Applicability of the alkaline elution procedure as modified for the measurement of DNA damage and its repair in nonradioactively labeled cells.
We have critically evaluated various modifications of the alkaline elution methodology that were required to adapt the method for measuring DNA damage in cells from animal tissues treated in vivo. These modifications involved the use of a fluorometric assay for the eluted DNA using the dye Hoechst 33258, which in turn required the use of a different combination of filter and lysis conditions than those used in conventional assays. This protocol was compared with the conventional protocols by examining the DNA damage produced in cultured Chinese hamster ovary cells after treatment with three agents (gamma-rays, cis-dichlorodiammineplatinum (DDP) and trans-DDP) that differ widely in the type and repairability of the DNA lesions that they induce. For both gamma-rays and trans-DDP, the results obtained by the various protocols were equivalent with respect to the amount, type, and rate of repair of the DNA damage produced. On the other hand, for cis-DDP, where the repair time for DNA crosslinks was significantly long relative to the cell-cycle time, DNA replication appeared to be a potentially complicating factor in the measurement of crosslink repair. However, even after treatment of rapidly dividing cultured cells, where any discrepancy between the radioactivity and Hoechst assays due to DNA replication should be maximal, the resulting difference in the amount of repair measured using the two assays was relatively small. Finally, in experiments using cis-DDP and trans-DDP, the data suggested that when polycarbonate and polyvinyl chloride filters were compared using the same cell lysis conditions, their relative sensitivity to detect DNA-protein versus DNA-interstrand crosslinking were comparable. The modified alkaline elution protocol for the measurement of DNA damage in vivo therefore appears, in most cases, to produce results comparable with those obtained by the conventional protocols.
D000818:Animals; D006690:Bisbenzimidazole; D002460:Cell Line; D002945:Cisplatin / Q000494:pharmacology; D004247:DNA / Q000187:drug effects / Q000302:isolation & purification* / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D005720:Gamma Rays; D006863:Hydrogen-Ion Concentration; D013050:Spectrometry, Fluorescence / Q000379:methods; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
D014316:Tritium; C034697:transplatin; D004247:DNA; D006690:Bisbenzimidazole; D002945:Cisplatin; D013936:Thymidine