Date of Award

Fall 12-15-2017

Degree Type

Thesis

Degree Name

MS Microbiology

Department

Biology

Advisor

Carolyn S. Bentivegna, Ph.D.

Committee Member

Heping Zhou, Ph.D.

Committee Member

Jessica Cottrell, Ph.D.

Committee Member

Jane Ko, Ph.D.

Keywords

Excitation-emission matrix spectroscopy, PCR-SSCP, Atlantic killifish, polycyclic aromatic hydrocarbons

Abstract

Polycyclic aromatic hydrocarbons (PAHs), constituents of crude oil, are implicated as a potent source of adverse toxicological effects on living organisms. To model the effects of PAHs in response to environmental oil spill disasters a species of killifish (Fundulus heteroclitus) was captured and exposed to crude oil in a laboratory-controlled setting. Over a period of 7 days, fish were dosed with crude oil by gavage, culled, and organs were harvested for analysis. Excitation-emission matrix spectroscopy (EEMS) of gall bladder tissue homogenates was used to verify exposure. Effects of PAHs on the p53 gene were evaluated as an indicator of genotoxicity. P53 is a tumor suppressor protein that when mutated is implicated in a variety of cancers and genomic instability. Specifically, the p53 DNA binding domain of the gene was amplified from liver tissue using PCR and analyzed using single-stranded conformational polymorphism (SSCP). SSCP showed that across control and treated killifish, 8 unique genetic profiles could be identified, indicating a combination of native polymorphisms with two unique profiles only seen in Day 7 exposed fish. EEMS analysis on samples from Days 3 and 7 confirmed PAH exposure. Several experimental profiles found in both control and treated fish were cloned and re-subjected to SSCP. Results showed masked polymorphisms in experimental and control fish. Subsequent DNA sequencing of clones showed multiple point nucleotide changes associated with different SSCP profiles. These polymorphisms were found primarily in introns but also exons, where 4 of 6 changes in exposed fish were substitutions of the amino acid asparagine (Asn) for aspartate (Asp). These findings suggest the possible occurrence of crude oil-related mutagenesis in critical gene targets and provide a process for identifying them.

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