Role of superoxide in radiation-killing of Escherichia coli and in thymine release from thymidine

The role of superoxide and hydroxyl radicals in gamma-radiation-killing of Escherichia coli K12 was studied in aerated suspensions supplemented with formate, phosphate, superoxide dismutase, catalase and saturated with nitrous oxide. Nitrous oxide, which converts e^-_aq to ^•OH, caused decreased radiosensitivity. On the other hand, formate, which results in conversion of ^•OH to ^•O₂^-, resulted in an increased radiosensitivity. The results implicated ^•O₂^- as a major cause of radiation-mediated cell-killing. The addition of the enzymes, superoxide dismutase or catalase to the E. coli suspensions prior to and during irradiation had no effect on cell survival, indicating that the biologically significant site of generation and action of ^•O₂^- is an intracellular one. Further studies were undertaken to examine the role of superoxide in DNA damage. The release of thymine from the DNA base, thymidine was studied as a result of gamma-irradiation and of chemically generated superoxide (using KO₂ in dimethyl sulfoxide). Thymine was identified by HPLC and mass spectrometry. C-13 NMR analysis of the reaction mixture of thymidine with KO₂ in dimethyl sulfoxide provided evidence for attack of ^•O₂^- at the ribosyl Cl′ atom.