The female MG undergoes most of its development postnatally, achieving a fully differentiated state late in pregnancy. This process includes numerous events that can be disrupted by exposure to EDCs. Gestation, puberty, and pregnancy are the critical periods during which EDC exposure may most affect MG development (Fenton 2006
). Critical events include mammary bud development in the fetus, exponential epithelial outgrowth during puberty, and the rapid transition to lactational competency that occurs during late pregnancy (). These stages occur in both rodents and humans.
Figure 1 Stages of normal rat MG development and effects of environment on subsequent events. Effects of early life EDC exposures can lead to altered developmental programming in the breast and have been reported neonatally, at puberty, and well into adulthood, (more ...) Normal MG development.
Normal female MG development involves a well-orchestrated sequence of events marked by extensive proliferation at puberty and by proliferation and differentiation during pregnancy. This process is regulated by hormones, growth factors, and stromal factors and is similar between rodents and humans, although rodent MG development is more completely described (Kleinberg et al. 2009
; Medina 2005
). Female human MG development begins with budding and branching between 6 and 20 weeks of gestation, yielding at birth a primitive gland composed of ducts ending in ductules. During childhood, MG growth keeps pace with overall body growth; at puberty it accelerates dramatically.
In rodents, the epithelial bud is formed at the site of the nipple around gestation days (GDs) 12–16, and by birth the epithelium has entered the fat pad and formed a ductal tree. The fat pad and mammary epithelium grow at the same pace as the body for the first 2–3 weeks of life, and just before puberty, an exponential growth phase begins. In rodents, and presumably in humans, this phase of ductal development is characterized by formation of terminal end buds (TEBs), which lead the epithelial extension through the fat pad, leaving behind a network of branched ducts. After the fat pad is filled, TEBs differentiate into terminal ductal structures, namely, terminal ductal lobular units in humans, lobules and alveolar buds in rats, and terminal ducts in mice. In humans and rodents, additional MG proliferation and regression events occur with each luteal phase of the ovulatory cycle, and at pregnancy there is significant differentiation of the terminal structures with lobular–alveolar development (Kleinberg et al. 2009
; Russo and Russo 2004a
). Mammary epithelial growth also occurs in male rats and men, whereas male mice lack mammary epithelium. Male mice and rats do not normally possess nipples because androgens during gestation induce regression. Retained nipples in male rats is a characteristic effect of prenatal antiandrogen exposure (Foley et al. 2001
Assessment of altered MG development.
Whole mounts and other techniques. Early life treatment with some hormonally active agents results in altered development of the MG in male and female rodents. Although laboratories vary in their methods for reporting altered MG development, the primary approach has been morphological assessment of the entire fourth or fifth abdominal MG fat pad mounted flat on a slide, fixed, stained, defatted, and permanently affixed to the slide as a “whole mount.” Whole mounts allow an assessment of total and relative abundance of mammary terminal ductal structures (i.e., TEBs, terminal ducts, alveolar buds, and lobules), extension of the epithelial cells through the fat pad, and branching patterns and density at different times during development [e.g., Fenton et al. 2002
; see also Supplemental Material, (doi:10.1289/ehp.1002864)]. A common measurement in mammary whole mounts is the number of TEBs. A TEB is a teardrop-shaped duct end with a diameter of about 100 µm in the rat compared with about 70 µm for a terminal duct (Russo and Russo 1978
Female MG outcomes after developmental environmental exposures: rodent–human concordance for selected agents.
Several rodent studies have reported altered MG development after prenatal, neonatal, or peripubertal exposure to a range of hormonally active agents, including pharmaceutical hormones, dietary constituents, and EDCs [see Supplemental Material, (doi:10.1289/ehp.1002864)]. These studies typically include histopathological evaluation of MG whole mounts of developing animals and report morphological features such as branching, extent of growth, and relative proportion of structures (e.g., TEBs, lobules, and terminal ducts). Other studies report changes in morphology or immunohistochemistry of tissue sections or gene expression in tissue homogenates. Methods and data reporting vary between laboratories, making it difficult to compare findings across studies. More uniform approaches will facilitate progress; however, unanticipated end points should continue to be reported because this field is still developing.
