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

Abstract

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.

Introduction

The mammary gland is a dynamic organ that undergoes extensive changes in histomorphology, complexity, and function throughout the process of pregnancy, lactation, and involution (1). These changes are driven by a series of regulated hormonal signals that induce the gland to grow and differentiate to provide milk for newborn offspring. Upon weaning, the mammary gland is reorganized and the epithelial network of alveolar structures regresses, returning to a morphology similar to but distinct from a nonparous female, via apoptosis and processes mediated by immune cells (2, 3). The parous (postlactating) mammary gland is distinguished from the nulliparous mammary gland by several features, including altered populations of ductal epithelium cell types (4), changes in gene expression (5, 6), long-lasting increases in the number and activity of several populations of immune cells (7, 8), and decreased proliferation in epithelial cell populations (9).

The relationship between parity and breast cancer is complex, having a “dual effect” (10, 11) where parity seemingly is long-term protective (12, 13), but has a short-term inductive effect on breast cancer during or shortly after pregnancy (termed pregnancy-associated breast cancer) (14). Furthermore, there appears to be an age-dependent relationship such that first pregnancy early in life decreases lifetime risk of breast cancer (15), whereas pregnancy after age 35 increases lifetime risk compared with nulliparous women (12, 16).

Studies from both humans and animal models have worked to elucidate the mechanism by which pregnancy mediates age-dependent effects on mammary cancer risk. One such mediator appears to be parity-induced alterations to estrogen signaling in the mammary gland (9). Estrogens are key drivers of mammary gland development through puberty, pregnancy, and lactation (1). Serum concentrations of 17β-estradiol increase throughout pregnancy (17) and estradiol promotes proliferation and reduces apoptosis and expression of several apoptosis-associated genes in cancer cells lines (18-20), and is considered a carcinogen in certain contexts (21, 22). Epidemiological studies have also reported that lifetime exposure to estrogen is positively associated with breast cancer development (23). Indeed, women who experience early age of menarche (24), late age of menopause (25, 26), or undergo some hormone replacement therapies after onset of menopause (26) have higher risk of breast cancer incidence.

If lifetime exposure to estrogens is a risk for breast cancer, how do we explain the association between early life parity (marked by increasing levels of estrogen throughout) and reduced breast cancer risk? Hormones associated with pregnancy, including estradiol, induce permanent reorganization and differentiation of mammary epithelial cells (27). There is a decrease in the response of mammary epithelial cells to estrogen (28) via a decrease in the number of estrogen receptor (ER)-positive cells, reducing overall proliferation within the mammary gland (27, 29). Further studies in rodents revealed that treatment of mice with estrogen and progesterone, at levels similar to those measured during pregnancy, provides a protective effect from tumorigenesis induced by chemical carcinogens (30) and in genetically susceptible mouse models (31), in the absence of an actual pregnancy.

The hormone-mediated reorganization of the pregnant and lactating mammary gland is a delicate process that is vulnerable to environmental influences. Because estrogens can promote breast cancer, but pregnancy reduces the risk of breast cancer, our work has begun to explore whether environmental chemicals with ER-agonist properties can promote or reduce the protective effects of pregnancy on breast cancer. Exposures to endocrine-disrupting chemicals (EDCs) during pregnancy and lactation can induce functional and morphological changes in the rodent mammary gland (32-36). Similarly, epidemiologic studies suggest associations between EDC exposures and reduced milk production (37-40) and increased breast cancer risk (41-43), although the most sensitive periods of exposure are more difficult to identify in human populations (44). A recent study from our lab (33) showed that exposures to oxybenzone (an EDC that interacts with ER) during pregnancy and lactation produced long-term alterations in mammary gland morphology and gene expression in mice, resulting in mammary phenotypes intermediate to nulliparous and parous animals.

In recent decades, researchers have begun to evaluate whether parabens, a class of EDCs, could have an effect on breast cancer risk, especially because many parabens are ER agonists (45). Parabens are alkyl-esters of p-hydroxybenzoic acid, produced in high quantities since the early 20th century due to their preservative and antimicrobial properties (46). Parabens are included in many commonly used cosmetics, pharmaceuticals, industrial products, and processed foods (47). An early study showed that 77% to 99% (depending on type) of personal care products contained parabens (48), while a more recent study from China detected at least one paraben in 77% of all personal care products tested (49). Human exposure to parabens is ubiquitous, with spot urine tests of 300 people from 9 different countries revealing detectable concentrations in all samples, with methylparaben and propylparaben found in 98% and 80% of samples, respectively (50).

In the early 2000s, work by Darbre and colleagues raised concerns about the safety of parabens when these chemicals were measured in cancerous breast tissues (51). Darbre et al suggested that use of underarm cosmetics (eg, deodorants and anti-perspirants), which commonly contain parabens and are topically applied near the breast, could play a causal role in breast cancer (52, 53). Yet, larger epidemiology studies have not confirmed this relationship, instead concluding there is insufficient evidence to address this potential risk factor (54). More recent studies have shown that parabens promote genotoxicity at high concentrations (≤1mM; (55)) and increase breast cancer cell invasiveness in vitro (56), a hallmark of tumorigenesis. Unfortunately, there are relatively few controlled rodent studies of parabens, leading the European Commission on Consumer Safety to conclude that there is inadequate evidence to support the safety of several of these chemicals (propylparaben, isopropylparaben, butylparaben, and isobutylparaben) in personal care products (57).

Propylparaben (CAS # 94-13-3) is detected in 44% of tested rinse-off and 53% of leave-on personal care products, in 55% of baby products in the United States (58) and in 73% of all personal care products tested in China (49). In a case-control study from the Long Island Breast Cancer Study Project, the highest quartile of urinary concentrations of propylparaben was associated with a nonsignificant increased odds for breast cancer development (odds ratio = 1.31; CI, 0.90-1.90 compared with the lowest quartile (59)). Propylparaben displays ER-agonist activity in cultured cells (45) and induces increased uterine weight in the uterotrophic assay (45, 60). Propylparaben can also induce gene expression profiles similar to estradiol in breast cancer cells (61), and our recent work demonstrates that it can induce ERα-dependent R-loop formation and DNA damage (62).

