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Seven nitroxides of different structures were studied for the ability to prevent glycoxidation of bovine serum albumin incubated with three monosaccharides (glucose, fructose, and ribose). Glycoxidation was estimated by fluorimetric parameters of protein modifications (formation of advanced glycation end products [AGEs], dityrosine, N'-formylkynurenine, and kynurenine) and enzyme-linked immunosorbent assay for AGEs. From among the nitroxides tested, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO), 4-carboxy-TEMPO, and 4-hydroxy-TEMPO offered significant protection against glycoxidation induced by glucose and fructose, while 3-carbamoyl-PROXYL was not protective, enhancing glycoxidation. Lower protection was observed for glycoxidation induced by ribose where only 3-carbamoyl-PROXYL and 4-amino-TEMPO showed some protection. Loss of electron spin resonance signal of the nitroxides was observed during glycoxidation indicating occurrence of free radical reactions in this process. These results suggest for the first time that nitroxides may be promising compounds for preventing glycoxidation. [ABSTRACT FROM AUTHOR]

Proteins are subject to various posttranslational modifications, some of them being undesired from the point of view of metabolic efficiency. Prevention of such modifications is expected to provide new means of therapy of diseases and decelerate the process of aging. In this review, modifications of proteins by reactive nitrogen species and reactive halogen species, is briefly presented and means of prevention of these modifications and their sequelae are discussed, including the denitrase activity and inhibitors of myeloperoxidase. [ABSTRACT FROM AUTHOR]

Garlic is a vegetable with numerous pro-health properties, showing high antioxidant capacity, and cytotoxicity for various malignant cells. The inhibition of cell proliferation by garlic is mainly attributed to the organosulfur compounds (OSCs), but it is far from obvious which constituents of garlic indeed participate in the antioxidant and cytotoxic action of garlic extracts. This study aimed to obtain insight into this question by examining the antioxidant activity and cytotoxicity of six OSCs and five phenolics present in garlic. Three common assays of antioxidant activity were employed (ABTS ● decolorization, DPPH ● decolorization, and FRAP). Cytotoxicity of both classes of compounds to PEO1 and SKOV-3 ovarian cancer cells, and MRC-5 fibroblasts was compared. Negligible antioxidant activities of the studied OSCs (alliin, allicin, S -allyl-D-cysteine, allyl sulfide, diallyl disulfide, and diallyl trisulfide) were observed, excluding the possibility of any significant contribution of these compounds to the total antioxidant capacity (TAC) of garlic extracts estimated by the commonly used reductive assays. Comparable cytotoxic activities of OSCs and phenolics (caffeic, p -coumaric, ferulic, gallic acids, and quercetin) indicate that both classes of compounds may contribute to the cytotoxic action of garlic.

Tardigrades are unique among animals in their resistance to dehydration, mainly due to anhydrobiosis and tun formation. They are also very resistant to high-energy radiation, low and high temperatures, low and high pressure, and various chemical agents, Interestingly, they are resistant to ionizing radiation both in the hydrated and dehydrated states to a similar extent. They are able to survive in the cosmic space. Apparently, many mechanisms contribute to the resistance of tardigrades to harmful factors, including the presence of trehalose (though not common to all tardigrades), heat shock proteins, late embryogenesis-abundant proteins, tardigrade-unique proteins, DNA repair proteins, proteins directly protecting DNA (Dsup and TDR1), and efficient antioxidant system. Antioxidant enzymes and small-molecular-weight antioxidants are an important element in the tardigrade resistance. The levels and activities of many antioxidant proteins is elevated by anhydrobiosis and UV radiation; one explanation for their induction during dehydration is provided by the theory of "preparation for oxidative stress", which occurs during rehydration. Genes coding for some antioxidant proteins are expanded in tardigrades; some genes (especially those coding for catalases) were hypothesized to be of bacterial origin, acquired by horizontal gene transfer. An interesting antioxidant protein found in tardigrades is the new Mn-dependent peroxidase.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl - and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

The modification of the replicative lifespan (RLS) of fibroblasts is of interest both from a knowledge point of view and for the attenuation of skin aging. The effect of six antioxidants at a concentration of 1 μM on the replicative lifespan of human dermal fibroblasts was studied. The nitroxide 4-hydroxy-TEMPO (TEMPOL), ergothioneine, and Trolox extended the replicative lifespan (RLS) (40 ± 1 population doublings (PD)) by 7 ± 2, 4 ± 1, and 3 ± 1 PD and lowered the expression of p21 at late passages. Coumaric acid, curcumin and resveratrol did not affect the RLS . The level of reactive oxygen species (ROS) was decreased or not affected by the antioxidants although TEMPOL and coumaric acid decreased the level of glutathione. Only ergothioneine and resveratrol decreased the level of protein carbonylation. The antioxidants that could prolong the RLS elevated the mitochondrial membrane potential. Protecting the activity of mitochondria seems to be important for maintaining the replicative capacity of fibroblasts.

Nitroxides are stable free radicals that have antioxidant properties. They react with many types of radicals, including alkyl and peroxyl radicals. They act as mimics of superoxide dismutase and stimulate the catalase activity of hemoproteins. In some situations, they may exhibit pro-oxidant activity, mainly due to the formation of oxoammonium cations as products of their oxidation. In this review, the cellular effects of nitroxides and their effects in animal experiments and clinical trials are discussed, including the beneficial effects in various pathological situations involving oxidative stress, protective effects against UV and ionizing radiation, and prolongation of the life span of cancer-prone mice. Nitroxides were used as active components of various types of nanoparticles. The application of these nanoparticles in cellular and animal experiments is also discussed.

