Environmental exposures and mammary gland development: state of the science, public health implications, and research recommendations.
OBJECTIVES: Perturbations in mammary gland (MG) development mayincrease risk for later adverse effects, including lactation impairment,
gynecomastia (in males), and breast cancer. Animal studies indicate that
exposure to hormonally active agents leads to this type of developmental
effect and related later life susceptibilities. In this review we
describe current science, public health issues, and research
recommendations for evaluating MG development.
DATA SOURCES: The Mammary Gland Evaluation and Risk Assessment
Workshop was convened in Oakland, California, USA, 16--17 November 2009,
to integrate the expertise and perspectives of scientists, risk
assessors, and public health advocates. Interviews were conducted with
18 experts, and seven laboratories conducted an MG slide evaluation
exercise. Workshop participants discussed effects of gestational and
early life exposures to hormonally active agents on MG development, the
relationship of these developmental effects to lactation and cancer, the
relative sensitivity of MG and other developmental end points, the
relevance of animal models to humans, and methods for evaluating MG
effects.
SYNTHESIS: Normal MG development and MG carcinogenesis demonstrate
temporal, morphological, and mechanistic similarities among test animal
species and humans. Diverse chemicals, including many not considered
primarily estrogenic, alter MG development in rodents. Inconsistent
reporting methods hinder comparison across studies, and relationships
between altered development and effects on lactation or carcinogenesis
are still being defined. In some studies, altered MG development is the
most sensitive endocrine end point.
CONCLUSIONS: Early life environmental exposures can alter MG
development, disrupt lactation, and increase susceptibility to breast
cancer. Assessment of MG development should be incorporated in chemical
test guidelines and risk assessment.
KEY WORDS: breast cancer, carcinogen susceptibility, development,
endocrine disruptors, human health, lactation, mammary gland, risk
assessment, rodent model, whole mount. Environ Health Perspect
119:1053-1061 (2011). doi:10.1289/ehp. 1002864 [Online 22 June 2011]
The trend toward earlier breast development initiation in U.S.
girls (Euling et al. 2008) may put them at increased risk of later life
outcomes such as breast cancer--already the most common cancer in U.S.
women (American Cancer Society 2010) and a leading cause of death for
U.S. women in midlife (Brody et al. 2007). Although many factors, such
as nutritional status and body size, may contribute to maturation trends
(Kaplowitz 2008) and breast cancer (Renehan et ai. 2008), environmental
chemicals have been hypothesized to contribute as well (Birnbaum and
Fenton 2003; Brody et al. 2007; Euling et al. 2008). Animal studies
demonstrate that early life exposure to hormonally active agents can
lead to effects on mammary gland (MG) development, impaired lactation,
and increased susceptibility to cancer (Fenton 2006). However, the
influence of environmental exposures on breast development outcomes is
poorly understood, as is the relationship between breast development,
lactational deficits, and breast cancer. Few chemicals coming into the
marketplace are evaluated for these effects. The findings in animal
studies raise concerns that perturbations to human breast development
may increase the risk for later life adverse effects including lactation
impairment, gynecomastia (in males), and breast cancer in either sex.
This review is the result of the Mammary-Gland Evaluation and Risk
Assessment Workshop held in Oakland, California, USA, on 16-17 November
2009 to review and discuss recent findings of altered MG development
after gestational/perinatal exposure to certain endocrine-disrupting
chemicals (EDCs). Workshop participants included research scientists
from multiple disciplines, public health advocates, and risk assessors.
Many of the participants are leading internationally recognized MG
experts.
The goal of the workshop was to improve assessment of MG
developmental end points and their integration into human health risk
assessment. Workshop participants discussed current research on the
effects of developmental exposures to EDCs on MG development, the
relationship of these effects to later life lactation and cancer
outcomes, relative sensitivity of MG development and other developmental
reproductive end points, relevance of effects in animal models to
humans, and MG assessment in current toxicology protocols. Data gaps and
research recommendations were identified at the workshop and through
interviews with 18 risk assessors and toxicologists. Presentation slides
and other workshop materials are available online (Silent Spring
Institute 2011). In conjunction with the workshop, experts from seven
laboratories in Canada, the United States, and Argentina participated in
a round-robin evaluation of MG whole mounts.
Factors Affecting Mammary Gland Development
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 (Figure 1).
These stages occur in both rodents and humans.
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
lobularalveolar development (Kleinberg et al. 2009; Russo and Russo
2004a, 2004b). Mammary epithelial growth also occurs in male rats and
men, whereas male mice lack mammary epithelium. Male mice and rats do
not normally possess nippies 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, Table 1 (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 um in the rat
compared with about 70 um for a terminal duct (Russo and Russo 1978).
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, Table 1 (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.
Steroid hormones. 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 tumorigencsis. 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, Table 1 (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 I.amartiniere 1995). Treatment of
postpubcrtal 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).
Phytoestrogens. Effects of treatment with phytoestrogens such as
genistein are similar to those observed after estrogen reccpror 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,
Table 1 (doi: 10.1289/ehp. 1002864)].
Environmental chemicals. Altered MG development after perinatal
exposure has also been observed for numerous EDCs, including atrazine,
bisphenol A (BPA), dibutylphthalate, dioxin, methoxychlor, nonylphenol,
poly-brominated 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, Table 1 (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 GDI5, but not after GDI9, led toMG
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, Zlfacin, 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 epigenctic changes, and this may differ
by compound.
Mechanisms. 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, bro-minated diphenyl ethers, and dioxin all have
been shown to induce a phenotypically similar response of delayed MG
development after neonatal exposures [see Supplemental Material, Table 1
(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 epigenctic 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).
Sex differences. 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 methoxycior 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.
Table 1. Female MG outcomes after developmental environmental
exposures: rodent-human concordance for selected agents.
Environmental Human study Animal study
factor MG outcomes MG outcomes
Development Lactation Cancer Development
risk
Hormonal [DELTA] [DELTA] [DELTA] [DELTA]
milieu:
dosing
(animals) or
surrogates
(humans)
DES [DELTA](c) [DELTA]
Genistein / [DELTA] [DELTA] A
soy
DDT/DDE [DELTA](c) [DELTA] [DELTA]
Dioxins / [DELTA] [DELTA](d) [DELTA]
furans
Environmental
factor
Lactation Cancer
susceptibility
Hormonal [DELTA] [DELTA]
milieu: (EE (2))-dams)(a)
dosing --
(animals) or (EE (2))-offspring(b)
surrogates
(humans)
DES [DELTA] (Dams) [DELTA]
Genistein / [DELTA] (Dams)
soy
[DELTA] (Offspring) [DELTA]
DDT/DDE -(Dams)
Dioxins / [DELTA] (Dams) [DELTA]
furans
Abbreviations:--, no effect on this end point; A, at least one study
has reported an association between the exposure and altered outcomes
[see details and citations in Supplemental Material, Table 2 (doi:
10.1289/ehp. 1002864)]; DDE, dichlorodiphenyldichloroethylene; DDT,
dichlorodiphenyltrichloroethane; DMBA, dimethylbenzanethrene;
EE (2), ethinyl-estradiol. Examples of concordance between
rodents and humans for MG effects are included here. In some cases,
findings are mixed or conflicting; in human studies, exposure
measures are often imprecise. (a)Lactation effect in animals dosed
continuously or during pregnancy and/or lactation. (b)Lactation
effect in animals dosed only in utero and/or preweaning.
(c)Conflicting findings.[d]Exposure may not have been during early
life/development.
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).
