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Nonrandom Nature of in Vivo Methylation by Dimethylnitrosamine and the Subsequent Removal of Methylated Products from Rat Liver Chromatin DNA¹

Fels Research Institute [R. R., S. R., D. S. R. S., E. F.] and Departments of Pathology [D. S. R. S., E. F.] and Biochemistry [S. R., E. F.], Temple University Medical School, Philadelphia, Pennsylvania 19140

This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-dgestible and nondigestible material 4 hr following the administration of [³H]DMN (0.5 mg/250 µCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region.

The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nucleaseaccessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.

¹ This investigation was supported in part by USPHS Research Grants CA 14689, CA 12218, and CA 12227 from the National Cancer Institute and American Cancer Society Grant BC-7P.

This work was presented at the annual meeting of American Association for Cancer Research held at San Diego, Calif., May 8 to 11, 1975 (29).

² Present address: Frederick Cancer Research Center, Post Box "B," Frederick, Md. 21701.

³ To whom requests for reprints should be addressed.

⁴ Present address: Department of Pathology, University of Toronto, 100 College Street, Toronto, Ontario, Canada M5G 1L5.

Received 12/11/75. Accepted 3/10/76.

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