To gauge the significance of metabolite changes, measurements from 0 hours to 96 hours postdose were used PLX-4720 concentration to estimate the area under the curve (AUC). AUC Testing allows pooling of the data across time for a single test of differences in trend. The

R package PK was utilized to estimate metabolite AUC for each sample. A t test was then performed to test for differences in AUC between cases and controls. Microarray data were obtained using Agilent’s Feature Extraction software (v. 7.5), using defaults for all parameters. The Feature Extraction Software performs error modeling before data are loaded into a database system. Images and GEML files were exported from the Agilent Feature Extraction software and deposited into Rosetta Resolver (v. 5.0, build 5.0.0.2.48) (Rosetta Biosoftware, Kirkland, WA). Rosetta Resolver combines data hybridizations using an error-weighted average that adjusts for additive and multiplicative noise.7 The resultant universal control profiles

were then exported as normalized log ratios, median centered across subjects and utilized for further statistical analyses by the R-project software.8 Principal component analysis was performed to investigate the presence of experimental artifacts. The first component of variation was defined by sample ethnicity, and this component was removed to produce an adjusted dataset that did not contain an ethnicity bias.9 The Protein Tyrosine Kinase inhibitor resultant ratio profiles from both the ethnically unadjusted and adjusted datasets were analyzed for differential gene expression. First, a two-tailed t test was utilized comparing universal control profiles with time-matched sham controls and statistically significant DEGs were identified at the P < 0.05 confidence level. DEGs from both datasets were then analyzed with ingenuity

pathways analysis (IPA) (Ingenuity Systems, www.ingenuity.com). Canonical pathways analysis identified the pathways Thalidomide from the IPA library of canonical pathways that were most significant to the dataset. The significance of the association between the dataset and the canonical pathway was measured in 2 ways: 1) A ratio of the number of genes from the dataset that map to the pathway divided by the total number of genes that map to the canonical pathway was obtained. 2) Benjamini-Hochberg testing corrected P-values were used to determine the probability that association between genes in the dataset and the canonical pathway is explained by chance alone. To increase our confidence in the IPA canonical pathway analysis, we utilized the more stringent gene set analysis (GSA) methodology on both the adjusted and unadjusted datasets comparing the cases to controls at each timepoint.12 For the five human overdose subjects, universal control profiles were normalized to five ethnically and gender-matched controls. A one-way analysis of variance (ANOVA) analysis with a Bonferroni multiple test correction was performed to identify DEGs.