Consequences of Altered Mammary Gland Development
Developmental exposures to certain EDCs can lead to MG
developmental effects, lactational deficits, or cancer, but little is
known about the relationships between the developmental and adult end
points. The morphological changes in MG development, particularly
effects on TEBs, suggest the potential for functional outcomes such as
lactational insufficiency, altered pubertal timing, preneoplasia, or
increased susceptibility to carcinogens (Fenton 2006). Table 1 and
Supplemental Material, Table 2 (doi: 10.1289/ehp. 1002864) show these
types of effects occurring across rodent and human studies for selected
compounds for which there are data. However, there are data gaps
regarding the relationships among the various MG outcomes because a)
only a handful of chemicals have been studied; b) there has not been a
standard procedure to assess MG developmental changes; c) MG assessment
in multigcnerational studies has been limited; and d) few studies
include full assessment of dose response.
Carcinogenesis. Reported changes in patterns of breast development
in U.S. girls (reviewed by Euling et al. 2008) raise concerns about
whether earlier onset of breast development is associated with breast
cancer or other adult diseases, because earlier menarche is an
established risk factor for breast cancer (Kelsey et al. 1993).
Furthermore, studies in humans and rodent models demonstrate that
hormonal factors that affect MG development also influence
susceptibility to carcinogens.
Hormonal factors alter susceptibility to carcinogens. Ovarian,
pituitary, and placental hormones, which vary by life stage and with
pregnancy events, are important determinants of breast cancer
susceptibility in humans and rodents (Russo and Russo 2004b). In both
mice and rats treated with chemical carcinogens, hormone withdrawal
(ovariectomy) inhibits tumor development, whereas hormone
supplementation increases the incidence of adenocarcinoma. In humans,
removal of ovaries by 35 years of age dramatically reduces breast cancer
risk (Eisen et al. 2005; Trichopoulos et al. 1972), and antiestrogens
are effective in breast cancer treatment and chemoprevention (Vogel et
al. 2010).
Susceptibility to carcinogens depends on life stage. The influence
of life stage on susceptibility to carcinogen exposure has been
demonstrated in rats and humans. For example, ionizing radiation is
maximally potent as a human breast carcinogen when exposure occurs
during childhood or adolescence (Henderson et al. 2010; Land 1995); this
observation is consistent with findings in rodents (Imaoka et al. 2009).
The increased tumor response from carcinogen exposure early in life is
attributed to the ptesence of proliferating and undifferentiated
structures such as TEBs, which are present during the pubertal mammary
epithelial expansion and display elevated DNA synthesis compared with
other MG structures (Kleinberg et al. 2009). TEBs are considered the
most vulnerable MG target structure for carcinogen exposure (Medina
2007; Russo and Russo 1996). In animals and humans, tumor response from
carcinogen exposure is highest when exposure occurs during adolescence,
when TEBs are still abundant (Henderson et al. 2010; Imaoka et al. 2009;
Land 1995; Russo and Russo 2004b). As a result, there is concern that
exposures to xenobiotics that increase the number or longevity of
proliferating TEBs might increase susceptibility to breast cancer
(Birnbaum and Fenton 2003; Fenton 2006). After the pubertal growth spurt and throughout adult life, it is the terminal ductal structures that
give rise to breast cancers (Kleinberg et al. 2009; Medina 2007; Russo
and Russo 2004b). During pregnancy, differentiation of terminal
structures increases, and this differentiation has been hypothesized to
account for lower MG sensitivity to carcinogens post-pregnancy (Russo
and Russo 2004b).
Early life exposures to (noncarcinogenic) chemicals may affect
response to carcinogens in later life. Experimental models involving
carcinogen challenge have been used widely to demonstrate that hormones
and growth factors influence MG development, differentiation, and
carcinogenesis; these models could readily be extended to evaluate
increased cancer risk from early life environmental exposures. These
models have been used, for example, to investigate potential
chemopreventive agents that accelerate MG differentiation (e.g., by
mimicking pregnancy hormones) and decrease tumor susceptibility
(Cotroneo et al. 2002; Kleinberg et al. 2009; Russo and Russo 1996).
Rodent models used in these studies include dimethylbenz [a] anthracene (DMBA) and nitrosomethylurea (NMU) challenge in rats and mice, and the
mouse mammary tumor virus (MMTV) model. More recently, genetically
modified mouse models have been used to study mammary tumors that are
comparable with human breast tumors in their latency, histotypes, and
endocrine responsiveness (Cardiff et al. 2000; Kamiya et al. 1995;
Medina 2007; Russo and Russo 2004b; Thompson and Singh 2000).
In rodents, early life exposure to hormon-ally active agents
affects MG tumor formation in carcinogen-challenge models. For example,
neonatal estrogen (or androgen) treatment of mice (MMTV model) or rats
(DMBA model) induced MG developmental changes and increased tumors
(Lopez et al. 1988; Mori et al. 1976, 1979). In addition, early life
exposures to genistein (Hilakivi-Clarke et al. 1998, 1999), alcohol
(Hilakivi-Clarke et al. 2004), dioxin (Brown et al. 1998; Desaulniers et
al. 2001; Jenkins et al. 2007), and oral BPA (Jenkins et al. 2009)
caused increased MG tumor multiplicity and decreased latency after DMBA
challenge at PND50. These effects were accompanied by altered MG
development observed in whole mounts (genistein, alcohol, dioxin) or
altered protein expression (BPA). Lifetime exposures (beginning
pre-natally) to genistein, ethinyl estradiol, and BPA have been reported
to alter MG development and increase incidence of preneoplastic lesions
in the MG, with a stronger effect in early adulthood than at 2 years of
age, when MG histopathology is typically performed (Latendresse et al.
2009; Murray et al. 2007;
Vandenberg et al. 2008). Short-term genistein treatment during the
peripubertal period reduces MG tumors after carcinogen challenge,
whereas perinatal or lifetime exposure seems to increase them, although
studies are not consistent (reviewed by Warri et al. 2008). Similarly,
both gestational (Brown et al. 1998) and prepubertal (Desaulniers et al.
2001) dioxin exposure caused increased MG tumors after carcinogen
challenge, whereas later life exposure decreased spontaneous MG tumors
(Kocibaetal. 1978).
In humans, maternal factors that affect the fetal hormone
environment also appear to affect later breast cancer risk in daughters,
possibly by imprinting the developing MG, thereby altering future tissue
responsiveness to hormonal stimulation (e.g., altering estrogen receptor levels or sensitivity) or to genotoxic insult (e.g., by increasing cell
proliferation or diminishing differentiation). The hypothesis that in
utero endocrine-related factors influence breast cancer risk of a
daughter is supported by epidemiology studies that have found d)
preeclampsia associated with reduced breast cancer risk in offspring and
b) high birth weight correlated with higher breast cancer risk (Hoover
and Troisi 2001; Troisi et al. 2007; Xue and Michels 2007). In addition,
there is some evidence that in utero exposure to DES is associated with
higher breast cancer risk in women [Palmer et al. (2006); however,
Verloop et al. (2010) did not find an association] and with increased MG
tumor incidence in rats (Rothschild et al. 1987). Furthermore, the
single epidemiologic study of the EDC dichlorodiphenyltrichloroethane (DDT) that used prospective measures of adolescent/young adult exposure
in relation to breast cancer risk (Cohn et al. 2007) found significant
associations, whereas many studies in which DDT or its metabolite
dichlorodiphenyldichloro-ethylene (DDE) were measured in older women did
not observe an association with breast cancer.
Pregnancy is another critical window corresponding to a time of
extensive MG proliferation and differentiation. DES exposure in pregnant
women has been associated with increased breast cancer risk in the
mother as well as her daughter (Tirus-Ernstoff et al. 2001). A study of
DMBA-challenged rats fed a high-fat diet during pregnancy showed an
increase in circulating estrogen during pregnancy and increased mammary
tumors (Hilakivi-Clarke et al. 1996).
