCDD exposure. Our data suggest that exposure to TCDD prior to migration of the mammary bud into the fat pad permanently alters mammary epithelial development in female rat offspring. Key Words: dioxin; fetus; mammary development; mammary gland; prolactin; rat; TCDD; thyroid; vaginal opening; vaginal thread.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transplacental and lactational transfer of environmental toxicants to a developing human fetus and infant has the potential to cause adverse effects. The fetus is particularly prone to damage by maternally derived toxins, as the blood-brain barrier does not completely form in the child until nearly 6 months after birth (reviewed by Rodier, 1995
). Transfer of toxic compounds to the infant may also occur postnatally during lactation, especially in the case of polychlorinated biphenyls (PCB) and polychlorinated dibenzo-para-dioxins (i.e., TCDD: Chen et al., 2001; Kacew, 1994; Li et al., 1995; Rogan and Ragan, 1994; Yakushiji, 1988). TCDD, or dioxin, is the most toxic of the polyhalogenated aromatic hydrocarbons, and as a lipophilic compound it bioaccumulates in higher vertebrates and has a half-life ranging from 6 to 12 years (Portier et al., 1999). The infant ingests 12–14% of a 25-year cumulative TCDD toxic-equivalent intake from a 6-month period of breastfeeding (Patandin et al., 1999). The TCDD toxic equivalent daily intake per kilogram body weight is 50 times higher in breast-fed infants than in adults. Children of 1 to 5 years also orally acquire nearly three times the adult dietary intake of dioxin-like compounds on a body-weight basis (Patandin et al., 1999).
TCDD is a well-studied ligand for the aryl hydrocarbon receptor (AhR). The AhR and its signaling partner, ARNT, are readily detected in the rodent fetus between gestation days 10–16 (Abbott and Probst, 1995; Abbott et al., 1995). The expression of the AhR during this important developmental period suggests appreciable physiological functions. Reproductive system aberrations found in the female rat following gestational exposures to TCDD include, but are not limited to, decreased pup body weights, presence of vaginal threads, delayed vaginal opening, cleft phallus, decrease in the number of pups at weaning (Flaws et al., 1997; Gray and Ostby, 1995; Gray et al., 1997), and possible retarded development of the mammary epithelium (Brown et al., 1998; Lewis et al., 2001). The AhR (along with AhR nuclear translocator, cytochrome P450 (CYP1A1 and CYP1B1) is expressed and immunohistochemically localized in the epithelial portions of the mouse mammary gland during periods of ductal proliferation. Absence of the AhR (Ahr-null mice) or treatment of mammary gland explants with tetrachlorodibenzofuran (a potent AhR agonist) resulted in suppressed lobule development of the glands (Hushka et al., 1998), suggesting an important physiological role of the AhR in normal mammary gland development. Rats and humans also possess functional AhR in both epithelial and stromal cells of the mammary gland (Christou et al., 1995; Larsen et al., 1998). Furthermore, whereas normal rat mammary epithelium expresses modest levels of AhR, high levels of nuclear localized AhR were detected in the epithelium of chemical carcinogen-induced rat mammary tumors (Trombino et al., 2000). The adverse effect of PCBs or TCDD on a mother's duration of lactation or ability to raise offspring to weaning has been reported in both human and wildlife species (Heaton et al., 1995; Restum et al., 1998; Rogan et al., 1987). In fact, the measurable level of PCBs in breast milk was inversely correlated with the duration of lactation in a study of North Carolina women (Rogan et al., 1987).
Mammary gland development in rodents and humans takes place in several phases. In rats, the mammary epithelial bud is formed at the site of the nipple by GD 12–14, and the gland is fairly inactive until approximately GD 16–17 when the migration of the bud begins to fill the stromal portion of the gland (Sakakura, 1987). At birth, the epithelium has entered the fat pad and formed a ductal tree, with several ducts and lateral branches from each primary duct. From birth to puberty, the gland undergoes allometric growth, slowly extending the fat pad and epithelium from the nipple toward the back of the animal. At puberty, the gland undergoes exponential growth, with rapid development of terminal end buds (TEB). The TEB are club-like clusters of cells, several layers thick, at the ends of the lateral ductal branches. The rapid growth of the gland at this point results from differentiation or division of the cells in the TEB and continues until the epithelium reaches the limits of the fat pad. Following puberty, the gland transitions to a differentiated resting phase, at which time TEB are absent and terminal ducts and small lobules or aveolar buds (which are the precursors to the large lobuloalveolar structures found during pregnancy) are common (Daniel and Silberstein, 1987; Richert et al., 2000). The periods of active egress (GD 16–20 and puberty) are critical periods of mammary development that have been shown to be altered by hormones, growth factors (Darcy et al., 1995; Dunbar and Wysolmerski, 2001; Fendrick et al., 1998; Imagawa et al., 1990; Topper and Freeman, 1980; Turner, 1970), and environmental agents (Brown and Lamartiniere, 1995; Brown et al., 1998; Chapin et al., 1997; Fritz et al., 1998; Lewis et al., 2001; Singletary and McNary, 1992).
