* Department of Pharmaceutical Sciences, Washington State University, Pullman, Washington 99164; Reproductive Toxicology Division, US EPA, ORD/NHEERL, Research Triangle Park, North Carolina 27711;
Department of Veterinary Clinical Sciences and
Center for Reproductive Biology, Washington State University Pullman, Washington 99164
Received October 14, 2003; accepted December 16, 2003
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ABSTRACT |
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Key Words: TCDD; mammary; pregnancy; prolactin; estrogen; progesterone.
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INTRODUCTION |
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The AhR is unique among the PAS proteins in that it binds a variety of xenobiotics, in particular planar aromatic compounds (Tian et al., 2002; Whitlock, 1999
). In fact, there has been considerable debate regarding the identity of endogenous ligands of the AhR, but its binding to a large number of exogenous compounds is well documented (Behnisch et al., 2001
; Denison et al., 2002
). Of the AhR ligands identified to date, the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has the highest affinity for the receptor and therefore has been used for the majority of studies characterizing the toxic and biological effects of AhR activation. TCDD belongs to a family of structurally related chemicals known as polyhalogenated aromatic hydrocarbons (PHAH). PHAH are ubiquitous environmental contaminants that include other congeners of chlorinated dibenzodioxins, as well as polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs). Additional less robust ligands for the AhR include polycyclic aromatic hydrocarbons (PAH), a large class of environmental and industrial pollutants that arise from cigarette smoke and vehicle exhaust.
Toxicity studies, conducted primarily in rodents, have described numerous detrimental effects resulting from exposure to AhR ligands, including developmental abnormalities, hepatotoxicity, and immunotoxic and carcinogenic outcomes (Birnbaum and Tuomisto, 2000; Couture et al., 1990
; Kerkvliet and Burleson, 1994
; Teeguarden and Walker, 2003
). Developing tissues are particularly sensitive to perturbation by TCDD exposure, as deformities in formation of the palate, kidney, and reproductive organs are often seen in the offspring of exposed animals (Birnbaum et al., 1985
; Couture-Haws et al., 1991
; Dienhart et al., 2000
; Gray and Kelce, 1996
; Lin et al., 2002
). Additionally, AhR agonists such as dioxins and PCBs are known to have endocrine disrupting activities. Changes in numerous endocrine and growth factor pathways have been described, including alterations in levels or actions of thyroid hormones, prolactin, estrogen, epidermal growth factor (EGF), and the EGF receptor (Abbott and Birnbaum, 1990
; Birnbaum and Fenton, 2003
; Bryant et al., 1997
; Jones et al., 1987
; Romkes et al., 1987
; Russell et al., 1988
; Van Birgelen et al., 1995
). The antiestrogenic effects of TCDD are particularly well-characterized, and evidence for disruption of estrogen-mediated functions includes inhibition of estrogen-induced uterine growth and breast cancer cell proliferation, and reduced levels of progesterone and prolactin receptors in MCF-7 cells (Safe, 1995
, 2001
). Mechanistically, AhR-mediated interference with hormonal responses may involve altered hormone metabolism or synthesis, down-regulation of receptor levels, dysregulation of normal receptor cross-talk, or altered expression of hormone-regulated autocrine/paracrine factors.
The AhR and its partner the AhR nuclear translocator protein (ARNT) are present in mammary tissues, and there is evidence that inactivation of these proteins results in impaired mammary development and lactation (Abbott et al., 1999; Hushka et al., 1998
; Le Provost et al., 2002
). However, our understanding of the effects of inappropriate AhR activation on breast tissue is very limited. The existing data in laboratory animals support the idea that exposure to AhR ligands affects the differentiation of mammary tissue. For example, decreased branching and diminished alveolar and terminal end buds were observed in rats exposed to TCDD during gestation (Fenton et al., 2002
; Lewis et al., 2001
). Similarly, weanling rats treated with TCDD at age 45 weeks showed decreased gland size and formation of terminal end buds (Brown and Lamartiniere, 1995
). In these studies, rats were exposed to TCDD during periods where differentiation of mammary tissue is active, that is, during prenatal development and puberty. After puberty, the gland undergoes only minor, cycle-induced periods of growth and regression until pregnancy, at which time a tremendous amount of growth and differentiation occurs in preparation for lactation (Hovey et al., 2002
; Richert et al., 2000
).
