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

Abstract

AbstractBackgroundCLARITY-BPA is a rare collaboration of guideline-compliant (core) studies and academic hypothesis-based studies to assess the effects of bisphenol A (BPA).Objectives1) 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.MethodsSprague 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.ResultsThe 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.ConclusionsBoth 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.