Male Sprague-Dawley VAF+ albino rats (CRL:CD(SD) BR; Charles River, Kingston, NY) approximately 57 weeks old were maintained on certified rodent chow (PMI Feeds, Inc., Brentwood, MO) ad libitum in individual stainless steel wire bottom cages suspended on racks. The animals were kept under controlled lighting (12-h light-dark cycle), temperature (720 1 50F), and humidity (50 1 20%) and were acclimated to this environment for 47 days prior to the start of the study. Healthy rats were randomly assigned to dose groups (3 rats/group) by a computerized method. For the 2-week studies, a 1-week pretest involved dosing of all animals via oral gavage with 10 ml/kg vehicle. The 24-h studies did not have any pretest period. Clofibrate (CAS # 637070), gemfibrozil (CAS # 25812300), and phenobarbital (CAS # 57307) were obtained from Sigma (St. Louis, MO); Wyeth 14,643 (CAS # 50892234) was obtained from ChemSyn Laboratories (Lenexa, KS). Dosing suspensions of all compounds were prepared using a high speed homogenizer, and all dose suspensions were continuously stirred until completion of dosing. The peroxisome proliferators (clofibrate, gemfibrozil, and Wyeth 14,643) were prepared using 1% carboxymethylcellulose/0.2% Tween 80 as vehicle and phenobarbital was prepared using water as vehicle. Drug concentration and identity were verified via HPLC, as per United States Pharmacopeia (USP) methods for clofibrate, gemfibrozil, and phenobarbital. For Wyeth 14,643, a Waters 600E HPLC was equipped with a variable wavelength detector set at 235 nm, and a Symmetry Cs 4.6 x 250 mm column. Equal portions (approximately 10 ml) of standard and test solutions were injected separately into the column. Parameters of the run were as follows: mobile phase, 0.0738 M sodium acetate:acetonitrile (55:45 v/v); flow rate, 1 ml/min; column temperature, ambient; and run time, 6 min. The HPLC retention time of Wyeth 14,643 under these parameters was 3.4 1 0.1 min. Body weights and food consumption were measured weekly. Based on the most recently recorded body weights, the volume of drug administered was adjusted. Animals were observed 23 times daily for signs of overt toxicity. Experiments were performed according to the guidelines established in the NIH Guide for the Care and Use of Laboratory Animals. At the end of the drug phase of the study, each animal was fasted overnight before necropsy. Animals were taken to a deep plain of anesthesia with CO2, sacrificed by axillary vessel incision; exsanguination and necropsy immediately followed. A cross section of the left lateral lobe of the liver was collected in 10% neutral buffered formalin for histopathology. The remaining portions of liver were collected in RNase-free tubes and snap frozen in liquid nitrogen. Frozen tissues were stored at 700C until processed for RNA extraction. A control sample was generated by pooling livers of 9 vehicle-treated rats. The liver tissues collected in formalin at necropsy were processed, embedded in paraffin, sectioned at 5 microns, and stained with hematoxylin and eosin (H&E). Histopathologic examinations of the liver sections were conducted by a pathologist and peer-reviewed. Total RNA was isolated using QIAGEN (Qiagen, Valencia, CA) RNeasy kits. Liver sections of 130250 mg were used for midipreps and liver sections of approximately 800 mg were used for maxipreps. Homogenization buffer was added to frozen liver sections, and the tissue was immediately homogenized on ice (tissue did not thaw prior to homogenization) using a Cyclone homogenizer equipped with a rotor/stator shaft (VirTis Company, Gardiner, NY). Homogenates were processed as per the standard QIAGEN 3/99 protocol. Final product yielded 260 nm/280 nm ratios of 1.62.0, purity was confirmed via gels, and concentration was determined based on 260 nm absorbances. For microarray hybridizations, each total RNA sample (3575 5g) was labeled with Cyanine 3 (Cy3)-or Cyanine 5 (Cy5)-conjugated dUTP (Amersham, Piscataway, NJ) by a reverse transcription reaction using reverse transcriptase, SuperScript (Invitrogen, Carlsbad, California), and the primer, Oligo dT (Amersham, Piscataway, NJ). Control samples were labeled with Cy3 while samples derived from chemically exposed animals were labeled with Cy5. The fluorescently labeled cDNAs were mixed and hybridized simultaneously to the cDNA microarray chip. Each RNA pair was labeled and hybridized independently in triplicate to a total of 3 arrays. The cDNA chips were scanned with an Axon Scanner (Axon Instruments, Foster City CA) using independent laser excitation of the 2 fluors at 532 and 635 nm wavelengths for the Cy3 and Cy5 labels, respectively. The raw pixel intensity images were analyzed using the ArraySuite v1.3 extensions of the IPLab image processing software package (Scanalytics, Fairfax, VA). This program uses methods that were developed and previously described by Chen et al. (1997) to locate targets on the array, measure local background for each target, and subtract it from the target intensity value, and to identify differentially expressed genes using a probability-based method. After pixel intensity determination and background subtraction, the ratio of the intensity of the treated cells to the intensity of the control was calculated. We have previously determined that significant autofluorescence of the gene features on the array, attributed to spotting solution, occurs at high scanning power (Tucker et al., unpublished). We measured the pixel intensity level of "blank" spots comprised of spotting solution. The data was then filtered to provide a cut off at the intensity level just above the blank measurement values in order to remove from further analyses those genes having one or more intensity values in the background range. -- taken largely verbatim from Hamadeh et al., Toxicological Sciences 67, 219-231 (2002)
