The Cellular and Organismal Effects of Nitroxides and Nitroxide-Containing Nanoparticles.

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.

Antioxidant Defense in the Toughest Animals on the Earth: Its Contribution to the Extreme Resistance of Tardigrades.

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.

Effect of Selected Antioxidants on the In Vitro Aging of Human Fibroblasts.

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.

Which Constituents Determine the Antioxidant Activity and Cytotoxicity of Garlic? Role of Organosulfur Compounds and Phenolics.

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.

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

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.

Effect of Low Concentration of Nitroxides on SH-SY5Y Cells Transfected with the Tau Protein.

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 Fe3+, 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.

Formation of a Purple Product upon the Reaction of ABTS Radicals with Proteins.

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.

Comparison of Antioxidant and Antiproliferative Effects of Various Forms of Garlic and Ramsons.

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 (IC50 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.

Induction of Oxidative Stress in SH-SY5Y Cells by Overexpression of hTau40 and Its Mitigation by Redox-Active Nanoparticles.

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.

Comparison of the Effects of Resveratrol and Its Derivatives on the Radiation Response of MCF-7 Breast Cancer Cells.

Radiotherapy is among the most important methods for breast cancer treatment. However, this method's effectiveness is limited by radioresistance. The aim of this study was to investigate whether the stilbene derivatives piceid, resveratrol, and piceatannol have a radiosensitising effect on breast cancer cells (MCF-7). The conducted research enabled us to determine which of the tested compounds has the greatest potential in sensitising cells to ionising radiation (IR). Among the stilbene derivatives, resveratrol significantly increased the effect of IR. Resveratrol and IR used in combination had a higher cytotoxic effect on MCF-7 cells than using piceatannol, piceid, or radiation alone. This was due to a significant decrease in the activity of antioxidant enzymes, which resulted in the accumulation of formed reactive oxygen species (ROS). The effect of resveratrol and IR enhanced the expression of apoptotic genes, such as Bax, p53, and caspase 8, leading to apoptosis.

Delphinidin Increases the Sensitivity of Ovarian Cancer Cell Lines to 3-Bromopyruvate.

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 (IC50) 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.

Biological Properties and Applications of Betalains.

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 Potential Effects of Phytoestrogens: The Role in Neuroprotection.

Phytoestrogens are naturally occurring non-steroidal phenolic plant compounds. Their structure is similar to 17-ß-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.

pH-Responsive Redox Nanoparticles Protect SH-SY5Y Cells at Lowered pH in a Cellular Model of Parkinson's Disease.

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.

Possible artefacts of antioxidant assays performed in the presence of nitroxides and nitroxide-containing nanoparticles.

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 Fe2+ 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.

Comparison of Antioxidants: The Limited Correlation between Various Assays of Antioxidant Activity.

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.

Interaction of Catechins with Human Erythrocytes.

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.

Does gender influence the outcome of ischemic heart disease?

Diseases of the cardiovascular system (myocardial infarction, stroke, heart failure, hypertensive heart disease, cardiomyopathy) account for 40% of all deaths in men and up to 49% of all deaths in women. For a long time it was thought that the clinical picture of ischemic heart disease in men and women was similar. Now, however, there are more reports suggesting that diverse manifestations of the symptoms of ischemic disease may be related to differences between sexes. The disparity between women and men is also evident in the diagnostic process, and various pathological mechanisms of cardiovascular diseases, in particular myocardial ischemia in men and women, affect the differences in the results of diagnostic tests. Vasomotor dysfunction is particularly frequent in women, as their coronary vessels are more sensitive to the catecholamines released during mental stress, resulting in spasm and ischemic myocardium. Moreover, a much lower dose of acetylcholine induced vasoconstriction, which indicates that women are more sensitive to this neurotransmitter. Therefore, coronary vasomotor disorders in the form of epicardial and microvascular dysfunction are more often seen in women. All these mentioned factors resulted in higher mortality and poorer quality of life of women suffering from ischemic heart disease.

Redox nanoparticles: synthesis, properties and perspectives of use for treatment of neurodegenerative diseases.

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, Gd3N@C80 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.

Leishmania tarentolae as a host for heterologous expression of functional human ABCB6 transporter.

The need for large amounts of reproducibly produced and isolated protein arises not only in structural studies, but even more so in biochemical ones, and with regard to ABC transporters it is especially pressing when faced with the prospect of enzymatic/transport activity studies, substrate screening etc. Thus, reliable heterologous expression systems/model organisms for large and complex proteins are at a premium. We have verified the applicability of the recently established novel eukaryotic expression system, using Leishmania tarentolae as a host, for human ABC protein overexpression. We succeeded in overexpressing human ABCB6, a protein with controversial subcellular localization and multiple proposed cellular functions. We were able to demonstrate its efficient expression in the expected subcellular locations as well as biochemical activity of the overexpressed protein (ATPase activity and porphyrin-like substrate transport). This activity was absent in cells overexpressing the catalytically inactive variant of ABCB6 (K629M). We demonstrate the possibility of applying a cost-effective expression system to study the activity of membrane transporters from the ABC superfamily.