A combined morphometric and statistical approach to assess non-monotonicity in the developing mammary gland of rats in the CLARITY-BPA study
Environmental Health Perspectives
We can and should take advantage of nonmonotonic properties to perform statistical analysis rigorously by new statistical and morphometric methods.
We aimed to a) determine whether BPA showed effects on the developing rat mammary gland using new quantitative and established semiquantitative methods in two laboratories, b) develop a software tool for automatic evaluation of quantifiable aspects of the mammary ductal tree, and c) compare those methods. Conclusions: Both the semiquantitative and the quantitative methods revealed nonmonotonic effects of BPA. The quantitative unsupervised analysis used 91 measurements and produced the most striking nonmonotonic dose–response curves. At all time points, lower doses resulted in larger effects, consistent with the core study, which revealed a significant increase of mammary adenocarcinoma incidence in the stop-dose animals at the lowest BPA dose tested.
A combined morphometric and statistical approach to assess non-monotonicity in the developing mammary gland of rats in the CLARITY-BPA study
Background: The Consortium Linking Academic and Regulatory Insights on Bisphenol-A (CLARITY-BPA) is a rare collaboration of guideline-compliant (core) studies and academic hypothesis-based studies to assess the effects of bisphenol A (BPA).
Objectives: We aimed to a) determine whether BPA showed effects on the developing rat mammary gland using new quantitative and established semiquantitative methods in two laboratories, b) develop a software tool for automatic evaluation of quantifiable aspects of the mammary ductal tree, and c) compare those methods.
Methods: Sprague-Dawley rats were exposed to BPA, vehicle, or positive control [ethinyl estradiol (EE2)] by oral gavage beginning on gestational day (GD)6 and continuing with direct dosing of the pups after birth. There were two studies: subchronic and chronic. The latter used two exposure regimes, one stopping at postnatal day (PND)21 (stop-dose) the other continuing until tissue harvest (continuous). Glands were harvested at multiple time points; whole mounts and histological specimens were analyzed blinded to treatment.
Results: The subchronic study’s semiquantitative analysis revealed no significant differences between control and BPA dose groups at PND21, whereas at PND90 there were significant differences between control and the lowest BPA dose and between control and the lowest EE2 dose in animals in estrus. Quantitative, automatized analysis of the chronic PND21 specimens displayed nonmonotonic BPA effects, with a breaking point between the 25 and
Conclusions: Both the semiquantitative and the quantitative methods revealed nonmonotonic effects of BPA. The quantitative unsupervised analysis used
The Consortium Linking Academic and Regulatory Insights on Bisphenol-A (CLARITY-BPA) is a collaboration between academic and federal government scientists, organized by the National Toxicology Program (NTP), the National Institute of Environmental Health Sciences (NIEHS), and the U.S. Food and Drug Administration (FDA) National Center for Toxicological Research (NCTR). This research consortium was “expected to significantly improve the interpretation of the wealth of data that is being generated by all consortium partners, including the characterization of the dose response of the effects observed and their interpretation in an integrated biological context” ( Schug et al. 2013; Heindel et al. 2015).
The endocrine disruptor bisphenol A (BPA) is widely employed in the manufacture of polycarbonate plastics and epoxy resins. It is present in various consumer products used on a daily basis ( Vandenberg et al. 2013b), such as thermal paper ( Thayer et al. 2016; Hehn 2016). BPA had a
Regarding the etiology of breast cancer, exposure to estrogens during a woman’s lifetime has long been considered a main risk factor (IBCERCC 2013; Kotsopoulos et al. 2010). Developmental exposure (fetal and neonatal) to natural estrogens and estrogen mimics has long been proposed to increase the risk of developing breast cancer (Trichopoulos 1990). This hypothesis is backed by more recent data showing that iatrogenic exposure to diethylstilbestrol (DES) as well as environmental exposure to the estrogenic pesticide dichlorodiphenyltrichloroethane (DDT) during fetal life increases the risk of developing breast cancer ( Hoover et al. 2011; Palmer et al. 2002; Cohn et al. 2015). Likewise, the ubiquitous xenoestrogen BPA increased the propensity of developing mammary lesions in rodents ( Murray et al. 2007; Acevedo et al. 2013; Durando et al. 2007; Lamartiniere et al. 2011; Jenkins et al. 2011). These data were gathered using different rat and mouse strains, different routes and timing of exposure, and different diets. Despite all these differences, the increased risk of effect attributed to BPA was consistent.
Our previous work on BPA-induced mammary gland carcinogenesis used the mouse model to address the effect of fetal and neonatal exposure on mammary gland morphogenesis ( Vandenberg et al. 2007; Markey et al. 2001; Muñoz-de-Toro et al. 2005; Wadia et al. 2013; Sonnenschein et al. 2011). The tissue organization field theory (TOFT) of carcinogenesis posits that carcinogenesis is akin to development gone awry (Soto and Sonnenschein 2011; Sonnenschein and Soto 2016). With this theoretical framework and previous data in mind, the main objective of the present study was to explore the effects of gestational and postnatal exposure to BPA on the morphogenesis of the rat mammary gland, as part of the CLARITY-BPA program.
Although the mouse mammary gland is easily amenable to morphometric measurements from its earliest developmental stage to full maturity due to the flat, planar structure of the ductal tree, the rat mammary gland poses challenges due to the florid structure of the ductal tree, which grows more conspicuously into the third dimension. This feature of the rat mammary gland hinders the application of conventional morphometric tools to the analysis of the rat mammary ductal system (Stanko et al. 2015). Hence, the second, subordinate objective of this work was to develop a proper software tool to perform computer driven, unsupervised analysis of the structure of the rat mammary ductal tree. An associated objective was to provide a comparison between the semiquantitative methods used to analyze the rat mammary gland ( Davis and Fenton 2013) and the novel quantitative methods we are describing herein.
The use of five BPA doses over a wide dose range, allowed us to explore the shape of the dose–response curve to BPA for the mammary gland end points examined in this study. Traditional toxicological methods assume a linear response at low doses in order to infer the lack of adverse effects at low dose from responses at higher doses. By contrast, endocrinology acknowledges nonmonotonic responses, that is to say, situations where a compound can lead to an effect at low dose and no effect or its opposite at a higher dose. Nonmonotonic effects are more difficult to analyze statistically, in part because the shape of nonmonotonic response curves is diverse—by definition they only need to display at least one change of trend (Vandenberg et al. 2013a). The last objective of this study was to assess and characterize the nonmonotonicity of the dose response by a combination of methods.
The American Statistical Association (ASA) and, later, a broad group of statisticians have recently criticized the overreliance on p-values to decide whether a result is scientifically significant ( Amrhein et al. 2019) and the difficulty is peculiar in biology where organisms are different individuals ( Montévil 2019). We take into account these perspectives in several ways. First, we distinguish exploratory and confirmatory analysis. In this study, we first performed an exploratory analysis and subsequently a confirmatory analysis on a different data set. These analyses provide evidence of a peculiar feature in the response curve, where we build our argument by leveraging the nonlinearity of the response. However, following the ASA, the resulting statistical argument alone is insufficient and we also perform a second round of exploratory analyses to investigate more specific biological manifestations of the phenomenon. The latter will hopefully be investigated further, confirmed, and theorized in other studies.
Materials and Methods
This study was conducted as part of the CLARITY-BPA consortium and we were provided with uniquely identified samples. The methods for this consortium have been published in detail ( Delclos et al. 2014; Heindel et al. 2015; Churchwell et al. 2014) but are briefly described below.
The CLARITY-BPA studies used the NCTR-specific Sprague-Dawley rat model and included five BPA doses, as well as a vehicle control and two doses of a positive reference estrogen control [ethinyl estradiol (EE2)]. Although the entire study included a very large number of animals, we were provided with tissues from a subset of these animals (
BPA [Chemical Abstract Service number (CASN) 80-05-7; TCI America;
Timed-pregnant rats that generated offspring used in these studies were dosed by gavage at a rate of
90-d subchronic study design (pilot study).
Four doses of BPA (2.5, 25, 260,
Chronic study design.
Five doses of BPA (2.5, 25, 250, 2,500, and
All doses were administered at NCTR by daily gavage with a modified Hamilton Microlab 500 series programable pump (Lewis et al. 2010). Dosing was always conducted from the lowest to highest dose within a dosing pump, and cleaning and maintenance of the equipment were performed as described by Delclos et al.( 2014). The accuracy of dose delivery from the pumps was assessed every 3 months and established to be within 10% of the target volume accuracy.
