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The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine.

ABSTRACT: 1. RNA was isolated from rat liver at selected times after the intraperitoneal injection of either [(14)C]methyl methanesulphonate (50mg/kg) or [(14)C]dimethylnitrosamine (2mg/kg). These doses were chosen to minimize effects due to toxicity. 2. Two methods of extraction and purification of RNA were used and an analysis of the radioactivity present was made by column chromatography of acid hydrolysates of the purified RNA. 3. The extent of methylation of guanine, the principal site of alkylation in rat liver RNA, was determined at times up to 14 days after injection. Although dimethylnitrosamine is a potent liver carcinogen and methyl methanesulphonate is not carcinogenic to rat liver, the rate of disappearance of 7-methylguanine from RNA was similar for both compounds, with a half-life of about 3.5 days. 4. An estimate of the biological half-life of rRNA was made by using [(3)H]orotic acid. A half-life of 5 days was obtained and this was not affected by injecting animals with unlabelled methyl methanesulphonate at the same dosage of 50mg/kg used in the studies of RNA methylation. 5. After administration of labelled orotic acid, reutilization of labelled RNA degradation products probably results in an overestimation of the biological half-life for rRNA. It is suggested that non-toxic doses of methylating agents such as methyl methanesulphonate and dimethylnitrosamine may prove to be a more effective way of accurately estimating the biological turnover of RNA species.

SUBMITTER: McElhone MJ

PROVIDER: S-EPMC1178187 | biostudies-other | 1971 Dec

REPOSITORIES: biostudies-other

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Project description:1. N[(14)C]-Methyl-N-nitrosourea, [(14)C]dimethylnitrosamine, [(14)C]dimethyl sulphate and [(14)C]methyl methanesulphonate were injected into rats, and nucleic acids were isolated from several organs after various time-intervals. Radioactivity was detected in DNA and RNA, partly in major base components and partly as the methylated base, 7-methylguanine. 2. No 7-methylguanine was detected in liver DNA from normal untreated rats. 3. The specific radioactivity of 7-methylguanine isolated from DNA prepared from rats treated with [(14)C]dimethylnitrosamine was virtually the same as that of the dimethylnitrosamine injected. 4. The degree of methylation of RNA and DNA produced in various organs by each compound was determined, and expressed as a percentage of guanine residues converted into 7-methylguanine. With dimethylnitrosamine both nucleic acids were considerably more highly methylated in the liver (RNA, about 1% of guanine residues methylated; DNA, about 0.6% of guanine residues methylated) than in the other organs. Kidney nucleic acids were methylated to about one-tenth of the extent of those in the liver, lung showed slightly lower values and the other organs only very low values. N-Methyl-N-nitrosourea methylated nucleic acids to about the same extent in all the organs studied, the amount being about the same as that in the kidney after treatment with dimethylnitrosamine. In each case the RNA was more highly methylated than the DNA. Methyl methanesulphonate methylated the nucleic acids in several organs to about the same extent as N-methyl-N-nitrosourea, but the DNA was more highly methylated than the RNA. Dimethyl sulphate, even in toxic doses, gave considerably less methylation than N-methyl-N-nitrosourea in all the organs studied, the greatest methylation being in the brain. 5. The rate of removal of 7-methylguanine from DNA of kidneys from rats treated with dimethylnitrosamine was compared with the rate after treatment of rats with methyl methanesulphonate. No striking difference was found. 6. The results are discussed in connexion with the organ distribution of tumours induced by the compounds under study and in relation to the possible importance of alkylation of cellular components for the induction of cancer.

Project description:1. The incorporation of methyl groups into histones from dimethylnitrosamine and from methionine was studied by injection of the labelled compounds, isolation of rat liver and kidney histones, and analysis of hydrolysates by column chromatography. 2. Labelled methionine gave rise to labelled in-N-methyl-lysine, di-in-N-methyl-lysine and an amino acid presumed to be omega-N-methyl-arginine. 3. Administration of labelled dimethylnitrosamine gave rise to labelled S-methylcysteine, 1-methylhistidine, 3-methylhistidine and in-N-methyl-lysine derived from the alkylating metabolite of dimethylnitrosamine. In addition, labelled formaldehyde released by metabolism of dimethylnitrosamine leads to the formation of labelled S-adenosylmethionine, and hence to labelling of in-N-methyl-lysine, di-in-N-methyl-lysine and omega-N-methylarginine by enzymic methylation. 4. The formation of in-N-methyl-lysine by alkylation of liver histones was confirmed by using doubly labelled dimethylnitrosamine to discriminate between direct chemical alkylation and enzymic methylation via S-adenosylmethionine. These experiments also suggested the possibility that methionine residues in the histones were alkylated to give methylmethionine sulphonium residues. 5. The extent of alkylation of liver histones was maximal at about 5h after dosing and declined between 5 and 24h. The methylated amino acids resulting from direct chemical alkylation were preferentially lost: this is ascribed to necrosis of the more highly alkylated cells. 6. Liver histones were about four times as alkylated as kidney histones; the extent of alkylation of liver histones was similar to that of liver total nuclear proteins. 7. Methyl methanesulphonate (120mg/kg) alkylated liver histones to a greater extent than did dimethylnitrosamine. Diethylnitrosamine also alkylated liver histones. 8. The results are discussed with regard to the possible effects of alkylation on histone function, and the possible role of histone alkylation in carcinogenesis by the three compounds.

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The stability of rat liver ribonucleic acid in vivo after methylation with methyl methanesulphonate or dimethylnitrosamine.

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