Studies have described variable effects on mammary gland development in rat offspring exposed to TCDD on day 15 of gestation (Brown et al., 1998, Lewis et al., 2001) or during puberty (Brown and Lamartiniere, 1995). Oral administration of 2.5 µg TCDD/kg bw on PND 25, 27, 29, and 31 (just prior to puberty) in Sprague-Dawley females caused a decrease in the number of TEB on PND 32 and significantly smaller mammary glands (Brown and Lamartiniere, 1995). However, the same authors reported no significant alterations to mammary glands in 21-day-old Sprague-Dawley pups exposed to 1 µg TCDD/kg bw on day 15 of gestation. Littermates that were 50 days old did reveal delayed differentiation of the TEB (Brown et al., 1998), similar to the recent findings of Lewis and coworkers (2001) in Holtzman rats.
Therefore, the current studies were designed to address the extent to which a gestational and lactational exposure to TCDD can delay the developing mammary gland, in part, to explore the possibility that TCDD-induced delays in mammary gland differentiation could be a contributing factor in the increased number of dimethybenz[a]anthracene (DMBA)-induced tumors in TCDD-exposed female rats (Brown et al., 1998; Desaulniers et al., 2001). These studies also involved identification of the critical window of exposure for retarded development. Epithelial transplantation studies were designed to evaluate the role that the epithelium and stroma may play in regulating the effects of TCDD.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
TCDD (>98% purity) in acetone (1 mg/10 ml) was obtained from Radian Corp. (Austin, TX). For the preparation of dosing solution, a volume of stock TCDD was added to corn oil and the acetone was removed by evaporation using a Savant Speed Vac (Savant Instruments, Inc., Farmingdale, NY). Following acetone evaporation, additional corn oil was added to achieve the desired TCDD concentration. All other chemicals were from commercial sources and were of the highest purity available.
Animals.
Time-pregnant Long-Evans rats (GD 9 [day after mating = GD 0]) were obtained from Charles River Breeding Laboratories (Raleigh, NC). Females were housed in clear plastic cages containing heat-treated pine shavings (Beta Chips, North Eastern Products Inc., Warrensburg, NY) and given food (Purina 5008 Rodent Chow, Ralston Purina Co., St. Louis, MO) and water ad libitum. They were maintained in a room with a 14:10-h light cycle, a temperature of 20–24°C, and a relative humidity of 40–50%. All animals were treated as approved by the National Health and Environmental Effects Research Laboratory, Institutional Animal Care and Use Committee.
Experimental design and dosing.
Two groups of 10 pregnant dams were treated by oral gavage on GD 15 with either 1 µg TCDD/kg bw in corn oil, or vehicle, in a dosing volume of 5 ml/kg. Litters were weighed and equalized to three males and five females (when possible) on PND 4. At weaning (PND 25), animals were weighed and housed together as siblings, as described above, in unisexual groups of two to three rats per cage. Female offspring were sacrificed on PND 4, 33, 37, 45, 68, and 110 to visualize their mammary gland development (at least two female offspring per dam). A third group of eight dams were dosed as described above (n = 4 in each group) with pups equalized and weaned as above. The females remaining in these litters were examined for vaginal opening (VO) and estrous cyclicity.
The critical time of TCDD exposure leading to stunted mammary gland development in Long-Evans rats was determined by dosing timed-pregnant dams (n = 5/treatment time) with 1 µg TCDD/kg bw on GD 15 or 20 or PND 1, 3, 5, or 10. The glands of female pups were analyzed by whole-mount analysis on PND 4 (if dam was dosed prior to this time) and 25 (all time points).
All female offspring in these studies were observed for VO beginning on PND 29. The presence of a "pinhole" opening was recorded and VO was assessed by the presence of a fully opened vagina (whether a thread was present or not). All animals were appraised for the persistence of vaginal thread until the time of smearing or euthanization. Vaginal smears (as described by Cooper and Goldman, 1999) began on PND 42 on the animals described (n = 10 each treatment from 4 litters; PND 42 was the day of VO for the last animals in these groups) and continued until PND 60. Smear data was visualized by assigning the value of 1 to diestrus, 5 to proestrus, and 10 to estrus and plotting in Lotus 123.
Radioimmunoassays.
Animals used to evaluate serum hormone concentrations were decapitated, and trunk blood was obtained and separated by centrifugation. Serum was kept frozen at –80°C until assays were performed. Serum prolactin (PRL) and thyroid stimulating hormone (TSH) radioimmunoassays were performed as previously described (Cooper et al., 2000). Serum triiodothyronine (T3) and thyroxine (T4) concentrations were determined using antibody-coated tube kits from Diagnostic Products Corp. (Los Angeles, CA), as instructed by the manufacturer.
Transplantation study.