Litters were randomly culled to 3–5 female:3–5 male pups per litter on PND1. Direct gavage dosing of pups at the same dose level of vehicle, BPA, or EE2 as their dams started on PND1 (day of birth is PND0). Therefore, negligible lactational transfer of treatments was anticipated in this study (Doerge et al. 2010). Each dose group in the chronic study was split into two dosing arms, a continuous-dose (CD) group and a stop-dose (SD) group, with the latter having treatment terminated at weaning on PND21. Terminal body weight was assessed for all animals at time of humane euthanasia. Samples from each group (same end point and study arm) all come from different animals from different litters (one sample per litter).
Offspring were euthanized on PND21 and PND90 (both subchronic and chronic study), as well as at 6 months of age (chronic study only). One female per litter was necropsied [
The fourth inguinal mammary glands were collected per animal. One mammary gland was whole mounted to a charged glass slide and fixed in 70% ethyl alcohol (ETOH) while the contralateral was placed in a cassette and fixed in 70% ETOH in a sealed plastic bag. The fixed mammary glands were shipped from NCTR to Tufts University School of Medicine. The whole mounted glands were stained with carmine and processed as previously described ( Maffini et al. 2005), and the contralateral glands were processed through ethanol gradients, paraffin embedded, and sectioned at
The whole mounts were evaluated by morphometric analysis using semiquantitative methods (PND21P, PND90P, and PND21C), an automatic morphometric method (PND21C), and a standard, nonautomatic quantitative morphometric assay (PND90SD, PND90CD, 6MSD, and 6MCD). Sections of the PND90 fixed mammary glands were used to assess the time course of histoarchitectural changes and the emergence of preneoplastic and neoplastic lesions. All samples were received without knowledge of treatment group, and data were not decoded until data collection of histological and morphometric analyses were complete and the raw data were recorded in the NTP Chemical Effects in Biological Systems database.
Mammary Gland Scoring of Development
Semiquantitative mammary gland scoring.
In the subchronic BPA study (Delclos et al. 2014), the negative and positive control samples were identified a priori to the investigators and were evaluated to determine the range of response. A stereomicroscope (Nikon SMZ800; Nikon Instruments, Inc.) was used to develop the range of scores reported in Figure S1, which was modified for the range of responses in this study from the criteria reported by Davis and Fenton (2013). A score of 7 represents a gland that is most well developed, whereas a score of 1 suggests that few of the necessary developmental criteria are present. The scoring was adjusted to a 7-point scale, for both PND21 and PND90, because there was a dramatic difference between control and high-dose EE2 groups [
In a blinded manner, slides from PND21P-, PND21C-, and PND90P-treated and control animals were evaluated using the scoring criteria summarized in Table S1. Stacks of slides were created for each score and all mammary glands within each score were reviewed a second and third time to ensure that the scores were assigned consistently over the course of the evaluation. Two individuals with knowledge of rat mammary morphology independently evaluated all slide sets. Disagreement in score of more than a full point for any sample required reconciliation between the two scorers. When this occurred, the two scorers together revisited their scores and notes on those slides in question and decided a new score that was most appropriate given the criterion in Table S1. The new scores were less than a full point from each other.
The PND90P whole mounts were adjusted for age and stage of development. For instance, number of branch points, size, lobule formation, and density were important contributors to assigned scores.
Nonautomatic Quantitative Mammary Gland Morphometric Analysis
In the PND90 and 6-month mammary gland quantitative analysis, mammary glands from PND90P, PND90CD, PND90SD, 6MCD, and 6MSD animals were assessed for overall glandular development and density. Wet mammary gland weight was recorded at the NCTR at time of collection. The chronic study glands were imaged with a Stemi 2000 stereomicroscope (Carl Zeiss) and Axiovision software (Carl Zeiss). Image J (NIH) software ( Schneider et al. 2012) was used to process and analyze captured images to assess epithelial density of the gland (a measure of total fat pad area and epithelial area). Three standardized separate areas of each gland were measured to determine average density of the gland. Area 1 (rostral) was closer to the third mammary gland, Area 2 was in the middle of the gland, and Area 3 (caudal) was closer to the fifth mammary gland. The thickness of these whole mounts precluded a complete scan using a confocal microscope. Moreover, the thickness of these whole mounts was variable, preventing equivalent sampling. Therefore, PND90 and 6-month glands were visually scored for the following countable morphological parameters: number of leading edge/internal terminal ends, as well as incidence of lateral branching, lateral budding, alveolar budding, and lobuloalveolar development. Putative lesions identified in whole mounts were excised for histopathological assessment.
Automatic Morphometric Analysis of PND21 Mammary Glands in Chronic Study
In order to reduce ambiguity in the analysis due to overlapping branches, we obtained optical sections to generate a three-dimensional (3D) image instead of a bright field image of the gland. This method was only applicable to PND21 mammary glands due to their smaller size and thickness compared with the later time points. Samples were imaged with a Zeiss LSM 510 confocal microscope using the auto-fluorescence of carmine as the signal. Due to the large size of the whole glands, the imaging was done on a grid, leading to 150–600 partially overlapping stacks. The resolution used was
Identification of epithelium.
Segmentation separates a region of interest from the background. In the mammary gland, the region of interest is the epithelium, whereas the background includes the stroma and the blood and lymph vessels. Currently available algorithms for reconstructing branching structures in the vascular system (Luboz et al. 2005) cannot be used for the mammary gland owing to the presence of ductal buds (shown in Figure 2A). Therefore, we designed a custom automatic method. In addition, due to optical limitations, the presence of the lumen did not provide a consistent pattern that could be used for segmentation. Because of this limitation, we found it easier to segment the stroma first instead of focusing directly on the epithelium. The segmentation algorithm used the following steps:
To remove nuclei of stroma cells and noise from image acquisition, we used bilateral filtering (with spatial radius 4 and range 150) followed by the subtraction of local background. The resulting image was then used for the segmentation.
The image was inverted, and the stroma segmented as a bright connected region, with a uniform threshold. Then, 3D Gaussian blur (radius 2) was applied to the resulting binary image to remove small structures such as blood vessels or adipocytes. Next, the image was inverted and the epithelium was obtained as the connected region above a given brightness, which included a point in the epithelial tree that had been manually selected. Holes in the epithelium, which were due to the lumen, were filled in and another Gaussian blur was performed. Finally, we performed a second selection of the connected region corresponding to the epithelium and above a given brightness. This second segmentation reduced possible artifacts that mostly stemmed from small blood vessels and adipocytes.
Human intervention was required for comparing the segmented epithelium with the original image. Although this means that the method is semiautomatic in the strictest sense, we will continue to refer to it as automatic here to avoid confusion. The purpose of this comparison was, first, to assess whether all the epithelium was accurately segmented. Missing epithelium typically corresponds to a loss of brightness in deeper parts of the sample or particularly thin epithelial structures. Second, the user ensured that structures other than mammary epithelium were not segmented (such as blood and lymph vessels or lymph nodes). If the output was deemed acceptable, segmentation was complete; otherwise, human intervention was required to correct the segmentation issues. Intervention corrected the stacks that were used at the beginning of Step 2. Epithelial structures lost during segmentation were recovered by increasing the brightness. Nonepithelial structures were removed either completely or by decreasing the brightness around these structures. After this operation was performed, the program went back to Step 2, performing the segmentation and subsequent verification again.
Extraction of quantitative morphological features.
The result of segmentation was a 3D reconstruction of the epithelium, which is illustrated by the green layer of Figure 2B. This representation of the epithelium was then used to extract several quantitative morphological features of mammary glands. Analyses were performed using ImageJ. Before performing these analyses, we automatically standardized the orientation of the glands on the basis of the axes of inertia. We first analyzed the properties of the 3D epithelial reconstruction on the x–y plane, which was comparable with assessments performed on bright field microscopy, such as the semiquantitative scoring. The analysis included quantities such as the aspect ratio (AR; length/width), the epithelial area, and the fractal dimension of the epithelium in two-dimensional (2D) (the projection of its 3D image). The same kind of global analysis was also performed in 3D and included an evaluation of the surface of the epithelium, of its volume, and of its 3D fractal dimension (based on the box counting method) ( Longo and Montévil 2014).
Last, the analysis used several plugins from ImageJ: the 3D object counter (Bolte and Cordelières 2006), the plugin 3D shape ( Sheets et al. 2013), and the bonej plugins (Doube et al. 2010). The latter included an evaluation of the local thickness (Figure 2C), which was performed after the normalization of the scales of the three spatial dimensions. The epithelium was skeletonized (Figure 2D) and this skeleton was analyzed by generic methods (counting the number of branches, average branch length, and so on). The analysis was performed both with and without terminal branches (pruning) given that some of the terminal branches may not have corresponded to actual epithelial structures but may have been artifacts from the process of skeletonization.
