Effects of butyl benzyl phthalate exposure during pregnancy and lactation on the post-involution mammary gland.

The mammary gland undergoes comprehensive reorganization during pregnancy, lactation, and subsequent involution. Following involution, the mammary gland has structural and functional differences compared to the gland of a nulliparous female. These parity-associated changes are regulated by hormones and may be vulnerable to endocrine-disrupting chemicals (EDCs). In this study, we evaluated the long-term effects of butyl benzyl phthalate (BBP), an estrogenic plasticizer, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 3, 500, or 18000 µg/kg/day BBP throughout both pregnancy and the lactational period. The litters born to these females were evaluated for litter size and growth. The parous females were then kept for five weeks following weaning of the pups, during which period there was no exposure to BBP. After five weeks of post-weaning, mammary glands were collected and assessed for changes in histomorphology, steroid receptor expression, innate immune cell number, and gene expression. An unexposed age-matched nulliparous control was also evaluated as a comparator group. BBP increased male and female pup weight at puberty and female offspring in adulthood. BBP also altered innate immune cells in the post-involution mammary gland, reducing the effect of parity on macrophages. Lastly, BBP modestly increased mammary gland ductal complexity and periductal structure, but had no effect on expression of estrogen receptor, progesterone receptor, or a marker of proliferation. These results suggest that BBP may interfere with some effects of parity on the mouse mammary gland and induce weight gain in exposed offspring.

Benzophenone-3 exposure alters composition of tumor infiltrating immune cells and increases lung seeding of 4T1 breast cancer cells.

Environmental chemicals are a persistent and pervasive part of everyday life. A subset of environmental chemicals are xenoestrogens, compounds that bind to the estrogen receptor (ER) and drive estrogen-related processes. One such chemical, benzophenone-3 (BP3), is a common chemical in sunscreen. It is a potent UV protectant but also is quickly absorbed through the skin. While it has been approved by the FDA, there is a renewed interest in the safety of BP3, particularly in relation to breast cancer. The focus of this study was to examine the impact that BP3 has on triple negative breast cancer (TNBC) through alterations to cells in the immune microenvironment. In this study, we exposed female mice to one of two doses of BP3 before injecting them with a TNBC cell line. Several immune endpoints were examined both in the primary tissues and from in vitro studies of T cell behavior. Our studies revealed that in the lung tumor microenvironment, exposure to BP3 not only increased the number of metastases, but also the total area of tumor coverage. We also found that BP3 caused alterations in immune populations in a tissue-dependent manner, particularly in T cells. Taken together, our data suggest that while BP3 may not directly affect the proliferation of TNBC, growth and metastasis of TNBC-derived tumors can be altered by BP3 exposures via the alterations in the immune populations of the tumor microenvironment.

Optimizing Conjugation Chemistry, Antibody Conjugation Site, and Surface Density in Antibody-Nanogel Conjugates (ANCs) for Cell-Specific Drug Delivery.

Targeted delivery of therapeutics using antibody-nanogel conjugates (ANCs) with a high drug-to-antibody ratio has the potential to overcome some of the inherent limitations of antibody-drug conjugates (ADCs). ANC platforms with simple preparation methods and precise tunability to evaluate structure-activity relationships will greatly contribute to translating this promise into clinical reality. In this work, using trastuzumab as a model antibody, we demonstrate a block copolymer-based ANC platform that allows highly efficient antibody conjugation and formulation. In addition to showcasing the advantages of using an inverse electron-demand Diels-Alder (iEDDA)-based antibody conjugation, we evaluate the influence of antibody surface density and conjugation site on the nanogels upon the targeting capability of ANCs. We show that compared to traditional strain-promoted alkyne-azide cycloadditions, the preparation of ANCs using iEDDA provides significantly higher efficiency, which results in a shortened reaction time, simplified purification process, and enhanced targeting toward cancer cells. We also find that a site-specific disulfide-rebridging method in antibodies offers similar targeting abilities as the more indiscriminate lysine-based conjugation method. The more efficient bioconjugation using iEDDA allows us to optimize the avidity by fine-tuning the surface density of antibodies on the nanogel. Finally, with trastuzumab-mertansine (DM1) antibody-drug combination, our ANC demonstrates superior activities in vitro compared to the corresponding ADC, further highlighting the potential of ANCs in future clinical translation.

