Chemicals
2,4DNT (97%) was purchased from Sigma-Aldrich (St. Louis, MO).
Animals and treatment
Female Sprague-Dawley rats (175-225 grams) were from the in- house breeding colony (College of Pharmacy, University of Louisiana at Monroe [ULM] and treated in accordance with the Guide for Use and Care of Animals[23]. Breeders were from Harlan-Sprague Dawley in Madison, WI. Housing consisted of a 12 h light/dark cycle with ad libitum access to tap water and rodent chow (Harlan/Teklad 7012, Madison, WI). Rats were housed individually in polycarbonate cages on hardwood bedding (Sani-chips, Harlan/Tekland, Madison, WI) one week prior to treatment. Food was withdrawn the night before treatments [vehicle control (5% DMSO in corn oil), or one of four doses (4.98, 49.8, 99.5, or 199 mg/kg) of 2,4DNT in 5% DMSO in corn oil] which were administered by gavage between 8 and 10 AM. Study protocols were preapproved by the ULM Animal Care and Use Committee.
Microarray experimental design
Changes in gene expression were tested using Agilent commercial whole rat genome microarrays (4 X 44K). In this project, four doses plus a vehicle control were employed for 2,4-DNT at 24h. The dose selection was based on the LD 50 data for each compound. Except controls, the lowest dose, the second lowest dose, the second highest dose and the highest dose were selected. Four biological replicates of this design were conducted, each using different animals.
Total RNA extraction
Total RNA was extracted from about 30mg of liver tissue. Tissues were homogenized in the lysis buffer with FAST Prep-24 from MP before using RNeasy kits (Qiagen). Total RNA concentrations were measured using a NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The integrity and quality of total RNA was checked on an Agilent 2100 Bioanalyzer (Palo Alto, CA). The gel-like images generated by the Bioanalyzer show that total RNAs have two bands, representing the 18S and 26S RNA of mammalian RNA . Nuclease-free water (Ambion) was used to elute total RNA.
Microarray hybridization
Rat whole genome oligo arrays in the format of 4X44K were purchased from Agilent Technologies. Sample cRNA synthesis, labeling, hybridization and microarray processing were performed according to manufacturer’s protocol "One-Color Microarray-Based Gene Expression Analysis" (version 1.0). The labeling reactions were performed using the Agilent Low RNA Input Linear Amplification Kit in the presence of cyanine 3-CTP. The labeled cRNA from each labeling reaction was hybridized to individual arrays at 65 °C for 17 hours using Agilent’s Gene Expression Hybridization Kit. After washing, the arrays were scanned using a GenePix 4200AL scanner (Molecular Device Inc., Sunnyvale, CA). The Feature extraction software (V. 9.5.1) from Agilent was used to automatically find and place microarray grids, reject outlier pixels, accurately determine feature intensities and ratios, flag outlier pixels, and calculate statistical confidences.
Microarray data pre-processing
Microarray data analyses were processed with GeneSpring version 7.0 and 10.0. The sample quality control was based on the Pearson correlation of a sample with other samples in the whole experiment. If the average Pearson correlation with other samples was less than 80%, the sample was excluded for further analysis. If the scanned intensity was less than 5.0 for a probe, it was transformed to 5. A perchip (within) array normalization was performed using 50 percentile values of all the probe values in the array. Per gene (between) array normalization was also applied using the median value of a gene across all samples in the experiment. Probe features were first filtered using flags. A "present" or "absent" flag was defined using the Agilent Feature Extraction 9.5.1 software. Only a probe that had present flags in at least 50% samples of all the arrays was kept for further analyses. Data were subsequently log (base 2) transformed for statistical analyses.
Rat Microarray Analysis
We use genome-scale gene expression data of liver cells from rats treated with 4.98, 49.8, 99.5, or 199 mg/kg 2,4DNT to perform pathway analysis using human pathway database (HPD) (Chowbina, Wu et al. 2009), build dose-specific protein-protein association matrix and dose- response networks. Therefore, the driving motivation is that knowledge of these pathways / protein-protein interaction networks will help clarify and interpret physiological responses to 2,4DNT, which will advance our understanding of the health consequences of 2,4DNT treatment.
Significant Analysis of Microarray (SAM)
Significance analysis of microarrays SAM software [24] was used in two-class mode to determine the list of genes best able to distinguish genes in control and each of the three 2,4DNT dose groups which were run on separate Microsoft Excel spreadsheets. The log (base 2) normalized gene expression data of rat liver was imported into a SAM plug-in of Microsoft Excel. The SAM method (Tusher, Tibshirani et al. 2001), uses a modified t-test statistic, with sample-label permutations to evaluate statistical significance. Delta was chosen to limit the output gene list so that fewer than 5% predicted false-positives are included. Significant positively and negatively correlated genes whose mean expression in the 2,4DNT group is greater or lesser than those in the CONTROL group with a fold change of at least 2 and q-value less than 0.01 are selected for further analysis. Differentially expressed genes for each dose are uploaded into HPD [17] and these genes are visualized as a pathway – protein frequency count matrix (PPFCM) using the TIGR MeV (MultiExperiment Viewer) [25].