* Departments of Obstetrics and Gynecology and
Pathology and Molecular Medicine, McMaster University and Health Sciences Centre, Hamilton, Ontario, Canada;
Department of Obstetrics and Gynecology, Duke University Medical Center, Durham, North Carolina; and
Systemic Toxicology and Pharmacokinetics Section, Environmental and Occupational Toxicology Division, Environmental Health Science Bureau, Health Canada, Environmental Health Centre, Tunneys Pasture, Ottawa, Ontario, Canada
Received June 27, 2003; accepted September 11, 2003
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ABSTRACT |
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Key Words: mixtures; genistein; mammary; tumors; development.
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INTRODUCTION |
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Synthetic halogenated organic compounds, such as dichlorodiphenoxytrichloroethane (DDT) and its persistent metabolite p,p'-dichlorodiphenoxy-dichloroethylene (p,p'-DDE), hexachlorobenzene (HCB), dioxins and furans, polychlorinated biphenyls (PCBs), other organochlorine pesticides, and metals such as lead and cadmium have been found as contaminants in tissues collected from the human population globally (Davies and Mes, 1987; Frank et al., 1988
; Mes, 1992
; Mes et al., 1990
; Newsome et al., 1995
; Szymczynski et al., 1981a
,b
). Detectable levels of suspected endocrine toxic chemicals such as p,p'-DDE, a-hexachlorocyclohexane, and selected PCB congeners in second trimester human amniotic fluid have been reported (Foster et al., 2000
), indicating fetal exposure to these compounds. However, the effect of these toxicants, when present together in a mixture on development or reproductive function, are poorly understood. Cell culture experiments with mixtures of common organochlorines have provided evidence that these chemicals in combination can have additive and synergistic effects as measured by MCF-7 cell proliferation (Payne et al., 2000
, 2001
). Taken together, these data suggest that environmental toxicants can interact to augment the growth of mammary gland cells, function, and, ultimately, pathophysiology.
Animal experiments suggest that developmental-stage exposure to environmental toxicants is an important determinant of toxicant-induced changes in reproductive-tract and mammary-gland development and the pathogenesis of mammary tumors. Specifically, prenatal and perinatal exposures to environmental toxicants are associated with an increase in mammary tumor formation (Brown et al., 1998a; Desaulniers et al., 2001
; Fenton et al., 2002
; Markey et al., 2001
) while toxicant exposure after differentiation of the mammary gland has been linked with either no effect or decreased risk of mammary gland tumor development (Holcomb and Safe, 1994
; Nesaretnam et al., 1998
; Ramamoorthy et al., 1999
). Mammary gland morphogenesis begins in utero and proceeds through several discreet stages as the animal ages (Russo and Russo, 1978
). At birth, the mammary gland consists of an epithelial ductal rudiment embedded in stroma tissue from which terminal end buds (TEBs) are formed during the first week of life. Estrogen stimulation, acting through estrogen receptor-
(ER
) induces rudimentary ducts to elongate and bifurcate to generate increasing numbers of TEBs that reach a maximum at 21 days of age. Gene-knockout studies have demonstrated that mammary glands fail to develop beyond the prepubertal stage in mice lacking ER
or the enzyme aromatase (Fisher et al., 1998
; Korach et al., 1996
). ERß has also been demonstrated to be required for normal lobuloalveolar development as suggested by the finding of larger alveoli and less secretory epithelium in ERß -/- mice compared to wild-type controls (Förester et al., 2002
). With the onset of sexual maturity and the addition of progesterone and prolactin, increased side branching and formation of alveolar buds occurs with each estrous cycle until 63 days of age in the rat. During pregnancy, progesterone has been shown to contribute to ductal branching and lobuloalveolar development capable of milk production (Conneely and Lydon, 2000
; Humphreys et al., 1997
; reviewed by Hovey et al., 1999
). In virgin mice, prolactin has been shown to exert indirect effects on ductal side branching and terminal end bud regression, whereas in pregnant animals, it acts directly on the mammary epithelium to induce lobuloalveolar development (Brisken et al., 1999
). A prolactin receptor-knockout mouse model revealed that a functional PRLR is required for mammary gland development and milk production during pregnancy (Kelly et al., 2002
). Hence, estrogens promote ductal elongation and progesterone-receptor expression whereas progesterone and prolactin promote lobuloalveolar development. We therefore propose that in utero exposure and early postnatal exposure to environmental toxicants will act on the undifferentiated cells of the mammary gland to alter mammary development and increase risk of tumor formation. Indeed, in utero exposure to environmental toxicants such as TCDD and bisphenol A has been associated with delayed differentiation of the mammary gland, which has been associated with increased sensitivity to carcinogens (Fenton et al., 2002
; Markey et al., 2001
). Mammary gland tumor numbers were augmented by prenatal treatment with TCDD (Brown et al., 1998a
). Similarly, a recent study (Band et al., 2002
) demonstrated a significant increase in risk for breast cancer for women who commence cigarette smoking during the pubertal transition or before a first full-term pregnancy compared to those that start smoking later in life. These data suggest that the critical window for carcinogen-induced mammary tumors is prior to complete differentiation of the mammary gland. Hence, we propose that in utero exposure to environmental toxicants with a long half-life will result in exposure of the developing mammary gland prior to differentiation of the TEB into alveolar buds and subsequently into lobules.