Bioorthogonal nanozymes for breast cancer imaging and therapy.

Bioorthogonal catalysis via transition metal catalysts (TMCs) enables the generation of therapeutics locally through chemical reactions not accessible by biological systems. This localization can enhance the efficacy of anticancer treatment while minimizing off-target effects. The encapsulation of TMCs into nanomaterials generates "nanozymes" to activate imaging and therapeutic agents. Here, we report the use of cationic bioorthogonal nanozymes to create localized "drug factories" for cancer therapy in vivo. These nanozymes remained present at the tumor site at least seven days after a single injection due to the interactions between cationic surface ligands and negatively charged cell membranes and tissue components. The prodrug was then administered systemically, and the nanozymes continuously converted the non-toxic molecules into active drugs locally. This strategy substantially reduced the tumor growth in an aggressive breast cancer model, with significantly reduced liver damage compared to traditional chemotherapy.

Targeted Drug Delivery Using a Plug-to-Direct Antibody-Nanogel Conjugate.

Targeted drug delivery using antibody-drug conjugates has attracted great attention due to its enhanced therapeutic efficacy compared to traditional chemotherapy. However, the development has been limited due to a low drug-to-antibody ratio and laborious linker-payload optimization. Herein, we present a simple and efficient strategy to combine the favorable features of polymeric nanocarriers with antibodies to generate an antibody-nanogel conjugate (ANC) platform for targeted delivery of cytotoxic agents. Our nanogels stably encapsulate several chemotherapeutic agents with a wide range of mechanisms of action and solubility. We showcase the targetability of ANCs and their selective killing of cancer cells over-expressing disease-relevant antigens such as human epidermal growth factor receptor 2, epidermal growth factor receptor, and tumor-specific mucin 1, which cover a broad range of breast cancer cell types while maintaining low to no toxicity to non-targeted cells. Overall, our system represents a versatile approach that could impact next-generation nanomedicine in antibody-targeted therapeutics.

Induced mammary cancer in rat models: pathogenesis, genetics, and relevance to female breast cancer.

Mammary cancer, or breast cancer in women, is a polygenic disease with a complex etiopathogenesis. While much remains elusive regarding its origin, it is well established that chemical carcinogens and endogenous estrogens contribute significantly to the initiation and progression of this disease. Rats have been useful models to study induced mammary cancer. They develop mammary tumors with comparable histopathology to humans and exhibit differences in resistance or susceptibility to mammary cancer depending on strain. While some rat strains (e.g., Sprague-Dawley) readily form mammary tumors following treatment with the chemical carcinogen, 7,12-dimethylbenz[a]-anthracene (DMBA), other strains (e.g., Copenhagen) are resistant to DMBA-induced mammary carcinogenesis. Genetic linkage in inbred strains has identified strain-specific quantitative trait loci (QTLs) affecting mammary tumors, via mechanisms that act together to promote or attenuate, and include 24 QTLs controlling the outcome of chemical induction, 10 QTLs controlling the outcome of estrogen induction, and 4 QTLs controlling the outcome of irradiation induction. Moreover, and based on shared factors affecting mammary cancer etiopathogenesis between rats and humans, including orthologous risk regions between both species, rats have served as useful models for identifying methods for breast cancer prediction and treatment. These studies in rats, combined with alternative animal models that more closely mimic advanced stages of breast cancer and/or human lifestyles, will further improve our understanding of this complex disease.

Toward a digital analysis of environmental impacts on rodent mammary gland density during critical developmental windows.

