Mammary Gland

C H A P T E R

61 Mammary Gland Barbara Davis1, Suzanne Fenton2 1

Tufts University, Boston, MA, USA, 2National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

O U T L I N E 1. Introduction

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2. Mammary Gland Structure, Function, and Cell Biology 2.1. Mammary Gland Development 2.2. Mammary Gland Development across Species 2.3. Mammary Gland Cell Biology 2.4. Mammary Gland Pathobiology and Cancer

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3. Evaluation of Toxicity 3.1. Physiologic Evaluation 3.2. Biochemical and Biomarker Evaluation 3.3. Morphologic Evaluation 3.4. In Vitro Techniques 3.5. Use of Animal Models

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1. INTRODUCTION Breast cancer is the most common malignancy in women in the USA, and a leading cause of death. Major risk factors for breast cancer include lifetime exposures to endogenous or exogenous estrogens, and specific drugs and environmental carcinogens having estrogenic activity contribute to this risk. We also know from animal studies that the developing gland has windows of increased susceptibility to external influences which may increase the predisposition to cancer later in life. This fact has raised concern that the significant shift to an earlier age of puberty in young girls will result in an increased susceptibility to and Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Third Edition. http://dx.doi.org/10.1016/B978-0-12-415759-0.00061-3

4. Response to Injury 2683 4.1. Physiologic Response to Injury 2683 4.2. Molecular and Biochemical Response to Injury 2685 4.3. Morphologic Response to Injury 2685 5. Mechanisms of Toxicity

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6. Conclusion

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Suggested Reading

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Appendix I. Mammary Gland Whole Mount Preparation II. Fixing and Staining Procedure

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incidence of breast cancer later in life. Thus, it is critical that methods in safety assessment and hazard identification using animal models are sufficiently designed to detect subtle changes in morphology during development, as well as preneoplastic and neoplastic changes in adults. There is also an increasing need for experience and specialization in relevant test models and properly conducted histomorphological assessments in both female and male mammary tissue. The purpose of this chapter is to provide an overview of mammary gland biology of different species with relevance to humans, and review models, methods, mechanisms, and definitions of mammary gland toxicity and carcinogenicity.

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Copyright Ó 2013 Elsevier Inc. All rights reserved.

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2. MAMMARY GLAND STRUCTURE, FUNCTION, AND CELL BIOLOGY While there are marked anatomical differences in the mammary gland across and within species, its development and function in the female – production of milk for nourishment of offspring – is generally recapitulated across all mammals. Mammary glands are modified tubuloalveolar glands of the skin (see Integument, Chapter 55), and form a complex organ composed of epithelial lined ducts and alveoli within an adipose-based fat pad surrounded by fibroblasts, immune/ inflammatory cells, and lymphatic and blood vessels. The epithelium is arranged in a bilayer of luminal cells and basal myoepithelial cells lying within a basement membrane. The luminal cells are secretory and the basal myoepithelial cells have multiple functions, including contraction, adhesion, and structural formation of the ducts. Mammary development resembles that of epidermal appendage sweat glands, but is under control of pituitary and gonadal hormones, in addition to the influences of hormones produced by other cells of the mammary fat pad. These hormonal influences transition the gland through numerous phases of growth, differentiation, involution, and atrophy.

2.1. Mammary Gland Development While both subtle and dramatic differences are observed in the morphology and hormonal controls of mammary gland development across species, there are fundamental similarities that it is important initially to define. Mammary gland development is generally categorized by the distinct anatomical (and functional) changes during embryogenesis and pre-puberty that include bud formation and early ductal tree formation, and during post-puberty and pregnancy that include rapid expansion and lobuloalveolar differentiation, and pregnancy and lactation. Bud and Ductal Tree Formation Gland formation begins early during embryogenesis with the development of linear bilateral ectodermal thickenings, referred to as the mammary line or ridge, which overlie a specialized mesoderm. The ectodermal cells of the ridge migrate and aggregate, forming “mammary buds” at the location of the future mammary

gland. These solid ectodermal cords grow into the underlying mesenchyme, followed by limited epithelial branching and canalization, to form mammary sprouts. Developing from the mesenchyme along with the formation of the ectodermally derived mammary buds are support structures including the adipose tissue, which later forms the fat pad containing blood vessels, lymphatics, and connective tissue. Myoepithelial cells that eventually surround epithelial structures (ectoderm) and nerves (neuroectoderm) differentiate separately from, but simultaneously with, the mammary buds. There are some species differences in the number and complexity of sprouts prior to birth, as sprouts form the papillary ducts of each gland. Each sprout also communicates externally with epithelium that will form the teat/nipple. Some species have collecting ducts that drain multiple primary ducts into a teat (mice, cows), whereas the human has numerous ducts that drain directly into the nipple, simultaneously. Ductal Elongation Initial growth of the gland occurs through linear ductal elongation promoted by a distinct bulbous structure at the distal end of the duct called the terminal end bud (TEB) (Figure 61.1). Terminal end buds are composed of multiple layers of epithelial cells and myoepithelial cells lining the basement membrane that interact closely with the surrounding mesenchymal stroma of the fat pad to determine length and patterning of the gland. The leading portion of the terminal end bud is covered by a distinct layer of “cap cells” overlying more numerous layers of “body cells,” each containing stem cells with the capacity to form ductal or luminal alveolar cell types. Canalization occurs at the trailing end of the duct to form a single layer of luminal cuboidal epithelial cells and basally located myoepithelial cells supported by a distinct basement membrane. A continuous basement membrane is principally composed of Type IV collagen, laminin, nidogen, and heparin sulfate proteoglycan. Both luminal epithelial and myoepithelial cells produce laminin 1 subunit chains, distinguished by type: epithelial cells deposit a3 and a5 chains; myoepithelial cells deposit a1 chains. The stroma surrounding the ducts consists of fibroblasts and adipocytes, macrophages, and eosinophils. These inflammatory cells have been specifically

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implicated in supporting ductal elongation and branching. Functionally and microscopically, mammary gland morphology and duct elongation is characterized as both a proliferative process and an apoptotic process. Lobuloalveolar Differentiation In all species, there is extensive growth of the mammary gland and branching of terminal end buds into lobular structures during adolescence and young adulthood. Although there are species differences in promoting the growth, in all species exponential growth and development is ignited with the onset of puberty and burst of ovarian hormones. In the adult, mammary glands are arranged into lobules of compound branched alveolar glands, separated by dense interlobular connective tissue and fat (Figure 61.2). Lobules are arranged into distinct lobes each with its own excretory duct, also called the lactiferous duct, and its own opening on the teat or nipple via the papillary duct. The lactiferous duct is lined by cuboidal basal cells and superficial columnar cells. The secretory units are alveoli, which respond to hormonal signals of lactation. An alveolus is lined by a secretory cuboidal or columnar epithelium and an outer layer of myoepithelial cells that lie between the epithelium and the basement membrane. Pregnancy and Lactation Marked development and differentiation continues through pregnancy and lactation. However, there are species differences in the extent of postpubertal development and glandular complexities. For example, animals that

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FIGURE 61.1 Mouse mammary gland at 5 weeks showing terminal end bud (TEB) and duct morphology. (A) Carmine alum-stained whole mount of mammary gland with many TEBs at leading edges filling the surrounding fat pad. (B) Higher magnification of the TEBs showing bulbous end and bifurcation. (C) Hematoxylin and eosin (H&E)-stained section of a TEB showing the single layer of TEB cap cells (outer ring) and multilayered pre-luminal body cells. The cap cells often seem artifactually separate from the body cells during processing. There is also often an increased stromal cellularity (inflammatory cells and fibroblasts) along the neck. TEBs should not be confused with foci of hyperplasia in young animals. Photographs courtesy of Dr Jason P. Stanko, National Toxicology Program, NIEHS.

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develop spontaneous pseudopregnancy, such as the dog, also exhibit mammary gland development during the postpubertal period similar to that which occurs during pregnancy (see Female Reproductive System, Chapter 60). The nulliparous rat mammary gland has considerably less lobular development than in dogs or primates, and is more similar to the lobular development in the human breast than the simple branched structure of the adult mouse. During pregnancy and lactation, extensive growth and alveolar maturation occurs to form milk-producing glands. With growth of the gland, there is a concurrent reduction in the amount of intra- and interlobular connective tissue. The secretory alveoli become lined by cuboidal epithelium, surrounded by a layer of myoepithelial cells, basement membrane, and an intimate network of capillaries and lymphatics. The continued growth of the mammary gland during the second half of pregnancy is due to increases in the height of epithelial cells and an expansion of the lumen of the alveoli. These cuboidal cells produce and secrete the milk components, which are expelled from lobuloalveolar units by contraction of myoepithelial cells. After lactation or end of suckling, the mammary glands undergo an apoptotic process, called involution, during which remaining milk is phagocytized and epithelial cells degenerate. The few remaining alveoli are lined by low cuboidal non-secreting epithelial cells and prominent myoepithelial cells. Stromal cells reactivate and the amount of interstitial and connective tissue is increased as involution proceeds. Corpora amylacea, which are small concretions of protein, and apoptotic bodies may be found in alveoli, ducts, or interstitial areas during this stage of remodeling.

2.2. Mammary Gland Development across Species Species differences to consider include the relative timing of development, and the extent and

= FIGURE 61.2 Rat mammary gland at 3 weeks showing alveolar buds and duct morphology prior to puberty. (A) Carmine alum-stained whole mount of mammary gland with many branching ducts and

alveolar buds filling the fat pad. (B) Higher magnification of branching ducts with alveolar buds. (C) Hematoxylin and eosin counter-stained section of duct showing branched ducts (left) and ducts lined with numerous buds of various sizes (right). Photographs courtesy of Dr Jason P. Stanko, National Toxicology Program, NIEHS.