Lactation. The American Association of Pediatrics (AAP) recommends
that all infants receive breast milk during the first 6 months (AAP
1997) and, further, that they are fed breast milk exclusively during
this time (AAP 2005), because of the numerous demonstrated benefits of
breast-feeding. Although data are limited, reports estimate that 3-6
million mothers are unable to produce milk or have difficulty
breast-feeding each year (Lew et al. 2009). The reasons for this remain
unclear, especially given that lactation insufficiency can be the result
of psychosocial as well as biological factors. However, environmental
chemicals are one candidate explanation for inability to initiate and/or
sustain breast-feeding (Neville and Walsh 1995).
Impaired lactation may be associated with altered MG development
(decreased or unresponsive breast tissue) and/or endocrine disruption
(improper hormonal support for lactation). Critical windows include
pregnancy and lactation as well as puberty and the prenatal/perinatal
period. As such, exposure to an EDC during pregnancy has the potential
to disrupt lactation in the mother and the daughter. In human studies,
strong early findings of associations between serum DDE and shortened
lactation in two populations have been only partly replicated, and few
other agents have been studied (Cupul-Uicab et al. 2008; Gladen and
Rogan 1995; Rogan et al. 1987). In rodents, impaired lactation has been
observed in conjunction with altered MG development in one or more
generations after gestational exposure to dioxin (Vorderstrasse et al.
2004), PFOA (White et al. 2007, 2009), atrazine (Rayner et al. 2005),
BPA (California Office of Environmental Health Hazard Assessment 2009;
Matsumoto et al. 2004), genistein [National Toxicology Program (NTP)
2008], and the candidate pharmaceutical Ziracin (Poulet et al. 2005).
For example, atrazine fed to rats during gestation induced MG
developmental changes in offspring, characterized by stunted
development, and when these rats were bred, their offspring
(second-generation) had significantly reduced weight gain, suggesting
insufficient milk production (Rayner et al. 2005). In an example of
effects on lactation in the dam, exposure of pregnant mice to PFOA
decreased pup weight and survival, diminished differentiation/growth of
dam MG, and induced some alterations in gene expression for milk
proteins, which taken together suggest effects on lactation in the
exposed dams (White et al. 2007, 2009).
Treatment during pregnancy has the potential to affect lactation in
both the dam and offspring. Impaired lactation in the dams is typically
identified because of decreased pup weight or survival, and impaired
lactation in the offspring can be determined only in multigenerational
studies where offspring are followed through successful reproduction and
lactation (Makris 2011). The rodent models and assessment methods used
in guideline studies arc not adequate for identifying effects on
lactation because the surrogate markers of pup weight and postnatal
survival are not sensitive or specific indicators of impaired lactation
(Makris 2011).
Human Health Risk
Assessment Issues
MG assessment in chemical test guidelines. MG development can be
affected after early exposure to EDCs in rodents. However, few guideline
studies for testing environmental chemicals include prenatal or early
life dosing, and MG end points are limited primarily to indirect or
surrogate observations during lactation and to clinical and pathological
evaluation of adult mammary tissue (Makris 2011). For example, the
standard 2-year rodent cancer bioassay, initiating treatment in young
adult animals, is likely to be less sensitive to carcinogens than if
developmental exposures were used, and it cannot provide information on
altered susceptibility to carcinogens induced by early life exposures
affecting MG development (Hovey et al. 2002; Medina 2007; Rudel et al.
2007; Russo and Russo 2004b; Singh et al. 2000; Thayer and Foster 2007).
To strengthen MG assessment and chemical testing, it is a priority
to enhance histopathological evaluation of MG development (e.g., using
longitudinal rather than transverse sectioning so that a larger tissue
plane is evaluated), increase attention to evaluation of male MG tissue,
and incorporate early life exposures in rodent subchronic and
chronic/carcinogenicity studies. Consistent with these recommendations,
the NTP has begun including gestational and lactational dosing in rats
assigned to subchronic and carcinogenicity studies and is taking steps
to include early life male and female MG whole-mount preparations and
longitudinal MG sectioning in reproductive assessment and cancer studies
(NTP 2010; Thayer and Foster 2007). Use of these expanded protocols will
facilitate linking altered MG development with later life outcomes.
Table 2. MG as a sensitive end point of endocrine disruption after
developmental exposures in rodents.(a)
Compound Study Species, MG effect
exposure type(b)
timing
Females
BPA Jenkins et al. Rat, Proliferation(d)
2009 postnatal
(lactation)
Murray et al- Rat, Hyperplasia(e)
2007 prenatal
Munoz-de-Toro Mouse, Morphology(f),
et al. 2005 perinatal proliferation(d)
DDT Brown and Rat, Proliferation(d)
Lamartiniere peripubertal
1995
Genistein Fritz etal. Rat, prenatal Morphology(f)
1998 and
postnatal
Padilla-Banks Mouse, Morphology(f)
et al. 2006 neonatal
Males
Genistein Delclos etal. Rat, prenatal Size(g);
2001 and hyperplasia(e)
postnatal
Compound MG effect Basis for
Females LOEL inclusion(c)
BPA 250[micro]g
No effects on
/ kg / age of V0,
day body weight,
serum
progesterone,
or serum
estradiol at
250 [micro]g / kg
/ day (highest
dose tested)
2.5[micro]g No effects on
/ kg / body weight,
day age of V0,
litter size,
or sex ratio
at this or
higher doses
(2.5-1,000 ug
/ kg / day).
25 ng / kg No effects on
/ day plasma
estradiol at
first
proestrus at
this or higher
dose (250
ng/kg / day)
DDT 50 ng / kg Single-dose
/ day study; no
effects on
body weight or
uterine-
ovarian
weight
Genistein 25 No effects on
mg/kg/day body weight,
uterine
weight, AGD,
estrous
cyclicity, or
age at V0 at
this or higher
dose (250 mg /
kg / day)
0.5 Effects on
mg/kg/day ability to
deliver live
pups and
estrous
cyclicity at
50 mg / kg /
day (but not
at either 0.5
or 5 mg / kg /
day)
Males
Genistein 25 ppm Effects on
ventral
prostate
weight,
pituitary
weight, age of
eye opening
and age of ear
unfolding at
1,250 ppm
Abbreviations: AGD, anogenital distance; LOEL, lowest observed effect
level. (a)For inclusion, a study must have assessed other end points
in addition to MG; findings are based on statistically significant
effects observed. (b)Ali effects are relative to negative controls;
effects on protein or gene expression are omitted. For more detail,
see Supplemental Material, Table 1 (doi: 10.1289/ehp. 1002864).
(c)See articles for further study methods and results. (d)Changes in
markers of proliferation/mitotic activity (e.g., cell cycle marker
proliferating cell nuclear antigen, cell number). (e)Changes in
numbers or sizes of hyperplastic structures. (f)Changes in
numbers/ratios of structures, branching, and so on for given
developmental stage. (g)Changes in the area or weight of gland.
As a potential addition to some toxicity test guidelines, MG
whole-mount assessment can demonstrate morphological changes in
development and differentiation and define the temporal and spatial
progression of epithelial development. Another important reason to
include MG assessments in screening-level toxicology studies is to
ensure that MG effeers are identified and can be evaluated in more
comprehensive studies. Specifically, data generated using whole mounts
may be used to trigger further assessment, such as: a) sectioning tissue
blocks, b) evaluating subsequent (e.g., F(1)) generations for
lactational impairment, c) maintaining a population longer on study for
spontaneous neoplasia evaluation, or d) evaluating altered tumor
susceptibility using a carcinogen-challenge protocol. Furthermore, a
whole mount may be the only indication of abnormal development in the
male MG, which is sensitive to very low doses in some studies (Delclos
et al. 2001; Latendresse et al. 2009). However, currently there are no
standardized whole-mount procedures, and consideration of these data in
chemical risk assessment has been limited. The OECD test guideline for
an extended one-generation reproductive toxicity study (OECD 2010) could
be revised to include assessment of MG development using whole mounts
and/or more thorough histopathology (Hvid et al. 2010). In addition, MG
assessment of males and females could be added to the U.S. EPA Endocrine
Disruptor Screening Program (EDSP) pubertal development protocols (U.S.