While mammographic breast density is associated with breast cancer risk in humans, there is no comparable surrogate risk measure in mouse and rat mammary glands following various environmental exposures. In the current study, mammary glands from mice and rats subjected to reproductive factors and exposures to environmental chemicals that have been shown to influence mammary gland development and/or susceptibility to mammary tumors were evaluated for histologic density by manual and automated digital methods. Digital histological density detected changes due to hormonal stimuli/reproductive factors (parity), dietary fat, and exposure to environmental chemicals, such as benzophenone-3 and a combination of perfluorooctanoic acid and zeranol. Thus, digital analysis of mammary gland density offers a high throughput method that can provide a highly reproducible means of comparing a measure of histological density across independent experiments, experimental systems, and laboratories. This methodology holds promise for the detection of environmental impacts on mammary gland structure in mice and rats that may be comparable to human breast density, thus potentially allowing comparisons between rodent models and human breast cancer studies.

Interindividual variation contributes to differential PCB 126 induced gene expression in primary breast epithelial cells and tissues.

PCB 126 is a pervasive, dioxin-like chemical pollutant which can activate the aryl hydrocarbon receptor (AhR). Despite being banned from the market, PCB 126 can be detected in breast milk to this day. The extent to which interindividual variation impacts the adverse responses to this chemical in the breast tissue remains unclear. This study aimed to investigate the impact of 3 nM PCB 126 on gene expression in a panel of genetically diverse benign human breast epithelial cell (HBEC) cultures and patient derived breast tissues. Six patient derived HBEC cultures were treated with 3 nM PCB 126. RNAseq was used to interrogate the impact of exposure on differential gene expression. Gene expression changes from the top critical pathways were confirmed via qRT-PCR in a larger panel of benign patient derived HBEC cultures, as well as in patient-derived breast tissue explant cultures. RNAseq analysis of HBEC cultures revealed a signature of 144 genes significantly altered by 3 nM PCB 126 treatment. Confirmation of 8 targets using a panel of 12 HBEC cultures and commercially available breast cell lines demonstrated that while the induction of canonical downstream target gene, CYP1A1, was consistent across our primary HBECs, other genes including AREG, S100A8, IL1A, IL1B, MMP7, and CCL28 exhibited significant variability across individuals. The dependence on the activity of the aryl hydrocarbon receptor was confirmed using inhibitors. PCB 126 can induce significant and consistent changes in gene expression associated with xenobiotic metabolism in benign breast epithelial cells. Although the induction of most genes was reliant on the AhR, significant variability was noted between genes and individuals. These data suggest that there is a bifurcation of the pathway following AhR activation that contributes to the variation in interindividual responses.

Genetic modifiers regulating DNA replication and double-strand break repair are associated with differences in mammary tumors in mouse models of Li-Fraumeni syndrome.

Breast cancer is the most common tumor among women with inherited variants in the TP53 tumor suppressor, but onset varies widely suggesting interactions with genetic or environmental factors. Rodent models haploinsufficent for Trp53 also develop a wide variety of malignancies associated with Li-Fraumeni syndrome, but BALB/c mice are uniquely susceptible to mammary tumors and is genetically linked to the Suprmam1 locus on chromosome 7. To define mechanisms that interact with deficiencies in p53 to alter susceptibility to mammary tumors, we fine mapped the Suprmam1 locus in females from an N2 backcross of BALB/cMed and C57BL/6J mice. A major modifier was localized within a 10 cM interval on chromosome 7. The effect of the locus on DNA damage responses was examined in the parental strains and mice that are congenic for C57BL/6J alleles on the BALB/cMed background (SM1-Trp53+/-). The mammary epithelium of C57BL/6J-Trp53+/- females exhibited little radiation-induced apoptosis compared to BALB/cMed-Trp53+/- and SM1-Trp53+/- females indicating that the Suprmam1B6/B6 alleles could not rescue repair of radiation-induced DNA double-strand breaks mostly relying on non-homologous end joining. In contrast, the Suprmam1B6/B6 alleles in SM1-Trp53+/- mice were sufficient to confer the C57BL/6J-Trp53+/- phenotypes in homology-directed repair and replication fork progression. The Suprmam1B6/B6 alleles in SM1-Trp53+/- mice appear to act in trans to regulate a panel of DNA repair and replication genes which lie outside the locus.