It is unclear if the effects of environmental toxicant mixtures on mammary tumor development occur at concentrations below the no-effect level for the individual components of the mixture. In addition, concerns over the possible effects of environmental toxicants with estrogenic activity have led to questions about the safety of phytoestrogens in breast milk and soy-based infant formula (Irvine et al., 1995), as well as the use of isoflavone supplements. Maternal transfer of dietary genistein to offspring has been demonstrated in animal and human studies. In lactating rats fed genistein (250 mg/kg body weight [bw] in the diet), total genistein concentration in the serum and milk of dams 7 days postpartum were 418 ± 198 and 137 pmol/ml, respectively (Fritz et al., 1998
). The total genistein concentrations in stomach milk, serum, and mammary gland of 7-day-old offspring in this study were 4439 ± 1109 pmol/ml; 726 pmol/ml, and 440 ± 129 pmol/ml, respectively. Phytoestrogen concentrations in human milk can increase 10-fold when the mother consumes soy products (Slavin, 1996
). One woman who ingested a moderate challenge with 20 g of roasted soybeans (equivalent to 37 mg isoflavones) had total isoflavones (daidzein, genistein, and glycitein) in plasma, breast milk, and urine of approximately 0.2 mM/l, 2.0 mM/l, and 3.0 mM/l, respectively (Franke et al., 1998
). Because infants can digest and absorb dietary phytoestrogens in active forms (Irvine et al., 1998a
), and since neonates are generally more susceptible than adults to perturbations of the sex steroid milieu (Irvine et al., 1998b
), we propose that exposure to these naturally occurring estrogenic compounds may pose a developmental hazard to estrogen-sensitive target tissue functions later in life. Therefore, half the animals in this study were also administered genistein postnatally and potential interaction between the environmental toxicant mixture andgenistein was investigated. Our overall objective was to determine the effect of in utero exposure to a mixture of common chemical contaminants to which humans are exposed on reproductive function at four distinct developmental life stages. In this study, gross enlargement of the mammary glands was detected in animals at 200 days of age only. Therefore, the objective of the present study was to document the effects of in utero exposure to a mixture of environmental toxicants and postnatal genistein on mammary gland histology in the 200-day-old Sprague-Dawley rat.
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MATERIALS AND METHODS |
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Mixture formulation.
The formulation of the mixture and selection of dose levels is described elsewhere (Wade et al., 2002). Briefly, the contaminant mixture was formulated to reflect the types of persistent organic and inorganic contaminants for which there is evidence of exposure in Canadian populations. The dose levels of each component in the mixture reflect the currently promulgated safe levels of exposure as published by the ATSDR (minimum risk level, MRL), Canadian Environmental Protection Act Chemical Assessments (tolerable daily intake; TDI) or U.S. EPA (reference dose, RfD), or at the lowest NOEL available in the scientific literature (Table 1
). The dosing solutions provided a daily dose of each component equivalent to 1x their daily safe or no-effect levels of exposure.
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Mammary gland histomorphology.
