Total Oxyradical Scavenging Capacity Assay Research Articles

"Effects of recombinant human erythropoietin high mimicking abuse doses on oxidative stress processes in rats".

Although many studies highlight how long-term moderate dose of Recombinant Human Erythropoietin (rHuEPO) treatments result in beneficial and antioxidants effects, few studies take into account the effects that short-term high doses of rHuEPO (mimicking abuse conditions) might have on the oxidative stress processes. Thus, the aim of this study was to investigate the in vivo antioxidant activity of rHuEPO, administered for a short time and at high doses to mimic its sports abuse as doping. Male Wistar healthy rats (n=36) were recruited for the study and were treated with three different concentrations of rHuEPO: 7.5, 15, 30μg/kg. Plasma concentrations of erythropoietin, 8-epi Prostaglandin F2α, plasma and urinary concentrations of NOx were evaluated with specific assay kit, while hematocrit levels were analyzed with an automated cell counter. Antioxidant activity of rHuEPO was assessed analyzing the possible variation of the plasma scavenger capacity against hydroxylic and peroxylic radicals by TOSC (Total Oxyradical Scavenging Capacity) assay. Statistical analyses showed higher hematocrit values, confirmed by a statistically significant increase of plasmatic EPO concentration. An increase in plasma scavenging capacity against peroxyl and hydroxyl radicals, in 8-isoprostane plasmatic concentrations and in plasmatic and urinary levels of NOX were also found in all the treated animals, though not always statistically significant. Our results confirm the literature data regarding the antioxidant action of erythropoietin administered at low doses and for short times, whereas they showed an opposite incremental oxidative stress action when erythropoietin is administered at high doses.

Determination of β-sitosterol and total sterols content and antioxidant activity of oil in acai (Euterpe oleracea)

In order to establish a method for the determination of the sterols of the oil in the freeze-dried acai (Euterpe oleracea Mart.) and to evaluate its antioxidant activities, a saponification/extraction procedure and high performance liquid chromatography (HPLC) analysis method were developed and validated for the analysis of phytosterols in PEE (Petroleum ether extract). Separation was achieved on a Purosper STAR LP C18 column with a binary, gradient solvent system of acetonitrile and isopropanol. Evaporative light scattering detection (ELSD) was used to quantify β-sitosterol and the total sterols. Peak identification was verified by retention times and spikes with external standards. Standard curves were constructed (r = 0.999 2) to allow for sample quantification. Recovery of the saponification and extraction was demonstrated via analysis of spiked samples. The highest content of total sterols is β-sitosterol. The antioxidant activities of the extracts were evaluated using the total oxyradical scavenging capacity assay (TOSC assay). The result showed that the PEE exhibited significant antioxidant properties, sample concentration and the antioxidant capacity had a certain relevance.

Improved peroxyl radical scavenging TOSC assay to quantify antioxidant capacity using SIFT-MS

We report a new, fast, sensitive variation of the total oxyradical scavenging capacity (TOSC) assay for measuring the antioxidant capacity of pure compounds, plant extracts and biological fluids using selected ion flow tube mass spectrometry (SIFT-MS). The TOSC assay examines the partial inhibition of ethene formation in the presence of antioxidants that compete with α-keto-γ-methiolbutyric acid (KMBA) for reactive oxygen species. The SIFT-MS-TOSC assay takes 15 s for each ethene analysis and the time interval between consecutive analyses is 20 s. We demonstrate the method by monitoring the antioxidant capacity of several standard radical scavengers of peroxyl radicals. For peroxyl radicals the measured SIFT-MS-TOSC concentrations necessary to produce 50% inhibition of radical reaction with KMBA are 6.1 ± 0.3 μM for Trolox, 5.7 ± 0.3 μM for ascorbic acid, 8.4 ± 0.4 μM for uric acid and 38 ± 2 μM for reduced glutathione.