Nitroxides, stable synthetic free radicals, are promising antioxidants, showing many beneficial effects both at the cellular level and in animal studies. However, the cells are usually treated with high millimolar concentrations of nitroxides which are not relevant to the concentrations that could be attained in vivo. This paper aimed to examine the effects of low (≤10 μM) concentrations of three nitroxides, 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO), 4-hydroxy-TEMPO (TEMPOL) and 4-amino-TEMPO (TEMPAMINE), in pure chemical systems and on SH-SY5Y cells transfected with the human tau protein (TAU cells), a model of chronic cellular oxidative stress, and transfected with the empty plasmid (EP cells). All nitroxides were active in antioxidant-activity tests except for the 2,2'-azinobis-(3-ethylbenzthiazolin-6-sulfonate) radical (ABTS • ) decolorization assay and reduced Fe 3+ , inhibited autoxidation of adrenalin and pyrogallol and oxidation of dihydrorhodamine123 by 3-morpholino-sydnonimine SIN-1. TEMPO protected against fluorescein bleaching from hypochlorite, but TEMPAMINE enhanced the bleaching. Nitroxides showed no cytotoxicity and were reduced by the cells to non-paramagnetic derivatives. They decreased the level of reactive oxygen species, depleted glutathione, and increased mitochondrial-membrane potential in both types of cells, and increased lipid peroxidation in TAU cells. These results demonstrate that even at low micromolar concentrations nitroxides can affect the cellular redox equilibrium and other biochemical parameters.

Methods employing horseradish peroxidase (HRP) are popular for quantification of hydrogen peroxide. This communication reports interference of the Ampliflu Red-HRP assay by phenolic compounds, abundant in food and beverages of plant origin. Concentrations of catechin, propyl gallate, quercetin and gallic acid lowering the yield of the product, resorufin, in this system by 50% were lower than 10 μM. The extent of inhibition increased with decreasing hydrogen peroxide concentration. These results point to the necessity of a careful interpretation of results concerning the quantification of hydrogen peroxide in materials containing phenolic compounds with methods employing HRP, especially when low concentrations of hydrogen peroxide are concerned., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Garlic is known to be rich in antioxidants, inhibit the proliferation of various cancer cells, and hamper cancer formation and growth, but various forms of garlic can differ greatly in these respects. This study aimed to compare the antioxidant properties of acetone, ethanol, and aqueous extracts of fresh Polish and Spanish garlic, black and granulated garlic, as well as fresh and dried ramsons. Extracts of black and granulated garlic showed the lowest total antioxidant capacity (TAC). The content of phenolic compounds correlated with TAC measured by ABTS • decolorization and FRAP methods, and with the results of FRAP and DPPH • decolorization assays. Garlic extracts inhibited the proliferation of PEO1 and SKOV3 ovarian cancer cells and, usually to a smaller extent, MRC-5 fibroblasts. PBS extracts of fresh Spanish garlic showed the highest potency for inhibition of proliferation of PEO1 cells (IC 50 of 0.71 µg extract dry mass/100 µL medium). No significant correlation was found between the potency for inhibition of proliferation and the content of phenolics or flavonoids, confirming that phenolics are the main determinants of TAC but do not contribute significantly to the antiproliferative effects of garlic.

Treatment of human neuroblastoma SH-SY5Y cells with a catecholaminergic neurotoxin, 6-hydroxydopamine (6-OHDA) is an acknowledged in vitro experimental model of Parkinson disease (PD). A decrease in the glutathione content occurs in PD. Higher concentrations of 6-OHDA lowered the glutathione level in SH-SY5Y cells, nonetheless, we and other authors found a considerable increase in these cells' glutathione content after 24 h treatment with 60 μM 6-OHDA. A synthetic antioxidant, 4-aminotetramethylpiperidine-1-oxyl (4-AT) exerted a similar effect. The aim of the present study was to explain this surprising effect by monitoring the time course of changes in the levels of reduced (GSH) and oxidized glutathione (GSSG), total antioxidant activity (TAC) of human neuroblastoma cell SH-SY5Y extracts as well as the level of reactive oxygen species and activities of enzymes of glutathione metabolism after treatment of the cells with 60 µM 6-OHDA and/or 4-AT for 30 min - 24 h. A transient decrease in the level of GSH and TAC of cell extracts, increase in the level of GSSG, and decrease in the activities of glutathione peroxidase, glutathione reductase, glutathione S-transferase and γ-glutamyl-cysteine ligase activities were found followed by normalization or overshoot of the GSH level, TAC and enzyme activities. Increased activity of γ-glutamyl-cysteine ligase activity starting after 4-6 h was responsible for the elevation of the level of GSH and TAC in cells treated with 6-OHDA, 4-AT, and both compounds. The 6-OHDA-induced increase in the GSH content is a result of an overcompensatory response. The antioxidant 4-AT may be useful for the induction of an increase in the level of GSH in neural cells, without the negative effect of 6-OHDA.

The reaction of the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) free radical (ABTS ● ) with proteins (bovine serum albumin, blood plasma, egg white, erythrocyte membranes, and Bacto Peptone) leads not only to a reduction of ABTS ● but also to the appearance of a purple color (absorption maximum at 550-560 nm). The aim of this study was to characterize the formation and explain the nature of the product responsible for the appearance of this color. The purple color co-precipitated with protein, and was diminished by reducing agents. A similar color was generated by tyrosine upon reaction with ABTS ● . The most feasible explanation for the color formation is the addiction of ABTS ● to proteins' tyrosine residues. The product formation was decreased by nitration of the bovine serum albumin (BSA) tyrosine residues. The formation of the purple product of tyrosine was optimal at pH 6.5. A decrease in pH induced a bathochromic shift of the spectra of the product. The product was not a free radical, as demonstrated by electrom paramagnetic resonance (EPR) spectroscopy. Another byproduct of the reaction of ABTS ● with tyrosine and proteins was dityrosine. These byproducts can contribute to the non-stoichiometry of the antioxidant assays with ABTS ● . The formation of the purple ABTS adduct may be a useful index of radical addition reactions of protein tyrosine residues.