Given the potential for AhR ligands to disrupt hormonal signals and interfere with normal tissue development, pregnancy-induced gland development represents another potential target for the toxic or biological action of AhR ligands. This has not been tested directly; however, mammary explants treated with hormones to mimic the effects of pregnancy displayed decreased lobule development when cultured with another potent AhR ligand, TCDF (2,3,7,8-tetrachlorodibanzofuran; Hushka et al., 1998). This suggests that exposure to AhR ligands may disrupt gland differentiation during pregnancy, resulting in impaired lactation and decreased offspring survival.
Thus, the goal of the studies presented here was to test the hypothesis that activation of the AhR during pregnancy impairs mammary gland development and lactation. To this end, we exposed pregnant mice to TCDD and assessed mammary gland differentiation at various times during pregnancy. We also examined the consequences of exposure to TCDD during pregnancy on the production of hormones that are important for gland development and lactation. Finally, we evaluated milk production by analyzing message levels of whey acidic protein. Our results demonstrate severe defects in mammary gland development and suggest that lactation and neonatal survival are adversely affected by exposure to TCDD during pregnancy.
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MATERIALS AND METHODS |
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TCDD treatment.
TCDD was dissolved in anisole and diluted in peanut oil to a concentration for dosing at 10 µl per gram body weight. Vehicle control consisted of peanut oil containing an equivalent concentration of anisole. Female mice were treated with 5 µg/kg of TCDD or vehicle control by gavage on days 0 and 7 of pregnancy, and again on day 14 for mice to be sacrificed on day 17 or later. The dose of TCDD used in these studies is well below the LD50 for adult mice of this strain (160 µg/kg), and is not considered fetolethal (Birnbaum et al., 1989, 1990
; Holladay et al., 1991
). Due to the relatively short half-life of TCDD in mice (11 days, Gasiewicz et al., 1983
), mice were treated every seven days in order to maintain TCDD levels throughout pregnancy. To characterize the effects of TCDD on gland differentiation and hormone levels throughout pregnancy, mice were sacrificed in early pregnancy (day 9), mid-pregnancy (day 12), late pregnancy (day 17), and on the day of parturition (usually day 19). Day 12 was chosen because 50% of gland growth occurs by this time. Virgin mice were given two doses of 5 µg/kg TCDD seven days apart, and sacrificed two days after the second dose.
Euthanasia and blood collection.
Pregnant mice were sacrificed by decapitation between 1030 and 1230 h, and trunk blood was collected. For the study conducted on the day of parturition, mice were euthanized by carbon dioxide overdose after delivering pups, and blood was collected by cardiac puncture into heparinized syringes. Plasma or serum was separated by centrifugation, and stored at 80°C for evaluation of hormones.
Mammary gland whole mounts.
The preparation of mammary glands has been described previously (Fenton et al., 2002, and http://mammary.nih.gov/methodcd/methodcd.html). Briefly, abdominal mammary glands were collected on the indicated day of pregnancy. Glands were weighed, and one gland from each animal was mounted onto a glass slide under weight, and fixed in Carnoy's fixative (6:3:1 ethanol: chloroform: glacial acetic acid). Fixed glands were transferred to 70% ethanol with gradual dilution to water. They were then stained with carmine alum. After rinsing in water, and gradually dehydrating to 100% ethanol, glands were cleared with xylenes substitue, and permanently mounted on slides using Permount (Fisher Scientific, Pittsburgh, PA). Mammary gland whole mounts were evaluated without knowledge of treatment. Whole mounts were examined using a Leica WILD M420 Microscope, and given a development score based on a five-point scale, dependent on growth patterns and differentiation (1 = poor development/differentiation and 5 = excellent growth and development for that age group). The subjective scoring scales were specific to the stage of development at each time point examined. The gland scores considered epithelial branching, development of lobuloalveolar units, and the size of the structures. Mean scores for each group were computed and analyzed for differences due to treatment.
Hormone analysis.
Radioimmunoassays (RIA) to measure prolactin were conducted by Dr. A. F. Parlow at the National Hormone and Peptide Program (Harbor-UCLA Medical Center, Torrance CA). Progesterone and estradiol were assayed using double antibody RIA kits from Diagnostic Services Laboratories (Webster, TX). Depending on the experiment, serum or plasma was used for hormone detection.
RT PCR for whey acidic protein (WAP).
RNA was isolated by mechanical disruption of mammary tissue in Trizol Reagent (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. The concentration of RNA was determined by spectrophotometry, and an equivalent amount of RNA from each sample was reverse transcribed. RNA quality and the equivalency of starting material were assessed by amplification of ß-actin message at various dilutions of cDNA. For amplification of WAP message, cDNA was subjected to PCR (94°C for 5 min; 25 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min; then 72°C for 10 min) using primer sequences provided by Lothar Hennighausen (NIH, Bethesda, MD) (http://mammary.nih.gov/tools/molecular/WAP/MMD.html) (5' TAG CAG CAG ATT GAA AGC ATT ATG 3', and 5' GAC ACC GGT ACC ATG CGT TG 3'). These primers span an intron, and result in amplification of a 500 bp product.
