The purification of human DNA fragments containing benzpyrene adducts

Abstract

DNA was isolated from human diploid lung epithelial cells treated in culture with [ 3H]benzpyrene. The DNA contained one covalently bound benzpyrene group per 38 kb and it was digested with a series of restriction endonucleases giving decreasing fragment sizes, and also with DNAase I to 96% acid solubility. The digests were applied to octyl-Sepharose columns under conditions which promote hydrophobic interaction of the benzpyrene groups on the DNA with the octyl groups in the column matrix. Separation of fragments without and with benzpyrene groups was achieved with successive high salt and ethanediol washes. As DNA fragment size is decreased, more DNA-associated benzpyrene is eluted with ethanediol. Under these conditions, DNA from untreated cells is totally removed in the high salt wash and free benzpyrene metabolites are retained on the column. The separation of DNA fragments with covalently-bound hydrophobic benzpyrene groups, from less modified or unmodified DNA will facilitate examination of the distribution of benzpyrene adducts in defined regions of the human genome.