Current methods to unravel ROS biology Methods

Methods 109 (2016) 1–2

Contents lists available at ScienceDirect

Methods journal homepage: www.elsevier.com/locate/ymeth

Guest Editor’s Introduction

Current methods to unravel ROS biology

In eukaryotic organisms reactive oxygen species (ROS) are mainly generated as by-products of aerobic metabolism. These compounds are the metabolic cost derived from the use of oxygen as the driver force for an extremely efficient mode of ATP production coupled to redox reactions. ROS are characterized by a high chemical reactivity and are, therefore, very harmful for cell viability. As a consequence, living organisms in the aerobic landscape have developed a large collection of biochemical mechanisms to prevent the deleterious effects of ROS production. The accumulative effects of a continuous or deregulated ROS production are associated to several disease states and to the ageing process. However, a controlled intracellular ROS production by dedicated molecular mechanisms has also key roles in molecular signalling pathways that regulate essential cellular processes, including cell proliferation and differentiation, motility and programmed cell death. The realization of this multifaceted activity of ROS in biological systems has enormously contributed to boost the research on the physiological roles of these molecules. A critical drawback to check and accurately define the functions of ROS in living cells, tissues and whole organisms is the highly volatile nature of these molecules. A plethora of new experimental approaches have been developed in the last years to localize and quantify ROS in situ and in vivo in different experimental systems and biological models. In this special issue of the Methods journal we present a comprehensive collection of protocols and methodological reviews encompassing the study of the physiological roles of ROS in different experimental systems. Wieckowski and colleagues [1] provide a detailed description of current protocols to detect ROS in living mammalian cells using redox-sensitive low molecular-weight chemical reporters (5(and-6)-chloromethyl-20 ,70 -dichlorodihydrofluorescein diacetate), and protein-based sensors (HyPer and SypHer). The mitochondria are major sites of ROS production in eukaryotic cells and, therefore, a central target to analyse ROS dynamics. Wang and colleagues [2] introduce the background methodology to visualize in vivo and quantify mitochondrial ROS production dynamics in mammalian cells using the circularly permuted yellow fluorescent protein (cpYFP) as an efficient biosensor. Contributions by Davies and colleagues [3] and Fabienne and colleagues [4] exemplify robust applications of electron paramagnetic resonance (EPR) spectroscopy spin trapping and related methods to the detection of ROS and other radical species in living mammalian cells and tissues and other biological samples, a gold standard technique in the field. In this perspective, Meinke and colleagues [5] methodically discuss the influence of different methodological parameters that can affect the EPR-based measurement of radical production in biological samples using the free radical formation process in the http://dx.doi.org/10.1016/j.ymeth.2016.10.006 1046-2023/Ó 2016 Published by Elsevier Inc.

skin after solar irradiation as a model system. This research group also uses EPR and spin traps to gauge the oxidative stress induced on the viability of eukaryotic cells by different compositions of a new generation of antimicrobial Ag-based nanoparticles [6]. The development of new efficient probes to detect specific types of ROS is also a major challenge in the field. Molecular singlet oxygen and its immediate hydroxyl radical derivatives are supposed to be the ROS most actively involved in cellular signalling. Nonell and colleagues [7] present a novel, potentially striking anthracenebased nanoprobe to detect singlet oxygen in mammalian cells. Casas and colleagues [8] propose a useful method to evaluate ROS production, total oxidation status and phototoxicity of new potential photosensitizers obtained from plant extracts in eukaryotic and prokaryotic cells. Recent experimental approaches to induce a controlled and localized production of molecular singlet oxygen in single mammalian cells, including two-photon excitation of selected photosensitizers, the use protein encapsulated photosensitizers and direct excitation of intracellular molecular oxygen, are thoroughly reviewed by Ogilby and colleagues [9]. As expected in our aerobic environment, the roles of ROS, either as toxic molecules or as true physiologic signalling messengers, are equally evident in the plant kingdom. ROS signalling in plant cells is also an emerging research topic. In this perspective, OrtegaVillasante and colleagues [10] provide a comprehensive methodological review on the current experimental approaches to monitor ROS dynamics and redox changes in photosynthetic organisms using small fluorescent chemical probes and protein-based ROS sensors. Chakraborty and colleagues [11] present an optimized method for an accurate quantification of ROS in plant tissue lysates using the Amplex Red probe. Kuzniak and colleagues [12] present a clever image-processing based method using the public domain Fiji (ImageJ) environment to quantify ROS accumulation in plant leaves. A series of contributions provide innovative and smart experimental approaches to the analysis of physiological roles for ROS and redox dynamics in biological systems. Lippert and colleagues [13] introduce a chemiluminescent platform adapted for smartphone camera devices to visualize and measure hydrogen peroxide, used as a clinical oxidative marker, in the exhaled breath condensate of human subjects. In the same way, La Favor and colleagues [14] present a microdialysis method to measure in vivo hydrogen peroxide and superoxide amounts in various rodent tissues, including adipose tissue, skeletal muscle, kidney, liver and the penis. A novel and interesting proposal suggesting the use of ultrasounds to generate ROS, pointing to possible applications in biological systems, is theoretically discussed by Soltermann and colleagues [15].