EPA 2009a, 2009b). MG developmental assessment could also be extended to
include females in the OECD Test Guideline 407 pubertal protocol (OECD
2008).
Adding MG whole-mount procedures to EDSP or OECD test guidelines
has raised concerns that a) these assays could be redundant to
endocrine-sensitive end points assessed [e.g., anogenit.al distance,
timing of vaginal opening (VO), circularing hormones, and estrous cyclicity], and b) that the procedure is too difficult to be
consistently executed across laboratories. However, in some cases MG
effects have been observed at lower doses than other EDC outcomes (Table
2), and there is concern that EDSP assays, which identify chemicals
affecting estrogen, androgen, or thyroid activities, may not be
sensitive to the many mechanisms that can affect breasr development. It
is reasonable, therefore, to include the MG whole mount in screening
studies, at least on a provisional basis, to see if the information
gathered is redundant or unique.
Human relevance of rodent models. Rodent models have been widely
used to characterize the influence of susceptibility factors (e.g.,
ovarian, pituitary, and placental hormones; life-stage and reproductive
events) on malignant transformation of the MG, and parallels between
rodent and human MG structures and pathologies have been enumerated
(Medina 2007; Russo and Russo 2004b; Singh et al. 2000). Although a few
findings in the context of chemicals testing have gener-ared concern
about human relevance (reviewed by Rudel et al. 2007), an extensive body
of breast cancer research demonstrates similarities between rodent and
human MG development and carcinogenesis. These studies indicate that
rodent mammary tumors mimic the diversity of human breast cancers with
respect to important initiation processes, histopathology, hormone
dependence, and host-target cell interactions (Boylan and Calhoon 1983;
Imaoka et al. 2009; Medina 2007; Rudland et al. 1998; Russo et al. 2000;
Russo and Russo 1993, 2004a; Singh et al. 2000). In general, research
indicates greater cross-species similarities for MG development and
cancer than for human menstrual and rodent estrous cyclicity or for
human puberty and rodent VO--end points currently included in many EDC
test protocols (U.S. EPA 2011).
An expert panel on MG tumors concluded that existing rodent models
are useful as screening tools for identifying potential breast
carcinogens (Thayer and Foster 2007). Further, the majority of chemicals
that are positive for mammary tumors in the rodent cancer bioassay have
some evidence of geno-toxicity and many are mulrisite carcinogens,
supporting relevance to humans (Rudel et al. 2007). Although there are
many similarities in the hormonal control of lactation across species,
less is known about the utility of the rodent as a model for predicting
chemical effects on human lactation. In any case, many risk assessment
guidelines operate on the principle that animal effects are considered
relevant to humans in the absence of data to indicate otherwise (U.S.
EPA 1991, 1996, 2005).
A related issue is the consideration of carcinogen-challenge models
as indicators of altered carcinogen susceptibility. DMBA and NMU are
primary breast-specific carcinogens that have been widely used in
experiments designed to assess the alteration of the tumor response by
hormones or other factors (Kamiya et al. 1995; Medina 2007; Singh et al.
2000). Despite the long-standing use of such carcinogen challenge
experiments to assess effects of hormonal or developmental alterations
on tumor susceptibility, the protocols are not common in chemical
toxicity assessment. Risk assessors have not considered data from
carcinogen challenge experiments because of concerns about the protocol
representing a chemical mixture study and about the presumed lack of
relevance of DMBA or NMU exposure to humans. However, a number of
consistent findings of increased susceptibility have been observed in
human and rodent studies across multiple MG end points for endogenous
hormonal factors, DES, genistein, and dioxin, among others (Table 1).
Models that consider the interactive effects of endogenous hormones and
carcinogenic factors across multiple life stages are likely to be more
relevant to human health than those with simpler design, because they
better reflect the human experience.
Table 3. Priority questions, current views, and issues for improving
risk assessment for MG effects.(a)
Priority Current views Outstanding
question for issues
risk
assessment
application
Are the rat Current knowledge Lack of
and mouse suggests that the information
adequate rat and mouse are about human
models for reasonable pubertal
human MG surrogates. development;
development? mechanisms
may differ
among
species.
What is the In utero exposure Few EDC
sensitivity of in some studies studies
MG leads to assess both
developmental developmental MG
effects? effects at doses development
similar to or and another
lower than other sensitive end
developmental and point of ED;
reproductive end there is a
points. lack of human
data to
address dose
response and
a lack of
standardized
MG
development
protocol and
assessment
criteria.
Are MG These changes in Varied
developmental MG are considered definitions
changes adverse because of
adverse? they represent "adversity,"
alterations in depending on
growth and scientific
development(b)and discipline
may be a risk and context.
factor for
lactation and/or
cancer outcomes.
(a)Based on the majority viewpoint of the experts at the Mammary
Gland Evaluation and Risk Assessment Workshop. (b)According to
guidance from the U.S. EPA (1991,1996).
Relative sensitivity of MG effects. A limited set of studies
provide evidence that MG alterations may be more sensitive to some EDCs
than arc other hormonally responsive end points (Table 2). To precisely
determine the relative sensitivity of EDC effects requires studies that
include MG as part of a larger set of endocrine-sensitive end points. Of
the studies that simultaneously evaluated MG morphology and at least one
other EDC-sensitive end point after developmental dosing, a subset has
detected effects on the MG at dose levels or during exposure periods
that did not elicit observable changes in other end points.
Adversity of MG developmental changes. Hormonal factors either
increase or decrease MG tumor susceptibility, and both transient and
permanent effects have been observed on MG development. This raises the
question of what types of alterations to MG development should be
considered adverse. In the context of regulatory evaluation of
chemicals, one point of view is that MG developmental changes reflect
altered growth and development, effects considered adverse by the U.S.
EPA Developmental Toxicity Risk Assessment Guidelines (U.S. EPA 1991).
For comparison, there is also controversy in the risk assessment
community about whether other common markers of altered pubertal timing
(e.g., VO, preputial separation) have human relevance. These end points
have nevertheless been considered adverse, as they are responsive to
endogenous sex steroids, which are important regulators of sexual
development conserved across mammalian species. By this reasoning,
altered MG growth and develop-menr, which is known to have human
relevance, should be considered adverse as well. The question of
adversity was discussed by experts gathered at the workshop in the
context of risk assessment. In spite of the outstanding questions, the
majority perspectives among experts advance the view that MG development
and subsequent effects represent a public health outcome of concern and
are a priority for future research and assessment. Priority questions,
current views, and outstanding issues for risk assessment are summarized
in Table 3.
An important question is whether MG developmental alterations are
plausibly related to increased tumor susceptibility by d) epi-genetic
imprinting of tissue, b) alteration of stem cell populations, or c)
increased number or ontological duration of TEBs or other structures
known to be more vulnerable to carcinogens. Some experts suggest that
such agents should themselves be considered carcinogens. Indeed, the
International Agency for Research on Cancer (IARC) deems an agent
carcinogenic if it is "capable of increasing the incidence of
malignant neoplasms, reducing their latency, or increasing their
severity or multiplicity" (IARC 2006). The U.S. EPA defines an
effect as adverse if it "reduces the organism's ability to
respond to an additional environmental challenge" (U.S. EPA 2010).