TOSC 법을 이용한 당알코올의 항산화 활성 평가

Although animal and epidemiological studies have suggested oxidative stress as an etiological factor in pathogenesis including cancer, inflammation, sepsis, fibrosis, cardiovascularlneurodegenerative diseases and aging-related disorders, conflicting results have been obtained in clinical trial with antioxidants. The reason for this discrepancy remains unknown but may be due, in part, to the lack of a validated assay system for evaluating antioxidant capacity. The antioxidant activity of a series of sugar alcohols against peroxyl radicals, hydroxyl radicals and peroxynitrites was determined by the total oxy-radical scavenging capacity (TOSC) assay and cell-based assay using H4IIE cells. Specific TOSC values calculated from the slope of the linear regression for erythritol, xylitol, sorbitol or mannitol against peroxyl radicals was <TEX>$2.1{\pm}0.2,\;3.7{\pm}0.3,\;9.1{\pm}0.3$</TEX> or <TEX>$8.7{\pm}1.1$</TEX> TOSC/mM, respectively. Specific TOSC values for erythritol, xylitol, sorbitol or mannitol against peroxynitrite was <TEX>$1.9{\pm}0.3,\;3.9{\pm}0.4,\;7.8{\pm}0.7$</TEX> or <TEX>$7.7{\pm}0.5$</TEX> TOSC/mM, respectively. These results suggest that oxy-radical scavenging capacity is dependent on the number of aliphatic hydroxyl group in sugar alcohols of monosaccharide. Tert-butylhydroperoxide (t-BHP)-induced cell toxicity determined by MTT assay was marginally attenuated by 10 mM erythritol, but completely inhibited by 10 mM xylitol, 2 mM sorbitol or 0.75 mM maltitol, a disaccharide alcohol. Oxidative stress markers, such as glutathione (GSH) and malondial-dehyde (MDA) levels, were measured in t-BHP-treated cells using HPLC equipped with a fluorescence detector and a reverse phase column. Erythritol did not change the levels of GSH and MDA in H411E cells treated with t-BHP. The t-BHP-induced changes in cellular GSH and MDA levels were ameliorated by 10 mM xylitol and completely blocked by 10 mM sorbitol and maltitol. These results indicate that sugar alcohols protect cells against oxidative stress via scavenging oxy-radical and suggest that TOSC assay in conjunction with cell-based assay is a valid method for evaluating antioxidant capacity of natural and synthetic chemicals.

Total oxyradical scavenging capacity responses in Mytilus galloprovincialis transplanted into the Venice lagoon (Italy) to measure the biological impact of anthropogenic activities

Oxidative stress related investigations to monitor the impact of the pollutant discharges into the Venice lagoon (Italy) originating from anthropogenic activities (raw sewage water, agricultural and industrial effluents, oil tanker traffic), on marine organisms have classically been carried out by analyzing specific, single antioxidants (i.e. catalase, superoxide dismutase). In this paper, two studies are reported where the total oxyradical scavenging capacity assay (TOSC) was selected and measured toward peroxyl, and hydroxyl free radicals, and peroxynitrite in mussels ( Mytilus galloprovincialis) transplanted into the Venice city and throughout the lagoon to measure the biological effects of anthropogenic activities. In the first experiment, mussels from a clean site (farm) were transplanted to the urban area of Venice for 0, 1, 2 and 4 weeks; cytosolic TOSC toward peroxyl and hydroxyl free radicals, and peroxynitrite revealed that the transplantation process caused a stress (handling stress, anoxia, oxidative burst) resulting in a reduction of TOSC in both control and urban sites, therefore, preventing clear interpretation of the data after one week. At week 2, a significant TOSC reduction ( P<0.05) toward peroxyl and hydroxyl radicals in the urban site revealed that mussels experienced oxidative pressure exerted by pollutants. Most TOSC values returned to initial levels at week 4; however, TOSC induction was noticed in the control group toward peroxyl and hydroxyl radicals while in the exposed group it was not indicating an inhibition of the oxidative metabolism. In the second experiment, mussels were deployed at seven different sites throughout the lagoon. After five weeks of exposure, significant TOSC reduction was measured ( P<0.05) toward peroxyl for Palude della Rosa, Chioggia and Valle Millecampi, toward hydroxyl radicals for Valle Millecampi and Campalto and toward peroxynitrite for Valle Millecampi. Although these data indicate a depletion of the low molecular weight scavengers, additional biomarkers are needed to draw a conclusion on the health of the mussels. TOSC was proved to be an interesting health index parameter to measure pollution impact in a transplantation study provided that the mussels are exposed for two weeks and a control is run in parallel.

Total Oxyradical Scavenging Capacity as an Index of Susceptibility to Oxidative Stress in Marine Organisms

Formation of reactive oxygen species (ROS) is a natural event for aerobic organisms, but it can be exacerbated by both biological and environmental factors. Cellular oxidative damage is well documented in aquatic organisms, and variations of antioxidants can be sensitive in revealing a pro-oxidant condition. Nonetheless, such effects are difficult to predict according to chemicals, kind of exposure, and species sensitivity. Measuring the overall efficiency of cellular antioxidants toward oxyradicals provides a more integrated assessment of susceptibility of a tissue to oxidative stress. Among the methods for analyzing total antioxidant capacity, the total oxyradical scavenging capacity (TOSC) assay offers the possibility to discriminate different oxyradicals, indicating the roles of these species or their metabolic pathways of formation, in the onset of oxidative disease. The TOSC assay has been widely used on marine organisms under natural and disturbed condition suggesting temporal and causal relationships among increased ROS formation, onset of antioxidant responses, and potential occurrence of oxidative damages and toxicity to cellular targets.

