Reactive Oxygen Species (ROS)

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Introduction

Reactive Oxygen Species (ROS, 활성산소종) 이란?

Reactive oxygen species (ROS)[1] is well-established molecules responsible for the deleterious effects of oxidative stress. Accumulation of free radicals coupled with an increase in oxidative stress has been implicated in the pathogenesis of several disease states. The role of oxidative stress in vascular diseases, diabetes, renal ischemia, atherosclerosis, pulmonary pathological states, inflammatory diseases, cancer, as well as ageing has been well established. Free radicals and other reactive species are constantly generated in vivo and cause oxidative damage to biomolecules, a process held in check by the existence of multiple antioxidant and repair systems as well as the replacement of damaged nucleic acids, proteins and lipids. Measuring the effect of antioxidant therapies and ROS activity is crucial to suppressing or treating oxidative stress inducers.[2]

Oxidative stress has been identified as a major mechanism of toxicity for nanoparticles. The socalled oxidative stress paradigm describes how increased levels of reactive oxygen species (ROS) lead to various cellular responses, such as antioxidant response, inflammation and cytotoxicity, following nanoparticle-cell interactions. fore example, ROS and inflammation can for instance be a consequence of the released toxic metal ions or a reactive particle surface leading to lysosomal destabilization.

Type of reactive Oxygen species[3]

Cytotoxicity & Genotoxicity by ROS (ROS 에 의해 발생하는 독성)

Cellular response by ROS generation
1) DNA damage

DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. [4]

2) Protein oxidation

Protein oxidation is defined as the covalent modification of a protein induced either by the direct reactions with reactive oxygen species (ROS) or indirect reactions with secondary by-products of oxidative stress. ROS can cause oxidation in both amino acid side chains and protein backbones, resulting in protein fragmentation or protein-protein cross-linkages. Although all amino acids can be modified by ROS, cysteine, and methionine that are the most susceptible to oxidative changes due to high reaction susceptibility of the sulfur group in those amino acids. Oxidative modifications of proteins can change their physical and chemical properties, including conformation, structure, solubility, susceptibility to proteolysis, and enzyme activities. These modifications can be involved in the regulation of fresh meat quality and influence the processing properties of meat products. Oxidative stress occurs when the formation of oxidants exceeds the ability of antioxidant systems to remove the ROS in organisms. Increased levels of protein oxidation have been associated with various biological consequences, including diseases and aging, in humans and other animal species. [5]

3) Cell content leakage
4) Membrane degradation
5) Lipid peroxidation

Lipid peroxidation is one of the most widely used indicators of free radical formation, a key indicator of oxidative stress. Unsaturated fatty acids such as those present in cellular membranes are a common target for free radicals. Reactions typically occur as a chain reaction where a free radical will capture a hydrogen moiety from an unsaturated carbon to form water. This leaves an unpaired electron on the fatty acid that is then capable of capturing oxygen, forming a peroxy radical (Figure 5). Lipid peroxides are unstable and decompose to form a complex series of compounds, which include reactive carbonyl compounds, such as malondialdehyde (MDA).[3]

6) Reduced cell viability
7) Defective respiratory chain

genotoxicity of metal oxide NPs seems to occur mainly via oxidative stress rather than direct DNA binding with subsequent replication stress. exposure to CuO, NiO and ZnO nanoparticles as well as to quartz resulted in activation of the oxidative stress reporter, although only at high cytotoxicity for ZnO. NiO NPs activated additionally a p53-associated cellular stress response, indicating additional reactive properties. Conventional assays for genotoxicity assessment confirmed the response observed in the ToxTracker assay. We show for CuO NPs that the induction of oxidative stress is likely the consequence of released Cu ions whereas the effect by NiO was related to the particles per se.[6]

Measurement

ROS assessment Tool ( 일반적인 ROS 측정방법)

flow cytometry, plate reader

시약제품 정보

1) H2DCF-DA
Formation of fluorescent Compound DCF by ROS[7]

Originally, DCF was thought to be specific for hydrogen peroxide, but recent evidence has shown that other ROS such as nitrate and hypochlorous acid can oxidize H2DCF.  Most importantly is the fact that H2O2-dependent oxidation of H2DCF requires ferrous iron.  In addition, as H2DCF is no longer ionic it is not precluded from migrating out of the cell and accumulating in the media, where it is free to interact with oxidants.

2) DCFH-DA
3) Cell ROX Green
Mechanism of DCF assay[8]

Result

측정 결과 해석

결과 또는 독성 (ROS) 발생에 영향을 미칠 수 있는 요인

나노입자 종류에 따른 ROS 발생
Silver nanoparticle (Ag, 은나노입자)

A number of studies suggested that cytotoxic effects of silver nanoparticles in macrophages were mediated by the generation of oxidative stress [5,6,11]. Oxidative stress is induced when the generation of ROS exceeds the cell’s antioxidant capacity. Apart from the damaging effects to cellular proteins, lipids and DNA, an increasing level of ROS triggers the cell to respond by activating pro-inflammatory signalling cascades, and ultimately induces programmed cell death. [9]

나노입자 크기에 따른 ROS 발생

Ag NPs , 20/80/113 nm (Nanocomposix) with no capping agent smaller nanoparticles may display or release more silver ions from its surface than larger nanoparticles.