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complexities of the ductal and lobuloalveolar development. Understanding of these species variations is critical to interpreting the effects of xenobiotics. Such interpretations also need to be considered with respect to the tremendous morphologic changes that occur during puberty, estrous cycles, pregnancy, lactation, involution, and aging. The primitive state of the epithelial ductal tree and the presence of terminal end buds in the gland just before and well beyond the activation of the ovarian hormones of puberty are strikingly similar across these species. However, one of the most dramatic differences is found in terms of lobular development, which starts just after puberty in adolescent girls and rats, but is not apparent in mice until adulthood. Figure 61.3 compares these developmental landmarks by age between mouse, rat, and humans. Rodents In the mouse, mammary gland development begins about gestation day 10.5 with the appearance of the five placodes of the mammary buds. Further development occurs along each of the mammary lines from gestation day 11.5 to day 13.5. The mesenchyme adjacent to the mammary epithelium becomes dense and begins to regulate elongation of the mammary buds. Epithelial cords are formed and continue to elongate as terminal ends form and grow into the fat pad by gestation days 15–16.5. Just prior to birth, ductal branches and lumens form. The rat differs from the mouse in that mammary anlage development begins about gestation day 12 with six sets of mammary buds which develop into a more complex branched pattern around the time of parturition. Prior to vaginal opening, ductal budding is apparent and abundant in some rat strains, and the terminal end buds begin to cleave into clusters of three to five smaller alveolar buds, each with a centrally located lumen surrounded by a layer of cuboidal epithelial cells. Thus, histologically, the mammary gland of the prepubertal female rat is characterized by scattered ducts lined by a single or double layer of cuboidal epithelium, which, after branching a number of times, becomes multilayered to form the terminal end buds. Terminal end buds are comprised of three to six layers of medium-sized epithelial cells with scant cytoplasm and oval nuclei. Growth and branching of the mammary structure in rodents continues after birth with

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exponential development during puberty, reaching peak growth rate from 21 to 55 days of age, depending on the strain. With each successive cycle, the alveolar buds form complex alveoli of smaller diameter with a distinct lumen lined by a single layer of low cuboidal epithelium. The mammary gland of mature, virgin female rats varies little histologically from that of prepubertal animals except for more abundant branching, narrower ducts, and more numerous alveolar buds and lobules, referred to as tubuloalveolar structures. As is the case in young women, complex lobuloalveolar development is not a feature of the nulliparous cycling rodent, likely because of the quick drop in prolactin after ovulation in the short diestrus phase. As in primates, estrogens are necessary for duct development, and progesterone and estrogen, along with prolactin, are necessary for lobuloalveolar pattern during pregnancy. In all of these mentioned species, changes in the extent of lobule formation may vary with the stage of the estrous/menstrual cycle. In young rodents with 4- or 5-day estrous cycles there are only subtle changes in mammary gland morphology, but as rodents age or experience periods of progesterone-prominent pseudopregnancy, glands will have more extensive budding and lobular formation. In dogs that have longer estrogen- and progesterone-influenced estrous cycles, mammary gland morphology can vary dramatically from a ductular proliferative state during proestrus and estrus to an alveolar secretory state in diestrus. Because of this, experiments with cycling females should be conducted with consideration for cycle stage at necropsy in all species. As virgin female rats of some strains (e.g., Charles River Sprague-Dawley or Fisher 344) become middle aged (from 8 to 14 months of age) and reproductive senescence ensues, there is an increasing level of prolactin secretion that contributes to the development of a number of morphologic changes, including inappropriate secretory activity, ectasia of ducts leading to the formation of cysts or galactoceles, epithelial hyperplasia, and periductal fibrosis. In humans, such changes are considered dysplastic. Therefore, these considerations should be weighed prior to use in test guideline studies. Although the mammary gland becomes a vestigial organ in the male rat and other species, it may be more susceptible to endocrine modulation

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FIGURE 61.3 Schematic representation of important stages of mammary gland development in mouse, rat, and human. There are many similarities in the timing and morphological progression across species. GD ¼ gestation day; EW ¼ embryonic week; PND ¼ postnatal day.

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during development and certainly retains the capacity to develop cancer in the adult. The development of the mammary gland in the male follows the same temporal pattern of development as in the female, and may show considerable sexual dimorphism in adulthood. For example, in male mice the mammary rudiment is destroyed on or near gestational day 14 in response to androgendependent condensation of the surrounding mammary mesenchyme. However, CD-1 mice retain a small mammary epithelial remnant throughout life. Young male rats have considerably more acinar structures and few ducts, while female counterparts display numerous ducts and some lobuloalveolar structures. Most of the lobules in adult male rats are lined by a single layer of cuboidal vacuolated epithelial cells, or by pseudostratified or stratified epithelium. There are general statements in the early literature that mammary gland development in the male occurs through inhibition of growth or that, in animal models, mammary gland histology is comparable between prepubertal males and females or between postpubertal males and nulliparous females. However, there are now considerable examples of sexual dimorphism in adult males and females, particularly in rats. Early anatomic studies of ductal development in the rat mammary gland show that, until approximately the fifth postnatal week, the duct systems of males and females are parallel in development. Thereafter, females exhibit more branches and ducts with lumens than the male, and have extended growth patterns. Neither the male rat nor the male mouse exhibit nipple development. Dogs The variation in mammary gland morphology in young animals always presents a challenge forunderstanding experimentally produced changes. Moreover, when dogs are used in safety assessment studies, they are usually young and sexually immature, which may limit the assessment of toxicity. That is, as in other species, prior to puberty dogs have rudimentary glands composed of large interlobular ducts with few laterally projecting terminal end buds, all housed within a dense connective tissue stroma. Like other species, marked proliferation of terminal end buds, linear growth, and tertiary branching into lobes occurs with the onset of puberty and requires the influence of estrogens and progestins.

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In cycling bitches, the gland of proestrus is characterized by interlobular ducts, a few small lobules, and extensive connective tissue stroma. During estrus, there is marked intralobular ductal epithelial proliferation and formation of many small ductules lined by a multilayered epithelium within loose connective tissue. By default in the bitch, corpora lutea function is maintained by prolactin during the 2 months (about 63 days) post-ovulation regardless of whether the bitch is pregnant or not (pseudopregnancy). Mammary gland development during the early half of this period (early diestrus if not pregnant, or early pregnancy) is characterized by greater development of the ducts, the stroma becomes mucinous, and fibroblasts within the interlobular stroma demonstrate increased mitotic activity. During the latter half of this period, late diestrus or late pregnancy, secretory alveoli begin to develop. Alveoli are filled with bright eosinophilic proteinaceous secretion, and lined by cuboidal to flattened cells and elongated or stellate myoepithelial cells, supported by minimal intralobular stroma. Stroma remains prominent around the ducts. If pregnant, at the time of parturition the mammary gland is stimulated by oxytocin and prolactin and becomes secretory. If not pregnant, the mammary gland involutes. Involution is characterized as reduction and shrinkage of the alveoli with vacuolated epithelial cells and apoptotic changes associated with senescence. Non-Human Primates As in humans, the rudimentary ductal tree is formed early in life. Terminal end buds form the leading edge, surrounded by myxoid stroma. Rapid growth and the appearance of “invasive” epithelium are normal in non-human primates, particularly macaques. However, mammary gland development and structure in non-human primates also differs slightly from that in humans, and also between the different species of monkeys, much of it varying with the differences in reproductive cycles. For example, marmosets do not undergo menopause so older females will maintain more developed glands, while rhesus macaques reach menopause at about 22–24 years of age, accompanied by atrophic changes in the mammary gland. Most mammary gland development occurs during puberty. In rhesus macaques, puberty occurs between 2 and 3 years of age, and

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in cynomolgus monkeys it occurs slightly later. The epithelial cells are surrounded by myoepithelium and stromal support consisting of fibroblasts and adipocytes. Indeed, most of the developing gland is adipose and fibrous connective tissue. The lactiferous ducts are lined by stratified squamous epithelium as they approach the surface, and in the non-lactating animal they are typically plugged by keratin. As the mammary gland continues to develop, there is rapid elongation and branching of major ducts to form a dense nest of arborized lobuloalveolar units. Thus, even early in puberty with the onset of menstruation, there may be well-differentiated, densely branched lobuloalveolar units lined with secretory epithelial cells. Toxicology studies often employ few and younger, 2- and 3-year-old, monkeys that may be just entering puberty. Mammary gland development can be quite mixed, from dense branching ducts to lobulo-alveolarization, which may be misinterpreted as toxicant effects. There are also subtle cycle-related changes characterized by ductal proliferation during the luteal phase of the cycle and alveolar proliferation during the follicular phase. During pregnancy and lactation, extensive alveolar maturation and differentiation to a milk-producing phenotype occurs. Involution is typically extensive with regression of alveoli to a nulliparous state. The sex steroid receptor expression profile in the adult macaque mammary gland is similar to that in the human. Androgen receptor is also expressed in macaque mammary tissue and in the skin and tip of the nipples. Notably, histomorphological studies have demonstrated that ER-alpha and progesterone receptors often coexpress in the same cells, but do not co-express with the proliferation marker Ki67 – findings also observed in the mouse. Prolactin is not an obligate component of mammary growth and development in macaques, but is required for lactation. Experimental studies have shown that during aging and menopause, or with surgical castration, atrophic ducts maintain estrogen and progesterone receptors and respond to exogenous steroids for some time. Humans Mammary milk-lines develop as early as 4–5 weeks of gestation and form one pair of mammary placodes. The primary ectoderm bud is present by 12 weeks of gestation, and small

ductal outgrowths as a primitive gland are present at birth. Also, rather than forming a single ductal tree, each human anlagen forms several trees initiating at the nipple. Before birth, the specified mammary epithelium grows from the nipple into the fat pad to form a small, branched ductal network. The advancing margins of undifferentiated terminal end buds are surrounded by a loose myxomatous connective tissue matrix. Terminal end buds express both estrogen receptors by gestational week 30 and progesterone receptors by week 41, evidence of the role that steroid hormones play in normal breast development with implications extending to the potential deleterious influences of endocrine-disrupting compounds. During childhood and adolescence, breast growth keeps pace with overall body growth until greatly accelerating at puberty. The hallmark responses of exponential growth and rapid differentiation of the mammary gland at puberty and during pregnancy, in preparation for lactation, demonstrates that this tissue continues to respond to hormones and remains flexible in its differentiation potential. Thus, critical windows for breast development extend from the prenatal period, continuing through peripubertal years and into adult life. Age of puberty in girls is decreasing significantly across continents, with genetics, nutrition, or increasing exposure to natural and synthetic estrogenic compounds all implicated as potential causes. Pubertal development is classified by five stages (denoted as “Tanner” stages; named after the investigator that developed this system of evaluation), which describe breast and pubic hair growth prior to the onset of menarche. Using the correlation between Tanner stages, accelerated age of puberty in girls is characterized as menarche occurring possibly a few months earlier, while breast development can occur up to 1–2 years earlier than it did a couple of decades ago. Thus, not only is breast development significantly advancing in the human population, but there is also dissociation between the pubertal hormonal control of breast development and menarche. These observations underscore the enhanced sensitivity of the mammary gland to genetic and environmental influences compared to other reproductive tissues (see Female Reproductive System, Chapter 60). Importantly, the advancement of breast development increases the overall time that terminal end

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buds remain as susceptible targets in the breast, which in turn increases the risk for development of cancer. Indeed, earlier age of menarche and later age of menopause are known risk factors for breast cancer. Thus, assessing the maturation of the mammary gland in rodent toxicology studies provides an important means to assess physiological alterations of the gland and identify potential human health hazards.

2.3. Mammary Gland Cell Biology The complex cellular interactions of mammary gland growth, differentiation, and regression are initiated by stem cell developmental programs, mediated by paracrine signaling between the epithelium and mesothelium of the fat pad early in development, and orchestrated by reproductive hormones (Figure 61.4). Much of our fundamental knowledge of these interactions is

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defined by studies in rodents in response to various hormone treatments and in the use of transgenic mouse models (Table 61.1), but there is species variation in the specific factors and particularly the reproductive hormones that play major roles in determining the development of this gland. Formation of the Rudimentary Buds and Nipples Studies in mice have elegantly demonstrated the coincidental and necessary localization and activation of the canonical WNT/b-catenin signaling pathway in the embryonic mammary placodes to direct stem cells in the surface epithelium towards appendage formation (rather than stratified epidermis differentiation). The canonical WNT pathway involves binding of the Wnt ligand to Frizzled (FZ) receptors and obligate low-density lipoprotein receptor-related protein

FIGURE 61.4 Diagrammatic summary of key signaling molecules or pathways mediating developmental stages of mammary gland morphogenesis. Photographs adapted from Rudel et al. (2011) Environmental exposures and mammary gland development: state of the science, public health implications, and research recommendations, Environ. Health Perspect. 119, 1053–1061, with permission.

