Phenobarbital Induces Progressive Patterns of GC-Rich and Gene-Specific Altered DNA Methylation in the Liver of Tumor-Prone B6C3F1 Mice

Abstract

Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a nongenotoxic rodent carcinogen which induces global hypomethylation and regions of hypermethylation in mouse liver. Liver tumor-sensitive (B6C3F1) and -resistant (C57BL/6) male mice were administered 0.05% (wt/wt) PB in drinking water for 2 or 4 weeks, and a 2-week recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (nontarget) tissues. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primedpolymerase chain reaction, and capillary electrophoretic separation of products. PB-induced regions of altered methylation (RAMs) which carry forward from an early to a later time point are more likely to be mechanistically relevant as compared to those that do not. Twelve of 69 RAMs (17%) present in B6C3F1 liver at 2 weeks were also seen at 4 weeks, while only 1 of the 123 RAMs (< 1%) present in C57BL/6 liver was seen at 4 weeks. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 weeks, increasing to 86 at 4 weeks. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to that of kidney. Following 4 weeks PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate that (1) progressive, nonrandom changes in methylation provide an epigenetic mechanism underlying the ability of PB to cause mouse liver tumorigenesis and (2) susceptibility to tumorigenesis is related inversely to the capacity to maintain normal patterns of methylation.

5-Hydroxymethylcytosine Profiling as an Indicator of Cellular State

“…Exposure to the NGC phenobarbital (PB) can lead to an increase in the incidence of spontaneously and chemically induced liver tumors in rodents and significantly promotes hepatic tumor incidence in B6C3F1 mice, as well as increasing the size of the tumors themselves [95][96][97][98][99].…”

BCL2 promotor methylation and miR-15a/16-1 upregulation is associated with sanguinarine-induced apoptotic death in rat HSC-T6 cells

“…Thus, several nongenotoxic chemicals have been found to affect gene function through changes in DNA methylation (27). For example, the nongenotoxic rodent carcinogen phenobarbital induces global hypomethylation and regional hypermethylation in rodent livers (28,3,29). MiRNAs are a group of regulatory RNAs of 19e22 nucleotides involved in posttranscriptional gene regulation (30).…”

Epigenetic effects of the continuous exposure to peroxisome proliferator WY-14,643 in mouse liver are dependent upon peroxisome proliferator activated receptor α

“…Short-term treatment with non-genotoxic rodent carcinogens, including peroxisome proliferators, is known to lead to hypomethylation of DNA and proto-oncogenes in mouse liver, events that were suggested to correlate with a burst of cell proliferation [11][12][13]. Our previous studies using a methyl-deficient model of non-genotoxic hepatocarcinogenesis in rats showed that alteration of cellular epigenetic processes, such as DNA hypomethylation and loss of histone H4 lysine 20 trimethylation, are key steps in the carcinogenic process induced by methyl deficiency [14,15].…”