A combined morphometric and statistical approach to assess non-monotonicity in the developing mammary gland of rats in the CLARITY-BPA study

Abstract

Background: 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: 1) determine BPA's effects on the developing rat mammary gland using new quantitative and established semi-quantitative methods in two labs, 2) develop a software tool for semi-automatic evaluation of quantifiable aspects of the mammary ductal tree, and 3) 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 6 and continuing with direct dosing of the pups after birth. There were two studies; sub-chronic and chronic. The latter used two exposure regimes, one stopping at PND21 the other continuing until tissue harvest. 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 non-monotonic BPA effects with a breaking point between the 25 and 250µg/kg/day doses. This breaking point was confirmed by a global statistical analysis of chronic study animals at PND90 and 6 months analyzed by the quantitative method. The BPA response was different from the EE2 effect for many features.Conclusions: Both the semiquantitative and the quantitative methods revealed non-monotonic effects of BPA. The quantitative unsupervised analysis used 91 measurements and produced the most striking non-monotonic 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. 4 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. The use of five BPA doses over a wide dose range, allowed us to explore whether the dose response curve to BPA is monotonic for the mammary gland end points examined in this study. A third objective of this study was to assess the non-monotonicity of the dose response using rigorous statistical tools. Our last objective was to provide a comparison between the semi-quantitative methods used to analyze the rat mammary gland (Davis and Fenton 2013) and the novel quantitative methods we are describing herein. Materials and Methods Experimental DesignThis study was conducted as part of the CLARITY-BPA Conso...

Safeguarding Female Reproductive Health Against Endocrine Disrupting Chemicals—The FREIA Project

“…We will improve whole mount examination in the rat mammary gland. An unbiased and sensitive software tool will be applied for examination of mammary gland whole mounts collected at young age [40]. (4) Puberty is a time with great hormonal changes, including a dramatic increase in DHEA over the adrenarche, yet this is largely overlooked in current test strategies dealing with EDCs.…”