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TABLE 61.1 Mammary Gland Morphologies Associated with Selected Receptor-Deficient Mice Genotype defect

Mammary gland phenotype

ER-a

Vestigial ducts at nipple

ER-b

No significant pathology

AR

Rudimentary ducts

EGFR/ErbB2

Deficient TEB, deficient ductal growth

ErbB2,3,4

Deficient lobuloalveolar differentiation and lactation

IGFR

Decreased branching

PR

Deficient transition in ductular to lobuloavelolar differentiation

PrlR

Decreased ductal outgrowth during pregnancy

GH

Decreased ductal outgrowth during pregnancy

co-receptors. This binding leads to inactivation of the multiprotein axin complex A proteins that function to degrade cytoplasmic b-catenin. As a result of the inhibition, b-catenin accumulates, translocates from the cytoplasm to the nucleus, and forms active transcriptional complexes with LEF/TCF family members. In the formation of mammary sprouts, WNT gene signaling interacts with several other genes including members of the fibroblast growth factor family, T-box transcription factor TBX3, and estrogen-regulated homeobox transcription factors MSX1 and MSX2. WNT-mediated transcription is also important later in the development of alveolar buds that sprout from the branching terminal end buds. There is extensive experimental and clinical evidence that dysregulation of WNT signaling is a key feature of mammary cancers. Nipple formation occurs after formation of the mammary bud epithelium and as an example of one of the earliest mediators of epithelial– mesenchymal crosstalk in mammary gland development. The mammary bud epithelium produces parathyroid hormone-related protein (PTHrP), which is an androgen-regulated protein. Its receptor, PTHR1, is on adjacent mesenchymal cells, and activation of this pathway triggers sexspecific development of the nipple/areolae in

rodents. The interaction of these molecules is also required for forming the mammary-specific mesenchyme, which in turn is required to form a rudimentary ductal tree. In male mice and rats the fetal androgen surge, which occurs around gestation day 14–15, causes the mesenchyme to condense and inhibits the formation of nipples/ areolae. In male mice the androgen surge also causes regression of the sprout so that male mice have no mammary tissue, with the single reported exception of CD-1 mice. However, the male rat of all strains investigated does form a ductal tree, and the male rat mammary gland remains responsive to endogenous and exogenous hormone agonists and antagonists throughout its life. In the female, the ductal tree continues to form through androgen- and estrogen-mediated signaling. Androgens in the female serve as the precursor hormone converted to estrogens. Mammary glands of female mice deficient in estrogen receptor (ER)-alpha develop only rudimentary ductal trees and thus demonstrate the critical role for estrogen-mediated signaling through ER-a in early mammary gland development. Mice lacking aromatase, the enzyme necessary for endogenous estradiol production, fail to develop mature glands during puberty. Recently, activation of the Snail pathway, which controls aromatase expression, has also been implicated in ductal growth and thus provides a mechanism to support estrogen production from androgens within the mammary compartment. Duct Elongation and Branching Epithelial–mesenchymal interactions are equally important in the formation of ductal elongation and branching patterns. Ultimately, the length and complexity of the ducts and the extent of lobuloalveolar proliferation is controlled by the fat pad. The fat pad produces regulatory molecules that promote growth, while inhibiting ducts from overlapping or penetrating adjacent fields by contact inhibition. Among the molecules defined in these interactions, LEF-1, hepatocyte growth factor, keratinocyte growth factor, and neuregulin are factors produced in the mesenchyme that interact with receptors on the epithelial cells to influence mammary epithelial growth. Considerable evidence also indicates that transforming growth factor (TGF)-b1 acts as a key

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negative regulator of mammary branching by limiting epithelial proliferation and by stimulating extracellular matrix production. Both inhibin and activin growth factors are also involved in epithelial–mesenchymal signaling in mammary gland development. These growth factors are produced in the stroma to modulate epithelial development, but it is the inhibin beta B subunit signaling that is required for epithelial ductal growth, as well as later alveolar development. Lobuloalveolar Development The bulk of growth of the mammary gland occurs during the postpubertal period, thus highlighting the critical role of ovarian and pituitary-derived hormones. Terminal end buds express both estrogen receptors and progesterone receptors, and are primed for response to steroid hormones at the onset of puberty and through the reproductive cycle. Estrogens and progesterone are major controllers of lobuloalveolar development of the mammary gland through receptor-mediated interactions with each other, and in the stimulation of and interaction with growth factors. Estrogen mediates its effects through ER-a located in both epithelial and stroma in rodents, although it appears to be epithelial-specific in humans. Progesterone mediates its effects through both progesterone receptors A and B (PRA and PR) interspersed on some, but not all, epithelial cells. Most of the progesterone receptor expressing epithelial cells in the developing gland also co-express estrogen receptor. These steroid receptor-positive epithelial cells are not within the proliferative pool of epithelial cells themselves, but regulate growth and lobuloalveolar differentiation through paracrine effects on adjacent steroid receptornegative cells. This paracrine interaction appears to involve Wnt signaling. The decrease in progesterone at the time of parturition and increases in oxytocin and prolactin are stimulatory for milk production. Although there are species differences, protein hormones, including growth hormone (GH), prolactin, and insulin-like growth factor (IGF)1, also play roles in growth and development. In mice, and likely most species, prolactin and GH are critical for lobuloalveolar development because lobuloalveolar development is deficient and impaired in mice lacking prolactin or GH

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receptors. In young rodents, prolactin and GH can, with high enough levels, stimulate some growth without steroids. Both EGFR and ErbB2 are functionally activated in mouse mammary tissue at puberty, and elimination of Erb2 in the virgin mammary gland resulted in structural alterations in the terminal end buds and delayed ductal growth during puberty and adolescence. In 8-week-old mice, ErbB2 is especially prominent in the epithelium and reduced in the stroma, whereas EGFR is localized to the stroma. One of the EGFR ligands, amphiregulin, is specifically isolated to the terminal end bud cells. There are numerous other factors that interact and affect the cell biology of the mammary gland. Interestingly, the Vitamin D receptor (VDR) serves as a negative growth regulator during mammary gland development via suppression of branching morphogenesis during puberty and modulation of differentiation and apoptosis during pregnancy, lactation, and involution. The many matrix metalloproteinases (MMPs) and cell adhesion molecules also modulate both negative and positive growth. For example, during puberty and pregnancy, MMP2 is necessary for normal terminal end and lateral ductal branching and extensive branching in mid-pregnancy, while MMP-3 is required for secondary and tertiary lateral branching of ducts. Studies with hormone receptor-deficient knockout mice reveal critical roles for receptormediated pathways in mammary gland development (Table 61.1).

2.4. Mammary Gland Pathobiology and Cancer There are some key aspects concerning mammary cancer in animals and humans that need to be understood in reference to mammary gland development and cell biology, and are particularly relevant to toxicologists. Breast cancer remains the most common invasive cancer and the leading cause of cancer-related mortality in women. As such, there remains heightened concern about the potential causes and risk factors, including those from environmental exposures, drugs, or dietary intake, that may contribute to the development or progression of this deadly disease. It is well supported that lifetime exposure to estrogens (early age of menarche, older age at menopause, nulliparity,

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unopposed estrogen) and genetic factors (family history of breast/ovarian cancer) are major factors that increase risk for breast cancer, and early age of first pregnancy (< 22 years old) is associated with decreased risk. Thus, exposure of breast tissue to exogenous or unopposed endogenous estrogens and estrogenic compounds is clearly linked to the development of breast cancer in women, while pregnancy is protective. Estrogen treatment alone, or combined with progestins in hormone-replacement therapies, also increased the risk for breast cancer in menopausal women. This interplay of reproductive and hormonal influences generally holds across species. For example, mammary cancer is the most common malignant cancer in intact bitches greater than 6 years old. Neutering prior to first estrus essentially negates the risk, while risk increases with neutering after consecutive cycles. In Old World macaques, the role of reproductive status is not clearly defined, but mammary gland biology and breast cancer development are generally comparable to those in women. In a population of captive macaques, the lifetime risk for breast cancer was estimated to be about 6% and considered comparable to women. The types of cancer, intraductular carcinomas and invasive and metastatic cancers, are also morphologically similar to those in women. Importantly, the role of estrogens and progestins in mammary proliferation is also comparable in macaques and women. In experimental studies in castrated cynomolgus and rhesus macaques, estrogen treatments alone stimulated epithelial proliferation, progesterone treatments alone had negligible effects, while both estrogen and progesterone treatment together stimulated proliferation exceeding that of estrogen alone. Hormonal and reproductive status and genetics also have significant effects on the development of mammary cancer in rodents. Mammary cancer can be induced with estrogen treatment in both rats and mice, and incidence of mammary cancer increases with increasing age in many strains of rats. Rising prolactin levels in aging rats have been associated with increases in both fibroadenomas and adenocarcinomas, with such mechanisms thought to be irrelevant to mechanisms of breast cancer in women. However, recent studies show a greater importance of prolactin in promoting breast

cancer in women, and therapies currently developed to inhibit prolactin-mediating signaling pathways show some efficacy. In rats and women, pregnancy is protective, although pregnancy does not have the same protective effect in mice. Mice also deviate from rats, monkeys, and women in that the most common types of mammary cancer in mice are alveololobular, and are non-invasive and not metastatic. In contrast, mammary cancers in women, monkeys, dogs, and rats are ductular, derived from the most primitive epithelial cells of the terminal ductular units, and are invasive and malignant.

3. EVALUATION OF TOXICITY Bioassays remain the mainstay of toxicity testing. However, traditional bioassays have limitations with respect to detection of mammary gland toxicants and carcinogens. For example, interpretation of an “effect” can be complicated by the strain susceptibility and high incidence of spontaneous mammary tumors in strains typically used. Species used in the National Toxicology Program (NTP) bioassays in past years, F344 female nulliparous rats, have less robust reproductive hormonal patterns, which certainly influences mammary gland morphology and sensitivity to test compounds. Current NTP studies use a rat strain (Harlan Sprague Dawley) that demonstrates a normal reproductive profile and has a low background mammary tumor rate. In fact, in reproductive assessment studies the NTP uses different strains of rats and mice such as Sprague-Dawley and CD-1 or Swiss mice, respectively, in part due to the poor performance of the F344 in breeding studies. Industry and European-based assessments typically rely on Charles River CD-1 Sprague-Dawley, Harlan Sprague-Dawley or Wistar rats, and CD1 mice. It is also important to consider that the windows of mammary gland susceptibility or mammary gland sensitivity are missed when exposure starts in adult nulliparous rodents, as is typical in bioassays. Thus, it is possible that potential mammary gland toxicants and carcinogens may have been missed. Additionally, there has always been concern that use of maximum-tolerated dose-based exposures may not be a relevant testing condition or produce realistic results. With regard to the guidelines for mammary gland studies (see Suggested Reading

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list; Makris 2011), there are scant requirements for mammary gland assessments, and study designs for carcinogenicity studies performed for biopharmaceutical agents are often still performed in adult nulliparous rodents. However, following an NTP-sponsored expert panel workshop, and with the accumulation of data linking early exposures with later-life mammary gland lesions, recent NTP carcinogenicity bioassays have incorporated early-life or multigenerational exposures along with morphologic evaluation of the mammary gland in the form of mammary gland whole mounts and advanced sectioning techniques such as those demonstrated in Figure 61.5. Moreover, as our understanding of how endocrine-disrupting compounds mediate their effects on cells, there has also been a greater emphasis

FIGURE 61.5 Mammary gland, rat, H&E stain, prepared in (A) the classical method (cross-section or transverse) and (B) the more contemporary and revealing frontal (coronal) method of sectioning. Classical cross-sections contained skin, but very little mammary tissue was available for evaluation. Using the more contemporary frontal sectioning, a much greater area of the mammary epithelia can be evaluated and hyperplastic lesions such as ductal bridging would be apparent. This method is also able to be compared to whole mount preparations of the tissue from the contralateral gland. Photographs submitted by J. R. Latendresse, Toxicologic Pathology Associates, National Center for Toxicological Research, Jefferson, Arkansas, USA.