Eight time-pregnant Long-Evans dams were treated as above on GD 15 with corn oil (n = 4) or TCDD (n = 4), and four to six females of each dam (total of 37) were used for epithelial transplantation studies to evaluate the source of the delayed mammary gland development. Sterile procedures were used throughout the surgeries. Control and TCDD-exposed pups of similar weights were anaesthetized (87 mg/kg ketamine and 13 mg/kg xylazine, in a dose volume of 10 ml/kg) on PND 22. Sedated animals were secured with tape, abdomens were washed with 70% ethanol, and nipples were outlined with a black marker. A "Y" incision was created in the lower inguinal region in both animals, and the #4 and #5 nipples, the blood vessels on either side of the inguinal gland lymph nodes, and the artery between the 5th and 6th glands were sealed using cautery (Harvard Appartus, Holliston, MA). The tissue containing the mammary epithelium between the nipple and extending just past the lymph node on the rat's right side was removed to sterile phosphate-buffered saline. An approximately 1-mm2 piece of tissue from the nipple area was embedded into a pocket created in the remaining fat pad of the recipient animal (control into TCDD-exposed, and vice versa). The left side was used as internal control. A portion of the animals in each group received their own epithelium into the cleared fat pad (to evaluate the efficiency of the epithelial transfer), others were left with a cleared fat pad only (to evaluate the efficiency of the clearing process), and the rest were left intact and were sham operated. The incision was stapled shut and the animals were allowed to recover in a clean, quiet cage for 4 h. The animals received a single injection of buprenorphine (0.05 mg/kg, im) to alleviate any potential pain. The transplants were allowed to outgrow for 6 weeks, at which time the rats were euthanized and the glands removed for whole-mount analysis. The weights of control and TCDD-exposed animals used for epithelial transplantation studies were not different, based on an unpaired t-test (246 vs. 255 g, respectively).
Mammary whole mounts.
The fourth and/or fifth inguinal mammary glands were removed in all experiments and pressed between a microscope slide and Parafilm® (under weight) for 1–12 h, depending on the thickness of the gland. Glands flattened to slides were then fixed at room temperature in Carnoy's solution (6:3:1, v:v:v, ethanol, chloroform, and glacial acetic acid) overnight. Glands were transferred to 70% ethanol, which was removed by gradual dilution to water. The fixed glands were stained overnight in 0.2% carmine and 0.5% aluminum potassium sulfate. Glands were rinsed in water and dehydrated to absolute alcohol, cleared in xylene, and permanently mounted; whole mounts were visualized/photographed on a Leica WILD M420 Macroscope at the magnifications indicated in the text. The effects of TCDD exposure on mammary epithelial proliferation were detectable in the #3 glands also, but the larger set of inguinal glands, free of muscle and comparable to the glands used in transplantation studies, were preferred in our studies.
Histology.
The fourth and/or fifth inguinal glands were removed for histological analyses to confirm identity of TEB and other terminal structures. Glands were compressed in cassettes in two changes of sodium phosphate-buffered 4% paraformaldehyde for 48 h each, at 4°C. Fixed glands were transferred to 70% ethanol and glands were processed in paraffin. The 5-µm sections (University of North Carolina, Histopathology Core Facility, Chapel Hill, NC) were stained in hematoxylin and eosin, permanently mounted, and viewed/photographed by light microscopy on a Leitz Laborlux D.
Statistical analysis.
InStat (GraphPad Software, San Diego, CA) was used in analysis of the data sets and p < 0.05 was used to indicate significant differences. The PND 4 whole mounts were photographed at 10x magnification. For ease of accurate measurements, all alveolar buds/terminal branches and primary ducts in the 4th and 5th glands were counted, and the length of epithelial extension from the nipple was determined (in cm) from 5 x 7 inch prints. Absolute distances were analyzed for significance, using an unpaired t-test (8.4 ± 0.4 cm, control; 6.3 ± 0.3 cm, treated). Epithelial growth data are expressed as a percent of control. The number of terminal/alveolar buds in PND 4 glands of control vs. TCDD-exposed rats was log-transformed, due to unequal variance in the two treatment groups, then evaluated by an unpaired t-test. Simple comparisons of the number of primary branches in PND 4 mammary glands, day of vaginal opening, days spent in diestrus or estrus, and serum PRL, T3, T4, and TSH concentrations of control and TCDD-exposed animals were also individually compared using unpaired t-tests. Rat body weight over time was evaluated using one-way analysis of variance (ANOVA) and a weighted least-squares regression model, due to modest variance over this growth period. A dam effect was apparent in regression analysis due to the small number of dams represented on PND 68 (six pups observed, three dams). The differences in body weights and serum PRL, T3, T4, and TSH concentrations of the animals exposed to TCDD or oil on various days of development and sacrificed on PND 25 (Fig. 6) were determined using one-way ANOVA.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mammary Gland Development
The mammary epithelia of TCDD-exposed and control animals were compared in whole-mount analyses on PND 4, 33, 37, 45, 68, and 110. Glands from PND 4 females were examined to discern if early signs of TCDD exposure were detectable. Mammary tissue from TCDD-exposed females contained significantly fewer primary branches from the collecting duct, delayed migration of the epithelium through the fat pad (75% of control), and fewer terminal branches and alveolar buds (terminal structures) when compared to corn oil-treated controls (Table 1
). In fact, when the number of terminal structures from all animals were compared (regardless of the specific structure type), the animals exposed to TCDD during gestation had less than 50% that of controls. These developmental defects and migratory delays were consistently detectable in whole mounts (Fig. 1) and histological sections (Fig. 2) of the fourth and fifth inguinal glands from 4-day-old females.