Finally, a more specialized approach to reconstructing the epithelial tree was performed by a custom plugin. This plugin started from the skeleton generated as discussed above and a manual selection of the starting point of the gland (the point of attachment). The plugin then reconstructed the mammary tree with the main duct as the root. To identify secondary branching, we performed the following operation recursively: For each branching, the size (depth) of the two subtrees was assessed; if the ratio between these depths was smaller than 0.3, then the branch associated with the smaller subtree, A, was identified as a secondary branch and the other, B, was identified as a part of the parent duct. In this case, the parent branch and B were merged. This reconstruction was then used as the basis for evaluating various properties. When quantities are defined per branch, the average over all branches is reported. To filter biologically relevant shapes, we report two versions of these quantities: one where we excluded branches smaller than
Overall our method assessed 91 structural features of mammary glands (see Table S2). Three complementary features were added: animal weight, mammary gland weight, and manual assessment of the number of TEBs.
Rationale of the statistical analysis.
In the automatic analysis, six dose groups (vehicle and five BPA doses) containing 10 animals each were used and all animals came from different litters. More than 90 end points for each animal were measured to assess dose responses. This is quite different than the customary situation when one animal provides a much smaller number of end points, with the exception being certain types of -omics studies (transcriptomics-metabolomics). These situations (our quantitative measurements and the -omics) are more conducive to different types of analysis, such as principal component analysis (PCA), the permutation tests, and so on ( Goh and Wong 2018). In addition, classical statistical tests such as Dunnett’s t and Student’s t lose power rapidly when the numbers of end points and doses increase. With the number of tests necessary for our quantitative experimental designs, these tests are not generally useful.
Another consideration is that there are neither theoretical nor empirical bases that predict a specific type of dose–response curve for BPA. Empirically, BPA dose–response curves could be monotonic for some end points and nonmonotonic for other end points (Villar-Pazos et al. 2017); this is also the case with natural estrogens when comparing the effects on the uterus (monotonic) and the mammary gland (nonmonotonic), using the same animal set (Vandenberg et al. 2006). Here, we consider morphological features that are the result of nonlinear processes of morphogenesis at the tissue level in vivo, where many levels of organization are entangled.
We focus on showing that a specific dose is the locus of a breaking point that is a specific kind of nonlinear behavior and use the permutation test to assess this hypothesis. In our analysis, we distinguish exploratory and confirmatory statistics and perform both to show the presence of this breaking point. Then, we performed exploratory analysis to further characterize this response, the main biological features involved, and the shape of their response by phenomenological curve-fitting.
Principal component analysis.
We performed PCA with R (version 3.5.3; R Development Core Team) and the FactoMineR package (Lê et al. 2008). We use the dimdesc function of this package to assess the meaning of dimensions resulting from PCA and the effect of treatments; for further details see “Supplementary Analysis by PCA” in the Supplemental Material.
Global analysis to identify a breaking point.
In PND21C, we noted that the response curve of many features seemed to possess a specific property. However, there are several problems to analyzing this situation. a) In many cases, individual features and specific doses are not statistically significant alone because variability is high and generic corrections of multiple comparisons cripple statistical significance. b) The recurrence of this pattern could very well stem from a common, random origin because different features of an animal can be correlated. c) Introducing a specific target of statistical analysis always bears the risk of choosing a pattern specific to the data—provided that even purely random data will have patterns.
To build on the diversity of features measured above (a) and avoid errors stemming from multiple comparisons of nonindependent variables (a and b), we designed an analysis at the level of whole data sets combined. To validate the analysis beyond a single data set (c), we used the PND21C data set to formulate a precise statistical hypothesis and we used the four other animal sets of the chronic study (PND90CD, PND90SD, 6MCD, and 6MSD) together for a confirmatory analysis.
Each data set stemmed from a unique set of animals. However, the different features observed for an animal are not independent a priori (b); for example, the total length of the epithelial branches and their volume are correlated. To accommodate this complex structure, we use the permutation test. Unlike traditional tests that use a standard distribution, the permutation test builds the statistic of the intended random variable on the basis of the data and the statistical hypothesis. Because some animals of different data sets come from the same litter, we also evaluated possible litter effects by the permutation test.
Defining the random variable X.
The responses detected in PND21C were not U-shaped; instead they seem to be characterized by a sudden drop or a breaking point—these two latter patterns cannot be set apart on purely empirical bases. We used the PND21C data set to propose a variable X describing the presence of an overall breaking point between the consecutive treatments,
More precisely, for each variable measured (see list in Table S2 for PND21C), we identified the consecutive concentrations where the difference was the largest. To avoid differences that stemmed from noise, we added an algorithmic criterion to check whether a difference was large enough to be included. We proposed two types of criteria:
The first series of criteria, A(
The second series of criteria, B(
We considered the variables:
The null hypothesis (
To assess whether our results were significant, we used the Monte Carlo permutation test (Nichols and Holmes 2002). Like most statistical tests, the permutation test assesses whether
No effect of the treatment means that randomly shuffling the exposure group label in the data set yields an outcome that is equally probable as that of the initial data set. Therefore, performing several such permutations and computing the resulting value of X every time generates an estimation of the statistic of X:
This operation was iterated 10,000 times to obtain the distributions of
Next, we looked at the threshold (
For this method, we assessed type 1 and type 2 error rates by simulations. To this end we used distributions mimicking our data. First we used Gaussian distributions with standard deviation 1 and mean 0, a/2, a, 0, a/2, a, where a is a parameter that determines the magnitude of the breaking point (the standard deviation being 1,
Mean comparisons and correlations.
The semiquantitative developmental score is a synthetic quantity where the problems mentioned above do not apply, and its use corresponds to the assumption that BPA effects are similar to EE2 effects. Analysis of variance was used to assess the effect of BPA treatment on body weight and mammary gland weight, as well as the interaction of body weight or mammary gland weight with semiquantitative developmental scores. The effect of BPA or EE2 on semiquantitative developmental scores were analyzed by Kruskal-Wallis nonparametric tests and a Dunn’s post hoc comparison of vehicle vs. treated glands [all at PND21, and at PND90 from females in the estrus stage only, and in all stages not including diestrus and metestrus (GraphPad Prism, version 7.05)]. Trends in effect were indicated at
For other quantitative and unsupervised measures, we used the Student’s t-test to perform an exploratory comparison of means between control, 0.5EE2 and a dose singled out by the other analyses on PND21. The normality of the distributions was assessed by the Shapiro test. When this criterion was not met, we used a simple permutation test for the absolute difference in mean. We control the false discovery rate due to multiple comparisons, q, by the location-based estimator method described in and implemented in the LBE package for R (Dalmasso et al. 2005).
We also performed multiple comparisons between control and the other doses using Dunnett’s t-test, controlling normality with the Shapiro test. To analyze correlations, we used Pearson’s product-moment correlation implemented in R (version 3.5.3; R Development Core Team). In the box plots, we define outliers using the 1.5 interquartile range method.
To perform regression, we used the linear model (lm function of R) on variables of interest. We compared the performance of the chosen model with simpler models (having fewer parameters) with a likelihood ratio (LR) test (using the lrtest function of R). We produced graphs for the qualitative assessment of normality and the distribution of residuals in supplementary materials. Given that we were performing multiple regressions, we used the LBE package to control the false discovery rate q.
We considered three hypotheses: (i) BPA effects were qualitatively similar to the effects of 0.5EE2, (ii) BPA impacted different features and/or had opposite effects of 0.5EE2, and (iii) BPA had no effect on mammary gland development.
Semiquantitative Developmental Scoring of Glands
PND21 mammary gland development.
Assessment of PND21P mammary gland development parameters showed that EE2 5.0 produced extensive ductal growth, twice the average semiquantitative developmental score of vehicle controls (see Figure S1A). These weanling mammary ductal trees displayed developmental characteristics akin to adult mammary glands (see Figure S1D). Therefore, this dose was deemed inappropriate as a positive control and doses were reduced to 0.5 and 0.05 for the chronic study. Although the effects of EE2 (at both 0.5 and 5.0) were significant, there were no statistically significant effects of BPA on the PND21P mammary glands in the subchronic study (see Figure S1A).
In the chronic study, analysis of variance did not show significant effects of BPA and EE2 exposures on the body weight of female weanlings nor on the weight of the excised mammary fat pad (see Figure S3). Scoring of PND21C mammary gland morphology revealed that only treatment with 0.5EE2 resulted in significantly different glandular development compared with vehicle control (
PND90 and 6-month mammary gland development.