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on using environmentally relevant dose (ERD) in place of MTD.

3.1. Physiologic Evaluation The most commonly described mammary gland toxicities have their foundation in alterations of the normal physiology of the mammary gland that result in structural alterations, from malformations to cancer. Strong experimental data suggest that chemicals, drugs, and dietary substances can interfere with branching and differentiation of mammary glands, resulting in either delayed or accelerated maturation, and transiently or permanently altering the morphology. There is also strong evidence demonstrating that the cells within terminal end buds are most susceptible or vulnerable to toxic or carcinogenic insult. Thus, conditions that alter the development and timing of maturation of terminal end buds may have a substantial impact on the health of the gland in adulthood. The susceptibility of the mammary gland to toxicants and carcinogens that perturb the hormonal milieu, or that interfere with the cellular responses to hormones, begets careful evaluation of serum hormones and other hormone-related endpoints such as timing of gonadal control of puberty (vaginal opening, heat, first estrus, etc.), cyclicity patterns, retention of nipples/ areolae in males, and possibly anogenital distance measurements. If alterations in any of these measured parameters are observed in studies, the mammary gland should be recognized as a potential target organ. Several chemicals are known to interfere with the full development of a lactational mammary gland (e.g., dioxin, atrazine, perfluorooctanoic acid). Such an effect should be investigated in reproductive studies reporting decreased postnatal survival, decreased litter weights, or altered growth curves in nursing pups. Nursing pups should have stomachs full of milk. When lactation deficiencies are suspected, functional assessments (often referred to as a “lactational challenge”) of dam–pup interactions should be made. Such evaluations incorporate timed nursing experiments in which dams are separated from pups for 2–8 hours. Immediately prior to reunion, the litter is weighed, the dam is reintroduced for a fixed amount of time (15–30 minutes), nursing behavior is assessed,

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and post-nursing litter weights are measured. The pre- and post-nursing litter weights serve as a surrogate for milk volume. Histological examination of the dams’ mammary glands is essential to help differentiate underlying morphological alterations vs function alterations (or both).

3.2. Biochemical and Biomarker Evaluation Measurements of serum protein and steroid hormones involved in reproduction are often used as biomarkers in assessing toxicity in the female. However, use of these as biomarkers specifically in relation to potential mammary gland toxicity may not be informative without correlative morphologic evaluation and, in the adult, comparison within the same stage of the cycle. Moreover, significant morphologic changes can occur in the gland early in development which may not be associated with altered circulating hormones in the adult. In tissues, measurements of cellular receptor levels, activation of receptors and their signaling pathways, and cell proliferation markers are typically regarded as sensitive biomarkers of toxicity. However, due to the complexity of the mammary tissue, it is often helpful or required to know the cell type that is conferring the noted change in the biomarker. For example, increases in epithelial density and cell proliferation in early development have been correlated with development of cancers later in life in both rodents and non-human primates. Thus, morphometric analysis of glands, coupled with immunohistochemical staining with PCNA or Ki67 cell proliferation markers, provides accurate and sensitive markers of mammary gland changes. Receptor levels or pathway activation can also be evaluated through immunohistochemical markers or molecular techniques, using the whole gland. Further analyses can be performed on digested and cell-sorted tissue samples, with increased time and cost investment. Lactational effects can be measured through surrogate analysis of pup or analysis of mammary gland sections of the exposed dam. Milk protein measurements from collected milk samples (collected at more than one time point, if possible) may provide valuable biomarkers of effect without having to sacrifice the animal. Lipid profiles, protein content, and other

nutritional information may be collected using expressed milk samples. The pioneering work of Cline with non-human primates (see Suggested Reading), among other critical contributions, illustrates the utility of longitudinal biopsies in studies with non-human primates to model mammographic studies in women. Temporal biopsies in non-human primates add the power of analyzing changes over time without, importantly, the need for sacrifice.

3.3. Morphologic Evaluation Thorough morphological evaluation of mammary glands incorporates routine assessment in histological hematoxylin and eosin (H&E) sections, quantitative morphometric analysis, and morphological evaluation of whole mounted mammary glands. Histological Assessment Routine histological assessment at the end of the study has been the mainstay in toxicological studies. In rodent studies all gross lesions are collected, and, routinely, the fifth mammary glands are dissected with or without skin from the same side in both sexes. Figure 61.5 demonstrates the major advantage of using the fourth and fifth glands of the rodent, cut in longitudinal sections, allowing a greater chance of detecting a lesion if it exists in the mammary tissue. This more contemporary method has recently been integrated into study designs of ongoing research at the National Toxicology Program (NIEHS, NIH) and National Center for Toxicological Research (ATSDR, FDA). Sampling in dogs includes the nipple and surrounding gland. More extensive collection is needed for non-human primates, as mammary epithelial growth extends far into the fat. Glands are placed on fiberboard or other material to fix them flat, and processed through formalin fixation to a 5-mm section on a glass slide stained with hematoxylin and eosin. Mammary glands from both sexes should be evaluated with respect to the normal sexual dimorphism between male and female rats. Most male mice lack mammary epithelium, and thus would not be evaluated unless nipple/areolae retention is noted early in life. Evaluation includes examination of a representative section for nonneoplastic and neoplastic changes. Diagnostic

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criteria and nomenclature are summarized in Section 4.3, on morphologic response to injury. Morphometric Analysis Increasingly, qualitative assessments are coupled with semi-quantitative morphometric analysis. Parameters evaluated include length of the ductal tree along the longitudinal axis, total area occupied by epithelial ducts, branching density, and total number and size of terminal end buds, all relative to the total area of mammary epithelium and total area of the fat pad. These parameters can be measured in the routine sections using image analysis software systems or with eyepiece reticules. These measurements on H&E sections are, of course, limited to the particular section(s) processed, and may not be representative of the gland, depending on the number and location of sections. Immunohistochemistry endpoints including cell proliferation and apoptosis measurements are similarly quantified with respect to location of cells (epithelial, myoepithelial, mesenchymal, ductal, bud, alveoli, and fat pad) and total number of cells evaluated. To date there does not seem to be a standard statistically based analysis of the number of sections that need to be evaluated or number of objects that need counted and power calculations, thus requiring such calculations on an individual study basis. Whole Mount Preparations While histologists and pathologists are wellversed in light-microscopic techniques and evaluation, whole mount preparation (the entire fourth and fifth gland mounted onto a slide) and evaluation is not yet a routine procedure in all laboratories. However, evaluation of mammary gland morphology in whole mounts allows for a better or more sensitive assessment of the branching complexities and glandular densities. Whole mounts are also more amenable to quantitative assessments of terminal end bud, alveolar buds, duct and branching development, and growth into the fat pad, because the entire gland is represented. As this technique is increasingly being used and incorporated into NTP study requirements, one of the goals in this chapter is to provide methods for whole mount preparation and assessment based on previously published peer-reviewed manuscripts (see

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Suggested Reading). Processing mammary gland for whole mounts requires some specialized techniques and reagents, detailed in the Appendix to this chapter. The whole mount should be prepared from the contralateral gland processed for standard H&E slide examination. Note, however, that masses or questionable lesions observed in whole mounts can be excised and reprocessed through paraffin for H&E microscopic examination (Figure 61.6). As in all assessments of the mammary gland, important factors to consider are the age of the animal and expected developmental maturity of the gland, species, strain, stage of cycle, pregnancy, lactation, or involution. Suggested age-specific factors to use in the evaluation of rat mammary tissues are listed in Table 61.2. Glands should be examined under a dissecting microscope first from control animals examined as a group to establish base-line morphology, such as is customarily done in scoring histopathological lesion severity. Then, in blinded fashion, mammary glands can be scored according to the criteria detailed in Table 61.2. If a chemical is known to have an estrogen-like effect in other tissues or cell studies, an ethinyl estradiol control may be used, but often the effects of a test compound on the gland are not known and opposite effects of estrogens may be detected (e.g., dioxin, perfluorooctanoic acid). As detailed in Table 61.2, key parameters to evaluate include: 1. The number of TEBs relative to the number of duct ends and the morphology of the terminal end buds; TEBs in a rat are end structures greater than 100 mm wide, and typically in the mouse TEBs are denoted as ends measuring equal to or greater than twice the duct width; 2. The degree of branching and amount of mammary tissue present, including the degree of lateral (side) branching and/or budding (branch density) and the number of primary ducts growing from the nipple (particularly in PND4 animals; this may not be possible in older animals); 3. The degree (relative amount compared to control samples) of alveolar bud and lobule formation; and 4. Growth of the gland, both length- and widthwise (Figure 61.5).

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FIGURE 61.6 (A) Rat mammary gland 5, whole mount preparation indicated a mass (arrows); Carmine-stained, 10. (B) Excised mass was reprocessed for histological examination and diagnosed as fibroadenoma. H&E stain, 100.

3.4. In Vitro Techniques A variety of in vitro techniques are useful as screening assays as well as in mechanistic studies. Screening assays are used to identify chemicals that may have endocrine active characteristics and thereby have the potential to cause mammary gland toxicities. The idea that chemicals can be screened for estrogenic activity in an in vitro assay was validated by Soto and Sonnescheim and colleagues describing the EScreen assay. This assay takes advantage of endogenous estrogen receptor-mediated events in breast epithelial cancer MCF7 cells. Briefly, MCF7 cells are plated in phenol red free growth medium (DMEM), then grown in charcoaldextran stripped 10% fetal bovine serum and with various (10-nm to 10-mm) concentrations of compounds of interest. Cell density depends on plate size – 24 or 96 wells can be used. After 3 days, levels of ER-dependent gene expression, such as pS2 and progesterone receptor, can be measured. After 6 days, cell proliferation can be assessed. Estradiol is used as a positive control in these assays. An “A-screen” has been similarly developed for assessing androgen receptor activity using prostate carcinoma or MCF7 cells transfected with androgen receptor.