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The TCDD-induced delay and stunting of mammary gland development was persistent over time; by PND 33 and PND 37 (peripubertal for control and TCDD-exposed, respectively), the sparse nature of the branching and the slowed epithelial migration through the fat pad were quite apparent (Fig. 1
). Evaluation of glands from 37-day-old control rats revealed differentiated ends (lobular structures) and filled fat pads, discerned by the fourth and fifth inguinal glands grown together. However, glands from age-matched TCDD-exposed females still retained their terminal end buds, as evidenced by the histological sections (Fig. 2). Only one of the 12 TCDD-exposed offspring examined at this point in time had a filled fat pad on the whole mounts. Furthermore, most glands of TCDD-exposed females evaluated at PND 45 retained a section of undifferentiated TEBs, whereas few control glands had sections of TEB remaining (not shown). This finding is significant as it is common to administer carcinogenic agents, such as DMBA, to rats to stimulate mammary tumors around this time, and the proliferating TEB are highly susceptible to such compounds (Russo and Russo, 1978).
Again, unlike controls, many undifferentiated terminal ducts were present in the glands from animals exposed to TCDD in utero and lactationally and that were euthanized on PND 68 (Fig. 1) and 110 (not shown). These TCDD-exposed glands were consistently characterized by spindly epithelial structures containing fewer lateral branches or lobules than their age-matched control counterparts, and they failed to completely fill the fat pad by 110 days.
Body Weights, Puberty, and Cyclicity
As anticipated, treatment of Long-Evans dams with 1 µg TCDD/kg bw on GD 15 resulted in offspring that were significantly smaller than corn oil-exposed pups when evaluated on PND 4, 33, 37, 45, and 68 (animals described above, Fig. 3). These comparisons were made using one-way ANOVA, and these differences in body weight are confirmed when using a more conservative analysis approach (weighted least-squares model). The estimated slope and standard errors in this regression analysis for control (4.39 ± 0.10) and TCDD-exposed (3.44 ± 0.14) pups indicated that the treated group continued to grow more slowly than control pups (p < 0.001).
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A subset of 20 female offspring (n = 10 control and 10 treated pups, each from 4 dams) was evaluated specifically for VO and cyclicity. The weight of the control and TCDD-exposed pups differed significantly at weaning (78.5 ± 3.0 and 68.1 ± 1.9 g, respectively, p < 0.01). Consistent with previous reports in Long-Evans and Holtzman rats (Gray and Ostby, 1995
; Gray et al., 1997), the day of vaginal opening was significantly delayed (p = 0.02) in TCDD-exposed animals (37.6 ± 1.2 vs. 34.2 ± 0.7 in controls). However, even though the TCDD-exposed animals were smaller and had delayed puberty, there was no significant difference between TCDD-exposed and control rats in their early estrous patterns (Fig. 4). The number of days in diestrus and estrus were similar for these animals during this early cyclicity period (Control = 8.7 + 0.6 diestrus, 6.3 + 0.4 estrus; TCDD = 7.6 + 0.4 diestrus, 6.9 + 0.5 estrus), although the control animals in our study were not all typical in their cyclicity. Also, similar to previous reports (Flaws et al., 1997; Gray and Ostby, 1995), significantly more animals exposed to TCDD (71%) retained vaginal threads until sacrifice or smearing in these studies, compared to age-matched controls (7%).
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Critical Period of Exposure
In a study designed to evaluate the critical period of exposure required to cause the mammary developmental perturbations seen with a single dose of TCDD, timed-pregnant dams (n = 5/dose group) received 1 µg TCDD/kg bw on GD 15 or 20 and PND 1, 3, 5, or 10. Mammary glands from all female pups exposed to TCDD at these times were examined as whole mounts on PND 4 (for PND 3 and earlier) or PND 25 (all exposure periods) to ascertain the critical window of development that was affected. Upon examination, the only dose day that triggered consistently underdeveloped epithelium was GD 15 (Fig. 5
). These glands lacked the epithelial migration and lateral branching patterns seen in controls. Glands from 20% of the animals exposed on GD 20 displayed sparse alveolar budding, specifically on PND 4 (as shown). However, their migration through the fat pad, lateral branch formation, and primary branching was similar to controls. TCDD exposure on GD 20 does not have a persistent effect on the mammary gland of offspring, like that shown by dosing on GD 15, as glands appeared similar to controls at weaning. The body weights of the animals in the GD 15 and GD 20 groups were statistically reduced (Fig. 6), although TCDD caused a decrease in body weight in all exposed groups. Gland development in animals exposed to TCDD on all postnatal dosing days was comparable to controls on PND 4 and 25 (those dosed on PND 3, 5, and 10 are not shown). These data demonstrate that exposure to TCDD during gestation lead to delays in mammary gland development of the female offspring. Exposure during lactation seems without effect.
