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Mutagenesis Advance Access originally published online on December 19, 2008
Mutagenesis 2009 24(2):183-189; doi:10.1093/mutage/gen070
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Accelerated 32P-HPLC for bulky DNA adducts

Eszter Nagy1, Michael G. Cornelius and Lennart Möller*

Department of Biosciences and Nutrition, Karolinska Institutet, SE-14157 Huddinge, Sweden

Many compounds can react with DNA forming covalent modifications, so-called DNA adducts, which can influence crucial biological processes. DNA adducts from various DNA-damaging agents can act both as biomarkers and as a measurement of the actual damage of the genome. Therefore, they are of value in determining exposed or sensitive individuals or populations and in identifying agents that can induce DNA damage. A common method to measure DNA adducts is through DNA hydrolysis, adduct enrichment, 32P-post-labelling and chromatographic separation. High-performance liquid chromatography (HPLC) with online radioactivity detection, the 32P-HPLC (direct injection of the 32P-labelled mixture into the HPLC with online 32P-detection) method, gives both high sensitivity and good resolution of complex mixtures of DNA adducts. One limitation with this method is the capacity when dealing with large numbers of samples. The aim of this study was therefore to increase the analytical capacity by reducing analysis time of the 32P-HPLC method. A change of HPLC columns to low backpressure columns and adaptation of elution conditions enabled a reduction in time per analysis from 100 to 20 min. This did not affect sensitivity but lowered chromatographic resolution, although bulky DNA adducts were still well resolved from other DNA components. This is useful when the total amount of DNA adducts is the primary interest. When high resolution is required, this can be achieved by gradient modifications, which increase the time required per analysis to 30 min. The accelerated 32P-HPLC increases the capacity in number of samples 3- to 5-fold, depending on resolution requirements, without any negative effect on sensitivity in both in vitro and in vivo samples.

* To whom correspondence should be addressed. Tel: +46 8 608 91 89; Fax: +46 8 608 15 01; Email: lennart.moller{at}ki.se

1 Present address: Department of Molecular Carcinogenesis, Institute of Cancer Research, 15 Cotswold Road, SM2 6BB Sutton, Surrey, UK

Received on February 27, 2008; revised on October 29, 2008; accepted on November 21, 2008.


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