Di-butyl Phthalate Targets Multiple Signaling Mechanisms to Down-Regulate Fetal Testosterone Biosynthesis.

Abstract

The in utero hormonal environment is critical for proper development of the male reproductive system and there is growing concern that increases in various human male reproductive tract abnormalities, including falling sperm counts, hypospadias, cryptorchidism, and testis germ cell cancer are the result of exposure to environmental endocrine disrupting chemicals (EDC). In utero exposure of male rats to these EDCs targets fetal testicular testosterone biosynthesis leading to adverse effects on the developing male reproductive system. This lab has demonstrated that phthalate esters, a widely used class of EDCs, induce reproductive abnormalities in part by down-regulating the transcription of testosterone biosynthesis genes. However, the mechanism and upstream signaling pathways by which DBP inhibits transcription is unknown and explored in this study. This study identified altered protein-DNA interactions in down-regulated genes by quantitative in vivo DNase footprinting and chromatin immunoprecipitation. Using RNAi, gene expression of these factors was knocked-down in MA-10 and R2C Leydig cells. Cells were stimulated and steroidogenic gene expression was quantified with real-time RT-PCR to confirm the role of each factor in testosterone biosynthesis. Additionally, phosphorylation state of each factor was determined following stimulation and subsequent phthalate exposure. Moreover, these results were compared to isolated primary fetal leydig cells to identify the role of these factors in fetal steroidogenesis. Results indicate that the transcription factors sp1, SF-1, and c/ebp β are differentially bound following toxic and non-toxic phthalate exposure. Further, results suggest that these factors work coordinately through multiple or redundant mechanisms to regulate transcription, which reflects the importance of testosterone biosynthesis during fetal testicular development. Taken together, this study characterized the role of each transcription factor in the regulation of steroidogenesis and the signaling mechanism regulating its activity.