Possible role of DNA hypomethylation in mutagenic activity of N'-ethyl-N'-nitrosourea (ENU) on subcutaneous rhabdomyosarcoma in rat

Purpose: The aim of the study was to find out the possible effects of DNA hypomethylation on N'-ethyl-N'-nitrosourea (ENU) mutagenity. Methods: Rats were injected with 300mg/kg body weight-body weight ENU (i.p.)for 45 weeks in order to induce tumour development and the control group received no treatment. Tissues from the ENU treated experimental group and epithelial tissues from non-treated control groups were processed for light microscopy and genomic DNA methyl pattern analysis. The genomic DNA from control and rhabdomyosarcomal tissues were isolated and digested with methylation-sensitive restriction enzymes, R.Msp1 and R.Hpall, to determine the methylation pattern differences in the enzyme cognate CCGG sequences. Results: Extensive tumour development was found in a wide range of tissues in the high dose ENU treated experimental group. Some of these tumours were determined histopathologically and immunohistochemically as subcutaneous rhabdomyosarcomas (RMS). In the normal genome the enzyme cognate CCGG sequence was methylated, but in the rhabdomyosarcoma genome, the target sequence was demethylated with high frequency at the cytosine residue of the CCGG sequences. Genomic DNA from rhabdomyosarcomal tissues showed high fragmentation sizes of R.Msp1 digestion when compared to R.Hpall digestion and controls. While control group DNA fragmentation condensed at approximately 21226 - 546 bps size for R.Msp1 digestion, at 21226 - 1000 bps for R.Hpall, the experimental group DNA fragmentation was condensed at