Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products

O G Lyublinskaya; V V Zenin; A N Shatrova; N D Aksenov; V I Zemelko; A P Domnina; A P Litanyuk; E B Burova; S S Gubarev; Y A Negulyaev; N N Nikolsky

doi:10.1016/j.freeradbiomed.2014.07.008

Intracellular oxidation of hydroethidine: compartmentalization and cytotoxicity of oxidation products

Free Radic Biol Med. 2014 Oct:75:60-8. doi: 10.1016/j.freeradbiomed.2014.07.008. Epub 2014 Jul 15.

Authors

O G Lyublinskaya¹, V V Zenin², A N Shatrova³, N D Aksenov⁴, V I Zemelko⁵, A P Domnina⁶, A P Litanyuk⁷, E B Burova⁸, S S Gubarev⁹, Y A Negulyaev¹⁰, N N Nikolsky¹¹

Affiliations

¹ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
² Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
³ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
⁴ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
⁵ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
⁶ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
⁷ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
⁸ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia. Electronic address: [email protected].
⁹ Department of Medical Physics and Bioengineering, Institute of Physics, Nanotechnology and Telecommunications, St. Petersburg State Polytechnical University, Polytechnicheskaya st. 29, St. Petersburg 195251, Russia. Electronic address: [email protected].
¹⁰ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia; Department of Medical Physics and Bioengineering, Institute of Physics, Nanotechnology and Telecommunications, St. Petersburg State Polytechnical University, Polytechnicheskaya st. 29, St. Petersburg 195251, Russia. Electronic address: [email protected].
¹¹ Department of Intracellular Signaling and Transport, Institute of Cytology, Russian Academy of Sciences, Tikhoretsky pr. 4, St. Petersburg 94064, Russia; Department of Medical Physics and Bioengineering, Institute of Physics, Nanotechnology and Telecommunications, St. Petersburg State Polytechnical University, Polytechnicheskaya st. 29, St. Petersburg 195251, Russia. Electronic address: [email protected].

PMID: 25035077
DOI: 10.1016/j.freeradbiomed.2014.07.008

Abstract

Hydroethidine (HE) is a blue fluorescent dye that is intracellularly converted into red-emitting products on two-electron oxidation. One of these products, namely 2-hydroxyethidium, is formed as the result of HE superoxide anion-specific oxidation, and so HE is widely used for the detection of superoxide in cells and tissues. In our experiments we exploited three cell lines of different origin: K562 (human leukemia cells), A431 (human epidermoid carcinoma cells), and SCE2304 (human mesenchymal stem cells derived from endometrium). Using fluorescent microscopy and flow cytometry analysis, we showed that HE intracellular oxidation products accumulate mostly in the cell mitochondria. This accumulation provokes gradual depolarization of mitochondrial membrane, affects oxygen consumption rate in HE-treated cells, and causes cellular apoptosis in the case of high HE concentrations and/or long cell incubations with HE, as well as a high rate of HE oxidation in cells exposed to some stimuli.

Keywords: 2-Hydroxyethidium; Ethidium; Flow cytometry; Fluorescent probes; Hydroethidine; Superoxide detection.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Apoptosis / physiology
Cell Line, Tumor
Ethidium / analogs & derivatives
Ethidium / chemistry
Flow Cytometry
Fluorescent Dyes / chemistry
Fluorescent Dyes / pharmacology*
Humans
Microscopy, Fluorescence
Mitochondria / metabolism*
Mitochondrial Membranes / physiology*
Oxidation-Reduction
Oxygen Consumption / physiology
Phenanthridines / chemistry
Phenanthridines / pharmacology*
Superoxides / chemistry
Superoxides / metabolism*

Substances

2-hydroxyethidium
Fluorescent Dyes
Phenanthridines
Superoxides
hydroethidine
Ethidium