Candidate genes responsible for early key events of phenobarbital-promoted mouse hepatocellular tumorigenesis based on differentiation of regulating genes between wild type mice and humanized chimeric mice

Abstract

Phenobarbital (PB) is a nongenotoxic hepatocellular carcinogen in rodents. PB induces hepatocellular tumors by activating the constitutive androstane receptor (CAR). Some previous research has suggested the possible involvement of epigenetic regulation in PB-promoted hepatocellular tumorigenesis, but the details of its molecular mechanism are not fully understood. In the present study, comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed in mouse hepatocellular adenomas induced by a single 90 mg kg intraperitoneal injection dose of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas. Of these, gene network analysis showed alterations of CAR signaling and tumor development-related genes. Pathway enrichment analysis revealed that differentially methylated or hydroxymethylated genes belong mainly to pathways involved in development, immune response and cancer cells in contrast to differentially expressed genes belonging primarily to the cell cycle. Furthermore, overlap was evaluated between the genes with altered expression levels with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma induced by DEN/PB and the genes with altered expression levels in the liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days. With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis. Interestingly, these genes did not overlap with genes altered by the PB treatment of humanized chimeric mice, thus suggesting a species difference between the effects of PB in mouse and human hepatocytes.

Case examples of an evaluation of the human relevance of the pyrethroids/pyrethrins-induced liver tumours in rodents based on the mode of action

“…80 Interestingly, these genes did not overlap with genes altered by PB treatment of the humanized chimeric mouse liver, suggesting a species difference between the effects of PB in mouse and human hepatocytes. 80 Therefore, these findings are consistent with the previous conclusion that the CARmediated MOA for rodent liver tumorigenesis is not relevant to humans. 15,17,19,20,[26][27][28][29][30][31][32][33][36][37][38]40,58,69,81…”

“…[24][25][26][27]40 Evidence in humans. 26,27,33,40,80 activator PB, it is considered that the proposed MOA for pyrethrins-produced rat hepatocellular tumour formation is not qualitatively plausible for humans. 30,55 Using mode of action information for regulatory decision-making…”

“…While epigenetics are suggested to be related to PB-mediated liver tumorigenesis, [72][73][74][75][76][77][78][79] our recent study focused on genes with alterations of DNA methylation and hydroxymethylation with concomitant alteration of expression levels. 80 Comprehensive analyses of DNA methylation, hydroxymethylation and gene expression using microarrays were performed on mouse hepatocellular adenomas induced by a single 90 mg kg −1 intraperitoneal injection of diethylnitrosamine (DEN) followed by 500 ppm PB in the diet for 27 weeks. DNA modification and expression of hundreds of genes are coordinately altered in PB-induced mouse hepatocellular adenomas.…”

“…Furthermore, overlap between genes with altered expression compared with 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) alterations in mouse hepatocellular adenoma and in liver of CD-1 mice or humanized chimeric mice treated with PB for 7 days was evaluated. 80 With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis while a functional analysis of the selected genes remains to be evaluated. 80 Interestingly, these genes did not overlap with genes altered by PB treatment of the humanized chimeric mouse liver, suggesting a species difference between the effects of PB in mouse and human hepatocytes.…”

“…80 With the integration of transcriptomic and epigenetic approaches, we detected candidate genes responsible for early key events of PB-promoted mouse hepatocellular tumorigenesis while a functional analysis of the selected genes remains to be evaluated. 80 Interestingly, these genes did not overlap with genes altered by PB treatment of the humanized chimeric mouse liver, suggesting a species difference between the effects of PB in mouse and human hepatocytes. 80 Therefore, these findings are consistent with the previous conclusion that the CARmediated MOA for rodent liver tumorigenesis is not relevant to humans.…”