Comet assay-based methods for measuring DNA repair in vitro; estimates of inter- and intra-individual variation.
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.
Department of Nutrition, Faculty of Medicine, University of Oslo, PB 1046, Blindern, 0316 Oslo, Norway.
Isabel
I
Gaivão
Anita
A
Piasek
Asgeir
A
Brevik
Sergey
S
Shaposhnikov
Andrew R
AR
Collins
D020552:Comet Assay / Q000379:methods*; D004260:DNA Repair / Q000187:drug effects; D004784:Environmental Monitoring; D006367:HeLa Cells; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D013997:Time Factors
D008274:Magnesium