Although concerns were raised over the safety of parabens, including propylparaben, 2 decades ago, even today very few studies have evaluated the effects of propylparaben exposures on animal models (63) and to our knowledge, no studies have examined the effects of propylparaben on the mammary gland in vivo (64). Here, we evaluate the long-term effects of propylparaben exposures during pregnancy and lactation on the mouse mammary gland. Our study reveals that environmentally relevant doses of propylparaben alter the parous mouse mammary gland morphology, gene expression, proliferation, and immune cell populations.

Methods

Animals

BALB/c mice (6 to 8 weeks old) were mated and housed in polysulfone cages at the University of Massachusetts Animal Facility, University of Massachusetts. The mice were provided ad libitum access to food (LabDiet Chow 5058; LabDiet, St. Louis, MO) and tap water in temperature- and light-controlled conditions. All experimental procedures and experimenters were approved by the University of Massachusetts Institutional Animal Care and Use Committee.

Prior to mating, dams were weighed and randomly assigned to treatment groups so that a normal distribution of weight was achieved across groups. Propylparaben (Sigma, Cat# 1577008, 99% purity) was dissolved in tocopherol-stripped corn oil to create dosing solutions. The parous treatment groups include tocopherol-stripped corn oil vehicle (referred to throughout the text as the parous control) or 1 of 3 doses of propylparaben (20, 100, or 10 000 μg/kg/day; abbreviated 20PP, 100PP, and 10 000PP throughout the text). The lowest dose is designed to approximate the intake of the 95^th percentile of pregnant American women (65) and the highest dose represents the toxicological no-observed adverse-effect level (NOAEL) for propylparaben (66). A nulliparous control was administered corn oil using the same dosing regimen and timing of tissue collection as the parous control group.

Dams were administered corn oil or corn oil with propylparaben once a day from pregnancy day 0 (PD0) until lactation day 21 (LD21) according to methods we have used previously (33). Briefly, each day, dams were weighed to adjust dosing volumes (1 µL/g body weight) and then orally exposed via pipette feeding. Dams were allowed to deliver naturally (with the day of birth considered LD0). Dams from all parous treatment groups were assessed for reproductive outcomes, including maternal body weight throughout pregnancy and lactation, length of gestation (number of days from when a sperm plug was observed until parturition), number of pups born per litter, and number of pups surviving to weaning.

On LD21, pups were weaned, and the dams were transferred to new cages to be co-housed with dams of the same treatment group. Dams were untreated for a period of 5 weeks to ensure the completion of mammary gland involution (females were then 17-20 weeks of age) and humanely euthanized via carbon dioxide asphyxiation. Dam estrous cycle stage was assessed at euthanasia using vaginal smears; no attempt was made to time necropsies to a specific stage of the estrous cycle.

Tissue Collection, Processing, and Storage

After euthanasia, the third (pectoral) pair of mammary glands was dissected from the skin, placed on a glass slide (Thermo Fisher Scientific, Waltham, MA), and fixed in 10% neutral buffered formalin (Thermo Fisher Scientific) overnight for whole-mount analysis. The fourth (inguinal) mammary gland pairs were also dissected from the skin and collected. The right inguinal gland was fixed in 10% neutral buffered formalin overnight, washed with phosphate-buffered saline, dehydrated in a series of alcohols, and then embedded in paraffin (Leica Biosystems, Richmond, IL) under a vacuum for histological and immunohistochemical evaluations. For the left fourth inguinal mammary gland, the lymph node was removed from the tissue, and the remaining gland was snap-frozen with dry ice and stored at −80 °C for quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). Finally, the fifth inguinal mammary glands were dissected from the skin and then processed for the mammosphere assay as described below.

Processing and Imaging of Whole-Mounted Mammary Glands

Whole-mounted pectoral mammary glands were stained using standardized methods (67). Briefly, glands were processed through a series of alcohols, cleared of fat with toluene, stained with carmine alum, dehydrated with alcohol and xylene, and then sealed in k-pax heat sealed bags (Thermo Fisher Scientific) containing methyl salicylate (Acros Organics, Morris Plains, NJ) as a preservative. Both the left and right whole-mounted mammary glands were imaged by an AxioImager dissection microscope (Carl Zeiss Microscopy, Jena, Germany) at 30× magnification and a Zeiss high-resolution color camera. Mammary gland morphology was evaluated using Zen software (Carl Zeiss Microscopy). To quantify the volume fraction of the gland comprised of mammary epithelium, unbiased stereology methods using a 13 × 16 grid (180 crosshairs, 0.3 mm apart) were used.

Histology Processing and Imaging

Paraffin-embedded mammary glands were sectioned with a Fisher rotary microtome (5 µm thickness) and mounted on positively charged glass slides (Thermo Scientific). Slides were then processed through a series of xylenes and alcohols to deparaffinize, stained, and then dehydrated with alcohols and xylene before being mounted and cover-slipped with permanent mounting medium. Histological stains included hematoxylin and eosin, toluidine blue, and Masson’s Trichrome. Slides were imaged using a Zeiss Axio Observer.Z1 inverted microscope with 10×, 20×, and 40× objectives and a high-resolution color camera (Carl Zeiss Microscopy).

Volume fraction of ductal epithelium was calculated from 4 randomly selected fields (imaged with the 10× objective) of sections stained with hematoxylin and eosin using Zen software (Carl Zeiss Microscopy). Briefly, an identical 10 × 13 grid (108 crosshairs, 100 µm apart) was placed on each image, and the structure at each crosshair was counted. Values from the 4 images were averaged to represent each sample. Periductal collagen thickness was calculated from at least 5 random images of major or secondary ducts in sections stained with Masson’s Trichrome per animal. Widths of collagen from epithelium outwards were taken at 5 different points for each duct. Values from the individual ducts were averaged to provide a single value for each animal.

Immunohistochemistry

Standard methods and commercial antibodies were used for immunohistochemistry for the following targets: estrogen receptor alpha (ERα; MilliporeSigma, St. Louis, MO), progesterone receptor (PR; Abcam, Cambridge, MA), Ki67, a marker of cell proliferation (Thermo Fisher Scientific), Wnt5a (Abcam), B cells (CD45R, eBioscience, San Diego, CA), CD8a cells (eBioscience), FoxP3-expressing cells (eBioscience), and CD3-positive cells (Abcam). Standard methods and commercial antibodies were also used for immunofluorescence co-staining of CD163/F480-positive cells, and mannose receptor CD206/F480-positive cells. Details about antibodies are included in Table 1.

Table 1.