Exposure to Propylparaben During Pregnancy and Lactation Induces Long-Term Alterations to the Mammary Gland in Mice.

The mammary gland is a hormone sensitive organ that is susceptible to endocrine-disrupting chemicals (EDCs) during the vulnerable periods of parous reorganization (ie, pregnancy, lactation, and involution). Pregnancy is believed to have long-term protective effects against breast cancer development; however, it is unknown if EDCs can alter this effect. We examined the long-term effects of propylparaben, a common preservative used in personal care products and foods, with estrogenic properties, on the parous mouse mammary gland. Pregnant BALB/c mice were treated with 0, 20, 100, or 10 000 µg/kg/day propylparaben throughout pregnancy and lactation. Unexposed nulliparous females were also evaluated. Five weeks post-involution, mammary glands were collected and assessed for changes in histomorphology, hormone receptor expression, immune cell number, and gene expression. For several parameters of mammary gland morphology, propylparaben reduced the effects of parity. Propylparaben also increased proliferation, but not stem cell number, and induced modest alterations to expression of ERα-mediated genes. Finally, propylparaben altered the effect of parity on the number of several immune cell types in the mammary gland. These results suggest that propylparaben, at levels relevant to human exposure, can interfere with the effects of parity on the mouse mammary gland and induce long-term alterations to mammary gland structure. Future studies should address if propylparaben exposures negate the protective effects of pregnancy on mammary cancer development.

The use of patient-derived breast tissue explants to study macrophage polarization and the effects of environmental chemical exposure.

Ex vivo mammary explant systems are an excellent model to study interactions between epithelium and stromal cell types because they contain physiologically relevant heterotypic interactions in the background of genetically diverse patients. The intact human mammary tissue, termed patient-derived explant (PDE), can be used to investigate cellular responses to a wide variety of external stimuli in situ. For this study, we examined the impact of cytokines or environmental chemicals on macrophage phenotypes. We demonstrate that we can polarize macrophages within human breast tissue PDEs toward M1 or M2 through the addition of interferon-γ (IFNγ) + lipopolysaccharide (LPS) or interleukin (IL)-4 + IL-13, respectively. Elevated expression levels of M(IFNγ + LPS) markers (HLADRA and CXCL10) or M(IL-4 + IL-13) markers (CD209 and CCL18) were observed in cytokine-treated tissues. We also examined the impact of the endocrine-disrupting chemical, benzophenone-3, on PDEs and measured significant, yet varying effects on macrophage polarization. Furthermore, a subset of the PDEs respond to IL-4 + IL-13 through downregulation of E-cadherin and upregulation of vimentin which is reminiscent of epithelial-to-mesenchymal transition (EMT) changes. Finally, we were able to show immortalized nonmalignant breast epithelial cells can exhibit EMT characteristics when exposed to growth factors secreted by M(IL-4 + IL-13) macrophages. Taken together, the PDE model system is an outstanding preclinical model to study early tissue-resident immune responses and effects on epithelial and stromal responses to stimuli found both endogenously in the breast and exogenously as a result of exposures.

Inter-Individual Variation in Response to Estrogen in Human Breast Explants.

Exposure to estrogen is strongly associated with increased breast cancer risk. While all women are exposed to estrogen, only 12% are expected to develop breast cancer during their lifetime. These women may be more sensitive to estrogen, as rodent models have demonstrated variability in estrogen sensitivity. Our objective was to determine individual variation in expression of estrogen receptor (ER) and estrogen-induced responses in the normal human breast. Human breast tissue from female donors undergoing reduction mammoplasty surgery were collected for microarray analysis of ER expression. To examine estrogen-induced responses, breast tissue from 23 female donors were cultured ex- vivo in basal or 10 nM 17ß-estradiol (E2) media for 4 days. Expression of ER genes (ESR1 and ESR2) increased significantly with age. E2 induced consistent increases in global gene transcription, but expression of target genes AREG, PGR, and TGFß2 increased significantly only in explants from nulliparous women. E2-treatment did not induce consistent changes in proliferation or radiation induced apoptosis. Responses to estrogen are highly variable among women and not associated with levels of ER expression, suggesting differences in intracellular signaling among individuals. The differences in sensitivity to E2-stimulated responses may contribute to variation in risk of breast cancer.