Mammary whole mounts of PND90P animals were assessed for semiquantitative developmental scoring. Females in the subchronic study were not necropsied at a predetermined stage of the estrous cycle. Analyses of semiquantitative developmental scores accounted for dose group and vaginal pathology-based cycle stage, and those data demonstrated significant estrous
Global Analysis: 25–250BPA as a Breaking Point
Exploratory PCA on PND21C computer-assisted morphological measurements.
PCA provided an overview of the 91 structural features (see Table S2) assessed in PN21SD mammary glands through our computational analyses, plus three additional features assessed separately (body and mammary weights and TEB number). PCA revealed that the high dose 0.5EE2 produced a strong change in mammary gland morphology (Figure 5A). Dimension (Dim) 1 represented the size of the gland and its highest correlation was with the number of branches and the surface area of the epithelium in 3D [correlation coefficient (CC): 0.97;
Because the dimensions of PCA depend on the data set used and EE2 conditions have a specific biological meaning, we assessed whether excluding them changes the results. We did not find a significant difference. For example, Dim 3 again separates 250BPA from other conditions (
PCA provides clues against hypothesis (i), BPA effects are similar to EE2, and for (ii), BPA treatment is associated with different morphological changes than that of 0.5EE2. Moreover, PCA shows significant differences between BPA treatments and vehicle, which suggests that the hypothesis that BPA has no effect (iii) does not hold.
In the following section we assess whether the nonmonotonic pattern around 25BPA and 250BPA is real, provided that it could be nonsignificant or an artifact from PCA.
Hypothesis formulation on the basis of PND21C data sets.
We used our PND21C results as the basis to formulate our statistical hypothesis, and we used the four other independent data sets (PND90CD, PND90SD, 6MCD, 6MSD) to test this hypothesis. As detailed in the “Methods” section, a simple way to formulate our question was to look at every feature we measured and for each of them to assess which consecutive concentrations have the largest difference. We used criteria to assess differences and decide whether they were sufficient to be taken into account. With the Criterion A(
In PND21C, with Criterion B(
|Control – 2.5BPA||2.5 – 25BPA||25–250BPA||250–2500BPA||2500BPA – 25000 BPA|
|1 (no threshold)||15||6||52||7||9|
Note: Differences are counted when the significance of this difference has a p-value lower than
On the basis of this result and the discussion above, we hypothesized that the consecutive concentrations 25–250BPA is the locus of the largest change for most variables. For a given data set t and a given Criterion, X(t) is the proportion of quantities whose largest consecutive difference meeting the criterion is between 25BPA and 250BPA, for example, for data set PND21C and Criterion B(0.5),
|Data set||Control to 2.5 BPA||2.5 to 25 BPA||25 to 250 BPA||250 to 2 500 BPA||2500 to 25 000 BPA||X ( t)||p-Value|
|PND21C (training set)||14||5||57||6||9||0.62||0.62||0.0094|
Note: For Criterion B with
To assess whether we should reject
Confirmatory analysis with PND90CD, PND90SD, 6MCD, and 6MSD data sets.
We used the remaining, independent data sets PND90CD, PND90SD, 6MCD, and 6MSD for a confirmatory analysis. For example, Table 2 provides details in the case of Criterion B(0.5). The latter leads to
|Criterion|| 95% of || 99.5% of |
Note: Results for other thresholds can be found in Table S3. Criterion A(1.2) [B(0.5)] means that the ratio (p-value of a t-test, respectively) between successive means has to be at least 1.2 [0.5] to be taken into account. BPA, bisphenol A; CD, continuous dose; M, month; PND, postnatal day; SD, stop-dose.
Taking litters into account did not change the result significantly, with
To conclude this global analysis, it is noteworthy that the semiquantitative scoring did, in fact, display graphically, but not robustly enough to show statistical significance, a nonmonotonic response with a slight breaking point between 25BPA and 250BPA-exposed glands in PND21C (Figure 4) and a more pronounced one in PND21P (see Figure S1) and PND90P (see Figure S4). PND21P and PND90P are animal sets that were not used in the global analysis, thus the fact that they reproduce the same pattern qualitatively is suggestive.
In simulations, the type 1 error rate remained close to the target with a loss of precision for
Further Exploratory Analysis of the Response Curve and Comparison with the Effect of EE2
PCA in PND90 and 6-month-old animals.
For PND90CD, the Dim 1 of PCA was correlated with the average density of the gland (0.88,
For PND90SD, Dim 2 was related to the number of TEB (0.57,
For 6MCD rats, Dim 1 was related to alveolar budding (0.78,
For 6MSD animals, Dim 1 was related to lobular alveolar budding (0.65,
A change in the trend of the response, nonmonotonicity, was observed in various measurements obtained from PND21 mammary glands. We analyzed them with a statistical model. For example, one end point was the mean variation of ductal thickness [standard deviation (SD) width 3D], which describes whether structures are more tubular or, the opposite, irregular. This measurement is associated with budding because small buds are not recognized as individual structures and lead instead to duct width variations in the automatic analysis. This quantity increased between control and 25BPA, dropped, and then increased again between 250BPA and 25000BPA ( Figure 8A).
The responses detected seemed to be characterized by a sudden drop or even a breaking point, which implies two changes of trend. The model chosen for describing these data was the sum of a linear response and a step function that models a breaking point:
where a, b, and c were found by linear regression. b represents a linear trend, whereas c quantifies the sudden change between 25 and 250BPA. If b and c have opposite signs, the change between 25 and 250BPA breaks monotonicity. Note that we are not interested in the significance of a because a being different from 0 means that the average response is not 0. To assess the significance of the model, it was not sufficient to show that it fit the data significantly, we also compared it systematically with simpler models: a constant model (no effect of BPA), a linear model, and a step model. We also assessed the quality of the regression graphically (see Figure S9).
At low doses, our model described a linear response for the considered variable (0–25BPA). Then, it led to a drop in the response, which was triggered at a critical concentration that we identified by maximum likelihood. In most cases, this negative effect was between 25BPA and 250BPA. Beginning at 250BPA, increasing BPA levels resulted in a linear response. We emphasize the significance of the nonlinearity of the response that took place between 25BPA and 250BPA ( Figure 8); however, we do not make strong claims on the equational form of the model. Our model is what is usually called a phenomenological or a heuristic model; it reproduced the trends of the dose response of several end points. The choice of a specific model may only be decided by a theoretical discussion (Montévil 2018).
Given that we performed exploratory statistics on all features observed, we assessed the false discovery rate for the comparison with a constant model with the threshold
For SD width 3D, the nonmonotonic model led to a significant fit (
Figure 8B represents the average of the local thickness. At a point, the local thickness equals the radius of the biggest sphere that contains this point and that is contained in the structure. The model’s fit of the data was significant (
Figure 8C represents the fractal dimension in 3D by the box counting method. The higher this quantity, the more the epithelium had filled the fat pad of the gland in 3D. This quantity was an assessment of the complexity of the gland. The fit was significant (
Figure 8D represents the average angle between the beginning and the end of ducts. This quantity assessed how much the ducts change direction during their growth. The fit was not entirely significant with
Figure 8E represents the third dimension constructed by PCA, which was associated with duct length. The linear part of the model was not significant (
Figure 8F represents the AR. This quantity equals the ratio between the largest axis of the gland and its smaller axis and was one aspect of how the epithelium invades the fat pad. For 250–2500BPA instead of 25–250BPA, our model was a good fit (
The existence of various end points exhibiting a nonmonotonic response was against hypothesis (iii), which postulated that BPA is devoid of effect. The conclusion of this exploratory analysis is that nonmonotonicity can be different than quadratic (U-shaped) responses, and that features related to thickness, duct width, fractal dimension in 3D are of interest. The AR also exhibited an interesting pattern, but not with respect to the 25–250BPA breaking point.
In PND90 and 6-month.
Nonmonotonic responses in PND90CD, PND90SD, 6MCD, and 6MSD were found which were similar to the ones in PND21C (Figure 9; Figure S10). More specifically, the gland weight (determined at necropsy) in PND90SD (Figure 9A) was a significant fit to our model (
Interestingly, the same quantity can be described by our model in PND90CD, 6MCD, and 6MSD (Figure 9B–D). This quantity was the branching density of the posterior region of the mammary gland, closest to the fifth mammary gland (Area 3). In PN90CD, the complete model was not a good fit (
In 6MCD, the model was a good fit (
In 6MSD, the situation was similar (0.11, 0.017 for b and c), was better than a constant and a linear model, and almost significantly better than a step model (
Comparison between negative control, BPA inflection point, and positive control (0.5EE2).
