Commentary“ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis”—a critical commentary
Section snippets
Intracellular DCF fluorescence arising from DCFH2-DA cannot be used to quantify intracellular hydrogen peroxide
DCFH2-DA is cell-permeative and is taken up into several cell types. DCFH2-DA ester is hydrolyzed inside the cells by esterases, forming DCFH2 (2′,7′-dichlorodihydrofluorescein, 2′,7′-dichlorofluorescin). DCFH2 is not strongly fluorescent; however, DCFH2 is oxidized to a green-fluorescent molecule, 2′,7′-dichlorofluorescein (DCF), a two-electron oxidation product of DCFH2. Contrary to common belief, DCFH2 does not readily react with H2O2 to form DCF [2], [3]. Trace levels of redox-active metal
Reaction between superoxide and hydroethidine (HE) or Mito-HE (MitoSOX red) does not result in the formation of ethidium or mito-ethidium
Existing literature data unambiguously show that superoxide/hydroethidine and superoxide/mito-hydroethidine reactions do not result in the formation of ethidium (E+) or mito-ethidium (Mito-E+) [14], [15], [16], [17], [18], [19], [20], [21]. Mito-HE, a triphenylphosphonium-conjugated mitochondria-targeted analog of HE, essentially reacts with superoxide in the same manner as does HE [17], [21]. Both HE and Mito-HE react with superoxide at similar rates (k = 2 × 106 M−1 s−1 [17], [18]) to form
Intracellular red fluorescence derived from HE cannot be used to quantitate superoxide formation
Ishikawa et al. state in their Science paper that the red fluorescence derived from Mito-HE is a quantitative measure of intracellular superoxide formation [1]. Both HE and Mito-HE are also oxidatively converted to their ethidium analogs (exhibiting red fluorescence) [15], [16], [20], [21]. The fluorescence spectra of E+ and 2-OH-E+ (2-OH-Mito-E+ and Mito-E+) have a significant overlap and it is nearly impossible to deduce the individual contributions of the superoxide/Mito-HE reaction product
Conclusion
In their Science paper, Ishikawa et al. did not appreciate the various methodological pitfalls for detecting ROS in cells using the fluorescent dyes DCFH2-DA and Mito-HE (MitoSOX red). We submit that Ishikawa et al. failed to provide evidence in support of enhanced ROS formation in cells overexpressing mitochondrial DNA mutations derived from metastatic cells and that their paper is one of many papers wherein the fluorescent probes have been used indiscriminately for ROS detection.
Publishers' Note:
The authors of the original article were shown this commentary and offered a response in print should they wish to make one. They have declined the offer.
Acknowledgment
Supported by the National Institutes of Health grants HL073056, NS039958 and R01HL067244.
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