Mg2+- and ATP-dependent inhibition of transient receptor potential melastatin 7 by oxidative stress

Transient receptor potential melastatin 7 (TRPM7) is a Ca²⁺- and Mg²⁺-permeable nonselective cation channel that contains a unique carboxyl-terminal serine/threonine protein kinase domain. It has been reported that reactive oxygen species associated with hypoxia or ischemia activate TRPM7 current and then induce Ca²⁺ overload resulting in neuronal cell death in the brain. In this study, we aimed to investigate the molecular mechanisms of TRPM7 regulation by hydrogen peroxide (H₂O₂) using murine TRPM7 expressed in HEK293 cells. Using the whole-cell patch-clamp technique, it was revealed that the TRPM7 current was inhibited, not activated, by the application of H₂O₂ to the extracellular solution. This inhibition was not reversed after washout or treatment with dithiothreitol, suggesting irreversible oxidation of TRPM7 or its regulatory factors by H₂O₂ under whole-cell recording. Application of an electrophile, N-methylmaleimide (NMM), which covalently modifies cysteine residues in proteins, also inhibited TRPM7 current irreversibly. The effects of H₂O₂ and NMM were dependent on free [Mg²⁺]_i; the inhibition was stronger when cells were perfused with higher free [Mg²⁺]_i solutions via pipette. In addition, TRPM7 current was not inhibited by H₂O₂ when millimolar ATP was included in the intracellular solution, even in the presence of substantial free [Mg²⁺]_i, which is sufficient for TRPM7 inhibition by H₂O₂ in the absence of ATP. Moreover, a kinase-deficient mutant of TRPM7 (K1645R) was similarly inhibited by H₂O₂ just like the wild-type TRPM7 in a [Mg²⁺]_i- and [ATP]_i-dependent manner, indicating no involvement of the kinase activity of TRPM7. Thus, these data suggest that oxidative stress inhibits TRPM7 current under pathological conditions that accompany intracellular ATP depletion and free [Mg²⁺]_i elevation.     

Redox evaluation in sepsis model mice by the in vivo ESR technique using acyl-protected hydroxylamine

In vivo electron spin resonance (ESR) spectroscopy is a noninvasive technique that measures the oxidative stress in living experimental animals. The rate of decay of the ESR signal right after an injection of nitroxyl radical has been measured to evaluate the oxidative stress in animals, although the probe’s disposition could also affect this rate. Because the amount of probes forming the redox pair of hydroxyl amine and its corresponding nitroxyl radical was shown to be nearly constant in most organs or tissues 10 min after the injection of 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in mice, we evaluated the oxidative stress in sepsis model mice induced by lipopolysaccharide (LPS) by intravenously injecting ACP as a precursor of redox probes. The in vivo ESR signal increased up to 7–8 min after the ACP injection and then decreased. Decay of the in vivo signal in LPS-treated mice was significantly slower than that in healthy mice, whereas no significant difference was observed in the rate of change in the total amount of redox probes in the blood and liver between these groups. ESR imaging showed that the in vivo signals observed at the chest and upper abdomen decayed slowly in LPS-treated mice. Suppression of the decay in LPS-treated mice was canceled by the administration of a combination of pegylated superoxide dismutase and catalase, or an inhibitor of nitric oxide synthase, or gadolinium chloride. These results indicate that the LPS-treated mouse is under oxidative stress and that reactive oxygen species, such as superoxide and peroxynitrite, related to macrophages are mainly involved in the oxidative stress.     

The complex interplay of iron metabolism,reactive oxygen species,and reactive nitrogen species: Insights into the potential of various iron therapies to induce oxidative and nitrosative stress

