Date of Award

1-1-2017

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Nanoscale Science and Engineering

Program

Nanoscale Engineering

Content Description

1 online resource (iii, v, 72 pages) : illustrations (some color)

Dissertation/Thesis Chair

Michael Fasullo

Committee Members

Yubing Xie, Thomas Begley, Xinxin Ding, Patrick Maxwell

Keywords

budding yeast, CYP1A2, DNA damage, Heterocyclic aromatic amines, NAT2, Aflatoxins, Heterocyclic compounds, Yeast, Cytochrome P-450, DNA repair

Subject Categories

Biomedical Engineering and Bioengineering | Microbiology | Molecular Biology

Abstract

The human response to environmental carcinogens, some of which require metabolic activation, is highly variable. Factors such as environment, lifestyle, and genetics all influence the rates of exposure to and ultimate bioactivation of these compounds. Genetic factors include mutations to cell-cycle regulation, cell proliferation, and DNA repair genes; however, epidemiological studies may lack significance due to inadequate patient numbers. We used budding yeast as a model organism to determine genetic susceptibility to food-associated carcinogens, including aflatoxin (AFB1) and heterocyclic aromatic amines (HAAs). Budding yeast does not contain P450s that activate these compounds, so expression vectors were induced that contain human CYP1A2 and NAT2 genes. CYP1A2 alone activates AFB1, while both CYP1A2 and NAT2 are required for the activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). To measure genotoxic effects, we first introduced P450 and NAT2 genes into a DNA repair double-mutant and a collection of strains with individual DNA repair (rad) gene deletions, and measured growth inhibition. To determine genes with possible resistance, a high-throughput approach for screening the yeast deletion collection expressing CYP1A2 and CYP1A2+NAT2 vectors was developed. In collaboration with Frank Doyle, a computer program was developed to analyze the outputs of these high-throughput analyses. Screens with aflatoxin, identified DNA repair genes that confer resistance to these activated carcinogens. Preliminary data into IQ exposures identified both recombinational repair and DNA damage tolerance genes. Further high throughput analysis were performed using 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), another bioactivatable food carcinogen. Upon exposure to our library with IQ and MeIQx, we identified genes responsible for base-excision repair, DNA damage tolerance, rDNA replication fork blocking, and telomere maintenance that may confer resistance to these activated HAAs. These studies thus open new avenues for exploring resistance to P450-activatable compounds and could be potentially utilized to better identify risk-factors for the development of various cancers.

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