Statistics.
Data were analyzed using StatView software (SAS Software, Cary, NC). Unless indicated otherwise, values obtained from vehicle- and TCDD-treated mice were compared using a Student's t-test, and a one-sided p value of 0.05 was considered significant.
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RESULTS |
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In contrast, severe developmental defects were seen in the glands taken from the TCDD-exposed mice (Fig. 2B). Stunted differentiation of the gland was evident as early as day 9 of pregnancy. Specifically, branching was decreased, and lobules were generally smaller and fewer in number than in the vehicle-treated animals. Poor lobulo-alveolar development was also evident on days 12 and 17, as glands from TCDD-treated mice had an increased number of ductal branches with little or no lobule development, giving them a spiked appearance. By the day of parturition there were extreme differences in the amount of differentiation between the two treatment groups. Relative to vehicle control, glands from the TCDD-treated mice sacrificed at this time point were small and sparsely filled, and lacked strong formation of lobular alveolar structures. Quantification of the observed differences showed statistically significant reductions in differentiation at each time point during pregnancy (Fig. 2C).
In contrast to effects seen in the rapidly developing glands in the pregnant animals, TCDD did not noticeably affect gland morphology in the adult virgin mice, where the glands were undergoing only minimal cycle-related changes in epithelial growth. All virgin mice, regardless of treatment, demonstrated a similar degree of growth and branching (Figs. 2A and 2B).
Analysis of Circulating Hormones
Given that AhR ligands are known endocrine disruptors, it was possible that suppressed glandular differentiation in the TCDD-treated dams resulted from diminished hormone levels. We therefore examined circulating progesterone, prolactin, and estradiol, three hormones critical for mammary development and milk production. As shown in Figure 3, progesterone levels increased during pregnancy and were slightly, but significantly, suppressed in TCDD-treated mice sacrificed on day 17. Expected increases in prolactin were seen in late pregnancy and on the day of parturition. Although levels appeared to be decreased by 50% in the TCDD-treated animals on day 17 and the day of parturition, this difference was not statistically significant. Estradiol levels did not change substantially during pregnancy, and were only slightly affected by treatment with TCDD. Estradiol was marginally suppressed on day 17 and after parturition. Taken together, these data are suggestive of a suppressive effect of TCDD on hormone levels during pregnancy. However, because suppressed gland differentiation was observed as early as day 9 of pregnancy, while no significant effect of TCDD on hormone levels was detected until day 17, these data do not suggest a causal relationship between alterations in these particular hormones and suppressed gland development.
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To address whether treatment with TCDD diminished milk production, we examined message levels of WAP, a predominant milk protein in mice. As shown in Figure 4A, TCDD treatment caused a 70% reduction in the amount of RNA recovered from mammary glands on the day of parturition. When the amount of RNA starting material was normalized for RT-PCR analysis of WAP, band intensities of the PCR products were similar (Fig. 4B). While this result may, at first glance, be interpreted to mean that TCDD did not affect WAP expression, it is important to consider that the amount of RNA starting material was normalized for the RT-PCR analysis. Thus, in the context of the profound suppression of RNA recovery, the amount of WAP was substantially diminished on a per gland basis. These results strongly suggest that exposure to TCDD impaired the ability of the dams to produce the quantity of milk, or in this case milk proteins, necessary to sustain their litters.
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DISCUSSION |
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Given the number of studies conducted during the past three decades that have examined the effects of prenatal exposure to AhR ligands on fetal development, it was surprising that this dramatic effect on mammary development had not previously been described. One explanation is that the majority of these studies examined effects on the fetus, and few postnatal endpoints were examined. A survey of published studies in which pregnancy did go to term and pups were born does, however, provide evidence that impaired mammary development has affected previous work. For example, Luster et al. (1980) found that dosing pregnant mice with 15 µg/kg TCDD on gestational day 14 resulted in 70% neonatal mortality. Likewise, studies conducted in rats exposed to TCDD during pregnancy reported that approximately 50% of pups died within two days of birth (Gehrs et al., 1997
; Gehrs and Smialowicz, 1999
). Finally, kits born to mink treated with AhR ligands weighed less and had a very high mortality rate compared to controls, and dehydration of the neonates was noted in one study (Brunstrom et al., 2001
; Restum et al., 1998
).