Production of minute concentrations of superoxide (O₂⁻) and nitrogen monoxide (nitric oxide, NO) plays important roles in several aspects of cellular signaling and metabolic regulation. However, in an inflammatory environment, the concentrations of these radicals can drastically increase and the antioxidant defenses may become overwhelmed. Thus, biological damage may occur owing to redox imbalance—a condition called oxidative and/or nitrosative stress. A complex interplay exists between iron metabolism, O₂⁻, hydrogen peroxide (H₂O₂), and NO. Iron is involved in both the formation and the scavenging of these species. Iron deficiency (anemia) (ID(A)) is associated with oxidative stress, but its role in the induction of nitrosative stress is largely unclear. Moreover, oral as well as intravenous (iv) iron preparations used for the treatment of ID(A) may also induce oxidative and/or nitrosative stress. Oral administration of ferrous salts may lead to high transferrin saturation levels and, thus, formation of non-transferrin-bound iron, a potentially toxic form of iron with a propensity to induce oxidative stress. One of the factors that determine the likelihood of oxidative and nitrosative stress induced upon administration of an iv iron complex is the amount of labile (or weakly-bound) iron present in the complex. Stable dextran-based iron complexes used for iv therapy, although they contain only negligible amounts of labile iron, can induce oxidative and/or nitrosative stress through so far unknown mechanisms. In this review, after summarizing the main features of iron metabolism and its complex interplay with O₂⁻, H₂O₂, NO, and other more reactive compounds derived from these species, the potential of various iron therapies to induce oxidative and nitrosative stress is discussed and possible underlying mechanisms are proposed. Understanding the mechanisms, by which various iron formulations may induce oxidative and nitrosative stress, will help us develop better tolerated and more efficient therapies for various dysfunctions of iron metabolism.     

GPx8 peroxidase prevents leakage of H2O2 from the endoplasmic reticulum

Unbalanced endoplasmic reticulum (ER) homeostasis (ER stress) leads to increased generation of reactive oxygen species (ROS). Disulfide-bond formation in the ER by Ero1 family oxidases produces hydrogen peroxide (H₂O₂) and thereby constitutes one potential source of ER-stress-induced ROS. However, we demonstrate that Ero1α-derived H₂O₂ is rapidly cleared by glutathione peroxidase (GPx) 8. In 293 cells, GPx8 and reduced/activated forms of Ero1α co-reside in the rough ER subdomain. Loss of GPx8 causes ER stress, leakage of Ero1α-derived H₂O₂ to the cytosol, and cell death. In contrast, peroxiredoxin (Prx) IV, another H₂O₂-detoxifying rough ER enzyme, does not protect from Ero1α-mediated toxicity, as is currently proposed. Only when Ero1α-catalyzed H₂O₂ production is artificially maximized can PrxIV participate in its reduction. We conclude that the peroxidase activity of the described Ero1α–GPx8 complex prevents diffusion of Ero1α-derived H₂O₂ within and out of the rough ER. Along with the induction of GPX8 in ER-stressed cells, these findings question a ubiquitous role of Ero1α as a producer of cytoplasmic ROS under ER stress.     

Overexpression of SOD in retina: need for increase in H2O2-detoxifying enzyme in same cellular compartment

In retinitis pigmentosa (RP), various mutations cause rod photoreceptor cell death leading to increased oxygen levels in the outer retina, progressive oxidative damage to cones, and gradual loss of cone cell function. We have been exploring the potential of overexpressing components of the endogenous antioxidant defense system to preserve cone cell function in rd10^+/+ mice, a model of RP. rd10^+/+ mice deficient in superoxide dismutase 1 (SOD1) showed increased levels of superoxide radicals and carbonyl adducts (a marker of oxidative damage) in the retina and more rapid loss of cone function than rd10^+/+ mice with normal levels of SOD1. This suggests that SOD1 is an important component of the antioxidant defense system of cones, but increased expression of SOD1 in rd10^+/+ mice increased oxidative damage and accelerated the loss of cone function. Coexpression of SOD1 with glutathione peroxidase 4 (Gpx4), which like SOD1 is localized in the cytoplasm, but not with catalase targeted to the mitochondria, reduced oxidative damage in the retina and significantly slowed the loss of cone cell function in rd10^+/+ mice. Gene transfer resulting in increased expression of SOD2, but not coexpression of SOD2 and mitochondrial Gpx4, resulted in high levels of H₂O₂ in the retina. These data suggest that to provide benefit in RP, overexpression of an SOD must be combined with expression of a peroxide-detoxifying enzyme in the same cellular compartment.     