Peroxiredoxin 2 (Prdx2) is the third most abundant erythrocyte protein. It was known previously as calpromotin since its binding to the membrane stimulates the calcium-dependent potassium channel. Prdx2 is present mostly in cytosol in the form of non-covalent dimers but may associate into doughnut-like decamers and other oligomers. Prdx2 reacts rapidly with hydrogen peroxide (k > 10 7 M -1 s -1 ). It is the main erythrocyte antioxidant that removes hydrogen peroxide formed endogenously by hemoglobin autoxidation. Prdx2 also reduces other peroxides including lipid, urate, amino acid, and protein hydroperoxides and peroxynitrite. Oxidized Prdx2 can be reduced at the expense of thioredoxin but also of other thiols, especially glutathione. Further reactions of Prdx2 with oxidants lead to hyperoxidation (formation of sulfinyl or sulfonyl derivatives of the peroxidative cysteine). The sulfinyl derivative can be reduced by sulfiredoxin. Circadian oscillations in the level of hyperoxidation of erythrocyte Prdx2 were reported. The protein can be subject to post-translational modifications; some of them, such as phosphorylation, nitration, and acetylation, increase its activity. Prdx2 can also act as a chaperone for hemoglobin and erythrocyte membrane proteins, especially during the maturation of erythrocyte precursors. The extent of Prdx2 oxidation is increased in various diseases and can be an index of oxidative stress.

Capsaicin (CAP) is a common food constituent, conferring a pungent taste to red peppers of the genus Capsicum. It has bactericidal and fungicidal activity. The study was aimed to test the hypothesis of whether oxidative stress mediates the toxicity of CAP to the baker's yeast Saccharomyces cerevisiae as a model yeast. CAP showed good antioxidant properties (1.30 and 1.10 mol Trolox equivalents/mol in the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate (ABTS) radical scavenging assay and the Ferric Reducing Antioxidant Power assay, respectively). However, its autoxidation generated hydrogen peroxide. CAP inhibited the growth of S. cerevisiae at concentrations ≥100 μM. Yeast mutants deficient in superoxide dismutase 1 or catalase T were more sensitive to CAP than wild-type yeast. CAP did not augment the ROS level in yeast cells. Standard antioxidants (N-acetylcysteine and ascorbate) did not protect significantly against CAP-induced yeast growth inhibition. Thus, oxidative stress does not mediate the CAP's inhibition of yeast growth. CAP did not decrease mitochondrial membrane potential of the yeast but induced a concentration-dependent decrease in membrane fluidity. These results indicate that the disturbance of membrane properties is the apparent cause of CAP toxicity to the yeast., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Purpose: Deinococcus radiodurans is an extremely radioresistant bacterium characterized by D 10 of 10 kGy, and able to grow luxuriantly under chronic ionizing radiation of 60 Gy/h. The aim of this article is to review the antioxidant system of D. radiodurans and its possible role in the unusual resistance of this bacterium to ionizing radiation., Conclusions: The unusual radiation resistance of D. radiodurans has apparently evolved as a side effect of the adaptation of this extremophile to other damaging environmental factors, especially desiccation. The antioxidant proteins and low-molecular antioxidants (especially low-molecular weight Mn 2+ complexes and carotenoids, in particular, deinoxanthin), as well as protein and non-protein regulators, are important for the antioxidant defense of this species. Antioxidant protection of proteins from radiation inactivation enables the repair of DNA damage caused by ionizing radiation.

Abnormally phosphorylated tau protein is the principal component of neurofibrillary tangles, accumulating in the brain in many neurodegenerative diseases, including Alzheimer's disease. The aim of this study was to examine whether overexpression of tau protein leads to changes in the redox status of human neuroblastoma SH-SY5Y cells. The level of reactive oxygen species (ROS) was elevated in tau-overexpressing cells (TAU cells) as compared with cells transfected with the empty vector (EP cells). The level of glutathione was increased in TAU cells, apparently due to overproduction as an adaptation to oxidative stress. The TAU cells had elevated mitochondrial mass. They were more sensitive to 6-hydroxydopamine, delphinidin, 4-amino-TEMPO, and nitroxide-containing nanoparticles (NPs) compared to EP controls. These results indicate that overexpression of the tau protein imposes oxidative stress on the cells. The nitroxide 4-amino-TEMPO and nitroxide-containing nanoparticles (NPs) mitigated oxidative stress in TAU cells, decreasing the level of ROS. Nitroxide-containing nanoparticles lowered the level of lipid peroxidation in both TAU and EP cells, suggesting that nitroxides and NPs may mitigate tau-protein-induced oxidative stress.

There are conflicting reports on the antioxidant activity of hispidulin. Antioxidant activity of hispidulin was evaluated using assays of ABTS • reduction, ferric ion reducing antioxidant power (FRAP) assay, DPPH reduction assay, and protection of erythrocyte membranes against lipid peroxidation and protein thiol oxidation. ABTS • reduction assay pointed to the involvement of all three phenol groups of hispidulin in ABTS • reduction. The reactivity of hispidulin in the FRAP assay and DPPH reduction assay was low (0.09 and 0.019 of the reactivity of Trolox). However, hispidulin was effective in protection against erythrocyte membrane lipid peroxidation and highly effective in protection against erythrocyte membrane protein thiol group oxidation (more effective than Trolox). These results point to the necessity of caution in extrapolating the antioxidant activity evaluated in simple cell-free systems on more complex systems.

There is recent interest in a diet that can be recommended for patients suffering from cancer. In this respect, the effects were studied of the extracts of several common fruits, herbs and vegetables on the viability of two human ovary cancer cell lines (SKOV-3 and PEO1) in vitro. Normal human MRC-5 fibroblasts were used as a control cell line. The extracts of garlic, horseradish and curly kale as well as green and black tea were the most effective in lowering the viability of ovarian cancer cells, while not affecting the viability of MRC-5 fibroblasts. Except for garlic and horseradish, the cytotoxic effects of the extracts correlated with their polyphenol content. The examination of changes in the content of ATP and glutathione, in the level of reactive oxygen species, mitochondrial potential and mitochondrial mass did not show a consistent pattern, suggesting that various extracts may act via different mechanisms. Although the extracts' toxicity to cells in vitro is a first and direct suggestion concerning their possible anticancer effects in vivo, these results point to potential vegetable candidates to become diet components recommended for ovary cancer patients.

