API

Event: 1632

Key Event Title

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Increase in reactive oxygen and nitrogen species (RONS)

Short name

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Increase in RONS

Biological Context

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Level of Biological Organization
Molecular

Cell term

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Organ term

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Key Event Components

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Process Object Action

Key Event Overview


AOPs Including This Key Event

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AOP Name Role of event in AOP
Increased DNA damage leading to breast cancer KeyEvent
RONS leading to breast cancer MolecularInitiatingEvent

Stressors

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Taxonomic Applicability

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Life Stages

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Sex Applicability

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Key Event Description

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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).


How It Is Measured or Detected

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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

Target

Name

Method

Strengths/Weaknesses

Hydrogen peroxide- extracellular

AmplexRed

Small molecule fluorescent probes

Can be applied to any tissue in vitro.

Hydrogen peroxide- mitochondrial

MitoPy1

Small molecule fluorescent probes

Can be applied to any tissue in vitro.

Hydrogen peroxide

HyPer

Protein-based fluorescent probes

Sensitive, can be targeted to specific cells and compartments. Slower and pH sensitive.

Hydrogen peroxide

HyPer3

Protein-based fluorescent probes

Rapid kinetics and larger dynamic range, can be targeted to specific cells and compartments. Sensitive to pH, less sensitive to H2O2.

Hydrogen peroxide

Boronate-based indicators

Small molecule fluorescent probe

Selective for H2O2 but can interact with peroxynitrite.

Superoxide- intracellular

DHE (dihydroethidium)

Small molecule fluorescent probe

Can be applied to any tissue in vitro, but not targeted to different compartments.

Superoxide- intracellular

cpYFP

Protein-based fluorescent probes

Reversible. Can be targeted to specific cells and compartments.

Superoxide- mitochondrial

MitoSox

Small molecule fluorescent probe

Can be applied to any tissue in vitro.

Superoxide- mitochondrial

mt-cpYFP

Protein-based fluorescent probes

Reversible. Can be targeted to specific cells and compartments.

Superoxide- extracellular

nitroblue tetrazolium

Small molecule fluorescent probe

Can be applied to any tissue in vitro.

Superoxide- intracellular or extracelluar

various trityl probes

EPR

Very specific, but requires specialized equipment, not as sensitive in tissue.

Nitric oxide

Fe[DETC]2 and

Fe[MGD]2,

EPR

Very specific, but requires specialized equipment, not as sensitive in tissue.

Nitric oxide

DAF-FM

Small molecule fluorescent probe

Can be applied to any tissue in vitro, but not targeted to different compartments

Peroxynitrite

EMPO

EPR

Very specific, but requires specialized equipment, not as sensitive in tissue.

Peroxynitrite

Boronate-based indicators

Small molecule fluorescent probe

Selective for H2O2 but can interact with (is inhibited by) peroxynitrite.

Peroxynitrite

8-nitroguanine (DNA) content

HPLC-MS/MS

Destruction of sample required for measurement.

Non-specific oxidation

DCHF

Small molecule fluorescent probe

Very non selective, and can produce false positive signals.

Non-specific oxidation

roGFP or FRET

Protein-based fluorescent probes

Slow acting. Good to look at steady state activity.

Non-specific oxidation

ratio of reduced to oxidized glutathione or cysteine

Redox state detectors

Slow acting. Good to look at steady state activity. Destruction of sample required for measurement.

Non-specific oxidation

8-oxoguanine (DNA) or protein carbonyl content

HPLC-MS/MS

Destruction of sample required for measurement.

Non-specific oxidation

TBARS (thiobarbituric acid reactive substance)

Lipid peroxidation

Destruction of sample required for measurement.


Domain of Applicability

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This KE is broadly applicable across species.


Evidence for Perturbation by Stressor


Overview for Molecular Initiating Event

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The following stressors increase this key event: ionizing radiation.



Ionizing Radiation

Reactive oxygen and nitrogen species are created by the interaction of ionizing radiation with tissue. When ionizing radiation encounters water or extracellular or intracellular components, it releases energy. This energy ejects electrons from atoms and molecules, and the ejected electrons pass energy on to neighboring molecules. Since the majority of biological tissue is composed of water molecules, ionizing radiation results in the radiolysis of water to hydroxyl radicals, which can interact to form additional reactive molecules. This reaction is generally accepted. Because RONS have such a short half-life, their appearance has been historically measured by their effect on the cell (e.g. in terms of DNA damage), and only more recently characterized using molecular probes that directly reflect their occurrence.

