Methods for Detection of Mitochondrial and Cellular Reactive Oxygen Species

Abstract

Significance: Mitochondrial and cellular reactive oxygen species (ROS) play important roles in both physiological and pathological processes. Different ROS, such as superoxide (O 2 -), hydrogen peroxide, and peroxynitrite (ONOO e ), stimulate distinct cell-signaling pathways and lead to diverse outcomes depending on their amount and subcellular localization. A variety of methods have been developed for ROS detection; however, many of these methods are not specific, do not allow subcellular localization, and can produce artifacts. In this review, we will critically analyze ROS detection and present advantages and the shortcomings of several available methods. Recent Advances: In the past decade, a number of new fluorescent probes, electron-spin resonance approaches, and immunoassays have been developed. These new state-of-the-art methods provide improved selectivity and subcellular resolution for ROS detection. Critical Issues: Although new methods for HPLC superoxide detection, application of fluorescent boronate-containing probes, use of cell-targeted hydroxylamine spin probes, and immunospin trapping have been available for several years, there has been lack of translation of these into biomedical research, limiting their widespread use. Future Directions: Additional studies to translate these new technologies from the test tube to physiological applications are needed and could lead to a wider application of these approaches to study mitochondrial and cellular ROS. Antioxid. Redox Signal. 20, 372-382.

Cytochrome P450 1B1 Is Critical for Neointimal Growth in Wire‐Injured Carotid Artery of Male Mice

“…Therefore, these observations, together with our finding that PDGF‐BB induced migration, proliferation, and H 2 O 2 production, were attenuated in VSMCs derived from Cyp1b1 −/− mice, or by treatment with Tempol of VSMCs from Cyp1b1 +/+ mice, indicate that neointimal growth and associated pathological changes are mediated most likely by superoxides generated via CYP1B1. However, further studies are required to confirm the measurement of superoxides using high‐performance liquid chromatography–based assay or electron spin resonance spectroscopy 42. Moreover, further studies are required to establish the contribution of ROS, generated by CYP1B1, to endogenous PDGF‐stimulated neointimal growth during vascular injury.…”

Mitochondrial Dysfunction and Redox Imbalance as a Diagnostic Marker of “Free Radical Diseases”

“…Since only the radical form is characterized by EPR/MRI contrast, this could be used as a quantitative marker for the as-sessment of the redox status in biological objects in vitro and in vivo (91). Many excellent review articles have described this possibility (92,(95)(96)(97)(98).…”

Regulation of Cellular Redox Signaling by Matricellular Proteins in Vascular Biology, Immunology, and Cancer

“…Aortic VSMCs from young WT mice displayed increased ROS levels compared with cells from arteries from Cd47 -/-mice, as characterized by Mitosox fluorescence (56). Additional chromatographic analysis of the fluorescent product would be required to confirm that CD47 regulates mitochondrial superoxide production rather than other mitochondrial ROS (42). Despite a higher mitochondrial density in skeletal muscle, young cd47 -/-mice consumed less oxygen and produced less heat than WT mice, suggesting that mitochondrial coupling is more efficient in the absence of CD47, which is consistent with the decreased ROS production.…”