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Serum Hormone Measurements
In an attempt to discern a potential reason for the delayed and irregular mammary gland development in animals exposed to TCDD on GD 15, trunk blood was taken from the 25-day-old offspring in the study just described. Radioimmunoassays were performed to determine serum PRL, TSH, T3, and T4 concentrations (hormones previously found altered following TCDD administration and also shown to alter mammary development). Although dosing with TCDD on GD 15 or 20 caused significantly decreased body weight at weaning, no correlation could be made with serum hormone status and delays in mammary epithelial development. As shown in Figure 6
, basal serum PRL levels were generally depressed following TCDD exposure, but this decrease was not significant, statistically or physiologically. Although there was a significant rise in serum TSH on most dosing days, there was no concomitant change in serum T3 or T4 levels detected, suggesting that the thyroids of these animals were compensating for the increased TSH levels. Trunk blood was also taken from 60-day-old animals that were used for vaginal smears. These animals were evaluated for the same serum hormone levels. Because all animals were decapitated on PND 60, regardless of the stage of the estrous cycle they were in, the basal serum PRL levels had quite a modest variability and were not found to be different (control = 15.4 ± 5.2 and TCDD = 15.0 ± 4.6 ng/ml). Serum TSH was again statistically elevated in TCDD-exposed animals (control = 1.5 ± 0.1, TCDD = 2.0 ± 0.2 ng/ml), and in this older group of animals, serum T4 was found to be decreased in the TCDD-exposed group (control = 45.8 ± 1.9, TCDD = 40.8 ± 1.6 ng/ml) at p < 0.05. Serum T3 was not different from controls in treated animals and was within normal levels.
Epithelial Transplants
Because the AhR is found expressed in both epithelial and stromal cells of the rat mammary gland (Christou et al., 1995), it was of interest to evaluate the role that each of those cellular compartments, which interact with each other in an intricate manner, may play in the TCDD-induced disruption of normal mammary gland development. To this end, mammary epithelium was transferred between cleared fat pads of 22-day-old pups that had been exposed to oil or TCDD on GD 15. As described in the Materials and Methods section, pairs of control and TCDD-exposed animals of similar weight were anesthetized, their fat pad was cleared of epithelium, and a small piece of that epithelium was transplanted into the recipient. Incisions were clipped and the animals were allowed to foster the transplant growth for a period of 6 weeks, at which time the animals were euthanized and mammary glands visualized by whole mount analysis. As shown in Figure 7, in the sham-operated row (top panels), on PND 64, mammary gland development progressed as expected. Control glands were filled with visible lobules and TCDD-exposed glands displayed sparse ductal development, including poor differentiation of terminal structures. When epithelium from a TCDD-exposed animal is transplanted into a control fat pad, it goes on to develop a proliferative ductal structure filled with lobules and differentiated ends (Fig, 7, lower right, differentiating ends shown with an arrow). On the contrary, control epithelium transplanted into a TCDD-exposed fat pad forms thickened, irregularly formed ducts, few lobules, and, in many, remnants of TEB (lower left, TEB shown by arrows). Only 40% of all the epithelial transplants grew in the host mammary gland. In those that grew, however, our data suggests that the stromal portion of the gland is no longer capable of appropriate signaling with the epithelium following a gestational TCDD-exposure. A similar, important role of the epithelial-stromal interaction for TCDD signaling has been demonstrated using mouse uterine tissue recombination grafted into nude mice (Buchanan et al., 2000).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The results of the studies presented here define the time period of disrupted mammary gland development and confirm its persistence in the virgin rat following a single exposure to TCDD during gestation. Our observations are consistent with similar TCDD-exposure strategies in previous reports citing delayed mammary development in 11-week-old Holtzman (Lewis et al., 2001
) or 50-day-old Sprague-Dawley rats (Brown et al., 1998). These results are also similar to the phenotype of mice null for the AhR (Hushka et al., 1998), in which 5 to 8 week-old –/– females displayed mammary epithelial development similar to that of 2-week-old controls (+/+). The glands were small with limited branches and decreased numbers of TEB, and they lacked substantial lobule development. However, our results also differ dramatically from the work of Brown and coworkers (1998) in which they demonstrate no difference in the number of TEB, terminal ducts, or lobules in an early stage of mammary gland development (21 days old). Delayed development of the mammary gland was detected in our studies as early as PND 4. This stunting of the gland due to the TCDD exposure was not compensated for during (PND 37) or following puberty (PND 45). In fact, it seemed that this was a permanent developmental delay, and this lasting effect of TCDD appears to be a factor of the developmental time of exposure.
