Development and validation of a modified comet assay to phenotypically assess nucleotide excision repair.
There is an increasing need for simple and reliable approaches to phenotypically assess DNA repair capacities. Therefore, a modification of the alkaline comet assay was developed to determine the ability of human lymphocyte extracts to perform the initial steps of the nucleotide excision repair (NER) process, i.e. damage recognition and incision. Gel-embedded nucleoids from A549 cells, pre-exposed to 1 microM benzo[a]pyrene-diol-epoxide, were incubated with cell extracts from frozen or freshly isolated lymphocytes. The rate at which incisions are introduced and the subsequent increase in tail moment is indicative for the repair capacity of the extracts. Freshly prepared extracts from lymphocytes of human volunteers (n = 8) showed significant inter-individual variations in their DNA repair capacity, which correlated with the removal of bulky DNA lesions over a period of 48 h determined by (32)P-post-labelling (R(2) = 0.76, P = 0.005). Repeated measurements revealed a low inter-assay variation (11%). Storage of cell extracts for more than 3 weeks significantly reduced (up to 80%) the capacity to incise the damaged DNA as compared to freshly isolated extracts. This reduction was completely restored by addition of ATP to the extracts before use, as it is required for the incision step of NER. In contrast, extracts freshly prepared from frozen lymphocyte pellets can be used without loss of repair activity. DNA repair deficient XPA-/- and XPC-/- fibroblasts were used to further validate the assay. Although some residual capacity to incise the DNA was observed in these cells, the repair activity was restored to normal wild-type levels when a complementary mixture of both extracts (thereby restoring XPA and XPC deficiency) was used. These results demonstrate that this repair assay can be applied in molecular epidemiological studies to assess inter-individual differences in NER.
Langie
Sabine A S
SA
Nutrition and Toxicology Research Institute Maastricht, NUTRIM, Department of Health Risk Analysis and Toxicology, Maastricht University, 6200 MD, PO Box 616, Maastricht, The Netherlands.
Knaapen
Ad M
AM
Brauers
Karen J J
KJ
van Berlo
Damien
D
van Schooten
Frederik-Jan
FJ
Godschalk
Roger W L
RW
D000255:Adenosine Triphosphate / Q000378:metabolism; D045744:Cell Line, Tumor; D020552:Comet Assay / Q000379:methods*; D015925:Cryopreservation; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D010641:Phenotype; D013997:Time Factors; D051760:Xeroderma Pigmentosum Group A Protein / Q000235:genetics
D004268:DNA-Binding Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; C086984:XPC protein, human; D000255:Adenosine Triphosphate