Abstract
The effect of dimethylnitrosamine on the nucleosomal structure of mouse liver chromatin was studied. After a single oral dose of dimethylnitrosamine (2–75 mg/kg body weight 45 min before sacrifice) liver nuclei were isolated and incubated with micrococcus nuclease. Nucleosomes were separated on sucrose density gradients. There were no differences in nucleosomal sedimentation velocities between preparations from control and dimethylnitrosamine treated animals. The supernatant obtained after centrifugation of the lysed nuclei (2 min at 4,000 g av) and nucleosomal peak fractions were used for isolation of DNA. DNA was heat denatured in 7 M urea or formamide. After electrophoresis on polyacrylamide gels areas under mononucleosomal DNA and smaller fragments were measured and compared with the total DNA area. The increase in DNA fragmentation was dimethylnitrosamine dose response dependent. When expressed as per cent of controls it amounted to 106% for 2 mg; 115% for 10 mg; 127% for 25 mg; 164% for 75 mg dimethylnitrosamine/kg body weight. A good correlation between mobility and log of chain length of Φ χ174 RF DNA-Hae III digest was obtained in nondenaturing 5% polyacrylamide gels and denaturing non-aqueous formamide polyacrylamide gels but not in 12% polyacrylamide gels containing 7 M urea. DNA of mononucleosomal peak fractions contained 200 and that of dinucleosomal peak fractions 400 nucleotides. Fragmentation of DNA was closely related to in vivo dimethylnitrosamine treatment but was not detected in measurements of protein-DNA complexes in the chromatin. It was disclosed on denaturation of DNA followed by polyacrylamide gel electrophoresis.
Similar content being viewed by others
Abbreviations
- DMN:
-
dimethylnitrosamine
- SDS:
-
sodium dodecyl sulfate
References
Bannon P, Verley W (1972) Alkylation of phosphates and stability of phosphate triesters in DNA. Eur J Biochem 31: 103–111
Burgoyne LA, Hewish DR, Mobbs J (1974) Mammalian chromatin substructure studies with the calcium-magnesium endonuclease and two-dimensional polyacrylamide-gel electrophoresis. Biochem J 143: 67–72
Cox R, Damjanov I, Abanobi SE, Sarma DSR (1973) A method for measuring DNA damage and repair in the liver in vivo. Cancer Res 33: 2114–2121
Damjanov I, Cox R, Sarma DSR, Farber E (1973) Patterns of damage and repair of liver DNA induced by carcinogenic methylating agents in vivo. Cancer Res 33: 2122–2128
Dynan WS, Burgess RR (1979) In vitro transcription by wheat germ ribonucleic acid polymerase II: Effects of heparin and role of template integrity. Biochemistry 21: 4581–4588
Floot GJ, Philippus EJ, Hart AHM, Den Engelse L (1979) Persistence and accumulation of (potential) single strand breaks in liver DNA of rats treated with diethylnitrosamine or dimethylnitrosamine: correlation with hepatocarcinogenicity. Chem Biol Interact 25: 229–242
Galbraith A, Itzhaki RF (1979) Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine. Chem Biol Interact 28: 309–322
Herzog J, Farber JL (1976) Inhibition of rat liver polymerases by action of the methylating agents dimethylnitrosamine in vivo and methyl methanesulfonate in vitro. Cancer Res 36: 1761–1770
Hultin T, Eriksson B, Nygård O, von der Decken A (1978) RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine. Chem Biol Interact 21: 45–58
Kornberg RD (1974) Chromatin structure: a repeating unit of histones and DNA. Science 184: 868–871
Magee PN, Nicoll JW, Pegg AE, Swann PF (1975) Alkylation intermediates in nitrosamine metabolism. Biochem Soc Trans 3: 62–65
Maniatis T, Ptashne M (1973) Multiple repressor binding at the operators in bacteriophage λ. Proc Natl Acad Sci USA 70: 1531–1535
Maniatis T, Jeffrey A, van de Sande H (1975) Chain length determination of small double and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14: 3787–3794
Noll M (1974) Internal structure of the chromatin subunit. Nucleic Acids Res 1: 1573–1578
Noll M (1974) Subunit structure of chromatin. Nature 251: 249–251
Noll M, Kornberg RD (1977) Action of micrococcal nuclease on chromatin and the location of histone H1. J Mol Biol 109: 393–404
Noll M, Thomas JO, Kornberg RD (1975) Preparation of native chromatin and damage caused by shearing. Science 187: 1203–1206
Staynov DZ, Pinder JC, Gratzer B (1972) Molecular weight determination of nucleic acids by gel electrophoresis in non-aqueous solution. Nature New Biol 235: 108–110
Stewart BW, Magee PN (1971) Effect of a single dose of dimethylnitrosamine on biosynthesis of nucleic acid and protein in rat liver and kidney. Biochem J 125: 943–952
Studier FW (1973) Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J Mol Biol 79: 237–248
von der Decken A, Andersson GM (1977) Template properties of rat liver chromatin after restricted protein supply; Transcription by homologous ribonucleic acid polymerase I and II. J Nutr 107: 949–958
Yu F-L (1975) An improved method for the quantitative isolation of rat liver nuclear polymerases. Biochim Biophys Acta 395: 329–336
Author information
Authors and Affiliations
Additional information
The work was supported by Grant Number 1 R0 1 CA26642-01, awarded to A.v.d.D. by the National Cancer Institute, DHEW
Rights and permissions
About this article
Cite this article
Näslund, B., von der Decken, A. Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration. Arch Toxicol 47, 169–177 (1981). https://doi.org/10.1007/BF00368677
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00368677