Our studies define the time around GD 15 as a critical period in fetal mammary gland development that may be sensitive to the effects of environmental toxicants such as TCDD. Offspring of dams that received TCDD on GD 15, which happens to coincide with the time that the mammary epithelial bud forms and begins its migration into the fat pad (Sakakura, 1989), were the only group of animals in our studies that displayed consistently impaired gland maturation. Not only were the number of primary ducts altered, but the lateral branching patterns and number of terminal structures in TCDD-exposed pups was also decreased from those of age-matched controls. Dosing the dams with TCDD on GD 20 or during lactation had no notable lasting effect on the development of the neonates' mammary glands. These findings suggest that long-term exposure of the developing offspring to TCDD (gestation and lactation) is not required for the effects of TCDD in the Long-Evans rat. Pups from dams exposed to TCDD on GD 15 or GD 20 weighed significantly less at weaning than those age-matched controls (21% and 16% reduction in weight, respectively, compared to about 10% for other dosing times). It has been previously suggested that reduced calorie intake and body weight is correlated with decreased or delayed mammary gland development in virgin mice (Engelman et al., 1994). It may also be hypothesized that alterations in puberty or circulating endocrine hormones in animals exposed to TCDD may predispose the rats to a delay in mammary epithelial proliferation. Our data, and those of others, do not support these hypotheses. In fact, in the pups exposed to TCDD on GD 15 and euthanized on PND 4, we did not observe dramatic differences in body weight when compared to controls, unlike those observed at later developmental time points (weaning, 21%, Fig. 6; and PND 33, 34%, Fig. 3). However, at this early time point, clear delays were seen in mammary gland development in TCDD-exposed animals. In recent data published by Lewis et al. (2001), pregnant Holtzman rats were exposed to TCDD on GD 15 (1 µg/kg) and offspring ovariectomized at 9 weeks. Although the authors noted a delay in mammary epithelial differentiation in TCDD-exposed females, they saw no difference in estrogen receptor-alpha (ER
) responsiveness to exogenous 17ß-estradiol (measured as increased mammary progesterone receptor expression and lobule formation) that could be attributed to the toxicant. In fact, the authors reported an increased expression of ER in the mammary glands of TCDD-exposed animals (likely due to increased numbers of terminal end bud structures). Previous studies have indicated that levels of circulating estradiol (Flaws et al., 1997; Gray et al., 1997) are not significantly altered in female rat pups following a gestational exposure to TCDD, even though delayed vaginal opening was reported and persistent vaginal threads were apparent. Furthermore, Holtzman rat dams exposed to TCDD on gestation days 4 to 15 (1 µg/kg bw), demonstrated no change in serum estradiol levels (Shiverick and Muther, 1983). Work by Chaffin and coworkers (1997), though they detected decreased serum estradiol levels in prepubertal females exposed to TCDD on GD15 (1 µg/kg), demonstrated similar serum progesterone and androstendione levels in exposed and control rats. Estrogen, progesterone (or adrenal corticoids), prolactin (or growth hormone), and EGF (or insulin) are known to be critical in branching morphogenesis, terminal end bud proliferation, migration, and differentiation of mammary epithelium of the peripubertal rodent (reviewed in Darcy et al, 1995; Imagawa et al., 1990; Silberstein, 2001).
Many studies have demonstrated altered serum PRL, TSH, T3, and T4 levels in adult rats exposed to large boluses of TCDD. Additionally, it was hypothesized that changes in serum PRL may be the cause of the drop in T4 seen in several studies, since PRL has been linked to the development and regulation of the thyroid and adrenal glands (Jones et al., 1987). Studies in adult male rats administered a 50 µg TCDD/kg bw injection were found to lose their circadian rhythm of PRL secretion and it was determined that a pimozide-reversible increase in dopamine in the median eminence was responsible, suggesting a hypothalamic mode of action for TCDD (Jones et al., 1987; Russell et al., 1988). These studies also detected decreased serum T4 in TCDD-exposed rats. A thorough study of the response of TSH, T3, and T4 in male rats administered single doses of TCDD (Potter et al., 1986) also found that high doses of the toxicant produced decreased T4 and increased TSH and T3. However, when low level TCDD exposures (0.1 µg/kg; Seo et al., 1995) were performed in timed-pregnant dams, a slight decrease in T4 was detected in weanling pups, but T3 and TSH levels were constant in TCDD- and PCB-exposed pups. Our studies suggest that baseline serum PRL levels are decreased, but not in a physiologically significant manner. The greatest drop we detected in weanling rats was from 3.9 ng/ml in controls to 2.7 ng/ml in animals exposed to TCDD on PND 3; a drop of 31% (and significantly different by unpaired t-test, p < 0.01). Both of those concentrations are well within the normal range for serum PRL levels in the young rat. Furthermore, a similar observation is made following RIAs for serum TSH and T4. Although serum TSH concentrations are significantly elevated in both instances where they were examined (critical period and cyclicity observation groups) and T4 concentrations were concordantly decreased in 60-day-old animals, the levels were still considered within the normal range for animals of this age. There is data in humans (Haddow et al., 1999) that provide a correlation between elevated maternal TSH (with/without noticeable changes in T4) and poorer performance by their children in attention, language, reading ability, school performance, and visual-motor skills. Therefore, although from our data it is not clear that relatively low levels of TCDD consistently alter anterior pituitary secretion of these important endocrine hormones in pups exposed during gestation, there is still a possibility that these hormones play a role in altered mammary development.
