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Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation

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Abstract

DNA repair is one of the important determinants of susceptibility to cancer. It is therefore useful to be able to measure DNA repair capacity in samples from population studies. Our aim was, first, to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects, to gain an impression of the degree of intra- and inter-individual variability. The in vitro assay consists of an incubation of lymphocyte extract with substrate nucleoid DNA from cells pretreated with specific damaging agent; either photosensitiser plus light to induce 8-oxoguanine, for BER, or short wavelength ultraviolet light irradiation for NER. In the new NER assay, which requires magnesium but not adenosine triphosphate, there was significant accumulation of UV-dependent incisions during a 30-min incubation of extract with DNA. We found significant correlations between individual repair rates from samples taken on different occasions; i.e. individuals have a characteristic repair capacity. There was also significant variation between individuals, to the extent of about fourfold for BER and tenfold for NER. There was no correlation between BER and NER rates. The BER and NER assays are simple to perform and can provide valuable information in molecular epidemiological studies in which DNA instability is an endpoint.

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Abbreviations

BER:

base excision repair

BPDE:

benzo(a)pyrene diolepoxide

FPG:

formamidopyrimidine DNA glycosylase

NER:

nucleotide excision repair

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Acknowledgments

We are grateful for help with the statistical analysis from Ladislava Wsólová. We acknowledge the support of ZESPRI International Ltd, EC contract LSHB-CT-2006–037575 (COMICS), FOOD-CT-2005–016320 (NewGeneris), and the Throne Holst Fund. Isabel Gaivão was supported by FCT (Portugal), and Anita Piasek by the EC Erasmus programme. Ro 19–8022 was a gift from F. Hoffmann-La Roche.

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Authors and Affiliations

  1. Department of Nutrition, Faculty of Medicine, University of Oslo, PB 1046, Blindern, 0316, Oslo, Norway

    Isabel Gaivão, Anita Piasek, Asgeir Brevik, Sergey Shaposhnikov & Andrew R. Collins

  2. Genetic and Biotechnology Department, University of Trás-os-Montes and Alto Douro, Quinta de Prados, 5001, Vila Real Codex, Portugal

    Isabel Gaivão

  3. Gdansk University of Technology, Gdansk, Poland

    Anita Piasek

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  1. Isabel Gaivão
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  2. Anita Piasek
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  3. Asgeir Brevik
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  4. Sergey Shaposhnikov
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  5. Andrew R. Collins
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Correspondence to Andrew R. Collins.

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Gaivão, I., Piasek, A., Brevik, A. et al. Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation. Cell Biol Toxicol 25, 45–52 (2009). https://doi.org/10.1007/s10565-007-9047-5

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  • DOI: https://doi.org/10.1007/s10565-007-9047-5

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