A modified total oxyradical scavenging capacity assay for antioxidants in plant tissues

The total oxyradical scavenging capacity (TOSC) assay of Winston et al. [Free Radical Biol. Med. 24 (1998) 480] was modified through the development and incorporation of a standard curve, yielding a relatively simple but reliable method to quantify the total water-soluble antioxidant capacity (TAC) of plant tissues. The basis of the TOSC assay is the oxidation of α-keto-γ-methiolbutyric acid (KMBA) by 2,2′-azobis-amidinopropane (ABAP) with the evolution of ethylene as the quantifiable end product. A standard curve was established by using a serial dilution (0–100 μM) of the antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) which scavenges peroxyl radicals formed from the thermal homolysis of ABAP, thereby reducing the rate of ethylene evolution. Regression analysis of the standard curve yielded a second-degree polynomial (R2=0.9975) that was used to predict the TAC (μMoles Trolox equivalents) of plant tissues. The precision and utility of the method was tested by determining TAC of skin tissue from ‘Delicious’ and ‘Empire’ apples (Malus domestica Borkh.) at various maturities, following cold storage, and after treatment with 1-methylcyclopropene (1-MCP). The TAC of ‘Delicious’ apples was twofold greater than that of ‘Empire’ apples, and remained relatively constant for up to 60 days in cold storage, then significantly decreased thereafter. This decrease during the latter stages of cold storage may be related to the development of superficial scald in this cultivar. ‘Delicious’ and ‘Empire’ fruit treated with 1-MCP had also a significantly higher TAC at various maturities after 120 days of cold storage. This is the first study to show that a treatment with 1-MCP can potentially maintain some intrinsic functional food quality parameters (i.e. antioxidant levels) in stored fruit. The establishment of a standard curve for use, in conjunction with the TOSC assay, resulted in a precise and highly reproducible predictor of TAC in apple fruit, while also permitting a rapid turnover of tissue samples for analysis.

Biomarker responses and PAH uptake in Mya truncata following exposure to oil-contaminated sediment in an Arctic fjord (Svalbard)

Expanding industrial activity (notably oil and gas exploration) in the Arctic requires assessment of the potential impact of chemicals on marine organisms living in seawater at low temperature. The bivalve Mya truncata is common in Svalbard fjord (Norway) where it experiences low temperature throughout the year. To measure the impact of polycyclic aromatic hydrocarbons (PAH) on M. truncata, the responses of three biomarkers [total oxyradical scavenging capacity-assay (TOSC), plasma membrane stability of haemocytes and respiration rates] were investigated from bivalves exposed to sediment contaminated with a PAH mixture (crude oil). After two weeks of exposure to the contaminated sediment, TOSC showed no change. The high TOSC value (4010±1339 unit mg −1 protein) of Mya truncata (control group) is thought to protect biomolecules with a low turnover rate efficiently in a low food availability environment. In the exposed bivalves, the haemocyte cellular membranes were significantly destabilised compared with controls ( P<0.05). Respiration rate of control and PAH-exposed individuals (0.055±0.020 mg O 2 dw −1 h −1) was similar and relatively low as is typical for polar bivalves, reflecting a strategy to minimise energy expenditure to cope with 9 months of starvation. Bioaccumulation of PAH by M. truncata was also low, due probably to a combination of low metabolic rate and reduced solubility of the oil compounds at low temperature. Data indicated an uptake of mainly low molecular weight compounds (two and three ring molecules). A good correlation of logBAF lipid (bioaccumulation factor) and log K ow (octanol/water partitioning coefficient) was shown ( r 2=0.87). Tissue sensitivity and/or functional differences (digestive gland vs. haemocytes), PAH uptake route (dietary vs. gills), the low metabolic rate of M. truncata and the low environmental temperature (reducing the bioavailability of PAH) are factors that help explain these findings.

Total oxyradical scavenging capacity toward different reactive oxygen species in seminal plasma and sperm cells.