Because an inflection point was detected between 25BPA and 250BPA for several features, such as sd width 3D or the fractal dimension in 3D (Figure 8), we systematically investigated differences between 250BPA and control using the t-test for all features observed. We assessed the false discovery rate due to multiple testing with the threshold
Glands of rats exposed to 250BPA were rounder (had a smaller AR) (
The proportion of small branches (buds and small ducts
Branch length measurements.
Branches were defined computationally as a path from a bifurcation to the next bifurcation. The branches were longer on average in 250BPA than in control (
Angle of branches.
When removing small structures (
The average of the number of branching points from a branch to a terminal end (average depth of subtrees) was high when the epithelial tree was more asymmetric and low when, to the contrary, it was more balanced or compact topologically. Epithelial trees were more symmetric (
Depth of the gland.
Similarly, the overall size of the epithelium along the z-axis were lower in 250BPA than in control (
We also performed multiple comparisons between all treatments and control using Dunnett’s test. Despite the important loss of statistical power when performing all comparisons, we found that the average branch length was significantly longer for 2.5BPA than for control, both without (
As illustrated in Figure 10, some of the features analyzed are consistent with hypothesis (i); the response to BPA and EE2 are similar. Others are consistent with hypothesis (ii), where BPA impacts features that EE2 does not impact (iia) and in some cases, BPA had opposite effects than EE2 (iib).
In PND90 and 6-month.
We used the same methodology as above (see Table S4). In PND90CD, the gland density was lower on average in 250BPA-exposed glands than in vehicle (
Performing comparisons between control and other continuously dosed treatment groups showed that gland density (Area 2) in 25BPA and 25000BPA was lower than in vehicle controls (
In PND90SD, lateral budding was higher in 250BPA than in vehicle, albeit not significantly (
In 6MCD, the fat pad area was larger in 250BPA than in vehicle controls (
In 6MSD, the standard deviation of gland density was higher in 250BPA than in control (
Comparison between the automatic measurements and the semiquantitative scoring of PND21C glands.
We compared the automated quantitative measurements of the glands with the semiquantitative developmental scores reported above for the chronic study and found correlations between this score and numerous morphological features. Quantities representative of the highest correlations with the score are the 2D fractal dimension of the gland (CC: 0.88,
The semiquantitative developmental score was also compared with the dimensions resulting from PCA. Table 4 shows that the scoring captured aspects of the two first dimensions of PCA (
|Correlation descriptors|| Dim 1 ( || Dim 2 ( ||Dim 3 (s)|
Note: Dim, dimension; PCA, principal component analysis; PND, postnatal day.
The standard deviation between the semiquantitative assessments of the two observers scoring the glands provided further insight on its relationship with the response to BPA. We interpret this standard deviation as the result of an ambiguity in evaluating the development of some glands when this development is altered. This standard deviation is negatively correlated with the proportion of small branches (
Histopathology in PND90 and 6-month.
Eight lesions were identified in whole mounts and histological sections from eight PND90 mammary glands across both continuous and SD-treatment groups. No lesions manifested in vehicle-treated animals and all lesions were diagnosed as benign or malignant, ranging from lobular hyperplasia, fibroadenoma, periductular fibrosis or ductal epithelial necrosis with lymphocytic infiltration to ductal carcinoma in situ (see Table S5A). Thirty-three total lesions were identified in whole mounts and excised from twenty-four 6-month mammary glands across both continuous and SD-treatment groups. Three malignant tumors (adenocarcinomas) were classified from continuous and SD 0.5EE2 treated females, and the remaining lesions/benign tumors were found in vehicle and 2.5BPA-, 25BPA-, and 25000BPA-treated females. The benign lesions included lobular or ductular alveolar dilatations (with and without secretions), periductular fibrosis (with and without lymphocytic infiltration), fibroadenomas, and adenomas (see Table S5B).
In a rare distribution of tissues from a very large guideline-compliant study to academic grantees, we had an opportunity to evaluate mammary gland specimens from female rats from two studies on the effects of exposure to BPA and EE2. In one study animals were exposed during fetal life until weaning (PND21) while in the other exposure ended at tissue collection at PND90 and 6 months of age.
The mammary gland is considered a sensitive target for endocrine disruption. Measurable effects manifest at low levels of exposure to endocrine disruptors, and these effects appear significantly earlier than the manifestation of mammary gland cancer. Thus, there is considerable interest in including its analysis in the animal tests used for regulatory purposes (Makris 2011; Rudel et al. 2011). At present it is mentioned in a footnote in Organisation for Economic Co-operation and Development (OECD) protocols [i.e., OETC TG443, which recommends that “end points involving pup mammary glands of both sexes be included in this Test Guideline” using validated methods (OECD 2018)]. However, the animal of choice for these regulatory studies was the NCTR-derived Sprague-Dawley rat. In contrast to mouse models (Soto et al. 2013; Paulose et al. 2015), there is a paucity of reports on the effect of fetal BPA exposure on rat mammary gland morphogenesis. This is in part due to the florid structure of the ductal tree which grows more conspicuously into the third dimension and makes quantitative assessment beyond weaning challenging ( Stanko et al. 2015). This feature of the rat mammary gland hinders the use of standard morphometric tools for the analysis of the rat mammary ductal system. Instead, conventional scoring methods are used. They are called semiquantitative because they construct a score from qualitative and countable morphological features, such as terminal end buds (see Table S1). These semiquantitative methods are reproducible, fast, and reliable. In addition, because the scoring method relies on the trained human eye to interpret structures in relation to function and pathology, the scores relate to biological outcomes.
A main objective of this study was to elucidate the shape of the dose–response curve and to test three alternative hypotheses: (i) that EE2 and BPA resulted in similar qualitative effects, (ii) that BPA and EE2 affected different features or had opposite effects, and (iii) that BPA had no effect on mammary gland development. In addition, because the size and thickness of the PND21 mammary glands were compatible with confocal scanning and complete 3D reconstruction of the ductal tree, the PND21 female rats were extensively evaluated for the effects of BPA and EE2 using a quantitative new methodology specially developed for this study.
Evaluation of Early Effects in the Mammary Gland
Consistent with our long history of evaluating both rat and mouse mammary whole mounts using semiquantitative and quantitative methods, we scored and measured glands from PND21 and PND90 in these studies, blinded to treatments. In addition, we postulated that an unsupervised, quantitative, and automated method may discover effects that are difficult to ascertain using the scoring methods. We developed and describe here a method consisting of optical confocal sections to reconstruct the gland and use of appropriate algorithms for its analysis. The choice of PND21 was motivated mostly by the size of these glands and the fact that this prepubertal age precedes the florid and fast development of the ductal system due to ovarian estrogens (Cowie 1949; Masso-Welch et al. 2000; Murray et al. 2007); thus, estrogenic responses, if induced by BPA and EE2, should be detected. The hypothesis behind this choice is that the effect of BPA would be qualitatively similar to that of EE2; that is, BPA will behave as a classical estrogen. We used the same set of mammary glands to compare this new quantitative method with the standard semiquantitative method (Davis and Fenton 2013). Both the semiquantitative and the quantitative methods were able to detect significant differences between the negative control (vehicle) and the positive control (0.5EE2) (Figures 4, 5, 10; Figure S4). However, the results obtained by both methods did not support the default hypothesis used in the experimental design, that BPA and EE2 would produce the same effect on the developing mammary gland.
Evidence for a Breaking Point between 25BPA and 250BPA and Nonmonotonicity in the Dose Response
An important motivation for the development of the quantitative assay was to obtain a precise evaluation of nonmonotonicity. There are inherent differences in assumptions about the shape of the dose–response curve in endocrinology and toxicology. The default assumption in toxicology is monotonicity. In contrast, nonmonotonic dose–response curves are a common occurrence in endocrinology. For instance, the proliferative response for estrogens and androgens follows inverted-U patterns (Stormshak et al. 1976; Amara and Dannies 1983; Soto et al. 1995; Maffini et al. 2002; Geck et al. 2000) and the effect of estradiol on the growth of the mammary ductal system is also nonmonotonic ( Vandenberg et al. 2006). Moreover, distinct end points show different estrogen dose–response curves in different organs of the same animal set: The uterotrophic assay and various other uterine morphological end points are clearly monotonic, whereas those pertaining to ductal mammary gland morphogenesis show mostly nonmonotonic dose–response curves ( Vandenberg et al. 2006).
As expected from examples in the literature, the BPA dose–response showed evidence for nonmonotonicity on data from the quantitative method in PND21C ( Jenkins et al. 2011; Cabaton et al. 2011). The dose–response curves observed for several features was not that of an inverted-U shape, instead it seemed to be characterized by a sudden drop or a breaking point located between 25BPA and 250BPA ( Jenkins et al. 2011).
