Increase in reactive oxygen and nitrogen species (RONS)

Reactive oxygen and nitrogen species (RONS) are highly reactive oxygen- and nitrogen-based molecules that often contain or generate free radicals. Key molecules include superoxide ([O2]•−), hydrogen peroxide (H2O2), hydroxyl radical ([OH]•), lipid peroxide (ROOH), nitric oxide ([NO]•, and peroxynitrite ([ONOO-]) (Dickinson and Chang 2011; Egea, Fabregat et al. 2017)

RONS are generated in the course of cellular respiration, metabolism, cell signaling, and inflammation (Dickinson and Chang 2011; Egea, Fabregat et al. 2017). Superoxide and hydrogen peroxide are commonly produced by the mitochondrial electron transport chain and cytochrome c and by membrane bound NADPH oxidases and related molecules. Hydrogen peroxide is also made by the endoplasmic reticulum in the course of protein folding. Nitric oxide is produced at the highest levels by nitric oxide synthase in endothelial cells and phagocytes. The other species are produced by reactions with superoxide or peroxide, or by other free radicals or enzymes.

RONS activity is principally local. Most reactive oxygen species (ROS) have short half-lives, ranging from nano- to milliseconds, so diffusion is limited, while reactive nitrogen species (RNS) nitric oxide or peroxynitrate can survive long enough to diffuse across membranes (Calcerrada, Peluffo et al. 2011). Consequently, local concentrations of ROS are much higher than average cellular concentrations and signaling is typically controlled by colocalization with redox buffers (Dickinson and Chang 2011; Egea, Fabregat et al. 2017). The effects of ROS and RNS are countered by cellular antioxidants, with glutathione and peroxiredoxins playing a major role (Dickinson and Chang 2011). Glutathione is slower but broad acting, while peroxiredoxins act quickly and are specific to peroxides. Peroxiredoxins are effective at low peroxide concentrations but can be deactivated at higher concentrations, suggesting the cellular response to peroxides may sometimes be non-linear.

Although their existence is limited temporally and spatially, reactive oxygen species (ROS) interact with other RONS or with other nearby molecules to produce more ROS and participate in a feedback loop to amplify the ROS signal, which can increase Reactive Nitrogen Species (RNS). Both ROS and RNS also move into neighboring cells and ROS can increase intracellular RONS signaling in neighboring cells (Egea, Fabregat et al. 2017).

RONS can modify a range of targets including amino acids, lipids, and nucleic acids to inactivate or alter target functionality (Calcerrada, Peluffo et al. 2011; Dickinson and Chang 2011; Go and Jones 2013; Ravanat, Breton et al. 2014; Egea, Fabregat et al. 2017). For example, phosphatases including the tumor suppressor PTEN can be reversibly deactivated by oxidation, and the movement of HDAC4 is peroxide dependent. Elevated ROS are implicated in proliferation and maintenance of stem cell population size (Dickinson and Chang 2011) and conversely in differentiation of stem cells and oncogene-induced senescence (Egea, Fabregat et al. 2017).

RONS is typically measured using fluorescent or other probes that react with RONS to change state, or by measuring the redox state of proteins or DNA (Dickinson and Chang 2011; Wang, Fang et al. 2013; Griendling, Touyz et al. 2016). Optimal methods for RONS detection have high sensitivity, selectivity, and spatiotemporal resolution to distinguish transient and localized activity, but most methods lack one or more of these parameters.

Molecular probes that indicate the presence of RONS species vary in specificity and kinetics (Dickinson and Chang 2011; Wang, Fang et al. 2013; Griendling, Touyz et al. 2016). Small molecule fluorescent probes can be applied to any tissue in vitro, but cannot be finely targeted to different cellular compartments. The non-selective probe DCHF was widely used in the past, but can produce false positive signals and is no longer recommended. Newer more selective small molecule probes such as boronate-based molecules are being developed but are not yet widely used. Alternatively, fluorescent protein-based probes can be genetically engineered, expressed in vivo, and targeted to cellular compartments and specific cells. However, these probes are very sensitive to pH in the physiological range and must be carefully controlled.  EPR (electron paramagnetic resonance spectroscopy) provide the most direct and specific detection of free radicals, but requires specialized equipment.

Alternative methods involve the detection of redox-dependent changes to cellular constituents such as proteins, DNA, lipids, or glutathione (Dickinson and Chang 2011; Wang, Fang et al. 2013; Griendling, Touyz et al. 2016). However, these methods cannot generally distinguish between the oxidative species behind the changes, and cannot provide good resolution for kinetics of oxidative activity.

Table 1. Common methods for detecting oxidative activity

This KE is broadly applicable across species.

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Griendling, K. K., R. M. Touyz, et al. (2016). "Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System: A Scientific Statement From the American Heart Association." Circulation research 119(5): e39-75.

Ravanat, J. L., J. Breton, et al. (2014). "Radiation-mediated formation of complex damage to DNA: a chemical aspect overview." Br J Radiol 87(1035): 20130715.

Wang, X., H. Fang, et al. (2013). "Imaging ROS signaling in cells and animals." Journal of molecular medicine 91(8): 917-927.