The time course of RONS following ionizing radiation has been described using molecular probes- primarily the non-specific fluorescent probe for ROS DCHF as well as non-specific lipid peroxidation. ROS levels increase at multiple time points: in vitro immediately following radiation (Denissova, Nasello et al. 2012; Yoshida, Goto et al. 2012; Martin, Nakamura et al. 2014), around 15 minutes later (Narayanan, Goodwin et al. 1997; Saenko, Cieslar-Pobuda et al. 2013), hours to days (Lyng, Seymour et al. 2001; Yang, Asaad et al. 2005; Choi, Kang et al. 2007; Du, Gao et al. 2009; Das, Manna et al. 2014; Werner, Wang et al. 2014; Ameziane-El-Hassani, Talbot et al. 2015; Manna, Das et al. 2015; Zhang, Zhu et al. 2017), and in vivo intestinal epithelial cells and bone marrow stem cells showed elevated ROS up to a year after IR exposure of the animal (Pazhanisamy, Li et al. 2011; Datta, Suman et al. 2012). In intestinal epithelial cells, widespread ROS expression over a period of weeks would require transgenerational expression of ROS, indicating that a cell with increased RONS can pass that characteristic to its daughter cells.

Multiple mechanisms underlie the increase in RONS after IR. The early (15 minute) and later (days to weeks) elevation in ROS is associated with increased NADPH-oxidase production of superoxide and H2O2 (Narayanan, Goodwin et al. 1997; Ameziane-El-Hassani, Talbot et al. 2015), and intermediate (hours to days) and chronic ROS elevation has been associated with mitochondrial respiration (Dayal, Martin et al. 2009; Datta, Suman et al. 2012; Saenko, Cieslar-Pobuda et al. 2013). The increase in mitochondrial respiration may be supported by nitric oxide, which increases around 8 hours after IR and remains elevated through at least day 2. A chronic (1 year) ROS effect of IR was not observed in cell culture when cell divisions were limited, potentially implicating cell division in sustaining chronic RONS (Suzuki, Kashino et al. 2009). RONS can also be indirectly initiated by ionizing radiation in neighboring cells via unknown soluble factors, possibly including extracellular H2O2, which is elevated immediately and in the first week following IR (Driessens, Versteyhe et al. 2009; Ameziane-El-Hassani, Boufraqech et al. 2010; Ameziane-El-Hassani, Talbot et al. 2015). Elevated intracellular ROS was observed in cells after exposure to media from IR-exposed cells (Narayanan, Goodwin et al. 1997; Lyng, Seymour et al. 2001; Yang, Asaad et al. 2005), and protein carbonylation and lipid oxidation reflecting RONS activity was elevated in cells 20 passages after exposure to media from IR cells (Buonanno, de Toledo et al. 2011), suggesting that the effect of IR on RONS can penetrate well beyond the directly exposed cells in both space and time. 

Few studies have measured RONS at multiple doses of ionizing radiation, and the time points, doses, and cell types tested for dose response vary between studies along with the dose-dependence. Two studies report dose-dependence of RONS measured with lipid peroxidation or DCHF in response to a few doses between 0.5 and 12 Gy IR (Jones, Riggs et al. 2007; Saenko, Cieslar-Pobuda et al. 2013), dose-dependence of ROS only at lower doses below 1 Gy (Werner, Wang et al. 2014), or non-linear dose-dependence (Narayanan, Goodwin et al. 1997). Dose-dependent RONS responses are also reported in extracellular media (Driessens, Versteyhe et al. 2009), and in bystander cells not directly exposed to IR (Narayanan, Goodwin et al. 1997), even after multiple generations in culture (Buonanno, de Toledo et al. 2011). ROS appears to be more dose-dependent immediately after IR and after 24 hours following IR with less dose-dependence at times in between (Narayanan, Goodwin et al. 1997; Saenko, Cieslar-Pobuda et al. 2013; Zhang, Zhu et al. 2017), possibly reflecting different mechanisms of ROS generation. These studies use probes for ROS or indicators of oxidation, but none that we are aware of explicitly measures indicators of RNS at different doses of IR.

 

Ameziane-El-Hassani, R., M. Boufraqech, et al. (2010). "Role of H2O2 in RET/PTC1 chromosomal rearrangement produced by ionizing radiation in human thyroid cells." Cancer Res 70(10): 4123-4132.

Ameziane-El-Hassani, R., M. Talbot, et al. (2015). "NADPH oxidase DUOX1 promotes long-term persistence of oxidative stress after an exposure to irradiation." Proceedings of the National Academy of Sciences of the United States of America 112(16): 5051-5056.