Hydrogen peroxide (H2O2) is generated under autoxidation of some components of beverages including flavonoids and ascorbate, especially in tea and coffee. As polyphenols are also present in solid food, especially in vegetables, we checked whether hydrogen peroxide is generated during cooking of several common vegetables. The formation of hydrogen peroxide was found in the decoctions of all cooked vegetables studied except for potato and in the homogenates of cooked vegetables except for garlic and purple potato. The highest concentration of hydrogen peroxide in 1:2 (w/v) homogenates was found for the broad bean (73.4±9.0 µM) followed by broccoli (18.6±0.3 µM), onion (10.4±1.6 µM) and leek (10.0±0.3 µM), while the H2O2 concentration in the decoctions was the highest for broccoli (24.4±0.8 µM), then for broad bean (21.4±1.1 µM), carrot (13.2±0.2 µM) and cauliflower (12.6±1.2 µM).

It has been argued that the mol/cell metric is more universal than concentration of the toxic agent since in many cases the effect of dose expressed as mol/cell is independent of ex-perimental setup. We confirmed it for hemolysis of erythrocytes in phosphate-buffered saline induced by hypochlorite where the amount of femtomoles/cell of hypochlorite needed for 50% hemolysis was independent of erythrocyte concentration. However, in the presence of blood plasma this metric became dependent on cell concentration. Similarly, the effect of 3-bromopyruvic acid (3-BP) on PEO1 cells as a function of mol/cell ratio depended on the volume of the 3-BP containing medium, due to the reaction of 3-BP with components of the medium. Hemolytic amounts of sodium dodecyl sulfate and Triton X-100 expressed as mol/cell decreased with increasing cell concentration while the effect of DMSO on the viability of a constant number of fibroblasts was independent of the volume of DMSO-containing medium. These results demonstrate that the mol/cell metric is still dependent on experimental conditions when the toxic agent interacts with components of the medium or when its physical state is modified by the target cells, and the effect is independent of the mol/per cell ratio for high excess of a cell damaging agent., (Copyright © 2021. Published by Elsevier Ltd.)

Phytoestrogens are naturally occurring non-steroidal phenolic plant compounds. Their structure is similar to 17-β-estradiol, the main female sex hormone. This review offers a concise summary of the current literature on several potential health benefits of phytoestrogens, mainly their neuroprotective effect. Phytoestrogens lower the risk of menopausal symptoms and osteoporosis, as well as cardiovascular disease. They also reduce the risk of brain disease. The effects of phytoestrogens and their derivatives on cancer are mainly due to the inhibition of estrogen synthesis and metabolism, leading to antiangiogenic, antimetastatic, and epigenetic effects. The brain controls the secretion of estrogen (hypothalamus-pituitary-gonads axis). However, it has not been unequivocally established whether estrogen therapy has a neuroprotective effect on brain function. The neuroprotective effects of phytoestrogens seem to be related to both their antioxidant properties and interaction with the estrogen receptor. The possible effects of phytoestrogens on the thyroid cause some concern; nevertheless, generally, no serious side effects have been reported, and these compounds can be recommended as health-promoting food components or supplements.

Betalains are water-soluble pigments present in vacuoles of plants of the order Caryophyllales and in mushrooms of the genera Amanita , Hygrocybe and Hygrophorus . Betalamic acid is a constituent of all betalains. The type of betalamic acid substituent determines the class of betalains. The betacyanins (reddish to violet) contain a cyclo-3,4-dihydroxyphenylalanine (cyclo-DOPA) residue while the betaxanthins (yellow to orange) contain different amino acid or amine residues. The most common betacyanin is betanin (Beetroot Red), present in red beets Beta vulgaris , which is a glucoside of betanidin. The structure of this comprehensive review is as follows: Occurrence of Betalains; Structure of Betalains; Spectroscopic and Fluorescent Properties; Stability; Antioxidant Activity; Bioavailability, Health Benefits; Betalains as Food Colorants; Food Safety of Betalains; Other Applications of Betalains; and Environmental Role and Fate of Betalains.

The damage to SH-SY5Y cells by 6-hydroxydopamine (6-OHDA) is an established cellular model of Parkinson's disease (PD). Redox nanoparticles are a promising tool for therapy, including neurodegenerative diseases. As pH of the brain tissue at sites affected by PD is lowered down to 6.5, we studied the effect of pH-responsive redox nanoparticles (poly(ethylene glycol)- b -poly[4-(2,2,6,6-tetramethylpiperidine-1-oxyl)aminomethylstyrene]), which change their structure in a pH-dependent manner and become active below pH 7 (NRNPs pH ), on the viability of SH-SY5Y cells treated with 6-OHDA at pH 6.5 and 7.4. Pretreatment of the cells with NRNPs pH (15-75 μM) prior to the 6-OHDA treatment increased their survival in a concentration-dependent manner at pH 6.5, but not at pH 7.4. Among several parameters studied (ATP and GSH content, the level of reactive oxygen species, mitochondrial potential, mitochondrial mass), only the mitochondrial mass was dose-dependently protected by NRNPs pH at pH 6.5, but not at pH 7.4. These results indicate that the action of NRNPs pH on mitochondria underlies their protective effect in this cellular model of PD. These results may have potential importance for future applications of NRNPs pH in preclinical and perhaps clinical studies.

