Xenobiotic effects on male mouse reproductive system and hepatic gene expression and epigenetics: studies with bisphenol A and TCPOBOP
Lodato, Nicholas John
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The nuclear receptor superfamily is a large group of related receptors that bind steroid hormones, signaling molecules, or xenobiotic chemicals and are expressed across many mammalian tissues. The impact of nuclear receptor activation using two different mouse model systems is explored in this thesis: (1) in utero exposure of the environmental xenoestrogen and proposed endocrine disruptor bisphenol A (BPA) and (2) short adult exposures to the mouse constitutive androstane receptor (CAR) specific agonist ligand 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). First, experiments involving the impact of in utero BPA exposure on the male mouse reproductive tract are described. Minimal changes to long-term mouse testis morphology and function were observed as mice treated with BPA in utero did not show significant changes in spermatozoa production or testis histopathology. Microarray analysis showed few persistently dysregulated genes, none of which were validated using qPCR due to high variability among biological replicates. Next, nuclear RNA-seq was used to characterize global changes in the mouse liver transcriptome following exposure to TCPOBOP, including changes in novel long non-coding RNAs that may contribute to xenobiotic-induced pathophysiology. Dysregulated protein coding genes were associated with a striking male-biased pro-tumor response, including activation of pro-tumor upstream regulators such as cyclin D1 and inhibition of tumor suppressors such as p21 and p53, consistent with the reported male-biased susceptibility to CAR-dependent mouse liver tumorigenesis. Novel long non-coding RNAs were identified in livers of mice exposed to TCPOBOP, including lncRNAs proximal to the CAR target genes like Cyp2b10. Then, DNase-seq was used to identify DHS in male and female mouse liver that open or close following TCPOBOP treatment proximal to CAR responsive coding and non-coding genes. Finally, a series of ChIP-seq experiments targeting the activating histone modifications H3K4me1, H3K4me3 and H3K27ac, and the repressive chromatin modification H3K27me3 were performed in male mice to characterize the corresponding changes in local chromatin environment around DHS and responsive genes. Using a combination of DNase-seq and ChIP-seq, several classes of DNA regulatory elements have been identified, including active enhancers and promoter regions that may play a function role in regulating nearby CAR-responsive protein-coding and lncRNA genes.