112(4).Tox.Pt2.MON.417-510

Abstract

Several studies using microarrays have shown that changes in gene expression provide information about the mechanism of toxicity induced by xenobiotic agents. Nevertheless, the issue of whether gene expression profiles are reproducible across different laboratories remains to be determined. To address this question, several members of the Hepatotoxicity Working Group of the International Life Sciences Institute Health and Environmental Sciences Institute evaluated the liver gene expression profiles of rats treated with methapyrilene (MP). Animals were treated at one facility, and RNA was distributed to five different sites for gene expression analysis. A preliminary evaluation of the number of modulated genes uncovered striking differences between the five different sites. However, additional data analysis demonstrated that these differences had an effect on the absolute gene expression results but not on the outcome of the study. For all users, unsupervised algorithms showed that gene expression allows the distinction of the high dose of MP from controls and low dose. In addition, the use of a supervised analysis method (support vector machines) made it possible to correctly classify samples. In conclusion, the results show that, despite some variability, robust gene expression changes were consistent between sites. In addition, key expression changes related to the mechanism of MP-induced hepatotoxicity were identified. These results provide critical information regarding the consistency of microarray results across different laboratories and shed light on the strengths and limitations of expression profiling in drug safety analysis. minimal expression of single-cell necrosis with minimal mononuclear infiltrate without associated changes in clinical chemistry parameters Male Sprague-Dawley rats were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Rats were 57 days old and weighed 233.4-274.0 g at the start of the treatment. Upon arrival to Abbott Laboratories (Abbott Park, IL), all rats were acclimated for 6 days before treatment began. The two treatment groups comprising four rats each received the test compound at a concentration of 10 or 100 mg/kg, respectively. Animals in the equally sized control group received vehicle only. Rats were dosed once daily by gavage for 7 days. The dose volume was 10 mL/kg. Doses were milligram salt per kilogram per day and were calculated for each rat on the basis of the most recent body weight data available. Rats were fasted overnight after their last treatment, euthanized under halothane anesthesia and submitted for necropsy. Each rat received its last treatment approximately 24 hr before scheduled necropsy. In vivo observations, pathology, and sampling. All rats were observed twice each day during the pretreatment and treatment periods for survival and general condition. Blood samples were drawn from all rats, and clinical chemistry parameters were obtained for alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKPHOS), total bilirubin (TBIL), glucose (GLU), and triglycerides (TRIG). At necropsy, liver was weighed and the percent of body weight of each organ was calculated. One part of the liver (left lateral lobe) was fixed for potential histopathology in 10% formalin and subsequently sectioned and stained with hematoxylin and eosin, while the rest of the organ was rinsed in phosphate-buffered saline, immediately flashfrozen in liquid nitrogen, and kept frozen for subsequent RNA isolation. RNA Isolation and Distribution Approximately 100 mg of tissue from each liver was placed into TRIzol reagent (Invitrogen Corp., Carlsbad, CA) and homogenized. Total RNA isolation was performed exactly according to the TRIzol reagent protocol. The remaining portion of the liver was retained frozen. Following isolation, the RNA was quantitated using a BioRad SmartSpec 3000 spectrophotometer (BioRad, Hercules, CA), and the integrity of the RNA was determined using an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). The RNA from the four animals in each treatment group was then pooled using equivalent amounts from each sample. The RNA was aliquoted and precipitated in ethanol and ammonium acetate for shipment to the participating DNA microarray users. In addition, RNA from individual animals was shipped to some of the DNA microarray analysis laboratories. DNA microarray analysis. RNA samples were analyzed independently by five different Affymetrix users: Boehringer-Ingelheim Pharmaceuticals, Novartis, Pfizer Inc, F. Hoffmann-La Roche AG, and Schering AG using rat RGU34A expression probe arrays (Affymetrix, Santa Clara, CA), containing 8,799 probe sets interrogating primarily annotated genes. The rat sequences used for the design of the RGU34A expression probe array were derived from Build 34 of the UniGene database (http://www.ncbi. nih.gov/UniGene/; created from Genbank 107/dbEST 11/18/98) and supplemented with additional annotated gene sequences from Genbank 110 (http://www.ncbi. nih.gov/GenBank/). UniGene clusters are represented by an example sequence that is the most complete and most 3´sequence in the cluster. The oligonucleotide probes are 25mers and 16 probe pairs per sequence are used. Processing of RNA and GeneChip experiments was carried out basically as recommended by Affymetrix, with some userspecific variations Data analysis. Tab-delimited files obtained from the Affymetrix Microarray Suite software, version 4.0, (*.chp files) and containing data on signal intensity [average difference (Avg Diff)] and categorical expression-level measurement (Absolute Call) were used for analysis. Data were normalized and further analyzed using Roche in-house developed software (RACE-A; F. Hoffmann-La Roche AG, Mannheim, Germany). Briefly, this software performs a normalization step on the signal intensities based on the average signal (Mean Avg Diff) of each microarray before calculating additional parameters. In the cases where biological replicates were included, RACE-A was also used to calculate the average signal (arithmetic mean), and SD for each probe set. Also, comparative analysis between control and treated was performed including fold change (Avg Diff Treated/Avg Diff Control) and a significance value (p-value), calculated using a two-tailed, unpaired t-test. Once the required statistical parameters were calculated, data were filtered and exported to MS-Excel 2002 (Microsoft, Corp., Bellevue, WA) or additional software for visualization and further analysis. In addition, methods comprising more sophisticated algorithms and designed et al. 1999). Unsupervised methods such as clustering algorithms and PCA are commonly used to determine if gene expression patterns allow the discrimination of natural subpopulations that might bear a biological meaning such as treated/untreated or healthy/diseased. PCA is a mathematical technique that reduces the dimensionality of highly multivariate data. The reduced dimensions (or components) actually describe the major part of the variation in the samples and separate natural subpopulations without a priori knowledge Cluster analysis is a method used to organize primary data. Pairwise average-linkage cluster is a form of unsupervised hierarchical clustering commonly used for the analysis of microarray data. Relationships among objects such as experimental conditions or genes are represented by a tree whose branch lengths reflect the degree of similarity between the objects as assessed by a pairwise similarity function based on correlation coefficients The complete data set is currently being submitted to ArrayExpress (EMBLEuropean Bioinformatics Institute, Hinxton, UK; http://www.ebi.ac.uk/arrayexpress) and will be available for public download by the second quarter of 2004. Accession numbers referencing this data set will be available on the HESI website (http://hesi.ilsi.org/ index.cfm?pubentityid=120). Results Clinical chemistry and histopathology. A significant change in both body weight and food consumption compared to that of control groups was seen in the high-dose, but not in the low-dose group (data not shown). Clinical chemistry values confirmed liver toxicity occurred in the high-dose rats No compound-related histopathological changes were found for the low-dose group, whereas several compound-related changes were seen in livers from rats treated at the high-dose level. These included cytoplasmic vacuolation of periportal hepatocytes, minimal to mild necrosis of periportal hepatocytes, increased infiltration of portal tracts by mononuclear inflammatory cells, and hyperplasia of oval cells along portal tracts. Comparisons across users. After microarray analysis of the RNA, it was determined that different users obtained comparable results despite possible variation in the sample processing [from total RNA up to fragmented IVT (in vitro transcript)] and microarray hybridization protocols. In a preliminary round of analysis using rigid cutoff values to assess which genes were modulated, the number of genes detected as regulated in the pooled samples (2-fold increase or decrease) by each user were strikingly different Further microarray results demonstrated that when individual animals were analyzed, as opposed to pooled samples, the number of genes detected as induced/ repressed was generally reduced