Park et al. suggested that silver nanoparticles dissolve into cytotoxic silver ions upon phagocytosis and transportation of the nanoparticles to lysosomes, mimicking a Trojan Horse.

다양한 입자크기(5, 15, 50 nm), 셀라인을 사용하여 그에 따른 독성 영향을 보았음. 하지만, 세포 주에 따른 독성을 비교를 보여주지 않고 HepG2에 대한 결과만 제시. discussion에서 입자 크기에 따른 ROS 발생을 서술하는 부분에 대해 가설은 있지만 그를 뒷받침할 만한 증거제시가 없음.  [10]

Oxidative stress in cells can be evoked by the cellular uptake behavior of extraneous particles (외부 입자) or by the redox and catalytic properties of the internalized particles.

To investigate the cellular oxidative stress induced by Ag NPs, we measured the changes in intracellular reactive oxygen species (ROS).

Results suggested their internalization of Ag NPs disturbed the cellular antioxidant defense system by evoking ROS.

In view of the large specific surface area, various nanoparticle have been found to churn out a plethora of ROS and provoke oxidative stress

(큰 표면적 때문에, 다양한 나노 입자들은 과잉의 ROS를 발생시키고, oxidative stress를 유도하는 것으로 알려져왔다.)

The ROS-generating capability of Ag NPs was also inversely proportional to the particle size. Under normal condition or mild stress, ROS can be easily neutralized by antioxidant enzymes ( such as SOD or GSH ). However, under violent stress, ROS generation may be overwhelming and antioxidant enzymes and molecules may be exhausted. The stronger oxidative activity of Ag NPs (5 nm) is probably dues to two factors : The more catalytically active sites and the easier internalization I cells. Both facts have a direct relationship with the particle size.

Others have suggested that the higher cytotoxicity of smaller nanoparticles compared to larger one was related to the amount of ROS generated at the relatively larger surface area if small nanoparticles. [11] Effect of Ag NPs is inflicting damage towards a range of different cell types, potentially resulting in a myriad of secondary effects, such as generation of ROS, DNA damage and inhibiting stem cell differentiation.

It is well known that the reactivity and toxicity of nanomaterials is frequently dependent on nanoparticle size. Ag NPs 10 nm resulted in DHR oxidation in a concentration dependent manner.

Theses authors tested two different coating agent, citrate and PVP and, in the same conditions, no differences in toxicity were observed, suggesting that the size rather than the capping agent was the property that triggered toxicity. All these findings corroborate the size- and time- dependent behavior of Ag NPs in our study with human neutrophils. We reported for the first time that the production of ROS by human neutrophils is stimulated by Ag NPs and is dependent on the particle size. The production of ROS in human neutrophils is initiated by the activation of the enzyme NADPH oxidase with subsequent production of O2`-, H2O2, HOCl among other ROS. [12]

나노입자 표면전하에 따른 ROS 발생
  1. https://en.wikipedia.org/wiki/Reactive_oxygen_species
  2. H.L.Karlsson et al. Particle and Fibre Toxicology 2014, 11 : 41, Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines
  3. 3.0 3.1 An introduction to Reactive Oxygen Species : measurement of ROS in cells from BioTek instruments Inc. http://www.biotek.com/resources/articles/reactive-oxygen-species.html
  4. J. NR et al. DNA damage by reactive species : mechanism, mutation and repair
  5. Z.W et al. Protein oxidation : basic principles and implications for meat quality.
  6. H.L.Karlsson et al. Particle and Fibre Toxicology 2014, 11 : 41, Mechanism-based genotoxicity screening of metal oxide nanoparticles using the ToxTracker panel of reporter cell lines
  7. Application Note : Using BioTeK's Synergy HT Reader to measure ROS generation in stimulated cells. http://www.biotek.com/resources/articles/reactive-oxygen-species-generation.html
  8. OxiSelect intracellular ROS assay kit (Green Fluorescence) http://www.cellbiolabs.com/sites/default/files/STA-342-ROS-assay-kit.pdf
  9. 9.0 9.1 M.V.D.Z Park et al. Biomaterials, The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles.
  10. W.Liu et al. Impact of silver nanoparticles on human cells : Effect of particle size
  11. M.V.D.Z. Park et al. The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles 
  12. T. Soares et al. Size-dependent cytotoxicity of silver nanoparticles in human neutrophils assessed by multiple analytical approaches