Information about antibodies

Peptide/protein target	Name of antibody	Manufacturer, catalog no., or name of source	Species raised in polyclonal or monoclonal	Dilution used	Secondary	Research resource identifier
ERα	Anti-estrogen receptor α (C1355)	Millipore, 06-935	Rabbit; polyclonal	1:1000	Biotinylated Goat Anti-Rabbit IgG Abcam 64256 Goat; Polyclonal Ready to use (5 µg/mL) RRID: AB_2661852	AB_31035
Ki67	Ki67	Thermo Fisher Scientific, RM-9106-S1	Rabbit; monoclonal	1:1000	Biotinylated Goat Anti-Rabbit IgG Abcam 64256 Goat; Polyclonal Ready to use (5 µg/mL) RRID: AB_2661852	AB_149792
Progesterone Receptor	Anti-progesterone receptor	Abcam, ab131486	Rabbit; polyclonal	1:500	Biotinylated Goat Anti-Rabbit IgG Abcam 64256 Goat; Polyclonal Ready to use (5 µg/mL) RRID: AB_2661852	AB_11156044
Wnt5a	Anti-Wnt5a	Abcam, ab174963	Rabbit; polyclonal	1:500	Biotinylated Goat Anti-Rabbit IgG Abcam 64256 Goat; Polyclonal Ready to use (5 µg/mL) RRID: AB_2661852	AB_2725803
CD45R	CD45R (B220) monoclonal antibody (RA3-6B2)	eBioscience, 14-0452	Rat monoclonal	1:200	Biotinylated Goat Anti-Rat IG BD Pharmingen 559286 Goat; polyclonal 1:50	AB_467254
CD8a	CD8a monoclonal antibody (4SM15)	eBioscience, 14-0808	Rat monoclonal	1:100	Biotinylated Goat Anti-Rat IG BD Pharmingen 559286 Goat; polyclonal 1:50	AB_2572861
FoxP3	FoxP3 monoclonal antibody (FJK-16s)	eBioscience, 14-5773	Rat monoclonal	1:100	Biotinylated Goat Anti-Rat IG BD Pharmingen 559286 Goat; polyclonal 1:50	AB_467576
CD3	Anti-CD3 antibody [SP7]	Abcam, ab16669	Rabbit monoclonal	1:100	Envision System HRP anti-Rabbit polymer (DAKO) K4003
CD163	Recombinant anti-CD163 antibody [EPR19518]	Abcam, ab182422	Rabbit monoclonal	1:200	Alexa Fluor 568 goat anti-rabbit (A-11011) 1:500
F4/80	F4/80 monoclonal antibody (BM8) Alexa fluor 488	eBioscience, 53-4801-82	Rat monoclonal	1:100		AB_469915
CD206	Anti-mannose receptor antibody	Abcam, ab64693	Rabbit polyclonal	1:200	Alexa Fluor 568 goat anti-rabbit (A-11011) 1:500
Secondary	Biotinylated Goat Anti-Rabbit IgG	Abcam, ab64256	Goat; polyclonal	Ready to use (5 µg/mL)		AB_2661852

For immunohistochemistry of ERα, PR, Ki67, and Wnt5a, 5-µm sections were deparaffinized with a series of xylenes and alcohols, microwaved in 10mM citrate buffer (pH 6) for antigen retrieval, and immersed in hydrogen peroxide to quench endogenous peroxidases. Nonspecific binding was blocked with 1% milk protein in 5% normal goat serum (Cell Signaling Technology, Danvers, MA), and then incubated overnight (14 to 16 hours) with primary antibodies (see Table 1) at 4 °C. Slides were then washed and incubated with biotinylated secondary antibody (goat anti-rabbit, Abcam, Cat# ab64256), incubated with streptavidin peroxidase complex (Abcam, Cat# ab24269), followed by diaminobenzidine chromogen (DAB; Abcam, Cat# ab64238), and then counter-stained with Harris’s hematoxylin (Thermo Fisher Scientific). Finally, slides were dehydrated in a series of alcohols and xylenes and cover-slipped with permanent mounting medium. Each run of immunohistochemistry included a negative control where the primary antibody was replaced by an equal volume of blocking solution.

Processed slides were imaged using a Zeiss Axio Obzerver.Z1 inverted microscope, a 40× objective, a Zeiss high-resolution color camera, and evaluated with Zen software (Carl Zeiss Microscopy). ERα, Ki67, and PR expression were represented by the percent ratio of the total number of epithelial cells counted. Wnt5a expression was analyzed using TissueQuant, a publicly available program that compares different color shades within an image to a DAB-stained reference color (68). TissueQuant calculates a color score based on the normalized hue, saturation, and color intensity of each pixel. The batch process option was used to calculate the overall concentration of positive staining and area density of positively stained areas for all images.

For analysis of immune cells, 4-µm mammary gland sections were processed using a DakoCytomation Autostainer (Dako, Carpinteria, CA) using antibodies listed in Table 1. These sections were deparaffinized with a series of xylenes, rehydrated in a series of alcohols, then rinsed in triphosphate-buffered saline (TBS). Antigen retrieval was performed in a 0.01M citrate buffer (pH 6), then slides were incubated with primary antibodies (Table 1) for 30 minutes at room temperature, followed by streptavidin (BD Pharmingen, Billerica, CA; Cat# 550946), and DAB chromogen and counter-stained with Harris’ hematoxylin. The slides were then dehydrated through a series of ethanols and xylenes, and cover-slipped with mounting medium.

Immunofluorescence co-stain was performed for CD163/F4/80 and Mannose Receptor (CD206)/F4/80. Sections were cut at 4 µm then deparaffinized with a series of xylenes, rehydrated in graded alcohols and rinsed in TBS. Antigen retrieval was performed in 0.01M citrate buffer (pH 6) before slides were cooled, rinsed with TBS buffer, and blocked with Dako protein block with 5% goat serum for 20 minutes at room temperature. Primary antibody rabbit monoclonal anti-CD163 (Abcam, 182422) 1:200 or rabbit polyclonal anti-mannose receptor (CD206) 1:200 and F4/80 monoclonal antibody conjugated with Fluor 488 (eBioscience, 53-4801-82) 1:100 were incubated overnight at 4 °C followed by fluorescently labeled secondary (Alexa Fluor 568 goat anti-rabbit [A-11011] 1:500) incubated for 1 hour at room temperature. The slides were counter-stained with DAPI (Vector H-1200) and mounted with Vectashield mounting medium for fluorescence before cover-slipping.

