Different sensitivities of cultured mammalian cells towards aphidicolin-enhanced DNA effects in the comet assay.
The comet assay in combination with the polymerase inhibitor aphidicolin (APC) has been used to measure DNA excision repair activity, DNA repair kinetics and individual DNA repair capacity. Since APC can enhance genotoxic effects of mutagens measured by the comet assay, this approach has been proposed for increasing the sensitivity of the comet assay in human biomonitoring. The APC-modified comet assay has mainly been performed with human blood and it was shown that it not only enhances the detection of DNA damage repaired by nucleotide excision repair (NER) but also damage typically repaired by base excision repair (BER). Recently, we reported that in contrast to blood leukocytes, A549 cells (a human lung adenocarcinoma cell line) seem to be insensitive towards the repair-inhibiting action of APC. To further elucidate the general usefulness of the APC-modified comet assay for studying repair in cultured mammalian cells, we comparatively investigated further cell lines (HeLa, TK6, V79). DNA damage was induced by BPDE (benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide) and MMS (methyl methanesulfonate) in the absence and presence of APC (3 or 15μM). APC was either added for 2h together with the mutagen or cells were pre-incubated for 30min with APC before the mutagen was added. The results indicate that the cell lines tested differ fundamentally with regard to their sensitivity and specificity towards the repair-inhibiting effect of APC. The actual cause for these differences is still unclear but potential molecular explanations are discussed. Irrespective of the underlying mechanism(s), our study revealed practical limitations of the use of the APC-modified comet assay.
Universität Ulm, Institut für Humangenetik, 89069 Ulm, Germany. Electronic address: guenter.speit@uni-ulm.de.
Günter
G
Speit
Universität Ulm, Institut für Humangenetik, 89069 Ulm, Germany.
Petra
P
Schütz
Universität Ulm, Institut für Humangenetik, 89069 Ulm, Germany.
Julia
J
Bausinger
D016590:Aphidicolin / Q000633:toxicity*; D002460:Cell Line; D020552:Comet Assay; D004247:DNA / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D004257:DNA Polymerase II / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism; D006801:Humans; D009153:Mutagens / Q000633:toxicity
D009153:Mutagens; D016590:Aphidicolin; D004247:DNA; D004257:DNA Polymerase II; D004258:DNA Polymerase III
Aphidicolin; Comet assay; Mutagens; Polymerases