We used the 91 distinct measurements obtained with the automated method for the analysis of PND21C glands to formulate a statistical test to assess whether 25–250BPA was the locus of a breaking point for a significant number of features. In this data set, our exploratory analysis by the permutation test led us to reject the hypothesis that BPA has no effect in favor of the existence of a breaking point between 25BPA and 250BPA ( Figure 6). To confirm this exploratory result, we used the smaller number of quantitative end points measured at PND90 and 6 months of age. This breaking point in the dose–response to BPA was confirmed by a single, global statistical analysis using the same permutation test (Figure 7). The key of this global analysis is the hypothesis that the breaking point between 25BPA and 250BPA is present at all time points. Again, the test leads us to reject the hypothesis that BPA has no effect in favor of a breaking point between 25BPA and 250BPA. We want to emphasize that performing this single test as a confirmatory analysis, when PND21C is used for the exploratory analysis, is a very rigorous analysis because it avoids making multiple comparisons for the many features and several data sets available. Moreover, the permutation test rigorously accommodates the individuality of each animal (i.e., the test takes into account that many features are correlated). Our overall strategy has been to build on the nonlinear feature observed in PND21C, the breaking point, in order to provide evidence that it remains valid in the other data sets taken together. Although there were no significant discernible effects in the subchronic PND21P mammary glands across BPA-treated groups, the reduced gland development in BPA260 compared with BPA25-exposed animals is consistent with the global analysis obtained from the chronic study animals (PND21C). In addition, in the 90-d subchronic pilot studies (see Figures S1 and S4), the increased development scores in the lowest BPA dose groups is another confirmation of this rationale, even though it failed to reach significance.
Once we have concluded that 25–250BPA is a breaking point, we could perform an exploratory analysis of which features are involved and the more specific shape of the dose response. Here, the analysis is performed with data sets already taken into account above; therefore, it cannot provide a confirmation of the presence of the breaking point. However, it is useful to assess the overall response curve and the features that best represent it. The model chosen was the sum of a linear response and step function because it is significantly better than either a linear model or a step function alone (Figure 8). In PND21C, for many variables, our model appears as a linear response at low doses; a drop in the response appears between 25BPA and 250BPA for most relevant features. At higher BPA doses, a linear response is observed again ( Figure 8). The most striking feature of the dose–response curve is the nonlinearity of the response that takes place between the 25BPA and 250BPA dose. In other data sets, specific quantities were also nonmonotonic such as gland weight in PND90SD and branching density in PND90CD, 6MCD, and 6MSD (Figure 9). Moreover, pair-wise and comparisons between 250BPA, 0.5EE2, and control revealed that some features are consistent with hypothesis (i), namely, the effects of EE2 and BPA are similar, and others with hypothesis (ii), namely, they are different, sometimes opposite ( Figure 10).
These results show the importance of establishing and using statistical methods appropriate for nonmonotonic responses. Linear models are a powerful tool to provide evidence of a causal relationship because they quantitatively relate the changes of a putative cause with the one of the effects. Moreover, linear responses to small causes are a common mathematical property albeit not universal. Therefore, exhibiting a linear response is a powerful method to provide empirical evidence of a causal relationship in a given context. However, this method is blind to nonmonotonic responses. The latter are common in endocrinology because the putative causes are involved in multilevel, complex regulations due to the evolutionary history of hormones and their functions. In this context, a more appropriate way to show the presence of causation is to show the prevalence of a specific nonmonotonic pattern, here a breaking point between 25BPA and 250BPA.
Semiquantitative Scoring and Quantitative Analyses
In PND21C, the scoring method captured the directionality of development as the arrow linking controls with 0.5EE2-treated (Figure 11) in the first two dimensions of PCA of data obtained by the quantitative method (correlated respectively with size and thickness). Indeed, the fact that the scoring method uses EE2 as the control implies that it would preferentially capture the effects of BPA when they mimic those of natural estrogens. Many of the features measured by the quantitative method do not relate directly to the features used for the scoring method, but still provide information about them. For example, the fractal dimension assesses the complexity of the ductal system and the mean variation of ductal thickness is associated with budding. Several of these unique features revealing significant BPA effects when evaluated in 3D are illustrated in Figure 8.
In PND21C, the data and analysis presented here demonstrate that the quantitative method, by using a multitude of automatic measurements, resulted in a greater sensitivity than the semiquantitative method to discriminate effect differences due to BPA dose. The difference between the methods reside in the fact that the quantitative method does not depend on a positive control to score development, and thus is blind to whether the effect of BPA is similar or not to that of EE2 and that the quantitative method interrogates effects existing in the third dimension (i.e., thickness, fractal dimension in 3D, angles).
In PND90 and 6-month-old animals, the scoring method revealed a significant effect of BPA in PND90P mammary glands exposed to BPA from gestation to tissue harvest. This effect was observed only at the BPA2.5 dose and only when the animals were humanely euthanized at estrus, a result consistent with the nonmonotonicity observed at all time points using the quantitative methods. The stage of the estrous cycle by itself appeared to affect the morphological outcomes, thereby validating the importance of assessing all the tissues at the same stage of the cycle for the determination of treatment effect. In fact, the effect of EE20.5 on an increased semiquantitative developmental score was not evident when glands from animals in all stages of the estrous cycle were considered (see Figure S4B). Moreover, these data also suggest that unlike the results in prepuberal PND21 animals, BPA may act in conjunction with endogenous estrogens in adult animals and thus produce a more estrogen-like pattern than that observed for BPA at PND21, that is, consistent with hypothesis (i).
The data presented here demonstrate that PND90 is an appropriate time point to assess the effect of low BPA doses and to reveal nonmonotonicity. They also highlight the importance of assessing the tissue at the same stage in the estrous cycle given that even the pronounced proliferative effect of the positive control on mammary epithelium was not evident when estrous stage was not taken into account in the data analyses. Our results also suggest that EE2 is not a good control for mammary gland end points because the effects of BPA and EE2 were distinct and there was no effect of the 0.05EE2, as expected; leaving us to suppose that the NCTR rat strain may be particularly estrogen insensitive. In summary, most of the results in these sets of mammary glands from cycling rats are consistent with hypothesis (ii), and inconsistent with hypothesis (iii).
Cancer: This Study and the Core Study
As in the mouse model, some effects of BPA are not similar to those of estrogens, for example, inhibition of ductal growth at puberty (Markey et al. 2001; Muñoz-de-Toro et al. 2005), enhanced ductal growth during fetal life ( Vandenberg et al. 2007; Speroni et al. 2017), others are clearly estrogen-like, such as the increased score at PND90P reported here and the accelerated expression of lateral branching in the mouse ( Muñoz-de-Toro et al. 2005). There are also other effects seemingly unrelated to estrogenicity; changes in the stromal fraction of the gland and inflammatory cell responses that have been noted in response to developmental BPA exposures (Tucker et al. 2018; Wadia et al. 2013).
Given that BPA is rapidly metabolized and does not bioaccumulate, the increased propensity of developing mammary cancer in animals exposed to BPA during organogenesis has been attributed to its direct effect on fetal mammary gland development and its indirect effects through the developing hypothalamic–pituitary–ovarian axis (HPOA) (Soto et al. 2013). In the present study, both PND90 and 6-month SD animals displayed a nonmonotonic response to BPA, which confirms the long-lasting effects of early BPA exposure (Table 2). The direct effect of BPA on fetal mammary gland development has been verified using fetal mammary gland explants in an ex vivo model (Speroni et al. 2017). Fetal exposure to BPA affects all the organs of the HPOA, altering ovarian steroidogenesis (Mahalingam et al. 2017; Peretz et al. 2011), hypothalamic controls of luteinizing hormone levels ( Rubin et al. 2006; Acevedo et al. 2018), and the gonadotroph number in the fetal pituitary (Brannick et al. 2012). These alterations, in turn, suggest altered regulation of mammotropic hormones ( Soto et al. 2013). Consistent with these findings, fetal exposure to BPA in mice not only affected the fetal period of mammary gland organogenesis, but also postnatal development, long after cessation of exposure. Alterations in ductal elongation at puberty and lateral branching and budding during adulthood were attributed to altered responses to mammotropic hormones such as estradiol and progesterone (Wadia et al. 2007; Ayyanan et al. 2011). Recent studies confirmed that developmental exposures to other BPA-related substances (Bisphenol S and Bisphenol AF) in mice also induce precocious development of the mammary epithelium and increased epithelial lesions and mammary tumors in adulthood (Tucker et al. 2018). However, these results were obtained in the mouse, which is not considered as good a model for mammary cancer as the rat. In spite of this widely held opinion, developmental exposure to BPA in mice also increased the incidence of mammary cancer in animals treated with a chemical carcinogen during adulthood or in MMTV-erbB2 mice exposed to BPA during adulthood ( Jenkins et al. 2011). It is remarkable that in this model, the effect of BPA was nonmonotonic. Several studies using different rat strains reported the development of hyperplasia, carcinoma in situ and palpable adenocarcinomas of the mammary gland after prenatal or neonatal exposure to BPA ( Mandrup et al. 2016; Murray et al. 2007). Not surprisingly, the CLARITY core study, run concurrently to this study, revealed a significant increase of adenocarcinomas as well as the combination of adenomas or adenocarcinomas in the SD animals treated with 2.5BPA at 2 years of age. EE2 induced a significant increase of adenocarcinomas only at the high dose and they were also detected in our animals (see Table S5) by 6 months of age.