Mammosphere Assay

Mammosphere formation was evaluated using standard methods (69). Briefly, the fifth inguinal mammary glands were collected from a subset of females, minced, and dissociated in Dulbecco’s Modified Eagle’s medium (DMEM):F12 (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum (Gibco, Paisley, UK), 2 mg/mL collagenase (Worthington Biochemicals, Freehold, NJ), 100U/mL Pen/Strep (Gibco), and 100 µg/nL gentamicin (Gibco) for 6 hours. The cell pellet was collected and further dissociated with 1 mL prewarmed 0.25% Trypsin-EDTA (Gibco) and 100 µL of 1mg/mL DNase I (Roche, Mannheim, Germany). Cell suspensions were sieved through a 40-µm cell strainer to obtain single cell suspension. These cells were seeded into ultra-low attachment dishes at a density of 125 000 cells/mL (20 000 viable cells/cm²). Cells were grown in a serum-free mammary epithelial growth medium supplemented with B27 (Gibco), 20 ng/mL mouse epidermal growth factor (mEGF) (Sigma), 20ng/mL Fibroblast Growth Factor-basic (bFGF) (Sigma), 4 μg/mL heparin (Sigma), 100 U/mL Pen/Strep, and 5 μg/mL gentamicin. After 7 days in culture, mammospheres were collected using gentle centrifugation (800 rpm for 5 minutes) and dissociated with 1 mL prewarmed 0.25% Trypsin-EDTA and 60 μL of 1 mg/mL DNase I for 5 to 8 minutes. Cell suspensions obtained from dissociation were sieved through a 40-μm cell strainer, diluted, and suspended in 96-well plates at a density of 1000 cells/well. The mammospheres that formed were considered secondary mammospheres. These were imaged in 96-well plates using a Keyence BZ-X700 microscope and counted by 2 experimenters who were blind to treatment group.

Gene Expression Analyses

Frozen mammary glands were analyzed for gene expression via quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) for Esr1, PgR, AREG, Igf1, and Tgfβ2 genes (see Table 2 for primer sequences). Total RNA was extracted from mammary glands using Trizol reagent (Thermo Fisher Scientific) and total RNA concentration was calculated using UV spectrophotometry with a Nanodrop 1000 (Thermo Fisher Scientific). For each sample, 1 µg of RNA was reverse transcribed to cDNA (Thermo Fisher Scientific reverse transcription) and the FastStart Universal SYBR Green Master kit (Roche Diagnostics, Indianapolis, IN) was used for qRT-PCR, along with 1 µL cDNA and 300nM forward and reverse primer cocktail for each target gene. All samples were run in duplicate and β-actin was used as a housekeeping gene for all target genes. A thermal cycler (Mastercycler Epgradient S model 5345, Eppendorf) used the following thermal parameters: 10 minutes at 95 °C, 40 cycles of 15 seconds at 95 °C, 30 seconds at 60 °C, and 15 seconds at 72 °C. Relative quantification was determined using the ΔΔCt to correct for differences in β-actin (70).

Table 2.

Information about primers

Gene	Forward Primer Sequence	Reverse Primer Sequence
Estrogen receptor α (Esr1)	TGC AAT GAC TAT GCC TCT GG (782-801)	CTC CGG TTC TTG TCA ATG GT (921-902)
Estrogen receptor β (Esr2)	TGT GTG TGA AGG CCA TGA TT	TCT TCG AAA TCA CCC AGA CC
Progesterone receptor (ProgR) total	AAA GGA TCC GCA GGT TCT C	GTT CCA TCT TCC AGC GGA TA
Wnt5a	GAA TCC CAT TTG CAA CCC CTC ACC	GCT CCT CGT GTA CAT TTT CTG CCC
β-actin (BA)	CAC ACC CGC CAC CAG TTC GC (89-108)	TTG CAC ATG CCG GAG CCG TT (162-143)

Statistical Analysis

Mouse experiments were run in 2 separate batches (separated by approximately 3 months) with all treatment groups included in each batch. A two-way analysis of variance (ANOVA) determined that there were no significant effects of batch on target outcomes, and thus both batches were combined for further analysis. For some outcomes, only dams from batch 1 were evaluated (eg, qRT-PCR, immune cell analyses). All analyses were conducted by observers who were blind to treatment groups. Graphs illustrate means ± SE unless otherwise stated.

Data were analyzed using SPSS version 26 (IBM, Inc. Armonk, NY). Continuous variable data were analyzed using one-way ANOVA General Linear Model analyses with treatment as the independent variable, followed by Fisher least significant difference (LSD) and Bonferroni post hoc tests. We report the Bonferroni post hoc results (the more conservative evaluation of differences between groups) whenever they were significant. When Fisher’s post hoc data are described, it is because the more stringent evaluation with Bonferroni did not reveal significant differences between specific groups.

Data were considered statistically significant at P < 0.05. Sample sizes for treatment groups and outcomes are provided in Table 3.

Table 3.

Sample sizes across treatment groups and outcomes

	Nulliparous	Parous control	20PP	100PP	10 000PP
Pregnancy outcomes	N/A	15	16	16	18
Whole mounts	15	12	16	16	16
Mammospheres	4	4	0	0	4
Immunohistochemistry (ER, PR, Ki67)	12	11	13	12	16
Analysis of immune cells	5	6	6	6	7
qRT-PCR	5	6	6	6	7

Results

Propylparaben Has Modest Effects on Pregnancy Outcomes

Females from all 4 parous groups were evaluated for differences in pregnancy outcomes and pup survivability (Fig. 1). The length of gestation, measured as the time from when a sperm plug was observed until the pups were born, was increased by about 12 hours (0.5 days) in all 3 propylparaben treatment groups, but this increase was not statistically significant (Fig. 1A). There were also no statistically significant effects of propylparaben on litter size (number of viable pups at birth) compared with the parous control (Fig. 1B). However, the number of pups in the 20PP and 100PP groups were significantly reduced compared with the 10 000PP group (Fig. 1B). Litter size was also evaluated at weaning to account for differences in survival (Fig. 1C). Fewer pups per litter survived to weaning in the 20PP and 100PP groups compared with the 10 000PP females, but pup survival was not different in any of the propylparaben groups compared with parous controls.

Figure 1.