Applying these definitions, compounds that cause cancer, either alone or
in combination with other factors at a variety of points in a biological
chain of events leading to tumor formation, may reasonably be considered
carcinogens, including chemicals that increase susceptibility to cancer.
Even if such agents are not designated as carcinogens, their profound
impacts should encourage the risk assessment community to consider the
increase in cancer susceptibility as an adverse effect and therefore to
characterize doses required to elicit the effect. In any case, applying
this approach to risk assessment requires a better understanding of the
relationship between altered MG development and carcinogen
susceptibility.
Conclusions and Research Recommendations
Research demonstrates many similarities between humans and rodents
in normal and perturbed MG development and carcinogenesis. In both
humans and rodents, developmental exposure to hormones affects MG
development and carcinogen susceptibility, and these findings are the
basis for ongoing research to identify chemo-preventative agents in
humans and to determine how EDCs may alter breast cancer risk, pubertal
timing, or lactation. EDCs with diverse mechanisms of action, including
many not considered primarily estrogenic, alter MG development in
rodents. In some cases, altered MG development can be the most sensitive
endocrine end point.
The lack of consistent methods for evaluating and reporting MG
changes makes it difficult to compare findings across studies, hindering
consideration of MG developmental effects in rodent removal services risk assessment. Continued
progress will require consistent approaches across laboratories, along
with a discussion of unique findings and unanticipated effects. In
addition, the relationships between altered development and effects on
lactation or carcinogenesis are still being defined. Addressing these
research needs [detailed in Supplemental Material (doi: 10.1289/ehp.
1002864)] is a priority, and enhanced chemical testing and risk
assessment are needed to characterize these effects.
Major research initiatives under way include The National
Children's Study, which has a number of EDC hypotheses proposed for
testing in its longitudinal study (Lewin Group 2003), and the NIEHS Breast Cancer and the Environment Research Centers (NIEHS 2010), which
have ongoing human studies focusing on the relationship between
environmental exposures and age of breast development onset and which
also support experimental animal research in this area. Research
priorities identified at the workshop [provided in detail in
Supplemental Material (doi: 10.1289/ehp. 1002864)] include further
development and validation of the MG whole-mount protocol, research to
establish the relationship between effects on MG development and later
life outcomes, and issues relevant to use of these data in risk
assessment.
REFERENCES
AAP (American Academy of Pediatrics). 1997. Breastfeeding and the
use of human milk. Pediatrics 100(6):1035-1039.
AAP (American Academy of Pediatrics). 2005. Policy statement:
breastfeeding and the use of human milk. Pediatrics 115(2):496-506.
American Cancer Society. 2010. Cancer Facts and Figures 2010.
Atlanta, GA:American Cancer Society. Available:
http://www.cancer.org/acs/groups/content/@nho/documents/document/acspc-024113.pdf [accessed 17 June 2011].
Andrews P, Freyberger A, Hartmann E, Eiben R, Loot I, Schmidt U, et
al. 2002. Sensitive detection of the endocrine effects of the estrogen
analogue ethinylestradiol using a modified enhanced subacute rat study
protocol (OECD Test Guideline no. 407). Arch Toxicol 76(4): 194-202.
Birnbaum LS, Fenton SE. 2003. Cancer and developmental exposure to
endocrine disruptors. Environ Health Perspect 111:389-394.
Boylan ES, Calhoon RE. 1983. Transplacental action of
diethyl-stilbestrol on mammary carcinogenesis in female rats given one
or two doses of 7,12-dimethylbenz(a)anthracene. Cancer Res
43(101:4879-4884.
Brody JG, Rudel RA, Michels KB, Moysich KB, Bernstein L, Attfield
KR, et al. 2007. Environmental pollutants, diet, physical activity, body
size, and breast cancer: where do we stand in research to identify
opportunities for prevention? Cancer 109(suppl 121:2627-2634.
Brown NM, Lamartiniere CA. 1995. Xenoestrogens alter mammary gland
differentiation and cell proliferation in the rat. Environ Health
Perspect 103:708-713.
Brown NM, Manzolillo PA, Zhang JX, Wang J, Lamartiniere CA. 1998.
Prenatal TCDD and predisposition to mammary cancer in the rat.
Carcinogenesis 19(91:1623-1629.
California Office of Environmental Health Hazard Assessment. 2009.
Meeting State of California Office of Environmental Health Hazard
Assessment. Proposition 65: Developmental and Reproductive Toxicant Identification Committee [meeting transcript, 15 July 2009). Available:
http://oehha.ca.gov/prop65/public_meetings/pdf/DARTICTranscript71509.
pdf [accessed 17 June 2011].
Cardiff RD, Anver MR, Gusterson BA, Hennighausen L, Jensen RA,
Merino MJ, et al. 2000. The mammary pathology of genetically engineered
mice: the consensus report and recommendations from the Annapolis
meeting. Oncogene 19(81:968-988.
Cohn BA, Wolff MS, Cirillo PM, Sholtz Rl. 2007. DDT and breast
cancer in young women: new data on the significance of age at exposure.
Environ Health Perspect 115:1406-1414.
Cotroneo MS, Wang J, Fritz WA, Eltoum IE, Lamartiniere CA. 2002.
Genistein action in the prepubertal mammary gland in a chemoprevention
model. Carcinogenesis 23(9):1467-1474.
Cupul-Uicab LA, Gladen BC, Hernandez-Avila M, Weber JP, Longnecker
MP. 2008. DDE, a degradation product of DDT, and duration of lactation
in a highly exposed area of Mexico. Environ Health Perspect 116:179-183.
Delclos KB, BucciTJ, Lomax LG, Latendresse JR, Watbritton A, Weis
CC, et al. 2001. Effects of dietary genistein exposure during
development on male and female CD (Sprague-Dawley) rats. Reprod Toxicol
15(61:647-663.
Desaulniers D, Leingartner K, Russo J, Perkins G, Chittim BG,
Archer MC, et al. 2001. Modulatory effects of neonatal exposure to TCDD,
or a mixture of PCBs, p,p'-DDT, and p-p'-DDE, on
methylnitrosourea-induced mammary tumor development in the rat. Environ
Health Perspect 109:739-747.
Doherty LF, Bromer JG, Zhou Y, Aldad TS, Taylor HS. 2010. In utero
exposure to diethylstilbestrol (DES) or bisphenol-A (BPA) increases EZH2
expression in the mammary gland: an epigenetic mechanism linking
endocrine disruptors to breast cancer. Harm Cancer 1:146-155.
Durando M, Kass L, Piva J, Sonnenschein C, Soto AM, Luque EH, et
al. 2007. Prenatal bisphenol A exposure induces preneoplastic lesions in
the mammary gland in Wistar rats. Environ Health Perspect 115:80-86.
Eisen A, Lubinski J, Klijn J, Moller P, Lynch HT, Offit K, et al.
2005. Breast cancer risk following bilateral oophorectomy in BRCA1 and
BRCA2 mutation carriers: an international case-control study. J Clin
Oncol 23(30):7491-7496.
Enoch RR, Stanko JP, Greiner SN, Youngblood GL, Rayner JL, Fenton
SE. 2007. Mammary gland development as a sensitive end point after acute
prenatal exposure to an atrazine metabolite mixture in female Long-Evans
rats. Environ Health Perspect 115-.541-547.
Euling SY, Selevan SG, Pescovitz OH, Skakkebaek NE. 2008. Role of
environmental factors in the timing of puberty. Pediatrics 121(suppl
3):S167-S171.
Fenton SE. 2006. Endocrine-disrupting compounds and mammary gland
development: early exposure and later life consequences. Endocrinology
147(suppl 6):S18--S24.