Effects of Benzophenone-3 and Propylparaben on Estrogen Receptor-Dependent R-Loops and DNA Damage in Breast Epithelial Cells and Mice.

BACKGROUND: Endocrine-disrupting chemicals have been shown to have broad effects on development, but their mutagenic actions that can lead to cancer have been less clearly demonstrated. Physiological levels of estrogen have been shown to stimulate DNA damage in breast epithelial cells through mechanisms mediated by estrogen-receptor alpha (ERα). Benzophenone-3 (BP-3) and propylparaben (PP) are xenoestrogens found in the urine of >96% of U.S. OBJECTIVES: We investigated the effect of BP-3 and PP on estrogen receptor-dependent transactivation and DNA damage at concentrations relevant to exposures in humans. METHODS: In human breast epithelial cells, DNA damage following treatment with 17ß-estradiol (E2), BP-3, and PP was determined by immunostaining with antibodies against γ-H2AX and 53BP1. Estrogenic responses were determined using luciferase reporter assays and gene expression. Formation of R-loops was determined with DNA: RNA hybrid-specific S9.6 antibody. Short-term exposure to the chemicals was also studied in ovariectomized mice. Immunostaining of mouse mammary epithelium was performed to quantify R-loops and DNA damage in vivo. RESULTS: Concentrations of 1µM and 5µM BP-3 or PP increased DNA damage similar to that of E2 treatment in a ERα-dependent manner. However, BP-3 and PP had limited transactivation of target genes at 1µM and 5µM concentrations. BP-3 and PP exposure caused R-loop formation in a normal human breast epithelial cell line when ERα was introduced. R-loops and DNA damage were also detected in mammary epithelial cells of mice treated with BP-3 and PP. CONCLUSIONS: Acute exposure to xenoestrogens (PP and BP-3) in mice induce DNA damage mediated by formation of ERα-dependent R-loops at concentrations 10-fold lower than those required for transactivation. Exposure to these xenoestrogens may cause deleterious estrogenic responses, such as DNA damage, in susceptible individuals. https://doi.org/10.1289/EHP5221.

Exposure to low doses of oxybenzone during perinatal development alters mammary gland morphology in male and female mice.

Oxybenzone (benzophenone-3) is an ultraviolet radiation filter commonly used in personal care products including sunscreens, textiles and inks, and food and beverage containers, among others. Due to its widespread use, human exposures to oxybenzone are widespread. Oxybenzone is considered an endocrine disrupting chemical due to its antiestrogenic and antiandrogenic properties. We evaluated the effects of oral exposures to oxybenzone on the growth and morphology of the mammary gland, body weight and anogenital distance in BALB/c mice exposed to 30, 212 or 3000 µg/kg/day in utero and during lactation. Developmental exposures to oxybenzone reduced the size and growth of mammary gland in males prior to and during puberty. In exposed females, oxybenzone reduced mammary cell proliferation, decreased the number of cells expressing estrogen receptor α, and altered mammary gland morphology in adulthood. These results suggest that even low doses of oxybenzone can disrupt hormone sensitive organs during critical windows of development.

The Mouse Mammary Gland: a Tool to Inform Adolescents About Environmental Causes of Breast Cancer.

