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. 1985 Oct;62:73–79. doi: 10.1289/ehp.856273

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

R Balhorn, J A Mazrimas, M Corzett
PMCID: PMC1568673  PMID: 4085450

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Balhorn R., Oliver D., Hohmann P., Chalkley R., Granner D. Turnover of deoxyribonucleic acid, histones, and lysine-rich histone phosphate in hepatoma tissue culture cells. Biochemistry. 1972 Oct 10;11(21):3915–3921. doi: 10.1021/bi00771a013. [DOI] [PubMed] [Google Scholar]
  2. Farmer P. B., Gorf S. M., Bailey E. Determination of hydroxypropylhistidine in haemoglobin as a measure of exposure to propylene oxide using high resolution gas chromatography mass spectrometry. Biomed Mass Spectrom. 1982 Feb;9(2):69–71. doi: 10.1002/bms.1200090205. [DOI] [PubMed] [Google Scholar]
  3. GITLIN D., KLINENBERG J. R., HUGHES W. L. Site of catabolism of serum albumin. Nature. 1958 Apr 12;181(4615):1064–1065. doi: 10.1038/1811064b0. [DOI] [PubMed] [Google Scholar]
  4. Galbraith A., Itzhaki R. F. Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine. Chem Biol Interact. 1979 Dec;28(2-3):309–322. doi: 10.1016/0009-2797(79)90171-6. [DOI] [PubMed] [Google Scholar]
  5. Gazit B., Panet A., Cedar H. Reconstitution of a deoxyribonuclease I-sensitive structure on active genes. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1787–1790. doi: 10.1073/pnas.77.4.1787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Groopman J. D., Busby W. F., Jr, Wogan G. N. Nuclear distribution of aflatoxin B1 and its interaction with histones in rat liver in vivo. Cancer Res. 1980 Dec;40(12):4343–4351. [PubMed] [Google Scholar]
  7. Gurley L. R., Hardin J. M. The metabolism of histone fractions. II. Conservation and turnover of histone fractions in mammalian cells. Arch Biochem Biophys. 1969 Mar;130(1):1–6. doi: 10.1016/0003-9861(69)90002-2. [DOI] [PubMed] [Google Scholar]
  8. Hancock R. Conservation of histones in chromatin during growth and mitosis in vitro. J Mol Biol. 1969 Mar 28;40(3):457–466. doi: 10.1016/0022-2836(69)90165-x. [DOI] [PubMed] [Google Scholar]
  9. Kootstra A. Effect of histone acetylation on the formation and removal of B(a)P chromatin adducts. Nucleic Acids Res. 1982 May 11;10(9):2775–2789. doi: 10.1093/nar/10.9.2775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Meyer M. C., Guttman D. E. The binding of drugs by plasma proteins. J Pharm Sci. 1968 Jun;57(6):895–918. doi: 10.1002/jps.2600570601. [DOI] [PubMed] [Google Scholar]
  11. Pereira M. A., Lin L. H., Chang L. W. Dose-dependency of 2-acetylaminofluorene binding to liver DNA and hemoglobin in mice and rats. Toxicol Appl Pharmacol. 1981 Sep 30;60(3):472–478. doi: 10.1016/0041-008x(81)90333-1. [DOI] [PubMed] [Google Scholar]
  12. Rechsteiner M., Kuehl L. Microinjection of the nonhistone chromosomal protein HMG1 into bovine fibroblasts and HeLa cells. Cell. 1979 Apr;16(4):901–908. doi: 10.1016/0092-8674(79)90105-3. [DOI] [PubMed] [Google Scholar]
  13. Weisbrod S., Weintraub H. Isolation of a subclass of nuclear proteins responsible for conferring a DNase I-sensitive structure on globin chromatin. Proc Natl Acad Sci U S A. 1979 Feb;76(2):630–634. doi: 10.1073/pnas.76.2.630. [DOI] [PMC free article] [PubMed] [Google Scholar]

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