Fenton SE, Hamm JT, Birnbaum LS, Youngblood GL. 2002. Persistent
abnormalities in the rat mammary gland following gestational and
lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).
Toxicol Sci 67(1):63-74.
Fielden MR, Fong CJ, Haslam SZ, Zacharewski TR. 2002. Normal
mammary gland morphology in pubertal female mice following in utevo and
lactational exposure to genistein at levels comparable to human dietary
exposure, Toxicol Lett 133(2-3):181-191.
Foley J, Dann P, Hong J, Cosgrove J, Dreyer B, Rimm D, et al. 2001.
Parathyroid hormone-related protein maintains mammary epithelial fate
and triggers nipple skin differentiation during embryonic breast
development. Development 128(4):513-525.
Fritz WA, Coward L, Wang J, Lamartiniere CA. 1998. Dietary
genistein: perinatal mammary cancer prevention, bioavailability and
toxicity testing in the rat. Carcinogenesis 19(12):2151-2158.
Gladen BC, Rogan WJ. 1995. DDE and shortened duration of lactation
in a northern Mexican town. Am J Public Health 85(4).504-508.
Henderson TO, Amsterdam A, Bhatia S, Hudson MM, Meadows AT, Neglia
JP, et al. 2010. Systematic review: surveillance for breast cancer in
women treated with chest radiation for childhood, adolescent, or young
adult cancer. Ann Intern Med 152(7):444-455.
Hilakivi-Clarke L, Cabanes A, de Assis S, Wang M, Khan G, Shoemaker
WJ, et al. 2004. In utero alcohol exposure increases mammary
tumorigenesis in rats. Br J Cancer 90(11):2225-2231.
Hilakivi-Clarke L, Cho E, Clarke R. 1998. Maternal genistein
exposure mimics the effects of estrogen on mammary gland development in
female mouse offspring. Oncol Rep 5(3):609-616.
Hilakivi-Clarke L, Cho E, Onojafe I, Raygada M, Clarke R. 1999.
Maternal exposure to genistein during pregnancy increases
carcinogen-induced mammary tumorigenesis in female rat offspring. Oncol
Rep 6(5): 1089-1095.
Hilakivi-Clarke L, Cho E, Raygada M, Kenney N. 1997. Alterations in
mammary gland development following neonatal exposure to estradiol,
transforming growth factor alpha, and estrogen receptor antagonist
IC1182,780. J Cell Physiol 170(3):279-289.
Hilakivi-Clarke L, Onojafe I, Raygada M, Cho E, Clarke R, Lippman
ME. 1996. Breast cancer risk in rats fed a diet high in n-6
polyunsaturated fatty acids during pregnancy. J Natl Cancer Inst
88(24):1821-1827.
Hoover RN, Troisi RJ. 2001. Understanding mechanisms of breast
cancer prevention. J Natl Cancer Inst 93(15):1119-1120.
Hovey RC, Asai-Sato M, Warri A, Terry-Koroma B, Colyn N, Ginsburg
E, et al. 2005. Effects of neonatal exposure to diethylstilbestrol,
tamoxifen, and toremifene on the BALB/c mouse mammary gland. Biol Reprod
72(2):423-435.
Hovey RC, Trott JF, Vonderhaar BK. 2002. Establishing a framework
for the functional mammary gland: from endocrinology to morphology. J
Mammary Gland Biol Neoplasia 7(1):17-38.
Hvid H, Thorup I, Oleksiewicz MB, Sjogren I, Jensen HE. 2010. An
alternative method for preparation of tissue sections from the rat
mammary gland. Exp Toxicol Pathol 63(4):317-324;
doi:10.1016/j.etp.2010.02.005 [Online 2 March 2010).
IARC (International Agency for Research on Cancer). 2006. Preamble
to the IARC Monographs (amended January 2006). Available:
http://monographs.iarc.fr/ENG/Preamble/index.php [accessed 17 June
2011].
Imaoka T, Nishimura M, leuka D.Daino K.Takabatake T, Okamoto M,
etal. 2009. Radiation-induced mammary carcinogenesis in rodent models:
what's different from chemical carcinogenesis? J Radiat Res (Tokyo)
50(4):281-293.
Jenkins S, Raghuraman N, Eltoum I, Carpenter M, Russo J,
Lamartiniere CA. 2009. Oral exposure to bisphenol A increases
dimethylbenzanthracene-induced mammary cancer in rats. Environ Health
Perspect 117:910-915.
Jenkins S, Rowell C, Wang J, Lamartiniere CA. 2007. Prenatal TCDD
exposure predisposes for mammary cancer in rats. Reprod Toxicol
23(31:391-396.
Jones LA, Bern HA. 1979. Cervicovaginal and mammary gland
abnormalities in BALB/cCrgl mice treated neonatally with progesterone
and estrogen, alone or in combination. Cancer Res 39(7 Pt 1):2560-2567.
Kamiya K, Yasukawa-Barnes J, Mitchen JM, Gould MN, Clifton KH.
1995. Evidence that carcinogenesis involves an imbalance between
epigenetic high-frequency initiation and suppression of promotion. Proc
Natl Acad Sci USA 92(5):1332-1336.
Kaplowitz PB. 2008. Link between body fat and the timing of
puberty. Pediatrics 121(suppl 3):S208-S217.
Kelsey JL, Gammon MD, John EM. 1993. Reproductive factors and
breast cancer. Epidemiol Rev 15(1):36-47.
Kleinberg DL, Wood TL, Furth PA, Lee AV. 2009. Growth hormone and
insulin-like growth factor-l in the transition from normal mammary
development to preneoplastic mammary lesions. Endocr Rev 30(1):51-74.
Kociba RJ, Keyes DG, Beyer JE, Carreon RM, Wade CE, Dittenber DA,
et al. 1978. Results of a two-year chronic toxicity and oncogenicity study of 2,3,7,8-tetrachlorodibenzo-p-dioxin in rats. Toxicol Appl
Pharmacol 46(2):279-303.
Land CE. 1995. Studies of cancer and radiation dose among atomic
bomb survivors. The example of breast cancer. JAMA 274(5):402-407.
Latendresse JR, Bucci TJ, Olson G, Mellick P, Weis CC, Thorn B, et
al. 2009. Genistein and ethinyl estradiol dietary exposure in
multigenerational and chronic studies induce similar proliferative
lesions in mammary gland of male Sprague-Dawley rats. Reprod Toxicol
28(3):342-353.
Lew BJ, Collins LL, O'Reilly MA, Lawrence BP. 2009. Activation
of the aryl hydrocarbon receptor during different critical windows in
pregnancy alters mammary epithelial cell proliferation and
differentiation. Toxicol Sci 111(1):151-162.
Lewin Group. 2003. Summary of Hypotheses Identified through The
Lewin Group's Targeted Literature Review (02SG0067). Available:
http://www.nationalchildrensstudy.gov/about/organization/advisorycommittee/2003Sep/Pages/NCS-Summary-of-Hyptheses.pdf [accessed 19 November
2010].
Li X, Warri A, Makela S, Ahonen T, Streng T, Santti R, et al.
EDCs and altered mammary gland development 2002. Mammary gland
development in transgenic male mice expressing human P450 aromatase.
Endocrinology 143(101:4074-4083.
Lopez J, Ogren L, Verjan R, Talamantes F. 1988. Effects of
perinatal exposure to a synthetic estrogen and progestin on mammary
tumorigenesis in mice. Teratology 38(2):129-134.
Makris SL. 2011. Current assessment of the effects of environmental
chemicals on the mammary gland in guideline rodent studies by the U.S.
Environmental Protection Agency, Organisation tor Economic Co-operation
and Development, and National Toxicology Program. Environ Health
Perspect 119:1047-1052.
