Elsevier

Free Radical Biology and Medicine

Volume 45, Issue 9, 1 November 2008, Pages 1217-1219
Free Radical Biology and Medicine

Commentary
“ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis”—a critical commentary

https://doi.org/10.1016/j.freeradbiomed.2008.07.025Get rights and content

Abstract

In a recent publication (K. Ishikawa et al., 2008, Science 320, 661–664), the authors described how replacing the endogenous mitochondrial DNA (mtDNA) in a weakly metastatic mouse tumor cell line with mtDNA from a highly metastatic cell line enhanced tumor progression through enhanced production of reactive oxygen species (ROS). The authors attributed the transformation from a low-metastatic cell line to a high-metastatic phenotype to overproduction of ROS (hydrogen peroxide and superoxide) caused by a dysfunction in mitochondrial complex I protein encoded by mtDNA transferred from the highly metastatic tumor cell line. In this critical evaluation, using the paper by Ishikawa et al. as an example, we bring to the attention of researchers in the free radical field how the failure to appreciate the complexities of dye chemistry could potentially lead to pitfalls, misinterpretations, and erroneous conclusions concerning ROS involvement. Herein we make a case that the authors have failed to show evidence for formation of superoxide and hydrogen peroxide, presumed to be generated from complex I deficiency associated with mtDNA mutations in metastatic cells.

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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