3-Bromopyruvic acid (3-BP) is a promising anticancer compound. Two ovary cancer (OC) cell lines, PEO1 and SKOV3, showed relatively high sensitivity to 3-BP (half maximal inhibitory concentration (IC 50 ) of 18.7 and 40.5 µM, respectively). However, the further sensitization of OC cells to 3-BP would be desirable. Delphinidin (D) has been reported to be cytotoxic for cancer cell lines. We found that D was the most toxic for PEO1 and SKOV3 cells from among several flavonoids tested. The combined action of 3-BP and D was mostly synergistic in PEO1 cells and mostly weakly antagonistic in SKOV3 cells. The viability of MRC-5 fibroblasts was not affected by both compounds at concentrations of up to 100 µM. The combined action of 3-BP and D decreased the level of ATP and of dihydroethidium (DHE)-detectable reactive oxygen species (ROS), cellular mobility and cell staining with phalloidin and Mitotracker Red in both cell lines but increased the 2',7'-dichlorofluorescein (DCFDA)-detectable ROS level and decreased the mitochondrial membrane potential and mitochondrial mass only in PEO1 cells. The glutathione level was increased by 3-BP+D only in SKOV3 cells. These differences may contribute to the lower sensitivity of SKOV3 cells to 3-BP+D. Our results point to the possibility of sensitization of at least some OC cells to 3-BP by D.

We found that the oxidation of cyanidin with hydrogen peroxide, tert -butyl hydroperoxide, 2,2'-azobis(2-methylpropionamidine), 3-morpholinosydnonimine hydrochloride, sodium hypochlorite, Fe 3+ , and Fe 2+ induced the appearance of a new fluorescence band, centered at 525-540 nm when excited at 470-490 nm. The intensity of this fluorescence was related to the oxidant concentration. The same fluorescence was induced by the oxidation of other anthocyanidins, an anthocyanin (cyanidin-3- O -glucoside) as well as anthocyanin-rich red cabbage extract, and blackcurrant and elderberry juices. Peroxidized blood plasma also induced the appearance of cyanidin fluorescence. We also showed that the formation of the fluorescent product of pelargonidin can be observed in a culture of the MRC-5 human fetal lung fibroblast cell line and the DU-145 human prostate carcinoma cell line. Our results suggest that this new anthocyanidin/anthocyanin fluorescence may be an indicator of oxidation, especially of food products, where these compounds are present or added as colorants, and can also be useful to detect oxidation in biomedical experiments.

Due to the progressive aging of the society, the prevalence and socioeconomic burden of neurodegenerative diseases are predicted to rise. The most common neurodegenerative disorders nowadays, such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis, can be classified as proteinopathies. They can be either synucleinopathies, amyloidopathies, tauopathies, or TDP-43-related proteinopathies; thus, nanoparticles with a potential ability to inhibit pathological protein aggregation and/or degrade already existing aggregates can be a promising approach in the treatment of neurodegenerative diseases. As it turns out, nanoparticles can be a double-edged sword; they can either promote or inhibit protein aggregation, depending on coating, shape, size, surface charge, and concentration. In this review, we aim to emphasize the need of a breakthrough in the treatment of neurodegenerative disorders and draw attention to nanomaterials, as they can also serve as a diagnostic tool for protein aggregates or can be used in a high-throughput screening for novel antiaggregative compounds., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2020 Monika Pichla et al.)

Replicative senescence is an unalterable growth arrest of primary cells in the culture system. It has been reported that aging in vivo is related to the limited replicative capacity that normal somatic cells show in vitro . If oxidative damage contributes to the lifespan limitation, antioxidants are expected to extend the replicative lifespan of fibroblasts. This article critically reviews the results of experiments devoted to this problem performed within the last decades under conditions of in vitro culture. The results of studied are heterogeneous, some papers showing no effects of antioxidants; most finding limited enhancement of reproductive capacity of fibroblasts, some reporting a significant extension of replicative lifespan (RLS). Both natural and synthetic antioxidants were found to extend the RLS of fibroblasts, either by a direct antioxidant effect or, indirectly, by activation of signaling pathways and activation of proteasomes or hormetic effects. Most significant prolongation of RLS was reported so far for nicotinamide, N -hydroxylamines, carnosine and Methylene Blue. These results may be of importance for the design of skin-protecting cosmetics., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Izabela Sadowska-Bartosz and Grzegorz Bartosz.)

The inhibitory effects a range of synthetic and natural antioxidants on lipid peroxidation of egg yolk and erythrocyte membranes induced by a free radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) was compared, with significant differences being found between both systems. When the protection by selected antioxidants against the effects of AAPH on erythrocytes (hemolysis, oxidation of hemoglobin and glutathione (GSH) and generation of reactive oxygen species (ROS)) was studied, most antioxidants were protective, but in some tests (oxidation of hemoglobin and GSH) some acted as prooxidants, inducing oxidation in the absence of AAPH and enhancing the AAPH-induced oxidation. These results demonstrate a diversified action of antioxidants in different systems and point to a need for careful extrapolation of any conclusions drawn from one parameter or experimental system to another.

