ToxSci Advance Access published online on March 14, 2006
Toxicological Sciences, doi:10.1093/toxsci/kfj155
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1 Department of Pharmacology and Toxicology, Michigan State Univ., East Lansing, MI, 48824
* To whom correspondence should be addressed. Altered DNA methylation contributes to tumorigenesis by affecting gene expression in a heritable fashion. Phenobarbital (PB) is a non-genotoxic 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% (w/w) PB in drinking water for 2 or 4wk, and a 2wk recovery was included following each dosing period. DNA was isolated from liver (target) and kidney (non-target) tissue. The methylation status of GC-rich regions of DNA was assessed via methylation-sensitive restriction digestion, arbitrarily primed PCR 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 wk were also seen at 4 wk while only 1 of the 123 RAMs (<1%) present in C57BL/6 liver was seen at 4 wk. In the B6C3F1 mice, 57 unique (as compared to the C57BL/6) regions of altered hepatic methylation (RAMs), predominantly hypomethylation, were observed at 2 wk, increasing to 86 at 4wk. Changes in methylation were largely reversible. Altered methylation in liver was highly dissimilar to kidney. Following 4 wk PB, bisulfite sequencing revealed hypomethylation of Ha-ras in B6C3F1, but not C57BL/6, which correlated with increased gene expression. These data indicate: 1) progressive, non-random 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.
Received January 6, 2006
Accepted March 2, 2006
Carcinogenicity
Phenobarbital Induces Progressive Patterns of GC-Rich and Gene-Specific Altered DNA Methylation in the Liver of Tumor-Prone B6C3F1 Mice
Ammie N. Bachman 1,
Jennifer M. Phillips 2,
and
Jay I. Goodman 1 *
2 Department of Biochemistry and Molecular Biology, Michigan State Univ., East Lansing, MI, 48824
Jay I. Goodman, E-mail: goodman3{at}msu.edu
Abstract 
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