Here we demonstrated that semiquantitative and quantitative methods were suitable to detect estrogenic effects in the mammary glands of NCTR SD rats, and both methods found BPA-induced mammary effects to be different from those of EE2. In addition, the semiquantitative method, by relying on the trained human eye, is better able to interpret structures in relation to function and pathology. The automatic quantitative method, by using a multitude of measurements in 3D, identified statistically significant differences and revealed a nonmonotonic BPA dose–response curve in mammary samples from PND21 animals. The nonmonotonic response was confirmed by a global analysis of quantitative assessment in mammary samples from older animals within the same study. These results show that we can and should take advantage of nonmonotonic properties to perform statistical analysis rigorously, and that these features are not limited to quadratic responses.
Consistent with our finding, the CLARITY core study, which used animals of the same cohort, found that EE2 and BPA are not similar. In the core study EE2 increased the incidence of neoplastic lesions only at the highest dose, whereas BPA only increased their incidence at the lowest dose. The BPA effect was nonmonotonic and differed between the SD and the continuous exposure regime. Thus, dose and duration of exposure contribute to the developmental and neoplastic outcomes. These data are consistent with the multiple non-GLP studies previously conducted demonstrating low-dose BPA exposures induce more adverse responses than high doses and that some low-dose BPA responses are different from those of estrogens and of high-dose BPA.
We acknowledge the technical help provided by L. Camacho and B. Delclos regarding the generation of animals and the dissection of the mammary glands examined in this study. We are also grateful to B. Davis for the histological assessment of the lesions and to our colleagues at Tufts University, C. Sonnenschein and B. Rubin, for their critical reading of the manuscript. We thank S. Baker (National Cancer Institute) for useful suggestions about the statistical design of this study. We are grateful to M. Tremblay-Franco (section of Statistics and Bioinformatics, Plateform MetaToul-AXIOM, INRA Toulouse) and K. Shockley [National Institute of Environmental Health Sciences (NIEHS)] for their critical reading and useful suggestions regarding statistical analysis. This work was supported by grant U01ES020888 from the NIEHS (A.M.S.) and NIEHS funding 1Z01ES102785 (S.E.F. and M.B.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS or the National Institutes of Health.
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Supplementary analyses by PCA
PCA is a method for dimensional reduction, i.e. for summarizing data sets where many quantities are assessed simultaneously. The starting point of PCA is to build new quantities called dimensions (Dim, named Dim 1, Dim 2, etc.) as linear combinations of the original quantities, for example if A, B, C are quantities measured, Dim 1= aA+bB+cC where a, b and c are determined by a computation. The new quantities are built to be independent of each other and to explain as much of the variance as possible. They are sorted by decreasing contribution to variance. The meaning of these dimensions with respect to the original quantities is proper to a given dataset because the coefficients a, b, c,… are different for different datasets. The strength of PCA is that it summarizes data in an automated fashion. Its limitation is that properties not included in the first or first few dimensions may still be biologically relevant (Section 7.5, Linear Algebra and Its Applications 5th Edition David C. Lay, Steven R. Lay, and Judi J. McDonald Pearson 2014). As an example, the subtended area of a gland is correlated to many variables since it is a way to assess the “size” of the gland, but the abundance of epithelial structures per unit volume conveys a different biological meaning. The latter can be more relevant to the understanding of the effect of the treatment even though it can be independent of some “size” variations that dominate spontaneous variability. As a result, the first dimension of PCA may not necessarily be of biological interest when discussing the response to a treatment. Since the dimensions of PCA depend on the entire data set, the results of PCA will be different depending on whether we include the positive controls (0.5EE2 and 0.05EE2) in the analysis, see Figure S5.
Supplementary figures and tables
|Age (PND)||Score||Criterion Used in Semiquantitative Scoring|
|21||1||Poor development, small epithelial growth, minimal branching and budding, few/no TEBs, poor development of cranial aspect of gland 4 (asymmetric)|
|2||Gland almost reaches the lymph node (LN) (retarded growth), little branching or budding, few TEBs, poor development of cranial aspect of gland 4|
|3||Gland touches LN, moderate branching and budding, external TEBs begin to appear around periphery, moderate development of cranial aspect of gland 4|
|4||Gland touches LN, wide with equal antral and dorsal development (symmetric), internal and external TEBs, excellent branching and budding throughout gland, symmetric|
|5||Excessive lateral growth, gland has grown past LN, dense budding with few gaps, internal and external TEBs, external TEBs around entire periphery|
|6||Excessive lateral growth, growth beyond LN, 4th and 5th gland have grown together, dense budding with very few gaps, fewer TEBs because they are beginning to differentiate into lobules (looks like typical development on PND 35 or 50)|
|7||Excessive lateral growth, gland has reached ends of fat pads and are terminally differentiating into lobules, 4th and 5th glands have grown over each other, very dense, difficult to see ducts (looks like young adult gland)|
|90||1||Small gland that fails to fill fat pad, moderate number of TEBs remain, moderate branching and budding with large gaps, minimal to no lobules L1, poor left side development of 4th gland (asymmetry)|
|2||Small to medium gland growth, with several TEB remaining, moderate branching and budding, asymmetry remains, many lobules L1|
|3||Medium sized gland with fair branching and growth, some TEBs, moderate budding with some gaps, small lobules L1-2. There is still some asymmetry of development|
|4||Growth extends in both directions without reaching ends of fat pad, asymmetry is absent, gaps are evident, but branching and budding are moderate, more lobules L1-2 present|
|5||Large gland almost reaching end of fat pad, few TEBs remain, dense branching, moderate budding with some gaps, many lobules L2-3|
|6||Gland extended to ends of fat pad nearly everywhere, dense branching, few TEB remnants remain, budding throughout branches, developed lobules L3, some gaps remain|
|7||Gland has reached ends of fat pad, terminally differentiated with no external or internal TEBs, dense branching and budding, no gaps, developed lobules L2-4, hard to see ducts|
Notes: PND=Postnatal Day, TEBs=Terminal End Buds, LN=Lymph Node, L=Lobule stage
Lobule stage defined in Russo IH and Russo J. 1996. Environ Health Perspect 104:938-967.
