Identification of Genes Implicated in Methapyrilene-Induced Hepatotoxicity by Comparing Differential Gene Expression in Target and Nontarget Tissue

Article excerpt

BACKGROUND: Toxicogenomics experiments often reveal thousands of transcript alterations that are related to multiple processes, making it difficult to identify key gene changes that are related to the toxicity of interest.

OBJECTIVES: The objective of this study was to compare gene expression changes in a nontarget tissue to the target tissue for toxicity to help identify toxicity-related genes.

METHODS: Male rats were given the hepatotoxicant methapyrilene at two dose levels, with livers and kidneys removed 24 hr after one, three, and seven doses for gene expression analysis. To identify gene changes likely to be related to toxicity, we analyzed genes on the basis of their temporal pattern of change using a program developed at the National Institute of Environmental Health Sciences, termed "EPIG" (extracting gene expression patterns and identifying co-expressed genes).

RESULTS: High-dose methapyrilene elicited hepatic damage that increased in severity with the number of doses, whereas no treatment-related lesions were observed in the kidney. High-dose methapyrilene elicited thousands of gene changes in the liver at each time point, whereas many fewer gene changes were observed in the kidney. EPIG analysis identified patterns of gene expression correlated to the observed toxicity, including genes associated with endoplasmic reticulum stress and the unfolded protein response.

CONCLUSIONS: By factoring in dose level, number of doses, and tissue into the analysis of gene expression elicited by methapyrilene, we were able to identify genes likely to not be implicated in toxicity, thereby allowing us to focus on a subset of genes to identify toxicity-related processes.

KEY WORDS: DNA microarray, gene expression, hepatotoxicity, liver, methapyrilene, toxicogenomics. Environ Health Perspect 115:572-578 (2007). doi:10.1289/ehp.9396 available via http://dx.doi.org/ [Online 17 January 2007]

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The advent of DNA microarray technology has spurred the recent growth of toxicogenomics studies [reviewed in Waters and Fostel (2004)]. By measuring changes in gene expression after toxicant exposure on a genomewide scale, investigators can attempt to identify genes or pathways involved in the mechanism of toxicity for that particular toxicant. However, because of the nature of global gene expression profiling, many of the genes found to be differentially expressed may not be related to toxicity. For example, some genes may change because of altered feeding schedules or diurnal rhythms (Boorman et al. 2005; Kita et al. 2002), whereas other gene changes may be related to the pharmacology but not toxicology of the administered substance. Careful design of toxicogenomics studies can reduce the complexities of analyzing gene expression data, such as using time-matched controls to remove those genes for which expression values change with diurnal rhythms. In addition, using different doses in toxicogenomics studies, ranging from pharmacologic/nontoxic to minimally toxic to highly toxic, can often identify genes that are responding to the pharmacologic properties of the administered toxicant.

Methapyrilene, an antihistaminic compound removed from the U.S. market after it was found to lead to the development of hepatic cancers in rats (Lijinsky et al. 1980), has been the focus of several toxicogenomics studies (Beekman et al. 2006; Hamadeh et al. 2002; Waring et al. 2004). The Hamadeh et al. and Waring et al. studies examined hepatic gene expression in rats treated from 1 to 7 days with methapyrilene at doses of 10 mg/kg and 100 mg/kg, whereas the Beekman et al. study examined expression changes in hepatocytes exposed in vitro to methapyrilene. The Hamadeh et al. study examined hepatic gene expression to try to correlate gene expression changes with alterations in histopathology after methapyrilene treatment to identify genes involved in methapyrilene-mediated hepatotoxicity (Hamadeh et al. …