Isolation and quantitation of DNA-bound benzo(a)pyrene metabolites: Comparison of hydroxyapatite and precipitation procedures

• Published in: Analytical biochemistry Vol. 123; no. 1; pp. 162 - 169
• Main Authors: Adriaenssens, Peter I., Bixler, Carol J., Anderson, Marshall W.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.1982
• Subjects: Animals; Benzopyrenes - isolation & purification; Benzopyrenes - metabolism; Binding Sites; Chemical Precipitation; Chromatography - methods; DNA - isolation & purification; DNA - metabolism; Female; Gastric Mucosa - metabolism; Hydroxyapatites; Liver - metabolism; Lung - metabolism; Mice; Mice, Inbred A
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(82)90637-6

The hydroxyapatite and precipitation procedures are two commonly used methods for isolating DNA, and both have been employed in studies on chemical carcinogenesis. A comparison was made of the specific activity (pmol/mg DNA) of benzo(a)pyrene metabolite-DNA adducts in DNA isolated by the two procedures from the lung, liver, and forestomach of A/HeJ mice given oral benzo(a)pyrene. Total DNA-associated radioactivity per milligram DNA was higher with the precipitation procedure than with the hydroxyapatite method. This was partly due to large amounts of early eluting peaks in the high-pressure liquid chromatography analyses of digests of DNA isolated by the precipitation procedure. However, the specific activities obtained for binding of benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide isomers to DNA isolated by the two methods were in close agreement. The hydroxyapatite procedure resulted in the isolation of purer DNA and required less time and sample handling than the precipitation method. For isolation of DNA from whole tissue for carcinogen-DNA adduct analysis, these properties make the hydroxyapatite procedure superior to the precipitation method. Our findings have important implications when comparing the results of DNA-binding studies which employ different DNA isolation procedures.