The scintillometric evaluation of DNA repair synthesis can be distorted by changes of thymidine pool radioactivity

The induction of DNA repair synthesis by UV radiation and methylmethane sulphonate (MMS) in mammalian cell lines of human (EUE, HeLa, FT, KB) and hamster (CHO, BHK) origin has been evaluated by means of autoradiography and the scintillometric procedure which implied the use of hydroxyurea (HU) to suppress DNA replication. While with UV radiation both methods produce concordant positive results, in the case of MMS the evidence of DNA repair synthesis obtained from the autoradiograms is occasionally accompanied by a lack of increase of DNA radioactivity in the treated cultures, as detected by scintillation counting. In such instances MMS is shown to reverse the enhancement of pool radioactivity in the cultures incubated with HU and even to reduce the radioactivity of thymidine pool below control values. By normalizing DNA radioactivities on the basis of pool variations, the discrepancy between autoradiography and scintillation counting is solved. The chromatographic analysis of thymidine pool components justifies the normalization procedure as it demonstrates that also in cultures treated with MMS or MMS + HU pool variations closely parallel the variations of thymidine triphosphate (dTTP) level. The normalization of DNA radioactivities based on the overall pool radioactivities gives an improved evaluation of the actual rate of DNA synthesis. It can be recommended for screening studies of DNA repair inducers because it allows one to correct false negative results without producing false positive data. Compared with the dTTP levels, overall pool radioactivities used as normalizing factors still produce an underestimate of DNA repair when high doses of MMS are applied to hamster cell cultures.