NONRANDOM NATURE OF INVIVO METHYLATION BY DIMETHYLNITROSAMINE AND SUBSEQUENT REMOVAL OF METHYLATED PRODUCTS FROM RAT-LIVER CHROMATIN DNA

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  • 1 January 1976
    • journal article
    • research article
    • Vol. 36  (6) , 2073-2079
Abstract
This investigation was designed to study whether methylation of liver chromatin DNA by the carcinogen dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 h following the administration of [3H]DMN (0.5 mg/250 .mu.Ci per 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 absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of radioactivity and 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 2 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 chromatin DNA further reveals that, of the methylated products present at 4 h, 62% is lost by 3 days, 87% is lost by 1 wk and 94% is lost by 2 wk. Loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 wk and 86% by 2 wk, suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin DNA. The results of this study indicated an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and decreased methylation and a slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. The distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.