Buonanno, M., S. M. de Toledo, et al. (2011). "Long-term consequences of radiation-induced bystander effects depend on radiation quality and dose and correlate with oxidative stress." Radiation research 175(4): 405-415.

Choi, K. M., C. M. Kang, et al. (2007). "Ionizing radiation-induced micronucleus formation is mediated by reactive oxygen species that are produced in a manner dependent on mitochondria, Nox1, and JNK." Oncol Rep 17(5): 1183-1188.

Das, U., K. Manna, et al. (2014). "Role of ferulic acid in the amelioration of ionizing radiation induced inflammation: a murine model." PLoS One 9(5): e97599.

Datta, K., S. Suman, et al. (2012). "Exposure to heavy ion radiation induces persistent oxidative stress in mouse intestine." PLoS One 7(8): e42224.

Dayal, D., S. M. Martin, et al. (2009). "Mitochondrial complex II dysfunction can contribute significantly to genomic instability after exposure to ionizing radiation." Radiation research 172(6): 737-745.

Denissova, N. G., C. M. Nasello, et al. (2012). "Resveratrol protects mouse embryonic stem cells from ionizing radiation by accelerating recovery from DNA strand breakage." Carcinogenesis 33(1): 149-155.

Driessens, N., S. Versteyhe, et al. (2009). "Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ." Endocrine-related cancer 16(3): 845-856.

Du, C., Z. Gao, et al. (2009). "Mitochondrial ROS and radiation induced transformation in mouse embryonic fibroblasts." Cancer Biol Ther 8(20): 1962-1971.

Jones, J. A., P. K. Riggs, et al. (2007). "Ionizing radiation-induced bioeffects in space and strategies to reduce cellular injury and carcinogenesis." Aviat Space Environ Med 78(4 Suppl): A67-78.

Lyng, F. M., C. B. Seymour, et al. (2001). "Oxidative stress in cells exposed to low levels of ionizing radiation." Biochemical Society transactions 29(Pt 2): 350-353.

Manna, K., U. Das, et al. (2015). "Naringin inhibits gamma radiation-induced oxidative DNA damage and inflammation, by modulating p53 and NF-kappaB signaling pathways in murine splenocytes." Free Radic Res 49(4): 422-439.

Martin, N. T., K. Nakamura, et al. (2014). "Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks." Cell Death Dis 5: e1130.

Narayanan, P. K., E. H. Goodwin, et al. (1997). "Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells." Cancer research 57(18): 3963-3971.

Pazhanisamy, S. K., H. Li, et al. (2011). "NADPH oxidase inhibition attenuates total body irradiation-induced haematopoietic genomic instability." Mutagenesis 26(3): 431-435.

Saenko, Y., A. Cieslar-Pobuda, et al. (2013). "Changes of reactive oxygen and nitrogen species and mitochondrial functioning in human K562 and HL60 cells exposed to ionizing radiation." Radiation research 180(4): 360-366.

Suzuki, K., G. Kashino, et al. (2009). "Long-term persistence of X-ray-induced genomic instability in quiescent normal human diploid cells." Mutation research 671(1-2): 33-39.

Werner, E., H. Wang, et al. (2014). "Opposite roles for p38MAPK-driven responses and reactive oxygen species in the persistence and resolution of radiation-induced genomic instability." PLoS One 9(10): e108234.

Yang, H., N. Asaad, et al. (2005). "Medium-mediated intercellular communication is involved in bystander responses of X-ray-irradiated normal human fibroblasts." Oncogene 24(12): 2096-2103.

Yoshida, T., S. Goto, et al. (2012). "Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation." Free Radic Res 46(2): 147-153.

Zhang, Q., L. Zhu, et al. (2017). "Ionizing radiation promotes CCL27 secretion from keratinocytes through the cross talk between TNF-alpha and ROS." J Biochem Mol Toxicol 31(3)


References

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Calcerrada, P., G. Peluffo, et al. (2011). "Nitric oxide-derived oxidants with a focus on peroxynitrite: molecular targets, cellular responses and therapeutic implications." Curr Pharm Des 17(35): 3905-3932.

Dickinson, B. C. and C. J. Chang (2011). "Chemistry and biology of reactive oxygen species in signaling or stress responses." Nature chemical biology 7(8): 504-511.

Egea, J., I. Fabregat, et al. (2017). "European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)." Redox biology 13: 94-162.

Go, Y. M. and D. P. Jones (2013). "The redox proteome." J Biol Chem 288(37): 26512-26520.

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.