Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse lymphoma L5178Y cells treated with Hydroxyurea

ABSTRACT: The multi-lab International Life Sciences Institute (ILSI) project on the application of genomics to risk assessment offered the unique opportunity to investigate the influence of variability within and between laboratories on identifying biologically relevant gene expression changes. We assessed the gene expression profiles of mouse lymphoma L5178Y cells treated with hydroxyurea (HU) in three independent studies from two different laboratories, Sanofi-Aventis and Procter and Gamble. Cells were dosed for 4 hr and harvested immediately at the end of the treatment or after a 20-hr recovery period. Cytotoxicity and genotoxicity were evaluated by standard assays. Statistical analysis of these data revealed that, while gene expression responses to HU treatment were markedly different at 4 hr vs. 24 hr, there was otherwise a consistent pattern of dose-response across the three studies. Therefore, the studies were merged and each time point was evaluated separately. At 4 hr, we identified 173 (P lt 0.0001) dose-responsive genes with a common trend in all three studies. These were mainly associated with the cell cycle, DNA repair and DNA metabolism, and in particular, the intra-S and G2/M phase checkpoints. At 24 hr, we identified 434 dose-responsive genes common across studies. These genes were involved in lymphocyte-specific activities and the activation of apoptosis via the caspase cascade. Our results show that despite inter-laboratory variability, combining the three studies in a single statistical analysis identifies more significantly-modulated genes than in any of the individual studies, due to improved statistical sensitivity. The genes identified in our study provide information that is relevant to HU biology.

ORGANISM(S): Mus musculus

SUBMITTER: Sanofi Aventis Performer

PROVIDER: E-TOXM-17 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The process for evaluating chemical safety is inefficient, costly, and animal intensive. There is growing consensus that the current process of safety testing needs to be significantly altered to improve efficiency and reduce the number of untested chemicals. In this study, the use of short-term gene expression profiles was evaluated for predicting the increased incidence of mouse lung tumors. Animals were exposed to a total of 26 diverse chemicals with matched vehicle controls over a period of three years. Upon completion, significant batch-related effects were observed. Adjustment for batch effects significantly improved the ability to predict increased lung tumor incidence. For the best statistical model, the estimated predictive accuracy under honest five-fold cross-validation was 79.3% with a sensitivity and specificity of 71.4 and 86.3%, respectively. A learning curve analysis demonstrated that gains in model performance reached a plateau at 25 chemicals, indicating that the size of the current data set was sufficient to provide a robust classifier. The classification results showed a small subset of chemicals contributed disproportionately to the misclassification rate. For these chemicals, the misclassification was more closely associated with genotoxicity status than efficacy in the original bioassay. Statistical models were also used to predict dose-response increases in tumor incidence for methylene chloride and naphthalene. The average posterior probabilities for the top models matched the results from the bioassay for methylene chloride. For naphthalene, the average posterior probabilities for the top models over-predicted the tumor response, but the variability in predictions were significantly higher. The study provides both a set of gene expression biomarkers for predicting chemically-induced mouse lung tumors as well as a broad assessment of important experimental and analysis criteria for developing microarray-based predictors of safety-related endpoints. Experiment Overall Design: Five-week-old female B6C3F1 mice were exposed for 13 weeks to 26 chemicals. The chemical and dose information are provided with the individual sample annotations. With each chemical treatment, a matched vehicle control group was run concurrently with the exposure. A subset of two chemicals, methylene chloride and naphthalene, were performed in a five-point dose response with a matched purified air control group. The concentrations for these exposures overlapped those in the original cancer bioassay. Gavage exposures were administered 5 days per week and feed exposures were provided 7 days per week. For inhalation exposures, mice were exposed 6 hr per day, 5 days per week. After 13 weeks, animals were euthanized and lungs were collected. The right lobe was used for microarray analysis. Microarray analysis was performed on the lungs of three to four mice per treatment group.

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Transcription profiling of mouse lymphoma L5178Y cells treated with Hydroxyurea

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