On PND 22, livers from five male and five female pups culled from the control and high dose groups were collected for toxicogenomic analysis (each pup came from a different dam). These animals were dosed through the day (PND 21) before liver tissue collection (PND 22). Liver samples were collected from 5 control female pups, 5 control male pups, 5 50 mg/kg female pups, and 5 50 mg/kg male pups. At week 13, 10 males from the 0 and 50 mg/kg groups were sacrificed and the liver tissue was collected for toxicogenomic analysis and histopathlogy (each animal came from a different dam in that dose group).
Growth protocol
Time mated female Wistar/Han dams (CRL:WI (HAN)) were obtained from Charles River Laboratories (Raleigh, NC). Dams were dosed by gavage with 0 or 50 mg/kg DE71 in corn oil (dosing volume 5 ml/kg) from GD 6 through PND 21, 7 days per week. Direct dosing of pups began on and continued through PND 21 for 7 days per week until weaning. Weaning occurred on PND 21, which was designated as Day 1 of the 13-week portion of the study, and the day the pups were assigned to the 13-week portion of the study. On day one of the 13-week portion of the study dosing began on a 5 days/week schedule. Pups designated for toxicogenomic study were dosed through PND 21, the day prior to PND 22 liver sample collection. Tap water and NTP-2000 diet (Zeigler Brothers, Inc. Gardners, PA) was made available ad libitum (National Toxicology Program 2005a, b).
Extracted molecule
total RNA
Extraction protocol
A section of the liver was collected at necropsy, frozen and sent to the microarray processing unit where the liver was placed into RNAlater (Ambion, Inc., Austin, TX). RNA was extracted from liver using the QIAGEN Rneasy (QIAGEN, Valencia, CA). The RNA was analyzed for quantity and purity by UV analysis using the NanoDrop ND-1000 (NanoDropTechnologies, Wilmington, DE). Samples were concentrated using Microcon filters (Millipore, Billerica, MA). All samples were evaluated for RNA integrity by gel electrophoresis using the Flash Gel RNA cassette system (Lonza, Rockland, ME).
Label
biotin
Label protocol
Total RNA (50 ng) was used to synthesize double-stranded cDNA for each sample using Affymetrix GeneChipExpression 3'amplication two-cycle target labeling and control reagents (Affymetrix Inc. Santa Clara, CA). The cDNA served as a template to synthesize biotin-labeled antisense cRNA using an in vitro transcription (IVT) labeling kit. Labeled cRNA was fragmented and hybridized to the Affymetrix Rat Genome 230 2.0 Genechip Array.
Hybridization protocol
Labeled cRNA was fragmented and hybridized to the Affymetrix Rat Genome 230 2.0 Genechip Array. Array hybridization, washing, and staining were performed according to the Affymetrix recommended protocol EuKGE_Ws2v5.
Scan protocol
Affymetrix GeneChip Scanner 3000
Description
liver from untreated female pup X100709.10_.Rat230_2..CEL
Data processing
Data were processed using R/affy software. In brief, the RMA method adjusts the background of perfect match (PM) probes, applies a quantile normalization of the corrected PM values, and calculates final expression measures with the Tukey median polish algorithm.