The first right thoracic mammary gland from each animal was carefully dissected and fixed in neutral buffered formalin for routine histopathological analysis. Briefly, mammary glands were dehydrated through a graded ethanol series, embedded in paraffin, and 5-µm-thick sections cut and stained with hematoxylin and eosin. Each slide was scored on a 4-point scale ranging from 0 (normal) to 4 (severe changes) by a pathologist blinded to treatment groups. Histopathological changes were further scored according to distribution of changes (0.25 focal), 0.5 (locally diffuse), and 0.75 (diffuse). Therefore, the most severely affected sections with severe diffuse pathological changes would be scored a maximum of 4.75.
Statistical analyses.
All statistical procedures were performed using SigmaStat (SPSS, Inc., Chicago, IL) Histopathological scores were summarized by descriptive statistics and checked for normality. Where the normality test failed, data were compared by ANOVA on ranks and treatment effects determined by multiple comparisons using Dunns method. The accepted level of significance was set at p 0.05.
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RESULTS |
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Genistein group (GEN).
The mammary glands of animals in the neonatal genistein-treatment group were characterized by lactational changes, prominent cystic ductal dilatations, florid and atypical epithelial hyperplasia (Fig. 1c), and prominent microcalcifications (Table 2
). Atypical cytological features with enlarged pleomorphic nuclei and occasional prominent nucleoli were found in the cells lining the ducts and the lobules. In two animals from the genistein group, in situ ductal carcinoma of the comedo type was detected, with focal microinvasion and stromal reaction. Expanded ducts with cancer cells showing high nuclear cytoplasmic ratio, nuclear pleomorphism, prominent nucleoli, and a high mitotic activity (with a mitotic rate of more than 20 per 10 high-power fields; x 40 objective) were also observed (Fig. 1d
). Atypical mitotic figures were also identified throughout sections of mammary glands from these animals. Comedo necrosis was very prominent and was accompanied by calcifications. Intraluminal secretions and lactational changes such as secretory changes in epithelial cells, lobular expansion, and secretory product in the lumen were observed in ducts surrounded by hyperplastic lobules lined by atypical cells.
Mixture plus genistein group (MIXGEN).
Cystic dilatation, adenosis, calcifications, lactational changes, and epithelial hyperplasia that ranged from mild (six animals) to atypical (six animals) were found in the prenatal mixture-plus-neonatal-genistein group (Fig. 1e). Atypical hyperplasia was noted with focal papillary growth and occasional regions with slight cribiforming. These latter features are highly suggestive of preneoplastic lesions (Fig. 1f
). However, compared to the GEN group, epithelial cell proliferation was less and no in situ or invasive carcinoma was detected.
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DISCUSSION |
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Some environmental toxicants are thought to promote the development of mammary tumors directly via estrogenic effects in vivo on breast epithelial cell proliferation (Bradlow et al., 1995; Davis et al., 1993
; Wolff and Weston, 1997
), whereas others act indirectly by altering the expression of genes responsible for estrogen synthesis and metabolism (Coumoul et al., 2001
; Mucci et al., 2001
). Alternative mechanisms involve induction of DNA adducts (Kuljukka-Rabb et al., 2001
; Rundle et al., 2000
) or direct effects of the toxicants on epidermal growth factor or insulin-like growth factor-I-regulated proliferation-signaling pathways (Tannheimer et al., 1997
; 1998
). Moreover, results from in vitro studies suggest that environmental toxicant mixtures can interact in an additive, synergistic, or inhibitory manner to modify the risk of breast cancer by altering cancer-cell proliferation and estrogen signaling (Du et al., 2000
; Payne et al., 2000
, 2001
; Suzuki et al., 2001
). Environmental toxicants may also inhibit tumor development or pathophysiology through antiestrogenic effects (Arcaro et al., 1999
). Thus, given similar or even competing mechanisms of action, it is difficult to predict the effect of environmental-toxicant exposure on mammary gland morphogenesis and cancer risk from studies in which toxicants have been tested in isolation. Therefore, we tested the effect of a mixture of environmental toxicants for which human exposure has been documented (Davies and Mes, 1987
; Frank et al., 1988
; Mes, 1992
; Mes et al., 1990
; Newsome et al., 1995
; Szymczynski et al., 1981a
,b
) and in utero exposure demonstrated (Foster et al., 2000
). Components of our mixture are known to possess divergent mechanisms of action with some know to be estrogenic, such as methoxychlor, while others such as TCDD have mixed estrogenic and antiestrogenic effects. Chemical components of our mixture may therefore either act in an additive or even synergistic manner to augment effects on mammary gland morphology. Alternatively, it is possible that these chemicals can inhibit the actions of each other with the result that there is no detectable effect. In the current study, histopathological changes documented in the mixture group were unremarkable and did not differ significantly from the control animals, indicating that the toxicant mixture does not produce morphological alterations in the mammary gland. We cannot exclude the possibility that, if we had followed the animals past 200 days of age, we may have seen morphological alterations and mammary tumor development. On the basis of several in vitro cell-based assays, it has been suggested that dietary intake of hormonally active toxicants such as organochlorine pesticide residues in food have little estrogenic activity (Gaido et al., 1998
; Safe, 1995
), and thus, it is unlikely that environmental toxicants would contribute markedly to the pathogenesis of breast cancer. Although our results appear to support the above view, evidence of interaction between our toxicant mixture and postnatal genistein are taken to suggest that treatments have disrupted mammary-gland morphogenesis directly, which are unmasked by postnatal phytoestrogen exposure prior to mammary gland differentiation and may involve changes in gonadal-steroid receptor or growth-factor receptor expression in the mammary gland.