Matsumoto C, Miyaura C, Ito A. 2004. Bisphenol-A suppresses the
growth of newborn pups through insufficient supply of maternal milk in
mice. J Health Sci 50(31:315-318.
Medina D. 2005, Mammary developmental fate and breast cancer risk.
Endocr Relat Cancer 12(31:483-495.
Medina D. 2007. Chemical carcinogenesis of rat and mouse mammary
glands. Breast Dis 28:63-68.
Moon HJ, Han SY, Shin JH, Kang IH, Kim TS, Hong JH, et al. 2007.
Gestational exposure to nonylphenol causes precocious mammary gland
development in female rat offspring. J Reprod Dev 53(2):333-344.
Mori T, Bern HA, Mills KT, Young PN. 1976. Long-term effects of
neonatal steroid exposure on mammary gland development and tumorigenesis
in mice. J Natl Cancer Inst 57(5):1057-1062.
Mori T, Nagasawa H, Bern HA. 1979. Long-term effects of perinatal
exposure to hormones on normal and neoplastic mammary growth in rodents:
a review. J Environ Pathol Toxicol 3(1-2):191-205.
Munoz-de-Toro M, Markey CM, Wadia PR, Luque EH, Rubin BS,
Sonnenschein C, et al. 2005. Perinatal exposure to bisphenol-A alters
peripubertal mammary gland development in mice. Endocrinology 146(9):
4138-4147.
Murray TJ, Maffini MV, Ucci AA, Sonnenschein C, Soto AM. 2007.
Induction of mammary gland ductal hyperplasias and carcinoma in situ following fetal bisphenol A exposure. Reprod Toxicol 23(3):383-390.
Neville MC, Walsh CT. 1995. Effects of xenobiotics on milk
secretion and composition. Am J Clin Nutr 61(suppl 3):687S-694S.
NIEHS (National Institute of Environmental Health Sciences). 2010.
Breast Cancer and the Environment Research Centers. Available:
http://www.niehs.nih.gov/research/supported/centers/breast-cancer/index.cfm [accessed 17 June 2011].
NTP (National Toxicology Program). 2008. Multigenerational
Reproductive Toxicology Study of Genistein (CAS NO. 446-72-0) in
Sprague-Dawley Rats (Feed Study). TR 539. Research Triangle Park,
NC:NTP. Available: http://ntp. niehs.nih.gov/files/539_FINAL_WEB_508.pdf
[accessed 17 June 2011].
NTP (National Toxicology Program). 2010. NTP Study Types:
Toxicology/Carcinogenicity. Available: http://ntp.niehs.nih.
gov/INDEX33B7.HTM?objectid=72015DAF-BDB7-CEBA-F9A7F9CAA57DD7F5 [accessed
21 June 2011].
Odum J, Pyrah IT, Soames AR, Foster JR, Van Miller JP, Joiner RL,
et al. 1999. Effects of p-nonylphenol (NP) and diethylstilboestrol (DES)
on the Alderley Park (Alpk) rat: comparison of mammary gland and uterus
sensitivity following oral gavage or implanted mini-pumps. J Appl
Toxicol 19(5):367-378.
OECD (Organisation for Economic Co-operation and Development).
2008. OECD Guideline for Testing of Chemicals. Repeated Dose 28-Day Oral
Toxicity Study in Rodents. TG 407. Paris:OECD.
OECD (Organisation for Economic Co-operation and Development).
2010. OECD Guideline for the Testing of Chemicals. Draft Proposal for an
Extended One-Generation Reproductive Toxicity Study. Parrs.OECD.
Available: http://www.oecd.org/dataoecd/23/10/46466062.pdf [accessed 17
June 2011].
Okazaki K, Okazaki S, Nishimura S, Nakamura H, Kitamura Y, Hatayama
K, etal. 2001. A repeated 28-day oral dose toxicity study of
methoxychlor in rats, based on the 'enhanced OECD test guideline
407' for screening endocrine-disrupting chemicals. Arch Toxicol
75(9):513-521.
Padilla-Banks E, Jefferson WN, Newbold RR. 2006. Neonatal exposure
to the phytoestrogen genistein alters mammary gland growth and
developmental programming of hormone receptor levels. Endocrinology
147(10):4871-4882.
Palmer JR, Wise LA, Hatch EE, Troisi R, Titus-Ernstoff L,
Strohsnitter W, et al. 2006. Prenatal diethylstilbestrol exposure and
risk of breast cancer. Cancer Epidemiol Biomarkers Prev 15(8):1509-1514.
Poulet FM, Veneziale R, Vancutsem PM, Losco P, Treinen K, Morrissey
RE. 2005. Ziracin-induced congenital urogenital malformations in female
rats. Toxicol Pathol 33(3):320-328.
Rayner JL, Enoch RR, Fenton SE. 2005. Adverse effects of prenatal
exposure to atrazine during a critical period of mammary gland growth.
Toxicol Sci 87(1):255-266.
Renehan AG, Tyson M, Egger M, Heller RF, Zwahlen M. 2008. Body-mass
index and incidence of cancer: a systematic review and meta-analysis of
prospective observational studies. Lancet 371 (9612):569-578.
Rogan WJ, Gladen BC, McKinney JD, Carreras N, Hardy P, Thullen J,
et al. 1987. Polychlorinated biphenyls (PCBs) and dichlorodiphenyl
dichloroethene (DDE) in human milk: effects on growth, morbidity, and
duration of lactation. Am J Public Health 77(10):1294-1297.
Rothschild TC, Boylan ES, Calhoon RE, Vonderhaar BK. 1987.
Transplacental effects of diethylstilbestrol on mammary development and
tumorigenesis in female ACI rats. Cancer Res 47(16):4508-4516.
Rudel RA, Attfield KR, Schifano JN, Brody JG. 2007. Chemicals
causing mammary gland tumors in animals signal new directions for
epidemiology, chemicals testing, and risk assessment for breast cancer
prevention. Cancer 109(suppl 12):2635-2666.
Rudland PS, Barraclough R, Fernig DG, Smith JA. 1998. Growth and
differentiation of the normal mammary gland and its tumours. Biochem Soc
Symp 63:1-20.
Russo IH, Russo J. 1978. Developmental stage of the rat mammary
gland as determinant of its susceptibility to
7,12-dimethylbenz[a]anthracene. J Natl Cancer Inst 61(6):1439-1449,
Russo IH, Russo J. 1996. Mammary gland neoplasia in long-term
rodent studies. Environ Health Perspect 104:938-967.
Russo J, Hu YF, Yang X, Russo IH. 2000. Developmental, cellular,
and molecular basis of human breast cancer. J Natl Cancer
lnstMonogr(27):17-37.
Russo J, Russo IH. 1993. Development pattern of human breast and
susceptibility to carcinogenesis. Eur J Cancer Prev 2(suppl 3):85-100.
Russo J, Russo IH. 2004a. Development of the human breast.
Maturitas 49(1):2-15.
Russo J, Russo IH. 2004b. Molecular Basis of Breast Cancer. New
York:Springer.
Silent Spring Institute. 2011. Silent Spring Institute Organizes
Scientific Workshop on Mammary Gland Evaluation and Risk Assessment.
Available: http://www.silentspring.org/mammary-gland-workshop [accessed
4 March 2011).
Singh M, McGinley JN, Thompson H J. 2000. A comparison of the
histopathology of premalignant and malignant mammary gland lesions
induced in sexually immature rats with those occurring in the human. Lab
Invest 80(2):221-231.
Sivaraman L, Stephens LC, Markaverich BM, Clark JA, Krnacik S,
Conneely OM, et al. 1998. Hormone-induced refractoriness to mammary
carcinogenesis in Wistar-Furth rats. Carcinogenesis 19(9):1573-1581.
