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Measurements of DNA double-strand break yields in E. coli after rapid irradiation and cell inactivation: the effects of inactivation technique and anoxic radiosensitizers.

A study has been made of methods for rapidly inactivating cells of E. coli at neutral pH to prevent enzymatic, chemical, or physical modification to DNA damaged by irradiation. The radiation was delivered in a fraction of a second using an electron accelerator. Cell inactivation was with ethanol or a solution (CSE) containing detergent, EDTA, and chloroform. It was found that DNA could be released from irradiated and inactivated cells simply by incubating them with the protease Pronase, and this DNA appeared to be in a form suitable for centrifugational analysis in neutral sucrose gradients. When cells were irradiated in the presence of oxygen, inactivation with ethanol gave a radiation-induced double-strand break (dsb) yield 1.8-fold higher than when inactivation was with CSE. Possible explanations of this are discussed. Using CSE inactivation, an oxygen enhancement ratio for dsb formation of 4.3 was observed and yields of dsb per Gray were in good agreement with results from other laboratories. At concentrations which enhanced cell killing 2.6-fold the "electron-affinic" type anoxic radiosensitizer misonidazole enhanced dsb formation 2.0-fold, whereas the nitroxyl free radical anoxic radiosensitizer norpseudopelletierine-N-oxyl had no significant effect on dsb yield although there was a possible slight enhancement at the higher doses used.