Clofibrate-induced gene expression changes in rat liver: a cross-laboratory analysis using membrane cDNA arrays.

Abstract

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity.

In vitro gene expression analysis of hepatotoxic drugs in rat primary hepatocytes

“…In contrast to a report in rat liver (Baker et al, 2004), no increases of Cyp4a1 or epoxide hydrase, a decrease of PPAR, or alterations of proliferation-associated genes were observed. Similarly, effects of CF on proliferationassociated genes in rat hepatocytes were not observed.…”

Variation in the Hepatic Gene Expression in Individual Male Fischer Rats

“…While several recent studies have addressed interlaboratory variation in microarray results, comparisons were between pooled samples (Baker et al, 2004;Chu et al, 2004;Ulrich et al, 2004;Waring et al, 2004). Thus, individual variation was not addressed.…”

Optimization of an Animal Test Protocol for Toxicogenomics Studies (Ii); A Cross-Laboratory Gene Expression Analysis

“…The observed discrepancies in expression profiles for individual genes might be due to chemical (for example, impure substance), animal, microarray, test reagent, procedure of animal testing and gene expression analysis. Baker et al (2004) have reported a cross-laboratory analysis using membrane cDNA arrays (Baker et al, 2004) and obtained essentially the same findings. Three separate animal tests were conducted and three separate gene expression analyses carried out under different conditions in their study, while three separate animal tests were conducted according to the same protocol and gene expression analysis carried out at one laboratory in our study.…”

“…They confirmed reasonable agreement in gene expression results between laboratories and across platforms, and the observed discrepancies in expression profiles for individual genes were largely due to platform differences and approaches to data analysis rather than to biological or interlaboratory variability. Baker et al also reported a cross-laboratory analysis of the same compounds using membrane cDNA arrays, including comparison of the gene expression profiles generated from pooled and individual RNA samples (Baker et al, 2004). They found results from all three laboratories to demonstrate a considerable degree of similarity.…”