Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical

The fluorogenic oxidation of hydroethidine (HE) to ethidium (E+) has been used as a measure of O2−. Evaluation of this method confirms that O2−, but not O2 or H2O2, rapidly oxidizes HE to E+. However the ratio of E+ produced per O2− introduced decreased as the flux of O2− was increased. This suggested that HE can catalyze the dismutation of O2− and this was affirmed. HE was oxidized to a red product, distinct from E+ by ferricytochrome c and a similar oxidation may occur within Escherichia coli. HE inhibited the growth and killed a SOD-null strain to a greater extent than the SOD-replete parental strain and these effects were much diminished under anaerobic conditions. This indicated that E+ was responsible for the toxicity of HE and indeed E+ was seen to be toxic under both aerobic and anaerobic conditions. In view of the data presented HE can be recommended as a qualitative but not as a quantitative measure of O2−.