Electronic Theses and Dissertations

Date of Award

1-1-2011

Document Type

Dissertation

Degree Name

Ph.D. in Pharmaceutical Sciences

Department

Biomolecular Sciences

First Advisor

Kristine L Willett

Second Advisor

Brian Scheffler

Third Advisor

David B. Murray

Relational Format

dissertation/thesis

Abstract

DNA methylation is one of the epigenetic mechanisms that controls gene expression and is vulnerable to early life environmental toxicant exposures. Our goal was to use two fish models, Fundulus heteroclitus and Danio rerio (zebrafish), to study the benzo(a)pyrene (BaP) effects on DNA methylation status and whether the alterations could contribute to BaP-mediated reproductive and developmental toxicities. Initially, we used Fundulus to study BaP effects on glycine N-methyltransferase (GNMT) expression throughout development. Fundulus embryos were exposed to waterborne BaP at nominally 10 or 100 μg/L and both GNMT mRNA expression and enzyme activity were measured. Quantitative PCR and whole mount in situ hybridization showed significant GNMT mRNA induction by BaP, whereas enzyme activity was significantly inhibited in the Fundulus embryos. In zebrafish embryos, however, neither significant changes in GNMT gene expression nor enzyme activity were detected after BaP (1, 10 or 100 μg/L) exposure. Similarly, BaP exposure did not change DNA methyltransferase 1 (DNMT1) mRNA expression at 48, 60 or 96 hours post fertilization (hpf) or nuclear DNMT1 enzyme activity at 96 hpf. Direct waterborne BaP treatment at 100 μg/L from 2.5 to 96 hpf to zebrafish embryos did significantly decrease global DNA methylation by 55% and gene-specifically reduced promoter CpG methylation in vasa by 17%. As a result, vasa mRNA expression was significantly increased by 33% after exposure. However, BaP at 100 μg/L did not change CpG island methylation or gene expression in Ras-association domain family member 1 (RASSF1), telomerase reverse transcriptase (TERT), c-Jun and c-Myca at 96 hpf. Teratogenic effects (e.g. delayed hatching, pericardial edema, head and tail deformity) were observed in the BaP-treated embryos. Parental zebrafish were also exposed to 23±2.3 or 42±1.9 μg/L BaP to measure the adverse impact on fish reproduction. Although BaP was detected in the exposed fish ovaries, egg production was not affected. However, when the offspring were continuously exposed, they demonstrated DNA hypomethylation, high mortality and various deformities (e.g. pericardial edema, tube heart, head and tail deformity). In conclusion, BaP can cause global and gene specific DNA methylation changes in zebrafish embryos, which may play a role in BaP-induced reproductive and developmental toxicities.

Concentration/Emphasis

Emphasis: Pharmacology

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