Although effects on mammary development resulting from activation of the AhR in the context of pregnancy have not been previously investigated, defects in glandular structures have been described in offspring of rodents exposed to AhR ligands during gestation or puberty. Specifically, Fenton et al. (2002) described an early-detected and persistent reduction in primary branching and alveolar buds in virgin rats exposed prenatally to TCDD. Likewise, Brown et al. (1998)
reported decreased lobule formation in 50-day old rats following prenatal TCDD exposure, although in apparent contrast with the aforementioned study, these effects were not detected at earlier time points. Finally, glands from virgin rats treated with TCDD during pubertal development showed decreased mammary gland size and number of terminal end buds, as well as decreased numbers of proliferating cells in end buds, terminal ducts, and lobules (Brown and Lamartiniere, 1995
).
PAS proteins play important regulatory roles in responses to external signals and in organogenesis. Activation of the AhR by exogenous ligands causes defects in the development and differentiation of multiple tissues, such as the heart, palate, kidney, and reproductive organs (Birnbaum et al., 1985; Couture-Haws et al., 1991
; Dienhart et al., 2000
; Hamm et al., 2000
; Ivnitski et al., 2001
; Lin et al., 2002
). Furthermore, AhR-deficient mice demonstrate structural defects in the vasculature of the kidney, liver, and eye, as well as lesions in the heart and uterus (Fernandez-Salguero et al., 1997
; Lahvis et al., 2000
). Based on these studies, an endogenous role of the AhR and ARNT in organogenesis has been postulated. However, with regard to mammary development, evidence for an endogenous role of the AhR is scant. Mammary development in AhR-deficient mice has only been addressed in one study (Hushka et al., 1998
). Although a 50% reduction in terminal end buds was reported, the number of mice assessed was quite small, and aberrant gland formation was observed in only one animal. Defects in lactogenesis in AhR-null mice have been inferred since, although fertile, these mice suffer from decreased reproductive success and neonatal mortality is often quite high (Abbott et al., 1999
; Fernandez-Salguero et al., 1997
). In fact, due to the poor reproductive success of the AhR-null mice, these colonies are generally maintained using heterozygous animals (Lahvis et al., 2000
; Schmidt et al., 1996
; Vorderstrasse et al., 2001
). As a result, empirical determination of the endogenous role of the AhR in mammary development has been difficult to ascertain.
The potential for AhR ligands to disrupt numerous endocrine signaling pathways is well documented, and we speculated that reductions in circulating hormone levels may provide a mechanistic explanation for the observed defects in mammary development. Numerous hormones are known to influence mammary differentiation during pregnancy and, in the mouse, critical hormones include prolactin, estrogen, progesterone, and placental lactogen (Brisken, 2002; Neville et al., 2002
). We were especially interested in examining estrogen levels due to the well-documented antiestrogenic properties of AhR ligands, and because these compounds induce metabolic enzymes that enhance estrogen degradation in in vitro systems (Safe, 1995
). However, we did not find that exposure to TCDD during pregnancy substantially reduced circulating estradiol, a result that is consistent with those from other studies examining effects of TCDD exposure on estrogen levels in virgin mice and pregnant rats (DeVito et al., 1992
; Shiverick and Muther, 1983
). Furthermore, suggestive decreases in estradiol were not evident until late in pregnancy, well after defects in gland differentiation were evident. Thus it is unlikely that diminished estrogen levels underlie suppressed mammary development in the TCDD-treated mice.
Suppression of prolactin was likewise an attractive mechanistic explanation due to the defects in lobulo-alveolar development and lactation observed in our studies. A number of pieces of evidence provide a basis for this hypothesis. First, prolactin is required for normal differentiation of the mammary gland, along with the induction of milk protein gene expression, and gland development is stunted in prolactin receptor knockout mice (Brisken, 2002; Horseman, 1999
; Ormandy et al., 1997
). Likewise, gland differentiation and lactation are impaired in prolactin knockout mice (Horseman et al., 1997
). Additionally, the pituitary contains functional AhR (Huang et al., 2000
, 2002
), and studies of TCDD-treated male rats and PCB-treated male mice have demonstrated suppression of circulating prolactin (De Krey et al., 1994
; Russell et al., 1988
). However, in our studies, we did not find that TCDD had substantial effects on circulating prolactin levels. Furthermore, the amount of prolactin detected in the pregnant mice was fairly low, rising only in very late pregnancy and after parturition. Although consistent with previous reports of prolactin kinetics in pregnant rodents (Barkley et al., 1978
; Murr et al., 1974
; Neville et al., 2002
; Soares and Talamantes, 1982
), this pattern is in contrast to that of pregnant women, where prolactin levels rise continuously throughout gestation. Thus, the low levels of prolactin detected in the pregnant mice and the lack of substantial effect of TCDD treatment argue against a mechanistic role for this hormone in TCDD-induced mammary defects.