2

Guest Editor’s Introduction / Methods 109 (2016) 1–2

Major targets of ROS inside the cell are proteins. Protein oxidation can trigger different responses ranging from cell death to the specific activation of selected signalling pathways. Oxidation of methionine residues, a major output of oxidative stress, can promote non-specific modifications in the peptide backbone inactivating protein function and disturbing methionine metabolic turnover. However, the regulation of the methionine redox status can have also different important physiological functions. In this perspective, Gladyshev and colleagues [16] provide a comprehensive guide to dynamically analyse protein oxidation in living cells using genetically encoded fluorescent biosensor of methionine sulfoxide. Photodynamic therapy combines a photosensitive compound or photosensitizer, light of adequate wavelength and molecular oxygen to induce the production and critical accumulation of ROS inside target cells promoting cell death. This therapeutic modality is widely used in the clinic to treat different skin pathologies, including cancer. Photodynamic therapy is also used as an efficient, antibiotic independent, antibacterial procedure. Photodynamic ROS production can occur through Type I or Type II photochemical pathways, each one having distinctive physic-chemical characteristics delimiting the efficiency of the involved photosensitizers. As indicated by Hamblin and colleagues [17], microbial cells may be more sensitive to Type I photosensitizers, while cancer cells to Type II photosensitizers. To better delineate the action mechanisms of photosensitizes, these authors present a method to distinguish between these photochemical pathways using two chemical probes, the singlet oxygen sensor green (SOSG) and 4-hydroxyphenyl-fluorescein (HPF) that detects the hydroxyl radical. Durantini and colleagues [18], discuss methodological approaches to characterize the photodynamic mechanisms involved in the sensitization of bacterial cells by a dicationic fulleropyrrolidinium derivative (DPC602+). As a useful complement to this contributions, a fresh methodological proposal to spot sites of transient ROS production after photodynamic treatment in mammalian cells using 3,30 -diaminobenzidine is posed by Stockert and BlázquezCastro [19]. Finally, Espada and colleagues [20] present a detailed methodology to induce a transient production of low ROS levels in living cells and tissues using precursors of the endogenous photosensitizer protoporphyrin IX. This photodynamic procedure, instead of inducing cell death, acts as a strong stimulatory signal in mammalian cultured cells. In mouse skin in vivo, a transient ROS production stimulates cell proliferation and differentiation programs, particularly in the hair follicle stem cell niche, promoting homeostatic responses in the tissue such as accelerated hair growth. This experimental approach emphasizes the physiological importance of ROS signalling at a systemic level in whole organisms. In a similar perspective, Jaen and colleagues [21] review clinical applications in the skin of standard ROS-dependent photodynamic therapy protocols that can efficiently promote tissue regeneration in human subjects. Both contributions highlight the physiological relevance of ROS biology in mammals pointing to these compounds as important targets in translational research and regenerative medicine. References [1] M. Oparka, J. Walczak, D. Malinska, L.M.P.E. van Oppen, J. Szczepanowska, W.J. H. Koopman, M.R. Wieckowski, Quantifying ROS levels using CM-H2DCFDA and HyPer, Methods 109 (2016) 3–11.

