Toxicogenomics : a transcriptomics approach to assess the toxicity of 4-nitrophenol to Sachharomyces cerevisiae

V 1. Toxicogenomics: Transcriptomics of yeast exposed to 4-nitrophenol . 1 1.1 General introduction . 1 1.2 Pollution & toxicity testing . 2 1.2. 1 Pollution of water and aquatic toxicology . 2 1.2. 2 Cellular effects of reactive and non-reactive toxins . 3 1.2. 3 Direct Toxicity Assessment . 4 1.2. 4 Toxicity testing using animals . 5 1.2. 5 In vitro cell culture toxicity testing . 6 1.3 Microbial toxicity tests . 6 1.3. 1 Advantages of using microbes in toxicity testing . 6 1.3. 2 Microbial tests for monitioring toxicity . 6 1.3. 3 Microorganism growth regulation in response to a changing external environment . 7 1.3. 4 Microbial growth inhibition assays . 7 1.3. 5 Microbial toxicity tests . 8 1.3. 6 The SOS chromotest for genotoxicity . 9 1.3. 7 The Ames test for genotoxicity . 10 1.3. 8 Eukaryotic genotoxicity tests . 10 1.3. 9 The use of S. cerevisiae in genomics . 10 1.4 The emergence of toxicogenomics: Combining genomics and toxicology . 11 1.4. 1 Transcriptomics.11 1.4. 2 The use of array technology for monitoring gene expression . 11 1.4. 3 Using transcriptomics and model organisms in toxicology . 12 1.4. 4 Toxicogenomics: Aiding cross species comparisons . 12 1.4. 5 Potential classification of chemicals using gene expression profiles . 12 1.4. 6 Applying toxicogenomics to genotoxicity . 13 1.4. 7. Response to stress in S. cerevisiae . 13 1.5 Genomic technologies; history, developments and standardisation . 13 1.5. 1 Expression technology: The emergence of macroarrays . 13 1.5. 2 Moving from macroarrays to microarrays . 14 1.5. 3 Sources of variation in microarray experiments . 14 1.5. 4 MIAME guidelines . 14 1.5. 5 Sources of technical variation in microarray experiments . 15 1.5. 6 Standardised microarray experimental procedures . 15 1.5. 7 Normalisation: theory, and importance in expression analysis . 16 1.5. 8 Probe summarisation . 16 1.5. 9 Checking the quality of microarray data . 17 1.6 Confirming microarray results using Real Time PCR . 17 1.6. 1 Development of Real Time PCR and Quantitative PCR . 17 1.6. 2 Benefits of Real Time PCR . 18 1.6. 3 How Real Time PCR works . 18 1.6. 4 Requirements for successful RT-PCR . 19 1.6. 5 Quantitative and semi quantitative RT-PCR without instrumentation . 19 1.6. 6 Normalisation of qPCR . 20 1.6. 7 Methods for calculating differential expression . 20 1.6. 8 How many genes need to be confirmed to validate a microarray experiment? 20 1.6. 9 Selecting genes for confirmation using qRT-PCR . 21 1.7 Analysing gene expression data . 22 1.7. 1 Assessing data quality using Principal Component Analysis (PCA) . 22 1.7. 2 Assessing data quality using Box Whisker plots . 22 1.7. 3 Identifying differentially expressed genes . 23 1.7. 4 Advantages of significant differential expression over fold changes in expression . 24 1.7. 5 Multiple Testing Correction . 24 1.7. 6 Types of MTC . 25 1.7. 6 Categorisation of genes using Gene Ontology . 25 1.7. 7 Clustering of gene expression data . 26 1.7. 8 Pathway analysis . 26 1.8 4-nitrophenol: A model toxicity testing compound . 26 1.8. 1 Phenolics are high priority pollutants . 26 1.8. 2 4-nitrophenol as a model organic pollutant . 27 1.8. 3 Sources of 4-nitrophenol in the environment . 27 1.8. 4 Toxicity of 4-nitrophenol . 28 1.8. 5 Toxicity of 4-nitrophenol to microbes . 28 1.8. 6 Toxicity of 4-nitrophenol to invertebrates . 28 1.8. 7 Toxicity of 4-nitrophenol to terrestrial vertebrates . 29 1.8. 8 Toxicity of 4-nitrophenol to aquatic vertebrates . 29 1.8. 9 Toxicity of 4-nitrophenol to plants. 29 Aims of this study . 31 Determining the effects of 4-nitrophenol on growth of Saccharomyces cerevisiae in different media: Identifying experimental conditions for subsequent toxicogenomic studies. 32 2.