tissue: liver pid: batch:protocol: 20118:201:NuGEN Sex: M treatment: CMPD E vehicle: 10% Polysorbate 80 dose (mkd): 200 days of administration: 4 covalent binding (pmol-eq/mg protein): 3969 ba-lra score: 0.4543102
Treatment protocol
Male Sprague Dawley, strain Crl:CD(SD), or Wistar, strain Crl:WI(HAN), rats were obtained from Charles River Laboratories (Raleigh, NC) for studies conducted in United States, or from Charles River Laboratories France Saint Germain sur l'Arbresle FRANCE, for studies conducted in Mirabel. Animals were 6–10 weeks of age, weighing 120–425 grams. The rats were housed individually in wire mesh cages (at 18°C–26°C, relative humidity of 50 ± 20% on a 12-h light/12-h dark cycle), fed PMI Certified Rodent Diet (SD-restricted [fed 16–22g shortly after dosing 1× per day]; Wistar ad libitum). Animals were acclimated for at least 4 days prior to randomization into treatment groups. All animal husbandry and experimental procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, 1996) and were approved by the Institutional Animal Care and Use Committee of the facility in which the studies were conducted. Animals were dosed daily via oral gavage with vehicle or with compounds for indicated dose and durations and sacrificed 24h later.
Extracted molecule
total RNA
Extraction protocol
Approximately 24hrs after dosing, animals were sacrificed, livers removed, a section (~50-100mg) frozen on dry ice and stored at ~-80C until total RNA was isolated. Frozen liver was pulverized in Trizol reagent, extracted with Chloroform, and further purified using the RNEasy column RNA isolation procedure (Qiagen, Chatsworth, CA).
Label
biotin
Label protocol
50 ng of each purified RNA sample was amplified and labeled using a custom automated version of the NuGEN Ovation WB protocol
Hybridization protocol
Hybridization to custom rat Affymetrix arrays (containing 43,686 probesets, GEO platform GPL28473)
Scan protocol
labeling and scanning were completed following the manufacturer's recommendations
Data processing
profiles were normalized using robust multi-array average followed by ratio’ing to corresponding vehicle control samples within each study.
Application of a Rat Liver Drug Bioactivation Transcriptional Response Assay Early in Drug Development that informs Chemically Reactive Metabolite formation
