Scintillometric determination of DNA repair in human cell lines: a critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [3H]thymidine the radioactivity incorporated into DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (CHU, THU). The ratios THU/CHU and THU/T:CHU/C, indicating absolute and relative increases in DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas no stimulation is inferred when both of them are .ltoreq. 1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations. Agents giving a relative but not an absolute increase in DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. The same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labeled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of the DNA radioactivity on the basis of pool alterations. The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value. Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintimetric procedure after normalization of DNA radioactivities. These agents were unable to stimulate repair synthesis, according to the results of autoradiography and isopycnic centrifugation. The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.