Yeast-based steroid hormone receptor gene transcription assays and in silico ligand-dependent reporter assays are also used in drug screening, drug development, and toxicology studies. Compounds showing activity in these screening assays should be considered as potential mammary gland toxicants. Cell culture experiments have provided important mechanistic knowledge of mammary gland biology and cancer. Neoplastic MCF7 and nonneoplastic MCF10 have been widely used, although there are many well-characterized mammary (breast) epithelial cells available. Another important and increasingly popular in vitro system is the three-dimensional (3D) mammary gland culture. 3D cultures were originally described by the Bissell Lab using MCF10A cells first suspended in medium containing 2% MatrigelÔ and then plated on a solid layer of Matrigel. Cultures are typically maintained for 10 days. When mammary epithelial cells (such as MCF10A cells) are cultured on laminin-rich extracellular matrix, they produce a basement membrane and arrange themselves in spherical acini with a centrally located lumen. Modulation of genes or stromal elements can affect the formation of the acini. For example,

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TABLE 61.2

Score

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Developmental Scoring System Used for Morphological Assessment of Mammary Gland Whole Mounts Following Early-Life Chemical Exposures A score of 4 represents a gland that is most well developed, while a score of 1 represents few of the necessary criteria needed Developmental criteria

PND 4 1

Large lateral branches off primary branches only, few ductal buds from lateral branches, undersized, one or few primary ducts, primary ducts lack buds

2

Small buds formed on primary branches and lateral branches, few branches, more than one primary duct with few buds

3

Good growth pattern with score of 1 and 2 surpassed, small terminal buds, moderate branching with moderate budding on both lateral and primary ducts

4

Growth exceeding above criteria is evident, distended terminal ends and abundant branching pattern, extensive budding on primary and secondary ducts, excellent growth

PND 22e28 1

Poor structure, clearly underdeveloped, no terminal end buds (TEBs) present (only terminal ducts), few branches off lateral ducts

2

Normal structure, few buds, few lateral branches, TEBs distended, many lateral branches on terminal ducts, but sparse in appearance (larger ducts may have few lateral branches)

3

Good branching, buds present throughout, large TEBs on all ducts and branches, all TEBs and terminal ducts distended

4

Generous branching, buds and TEBs on ducts, excellent migration and gland size, lobule formation starting throughout gland

PND 33 1

TEBs remain on all sides, two glands not grown together, growth pattern and differentiation impaired, no or few lobules apparent

2

TEBs on two or three sides, glands close together, growth impaired and sparse branching, lobule buds barely evident

3

Glands close together or touching, TEBs only on distal ends, branching sparse, one gland with lobules, one not, or half and half

4

Glands grown together, few TEBs remain, dense branching, lobules visible emulti-unit

PND 40e45 1

TEBs on both ends, sparse branching, limited growth pattern, glands not touching

2

TEBs on both ends, moderate growth, limited lobule development

3

TEBs on long end (MG#4), dense and large, lobules apparent throughout

4

Few to no TEBs remain, very large, dense branching, multi-unit lobules developed

PND 67e90 1

TEBs still present, epithelium doesn’t fill fat pad, sparse branching throughout, lobules evident

2

No TEBs evident, sparsely branched with many large lobuloalveolar units, may not fill fat pad

3

Moderate size, moderate filling and branching, some areas beginning to differentiate into static state, moderate lobules

4

Few complex lobules, dense branching throughout gland with buds on ducts, smalleno lobules

* The score criteria presented in the table above are for female Long-Evans rats of the indicated ages. The criteria may be used to detect delayed development following chemical exposures early in life. They may be used for other rat strains, mice, or males of any age, species, or strain with minor modifications.

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overexpression of oncogenes and anti-apoptotic genes in the epithelial cells in this culture system produces multiacinar structures lined by piles of cells with no lumen, characteristic of carcinoma in situ. Overexpression of extracellular matrix proteins can produce similar morphologies in epithelial cells. Methods have evolved that included various matrices and non-neoplastic and neoplastic epithelial cells, including cells derived from human breast. Cells can be grown on top of the matrix for easier imaging studies and shorter cultures. Such 3D cultures provide an advantage in toxicology studies by providing capabilities to assess both morphological and molecular changes, and may demonstrate relevant cell-type interactions.

3.5. Use of Animal Models Although there are developmental and biological differences between various species of animals and humans, modeling effects of chemicals in animals has proved necessary and critical to advance our understanding of mammary gland biology, response to injury, and cancer. As we’ve learned more about underlying mechanisms of toxicity and carcinogenicity, the types of animal models and the approaches to assessing toxicity have evolved. Spontaneous Models Chronic bioassays conducted by industry and agencies, such as the National Toxicology Program (NTP), remain a standard approach for hazard identification. Bioassays conducted by the NTP have historically relied on B6C3/F1 mice and F344 inbred rats exposed up to maximally tolerated doses (MTD) from sexual maturity (6–8 weeks old) up to 2 years. Industry-sponsored studies more often use CD1 mice and CD1 Sprague-Dawley, Harlan Sprague-Dawley, or Wistar rats. These different strains of animals have different sensitivities and tumor susceptibilities, which all need to be taken into account in study design and study interpretation. Mammary gland fibroadenomas are the most common spontaneous tumor in female Sprague-Dawley rats, with incidences reported as high at 70% in chronic studies. Female Fischer rats have reported incidences of about 40%. To note, fibroadenomas are not considered a premalignant lesion in humans, and neither are

rat mammary fibroadenomas considered predictive of carcinoma in women. Spontaneous mammary adenocarcinomas, which are considered relevant in studies, are more common in Sprague-Dawley rats, with reported incidences of 11% in Harlan Sprague-Dawley rat. However, Fisher 334 rats are relatively resistant to spontaneous mammary adenocarcinomas and, as a consequence, chemicals that induce adenocarcinomas in Fisher 344 rats are readily distinguishable from the background mammary gland fibroadenomas that develop spontaneously. Mice, as compared to rats, have a lower background incidence of mammary tumors. B6C3F1 mice are relatively resistant to spontaneous mammary gland tumor development, and the parental strains, C3H/He and C57BL/6, are relatively resistant to spontaneous and carcinogen-induced mammary gland tumorigenesis. Chemical Carcinogenesis (See also Carcinogenesis: Mechanisms and Manifestations, Chapter 5.) Genetic susceptibilities of the various species and strains of rodents have been exploited in development of animal models for mammary gland cancer. For example, spontaneous mammary gland cancers in rats, generally adenocarcinomas, can be enhanced by treatment with genotoxic carcinogens like 12-dimethylbenz[a] anthracene (DMBA), N-nitrosomethylurea (NMU), and ENU, or by irradiation. These induced mammary tumors are hormone-dependent and can be modulated by a number of factors, including reproductive history, estrogen treatment, diet, and dose and timing of carcinogen administration. Postpubertal administration of DMBA is most effective in enhancing mammary cancer development, while prepubertal administration of NMU is most effective in enhancing mammary cancer development. Part of the agesusceptibility pattern to DMBA is attributed to the development of the enzymes necessary to metabolize DMBA to its carcinogenic form. The prepubertal effects of NMU, on the other hand, are attributed to the deficiency of a DNA repair enzyme in the immature rat gland and the induction of H-ras mutations in the mammary epithelial cells. Also, the mutagenic effects of NMU are not dependent on metabolism. Irradiation is most effective when administered in the

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postpubertal period and enhanced by short-term estrogen treatment during this time. The coadministration of these cancer-inducing agents with a test article of interest is a commonly used and well-accepted method in toxicology studies in academic laboratories. Genetically Engineered Mice (GEM) (See also Genetically Engineered Animals in Product Discovery and Development, Chapter 12.) Mice, which tend to have a lower incidence of spontaneous mammary cancers that rarely are malignant, have nonetheless been exploited as models for breast cancer. Historically, it was discovered that infection with lactationally transmitted mouse mammary tumor virus (MMTV) caused hyperplastic lesions and mammary tumors through activation of Wnt, FGF, and notch signaling pathways that are also critical in early mammary gland morphogenesis. Hyperplastic lesions and spontaneous tumors that develop in naturally MMTV-infected mice are characteristic of well-differentiated alveolar nodules (HANS) and alveolar tumors. Some have promoted these lesions to represent preneoplastic lesions comparable to such lesions in women’s breast tissue, but others maintain that the biology and morphology of these hyperplasias and benign tumors are features that should be considered mouse-specific. Nonetheless, MMTV has also been exploited in the development of many GEM models of mammary cancer by targeting and driving gene expression using MMTV as a promoter. Another commonly used promoter is the whey acidic protein (WAP) promoter. A tremendous effort has gone into characterizing GEM to bridge morphologic and genotypic variations of mouse mammary cancer to breast cancers in women. More than 40 models have been classified and categorized according to (1) lesions that resemble spontaneous mouse mammary tumors; (2) lesions that are unique and specific for the transgene; and (3) lesions that resemble human breast lesions. However, for most models and transgenes tested there is limited evidence that these show malignant characteristics and they are not transplantable or immortal. Nonetheless, these models remain informative, and recent characterizations further support their importance in understanding mammary gland biology and cancer.

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In summary, the use of animal models (especially rodents) has demonstrated three critical factors that it would have taken decades to discern in humans. First, sensitive life stages (especially fetal development, puberty, and pregnancy) impart a unique sensitivity to some chemical exposures leading to later-life disease risk. Second, toxicants can act by mechanisms other than as frank carcinogens (i.e., as endocrine disruptors) to confer an increased risk of cancer or other later-life effect, such as insufficient lactation. The use of transgenic, knockout, and other gene-modified rodents (primarily mice) has identified highly important details of mechanisms in disease development and progression. Finally, the non-human primate is an excellent model to understand pleiotropic effects of toxicants or drugs on mammary gland development as well as carcinogenic potential. It is often overlooked because of costs or availability.

4. RESPONSE TO INJURY 4.1. Physiologic Response to Injury The most recent emphasis in toxicology has been placed on understanding the direct or modifying effects of endocrine-disrupting compounds (see Endocrine Disruptors, Chapter 37). These are often reported as causing either accelerated maturation or delayed differentiation of the gland in females (Figure 61.7), or in altering glands in the male from a lobular phenotype to a more female-like ductular morphology. Retained nipples in male rodents are also characteristic of anti-androgen effects. Accelerated mammary growth may be characterized as increased terminal end bud formation at early time points and decreased numbers at later time periods. Delayed maturation may be characterized by the presence of terminal end buds until much later in development, extending the window of time that these carcinogensensitive structures are present in the gland. Inhibition of development may be reflected as a decrease in number of ductal branches or branching density of the gland, as well as an overall decrease in terminal end buds formed. Ductal hyperplasias and dysplasias can be induced with various estrogenic-like compounds, but, as mentioned previously, many of these

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FIGURE 61.7 Stages of mammary gland development in the rat; normal (top) and altered (bottom). Numerous examples of early-life endocrine disruptor effects on mammary gland development have been associated with later-life adverse outcomes such as altered breast developmental timing during puberty, insufficient lactation or increased risk for mammary tumors. Figure adapted from Rudel et al. (2011) Environmental exposures and mammary gland development: state of the science, public health implications, and research recommendations, Environ. Health Perspect. 119, 1053–1061, with permission.

effects depend on timing of exposure and the extent of mammary gland development within the animal of interest. For example, exposure to estrogenic compounds through the perinatal period in rats, when terminal end buds are developing, is associated with hyperplasia of the buds, while exposure during the peripubertal period, when ducts are developing, is associated with ductal hyperplasia. Neonatal exposure to estrogen, progesterone, or both in mice causes irreversible effects in adults, including secretory stimulation, dilated ducts, and abnormal lobuloalveolar development. Phytoestrogens, such as genistein, and resveratrol, and the mycoestrogen zearalenone act similarly to estrogen agonists in their effects on the gland. Changes include delayed development, ductal hyperplasia, alveolar hypoplasia, reduced apoptosis in TEBs, increased or decreased numbers of terminal ducts or lobules, and accelerated

alveolar differentiation, again depending on time of exposure. Altered mammary gland development following perinatal exposure has also been observed for other endocrine-disrupting compounds, including atrazine, bisphenol A (BPA), dibutylphthalate, dioxin, methoxychlor, nonylphenol, polybrominated diphenyl ethers, and PFOA. Some of these compounds, such as methoxychlor, act as estrogen agonists, but most of them have pleomorphic effects on hormone receptors or hormone signaling in many tissues; thus, correlating a specific physiological and morphological response to classes or specific compounds is certainly complex. In contrast, systemic hormonal changes and correlative mammary morphologies related to spontaneous aging and testing of pharmaceutical-based hormone receptor-specific agonists and antagonists have been nicely characterized