Despite the lack of conclusive evidence that diminished levels of circulating hormones play a role in the suppressed mammary development, it remains possible that impaired receptor expression underlies the observed defects. In fact, evidence suggests that the antiestrogenic properties of AhR agonists may result, at least in part, from diminished levels of the estrogen receptor. This is supported by decreased levels of estrogen receptor found in uteri and livers of TCDD-treated mice and rats (DeVito et al., 1992; Romkes et al., 1987
). Likewise, TCDD treatment of human breast cancer cells affects steady state and estrogen-induced expression of prolactin receptor and of estrogen-induced progesterone receptor (Harper et al., 1994
; Lu et al., 1996
). Therefore, it is possible that AhR activation affects mammary development via defects in receptor expression or other components of endocrine signaling pathways.
Alternatively, it is possible that suppressed gland differentiation is not secondary to defective hormonal signaling, but instead results from a direct insult to the mammary tissue or altered regulation of local growth factor signaling. This interpretation is supported by the fact that the AhR is present in the mammary gland, and that mammary explants exposed to TCDF in the culture media demonstrated suppressed lobule development (Hushka et al., 1998). Furthermore, several pieces of evidence suggest that the defective branching and lobule formation observed in our studies may result from activation of the AhR directly in the mammary epithelium. First, localization of the AhR within the mammary tissue demonstrated highest levels within the ductal and lobular epithelial cells (Hushka et al., 1998
), suggesting that these cells may be direct targets for AhR ligands. Second, other epithelial tissues are affected by TCDD exposure during embryonic differentiation, including the palate, kidney, seminal vesicle, and prostate (Bryant et al., 2001
; Hamm et al., 2000
; Lin et al., 2002
). Additionally, TCDD has been shown to affect the status and activity of the EGF receptor in several tissues of the rodent (Abbott et al., 1992
, 2003
; Hurst et al., 2002
). The EGF receptor and its ligands are important for mammary gland development and are found in the lactating gland (Luetteke et al., 1999
; Wiesen et al., 1999
). The local regulation of EGF receptor or its ligands by the AhR signaling pathway represents yet another mechanism by which dioxin can disrupt gland development. Finally, AhR ligands may also affect nonepithelial components within the gland, since stromal elements of the gland appear critical for disrupted mammary development induced by prenatal exposure to TCDD (Fenton et al., 2002
). Transplantation studies using AhR-null animals should help elucidate the mechanism by which TCDD disrupts gland development and lactation.
Many compounds that activate the AhR are ubiquitous and persistent environmental contaminants, to which both humans and animals are routinely exposed. In fact, the primary route of human exposure to dioxins and PCBs is via the consumption of foodstuffs derived from animals that have accumulated these chemicals in their fat (Schecter et al., 2001; Startin and Rose, 2003
). Thus, our novel finding that activation of the AhR impairs mammary differentiation and lactation is relevant to human health as well as to the reproductive success of exposed wildlife and domestic animals. There is suggestive evidence from studies in mink that dietary exposure to PHAH-contaminated fish diminishes reproductive success (Restum et al., 1998
). However, to our knowledge the potential for PHAH exposure to affect lactation and breast development in highly exposed women has not been studied. For example, it would be of interest to determine lactational success in a group of Canadian Inuit who are exposed to relatively high concentrations of these chemicals via their diet (Ayotte et al., 1996
). Additionally, the accidental exposure of several thousand women in Seveso, Italy; western Japan; and Taiwan (Warner et al., 2002
; Yoshimura, 2003
; Yu et al., 2000
) could provide critical epidemiological data on the effects of dioxin and PCBs on breast development and function. Given the prevalence of AhR agonists in the environment and the fact that the AhR is present in mammary tissue, it is possible that exposure to these chemicals leads to defects in mammary differentiation in humans.
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ACKNOWLEDGMENTS |
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NOTES |
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Portions of these data were presented at the 42nd annual meeting of the Society of Toxicology, March 2003, Salt Lake City, UT.
1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, Wegner Hall, Washington State University, Pullman, WA 99164-6534. Fax: (509) 335-5902. E-mail: bpl{at}mail.wsu.edu
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