Nitroxides and nitroxide-containing nanoparticles (RNP) are excellent antioxidants. However, they have relatively high reduction potentials, which make them behave like oxidants or show little activity in some antioxidant assays. We found that stable nitroxyl radicals (TEMPO and 4-amino-TEMPO) has low reactivity in the test of scavenging of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical (ABTS • ). As a result, supplementation of blood plasma with nitroxides may decrease its total antioxidant capacity assayed with ABTS • . Nitroxides oxidize Fe 2+ and in this way interfere with the ferric-Xylenol Orange assay of peroxides. Nitroxides as well as RNP directly oxidize glutathione and fluorogenic probes used for estimation of reactive oxygen species (ROS) (dihydro-2'7'-dichlorofluorescein diacetate, dihydroethidine and dihydrorhodamine 123) and thus produce artefacts in assays of glutathione and ROS in cell-free and cellular systems. These results point to the necessity of careful interpretation of antioxidant assays concerning nitroxides and RNP or performed in their presence., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) patients can benefit from antioxidant supplementation, and new efficient antioxidants are needed. The aim of this study was to evaluate the protective effect of selected nitroxide-containing redox nanoparticles (NRNPs) in a cellular model of PD. Antioxidant properties of NRNPs were studied in cell-free systems by protection of dihydrorhodamine 123 against oxidation by 3-morpholino-sydnonimine and protection of fluorescein against bleaching by 2,2-azobis(2-amidinopropane) hydrochloride and sodium hypochlorite. Model blood-brain barrier penetration was studied using hCMEC/D3 cells. Human neuroblastoma SH-SY5Y cells, exposed to 6-hydroxydopamine (6-OHDA), were used as an in vitro model of PD. Cells were preexposed to NRNPs or free nitroxides (TEMPO or 4-amino-TEMPO) for 2 h and treated with 6-OHDA for 1 h and 24 h. The reactive oxygen species (ROS) level was estimated with dihydroethidine 123 and Fluorimetric Mitochondrial Superoxide Activity Assay Kit. Glutathione level (GSH) was measured with ortho -phtalaldehyde, ATP by luminometry, changes in mitochondrial membrane potential with JC-1, and mitochondrial mass with 10-Nonyl-Acridine Orange. NRNP1, TEMPO, and 4-amino-TEMPO (25-150  μ M) protected SH-SY5Y cells from 6-OHDA-induced viability loss; the protection was much higher for NRNP1 than for free nitroxides. NRNP1 were better antioxidants in vitro and permeated better the model BBB than free nitroxides. Exposure to 6-OHDA decreased the GSH level after 1 h and increased it considerably after 24 h (apparently a compensatory overresponse); NRNPs and free nitroxides prevented this increase. NRNP1 and free nitroxides prevented the decrease in ATP level after 1 h and increased it after 24 h. 6-OHDA increased the intracellular ROS level and mitochondrial superoxide level. Studied antioxidants mostly decreased ROS and superoxide levels. 6-OHDA decreased the mitochondrial potential and mitochondrial mass; both effects were prevented by NRNP1 and nitroxides. These results suggest that the mitochondria are the main site of 6-OHDA-induced cellular damage and demonstrate a protective effect of NRNP1 in a cellular model of PD., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Monika Pichla et al.)

The aim of this study was to characterize the interaction of chosen catechins ((+)-catechin, (-)-epigallocatechin (EGC), and (-)-epigallocatechin gallate (EGCG)) with human erythrocytes and their protective effects against oxidative damage of erythrocytes. Uptake of the catechins by erythrocytes was studied by fluorimetry, their interaction with erythrocyte membrane was probed by changes in erythrocyte osmotic fragility and in membrane fluidity evaluated with spin labels, while protection against oxidative damage was assessed by protection against hemolysis induced by permanganate and protection of erythrocyte membranes against lipid peroxidation and protein thiol group oxidation. Catechin uptake was similar for all the compounds studied. Accumulation of catechins in the erythrocyte membrane was demonstrated by the catechin-induced increase in osmotic resistance and rigidification of the erythrocyte membrane detected by spin labels 5-doxyl stearic acid and 16-doxyl stearic acid. (-)-Epigallocatechin and EGCG inhibited erythrocyte acetylcholinesterase (mixed-type inhibition). Catechins protected erythrocytes against permanganate-induced hemolysis, oxidation of erythrocyte protein thiol groups, as well as membrane lipid peroxidation. These results contribute to the knowledge of the beneficial effects of catechins present in plant-derived food and beverages.

The study was aimed at evaluation of the role of secondary oxidative stress in the stress-induced premature senescence (SIPS) of human fibroblasts induced by H 2 O 2 . Two fibroblast lines were used: lung MRC-5 and ear H8F2p25LM fibroblasts. The lines differed considerably in sensitivity to H 2 O 2 (IC 50 of 528 and 33.5 μM, respectively). The cells were exposed to H 2 O 2 concentrations corresponding to IC 50 and after 24 h supplemented with a range of antioxidants. Most of antioxidants studied slightly augmented the survival of fibroblasts at single concentrations or in a narrow concentration range, but the results were not consistent among the cell lines. Chosen antioxidants (4-amino-TEMPO, curcumin, caffeic acid and p -coumaric acid) did not restore the level of glutathione decreased by H 2 O 2 . Hydrogen peroxide treatment did not induce secondary production of H 2 O 2 and even decreased it, decreased mitochondrial potential in both cell lines and induced changes in the mitochondrial mass inconsistent between the lines. Antioxidant protected mitochondrial potential only in H8F2p25LM cells, but attenuated changes in mitochondrial mass. These results speak against the intermediacy of secondary oxidative stress in the SIPS induced by H 2 O 2 and suggest that the small protective action of antioxidants is due to their effects on mitochondria.

Aims: 3-Bromopyruvate (3-BP), an alkylating agent and a glycolytic inhibitor, is a promising anticancer agent, which can be efficient also against multidrug-resistant cancer cells. The aim of this study was to examine how 3-BP affects the survival and mobility of rat (MAT-LyLu and AT-2) and human (DU-145 and PC-3) metastatic prostate cancer cell lines., Main Methods: Cytotoxicity was estimated with Neutral Red. Cell mobility was analyzed by time-lapse microscopic monitoring of trajectories of individual cells at 5-min intervals for 6h. ATP was estimated with luciferin/luciferase and glutathione (GSH) with o-phthalaldehyde. Actin cytoskeleton was visualized with phalloidin conjugated with Atto-488., Key Findings: All metastatic prostate cell lines studied were very sensitive to 3-BP (IC 50 of 4-26μM). 3-Bromopyruvate drastically reduced cell movement even at concentrations of 5-10μM after 1h treatment. This compound depleted also cellular ATP and GSH, and disrupted actin cytoskeleton., Significance: The data obtained suggest that 3-BP can potentially be useful for treatment of metastatic prostate cancer and, especially, be efficient in limiting metastasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Studies of 54 antioxidants revealed that 27 of them, mainly polyphenols, generated hydrogen peroxide (H 2 O 2 ) when added to Dulbecco's modified Eagle's medium (DMEM), other media used for culture of mammalian and yeast cells and phosphate-buffered saline. The most active antioxidants were: propyl gallate (PG), (-)-epigallocatechin gallate (EGCG) and quercetin (Q). Chelex treatment and iron chelators decreased H 2 O 2 generation suggesting that transition metal ions catalyze antioxidant autoxidation and H 2 O 2 production. Green tea also generated H 2 O 2 ; tea prepared on tap water generated significantly more H 2 O 2 than tea prepared on deionized water. Ascorbic acid decreased H 2 O 2 production although it generated H 2 O 2 itself, in the absence of other additives. Lemon added to the tea significantly reduced generation of H 2 O 2 . Hydrogen peroxide generated in the medium contributed to the cytotoxicity of PG, EGCG and Q to human prostate carcinoma DU-145 cells, since catalase increased the survival of the cells subjected to these compounds in vitro., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