Relationship between chloroplastic H2O2 and the salicylic acid response

Reactive oxygen species (ROS) act as signaling molecules for regulating plant responses to abiotic and biotic stress and there exist source- and kind-specific pathways for ROS signaling. Recently, we created a novel system for producing H₂O₂ in Arabidopsis chloroplasts by chemical-dependent thylakoid membrane-bound ascorbate peroxidase (tAPX) silencing using an estrogen-inducible RNAi method. Microarray analysis revealed that the expression of a large set of genes was altered in response to tAPX silencing, some of which are known to be involved in pathogen response/resistance. Furthermore, we found that tAPX silencing enhances the levels of salicylic acid (SA) and the response to SA, a central regulator for biotic stress response. In this addendum, we describe the relationship between chloroplastic H₂O₂ and SA in stress response, and discuss the function of the kind- and source-specific ROS signaling in SA-mediated stress response.     

Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all three treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.     

Variation of antioxidants and secondary metabolites in nitrogen-deficient barley plants

Barley (Hordeum vulgare cv. Bojos) plants cultured in low nitrogen (N) containing Hoagland solution (20 mg/l) were exposed to N deficiency (−N) over 15 days. Plants revealed relatively high tolerance to total N deficit because shoot length was not altered and dry biomass was depleted by ca. 30% while root length increased by ca. 50% and dry biomass remained unaffected. Soluble proteins and free amino acids decreased more pronouncedly in the roots. Antioxidants (glutathione and ascorbic acid) decreased in the shoots but increased or were not affected in the roots. Ascorbate peroxidase and glutathione reductase activities were depleted in shoots and/or roots while guaiacol peroxidase activity was stimulated in the shoots. In accordance, fluorescence signal of reactive oxygen species (ROS) and nitric oxide was elevated in shoots but no extensive changes were observed in roots if +N and −N treatments are compared. At the level of phenolic metabolites, slight increase in soluble phenols and some phenolic acids and strong elevation of flavonoid homoorientin was found in the shoots but not in the roots. Fluorescence microscopy in terms of detection of phenols is also discussed. We also briefly discussed accuracy of quantification of some parameters owing to discrepancies in the literature. It is concluded that N deficiency induces increase in shoot phenolics but also elevates symptoms of oxidative stress while increase in root antioxidants probably contributes to ROS homeostasis aimed to maintain root development.     

Key roles of the Escherichia coli AhpC C-terminus in assembly and catalysis of alkylhydroperoxide reductase,an enzyme essential for the alleviation of oxidative stress

2-Cys peroxiredoxins (Prxs) are a large family of peroxidases, responsible for antioxidant function and regulation in cell signaling, apoptosis and differentiation. The Escherichia coli alkylhydroperoxide reductase (AhpR) is a prototype of the Prxs-family, and is composed of an NADH-dependent AhpF reductase (57 kDa) and AhpC (21 kDa), catalyzing the reduction of H₂O₂. We show that the E. coli AhpC (EcAhpC, 187 residues) forms a decameric ring structure under reduced and close to physiological conditions, composed of five catalytic dimers. Single particle analysis of cryo-electron micrographs of C-terminal truncated (EcAhpC_1 -172 and EcAhpC_1 -182) and mutated forms of EcAhpC reveals the loss of decamer formation, indicating the importance of the very C-terminus of AhpC in dimer to decamer transition. The crystallographic structures of the truncated EcAhpC_1 -172 and EcAhpC_1 -182 demonstrate for the first time that, in contrast to the reduced form, the very C-terminus of the oxidized EcAhpC is oriented away from the AhpC dimer interface and away from the catalytic redox-center, reflecting structural rearrangements during redox-modulation and -oligomerization. Furthermore, using an ensemble of different truncated and mutated EcAhpC protein constructs the importance of the very C-terminus in AhpC activity and in AhpC–AhpF assembly has been demonstrated.     