Morphological changes reflect timing
of assessment. Because normal development involves a well-characterized, consistent progression of types and ratios of terminal structures and extension through the fat pad, alterations are sometimes reported as accelerated or delayed development relative to controls (e.g., Moon et al. 2007
). Some agents alter the pace at which differentiation occurs, leading to an increased or decreased number of TEBs depending on timing of assessment. If a perinatally administered EDC causes accelerated development, the number of TEBs in the treated group will be higher than that in vehicle-treated controls at weaning [postnatal day (PND) 21] because of increased proliferation, but lower in early adulthood (PNDs 45–50) because of accelerated differentiation, as is seen after exposure to estrogens (Hovey et al. 2005
). In the case of an EDC, such as dioxin, that causes delayed development, reduced differentiation leads to a higher number of TEBs in early adulthood and a longer period during which TEBs are present (Brown et al. 1998
; Fenton et al. 2002
). The number of TEBs present in the gland also depends on the number of ducts in the gland. Therefore, the number of TEBs at a particular time point can be altered by changes to the extent of growth as well as to the pace of differentiation. For example, if the overall number of ducts is decreased by an environmental exposure, then the overall number of TEBs in the gland will be decreased compared with those in controls at any time point, as demonstrated for perfluorooctanoic acid (PFOA) exposures in mice (White et al. 2009
). Some reports have not differentiated between changes in TEB number due to overall gland size and those due to altered developmental pace. Evaluation at multiple time points and consideration of the total number of terminal ends, as well as the absolute number of TEBs, alleviate this problem and convey the relative number of structures.
In one of the first studies
of neonatal exposure to estrogen, progesterone, or both in mice, Jones and Bern (1979)
reported irreversible adult MG effects, including secretory stimulation, dilated ducts, and abnormal lobuloalveolar development. Perinatal treatment with estrogens such as estradiol or diethylstilbestrol (DES) has been reported to produce accelerated development, characterized by increased pubertal TEB density, and to promote ductal proliferation during the peripubertal period in both rats and mice (Fielden et al. 2002
; Hilakivi-Clarke et al. 1997
; Hovey et al. 2005
; Tomooka and Bern 1982
; Warner 1976
). In addition, Doherty et al. (2010)
reported that prenatal DES exposure in mice altered expression in MG of genes that may be important in tumorigenesis. Ovariectomy has been reported to diminish or obviate the effect of neonatal ovarian steroids on mouse MG development (Jones and Bern 1979
; Mori et al. 1976
), and strain differences in sensitivity have also been reported (Mori et al. 1976
; Yang et al. 2009
). In rats exposed continuously beginning at conception, oral ethinyl estradiol exposure induced ductal hyperplasia in male rat MGs by PND50, and this effect was less apparent in rats assessed later in life (Latendresse et al. 2009
). Thus, morphological changes in MG reflect timing of exposure as well as timing of assessment, and so both of these variables must be considered when comparing results across studies. Supplemental Material, (doi:10.1289/ehp.1002864) compiles the methods and findings of studies that have evaluated the effects of hormone, dietary, or chemical exposures during the prenatal, neonatal, or peripubertal periods on MG development up to 10 weeks. Several additional endocrine-sensitive end points commonly assessed to indicate relative sensitivity are also included in the table.
In addition, whereas perinatal steroid hormone exposure alters proliferation and TEB number, peripubertal exposure that occurs after proliferation has begun affects mainly the differentiation of TEBs into mature structures. For example, pubertal DES treatment in rats increased the pace of lobule formation and decreased the number of terminal ducts and TEBs compared with vehicle-treated controls just after puberty (Odum et al. 1999
). Prepubertal DES treatment of rats (on PNDs 23–29) resulted in fewer TEBs, terminal ducts, and alveolar buds, with a concomitant increase in the more differentiated lobules, overall suggesting a faster differentiation pace (Brown and Lamartiniere 1995
). Treatment of postpubertal rodents with steroids or human chorionic gonadotropin increases differentiation of the MG in a manner thought to mimic development during pregnancy (Russo and Russo 2004b
; Sivaraman et al. 1998
Effects of treatment with phytoestrogens such as genistein are similar to those observed after estrogen receptor agonist exposure; perinatal exposure can lead to increased proliferation, and peripubertal exposure can lead to accelerated differentiation (reviewed by Warri et al. 2008
). For example, Hilakivi-Clarke et al. (1998)
and Padilla-Banks et al. (2006)
showed increased TEBs after perinatal genistein treatment, and Cotroneo et al. (2002)
showed accelerated development in MG after prepubertal exposure, as indicated by increased TEBs and ductal branching at an early time point, compared with untreated animals. After gestational and lactational genistein exposure, You et al. (2002)
observed enhanced glandular differentiation at weaning, and males were more sensitive to the effect than females. Male rats in a multigenerational genistein feeding study also showed ductal hyperplasia at PND50, a surprisingly early life stage for these effects (Latendresse et al. 2009
). Effects on MG development have also been observed after perinatal exposure to other phytoestrogens, including zearalanone and resveratrol, and to flaxseed [see Supplemental Material, (doi:10.1289/ehp.1002864)].