The AhR and ARNT (1 and 2) are expressed in regions of the rat brain controlling appetite and circadian rhythms, such as the arcuate nucleus, ventromedial hypothalamus, paraventricular nucleus, and the suprachiasmatic nucleus (Petersen et al., 2000). Furthermore, the tuberoinfundibular dopaminergic neurons, ultimately regulating pituitary PRL secretion, begin stably expressing tyrosine hydroxylase by GD 18 in the rat (Solberg et al., 1993), and either their development does not become complete until early lactation (Shah et al., 1988; Shyr et al., 1986) or the pituitary lactotropes are not responsive to the dopaminergic signals until several days postnatally (Yamamura et al., 1999). Data suggest that this neuronal development may be controlled by the hyperprolactinemic state of the dam during late gestation and early lactation. Therefore, alterations to the dams' circulating hormone status may be critical to the final endocrine milieu of the pups. Preliminary studies in Long-Evans dams dosed with TCDD on GD 15 indicate that serum PRL levels on GD 21 are dramatically decreased by this single exposure (S.E. Fenton, unpublished observations). Therefore, more dramatic endocrine changes may be apparent in pups if examined during a more appropriate time frame in future studies (as suggested by Shah et al., 1988).
There are many conflicting pieces of evidence in papers reviewed by Brown and coworkers (1998) that TCDD exposure may be related to breast cancer susceptibility. These authors, in particular, reported an increase in the mean number of terminal end buds and a decrease in the number of lobular structures on PND 50 in Sprague-Dawley females exposed gestationally to TCDD (1 µg/kg bw on GD 15). Additionally, they found an elevated number of tumors per animal and an increased incidence of tumor formation in TCDD-exposed animals treated with a chemical carcinogen, DMBA, on PND 50. The authors propose that the increased susceptibility to tumor formation may be directly correlated to the increased number of TEB present in the tissue at the time of exposure to the carcinogen. The substantial increase in time that TEB are present in the mammary glands of the Long-Evans rats in the present study (estimated at 20 days in controls and up to nearly 40 days following TCDD exposure), strengthen the suggestion that TCDD lengthens the "window of susceptibility" to carcinogens following a fetal exposure. A recent report (Desaulniers et al., 2001) also demonstrates that a single dose of TCDD on PND 18 (2.5 µg/kg bw) increases the number of methylnitrosourea-induced tumors in Sprague-Dawley rats. However, these authors failed to examine the morphology of the mammary tissue for an effect of TCDD exposure prior to administration of the carcinogen.
As the present studies found relatively "normal" endocrine hormone profiles following TCDD exposure, it was surprising to find, in epithelial transplant studies, that oil-treated rat mammary epithelium did not develop in a typical manner in the cleared fat pad of TCDD-exposed rats. Additionally, knowing that the mammary stroma of these rats is unable to support proper development of non-treated mammary epithelium, we suggest that early TCDD exposure may alter the stroma (receptor populations, basement membrane components, angiogenesis, etc.) and create an environment more conducive to tumor formation, given the appropriate signals. The stroma of the mammary gland plays an integral part in normal epithelial formation and tumor development (Cunha, 1994; Cunha and Hom, 1996). We hope to discover differences in gene expression between control and TCDD-exposed mammary tissue using cDNA microarray technology that will enlighten us as to the gene pathways or families involved in this stroma-mediated effect.
Early and permanent developmental delays in mammary glands of TCDD-exposed rat pups, without a clear cause, lead us to suggest that there is a critical window of fetal mammary development that was altered by this toxicant. Whether or not this is an "imprinting" or "fetal programming" effect is not known. These studies also raise the question as to the level of TCDD exposure necessary to trigger the mammary developmental delays seen. Thus, we cannot say if an in utero exposure (followed by milk from a control dam) differs from these results (gestational and lactational exposure). It is possible that the reason that Brown et al. (1998) did not see early developmental delays in their study is that the pups they used were transferred to untreated surrogate mothers at birth. However, in our study, dosing on GD 20 or during lactation alone did not cause a similar delay in mammary development to that seen by dosing on GD 15, when the tissues were examined on PND 4 or 25. It is not known at what concentration of TCDD the developmental delays seen in the mammary glands would not be observed, nor can we say that this effect is necessarily adverse. However, preliminary results from a study that followed TCDD-exposed pups for two generations demonstrate a significant effect on lactational performance in offspring exposed in utero and lactationally (Fenton, 2001). In hamsters, Wolf and coworkers (1999) found a dramatic drop in survival through weaning for pups whose mothers were exposed to TCDD during gestation (15% treated vs. 78% control), suggesting mammary development not able to support lactation in these dams. This finding is also similar to data published on mink following a dietary exposure to PCB-contaminated fish (Restum et al., 1998). Further studies are necessary to elucidate the full potential of TCDD exposure on lactational performance in our endangered wildlife that depend entirely on lactation for pup and species survival.
