Dissecting Mechanisms of Toxicity in HepG2 Cells Using Gene Expression Analysis

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Title: Dissecting Mechanisms of Toxicity in HepG2 Cells Using Gene Expression Analysis
Author: Colton, Heidi Muth
Advisors: Dr. Scott Laster, Committee Co-Chair
Dr. Warren Casey, Committee Co-Chair
Dr. Hosni Hassan, Committee Member
Dr. Steven Libby, Committee Member
Abstract: Reduction/oxidation (redox) balance is a critical component of cell viability. When oxidants reach levels that overcome the ability of antioxidants to eliminate them, it results in damage to cellular macromolecules. The damage to DNA, lipids and protein initiates a cascade of physiological events in response to the oxidative stress. Researchers have been studying these cellular responses by analyzing gene expression and protein activity for many years. Recently, new technologies have emerged that allow scientists to analyze the differential expression of extremely large sets of genes in response to oxidative stressors and other toxicants. Most experiments performed to date have involved a single dose of a chemical and a single timepoint for analysis. However, gene expression has proven to be a dynamic process with many transcriptional changes over a relatively short timecourse. In order to study the dynamic nature of gene expression and its effects on cellular physiology, experiments were performed to analyze the effects of oxidative stress on HepG2 cells over a 24 hour timecourse with a range of doses of the glutathione depletor, diethylmaleate (DEM). Using Clontech microarrays, TaqMan RT-PCR, and assays to measure reduced glutathione (GSH) concentrations and to determine cell cycle status, an overall picture of the effects of oxidative stress in relation to dose and time was created. DEM caused GSH depletion to the extent that cells treated with 1.25mM DEM for 4 hours contained less than 20% of the GSH levels in untreated cells. The redox imbalance caused the transcription of genes that initiate cell cycle arrest, DNA repair, and induction of stress proteins. The p53-independent induction of p21 initiated a cascade of events including the decreased transcription of cyclins that resulted in cell cycle arrest. Additionally, the transcription of stress induced genes such as HSP70 and heme oxygenase-1 exhibited significant time and dose-dependent increases in reponse to DEM. While the genes exhibiting differential expression remained generally the same between doses, it was the time taken for these gene changes to occur that varied greatly from the highest dose to the lowest dose of DEM. These experiments demonstrate the importance of analyzing an effective dose range over an extended time period when using differential gene expression to study the mechanisms of toxicity.
Date: 2002-10-15
Degree: MS
Discipline: Microbiology
URI: http://www.lib.ncsu.edu/resolver/1840.16/2867

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