Effects of propylparaben on pregnancy outcomes. A, There was no effect of treatment on length of gestation (ANOVA, P = 0.12). B, There was an effect of treatment on number of pups born (ANOVA, P = 0.007). Number of pups born per litter was not different in PP groups compared with controls. However, litter size was significantly higher in the 10 000PP dose group compared with the lower PP doses. C, There was also an effect of treatment on number of pups surviving until weaning (ANOVA, P = 0.031). No significances were observed between any PP treatment group and controls. More pups survived in the 10 000PP dose group compared with lower PP doses. In all panels, different letters between groups indicate significant differences, P < 0.05, Fisher LSD post hoc test.

Propylparaben Alters Morphology of the Post-Involution Mammary Gland

To determine if propylparaben exposure during pregnancy and lactation alters the three-dimensional morphology of the post-involution mammary gland, whole-mounted mammary glands were collected from females 5 weeks after weaning and age-matched nulliparous control females (Fig. 2A and 2B). As expected, the volume fraction of the gland comprised of ductal epithelium was increased in parous controls compared with nulliparous control females. Yet, the volume fraction of ductal epithelium was significantly decreased in 20PP and 100PP females compared with parous control animals, but significantly higher than nulliparous controls. This finding is consistent with an intermediate phenotype (eg, an appearance in-between parous and nulliparous animals). Females exposed to 10 000PP were not statistically distinguishable from parous control females.

Figure 2.

The morphology of the post-involution mammary gland is altered by exposure to propylparaben during pregnancy and lactation. A, Morphology of whole-mounted mammary glands. Scalebar = 0.5 mm. B, Volume fraction of epithelium measured in whole-mounted mammary gland is affected by treatment (ANOVA, P < 0.001). Volume fraction was significantly increased in parous controls. Mammary glands from females from the 20PP and 100PP exposure groups were significantly different from either nulliparous or parous controls, consistent with intermediate phenotype. Different letters indicate significant differences among groups, P < 0.05, Fisher LSD post hoc test. C, Histomorphology in mammary gland sections stained with hematoxylin and eosin. Scalebar = 100 μm. D, There were significant differences between treatment groups for volume fraction of epithelium measured in historical sections (ANOVA, P < 0.001). Quantification of epithelial density in histological sections revealed that volume fraction of epithelium was increased by parity. Mammary glands from all 3 propylparaben groups were significantly different from either control, indicating an intermediate phenotype. Different letters indicate significant differences among groups, P < 0.01, Bonferroni post hoc test. E, Treatment-dependent effects on periductal collagen thickness were also observed (ANOVA, P < 0.001). There were significant effects of parity, and propylparaben exposures produced periductal collagen thicknesses more consistent with nulliparous females. Different letters indicate significant differences among groups, P < 0.01, Bonferroni post hoc test.

Mammary gland histomorphology was further evaluated using paraffin sections stained with hematoxylin and eosin (Fig. 2C and 2D). Again, volume fraction of epithelium was significantly increased in parous controls compared with nulliparous controls, as expected. A statistically significant intermediate phenotype for volume fraction of epithelium was observed for all propylparaben groups.

Finally, periductal collagen thickness was measured in paraffin sections stained with Masson’s Trichrome. As expected, a significant increase in periductal collagen thickness was seen in parous controls compared with nulliparous controls (Fig. 2E). All 3 propylparaben groups had a periductal collagen thickness that was quantitatively more similar to the nulliparous controls than the parous controls. These results are consistent with a loss of parity-induced increases in periductal collagen width.

Propylparaben Exposure Alters Proliferation, but Not Mammary Stem Cell Formation

We next evaluated proliferation in the mammary epithelium via expression of Ki67 (Fig. 3A). As expected, a decrease in mean Ki67 expression was seen in parous controls compared with nulliparous controls, although these groups were not statistically different. Yet, both the 20PP and 100PP groups had a significant increase in Ki67-positive cells compared with parous controls. These results are consistent with a loss of the typical parity-associated decrease in epithelial cell proliferation in these propylparaben-exposed females.

Figure 3.

Propylparaben alters proliferation but not stem cell number. A, Immunohistochemistry for Ki67 reveals differences in cell proliferation across treatment groups. Scalebar = 20 μm, arrows indicate positive cells. B, Quantification of Ki67 expression reveals differences between treatments (ANOVA, P < 0.001). Although the decrease in Ki67-positive cells is not significantly different in the parous vs nulliparous controls, the percent of Ki67-positive cells in the 20PP and 100PP exposure groups was increased compared with parous controls. Different letters indicate significant differences among groups, P < 0.05, Bonferroni post hoc test. C, Quantification of secondary mammospheres revealed no effect of parity or exposure to 10 000PP.

To further assess effects of parity and propylparaben exposure on proliferation parameters, secondary mammospheres, which provide a measure of stem cells, were quantified (Fig. 3B). Surprisingly, we observed no effect of parity on mammosphere number, and no effect was observed in the 10 000PP group, either.

Propylparaben Alters Hormone Receptors in the Mammary Gland

Because propylparaben is an ER agonist, we next evaluated if exposure to propylparaben during pregnancy and lactation produced long-term effects on ERα or expression of its downstream targets in the mammary gland. The percent of epithelial cells that were ERα-positive was increased in all parous groups (eg, the parous control and all 3 propylparaben treatments) compared with the nulliparous controls (Fig. 4A and 4B). The qRT-PCR analysis for expression of Esr1, the gene encoding ERα, showed a similar pattern, but there were no statistically significant differences between any treatment groups (Fig. 4C).

Figure 4.

Number of epithelial cells expressing PR, but not ER, is affected by parity. A, Quantification of ERα-positive cells using immunohistochemistry demonstrated differences between treatment groups (ANOVA, P = 0.028). Post hoc comparisons revealed a significant increase in parous compared with nulliparous controls. Different letters indicate significant differences among groups, P < 0.05, Fisher LSD post hoc test. B, An example of mammary tissue with ERα-positive epithelial cells, indicated by arrows. Scalebar = 20 μm. C, Relative expression of Esr1, the gene encoding ERα. There were no significant differences between any treatment groups. D, Quantification of PR-positive epithelial cells revealed treatment-related effects (ANOVA, P = 0.027). PR expression was reduced in all parous groups compared with nulliparous controls, and expression was not affected by propylparaben exposure. Different letters indicate significant differences among groups, P < 0.01, Bonferroni post hoc test. E, An example of mammary tissue with PR-positive epithelial cells, indicated by arrows. Scalebar = 20 μm. F, Quantification of relative expression of PgR mRNA with differences between treatment groups (ANOVA, P = 0.019). PgR expression was reduced in all parous groups compared with nulliparous controls. PP exposure resulted in intermediate phenotypes with 10 000PP significantly different from the parous control group. Different letters indicate significant differences among groups, P < 0.05, Fisher LSD post hoc test.