Adolescence is a vulnerable period of breast development, and environmental chemical exposures that occur during this period can increase the risk of breast cancer in adulthood. Discussing breast health with adolescent girls can be difficult for several reasons. In this project, we worked to not only inform adolescent researchers about environmental risks for breast cancer but to also involve them in research studies. We taught adolescents about the stages of mammary gland development using samples collected from mice, with a specific focus on pre-pubertal and pubertal stages of development. Our analysis shows that adolescent researchers, with relatively modest training, can collect reliable and reproducible data on aspects of mammary gland biology that are known to be disrupted by environmental chemicals, with coefficients of variation < 2.5% for basic mammary gland parameters and 5-7% for more complex measures. Finally, we provided these adolescents with information about environmental risk factors for breast cancer that they could share with their peers and community and action items to potentially modify their individual risk. We hope that researchers working in this field will engage adolescent researchers in projects to evaluate chemicals that influence breast cancer risk. Summer research programs that inform young adolescents about breast cancer risk factors not only benefit these novice researchers individually but also benefit their communities when they are encouraged to talk about the value of basic science studies, discuss vulnerable periods of mammary gland development, and share what they have learned about cancer and the environment.

Environmental exposures during windows of susceptibility for breast cancer: a framework for prevention research.

BACKGROUND: The long time from exposure to potentially harmful chemicals until breast cancer occurrence poses challenges for designing etiologic studies and for implementing successful prevention programs. Growing evidence from animal and human studies indicates that distinct time periods of heightened susceptibility to endocrine disruptors exist throughout the life course. The influence of environmental chemicals on breast cancer risk may be greater during several windows of susceptibility (WOS) in a woman's life, including prenatal development, puberty, pregnancy, and the menopausal transition. These time windows are considered as specific periods of susceptibility for breast cancer because significant structural and functional changes occur in the mammary gland, as well as alterations in the mammary micro-environment and hormone signaling that may influence risk. Breast cancer research focused on these breast cancer WOS will accelerate understanding of disease etiology and prevention. MAIN TEXT: Despite the plausible heightened mechanistic influences of environmental chemicals on breast cancer risk during time periods of change in the mammary gland's structure and function, most human studies of environmental chemicals are not focused on specific WOS. This article reviews studies conducted over the past few decades that have specifically addressed the effect of environmental chemicals and metals on breast cancer risk during at least one of these WOS. In addition to summarizing the broader evidence-base specific to WOS, we include discussion of the NIH-funded Breast Cancer and the Environment Research Program (BCERP) which included population-based and basic science research focused on specific WOS to evaluate associations between breast cancer risk and particular classes of endocrine-disrupting chemicals-including polycyclic aromatic hydrocarbons, perfluorinated compounds, polybrominated diphenyl ethers, and phenols-and metals. We outline ways in which ongoing transdisciplinary BCERP projects incorporate animal research and human epidemiologic studies in close partnership with community organizations and communication scientists to identify research priorities and effectively translate evidence-based findings to the public and policy makers. CONCLUSIONS: An integrative model of breast cancer research is needed to determine the impact and mechanisms of action of endocrine disruptors at different WOS. By focusing on environmental chemical exposure during specific WOS, scientists and their community partners may identify when prevention efforts are likely to be most effective.

Gene expression signature of atypical breast hyperplasia and regulation by SFRP1.