Effect of Short-Term Caloric Restriction on H2O2 Production and Oxidative DNA Damage in Rat Liver Mitochondria and Location of the Free Radical Source

Oxygen free radicals (ROS) of mitochondrial origin seem to be involved in aging. Whereas in other tissues complexes I or III of the respiratory chain contain the ROS generators, in this study we find that rat liver mitochondria generate oxygen radicals at complexes I, II, and III. Short-term (6 weeks) caloric restriction significantly decreased H₂O₂ production in rat liver mitochondria. This decrease in ROS production was located at complex I because it occurred with complex I-linked substrates (pyruvate/malate), but did not reach statistical significance with the complex II-linked substrate succinate. The mechanism responsible for the lowered ROS production was not a decrease in oxygen consumption. Instead, the mitochondria of caloric-restricted animals released less ROS per unit electron flow. This was due to a decrease in the degree of reduction of the complex I generator. Furthermore, oxidative damage to mitochondrial and nuclear DNA was also decreased in the liver by short-term caloric restriction. The results agree with the idea that caloric restriction delays aging, at least in part, by decreasing the rate of mitochondrial ROS generation and thus the rate of attack to molecules, like DNA, highly relevant for the accumulation of age-dependent changes.     

Mitochondrial free radical overproduction due to respiratory chain impairment in the brain of a mouse model of Rett syndrome: protective effect of CNF1

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and highlight CNF1 efficacy in counteracting RTT-related mitochondrial defects.     

Structural studies of an eukaryotic cambialistic superoxide dismutase purified from the mature seeds of camphor tree

Inflammation is one of the leading causes of the many pathological states associated with oxidative stress. A crucial role in the development of inflammation-induced oxidative stress is played by reactive oxidant species (ROS), which are very difficult to detect in vivo. One of the most sensitive and definitive methods in the detection of ROS is electron spin resonance, especially as used in conjunction with spin trapping. Unfortunately, the commonly used nitrone spin traps have a very low efficacy for trapping superoxide radicals, and their radical adducts are not stable. To address this deficiency, we have developed negatively charged cyclic hydroxylamines such as 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidine (PP-H) for the detection of reactive oxidant species as a diagnostic tool for extracellular inflammation-induced oxidative stress. We used inflammation induced by a bacterial endotoxin lipopolysaccharide (LPS) as a model. ROS formation was tested in cultured macrophages, in blood and in vivo. PP-H reacts with reactive oxidant species generating the stable nitroxide radical 4-phosphonooxy-TEMPO. It was shown that a 5-h treatment of macrophages with LPS (1 microg/ml) leads to a threefold increase in superoxide formation as demonstrated using superoxide dismutase. Formation of reactive oxidant species 5 h after LPS (1 mg/kg) treatment of Fischer rats was analyzed in arterial blood; formation of reactive oxidant species in LPS-treated animals increased by a factor of 2.2 and was dependent upon the LPS dose. Diphenyleneiodonium (0.1 mM) inhibited formation of LPS-stimulated reactive oxidant species by 80%. We suggest that this test could be used as a noninvasive diagnostic tool for inflammation-induced oxidative stress.     

Redox activation of DUSP4 by N-acetylcysteine protects endothelial cells from Cd2+-induced apoptosis

Redox imbalance is a primary cause of endothelial dysfunction (ED). Under oxidant stress, many critical proteins regulating endothelial function undergo oxidative modifications that lead to ED. Cellular levels of glutathione (GSH), the primary reducing source in cells, can significantly regulate cell function via reversible protein thiol modification. N-acetylcysteine (NAC), a precursor for GSH biosynthesis, is beneficial for many vascular diseases; however, the detailed mechanism of these benefits is still not clear. From HPLC analysis, NAC significantly increases both cellular GSH and tetrahydrobiopterin levels. Immunoblotting of endothelial NO synthase (eNOS) and DUSP4, a dual-specificity phosphatase with a cysteine as its active residue, revealed that both enzymes are upregulated by NAC. EPR spin trapping further demonstrated that NAC enhances NO generation from cells. Long-term exposure to Cd²⁺ contributes to DUSP4 degradation and the uncontrolled activation of p38 and ERK1/2, leading to apoptosis. Treatment with NAC prevents DUSP4 degradation and protects cells against Cd²⁺-induced apoptosis. Moreover, the increased DUSP4 expression can redox-regulate the p38 and ERK1/2 pathways from hyperactivation, providing a survival mechanism against the toxicity of Cd²⁺. DUSP4 gene knockdown further supports the hypothesis that DUSP4 is an antioxidant gene, critical in the modulation of eNOS expression, and thus protects against Cd²⁺-induced stress. Depletion of intracellular GSH by buthionine sulfoximine makes cells more susceptible to Cd²⁺-induced apoptosis. Pretreatment with NAC prevents p38 overactivation and thus protects the endothelium from this oxidative stress. Therefore, the identification of DUSP4 activation by NAC provides a novel target for future drug design.     

