The plasticizer butyl benzyl phthalate induces genomic changes in rat mammary gland after neonatal/prepubertal exposure

Abstract

Background: Phthalate esters like n-butyl benzyl phthalate (BBP) are widely used plasticizers. BBP has shown endocrine-disrupting properties, thus having a potential effect on hormone-sensitive tissues. The aim of this study is to determine the effect of neonatal/prepubertal exposure (post-natal days 2-20) to BBP on maturation parameters and on the morphology, proliferative index and genomic signature of the rat mammary gland at different ages of development (21, 35, 50 and 100 days).

Results: Here we show that exposure to BBP increased the uterine weight/body weight ratio at 21 days and decreased the body weight at time of vaginal opening. BBP did not induce significant changes on the morphology of the mammary gland, but increased proliferative index in terminal end buds at 35 days and in lobules 1 at several ages. Moreover, BBP had an effect on the genomic profile of the mammary gland mainly at the end of the exposure (21 days), becoming less prominent thereafter. By this age a significant number of genes related to proliferation and differentiation, communication and signal transduction were up-regulated in the glands of the exposed animals.

Conclusion: These results suggest that BBP has an effect in the gene expression profile of the mammary gland.

Figure 1

Proliferative index analyses of the mammary gland. A: Immunohistochemical detection of BrdU incorporation in proliferating cells (brown cells) in the epithelial structures TEB, TD and ducts, AB and Lob1. Olympus BX40 microscope with 40× objective. B: Proliferative index (mean ± SEM) in each epithelial structure at 21, 35, 50 and 100 days of age. *: significantly different compared to control group (p < 0.05).

Figure 2

Genomic changes induced by BBP. Number of differentially expressed genes in the mammary glands of rats exposed neonatally/prepubertally to BBP when compared to controls at different ages. Genes were obtained by confident analysis of microarrays data at p < 0.05.

Figure 3

Comparative expression analyses of selected genes. Gene expression analyses by real time RT-PCR in comparison with results from microarrays. Grey bars represent results obtained in the microarrays, black bars represent results obtained in the real time RT-PCR. Results show the mean fold expression of the treated group related to control group. The functional categories significantly over-represented are indicated (21 days). *: significant differences compared to control group (p < 0.05).