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in rats (see Lucas and colleagues, in Suggested Reading). Common changes in the aging adult male rat include a tubular alveolar pattern with formation of central lumens as opposed to lobular alveolar with formation of acini and few ducts. This is occasionally referred to as mammary gland “feminization,” and is attributed to increased prolactin and growth hormone levels. Because both these hormones increase in aging rats, particularly strains with a high incidence of pituitary tumors, such changes in male rat morphology can be an indirect effect of a treatment as well as represent an adverse effect of endocrine disruption. In the adult female rat mammary gland, lobuloalveolar hyperplasias with or without ductal ectasia and secretory activity are associated with increased levels of circulating prolactin, growth hormone, or estrogen levels often associated with endocrine disruptors. Lobuloalveolar morphology, sometimes referred to as “virilization” in the female gland, occurs with androgen stimulation or higher levels of circulating testosterone. The varied effects of pharmaceutical agents are consistent and predictable. For example, Era agonists cause lobuloalveolar hyperplasia and secretory activity in female rats, and feminization of the mammary gland in male rats. AR agonists have no effect in the male, but will cause virilization and increased secretions in the female rat. In contrast, AR antagonists will cause atrophy of the male rat mammary gland and have no effect on the female rat mammary gland. PR antagonists cause lobuloalveolar hyperplasia and secretions in female rat mammary glands, but have no effect on male rat mammary gland. Importantly, dopamine receptor antagonists, which stimulate prolactin secretion, will cause hyperplasia in female and feminization in male rat mammary glands – an effect often seen in aging rats with elevated prolactin levels. Thus, when such morphologies are observed in the mammary gland of rats in study, careful consideration should be given to determining hormonal effects as well as the potential for direct effects on mammary gland development.

4.2. Molecular and Biochemical Response to Injury At the biochemical and molecular levels, complex and varied responses occur after injury.

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Molecular signaling through hormone and growth-factor receptors is altered by changes in hormone receptor expression, receptor levels, receptor affinity to ligands, or receptor localization. These in turn are further altered by production of local growth factors and hormones as well as genetic mutations that result from injury. To date, a few efforts are underway to characterize molecular signatures of chemically induced mammary lesions and cancers from spontaneous lesions. For example, gene expression profiles from chemically induced mammary gland cancers in Sprague-Dawley rats show distinct differences from spontaneous mammary tumors. Compared to spontaneous carcinomas, carcinomas induced by 2-amino-1-methyl-6phenylimidazo[4,5-b]pyridine (PhIP), DMBA, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), NMU, or 4-aminobiphenyl (4ABP) show higher expression of genes associated with mammary epithelial cell growth and proliferation, such as cyclin D1 and PDGF-a, and relatively lower expression of differentiation marker genes, such as b-casein, whey acidic protein, and transferrin. Additionally, several components of the prolactin/prolactin receptor/Stat5a/cyclin D1 signaling pathways are found in the chemically induced rat mammary gland carcinomas. Mammary cancer in DMBA-treated FVB mice shows elevated expression of the aryl hydrocarbon receptor (AhR), c-myc, cyclin D1, and hyperphosphorylated retinoblastoma (Rb) protein compared to normal mammary gland tissue. Mammary cancer associated with benzene and ethylene oxide exposure to mice had increased mutations in Tp53 protein and Hras mutations in a chemically related pattern distinguishable from spontaneous mutations. However, for most of the compounds associated with mammary gland injury and dysmorphogenesis, the molecular pathways remain to be defined.

4.3. Morphologic Response to Injury The response of the mammary gland to injury recapitulates a wide spectrum of non-neoplastic and neoplastic changes. It is essential to adopt standardized nomenclature in order to provide consistency of diagnoses across studies, and capture and identify patterns of lesions that

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represent xenobiotic effects with biological significance. This is commonly done for rodents used in toxicity studies, but should be done for all species, including non-human primates – for which there appears to be a propensity for medical pathologists to diagnose malignancy and veterinary pathologists to diagnose benign lesions. Rodents The Mammary Gland Organ Systems Working Group of the International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice has standardized the nomenclature summarized in this chapter for rodents. An important note is that the various strains of mice and rats will have their own classifications of background lesions for which an effect of chemical needs to be evaluated. Historical background incidences from various studies are often available from the supplier or study site. Non-neoplastic changes manifest as degenerative, necrotic, inflammatory, and vascular lesions, or, in relation to alterations in growth, manifest as atrophy, hypertrophy, or hyperplasia. Degenerative changes affecting the epithelial and myoepithelial cells of the ducts and alveoli are most commonly associated with aging or are occasionally observed as a test article effect. The changes are characterized by epithelial vacuolization, loss of cell layers and ductal dilation with accumulation of proteinaceous material. Cellular necrosis within the mammary gland is rarely observed, but fat necrosis and inflammation occur as incidental findings. Regeneration of epithelial cells is usually observed in areas of degeneration as well, and degeneration, necrosis, and regeneration typically present together in repeated mammary gland injury. Inflammation in rodent mammary glands is usually limited to small infiltrates of leukocytes, and should be differentiated from the lymphocytic and eosinophilic infiltrates that accompany ductular morphogenesis. Acute inflammation is characterized by epithelial degeneration, vascular congestion, edema, and an admixture of neutrophils, lymphocytes, and few plasma cells. In chronic inflammation, infiltrates of macrophages and fibrosis will accompany epithelial regeneration, hyperplasia, or metaplasia. Older rats occasionally develop granulomatous inflammation associated with ruptured

galactoceles or dilated/ectatic ducts. Periductular fibrosis is a common age-related change in rats, and has been associated with EGF treatment in mice. Congestion, edema, hemorrhage, and thrombosis are also associated with inflammatory responses. Recent studies have demonstrated the increasing incidence of toxicants affecting the stromal and adipose-rich areas of the mammary gland; specifically, enhanced macrophage infiltration, stromal hyperplasia, and altered fat cell size or number have been noted. Dilation and ectasia or galactoceles of ducts or alveoli with or without epithelial hypertrophy or hyperplasia occur as age-related changes (Figure 61.8). However, ductular ectasia with alveolar epithelial hypertrophy and hyperplasia has been observed as a test article-related effect in younger animals. The lesion may be considered secondary to effects on the hypothalamic– pituitary–ovarian (HPO) axis. Neoplastic changes in rodent mammary glands occur as spontaneous and test article-related benign and malignant tumors. Ductal epithelial atypia and ductal carcinomas in situ (DCIS) are increasingly being recognized in studies, particularly in studies of rats exposed to chemical carcinogens or endocrine disruptors during development. These lesions are characterized using the same criteria as used for human breast. DCIS shows disruption of the epithelial bilayer, and atypical disorganized masses of cells bridge across or fill the ductal lumens. The basement membrane remains intact (Figure 61.9). Benign neoplasms of epithelial origin include adenomas, fibroadenomas, and benign mixed tumors; malignant tumors include adenocarcinomas, adenocarcinomas arising in fibroadenomas, and malignant mixed tumors. In the chemical carcinogen-treated rat model, benign tumors arise from alveolar buds and lobules while carcinomas arise from epithelial cells in less-differentiated terminal end buds and terminal ducts. Adenomas are usually visible as nodules. Histologically, they are characterized as welldemarcated, encapsulated, and expansive masses that compress surrounding normal tissue. They are composed of cysts, alveoli, or papillary fronds of single or multiple layers of epithelial cells aligned on a fine fibrovascular stroma. The epithelial cells are cuboidal, well differentiated, and may show secretory activity. Focal areas of squamous metaplasia may occur. Although there

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FIGURE 61.8 Mammary gland epithelial hyperplasias. H&E stain. (A, B) Young rat treated with an endocrine disruptor. (A) Low magnification of mammary gland with widely and irregularly dilated duct within the fat pad. 20. (B) Higher magnification of (A) to demonstrate clusters of tubuloalveolar formation characteristic of alveolar epithelial hyperplasia. 100. (C, D) Mammary glands showing that the extent of the alveolar hyperplasia can be quite dramatic. 100. Photographs courtesy of D. Rudmann.

are morphological subtypes, it is not standard to subclassify tumors in toxicity studies. Fibroadenomas (see Figure 61.6) represent a proliferation of glandular epithelium surrounded by layers of proliferated fibrous tissue. Fibroadenomas are lobular, well-demarcated, and expansile, with varying proportions of welldifferentiated epithelial tubules or glands and dense fibrous stroma. Mitotic activity is low. Rarely, carcinomas can arise within fibroadenomas and should be diagnosed appropriately as “adenocarcinoma arising in fibroadenoma.”

Benign mixed tumors are rare in rodents but the most common spontaneous tumor in dog. In rodents, these mammary tumors are neoplastic proliferations of both epithelial and myoepithelial cells with differentiation of the latter into islands of cartilage, bone, adipose, or, sometimes, hematopoietic marrow, and diagnostically classified differently from fibroadenomas because benign mixed tumors do not have a predominant fibrous component. The distinction between benign and malignant mixed tumors can be difficult, but typically is based

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FIGURE 61.9 Mammary gland intraductular lesions, rat. H&E stain. (A, B) Ductular atypical proliferation. Ductal epithelial atypia is characterized by proliferation of ductular epithelial cells into single to two layers along a basement membrane. The epithelial cells vary mildly in size and shape, and mitotic figures can be recognized. 200 and 400. (C, D) Ductal carcinomas in situ (DCIS). These lesions are characterized using the same criteria as used for human breast showing disruption of the epithelial bilayer and atypical disorganized masses of cells bridging across the ductal lumens. The basement membrane remains intact. 100 and 400. Photographs (A) and (B) courtesy of D. Rudmann.

on the extent of invasion of the surrounding tissues by the epithelial cells or resemblance of the mesenchymal component to osteosarcoma or chondrosarcoma.

Adenocarcinomas are malignant proliferation of pleomorphic columnar or cuboidal epithelial cells arranged in papillary, cystic, tubular, solid, or cribriform or comedo patterns (Figure 61.10).

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FIGURE 61.10 Mammary gland, rat, with adenocarcinoma. H&E stain. (A) Low magnification of an expansile unencapsulated mass effacing the normal mammary gland. 10. (B) At higher magnification, the mass is composed of malignant proliferation of pleomorphic cuboidal epithelial cells arranged in solid and cribiform patterns. 400.

