Hypomethylation of the c-myc promoter region induced by phenobarbital in rat liver

Background: The changes in DNA methylation are considered as one of the early events in hepatocarcinogenesis.

Objective: We evaluated the ability of phenobarbital (PB) – the most widely used anticonvulsant worldwide and classical rodent liver carcinogen – to cause the promoter region of the c-myc protooncogene hypomethylation as well as changes of mRNA level of this gene. Moreover, the expression of Dnmt1 protein in rat treated with this compound was analyzed.

Material and Methods: Male Wistar rats received PB in daily oral doses of 92.8 mg kg-1 b.w. day-1 (at 24-h intervals; for one, three and fourteen days). Methylation of the c-myc promoter region was measured by PCR-based methylationsensitive restriction enzyme analysis (MSRA). Levels of mRNA for c-myc and protein Dnmt1 were assayed using Real-Time PCR and Western Blot, respectively.

Results: The study showed that phenobarbital stimulated persistent changes in DNA methylation, i.e. loss of methylation in the promoter region of the c-myc gene and up-regulated its mRNA level. In addition, a significant increase in protein level of Dnmt1 in the c-myc over-expressing liver cells was observed.

Conclusion: The oppose relationship between Dnmt1 activity and methylation status of c-myc gene was demonstrated. The c-myc over-expression by demethylation might represent an important, early events in the mechanism of action (MOA) of phenobarbital.