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Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: A rapid method for its analysis using hydroxylapatite column chromatography

Authors
Journal
Chemico-Biological Interactions
0009-2797
Publisher
Elsevier
Publication Date
Volume
25
Issue
1
Identifiers
DOI: 10.1016/0009-2797(79)90070-x
Disciplines
  • Biology
  • Physics

Abstract

Summary A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[ 3H]dimethylbenz[a] anthracene ([ 3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.

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