BACKGROUND: Atypical breast hyperplasias (AH) have a 10-year risk of progression to invasive cancer estimated at 4-7%, with the overall risk of developing breast cancer increased by ~ 4-fold. AH lesions are estrogen receptor alpha positive (ERα+) and represent risk indicators and/or precursor lesions to low grade ERα+ tumors. Therefore, molecular profiles of AH lesions offer insights into the earliest changes in the breast epithelium, rendering it susceptible to oncogenic transformation. METHODS: In this study, women were selected who were diagnosed with ductal or lobular AH, but no breast cancer prior to or within the 2-year follow-up. Paired AH and histologically normal benign (HNB) tissues from patients were microdissected. RNA was isolated, amplified linearly, labeled, and hybridized to whole transcriptome microarrays to determine gene expression profiles. Genes that were differentially expressed between AH and HNB were identified using a paired analysis. Gene expression signatures distinguishing AH and HNB were defined using AGNES and PAM methods. Regulation of gene networks was investigated using breast epithelial cell lines, explant cultures of normal breast tissue and mouse tissues. RESULTS: A 99-gene signature discriminated the histologically normal and AH tissues in 81% of the cases. Network analysis identified coordinated alterations in signaling through ERα, epidermal growth factor receptors, and androgen receptor which were associated with the development of both lobular and ductal AH. Decreased expression of SFRP1 was also consistently lower in AH. Knockdown of SFRP1 in 76N-Tert cells resulted altered expression of 13 genes similarly to that observed in AH. An SFRP1-regulated network was also observed in tissues from mice lacking Sfrp1. Re-expression of SFRP1 in MCF7 cells provided further support for the SFRP1-regulated network. Treatment of breast explant cultures with rSFRP1 dampened estrogen-induced progesterone receptor levels. CONCLUSIONS: The alterations in gene expression were observed in both ductal and lobular AH suggesting shared underlying mechanisms predisposing to AH. Loss of SFRP1 expression is a significant regulator of AH transcriptional profiles driving previously unidentified changes affecting responses to estrogen and possibly other pathways. The gene signature and pathways provide insights into alterations contributing to AH breast lesions.

Sterilization of Silastic Capsules Containing 17ß-Estradiol for Effective Hormone Delivery in Mus musculus.

Silastic capsules are frequently used to study the physiologic effects of estrogen exposure in animal models. The Officeof Laboratory Animal Welfare requires the sterilization of nonpharmaceutical-grade compounds before use. We compared 2commonly used terminal sterilization methods-ionizing radiation (IR) and ethylene oxide (EO)-for their utility in sterilizingsilastic capsules containing 0.05 or 0.1 mg 17ß-estradiol (E2). E2-specific ELISA demonstrated that serum estrogen levelsdid not differ between mice implanted with 0.05-mg E2 capsules that were sterilized with IR or EO and those implanted withnonsterilized capsules. Likewise, mammary gland morphology and progesterone receptor expression and proliferation inmammary epithelium were similar among mice treated with E2 capsules, regardless of sterilization method, and pregnant day15 mice. In addition, IR-sterilized 0.1-mg E2 pellets provided high serum E2. We conclude that neither ionizing radiation norethylene oxide degraded E2 or the cellulose matrix, suggesting that these methods of sterilization are appropriate to provideeffective sterile hormone capsules for animal research.

Oxybenzone Alters Mammary Gland Morphology in Mice Exposed During Pregnancy and Lactation.

Hormones and endocrine-disrupting chemicals are generally thought to have permanent "organizational" effects when exposures occur during development but not adulthood. Yet, an increasing number of studies have shown that pregnant females are disrupted by endocrine-disrupting chemical exposures, with some effects that are permanent. Here, we examined the long-term effects of exposure to oxybenzone, an estrogenic chemical found in sunscreen and personal care products, on the morphology of the mammary gland in mice exposed during pregnancy and lactation. Female mice were exposed to vehicle or 30, 212, or 3000 µg oxybenzone/kg/d, from pregnancy day 0 until weaning. A nulliparous group, receiving vehicle treatment, was also evaluated. Mammary glands were collected 5 weeks after involution for whole-mount, histological, immunohistochemical, and molecular analyses. Exposure to 3000 µg oxybenzone/kg/d induced permanent changes to ductal density that was significantly different from both the nulliparous and vehicle groups. The two highest doses of oxybenzone similarly induced an intermediate phenotype for expression of progesterone receptor. A monotonic, dose-dependent increase in cell proliferation was also observed in the oxybenzone-treated females, becoming statistically significant at the highest dose. Finally, oxybenzone exposure induced an intermediate phenotype for Esr1 expression in all oxybenzone-treated groups. These data suggest that oxybenzone, at doses relevant to human exposures, produces long-lasting alterations to mammary gland morphology and function. Further studies are needed to determine if exposure to this chemical during pregnancy and lactation will interfere with the known protection that pregnancy provides against breast cancer.