In the current study, postnatal genistein treatment on PND 28 induced profound alterations in mammary gland morphology of the adult rat consistent with prolonged estrogen exposure. The effects of genistein on mammary gland development and tumorigenesis are equivocal, with several studies documenting protective effects (Cotroneo et al., 2002; Fritz et al., 1998
; Murrill et al., 1996
; Rowlands et al., 2002
), whereas others have demonstrated genistein-induced increased breast cancer cell-line proliferation (Allred et al., 2001
; Dees et al., 1997
) and no difference in tumor number but an increased time-to-tumor appearance (Jin et al., 2002
). A recent study has also demonstrated that high-level genistein exposure in the diet (300 and 800 ppm) alters mammary gland development in male but not female rats (You et al., 2002
) and modulates the toxicological behavior of methoxychlor. The effect of diet on breast cancer risk is difficult to determine, as the same dietary component might have a different or even opposing effect on breast cancer, depending on the time of exposure (Hilakivi-Clarke et al., 2001
). For example, it has been suggested that soy phytoestrogens are protective against breast cancer during premenopausal years, but might have adverse effects after menopause, when endogenous estrogens are low (Willett, 2001
). Results of the present study suggest that early postnatal exposure to pharmacological levels of genistein adversely affect mammary gland morphogenesis. In the present study, rats were administered 10 µg genistein/g bw/day between postnatal days 2 and 8, which resulted in significant alteration in mammary gland morphology. Our findings are in contrast to a prior study (Fielden et al., 2002
) in which developmental exposure to genistein at levels between 0.1 and 10 µg/g/day was shown to have no effect on mammary gland morphology. Lewis et al. (2002)
have demonstrated estrogenic effects on uterine weight, vaginal opening, and induction of persistent estrus from developmental exposure (postnatal days 17) to genistein at a dose of 40 mg genistein/kg/day, whereas a dose of 4 mg genistein/kg/day, a dose considered to be representative of infant exposure to genistein in soy formula, was without effect. The dose of genistein used in the current study, while greater than that used in previous studies in which no effect of genistein has been found (Fielden et al., 2002
), was markedly lower than the levels (500 µg genistein/g bw administered by sc injection on postnatal days 16, 18, and 20) used by Cotroneo and coworkers (2002)
that were found to inhibit carcinogen-induced mammary tumor formation. Differences in the pharmacokinetics of the sc-vs.-oral route of administration could account for differences in our findings and those of Fielden and coworkers (2002)
. Specifically, genistein is metabolized in the liver where it is glucuronidated and sulfonated, resulting in a loss of estrogenic activity, whereas genistein given by sc injection would bypass the liver, resulting in higher concentrations of genistein being delivered to the target tissue. However, route of administration cannot explain the divergent findings between ours and Cotroneo and colleagues (2002)
. Hence our data suggest that both the dose and timing of exposure are critical factors in altering mammary-gland sensitivity to genistein-induced changes in mammary gland morphogenesis and potentially tumorigenesis. Taken together, we propose that neonatal exposure to genistein induces morphological alterations in mammary glands and increased risk of mammary tumorigenesis, whereas prepubertal exposure to pharmacological doses of genistein are associated with increased differentiation of the mammary gland and decreased risk of mammary tumors (Cotroneo et al., 2002
). Since mammary gland morphogenesis is a hormone-dependent process (Conneely and Lydon, 2000
; Fisher et al., 1998
; Förester et al., 2002
; Hovey et al., 1999
; Humphreys et al., 1997
; Korach et al., 1996
) that progresses from birth into adulthood (Russo and Russo, 1978
), with cyclical changes occurring with each estrous cycle as well as pregnancy and lactation (Hovey et al., 1999
), defining the potential risks vs. benefits of genistein, will require attention to the developmental stage of the mammary gland during exposure as well as dosage and route of administration.