Altered MG development after perinatal exposure has also been observed for numerous EDCs, including atrazine, bisphenol A (BPA), dibutylphthalate, dioxin, methoxychlor, nonylphenol, polybrominated diphenyl ethers, and PFOA. Changes include delayed MG development, ductal hyperplasia, alveolar hypoplasia, reduced apoptosis in TEBs, altered gene or protein expression, increased or decreased numbers of terminal ducts or lobules, and accelerated alveolar differentiation [see Supplemental Material, (doi:10.1289/ehp.1002864)], as well as increased MG tumors after carcinogen challenge (Brown et al. 1998
; Durando et al. 2007
; Jenkins et al. 2007
). In addition, late-gestational treatment with Ziracin, a candidate antibacterial drug, induced hypoplasia (ducts without any acinar development) in rats (Poulet et al. 2005
Critical exposure windows and reversibility.
Studies of the ubiquitous industrial pollutant dioxin and the high-use herbicide atrazine have investigated critical periods of exposure associated with MG effects. Atrazine delayed MG development when exposure occurred around GD17–19 but had less of an effect after earlier 3-day windows, and dioxin exposure at GD15, but not after GD19, led to MG underdevelopment (Fenton et al. 2002
; Rayner et al. 2005
). More recent studies on the industrial surfactant PFOA (White et al. 2009
) demonstrate a similar critical period. The heightened sensitivity during this time period is attributed to the formation of the mammary bud and initial branching that occurs during late pregnancy. As discussed above, exposure timing and dose influence the pattern of MG changes (Warri et al. 2008
Although numerous studies have shown persistent effects on the MG, few have evaluated whether the changes could be reversible. For example, in utero
exposure to dioxin, Ziracin, PFOA, or BPA led to permanent changes in the adult MG (Fenton et al. 2002
; Poulet et al. 2005
; Vandenberg et al. 2007
; White et al. 2009
). In contrast, effects of genistein and ethinyl estradiol in male MG appeared to reverse after treatment withdrawal (Latendresse et al. 2009
). It is unclear whether the persistence of alterations reflects the biological half-life and lipophilicity of the chemical or epigenetic changes, and this may differ by compound.
It is striking that MG developmental changes have been observed after exposure to diverse agents, including estrogens, androgens, antiandrogens, thyroid-active chemicals, and aryl hydrocarbon receptor agonists. Data do not indicate a similar mode of action for atrazine, but PFOA, brominated diphenyl ethers, and dioxin all have been shown to induce a phenotypically similar response of delayed MG development after neonatal exposures [see Supplemental Material, (doi:10.1289/ehp.1002864)]. Novel mechanisms continue to be discovered. In a recent study, Doherty et al. (2010)
found that in utero
exposure of mice to DES or BPA increased protein expression and functional activity of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) in the MG. EZH2 has been linked to breast cancer risk and epigenetic regulation of tumorigenesis. Its up-regulation is a potential mechanism through which in utero
exposure to these chemicals may produce epigenetic changes leading to increased breast cancer risk (Doherty et al. 2010
The few studies that have evaluated effects on male MG have indicated that male rats could be more sensitive. For example, one study found altered MG in males, but not females, treated with methoxyclor during gestation (You et al. 2002
), and MG effects of genistein and ethinyl estradiol have been reported in males at lower doses than in females (Delclos et al. 2001
; Latendresse et al. 2009
). Study of sex differences in responsiveness can provide information about mechanisms of action for the test agents. Although male mice lack mammary epithelia, there are transgenic mouse models in which mammary epithelial growth can be induced in males (Li et al. 2002
). Mouse models are needed to study some chemicals, such as PFOA, whose pharmacokinetics in mice and humans are most similar.
The Organisation for Economic Co-operation and Development (OECD) guidelines for subchronic oral toxicity testing (OECD 2008) include evaluation of the male, but not female, MG as an optional end point. In some studies using these guidelines, the male MG appears to be among the most sensitive end points evaluated (Okazaki et al. 2001
), and at least one such study has found it to be the most sensitive end point in males (Andrews et al. 2002