Although no changes in ER expression were found between the propylparaben and parous control groups, propylparaben has been previously shown to have effects on ER-mediated gene expression (61). We next evaluated several targets known to be downstream of ER signaling pathways. First, we quantified epithelial cells that were positive for PR, and found that expression of the protein was significantly decreased in parous controls compared with nulliparous controls (Fig. 4D and 4E). A similar decrease was observed in all propylparaben groups, consistent with an effect of parity, but no effect of the chemical. Using qRT-PCR, we observed that expression of the gene encoding progesterone receptor (PgR) was significantly decreased in parous controls compared with nulliparous controls (Fig. 4F). Intermediate levels of PgR expression were seen for all propylparaben groups (higher than parous controls, lower than nulliparous controls), with a statistically significant increase from parous controls observed only in the 10 000PP group.

Propylparaben Alters Downstream Targets of ER

We examined expression of a small number of ER-mediated genes, including amphiregulin (AREG) and Insulin-like growth factor 1 (IGF1). No effect of parity or propylparaben treatment was observed for AREG expression (Fig. 5A). In contrast, IGF1 expression was reduced in parous controls compared with nulliparous controls (Fig. 5B) and intermediate levels of 1 expression were seen in all propylparaben doses.

Figure 5.

Propylparaben disrupts expression of several, but not all, ER-mediated targets. A, Relative mRNA expression of AREG mRNA. Neither parity nor propylparaben had an effect on ARexpression. B, A trend toward treatment-related effects was observed on relative mRNA expression of Igf1 mRNA (ANOVA, P = 0.078). The results are consistent with a reduction in mean Igf1 expression in parous controls compared to nulliparous females and somewhat higher expression in the PP groups. Different letters indicate P < 0.05, Fisher LSD post hoc test. C, Quantification of Wnt5a protein expression revealed no effect of parity or propylparaben exposure. D, Relative expression of TGFβ2 mRNA revealed a trend toward treatment-related effects (ANOVA, P = 0.057). TGFβ2 expression was increased in the 20PP and 10 000PP groups compared with the nulliparous controls. Different letters indicate P < 0.05, Fisher LSD post hoc test. All qRT-PCR expression data were normalized using the ΔΔCt method.

Wnt5a is an ER-mediated secreted molecule localized to both the mammary epithelium and stroma. Using Wnt5a antibodies and TissueQuant, a method to quantify tissue expression of immunohistochemical signals, we found no effect of either parity or propylparaben on the intensity of Wnt5a expression (Fig. 5C). Using qRT-PCR, we observed no effect of parity on TGFβ2 gene expression, another ER-mediated gene. However, treatment with either 20PP or 10 000PP induced an increase in TGFβ2 expression compared with nulliparous, but not parous, controls (Fig. 5D).

Propylparaben Exposure Alters Several Immune Cell Populations in the Mammary Gland

Immune cells serve an important regulatory function in the mammary gland, and immune cell populations have been documented to be affected by parity (7, 8). We next quantified several immune cell populations, including mast cells, neutrophils, B cells, T cells, regulatory T cells, and macrophages, to determine if any effect of parity was altered by propylparaben exposure. Quantification of B cells revealed a higher number in parous controls compared with nulliparous controls (Fig. 6A) and intermediate numbers of B cells in all 3 propylparaben groups. However, there were no statistically significant differences across treatment groups. A similar pattern was observed for CD8-positive cells (T cells). Again, there was a non-statistically significant increase in their number in the parous controls, and an intermediate number in all 3 propylparaben groups (Fig. 6B). A statistically significant parity-associated signature was observed for CD3-positive cells (mature T cell lymphocytes and pro-thymocytes), which were significantly increased in parous controls compared with nulliparous controls (Fig. 6C). Females from the 20PP group had significantly more CD3-positive cells compared with all other treatment groups, including both control groups.

Figure 6.

Both parity and propylparaben alter mammary gland immune cell populations. A, Although no significant differences were observed based on treatment (ANOVA, P = 0.26), quantification of B cells reveals that the number of B cells tended to increase with parity, while PP exposure induced an intermediate phenotype. B, Again, there were no statistically significant effects of treatment on quantification of CD8+ T cells (ANOVA, P = 0.20). The number of CD8+ cells appears to be increased due to parity, with intermediate numbers in the PP treated groups. C, Quantification of CD3+ T cells revealed effects due to treatment (ANOVA, P = 0.013). The number of CD3+ cells was increased due to parity. 20PP exposure further increased the number of CD3+ cells. Different letters indicate significant differences among groups, P < 0.05, Fisher LSD post hoc test. D, Effects of treatment were observed on number of CD163+/F480+ M2 macrophages (ANOVA, P < 0.001). CD163+/F480+ cells were increased by parity and PP exposure compared with nulliparous controls. The 100PP group was statistically distinct from both control groups. Different letters indicate significant differences among groups, P < 0.05, Fisher LSD post hoc test. E, A trend for treatment-related effects on number of CD+ 206/F480+ M2 macrophages (ANOVA, P = 0.061). CD206+/F480+ were increased by parity and PP exposure compared with nulliparous controls.

Next M2 macrophages were identified and counted via the double labeling of either CD163+ and F480+ cells or CD206+ cells and F480+ cells, dual markers that are suggestive of polarization toward a subtype of macrophage previously found to have a high range of invasion in tumorigenic mammary glands (71). CD163+/F480+ cells were significantly increased in parous compared with nulliparous controls (Fig. 6D). The 100PP group had intermediate levels of this macrophage population, which was statistically different from both controls. CD206+/F480+ cells were also increased in parous controls compared with nulliparous controls (Fig. 6E). There was no obvious effect of propylparaben, with only the 100PP group displaying an intermediate number of CD206+/F480+ cells compared with both controls.

Toluidine blue staining reveals all granulated cells of myeloid lineage including mast cells and neutrophils. Toluidine blue–positive cells were counted but no quantitative differences were observed due to parity or propylparaben treatment (data not shown). Finally, the number of T-regulatory cells, marked by Foxp3 expression, was not affected by either parity or propylparaben treatment (data not shown).