The mechanism of genistein action in the present study is difficult to determine, due to both the multiple mechanisms through which genistein might act as well as the long latency between exposure and assessment of histological changes in the mammary gland. Genistein is recognized as a preferential ERß agonist that has also been shown to increase the synthesis of sex hormone-binding globulin, inhibit the activity of different enzymes including aromatase, DNA-topoisomerase, and protein kinases, and possess antioxidant properties (Aldercreutz et al., 1987; Barnes, 1998
; Kao et al., 1998
; Petersen and Barnes, 1991
). Dietary genistein (15, 150, and 300 ppm) has been shown to dose-dependently increase the growth of estrogen-dependent breast cancer cells (MCF-7) transplanted to athymic nude mice, indicating a potential estrogenic mechanism (Allred et al., 2001
). Since all of the foregoing mechanisms are acute effects of genistein, it is unlikely that they contributed significantly to the histological changes in the mammary glands in our study. Therefore, while multiple mechanisms likely contribute to the mammary gland development and pathogenesis of breast cancer, we propose that in the current study, genistein exposure during the first week of life altered mammary gland programming through local changes in gonadal steroid or growth factor receptor expression in the developing mammary gland. This view is supported by several distinct reports. First, in rats fed soy proteins from PND 4 onward, there was an increase in the number of cells expressing progesterone receptors in the mammary gland TEBs; however, there were no differences in the number of cells expressing ER
or ERß (Rowlands et al., 2002
). In addition, dietary genistein exposure (300 and 800 ppm), in combination with methoxychlor (800 ppm) during gestation and lactation, has been shown to alter mammary-gland development and to increase the expression of ER
, PR, and insulin-like growth factor-1 receptor (IGF-1R) in male, but not female rats (You et al., 2002
). Furthermore, we have recently demonstrated that gestational and lactational exposure to 15 mg genistein/kg bw increased progesterone-receptor expression in the glandular epithelium of the uterus (Hughes et al., 2003
). These data demonstrate that early postnatal exposure to physiologically relevant concentrations of soy proteins in the diet can alter gonadal steroid-receptor expression in estrogen-sensitive target tissues, including the mammary gland.
The mechanism of the interaction between environmental toxicants and genistein is not known. Combining the chemical mixture insult in utero with exposure to pharmacological levels of genistein during early postnatal development in the current study, augmented genistein-alone-induced secretory changes and produced pronounced cystic dilatation, focal fibrosis, and calcification in mammary glands of the adult rat. Our results, therefore, demonstrate that prenatal exposure to concentrations of environmental toxicants currently considered to be without adverse effect does not induce statistically significant changes in mammary gland morphology or induce mammary gland tumors but does interact with exposure to dietary estrogens during mammary gland development to induce profound morphological alterations. The interaction of diet and prior exposure in utero to environmental toxicants has not yet been explored empirically. However, the findings of this study, although preliminary, suggest that exposure to dietary phytoestrogens at levels consistent with use of dietary supplements, during mammary gland development, induces profound changes in mammary gland morphology, and in utero exposure to estrogenic agents present in the environmental toxicant mixtures modified mammary gland response to the adverse effects of genistein.
In summary, results of the present study demonstrate that gestational exposure to mixtures of environmental toxicants on their own, at the upper range of levels currently considered to be without adverse effect, does not induce changes in mammary gland structure or increase mammary gland tumor development. However, the combination of gestational exposure to environmental toxicant mixtures and neonatal exposure to pharmacological concentrations of the dietary phytoestrogen genistein increases morphological alterations in the mammary glands of adult rats. Collectively, although preliminary, our data suggest that in utero exposure to low levels of hormonally active chemicals can produce morphological changes that may increase mammary gland susceptibility to breast cancer in adulthood.
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ACKNOWLEDGMENTS |
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NOTES |
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