Oxidative stress and androgen receptor signaling in the development and progression of castration-resistant prostate cancer

Aberrant androgen receptor (AR) signaling plays a critical role in androgen-dependent prostate cancer (PCa), as well as in castration-resistant PCa (CRPC). Oxidative stress seems to contribute to the tumorigenesis and progression of PCa, as well as the development of CRPC, via activation of AR signaling. This notion is supported by the fact that there is an aberrant or improper regulation of the redox status in these disorders. Additionally, androgen-deprivation-induced oxidative stress seems to be involved in the pathogenesis of several disorders caused by androgen-deprivation therapy (ADT), including osteoporosis, neurodegenerative disease, and cardiovascular disease. Oxidative stress can be suppressed with antioxidants or via a reduction in reactive oxygen species production. Thus, developing new therapeutic agents that reduce oxidative stress might be useful in preventing the conversion of androgen-dependent PCa into CRPC, as well as reducing the adverse effects associated with ADT. The objective of this review is to provide an overview regarding the relationship between oxidative stress and AR signaling in the context of PCa and especially CRPC. Additionally, we discuss the potential use of antioxidant therapies in the treatment of PCa.     

The role of reactive oxygen species and oxidative stress in carbon monoxide toxicity: An in-depth analysis

Abstract

The underlying mechanism of the central nervous system (CNS) injury after acute carbon monoxide (CO) poisoning is interlaced with multiple factors including apoptosis, abnormal inflammatory responses, hypoxia, and ischemia/reperfusion-like problems. One of the current hypotheses with regard to the molecular mechanism of CO poisoning is the oxidative injury induced by reactive oxygen species, free radicals, and neuronal nitric oxide. Up to now, the relevant mechanism of this injury remains poorly understood. The weakening of antioxidant systems and the increase of lipid peroxidation in the CNS have been implicated, however. Accordingly, in this review, we will highlight the relationship between oxidative stress and CO poisoning from the perspective of forensic toxicology and molecular toxicology.     

Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

Intranasal administration of a combination of choline chloride,vitamin C,and selenium attenuates the allergic effect in a mouse model of airway disease

Respiratory allergic disease is an inflammatory condition accompanied by oxidative stress. Supplementation of an anti-inflammatory agent with antioxidants may have a therapeutic effect. In this study, the effects of choline chloride in combination with antioxidants were evaluated via the intranasal route in a mouse model of allergic airway disease. Balb/c mice were sensitized on days 0, 7, and 14 and challenged on days 25–30 with cockroach extract (CE) and with a booster challenge on day 38. They were treated with choline chloride (ChCl; 1 mg/kg), vitamin C (Vit C; 308.33 mg/kg), and selenium (Se; 1 mg/kg) alone or in combination via the intranasal route on days 31, 33, 35, 37, and 39. The mice were sacrificed on day 40 to collect blood, bronchoalveolar lavage fluid, lungs, and spleen. Mice immunized with CE showed a significant increase in airway hyperresponsiveness (AHR), lung inflammation, Th2 cytokines, and the oxidative stress markers intracellular reactive oxygen species and 8-isoprostanes compared to the phosphate-buffered saline control group. A significant decrease was observed in these parameters with all the treatments (p<0.01). The highest decrease was noticed in the ChCl+Vit C+Se-treated group, with AHR decreased to the normal level. This group also showed the highest decrease in airway inflammation (p<0.001), IL-4 and IL-5 (p<0.001), IgE and IgG1 (p<0.001), NF-κB (p<0.001), and 8-isoprostane levels (p<0.001). Glutathione peroxidase activity, which was decreased significantly in CE-immunized mice, was restored to normal levels in this group (p<0.001). IL-10 level was decreased in CE-immunized mice and was restored to normal by combination treatment. The combination treatment induced FOXP3⁺ cells in splenocyte culture, responsible for the upregulation of IL-10. In conclusion, the combination of choline chloride, vitamin C, and selenium via the intranasal route reduces AHR, inflammation, and oxidative stress, probably by causing IL-10 production by FOXP3⁺ cells, and possesses therapeutic potential against allergic airway disease.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Are antioxidants useful for treating skeletal muscle atrophy?