Discussion

Here we have evaluated the effects of propylparaben exposure during pregnancy and lactation in the mouse model and found evidence that it induces long-term effects in the mammary gland. Specifically, effects of propylparaben were observed on the volume of mammary epithelium, thickness of periductal collagen, epithelial cell proliferation, and immune cells populations. In several of these outcomes, propylparaben reduced the effect of parity, creating intermediate phenotypes (ie, distinguishable from both parous and nulliparous controls). Taken together, our study indicates that propylparaben exposure during pregnancy and lactation alters the remodeling of the mammary gland that normally occurs during lactation and involution.

Our analyses of the effects of propylparaben on the mammary gland started with an evaluation of mammary gland morphology. Quantification of both the whole-mounted mammary gland and histological sections revealed that parity increased the volume of ductal epithelium in the mammary gland, but propylparaben diminished this effect (Fig. 2A-2D). A similar pattern was observed in measurements of periductal collagen in the propylparaben-exposed dams (Fig. 2E). We previously reported alterations of mammary gland histomorphology in females exposed to other estrogenic chemicals during pregnancy and lactation. In mice exposed to the sunscreen additive oxybenzone, a reduced fraction of epithelium and decreased width of the periductal collagen were observed following involution (33). Similarly, mice exposed to the ER agonist bisphenol-S during pregnancy and lactation displayed a reduced volume fraction of epithelium during late lactation, consistent with early involution (32).

Epithelial stem cell proliferation has received much attention as a potential driver of tumorigenesis in the mammary gland (72). Decreased rates of proliferation postparity may be a key factor in the protection from breast cancer observed in parous women, and this is a characteristic of the parous mammary gland in rodents (27). Here, we saw a decrease in the mean number of Ki67-positive cells in the parous controls compared with the nulliparous, although this decrease did not reach statistical significance. Still, we found that proliferation (eg, Ki67-positive cells) was increased in propylparaben-exposed animals; proliferation rates in the 2 lowest exposure groups (20PP and 100PP) were distinct from the parous controls and similar to the nulliparous group (Fig. 3). In recent years, it has become well-understood that many EDCs have non-monotonic dose responses, where low dose exposures can induce more severe effects than intermediate or even high doses (73). Future studies should examine whether the long-term consequences of propylparaben exposure are more severe in the low dose groups. Those studies should also evaluate whether long-term shifts in proliferation observed in propylparaben-exposed females alter the latency or incidence of mammary gland tumor development in vivo.

It is interesting that we observed both decreases in ductal epithelium volume and increased epithelial proliferation simultaneously in the females exposed to the lower doses of propylparaben. On the surface these results may seem counterintuitive, but they could be explained by propylparaben-induced increases in the degenerative processes of involution (74). Future studies should examine the effects of propylparaben during the actual process of involution to determine its influence on rates of epithelial “trimming” including processes such as increased apoptosis, metalloprotease activity, and immune cell invasion, each of which could contribute to reduced epithelial density and periductal collagen (75).

Several studies suggest that parity reduces the number of mammary stem cells (27, 76), with some researchers hypothesizing that reduced numbers of mammary stem cells in parous animals could be responsible for the protective effect of pregnancy on breast cancer risk (77). Other groups have not observed a parity-induced reduction of mammary stem cell number, suggesting that this effect may be limited to young animals or only observed when using specific markers to identify stem populations (78, 79). Here, we evaluated stem cell number using mammosphere assays conducted on mammary glands from both the nulliparous and parous controls, and the 10 000PP exposure group. Our results did not indicate any difference in mammosphere number based on either parity or propylparaben exposure (Fig. 3C), suggesting no lasting effects on stem cell colonies. Although our evaluation of mammosphere numbers is limited based on the small number of samples used in these experiments for feasibility reasons (n = 4 per group), our results are in contrast to a study concluding that methylparaben exposure increases mammosphere size in experiments conducted with primary breast cells and breast cancer cell lines (80). Unfortunately, for feasibility reasons, mammary glands from the 20PP and 100PP exposure groups were not included in the mammosphere assay, yet these groups displayed increases in cell proliferation, again suggesting that these low doses warrant further study.

The process of involution requires macrophages (81), which clear the cellular debris when secretory epithelial cells undergo apoptosis, and the gland undergoes extensive and irreversible remodeling and reorganization to return to a pre-pregnant appearance (74). Furthermore, parity is associated with permanent changes in the immune cells that reside in the mammary stroma (7, 8). Propylparaben, and parabens as a class, have been relatively understudied for immunomodulatory effects, however, epidemiological data report associations between paraben exposures and immunological biomarkers or allergen sensitivities. For example, a cross-sectional study evaluating 860 children form the 2005–2006 National Health and Nutrition Survey (NHANES) found that subjects in the highest tertile of urinary propylparaben levels had increased odds of developing aeroallergen sensitivities compared with children in the lowest tertile (OR = 2.04; CI, 1.12-3.74) (82). Increases in urinary propylparaben concentrations have also been associated with decreases in the inflammatory biomarker C-protein, and increases in the oxidative stress marker, isoprostane, in pregnant women in a prospective birth cohort study from Northern Puerto Rico (83). Similarly, work in animal models has found that paraben exposures alter important inflammatory response genes. For example, propylparaben exposure in zebrafish increased expression of tnfa (84), the gene that encodes the macrophage-produced TNFα protein. Another study of cultured human peripheral lymphocytes reported increased genotoxicity and cytotoxicity in these cells when exposed to high concentrations of parabens, including propylparaben (85).

To address the possibility that propylparaben exposure might influence parity-induced changes in immune cell numbers, we evaluated several resident immune cell populations. In all cases, parity induced significantly higher counts of immune cells, although for feasibility reasons, some of these effects were not statistically significant. Although propylparaben exposure had mixed effects based on cell type and dose, overall, a pattern emerged: for many immune cell populations, propylparaben appeared to blunt the parity-induced increases in immune cell number (Fig. 6). The only exception was a significant increase in mature T cell lymphocytes (CD3+ cells) in animals from the 20PP dose group (Fig. 6C) compared with both nulliparous and parous control groups. This effect was not observed after exposure to the higher doses. Importantly, the immune cell populations evaluated in this study were limited to those that reside in the mammary stroma several weeks after recovery from involution, as well as several weeks following the last administration of propylparaben. Any effect of propylparaben, even a transient one, on the presence of immune cells could have profound implications for the long-term health of the mammary gland, as changes in immune cell function have been linked to breast cancer and tumor growth (86).