An alkylating compound, 3-bromopyruvic acid (3-3-bromopyruvic acid (BP)) is a promising anti-cancer agent, potentially able to act on multidrug-resistant cells. Its action has been attributed mainly to inhibition of glycolysis. This compound induces also oxidative stress at a cellular level. The effects of 3-BP on gene expression have not been studied although they may determine the survival of cells exposed to 3-BP. The aim of this paper was to examine the effect 3-BP on gene expression pattern in breast MCF-7 cancer cells. Detection of the differences in gene expression was performed using microarrays and dysregulated genes were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Exposure of cells to 100 µM 3-BP for 6, 12 and 24 increased expression and diminished expression of 39 and 6 genes, respectively. Among the induced genes, 22 belong to general cellular stress response genes, maintenance genes involved in redox homeostasis, responding to oxidative stress (among them metallothioneins, low-molecular-weight thiol homeostasis enzymes and genes coding for NAD(P)H-dependent oxidoreductases operating on complex organic substrates, including aldo-keto reductases). These results demonstrate that transient oxidative stress in cells exposed to 3-BP is followed by antioxidant response.

Interaction of metal, especially iron ions with flavanols is considered as an important feature of these compounds and is believed to contribute to their both antioxidant and prooxidant properties. The aim of this study was to examine how Fe 2+ binding to form a 4:1 (flavanol:Fe 2+ ) mixtures affects the antioxidant properties of flavanols. ABTS ∗ scavenging, protection against fluorescence bleaching induced by AAPH and hypochlorite, protection against lipid peroxidation and protection against hypochlorite-induced hemolysis demonstrated that flavonol-Fe 2+ mixtures retain antioxidant properties, although, in most cases, they are lower with respect to the flavanols alone. No superoxide dismutase-like or catalase-like activity of the mixtures was revealed., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Oxidative stress (OS) and nitrative stress (NS) accompany many diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Antioxidants have been proposed to counteract OS/NS in these diseases. Nevertheless, the effects of antioxidants are limited and new, more efficient antioxidants are searched for. Redox-active nanoparticles (RNPs), containing antioxidants create a new therapeutical perspective. This review examines the recent literature describing synthesis and potential applications of cerium oxide RNPs, boron cluster-containing and silica containing RNPs, Gd 3 N @ C 80 encapsulated RNPs, and concentrates on nitroxide-containing RNPs. Nitroxides are promising antioxidants, preventing inter alia glycation and nitration, but their application poses several problems. It can be expected that application of RNPs containing covalently bound nitroxides, showing low toxicity and able to penetrate the blood-brain barrier will be more efficient in the treatment of neurodegenerative disease, in particular AD and PD basing on their effects in cellular and animal models of neurodegenerative diseases.

Non-enzymatic protein modifications occur inevitably in all living systems. Products of such modifications accumulate during aging of cells and organisms and may contribute to their age-related functional deterioration. This review presents the formation of irreversible protein modifications such as carbonylation, nitration and chlorination, modifications by 4-hydroxynonenal, removal of modified proteins and accumulation of these protein modifications during aging of humans and model organisms, and their enhanced accumulation in age-related brain diseases.

TEMPO-phosphate has been introduced as a phosphate analogue to study phosphate transport in erythrocytes. The nitroxide is reduced intracellularly upon entering the cells, the membrane transport being the rate-limiting step of the loss of ESR signal. The use of TEMPO-phosphate is convenient and avoids the hazard of radioactivity. We studied the inhibition of TEMPO-phosphate transport to human erythrocytes by various compounds. DIDS and SITS, inhibitors of Band 3, inhibited the TEMPO-phosphate transport. 1-cyano-4-hydroxycinnamic acid, inhibitor of monocarboxylate transporters, did not affect the permeation of TEMPO-phosphate. The transport of TEMPO-phosphate was inhibited by various polyphenols, especially curcumin, naringin, quercetin, luteolin and kaempferol. Interestingly, 3-bromopyruvic acid, an alkylating agent and potential anticancer agent, induced an apparent enhancement of TEMPO-phosphate transport into erythrocytes.