Changes in the skeletal muscle protein mass frequently occur in both physiological and pathological states. Muscle hypotrophy, in particular, is commonly observed during aging and is characteristic of several pathological conditions such as neurological diseases, cancer, diabetes, and sepsis. The skeletal muscle protein content depends on the relative rates of synthesis and degradation, which must be coordinately regulated to maintain the equilibrium. Pathological muscle depletion is characterized by a negative nitrogen balance, which results from disruption of this equilibrium due to reduced synthesis, increased breakdown, or both. The current view, mainly based on experimental data, considers hypercatabolism as the major cause of muscle protein depletion. Several signaling pathways that probably contribute to muscle atrophy have been identified, and there is increasing evidence that oxidative stress, due to reactive oxygen species production overwhelming the intracellular antioxidant systems, plays a role in causing muscle depletion both during aging and in chronic pathological states. In particular, oxidative stress has been proposed to enhance protein breakdown, directly or by interacting with other factors. This review focuses on the possibility of using antioxidant treatments to target molecular pathways involved in the pathogenesis of skeletal muscle wasting.     

Enhancing effect of melatonin on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper

The oxidation of melatonin (MEL) using the Cu(II) + H₂O₂ + HO⁻ (the Fenton-like reaction) system was investigated by chemiluminescence (CL), fluorescence, spectrophotometric, and EPR spin trapping techniques. The reaction exhibits CL in the 400–730 nm region. The light emission from the Fenton-like reaction was greatly enhanced in the presence of MEL and was strongly dependent on its concentration. The spectrum measured with cut-off filters revealed maxima at around 460, 500, 580–590, 640–650, and 690–700 nm. The band at 460 nm may be due to the excited cleavage product, N¹-acetyl-N²-formyl-5-methoxykynuramine, whereas the bands at 500, 580–590, 640–650, and 700 nm were similar to those observed for singlet molecular oxygen (¹O₂). The effect of reactive oxygen species (ROS) scavengers on the light emission was studied. The CL was strongly inhibited by the ¹O₂ scavengers in a dose-dependent manner; at concentration 1 mM the potency of ¹O₂ scavenging was 5,5-dimethylcyclohexandione-1,3 > methionine > histidine > hydroquinone. The potency of HO^• scavenging by thiourea, tryptophan, cysteine at concentration 5 mM was 79–94%, by 1 mM glutathione and trolox 75 and 94%, respectively, and by 10 mM cimetidine 18%. Specific acceptors of O₂^•− such as p-nitroblue tetrazolium chloride and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) at concentration 5 mM decreased the CL by 51 and 95%, respectively, whereas superoxide dismutase (SOD) does not reduce the emission at concentration 2.8 U/ml. At higher concentration SOD substantially enhanced the light emission. Addition of 1360 U/ml catalase and 100 μM desferrioxamine strongly inhibited CL (96 and 90%, respectively). The increased generation of ¹O₂ from the Cu/H₂O₂ system in the presence of MEL was confirmed using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline and by trapping experiments with 2,2,6,6-tetramethylpiperidine (TEMP) and subsequent electron paramagnetic (EPR) spectroscopy. These findings suggest the increased production of reactive oxygen species (O₂^•−, HO^•, ¹O₂) from the Fenton-like reaction in the presence of MEL. This means that the hormone is not able to act as classical chain-breaking antioxidant even at low concentration, and may show clear prooxidant activity at higher concentrations. In addition, long-lived carbonyl product of the MEL transformation in the triplet state can also be toxic by transferring its energy to organelles and causing a photochemical process.