A comparison of the techniques of alkaline filter elution and alkaline sucrose sedimentation used to assess DNA damage induced by 2-nitroimidazoles

Abstract

The induction of DNA single-strand breaks (DNA-SSB) in Chinese hamster V79-379A lung fibroblasts by misonidazole or RSU-1069 under both aerobic and hypoxic conditions was examined following incubations for up to 4hr at 310°K using the technique of alkaline filter elution. Incubation with RSU-1069 induces DNA-SSB under both hypoxic and aerobic conditions, whereas incubation with misonidazole induces DNA-SSB only under hypoxia. The yield of breaks is dependent on both agent concentration and contact time. Following identical treatments with these agents, the yield of DNA-SSB (expressed in radiation dose equivalents) determined by alkaline filter elution is about one order of magnitude less than that previously determined by alkaline sucrose gradient sedimentation. In contrast to radiation induced DNA-SSB, alkaline elution is less sensitive than alkaline sucrose gradient sedimentation when determining DNA-SSB induced by RSU-1069 and misonidazole. During the filter elution assay, either increasing cell lysis from 2 to 4 hr, the pH of the lysing buffer from pH 8.7 to 12.5 or the elution buffer from pH 12.2 to 12.5 does not significantly effect the yield of DNA-SSB. Increasing the pH of the lysing or elution buffers to > pH 13 however results in considerable degradation of the DNA, whereby 50–85% of the total DNA passes through the filter with the lysing solution. This effect was similar for DNA from both control and chemically insulted cells. In conclusion, it is apparent that incubation with these agents results in the induction of DNA damage which is expressed as a DNA-SSB only after prolonged treatment under alkaline conditions. Further, the use of alkaline elution to study DNA-SSB damage induced chemically must be treated with caution in the light of these findings.