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

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Abstract

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.

Introduction

Hydroethidine (HE)1 is oxidized to the fluorescent ethidium (E+) by O2 whether added as KO2, or produced by activated leukocytes [1]. Moreover, the oxidation of HE to E+ was shown to be rapid when the oxidant was O2; but not when O2, H2O2, HOCl or ONOO were used [2]. Consequently HE has been applied as a detector of intracellular O2 [3].

We have investigated chromogenic and lumigenic detectors of O2, such as the tetrazoliums [4] and lucigenin [5], and have found them to be prone to artifact in that they can cause O2 production even in systems not producing this radical in the absence of these compounds. We wondered whether in hydroethidine we might finally have a convenient measure of O2, free from such problems.

In the experiments reported herein we verify earlier reports [1] that the oxidation of HE to E+ is rapidly caused by O2 but not by H2O2 [2]. However we note that HE can be oxidized by cytochrome c and more importantly that HE can catalyze the dismutation of O2. This latter process interferes with the fluorimetric measurement of O2 with HE.

Section snippets

Materials and methods

HE was purchased from Polysciences Inc. and was dissolved, to 63.5 mM, in either N,N-dimethyl formamide or dimethyl sulfoxide. This stock solution was stored under N2 at −80°C. Working solutions were prepared by diluting this stock into 50 mM potassium phosphate at pH 7.8. This was done immediately prior to use. DNA and E+ were from Sigma. The oxidation of HE to E+ was monitored fluorimetrically by exciting at 470 nm and following emission at 590 nm.

The sodA sodB strains of Escherichia coli

Oxidation of HE to E+ by O2

Line 1, Fig. 1 presents the rate of oxidation of HE to E+ by the flux of O2 generated by the xanthine oxidase reaction. That this oxidation was due to O2, and not to H2O2 or to O2, is shown by the dose-dependent inhibition by SOD (lines 2, 3 and 4). Catalase, in contrast was without effect (not shown). These results are in full accord with previous reports [1], [2], [3]. It should be noted that xanthine oxidase plus xanthine did not cause the reduction of E+ to HE, even under anaerobic

Discussion

In accord with previous reports [1], [2] we find HE to be oxidized to E+ by O2, whereas the rates of oxidation by O2 or by H2O2are much slower. The reduction of E+ to HE, by glucose oxidase plus glucose, does increase cyt c reduction, but that is due to a direct effect of HE and not to HE-mediated O2 production; since this cyt c reduction was not SOD-inhibitable. The oxidations of HE by cyt c consumed more than the theoretical 2 cyt c/HE and the product was not E+. This indicates that cyt c

Acknowledgements

This work was supported by grants from the Council for Tobacco Research, U.SA., Inc. (2871BR1) and the National Institutes of Health (HL56025-03).

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