There may be complete loss of lobular-alveolar structures, and acini may become cystic or blood-filled. Adenocarcinomas can induce a marked schirrous response. These cancers can be locally invasive and are metastatic. There may some squamous differentiation, but this should not compose more than 25% of the lesion. If more than about 25% of the tumor is squamous, then it should be diagnosed as an adenosquamous carcinoma, more commonly seen in mice than in rats. Non-Human Primates Non-neoplastic lesions manifest as cystic changes in lobules and ducts, and apocrine metaplasia with alveolar dilation and secretory changes resembling apocrine sweat glands. These are generally incidental, and related to aging and reproductive status. Hyperplastic lesions are focal and multifocal lobular proliferations seen in older rhesus, pigtail, and cynomolgus macaques. The lesions are characterized as enlarged or distinct nodules of well-differentiated alveoli, but proliferate independently of hormonal stimulation. They can be found within generalized lobular hyperplasia of lactation or estrogen and progestogen treatment. Ductal hyperplasias are characterized

by focal increased epithelial cells into two to three layers, with maintenance of polarity and size. These are common spontaneous lesions in intact middle-aged rhesus macaques. Ductal carcinoma in situ is distinguished from hyperplasias by increased layers of disorganized, pleomorphic epithelial cells that bridge and occlude the lumens of tubules. The basement membrane remains intact. Loss of basement membrane is diagnostic of intraductular carcinomas, which can be invasive and metastatic. Ductal carcinomas in situ occur commonly in mammary gland of cynomolgus and rhesus macaques, but development of larger carcinomas is not common. Malignant cancers include adenocarcinomas, most often of the comedo carcinoma type.

5. MECHANISMS OF TOXICITY Mammary gland toxicity and carcinogenicity have been observed in animal models exposed to a wide variety of agents, including estrogens, androgens, anti-androgens, and thyroid-active chemicals, and aryl-hydrocarbon receptor agonists, genotoxic compounds, and mutagens. However, the mechanisms of toxicity and carcinogenicity are generally unknown. A number of the

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rodent mammary gland carcinogens are epoxides or epoxide metabolites. These include such chemicals as 1,3-butadiene and related chloroprene and isoprene, which are metabolized to epoxides. These epoxides are mutagens and are associated with K-ras mutations in rodents. Isoprene and 1,3-butadiene also cause chromosomal aberrations in rodent bone marrow cells. Although the mechanisms related to epoxide-induced mammary cancer are unknown, one hypothesis is that the mammary gland is efficient in metabolizing the chemicals to their epoxides. Brominated species are also associated with rodent mammary cancers. Chemicals, such as 2,2-bis(bromomethyl)-1,3- propanediol are proposed to act through either oxidative damage or formation of DNA adducts and then DNA damage. Many of these chemicals also induce cytochrome P450 metabolizing enzymes, which, like DMBA, can activate chemical reactivity or be metabolized to electrophilic oxygenated species that bind DNA. It has been proposed that halogenated mammary gland carcinogens, which tend to be lipophilic, accumulate in breast fat, resulting in exposures for prolonged periods of time. Atrazine, brominated diphenyl ethers, and dioxin have all been shown to induce a delayed mammary gland development following neonatal exposures through interactions with hormone responses. At the molecular level, in utero exposure of mice to DES or BPA shows that such estrogenic compounds increase protein expression and functional activity of the histone methyltransferases, which have been linked to breast cancer risk and epigenetic regulation of tumorigenesis. It is also common for endocrine-disrupting compounds to act via indirect mechanisms to induce persistent effects in mammary tissue. Furthermore, recent data suggest that epigenetic mechanisms – those changes to DNA that cause a heritable modification without changing the DNA code – may have important roles not only in mammary cancer but also in many other cancers over the lifetime.

6. CONCLUSION The high incidence of breast cancer in women has brought attention to the need to incorporate appropriate and sensitive methodologies with

which to evaluate the mammary gland in animal models as applicable to hazard identification for humans. We now have a substantial number of genetically modified and spontaneously occurring mice and rat mammary cancer models to employ, and various approaches to assess gland morphology. Evaluation of mammary tissue in studies with non-human primates can also serve as an important tool. Of the lessons learned, we know that hormonal perturbations, including chemicals that act as endocrine disruptors, pose a significant risk, and that such risk may be increased when exposures occur during development. Given this information, animal studies designed for hazard identification should include exposures during development and enhanced methods for histological and morphometric mammary gland evaluation, such as whole mount evaluations. In vitro screens also continue to serve as important tools for identifying potential hazards and supporting mechanistic understanding of effects. Indeed, discovering genetic and environmental causes of breast cancer goes hand-in-hand with discoveries of therapies to treat breast cancer and strategies to prevent breast cancer.

SUGGESTED READING Mammary Gland Development and Biology Cardy, R.H., 1991. Sexual Dimorphism of the Normal Rat Mammary Gland. Vet. Pathol. Online 28 (2), 139–145. Chandra, S.A., Mark Cline, J., Adler, R.R., 2010. Cyclic Morphological Changes in the Beagle Mammary Gland. Toxicol. Pathol. 38 (6), 969–983. Cline, J.M., Wood, C.E., 2008. The Mammary Glands of Macaques. Toxicol. Pathol. 36 (Suppl. 7), 130S–141S. Cooper, T.K., Gabrielson, K.L., 2007. Spontaneous lesions in the reproductive tract and mammary gland of female nonhuman primates. Birth Defects Res. B Dev. Reprod. Toxicol. 80 (2), 149–170. Jo¨chle, W., Andersen, A.C., 1977. The estrous cycle in the dog: A review. Theriogenology 7 (3), 113–140. Lamote, I., Meyer, E., Massart-Leen, A.M., Burvenich, C., 2004. Sex steroids and growth factors in the regulation of mammary gland proliferation, differentiation, and involution. Steroids 69 (3), 145–159. Malhotra, G.K., Zhao, X., Band, H., Band, V., 2010. Histological, molecular and functional subtypes of breast cancers. Cancer Biol. Ther. 10 (10), 955–960. Masso-Welch, P.A., Darcy, K.M., Stangle-Castor, N.C., Ip, M.M., 2000. A Developmental Atlas of Rat Mammary

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Gland Histology. J. Mammary Gland Biol. Neoplasia 5 (2), 165–185. Rehm, S., Stanislaus, D.J., Williams, A.M., 2007. Estrous cycledependent histology and review of sex steroid receptor expression in dog reproductive tissues and mammary gland and associated hormone levels. Birth Defects Res. B Dev. Reprod. Toxicol. 80 (3), 233–245. Rudland, P.S., Barraclough, R., Fernig, D.G., Smith, J.A., 1998. Growth and differentiation of the normal mammary gland and its tumours. Biochem. Soc. Symp. 63, 1–20. Rudmann, D.G., Cohen, I.R., Robbins, M.R., Coutant, D.E., Henck, J.W., 2005. Androgen Dependent Mammary Gland Virilism in Rats Given the Selective Estrogen Receptor Modulator LY2066948 Hydrochloride. Toxicol. Pathol. 33 (6), 711–719. Russo, J., Russo, I.H., 2004. Development of the human breast. Maturitas 49 (1), 2–15. Sorenmo, K.U., Rasotto, R., Zappulli, V., Goldschmidt, M.H., 2011. Development, Anatomy, Histology, Lymphatic Drainage, Clinical Features, and Cell Differentiation Markers of Canine Mammary Gland Neoplasms. Vet. Pathol. Online 48 (1), 85–97.

Animal Models Haseman, J.K., Hailey, J.R., Morris, R.W., 1998. Spontaneous Neoplasm Incidences in Fischer 344 Rats and B6C3F1 Mice in Two-Year Carcinogenicity Studies: A National Toxicology Program Update. Toxicol. Pathol. 26 (3), 428–441. Johnson, A.N., 1989. Comparative Aspects of Contraceptive Steroids – Effects Observed in Beagle Dogs. Toxicol. Pathol. 17 (2), 389–395. Makris, S.L., 2011. Current assessment of the effects of environmental chemicals on the mammary gland in guideline rodent studies by the US Environmental Protection Agency (U.S. EPA), Organisation for Economic Co-operation and Development (OECD), and National Toxicology Program (NTP). Environ. Health Perspect. 119 (8), 1047–1052. Shan, L., Yu, M., Snyderwine, E.G., 2005. Global Gene Expression Profiling of Chemically Induced Rat Mammary Gland Carcinomas and Adenomas. Toxicol. Pathol. 33 (7), 768–775. Singh, M., McGinley, J.N., 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. Sinkevicius, K.W., Burdette, J.E., Woloszyn, K., Hewitt, S.C., Hamilton, K., Sugg, S.L., Temple, K.A., Wondisford, F.E., Korach, K.S., Woodruff, T.K., Greene, G.L., 2008. An Estrogen Receptor-a Knock-In Mutation Provides Evidence of Ligand-Independent Signaling and Allows Modulation of Ligand-Induced Pathways in Vivo. Endocrinology 149 (6), 2970–2979. Russo, I.H., Russo, J., 1978. Developmental stage of the rat mammary gland as determinant of its susceptibility to 7,12dimethylbenz[a]anthracene. J. Natl. Cancer Inst. 61 (6), 1439–1449.

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Russo, I.H., Russo, J., 1996. Mammary gland neoplasia in long-term rodent studies. Environ. Health Perspect. 104 (9), 938–967. Son, W.-C., Gopinath, C., 2004. Early Occurrence of Spontaneous Tumors in CD-1 Mice and Sprague-Dawley Rats. Toxicol. Pathol. 32 (4), 371–374. Thayer, K.A., Foster, P.M., 2007. Workgroup report: National Toxicology Program workshop on Hormonally Induced Reproductive Tumors – Relevance of Rodent Bioassays. Environ. Health Perspect. 115 (9), 1351–1356.

Genetically Modified Mouse Models Blackshear, P.E., 2001. Genetically Engineered Rodent Models of Mammary Gland Carcinogenesis: An Overview. Toxicol. Pathol. 29 (1), 105–116. Cardiff, R.D., Rosner, A., Hogarth, M.A., Galvez, J.J., Borowsky, A.D., Gregg, J.P., 2004. Validation: The New Challenge for Pathology. Toxicol. Pathol. 32 (Suppl. 1), 31–39. Cardiff, R., Anver, M., Gusterson, B.A., Hennighausen, L., Jensen, R.A., Merino, M.J., Rehm, S., Russo, J., Tavassoli, F.A., Wakefield, L.M., Ward, J.M., Green, J.E., 2000. The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting. Oncogene 19 (8), 968–988.

Endocrine Disruptors’ Health Effects Euling, S.Y., Herman-Giddens, M.E., Lee, P.A., Selevan, S.G., Juul, A., Sørensen, T.I., Dunkel, L., Himes, J.H., Teilmann, G., Swan, S.H., 2008a. Examination of US puberty-timing data from 1940 to 1994 for secular trends: panel findings. Pediatrics 121 (Suppl. 3), S172–S191. Euling, S.Y., Selevan, S.G., Pescovitz, O.H., Skakkebaek, N.E., 2008b. Role of environmental factors in the timing of puberty. Pediatrics 121 (Suppl. 3), S167–S171. Fenton, S.E., 2006. Endocrine-disrupting compounds and mammary gland development: early exposure and later life consequences. Endocrinology 147 (Suppl. 6), S18–S24. Fenton, S.E., 2009. The mammary gland: a tissue sensitive to environmental exposures. Rev. Environ. Health 24 (4), 319–325. Fenton, S.E., Beck, L.M., Borde, A.R., Rayner, J.L., 2012a. Developmental Exposure to Environmental Endocrine Disruptors and Adverse Effects on Mammary Gland Development. In: Diamanti-Kandarakis, E.a.G. (Ed.), Contemporary Endocrinology Series: Endocrine Disruptors and Puberty. A.C., Springer/Humana Press, pp. 201–224. Fenton, S.E., Reed, C., Newbold, R.R., 2012b. Perinatal Environmental Factors Affect Breast Development: Is precocious thelarche a marker of endocrine disruption? Ann. Rev. Pharmacol. Toxicol. 52, 455–479. Latendresse, J.R., Bucci, T.J., Olson, G., Mellick, P., Weis, C.C., Thorn, B., Newbold, R.R., Delclos, K.B., 2009. Genistein and ethinyl estradiol dietary exposure in multigenerational and chronic studies induce similar proliferative lesions in

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mammary gland of male Sprague-Dawley rats. Reprod. Toxicol. 28 (3), 342–353.

