Date of Award

2020

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Pharmaceutical Sciences

First Advisor

Frank A Barile

Second Advisor

Blase C Billack

Third Advisor

Jeanette C Perron

Abstract

Examining epigenetic (EG) manifestations and genomic heterogeneity is a novel perspective to understand opioid induced toxicity. Aberrations in histone protein post-translational modifications (HP-PTM) induce perturbations in chromatin integrity resulting in consequences for genomic expression patterns. In the current study, we hypothesize that chronic exposure to morphine sulfate (MS) alters histone-3-protein (H3)-PTM and disrupts STAT3 gene expression in human induced pluripotent stem cells (iPSC). We analyzed 21 genomic H3-PTM following exposure to 1 and 10 uM MS for 2 and 5 days. The results showed decreases in levels of repressive H3-PTM, namely H3K9me1 (2 day) and H3K27me3 (5 day). To confirm if these changes were reversible, cells were allowed to recover for 3 days in the absence of MS; genomic levels of both H3K9me1 and H3K27me3 rebounded to control levels, suggesting that MS induced EG effects are reversible and not heritable. Additionally, decreases in levels of H3K9me1 and H3K27me3 were concentration dependent and were not antagonized by pre-exposure of iPSC to naltrexone indicating that EG effects are independent of opioid receptor antagonism. Continuous chronic MS exposure for through 10 passages rendered the levels of histone modifications to increase by day 26. In addition, exposure for 2 days resulted in significant up-regulation of STAT3 gene expression which plunged with continuing MS exposure. This characteristic transcriptional up-regulation coupled with translational downregulation of STAT3 demonstrates the ability of MS mediated gene expression disruption. Interestingly, STAT3 protein levels remained at control levels when iPSC were pretreated with naltrexone prior to MS exposure. Controlled regulation of STAT3 signaling pathway is pivotal in sustaining and propagating pluripotency phenotype in stem cells. Furthermore, the levels of phosphorylated STAT3 at residues –tyrosine705 (STAT3-pTyr-705) and –serine727 (STAT3-pSer-727) were down-regulated on day 5 and day 2, correspondingly, following MS exposure. Together, the results indicate that MS alters pre-programmed genomic H3-PTM and induces STAT3 gene expression perturbations in iPSC.

Download

Share

COinS