The objective of this work was to evaluate the capability of both seminal plasma and sperm cells to scavenge different forms of oxyradicals and the possible correlation with sperm motility parameters. A total of 14 individuals were analyzed by computer-assisted sperm analysis (CASA) and the results integrated with the measurement of total oxyradical scavenging capacity (TOSC) toward peroxyl radicals, hydroxyl radicals and peroxynitrite in seminal plasma and spermatozoa. TOSC values revealed some significant correlation with kinetic sperm cell parameters, including curvilinear velocity (VCL), straight-line velocity (VSL) and linearity (LIN). A lower antioxidant capacity toward hydroxyl radical was found in the seminal fluid of men with reduced sperm motility. Such correlations were not found with peroxyl radicals and peroxynitrite, neither when TOSC values were analyzed in spermatozoa. The TOSC assay is a useful tool for studying the relationship between oxyradical toxicity and abnormal sperm cell motility. Although further investigations are needed, the data clearly establish different role for various forms of oxyradicals, i.e., hydroxyl radicals, in altering sperm motility. Measurement of TOSC is suggested as a useful means of indicating relationship between reactive oxygen species and sperm cell kinetics in clinical trials and antioxidant-based treatments.

Susceptibility to oxidative stress in Adélie and emperor penguin

The antioxidant defences in aerobic organisms represent the detoxification pathway against toxicity of reactive oxygen species (ROS). These highly reactive molecules are normally produced during the 4-electrons reduction of molecular oxygen to water coupled with oxidative phosphorylation, and during the activity of several enzymatic systems which produce ROS as intermediates. However, the endogenous generation of oxyradicals may be influenced by different environmental and biological factors, and the basal efficiency of antioxidant systems generally reflects the normal prooxidant pressure to which organisms are exposed. If the antioxidant capacity is exceeded (i.e. as a consequence of enhanced intracellular formation of ROS), a pathological condition, generally termed oxidative stress, may arise. In this preliminary work, susceptibility to oxidative stress has been compared in plasma of Adelie penguin (Pygoscelis adeliae), emperor penguin (Aptenodytes forsteri), south polar skua (Catharacta maccormicki) and snow petrel (Pagodroma nivea). Within the framework of the Italian Research Program in Antarctica, blood samples were collected during the austral summer 1998–1999 and the Total Oxyradical Scavenging Capacity (TOSC) analysed. The TOSC assay, measuring the capability of biological samples to neutralise different oxyradicals, has been recently standardised to provide a quantifiable value of biological resistance to toxicity of ROS. Penguins exhibited higher scavenging capacity towards peroxyl radicals than south polar skua and snow petrel. The greater resistance to toxicity of oxyradicals might suggest that penguins are naturally exposed to a higher basal prooxidant pressure in comparison to other analysed Antarctic birds.

DNA integrity and total oxyradical scavenging capacity in the Mediterranean mussel, Mytilus galloprovincialis: a field study in a highly eutrophicated coastal lagoon

In Mediterranean coastal lagoons, the combination of human impact and wide variability of natural environmental factors can lead to upsets in ecosystem homeostasis resulting in biodiversity decline. Oxidative damage has been causally linked to various kinds of environmental stress, both natural and artificial, the result being impairment of cellular functions. DNA damage and the efficiency of antioxidant defences in Mytilus galloprovincialis from the highly eutrophicated Orbetello Lagoon (Tuscany, Italy) were investigated, respectively by the single cell gel electrophoresis (or Comet test) and the total oxyradical scavenging capacity assay. Results showed significantly higher levels of DNA damage in mussels collected from the inner parts of the lagoon compared to specimens from more external sites. Specimens with the lower genetic integrity also exhibited a reduced efficiency in neutralizing three potent cellular oxidizing species, namely peroxyl radicals (ROO ), hydroxyl radicals ( OH) and peroxynitrite (HOONO), suggesting the involvement of reactive oxygen species in mediating the genetic damage. The analyzed biological parameters also showed a seasonal variability with a minimum of both DNA integrity and antioxidant scavenging efficiency during the warm months and an opposite trend in winter. The potential of analyzed techniques is discussed for the assessment of both anthropogenic and natural disturbance.

Total antioxidant capacity and nuclear DNA damage in keratinocytes after exposure to H2O2.

Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione S-transferase, glutathione peroxidase) were studied. Exposure to H2O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.

