Application of HPLC to the isolation of molecular targets in dosimetry studies

Abstract

High-performance liquid chromatography (HPLC) methods are described which permit the rapid isolation of multiple target macromolecules from the tissues of animals exposed to chemical mutagens. DNA and selected chromosomal proteins are isolated in a simple two step separation scheme. Isolated nuclei are dissolved in 3 M guanidine hydrochloride and the DNA and chromosomal proteins separated on a TSK 3000 SW column. The DNA peak is retained for analysis and the chromatin proteins are dialyzed, lyophylized, and rechromatographed on a PRP-1 column to separate individual histones. Hemoglobin and albumin, two proteins that may prove useful for monitoring mutagen exposure, are isolated from 100 microL of blood by HPLC on a Poly Cat A cation exchange column. Using this approach, we have monitored the kinetics and dose response of adduct formation (and repair) to DNA, histone, hemoglobin and albumin in mice exposed to 7-bromomethylbenzanthracene and benzo[a]pyrene. The results of these studies are described and briefly discussed. Experiments with other mutagens demonstrate the limits to which DNA adduct quantification may be pushed using radioisotopes. Exposures to very high specific activity (200 Ci/mmole) benzo(a)pyrene have allowed DNA adduct quantification down to a few adducts per cell.