In Vitro Assays Krause, S., Maffini, M., Soto, A.M., Sonnenschein, C., 2010. The microenvironment determines the breast cancer cells’ phenotype: organization of MCF7 cells in 3D cultures. BMC Cancer 10 (1), 263. Lee, G.Y., Kenny, P.A., Lee, E.H., Bissell, M.J., 2007. Threedimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4 (4), 359–365. Szelei, J., Soto, A.M., Geck, P., Desronvil, M., Prechtl, N.V., Weill, B.C., Sonnenschein, C., 2000. Identification of human estrogen-inducible transcripts that potentially mediate the apoptotic response in breast cancer. J. Steroid Biochem. Mol. Biol. 72, 89–102.

Physiologic Response to Injury Cline, J.M., 2007. Assessing the Mammary Gland of Nonhuman Primates: Effects of Endogenous Hormones and Exogenous Hormonal Agents and Growth Factors. Birth Defects Res. B Dev. Reprod. Toxicol. 80, 126–146. Lucas, J.N., Rudmann, D.G., Credille, K.M., Irizarry, A.R., Peter, A., Snyder, P.W., 2007. The Rat Mammary Gland: Morphologic Changes as an Indicator of Systemic Hormonal Perturbations Induced by Xenobiotics. Toxicol. Pathol. 35 (2), 199–207.

Hovey, R.C., Coder, P.S., Wolf, J.C., Sielken Jr., R.L., Tisdel, M.O., Breckenridge, C.B., 2011. Quantitative Assessment of Mammary Gland Development in Female Long Evans Rats Following In Utero Exposure to Atrazine. Toxicol. Sci. 119 (2), 380–390. Long, G.G., Reynolds, V.L., Dochterman, L.W., Ryan, T.E., 2009. Neoplastic and Non-neoplastic Changes in F-344 Rats Treated with Naveglitazar, a g-Dominant PPAR a/g Agonist. Toxicol. Pathol. 37 (6), 741–753. Rudel, R.A., Fenton, S.E., Ackerman, J.M., Euling, S.Y., Makris, S.L., 2011. Environmental Exposures and Mammary Gland Development: State of the Science, Public Health Implications, and Research Recommendations. Environ. Health Perspect. 119 (8). Souda, M., Umekita, Y., Abeyama, K., Yoshida, H., 2009. Gene expression profiling during rat mammary carcinogenesis induced by 7,12-dimethylbenz[a]anthracene. Int. J. Cancer 125 (6), 1285–1297. Vorderstrasse, B.A., Fenton, S.E., Bohn, A.A., Cundiff, J.A., Lawrence, B.P., 2004. A Novel Effect of Dioxin: Exposure during Pregnancy Severely Impairs Mammary Gland Differentiation. Toxicol. Sci. 78 (2), 248–257.

APPENDIX

Mechanisms of Toxicity

I. Mammary Gland Whole Mount Preparation

Ariazi, J.L., Haag, J.D., Lindstrom, M.J., Gould, M.N., 2005. Mammary glands of sexually immature rats are more susceptible than those of mature rats to the carcinogenic, lethal, and mutagenic effects of N-nitroso-N-methylurea. Mol. Carcinog. 43 (3), 155–164. Cameron, H.L., Foster, W.G., 2009. Developmental and Lactational Exposure to Dieldrin Alters Mammary Tumorigenesis in Her2/neu Transgenic Mice. PLoS One 4 (1) e4303. Currier, N., Solomon, S.E., Demicco, E.G., Chang, D.L., Farago, M., Ying, H., Dominguez, I., Sonenshein, G.E., Cardiff, R.D., Xiao, Z.X., Sherr, D.H., Seldin, D.C., 2005. Oncogenic Signaling Pathways Activated in DMBAInduced Mouse Mammary Tumors. Toxicol. Pathol. 33 (6), 726–737. Hoenerhoff, M.J., Hong, H.H., Ton, T.V., Lahousse, S.A., Sills, R.C., 2009. A Review of the Molecular Mechanisms of Chemically Induced Neoplasia in Rat and Mouse Models in National Toxicology Program Bioassays and Their Relevance to Human Cancer. Toxicol. Pathol. 37 (7), 835–848. Houle, C.D., Ton, T.-V.T., Clayton, N., Huff, J., Hong, H.H., Sills, R.C., 2006. Frequent p53 and H-ras Mutations in Benzene- and Ethylene Oxide-Induced Mammary Gland Carcinomas from B6C3F1 Mice. Toxicol. Pathol. 34 (6), 752–762.

A. Necropsy Equipment and Supplies 1. Fine curved scissors 2. Dissection boards 3. Straight scissors 4. Holding pins 5. Fine curved forceps 6. Charged slides 7. Curved, serrated forceps 8. 70% Ethanol 9. Baking or cafeteria-style trays for stacking slides B. Procedure 1. Pre-label all slides using a xylene-proof method. Pencil works and should be covered with mounting solution at the end to preserve label. 2. After euthanasia, place the animal on its back on a dissecting board. 3. Stretch and pin all 4 feet down, with the rear feet forming an inverted V (holding pins or small gauge needles work well). 4. Wet the animal’s abdomen and rear legs with 70% ethanol to avoid hair in the sample

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5. Make a skin incision from the pubis to the rib cage, being careful not to cut through the internal abdominal wall. 6. Continue incisions from the pubis to the medial aspect of each rear limb, forming an inverted Y. 7. Peel the skin back by grasping the skin, but not the attached gland, at the center of the inverted Y with serrated forceps, and pin the skin to the dissection board (or other flat surface such as a cooler top). This exposes the 4th and 5th mammary glands (and 6th in the rat). In the rat, the 5th and 6th glands may need to be separated by cutting through the gland along the inner thigh of the hindleg, being careful not to cut through the femoral artery. 8. Using the back side of the curved forceps (not the tips), gently separate the fat pad containing the 4th and 5th glands (leave the 6th gland in rats) from the skin at the point where the skin is pinned (make sure to obtain the 4th nipple area) and lift it away from the skin, cutting the attachments to the skin as you go (using fine scissors). Use long, smooth cuts rather than short snips to prevent alteration of the gland morphology and the formation of air bubbles on the slides. As progress is made, the forceps may need to be repositioned further down the fat pad to prevent the gland from tearing. 9. When the entire fat pad (to the back of the animal) with the 4th and 5th mammary glands has been separated from the skin, make a straight cut parallel to the animal’s body and detach the fat pad/mammary glands. 10. Spread the mammary tissue on a dry slide, with the side that was adjacent to the skin down, with the forceps (completely spread out). One fat pad/ mammary gland set (glands 4/5 as a single unit) per slide. The thickest part of the gland should be on the end nearest the slide label and the lymph node should be near the center of the slide. Extra large slides may be necessary for glands of older or lactating rats.

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II. Fixing and Staining Procedure A. Equipment and Supplies 1. Carnoy’s Fixative: a. 6 parts 100% ethanol b. 3 parts chloroform c. 1 part glacial acetic acid. 2. Carmine Alum Stain: a. 1 g carmine (Sigma C1022) b. 2.5 g aluminum potassium sulfate (Sigma A7167) c. Fill to 500 mL with distilled water d. Boil for 20 minutes e. Adjust final volume to 500 mL with distilled water f. Filter (to remove residues) into a dark or foil-wrapped bottle g. A small amount (1 crystal) of thymol may be added as a preservative h. Refrigerate. Discard stain after 3–4 months (6 months with thymol) from date of preparation. Carmine can be removed from glassware with 100% ethanol. 3. Glass microscope slides (use 2  3-inch slides for PND 70–90 rats; the same or standard sized slides may be used for all other animals). 4. Parafilm (2  3-inch). 5. Glass slide dishes. 6. 70% and 95% ethanol. 7. Xylene. 8. Permount (Fisher Scientific). 9. Glass disposable pipets. 10. Rubber suction bulbs 11. Cover slips (appropriate size for glands removed – will vary with animal age). 12. 50-mL conical tubes filled with water and capped, or another object with a flat surface that weighs 415–420 g (e.g., a beaker with water). 13. Refrigerator with space for cooling tissue (slide trays may be stacked). B. Procedure 1. Once the mammary tissue has been spread onto slide, press on the tissue with gloved fingers to remove bubbles that may be under the tissue, place a 2  3-inch rectangle of Parafilm on the gland and cover with another glass slide. Place an inverted 50-mL conical tube filled with water (or another object weighing

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415–420 g) on the mammary tissue/glass slide sandwich. 2. Compress the mammary tissue in a refrigerator for 30 minutes to several hours, depending on its thickness (PND 4 or 11 for 20–30 minutes; rat PND 45 for 1– 2 hours; PND 70–90 rats may require as much as 2–3 hours). 3. Remove the 50-mL conical tube and the top glass slide, and peel the Parafilm back from one end, being careful not to loosen the gland from the slide. Place the slides in a glass staining tray. Fix the tissue in Carnoy’s Fixative (see II.A.1. above) for 4–48 hours, depending on thickness, at room temperature. Most mammary tissue from PND 32–45 rats and smaller can be fixed for 18–24 hours (overnight). Tissue from PND 90 rats may require more time. If white areas are present in the mammary tissue after fixing (more opaque than rest of gland), change the fixative and allow those glands to fix for an additional 24 hours. 4. Remove fixative and soak in 70% ethanol for 15–30 minutes. 5. Change gradually to water. (Pour out onethird, add water, let sit 5 minutes, repeat three times). 6. Equilibrate in water for 5 minutes.

7. Stain in Carmine alum stain (see II.A.2. above) for 12–24 hours, depending on thickness (longer does not hurt it, but be consistent for all tissues of same age). The stain is reusable. 8. Soak in water for 30 seconds. 9. Soak in 70% ethanol for 15–30 minutes. 10. Soak in 95% ethanol for 15–30 minutes. 11. Soak in 100% ethanol for 20–30 minutes. 12. Clear in xylene for 4–72 hours, depending on the thickness of gland. The gland should be translucent after clearing. If any opaque (whitish) areas remain, place those slides in larger containers with xylene until translucent (clear). Xylene may be re-used within a week or so, if not lipid-filled. 13. Pipet enough Permount onto the tissue to cover the specimen and place a cover slip on top, being careful to avoid air bubbles. If bubbles form, lift the cover slip, pop the bubbles with the edge of a slide, and reapply the cover slip (additional Permount may be necessary). 14. After slides have dried, clean excess Permount from the outside of the slide with 100% EtOH or xylene on a cotton swab, and let dry in a ventilated area (hood) before placing on the dissecting microscope.

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