Ribonucleotide reductase (RNR) catalyses the rate limiting step of DNA synthesis utilising a mechanism that requires a tyrosyl radical. We have previously shown that superoxide can quench protein tyrosyl radicals in vitro, either by oxidative addition, or reduction of the radical to tyrosine. Here, we observe that Saccharomyces cerevisiae strains lacking either copper-zincSOD (SOD1) or manganese SOD (SOD2) had decreased RNR activity compared to SOD-competent yeast. When superoxide production was increased by treatment with paraquat, RNR activity was further decreased, with yeast lacking SOD1 being the most sensitive. The growth of yeast lacking SOD1 was also the most sensitive to paraquat treatment. Using expressed recombinant RNR, superoxide addition was not detectable using mass-spectrometry. This suggests that oxidative addition is not the major route of inhibition in our system, but does not rule out reduction by superoxide as a possible mechanism. Our results demonstrate that protection of RNR from inactivation by superoxide is an important function of SOD, particularly cytoplasmic SOD1., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Antioxidant properties of five catechins and five other flavonoids were compared with several other natural and synthetic compounds and related to glutathione and ascorbate as key endogenous antioxidants in several in vitro tests and assays involving erythrocytes. Catechins showed the highest ABTS-scavenging capacity, the highest stoichiometry of Fe 3+ reduction in the FRAP assay and belonged to the most efficient compounds in protection against SIN-1 induced oxidation of dihydrorhodamine 123, AAPH-induced fluorescein bleaching and hypochlorite-induced fluorescein bleaching. Glutathione and ascorbate were less effective. (+)-catechin and (-)-epicatechin were the most effective compounds in protection against AAPH-induced erythrocyte hemolysis while (-)-epicatechin gallate, (-)-epigallocatechin gallate and (-)-epigallocatechin protected at lowest concentrations against hypochlorite-induced hemolysis. Catechins [(-)-epigallocatechin gallate and (-)-epicatechin gallate)] were most efficient in the inhibition of AAPH-induced oxidation of 2'7'-dichlorodihydroflurescein contained inside erythrocytes. Excellent antioxidant properties of catechins and other flavonoids make them ideal candidates for nanoformulations to be used in antioxidant therapy., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Salivary gland tumors account for 3-6% of tumors of the head and neck. About 80% of salivary gland tumors occur in parotid glands. Oxidative stress (OS) is implicated in the origin, development, and whole-body effects of various tumors. There are no data on the occurrence of OS in the parotid gland tumors. The aim of this study was to ascertain if whole-body OS accompanies parotid gland tumors, based first of all on oxidative modifications of blood serum proteins and other markers of OS in the serum of the patients. The group studied included 17 patients with pleomorphic adenoma, 9 patients with Warthin's tumor, 8 patients with acinic cell carcinoma, and 24 age-matched controls. We found increased concentration of interleukin 4 in patients with acinic cell carcinoma, decreased plasma thiols, increased AOPP concentration, and decreased FRAP of blood serum in all groups of the patients while protein oxidative modifications assessed fluorimetrically, protein carbonyls, protein nitration, malondialdehyde concentration, and serum ABTS ⁎ -scavenging capacity were unchanged. These data indicate the occurrence of OS in patients with parotid gland tumors and point to various sensitivities of OS markers.

Myasthenia gravis (MG) is an autoimmune disease caused by production of antibodies against acetylcholine receptors of the neuromuscular junction (Ab). The aim of this study was to ascertain if oxidative stress accompanies MG by estimation of the several independent parameters of oxidative damage, mainly the levels of oxidative modifications of blood serum proteins. The group studied consisted of 50 MG patients (28 females and 22 males), 24 with ocular MG (OMG) and 26 with generalized MG (GMG), of mean age of 66.7 (30-81)years (y), mean disease duration of 9.5 (0.5-34)y, mean level of Ab of 8.9 (0.1-85)nmol/ml, and 25 age-matched healthy controls. MG patients were stratified into groups according to disease duration (<5y or >5y), Ab level (low, <3 or high, >3nmol/l) as well as symptoms (GMG or OMG). Glycophore fluorescence was increased in OMG a . Dityrosine was increased in both types of MG c , in patients ill <5 b and >5 c y, with low c and high c levels of Ab. N-formylkynurenine was increased in OMG a and GMG b , in both disease duration groups a , in the group of low Ab a . Kynurenine was increased in the group with high Ab a . Tryptophan fluorescence was decreased in OMG b and GMG c , in patients ill for <5 b and >5 a y, with low a and high c Ab. Serum thiol group concentration were decreased in GMG c , in patients ill for >5y b . AOPP level was elevated in OMG a , in patients ill for >5y a with high Ab a . Protein carbonyls were increased in both OMG c and GMG c , in patients ill for >5 a y, with low b and high b Ab. FRAP and ABTS • scavenging by fast antioxidants were unchanged, but ABTS • scavenging by slow antioxidants was lower in OMG b and GMG c , in patients ill for >5 c y, in patients with low c and high b Ab ( a p<0.05, b p<0.01, c p<0.001). These results demonstrate systemic oxidative stress in MG, suggesting therapeutic use of antioxidants., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Glycation is the cause of diabetic complications and contributes to the development of other diseases and aging. Numerous exogenous compounds have been tested for their anti-glycating activity. The aim of this study was to answer the question, which endogenous compounds at physiological concentrations can effectively prevent glycation. A set of endogenous compounds has been tested for the ability to protect albumin from glucose-induced glycation in vitro at a concentration of 1 mM and in a physiological concentration range. Only glutathione was found to protect significantly against glycation at physiological concentrations. Glutathione depletion increased the rate of hemoglobin glycation in erythrocytes incubated with high glucose concentrations. These results indicate that the level of glutathione is the main determinant of glycation of intracellular proteins.

Deoxyribose test has been widely used for determination of reactivities of various compounds for the hydroxyl radical. The test is based on the formation of hydroxyl radical by Fe 2+ complex in the Fenton reaction. We propose a modification of the deoxyribose test to detect strong iron binding, inhibiting participation of Fe 2+ in the Fenton reaction, on the basis of examination of concentration dependence of deoxyribose degradation on Fe 2+ concentration, at a constant concentration of a chelating agent.

Multiple sclerosis (MS) is a disease involving oxidative stress (OS). This study was aimed at examination of the effect of melatonin supplementation on OS parameters, especially oxidative protein modifications of blood serum proteins, in MS patients. The study included 11 control subjects, 14 de novo diagnosed MS patients with the relapsing-remitting form of MS (RRMS), 36 patients with RRMS receiving interferon beta-1b (250  μ g every other day), and 25 RRMS patients receiving interferon beta-1b plus melatonin (5 mg daily). The levels of N'-formylkynurenine, kynurenine, dityrosine, carbonyl groups, advanced glycation products (AGEs), advanced oxidation protein products (AOPP), and malondialdehyde were elevated in nontreated RRSM patients. N'-Formylkynurenine, kynurenine, AGEs, and carbonyl contents were decreased only in the group treated with interferon beta plus melatonin, while dityrosine and AOPP contents were decreased both in the group of patients treated with interferon beta and in the group treated with interferon beta-1b plus melatonin. These results demonstrate that melatonin ameliorates OS in MS patients supporting the view that combined administration of interferon beta-1b and melatonin can be more effective in reducing OS in MS patients than interferon beta-1b alone.