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ACKNOWLEDGMENTS |
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Financial support for J.T.H. was provided for this study by the U.S. Environmental Protection Agency, Cooperative Training Agreement (No. CT902908) with the University of North Carolina, Chapel Hill, NC 27599-7270. The authors would like to extend thanks to Selena Mistich and W. Keith McElroy (U.S. EPA) for running the PRL and TSH RIAs, to Judy Schmid (U.S. EPA) for statistical comparisons of body weight over time, and to Nannette Stangle-Castor (Roswell Park Cancer Institute, Buffalo, NY) for the mouse mammary gland transplantation protocol that was used as a guide for the procedure in the rat. The authors also appreciate the constructive review of this manuscript by Dr. Ralph Cooper (Reproductive Toxicology Division) and Dr. Mike DeVito (Experimental Toxicology Division) at the U.S. EPA.
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NOTES |
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This manuscript has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. Data within this manuscript were presented in part at the Society of Toxicology Meeting in San Francisco, CA, March 2001.
1 To whom correspondence should be addressed at the U.S. Environmental Protection Agency, ORD, National Health and Environmental Effects Research Laboratory, Reproductive Toxicology Division, MD-72, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: fenton.suzanne{at}epa.gov. 
2 Present address: Lorillard Tobacco Co., 420 N. English St., P.O. Box 21688, Greensboro, NC 27420-1688.
3 Present address: Stratagene, 11011 N. Torrey Pines Road, La Jolla, CA 92037.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. S. White, A. M. Calafat, Z. Kuklenyik, L. Villanueva, R. D. Zehr, L. Helfant, M. J. Strynar, A. B. Lindstrom, J. R. Thibodeaux, C. Wood, et al. Gestational PFOA Exposure of Mice is Associated with Altered Mammary Gland Development in Dams and Female Offspring Toxicol. Sci., March 1, 2007; 96(1): 133 - 144. [Abstract] [Full Text] [PDF]
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E. A. Andreasen, L. K. Mathew, C. V. Lohr, R. Hasson, and R. L. Tanguay Aryl Hydrocarbon Receptor Activation Impairs Extracellular Matrix Remodeling during Zebra Fish fin Regeneration Toxicol. Sci., January 1, 2007; 95(1): 215 - 226. [Abstract] [Full Text] [PDF]
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S. E. Fenton Endocrine-Disrupting Compounds and Mammary Gland Development: Early Exposure and Later Life Consequences Endocrinology, June 1, 2006; 147(6): s18 - s24. [Abstract] [Full Text] [PDF]
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H. M. Miettinen, R. Sorvari, S. Alaluusua, M. Murtomaa, J. Tuukkanen, and M. Viluksela The Effect of Perinatal TCDD Exposure on Caries Susceptibility in Rats Toxicol. Sci., June 1, 2006; 91(2): 568 - 575. [Abstract] [Full Text] [PDF]
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J. L. Rayner, R. R. Enoch, and S. E. Fenton Adverse Effects of Prenatal Exposure to Atrazine During a Critical Period of Mammary Gland Growth Toxicol. Sci., September 1, 2005; 87(1): 255 - 266. [Abstract] [Full Text] [PDF]
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 F. M. Poulet, R. Veneziale, P. M. Vancutsem, P. Losco, K. Treinen, and R. E. Morrissey Ziracin-Induced Congenital Urogenital Malformations in Female Rats Toxicol Pathol, April 1, 2005; 33(3): 320 - 328. [Abstract] [Full Text] [PDF]
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C. Emond, L. S. Birnbaum, and M. J. DeVito Physiologically Based Pharmacokinetic Model for Developmental Exposures to TCDD in the Rat Toxicol. Sci., July 1, 2004; 80(1): 115 - 133. [Abstract] [Full Text] [PDF]
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B. A. Vorderstrasse, S. E. Fenton, A. A. Bohn, J. A. Cundiff, and B. P. Lawrence A Novel Effect of Dioxin: Exposure during Pregnancy Severely Impairs Mammary Gland Differentiation Toxicol. Sci., April 1, 2004; 78(2): 248 - 257. [Abstract] [Full Text] [PDF]
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W. G. Foster, E. V. Younglai, O. Boutross-Tadross, C. L. Hughes, and M. G. Wade Mammary Gland Morphology in Sprague-Dawley Rats following Treatment with an Organochlorine Mixture in Utero and Neonatal Genistein Toxicol. Sci., January 1, 2004; 77(1): 91 - 100. [Abstract] [Full Text] [PDF]
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