|Type of analysis performed||Feature Label||Explanation of Feature Label|
|Weights||Necropsy Weight (g)||Body weight at necropsy (grams)|
|Mammary Gland Weight (mg)||Weight of mammary gland (milligrams)|
|Manual assessment||TEB||Number of terminal end buds|
|Analyses of the 2D projection of the mammary tree||Area (µm2)||Surface of 2D projection (square micrometers)|
|Major (µm)||Size of the major axis of the gland (micrometers)|
|Minor (µm)||Size of the minor axis of the gland (micrometers)|
|Feret (µm)||Feret diameter (micrometers)|
|AR||Aspect ratio of the gland|
|Round||Roundness (inverse aspect ratio)|
|Fractal Dimension||Self-explanatory (higher for denser glands, lower for sparse glands)|
|Extension LV (µm)||Farthest distance from the lymph vessels (LV); negative when it is not reached (micrometers)|
|Vesselp||Proportion of the gland beyond a specific lymph vessel|
|Nodep||Proportion beyond the lymph node|
|Global analyses in 3D||Width (µm)||Width of the gland along its main directions (micrometers)|
|Height (µm)||Height of the gland along its main directions (micrometers)|
|Depth (µm)||Depth of the gland along its main directions (micrometers)|
|Vol (µm3)||Raw volume of epithelium (cubic micrometers)|
|SA (µm2)||Surface of the epithelium (i.e., surface the boundary epithelium/stroma) (square micrometers)|
|Solidity 3D (µm3)||Volume / convex volume (cubic micrometers)|
|Encl Vol (µm3)||Volume with some corrections (cubic micrometers)|
|I1||Momentum of inertia along axis 1|
|I2||Momentum of inertia along axis 2|
|I3||Momentum of inertia along axis 3|
|Euler||Assessment of Euler characteristic, which provides information on the lack of convexity of the object|
|Holes||Number of topological holes.|
|Thickness (µm)||Average local thickness of the gland (estimates the diameter, but biased by the compression exerted on the gland) (micrometers)|
|SD Thickness (µm)||Average local thickness of the gland (estimates the diameter, but biased by the compression exerted on the gland) (micrometers)|
|Max Thickness (µm)||Average local thickness of the gland (estimates the diameter, but biased by the compression exerted on the gland) (micrometers)|
|Dimension 3D||Fractal dimension in 3D - high if the gland fills space in 3 dimension (thick, no lacunarity, high budding, ...)|
|Direct skeleton analysis (raw)||X Branches||Number of branches|
|X Junctions||Number of junctions|
|X Junction Voxels||Number of junction voxels|
|Average Branch Length (µm)||Branch length (micrometers)|
|X Triple Points||Number of bifurcation|
|X Quadruple Points||Number of triple branching|
|Maximum Branch Length (µm)||Maximum branch length (micrometers)|
|Direct skeleton analysis after pruning||X Branches1||Number of branches (only for non-terminal branches)|
|X Junctions1||Number of junctions (only for non-terminal branches)|
|X Junction Voxels1||Number of junction voxels (only for non-terminal branches)|
|X Slab Voxels1||Number of voxels (only for non-terminal branches)|
|Average Branch Length1 (µm)||Branch Length (micrometers) (only for non-terminal branches)|
|X Triple Points1||Number of bifurcation (only for non-terminal branches)|
|X Quadruple Points1||Number of triple branching (only for non-terminal branches)|
|Maximum Branch Length1 (µm)||Maximum branch length (micrometers) (only for non-terminal branches)|
Specialized analysis. When quantities are defined per branch the average over all branches is reported.
All branches larger than 20µm are taken into account.
|Size (µm)||Length of branch (micrometers)|
|Number of Neighbors||Number of disregarded connections|
|Depth from Root||Number of bifurcation from the nipple to the branch|
|Depth Subtree (µm)||Average depth of the subtree of each branch (micrometers)|
|Number of Children||Average number of sub branches|
|Euclidean Distance (µm)||Distance between beginning and end of each branch (micrometers)|
|Tortuosity||Ratio: length of branches /Euclidean distance|
|Angle Between Beginning and End||Angle between beginning and end of a branch|
|Angle with Parent Local||Angle between the end of the parent branch and the beginning the branch|
|Angle with Parent Global||Angle between the direction of the parent branch and the branch|
|Angle Wr Main Dir||Angle between the direction of the branch and the average direction of all branches|
|Length to Nipple (µm)||Distance in the tree between a branch and the nipple (micrometers)|
|Mean Width (µm)||Mean distance map of the branch without the z axis (i.e., 2D width of the branch) (micrometers)|
|Max Width (µm)||Max distance map of the branch without the z axis (i.e., 2D width of the branch) (micrometers)|
|SD Width (µm)||Standard deviation of the distance map of the branch without the z axis (i.e., 2D width of the branch) (micrometers)|
|Mean Width2 (µm)||Mean local thickness of the branch (micrometers)|
|Max Width2 (µm)||Max local thickness of the branch (micrometers)|
|SD Width2 (µm)||Standard deviation of the local thickness of the branch (micrometers)|
|Length Farthest Leaf (µm)||Distance in the tree between a branch and farthest leaf (micrometers)|
|Topodepth||Total depth (number of bifurcation from nipple to the farthest branch)|
|Nblarge||Putative bud clusters (structures with a wide end)|
|Secondary Bud||Putative number of budding from ducts|
|Nbbranchestree||Number of branches|
|Type1 (%)||Percent secondary bifurcation|
|Type2 (%)||Percent subbranches of secondary bifurcations|
Specialized analysis. When quantities are defined per branch the average over all branches is reported.
Only branches larger than 75µm are taken into account.
|Size1 (µm)||Length of branches (micrometers)|
|Number of Neighbours1||Number of disregarded connections|
|Depth from Root1||Number of bifurcation from the nipple to the branch|
|Depth Subtree1 (µm)||Average depth of the subtree of each branch (micrometers)|
|Number of Children1||Average number of sub branches|
|Euclidean Distance1 (µm)||Distance between beginning and end of each branch (micrometers)|
|Tortuosity1||Ratio: length of branches /Euclidean distance|
|Angle Between Beginning and End1||Angle between beginning and end of a branch|
|Angle with Parent Local1||Angle between the end of the parent branch and the beginning the branch|
|Angle with Parent Global1||Angle between the direction of the parent branch and the branch|
|Angle Wr Main Dir1||Angle between the direction of the branch and the average direction of all branches|
|Length to Nipple1 (µm)||Distance in the tree between a branch and the nipple (micrometers)|
|Mean Width1 (µm)||Mean distance map of the branch without the z axis (i.e., 2D width of the branch) (micrometers)|
|Max Width1 (µm)||Max distance map of the branch without the z axis (i.e., 2D width of the branch) (micrometers)|
|SD Width1 (µm)||Standard deviation of the distance map of the branch without the z axis (i.e., 2D width of the branch) (micrometers)|
|Mean Width2.1 (µm)||Mean local thickness of the branch (micrometers)|
|Max Width2.1 (µm)||Max local thickness of the branch (micrometers)|
|SD Width2.1 (µm)||Standard deviation of the local thickness of the branch (micrometers)|
|Length Farthest Leaf1 (µm)||Distance in the tree between a branch and farthest leaf (micrometers)|
|Topodepth1||Total depth (number of bifurcation from nipple to the farthest branch)|
|Nblarge1||Putative bud clusters (structures with a wide end)|
|Secondary Bud1||Putative number of budding from ducts|
|Nbbranchestree1||Number of branches|
|Type1.1 (%)||Percent secondary bifurcation|
|Type2.1 (%)||Percent subbranches of secondary bifurcations|
|Criterion||Xobserved||95 % of Xsim<||99 % of Xsim<||99.5% of Xsim<||Pestimated|
|PND90CD||Average gland density||32.0 ±14.1||18.1 ± 9.4||22.4 ± 7.0|
|PND90CD||Density in the rostral area (area 1)||36.6 ± 19.4||16.8 ± 12.03||28.6 ± 10|
|PND90CD||density in the middle of the gland (area 2)||27.1 ± 14.2||5.4 ± 17.6||11.9 ± 8.6|
|PND90CD||Lobuloalveolar budding||0.1 ± 0.32||0.9 ± 0.57||0.7 ± 0.67|
|PND90SD||lateral budding||1.3 ± 0.68||1.9 ± 0.57||2.4 ± 0.70|
|6MCD||fat pad area cm2||41.1 ± 6.4||47.31 ± 5.4||44.2 ± 4.7|
|6MCD||percent coverage||52.2 ± 4.7||47.1 ± 4.5||57.4 ± 9.9|
|6MSD||standard deviation of gland density||6.58 ± 3.2||14.0 ± 7.3||8.2 ± 5.8|
|6MSD||percent coverage||52.4 ± 7.5||45.8 ± 4.9||53.2 ± 3.8|
|6MSD||Lateral branching||2.6 ± 0.52||2.0 ± 0||2.4 ± 0.52|
|6MSD||Lateral budding||1.6 ± 0.70||1.0 ± 0.47||1.8 ± 0.42|
|6MSD||alveolar budding||1.5 ± 0.85||0.6 ± 0.84||1.7 ± 0.82|
|PND 90 Continuous Dose (PND90CD)|
|Treatment||Animals (n)||Lobular Hyperplasia||Fibroadenoma||Periductular Fibrosis (± lymphocytic infiltration)||Ductal epithelial necrosis with inflammatory infiltrate||DCIS|
|PND 90 Stop Dose (PND90SD)|
|Treatment||Animals (n)||Lobular Hyperplasia||Fibroadenoma||Periductular Fibrosis (± lymphocytic infiltration)||Ductal epithelial necrosis with inflammatory infiltrate||DCIS|
|6 Month Continuous Dose (6MCD)|
|Treatment||Animals (n)||Lobulo/Ductular-alveolar dilatation (± secretions)||Periductular Fibrosis (± lymphocytic infiltration)||Fibroadenoma||Adenoma||Adenocarcinoma (±cyst)|
|6 Month Stop Dose (6MSD)|
|Treatment||Animals (n)||Lobulo/Ductular-alveolar dilatation (± secretions)|| |
(± lymphocytic infiltration)