Phenobarbital Mediates an Epigenetic Switch at the Constitutive Androstane Receptor (CAR) Target Gene Cyp2b10 in the Liver of B6C3F1 Mice

Abstract

Evidence suggests that epigenetic perturbations are involved in the adverse effects associated with some drugs and toxicants, including certain classes of non-genotoxic carcinogens. Such epigenetic changes (altered DNA methylation and covalent histone modifications) may take place at the earliest stages of carcinogenesis and their identification holds great promise for biomedical research. Here, we evaluate the sensitivity and specificity of genome-wide epigenomic and transcriptomic profiling in phenobarbital (PB)-treated B6C3F1 mice, a well-characterized rodent model of non-genotoxic liver carcinogenesis. Methylated DNA Immunoprecipitation (MeDIP)-coupled microarray profiling of 17,967 promoter regions and 4,566 intergenic CpG islands was combined with genome-wide mRNA expression profiling to identify liver tissue-specific PB-mediated DNA methylation and transcriptional alterations. Only a limited number of significant anti-correlations were observed between PB-induced transcriptional and promoter-based DNA methylation perturbations. However, the constitutive androstane receptor (CAR) target gene Cyp2b10 was found to be concomitantly hypomethylated and transcriptionally activated in a liver tissue-specific manner following PB treatment. Furthermore, analysis of active and repressive histone modifications using chromatin immunoprecipitation revealed a strong PB-mediated epigenetic switch at the Cyp2b10 promoter. Our data reveal that PB-induced transcriptional perturbations are not generally associated with broad changes in the DNA methylation status at proximal promoters and suggest that the drug-inducible CAR pathway regulates an epigenetic switch from repressive to active chromatin at the target gene Cyp2b10. This study demonstrates the utility of integrated epigenomic and transcriptomic profiling for elucidating early mechanisms and biomarkers of non-genotoxic carcinogenesis.

Effects of Phenobarbital on Expression of UDP-Glucuronosyltransferase 1a6 and 1a7 in Rat Brain

“…Such epigenetic changes are involved in gene regulation prior to transcriptional activation. In a previous study, PB-mediated changes in DNA methylation and histone modification were shown to be associated with the upregulation of cytochrome P450 2b10 (a CAR target gene) in mice (Lempiäinen et al, 2011). Moreover, CAR activation has been shown to be related to epigenetic changes at the promoter region of CAR target genes (Chen et al, 2012).…”

“…PB can also alter DNA methylation and histone modifications (Watson and Goodman, 2002;Lempiäinen et al, 2011). Such epigenetic changes are involved in gene regulation prior to transcriptional activation.…”

Comprehensive analysis of DNA methylation and gene expression of rat liver in a 2-stage hepatocarcinogenesis model

“…MicroRNA34b and MicroRNA34c, which are two components of the p53 network, were hypermethylated in many colorectal cancer specimens (Toyota et al, 2008) resulting in abnormal p53 signal transmission. Finally, DNA methylation levels were higher in the gastric mucosa of Helicobacter pylori-negative patients with multiple gastric cancers than in those with a single cancers (Nakajima et al, 2006), which suggests a relationship between the accumulation of aberrant DNA methylation and carcinogenesis. As these data collectively suggest that epigenetic changes are involved in carcinogenesis, elucidating the role of epigenetic changes in chemically induced carcinogenesis might facilitate the assessment of the carcinogenic potential of drug candidates.…”

International regulatory needs for development ofan IATA for non-genotoxic carcinogenic chemical substances

“…Relevant information about DNA methylation, RNA and miRNA expression studies and chromatin structure and modification data can already start to be derived from the literature, together with analyses to identify markers for detection of NGTxC with epigenetic activity. This information can be used in combination with the key event assay blocks identified in Table 1, specific examples being endocrine receptor Ho, 2011, reviewed in Greally andJacobs, 2013) and xenobiotic receptor activation (Lempiainen et al, 2011). One of the major challenges when studying epigenetic changes inferred by NGTxC is to link these epigenetic changes to adverse outcomes.…”