Thayer KA, Foster PM. 2007. Workgroup report: National Toxicology
Program workshop on Hormonally Induced Reproductive Tumors--Relevance of
Rodent Bioassays. Environ Health Perspect 115:1351-1356.
Thompson HJ, Singh M. 2000. Rat models of premalignant breast
disease. J Mammary Gland Biol Neoplasia 5(4):409--420.
Titus-Ernstoff L, Hatch EE, Hoover RN, Palmer J, Greenberg ER,
Ricker W, et al. 2001. Long-term cancer risk in women given
diethylstilbestrol (DES) during pregnancy. Br J Cancer 84(f):126-133.
Tomooka Y, Bern HA. 1982. Growth of mouse mammary glands after
neonatal sex hormone treatment. J Natl Cancer Inst 69(6):1347-1352.
Trichopoulos D, MacMahon B, Cole P. 1972. Menopause and breast
cancer risk. J Natl Cancer Inst 48(3):605-613.
Troisi R, Potischman N, Hoover RN. 2007. Exploring the underlying
hormonal mechanisms of prenatal risk factors for breast cancer: a review
and commentary. Cancer Epidemiol Biomarkers Prev 16(9):1700-1712.
U.S. EPA (U.S. Environmental Protection Agency). 1991. Guidelines
for Developmental Toxicity Risk Assessment. Fed Reg 56: 63798-63826.
EPA/600/FR-91/Q01. Washington, DC:U.S. EPA. Available:
http://oaspub.epa.gov/eims/eimscomm. getfile?p_download_id=4560 [accessd
17 June 2011].
U.S. EPA (U.S. Environmental Protection Agency). 1996. Guidelines
for Reproductive Toxicity Risk Assessment. 630/R-96/009. Washington,
DC:U.S. EPA.
U.S. EPA (U.S. Environmental Protection Agency). 2005. Guidelines
for Carcinogen Risk Assessment. EPA/630/P-03/001F. Washington, DC:U.S.
EPA. U.S. EPA (U.S. Environmental Protection Agency). 2009a. OPPTS 890.1450. Pubertal Development and Thyroid Function in Intact
Juvenile/Peripubertal Female Rats. EPA 740-C-09-009. Washington, DC:U.S.
EPA.
U.S. EPA (U.S. Environmental Protection Agency). 2009b. OPPTS
890.1500: Pubertal Development and Thyroid Function in Intact
Juvenile/Peripubertal Male Rats. EPA 740-C-09-012. Washington, OC:U.S.
EPA.
U.S. EPA (U.S. Environmental Protection Agency). 2010. Integrated
Risk Information System. Available: http://www.epa.gov/IRIS/ [accessd 17
June 2011]. U.S. EPA (U.S. Environmental Protection Agency). 2011.
Endocrine Disruptor Screening Program assay status table. Available:
http://www.epa.gov/endo/pubs/assayvalidation/status.htm [accessed 22
June 2011].
Vandenberg LN, Maffini MV, Schaeberle CM, Ucci AA, Sonnenschein C,
Rubin BS, et al. 2008. Perinatal exposure to the xenoestrogen
bisphenol-A induces mammary intraductal hyperplasias in adult CD-I mice.
Reprod Toxicol 26(3-4):210-219.
Vandenberg LN, Maffini MV, Wadia PR, Sonnenschein C, Rubin BS, Soto
AM. 2007. Exposure to environmentally relevant doses of the xenoestrogen
bisphenol-A alters development of the fetal mouse mammary gland.
Endocrinology 148(1):116-127.
Verloop J, van Leeuwen FE, Helmerhorst TJ, van Boven HH, Rookus MA.
2010. Cancer risk in DES daughters. Cancer Causes Control
21(7):999-1007.
Vogel VG, Costantino JP, Wickerham DL, Cronin WM, Cecchini RS,
Atkins JN, et al. 2010. Update of the National Surgical Adjuvant Breast
and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial:
Preventing Breast Cancer. Cancer Prev Res (Phila) 3(61:696-706.
Vorderstrasse BA, Fenton SE, Bohn AA, Cundiff JA, Lawrence BP.
2004. A novel effect of dioxin: exposure during pregnancy severely
impairs mammary gland differentiation. Toxicol Sci 78(2):248-257.
Warner MR. 1976. Effect of various doses of estrogen to BALB/cCrgl
neonatal female mice on mammary growth and branching at 5 weeks of age.
Cell Tissue Kinet 9(5):429-438.
Warri A, Saarinen NM, Makela S, Hilakivi-Clarke L. 2008. The role
of early life genistein exposures in modifying breast cancer risk. Br J
Cancer 98(9):1485-1493.
White SS, Calafat AM, Kuklenyik Z, Villanueva L, Zehr RD, Helfant
L, et al. 2007. Gestational PFOA exposure of mice is associated with
altered mammary gland development in dams and female offspring. Toxicol
Sci 96(1):133-144.
White SS, Kato K, Jia LT, Basden BJ, Calafat AM, Hines EP, et al.
2009. Effects of perfluorooctanoic acid on mouse mammary gland
development and differentiation resulting from cross-foster and
restricted gestational exposures. Reprod Toxicol 27(3-4):289-298.
Xue F, Michels KB. 2007. Intrauterine factors and risk of breast
cancer: a systematic review and meta-analysis of current evidence.
Lancet Oncol 8(12):1088-1100.
Yang C, Tan YS, Harkema JR, Haslam SZ. 2009. Differential effects
of peripubertal exposure to perfluorooctanoic acid on mammary gland
development in C57BI/6 and Balb/c mouse strains. Reprod Toxicol
27(3-41:299-306.
You L, Casanova M, Bartolucci EJ, Fryczynski MW, Dorman DC, Everitt
Jl, et al. 2002. Combined effects of dietary phytoestrogen and synthetic
endocrine-active compound on reproductive development in Sprague-Dawley
rats: genistein and methoxychlor. Toxicol Sci 66(1):91-104.
Ruthann A.Rudel,(1) Suzanne E. Fenton, (2) Janet M. Ackerman, (1)
Susan Y. Euling, (3) adn Susan L. Makris(3)
(1)Silent Spring Institute, Newton, Massachusetts, USA; (2)National
Toxicology Program, National Institute of Environment Health Sciences,
National Institutes of Health, Department of Health and Human Services,
Research Triangle Park, North Carolina, USA; (3)National Center for
Environmental Assessment, U.S. Environmental Protection Agency,
Washington, DC, USA
Address correspondence to R. Rudel, Silent Spring Institute, 29
Crafts St., Newton, MA 02458 USA. Telephone: (617) 332-4288, ext 214.
Fax: (617) 332-4284. E-mail: rudel@silcntspring.org
Supplemental Material is available online (doi: 10.1289/ehp.
1002864 via http://dx.doi.org/).
We thank our workshop participants for their contributions to this
project [sec Supplemental Material, p. 2 (doi: 10.1289/ehp. l002864)].
We also thank C. Reed for careful review of the tables in Supplemental
Material.
This workshop was supported by the California Breast Cancer
Research Program, U.S. Environmental Protection Agency (U.S. EPA),
National Institute or Environmental Health Sciences (NIEHS), National
Toxicology Program (NTP), and Silent Spring Institute.
The views expressed in this paper are those of the authors and do
not necessarily reflect the views or policies of the U.S. EPA or NIEHS.
R.A.R. and J.M.A. are employed at Silent Spring Institute, a
scientific research organization dedicated to studying environmental
factors in women's health. The Institute is a 501(c)3 public chanty funded by federal grants and contracts, foundation grants, and private
donations, including donations from breast cancer organizations. The
authors declare they have no actual or potential competing financial
interests.
Received 16 August 2010; accepted 17 May 2011.