Related Experiment Videos
Chain length heterogeneity of nucleosomal DNA in mouse liver after dimethylnitrosamine administration
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 gav) 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 phi chi 174 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.
Insights
Dimethylnitrosamine exposure increases DNA fragmentation in mouse liver chromatin. This DNA damage is dose-dependent and detectable after denaturation, but does not affect nucleosome structure.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- Dimethylnitrosamine (DMN) is a known carcinogen.
- Chromatin structure plays a role in DNA accessibility and repair.
- Understanding DMN's effects on DNA is crucial for toxicology and cancer research.
Purpose of the Study:
- To investigate the impact of dimethylnitrosamine (DMN) on mouse liver chromatin structure.
- To determine if DMN alters nucleosomal DNA integrity.
- To quantify DNA fragmentation following DMN exposure.
Main Methods:
- Mice were administered single oral doses of DMN.
- Liver nuclei were isolated and treated with micrococcal nuclease.
- Nucleosomes were separated by sucrose density gradient centrifugation.
- DNA was isolated, denatured, and analyzed by polyacrylamide gel electrophoresis.
Main Results:
- No significant changes in nucleosomal sedimentation velocity were observed.
- A dose-dependent increase in DNA fragmentation was detected after DMN treatment.
- DNA fragmentation was evident upon denaturation and electrophoresis, correlating with DMN dosage.
- Mononucleosomal DNA contained approximately 200 nucleotides, and dinucleosomal DNA contained approximately 400 nucleotides.
Conclusions:
- Dimethylnitrosamine induces DNA fragmentation in mouse liver chromatin.
- The observed DNA fragmentation is dose-dependent and occurs without altering nucleosome structure.
- Denaturation is necessary to reveal DMN-induced DNA damage in chromatin.