Interlaboratory evaluation of rat hepatic gene expression changes induced by methapyrilene - Genomics and Risk Assessment: Mini-Monograph

Environmental Health Perspectives, March 15, 2004 by Jeffrey F. Waring, Roger G. Ulrich, Nick Flint, David Morfitt, Arno Kalkuhl, Frank Staedtler, Michael Lawton, Johanna M. Beekman, Laura Suter

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 gone 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. Key words: methapyrilene, microarray, support vector machine, toxicogenomics, unsupervised algorithms, variability. Environ Health Perspect 112:439-448(2004). doi:10.1289/txg.6643 available via http://dx.doi.org/[Online 15 January 2004]

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In recent years the field of toxicology has begun the process of integrating genomic technologies into drug safety evaluation to understand and possibly predict adverse drug side effects. New technologies allow for the identification and quantification of thousands of gene changes occurring in a cell in a single experiment. Currently, microarrays are state of the art technology for evaluation of global gene expression changes. Several studies using microarrays have shown that changes in gene expression provide crucial information regarding the mechanism of toxicity induced by xenobiotic agents, including methapyrilene (MP), Aroclor 1254, and acetaminophen (Hamadeh et al. 2002; Reilly et al. 2001; Waring et al. 2002; Waring and Halbert 2002). In addition several studies have shown that compounds associated with a particular mechanism of toxicity, such as DNA-damaging agents, Aryl hydoxylase (Ah)-receptor ligands, and peroxisome proliferators, yield similar gene expression profiles (Burczynski et al. 2000; Thomas et al. 2001; Waring et al, 2001). Despite the potential that microarray analysis offers to toxicology, many questions remain concerning the reliability and reproducibility of these assays. Perhaps of primary importance is the issue of whether gene expression profiles for a given compound will reproduce consistently from study to study and across different laboratories.

To begin to address this issue, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute (ILSI) (http://www.ILSI.org) formed a consortium. Over thirty pharmaceutical companies participate in this effort that focuses on three categories of toxicants, hepatotoxins, nephrotoxins, and genotoxins.

In the HESI Hepatotoxicity Working Group, one of the compounds used in the evaluation was MP. MP is a known hepatotoxin that causes periportal cell necrosis and carcinomas in rats (Cunningham et al. 1995; Ratra et al. 2000). MP is metabolized in liver mainly by phase I enzymes; it does not show mutagenic properties and does not induce DNA synthesis (Budroe et al. 1984). Hence, MP is believed to act through a nonmutagenic mechanism. In the current study we have investigated the effects of a low (presumably nontoxic) and a high (toxic) dose of this compound on hepatic gene expression, with the goal of evaluating changes across different time points and concentrations. Most important, we designed the study to assess the reproducibility of the gene changes across different laboratories. To minimize variation due to the performance of the in vivo studies that might be reflected on the gene expression results, animals were treated at one facility (Abbott Laboratories) and RNA was distributed to five different users of the Affymetrix GeneChip system: Boehringer Ingelheim Pharmaceuticals (BI), Novartis Pharma AG (Nov), Pfizer Inc (Pfi), F. Hoffmann-La Roche AG (RO), and Schering AG (Sch). The results showed that, despite some variability, robust gene expression changes were consistent between sites. In addition, key gene expression changes related to the mechanism of methapyrilene-induced hepatotoxicity were identified.