Date of Award
Consuelo J. Alvarez, Ph.D.
Creosote and pentachlorophenol (PtCP) are two commercial wood preservatives that are regulated by the EPA because of their toxicity to wildlife and humans. Cresote and PtCP have been suspected of causing cancer in humans, but that claim has not been proven. To observe changes in gene expression in organisms exposed to these compounds, a model system such as Saccharomyces cerevisiae (baker's yeast) is used. S. cerevisiae cells were exposed to a creosote concentration of 50ng/ml and to a PtCP concentration of 50uM. Since creosote and PtCP were suspended in methylene chloride and ethanol, respectively, yeast cells were also exposed separately to the solvents as controls. cDNA was prepared from a total RNA extraction of exposed and non-exposed S. cerevisiae cells and was hybridized onto microarray chips containing the entire yeast genome using Genisphere Array Kit procedures. A total of twenty microarray chips were tested (seven creosote chips, seven PtCP chips, three methylene chloride chips, and three ethanol chips) for this study. Analysis of the microarray data was done using MAGIC Tool software and Microsoft Excel to find statistical significance in gene expression. Genes showing significant changes in genes expression underwent real-time polymerase chain reaction (RT-PCR) to validate that their change in gene expression was correctly measured in the microarray experiment and that it is due to genetic regulation. Because creosote and PtCP have been indirectly linked to causing cancer in humans, clustering analysis in MAGIC Tool and BLAST analysis on the National Center for Biotechnology Information (NCBI) website compared genes with significant changes in gene expression to other genes in the S. cerevisiae genome and genes with the human genome. In both experimental treatments, creosote and PtCP, genes with roles in cell cycle regulation, drug transport, transcription regulation, and response to stress had significant changes in gene expression. RT-PCR analysis verified that the changes in gene expression could be validated. Clustering analysis in MAGIC Tool revealed highly correlated gene expression in genes associated with mitotic controls. BLASTn and BLASTp analysis confirmed that some genes with significant changes in gene expression had homology to human nucleotide and protein sequences. Overall, the results of this DNA microarray study of S. cerevisiae cells exposed to wood preservatives are a sign of the necessity for more studies to be done by the EPA and workers' health associations in order to establish job/health regulations. The results of this study could be a starting point for R-1 institutions that concentrate in cancer studies.
Stevens, Madison M., "Gene expression of Saccharomyces cerevisiae exposed to commercial wood preservatives by DNA Microarray Analysis and RT-PCR" (2009). Theses, Dissertations & Honors Papers. 39.