[2] W. Wang, H. Zhang, H. Cheng, Mitochondrial flashes: from indicator characterization to in vivo imaging, Methods 109 (2016) 12–20. [3] M.J. Davies, Detection and characterisation of radicals using electron paramagnetic resonance (EPR) spin trapping and related methods, Methods 109 (2016) 21–30. [4] K. Abbas, N. Babic´, F. Peyrot, Use of spin traps to detect superoxide production in living cells by electron paramagnetic resonance (EPR) spectroscopy, Methods 109 (2016) 31–43. [5] S. Albrecht, S. Ahlberg, I. Beckers, D. Kockott, J. Lademann, V. Paul, L. Zastrow, M.C. Meinke, Effects on detection of radical formation in skin due to solar irradiation measured by EPR spectroscopy, Methods 109 (2016) 44–54. [6] S. Ahlberg, F. Rancan, M. Epple, K. Loza, D. Höppe, J. Lademann, A. Vogt, B. Kleuser, C. Gerecke, M.C. Meinke, Comparison of different methods to study effects of silver nanoparticles on the pro- and antioxidant status of human keratinocytes and fibroblasts, Methods 109 (2016) 55–63. [7] R. Bresolí-Obach, J. Nos, M. Mora, M.L. Sagristà, R. Ruiz-González, S. Nonell, Anthracene-based fluorescent nanoprobes for singlet oxygen detection in biological media, Methods 109 (2016) 64–72. [8] L. Mamone, G. Di Venosa, D. Sáenz, A. Batlle, A. Casas, Methods for the detection of reactive oxygen species employed in the identification of plant photosensitizers, Methods 109 (2016) 73–80. [9] M. Westberg, M. Bregnhøj, C. Banerjee, A. Blázquez-Castro, T. Breitenbach, P.R. Ogilby, Exerting better control and specificity with singlet oxygen experiments in live mammalian cells, Methods 109 (2016) 81–91. [10] C. Ortega-Villasante, S. Burén, Á. Barón-Sola, F. Martínez, L.E. Hernández, In vivo ROS and redox potential fluorescent detection in plants: present approaches and future perspectives, Methods 109 (2016) 92–104. [11] S. Chakraborty, A.L. Hill, G. Shirsekar, A.J. Afzal, G.-L. Wang, D. Mackey, P. Bonello, Quantification of hydrogen peroxide in plant tissues using Amplex Red, Methods 109 (2016) 105–113. [12] J. Sekulska-Nalewajko, J. Gocławski, J. Chojak-Koz´niewska, E. Kuz´niak, Automated image analysis for quantification of reactive oxygen species in plant leaves, Methods 109 (2016) 114–122. [13] M.E. Quimbar, K.M. Krenek, A.R. Lippert, A chemiluminescent platform for smartphone monitoring of H2O2 in human exhaled breath condensates, Methods 109 (2016) 123–130. [14] J.D. La Favor, A.L. Burnett, A microdialysis method to measure in vivo hydrogen peroxide and superoxide in various rodent tissues, Methods 109 (2016) 131– 140. [15] W. Duco, V. Grosso, D. Zaccari, A.T. Soltermann, Generation of ROS mediated by mechanical waves (ultrasound) and its possible applications, Methods 109 (2016) 141–148. [16] Z. Peterfi, L. Tarrago, V.N. Gladyshev, Practical guide for dynamic monitoring of protein oxidation using genetically encoded ratiometric fluorescent biosensors of methionine sulfoxide, Methods 109 (2016) 149–157. [17] M. Garcia-Diaz, Y.-Y. Huang, M.R. Hamblin, Use of fluorescent probes for ROS to tease apart Type I and Type II photochemical pathways in photodynamic therapy, Methods 109 (2016) 158–166. [18] N.S. Gsponer, M.L. Agazzi, M.B. Spesia, E.N. Durantini, Approaches to unravel pathways of reactive oxygen species in the photoinactivation of bacteria induced by a dicationic fulleropyrrolidinium derivative, Methods 109 (2016) 167–174. [19] J.C. Stockert, A. Blázquez-Castro, Establishing the subcellular localization of photodynamically-induced ROS using 3,30 -diaminobenzidine: a methodological proposal, with a proof-of-concept demonstration, Methods 109 (2016) 175–179. [20] E. Carrasco, A. Blázquez-Castro, M.I. Calvo, Á. Juarranz, J. Espada, Switching on a transient endogenous ROS production in mammalian cells and tissues, Methods 109 (2016) 180–189. [21] P. Fonda-Pascual, O.M. Moreno-Arrones, A. Alegre-Sanchez, D. Saceda-Corralo, D. Buendia-Castaño, C. Pindado-Ortega, P. Fernandez-Gonzalez, K. VelazquezKennedy, M.I. Calvo-Sánchez, A. Harto-Castaño, B. Perez-Garcia, L. Bagazgoitia, S. Vaño-Galvan, J. Espada, P. Jaen-Olasolo, In situ production of ROS in the skin by photodynamic therapy as a powerful tool in clinical dermatology, Methods 109 (2016) 190–202.

Guest Editor Jesús Espada Ramón y Cajal Institute for Biomedical Research (IRYCIS), Ramón y Cajal University Hospital, Colmenar Viejo Rd. Km. 9, 100, 28034 Madrid, Spain Bionanotechnology Laboratory, Bernardo O’Higgins University, General Gana 1780, 8370854 Santiago, Chile