Total oxidant scavenging capacity of Antarctic, Arctic, and Mediterranean scallops

The Total Oxyradical Scavenging Capacity (TOSC) assay was used in three species of scallops for quantifying their ability to neutralise peroxyl (ROO*) and hydroxyl (*OH) radicals and per‐oxynitrite (HOONO). Adamussium colbecki and Chlamys islandicus represent key organisms for monitoring Antarctic and Arctic regions while Pecten jacobaeus was chosen for comparison with a related temperate species. TOSC values for ROO* were significantly higher in A. colbecki, indicating that this species is the most efficient ROO* scavenger. Mediterranean scallops had the lowest TOSC for ROO*. Adamussium colbecki also exhibited the highest scavenging capacity for *OH with values more than two‐fold greater than for C. islandicus and P. jacobaeus. TOSC values for HOONO were lower for all scallops as compared to those for ROO* or *OH; for microsomes they were not significantly different among the species for any reactive oxygen species studied, and the percentage contribution to the specific TOSC for the various oxidants of microsomes of all scallops accounted for 1–3% of the total TOSC of the post‐mitochondrial fraction. The specific TOSC of scallop microsomes for *OH was ‐10 times lower than that for ROO* or HOONO. The higher basal capability of the Antarctic scallop to neutralise different reactive oxygen species is discussed in terms of a possible adaptation to this extreme environment, and TOSC is validated as a quantifiable measure of susceptibility to oxidative stress in marine organisms. In a second part of this study, the seasonal fluctuations of susceptibility to oxidative stress were analysed in the Antarctic scallop Adamussium colbecki by the TOSC Assay. A marked increase in the capability to neutralise peroxyl radicals and hydroxyl radicals was demonstrated at the end of December, while resistance towards peroxynitrite did not show significant variations during the Antarctic summer. The increased resistance to these specific oxyradicals was almost contemporary with the period of highest feeding activity and the development of gonadic tissues. Sensitivity to hydroxyl radicals and peroxyl radicals toxicity was also influenced by UV‐B and UV‐? wavelengths, as indicated by the lower TOSC values in scallops exposed under laboratory conditions. Toxicity of UV radiation seems partly mediated by formation of reactive oxygen species caused by the direct exposure of mantle edges and/or the photochemical production of hydrogen peroxide in sea water.

Total oxyradical scavenging capacity in mussel Mytilus sp. as a new index of biological resistance to oxidative stress

The Total Oxyradical Scavenging Capacity (TOSC) has been characterized in various subcellular fractions from digestive gland of the mussel Mytilus sp.. A simple and sensitive gas-chromatographic assay is used for quantitating this new biochemical parameter. The method is based on the reaction between peroxyl radicals and α-keto-γ-methiolbutyric acid (KMBA) which is oxidized to ethylene: in the presence of cellular antioxidant competing with KMBA for peroxyl radicals, ethylene formation is quantitatively reduced. Whole cytosol from M. edulis exhibited slightly higher TOSC values than the Mediterranean species. Soluble antioxidants provided greater protection than proteins accounting for 70% of oxyradical scavenging capacity of whole cytosols. TOSC assay was suitable also for lipid-soluble antioxidants, and the analysis of the microsomal fraction confirmed the importance of membrane-associated antioxidants in protection against toxicity of reactive oxygen species (ROS). The analysis of TOSC in sentinel organisms is proposed as a new index of contaminant exposure, one which permits quantifiable assessment of the biological resistance to oxidative stress.

A Rapid Gas Chromatographic Assay for Determining Oxyradical Scavenging Capacity of Antioxidants and Biological Fluids

Herein, we report a new, rapid,and reliable method for measuring the protective antioxidant potential of pure antioxidant solutions or biological tissues. Peroxyl radicals generated by thermal homolysis of 2,2′-azobis-amidinopropane (ABAP) cause the oxidation of α-keto-γ-methiolbutyric acid (KMBA) to ethylene; ethylene formation is monitored by gas chromatographic analysis of head space from the reaction vessel. The partial inhibition of ethylene formation in the presence of antioxidants that compete with KMBA for oxyradicals is the basis of the Total Oxyradical Scavenging Capacity Assay (TOSCA). The assay is shown to be reliable for quantifying ROS scavenging potential. The quantifiable parameters are consistent with the relative order of those predicted by the fluorescence- and oxygen electrode-based assays reported in the literature. Antioxidants competing for peroxyl radicals influenced the rate of KMBA oxidation in different ways, but the calculation of TOSC was not affected by such variations. Responses were linear over a wide range of sample concentrations and the TOSC values of classical soluble antioxidants showed the following relative order: Trolox > uric acid > ascorbic acid > GSH. The KMBA method was reliable for biological tissues; the TOSC for 1 μg rat liver cytosolic protein was 0.40 ± 0.02 and for the microsomal membrane, 0.15 ± 0.03. Soluble antioxidants accounted for 77% of the protective antioxidant potential in rat liver cytosol. When incorporated into the microsomal membrane, α-tocopherol markedly enhances antioxidant protection against peroxyl radical; thus, the assay is suitable for the assessment of fat-soluble antioxidants.