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 O-2(-). Evaluation of this method confirms that O-2(-), but not O-2 Or H2O2, rapidly oxidizes HE to E+. However the ratio of E+ produced per O-2(-) introduced decreased as the flux of O-2(-) was increased. This suggested that HE can catalyze the dismutation of O-2(-) 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 El 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 O-2(-) (C) 1998 Elsevier Science Inc.