Despite few studies examining parabens and immune cell regulation, other EDCs have received attention for their immunomodulatory effects (reviewed in (87)). For example, xenoestrogens have been found to decrease movement of macrophages and T cells to tissues infected by Escherichia coli (88) and decrease neutrophil chemotaxis across chemoattractant gradients (89). ER agonists also induce apoptosis in RAW264.7 macrophage-like cells (90). Future studies are needed to determine how propylparaben alters immune cells during the period of exposure, or during the period of involution, which may help to understand the relationship between propylparaben-induced proliferation and decreased density of ductal epithelium. Furthermore, future studies should seek to elucidate the effects of propylparaben on the processes known to drive the initial stages of involution (eg, apoptotic and metalloprotease enzymatic activity) (75).

Several results of our mammary gland studies are consistent with estrogenic activity (72). We were also surprised to see that the parous controls, and all of the propylparaben-treated females had an increase in the number of cells that were ERα-positive (Fig.4C); this was surprising because our prior study revealed a decrease in Esr1 after first parity (33). Expression of some ERα-mediated genes were altered in propylparaben-exposed females (Figs 4, 5). For example, Igf-1 expression, known to be a target of ERα (91), was decreased by parity yet expression in the 20PP group more closely resembled expression in the nulliparous controls (Fig. 5B). Yet, other expected targets of ERα activation were not altered by propylparaben exposures. For example, no difference in AREG mRNA expression was induced either by parity, or after propylparaben exposure (Fig. 5A). Prior studies also concluded that propylparaben can bind to ERβ in vitro (92). Expression of TGF-β2, an ER-mediated gene which was not affected by parity was increased in the 20PP and 10 000PP groups (Fig. 5D). TGF-β signaling in cancers has drawn much attention, as dysregulation to this family of molecules (including TGF-β2) has been associated with tumor development in several tissues, including the breast (93, 94).

Several lines of evidence suggest that chemicals can have estrogenic or antiestrogenic effects with little or negligible affinity to ER. Instead, these chemicals alter the synthesis or regulation of estrogen signaling molecules, ultimately resulting in estrogenic effects (95). Previous studies indicate that propylparaben can disrupt estrogen signaling through a reduction in the activity of the estrogen-converting enzyme, 17β-hydroxysteroid dehydrogenase 2 (96), inhibition of estrogen sulfotransferases, eg, the enzymes that reduce endogenous estrogen activity (97), and activation of intracellular signaling cascades (eg, cAMP, Erk1/2, and PI3k/Akt) (98). A small number of rodent studies utilizing the uterotrophic assay have confirmed that propylparaben is indeed estrogenic in vivo (45, 60). Yet, it has also been argued that concern over propylparaben (and other parabens) is unnecessary because of its “weaker” activation of ER than estradiol (66, 99). Recent evaluation of propylparaben using the “key characteristics” approach (100) suggests that it should also be classified as an EDC (64). In vitro exploration of its endocrine-disrupting mechanisms indicates that propylparaben is estrogenic and acts as an agonist of both ER isoforms (ERα and ERβ) (101), promotes ER-dependent R-loop formation and DNA damage at levels lower than those needed for ER transactivation (62), and alters estrogen-dependent gene transcription in breast cancer cell lines (61).

Propylparaben may have effects beyond the mammary gland. We evaluated pregnancy outcomes and observed modest, but nonsignificant increases in length of gestation (Fig. 1A). Furthermore, both the number of pups born and the number of pups surviving to weaning had a similar (but not statistically significant) pattern of dose-dependent effects (Fig. 1B and 1C), in which low doses saw modest decreases, but the high dose exhibited increases in both outcomes. Future studies with larger sample sizes are needed to investigate these effects further. Previous data have shown EDCs can alter maternal behaviors (102), such as grooming (103), time spent nursing (104), pup retrieval behaviors (105), as well as maternal milk protein expression (35, 106), which could explain the decreased pup survival at low doses of propylparaben. Future studies are needed to parse out effects of propylparaben on maternal-pup interactions and describe risks to developing offspring.

Conclusions

The Endocrine Society defines an EDC as “an exogenous chemical, or mixture of chemicals, that interferes with any aspect of hormone action” (107). Our study provides strong evidence that propylparaben disrupts the endocrine system by altering mRNA expression of hormone pathways, immune cell populations, rates of epithelial cell proliferation, and hormone responsive morphology in the parous mouse mammary gland, a hormonally responsive organ (44). Furthermore, our results indicate that propylparaben exposure during pregnancy and lactation alters mammary gland development, potentially diminishing the protective effects of parity. Importantly, many of these effects were observed at doses that are relevant to exposure in pregnant American women (eg, the 20PP group) (65).

This study confirms that pregnancy and lactation are indeed vulnerable periods of development that are sensitive to effects of EDCs. Using the foundation laid in this study, future research should focus on the effects of propylparaben exposure in animal models to elucidate the mechanisms of action and evaluation of adverse outcomes in the mammary gland during other critical periods of development (gestation, puberty, etc.). More work is needed to better assess the risk of propylparaben exposures, especially for susceptible populations and during critical windows of development, and to understand whether propylparaben can undermine the protective effects of parity against breast cancer.

Glossary

Abbreviations

20PP/100PP/10 000PP

20/100/10 000 μg propylparaben/kg/day

ANOVA

analysis of variance

DAB

diaminobenzidine

EDC

endocrine-disrupting chemical

ER

estrogen receptor

LD#

lactation day #

LSD

least significant difference

PD#

pregnancy day #

PgR

progesterone receptor

PR

progesterone receptor

qRT-PCR

quantitative reverse-transcriptase polymerase chain reaction

TBS

triphosphate-buffered saline

Additional Information

Disclosures: L.N.V. is a member of the US EPA’s Science Advisory Board Chemical Assessment Advisory Committee, a scientific advisor (unpaid) to 2 Horizon 2020 EDC grants and a paid scientific advisor to SUDOC, LLC. Her travel has been sponsored by various government, academic and industry groups to present findings of her research and her EDC-related research has been funded by US government agencies, the University of Massachusetts Amherst, and NGOs including the Cornell Douglas Foundation and the Great Neck Breast Cancer Coalition. The other authors have no conflicts of interest to disclose.

Data Availability

Some or all datasets generated during the current study are not publicly available but are available from the corresponding author on reasonable request.