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The protozoan parasite Leishmania spp. causes leishmaniases, a group of diseases creating serious health problems in many parts of the world with significant resistance to existing drugs. Insect derived antimicrobial peptides are promising alternatives to conventional drugs against several human disease-causing pathogens because they do not generate resistance. Halictine-2, a novel antimicrobial peptide from the venom of eusocial honeybee, Halictus sexcinctus showed significant anti-leishmanial activity in vitro, towards two life forms of the dimorphic parasite, the free-swimming infective metacyclic promastigotes and the intracellular amastigotes responsible for the systemic infection. The anti-leishmanial activity of the native peptide (P 5S ) was significantly enhanced by serine to threonine substitution at position 5 (P 5T ). The peptide showed a propensity to form α-helices after substitution at position-5, conferring amphipathicity. Distinct pores observed on the promastigote membrane after P 5T exposure suggested a mechanism of disruption of cellular integrity. Biochemical alterations in the promastigotes after P 5T exposure included generation of increased oxygen radicals with mitochondrial Ca 2+ release, loss of mitochondrial membrane potential, reduction in total ATP content and increased mitochondrial mass, resulting in quick bioenergetic and chemiosmotic collapse leading to cell death characterized by DNA fragmentation. P 5T was able to reduce intracellular amastigote burden in an in vitro model of Leishmania infection but did not alter the proinflammatory cytokines like TNF-α and IL-6. The ability of the P 5T peptide to kill the Leishmania parasite with negligible haemolytic activity towards mouse macrophages and human erythrocytes respectively, demonstrates its potential to be considered as a future antileishmanial drug candidate., (Copyright © 2020. Published by Elsevier Inc.)

The global tobacco epidemic is still a devastating threat to public health. Toxic reactive oxygen species (ROS) in the cigarette smoke cannot be efficiently eliminated by currently available cigarette filters. The resultant oxidative stress causes severe lung injury and further diseases. To tackle this challenge, herein, a novel copper tannic acid coordination (CuTA) nanozyme is reported as a highly active and thermostable ROS scavenger. The CuTA nanozyme exhibits intrinsic superoxide dismutase-like activity, catalase-like activity, and hydroxyl radical elimination capacity. These synergistic antioxidant abilities make the CuTA nanozyme a promising candidate for the improvement of commercial cigarette filters. Mouse model results show that commercial cigarettes loaded with CuTA nanozyme efficiently scavenge ROS in the cigarette smoke, reduce oxidative stress-induced lung inflammation, and minimize the resultant acute lung injury. The developed CuTA nanozyme offers an efficient ROS scavenger with multiple antioxidant ability and opens up new opportunities for the modification of cigarette filters to reduce the toxic effects of cigarette smoke., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rheumatoid arthritis (RA) has been associated with a higher risk of developing cardiovascular (CV) diseases. It has been proposed that systemic inflammation plays a key role in premature atherosclerosis development, and is therefore crucial to determine whether systemic components from RA patients promotes endothelial cell-oxidative stress by affecting reactive oxygen species (ROS) and nitric-oxide (NO) production. The aim of this study was to evaluate whether plasma from RA patients impair NO synthesis and ROS production by using the cell-line ECV-304 as a biosensor. NO synthesis and ROS production were measured in cells incubated with plasma from 73 RA patients and 52 healthy volunteers by fluorimetry. In addition, traditional CV risk factors, inflammatory molecules and disease activity parameters were measured. Cells incubated with plasma from RA patients exhibited reduced NO synthesis and increased ROS production compared to healthy volunteers. Furthermore, the imbalance between NO synthesis and ROS generation in RA patients was not associated with traditional CV risk factors. Our data suggest that ECV-304 cells can be used as a biosensor of systemic inflammation-induced endothelial cell-oxidative stress. We propose that both NO and ROS production are potential biomarkers aimed at improving the current assessment of CV risk in RA.

Metal nanozyme has attracted wide interest for biomedicine, and a highly catalytic material in the physiological environment is highly desired. However, catalytic selectivity of nanozyme is still highly challenging, limiting its wide application. Here, we show a trimetallic (triM) nanozyme with highly catalytic activity and environmental selectivity. Enzyme-mimicked investigations find that the triM system possesses multi-enzyme-mimetic activity for removing reactive oxygen species (ROS) and reactive nitrogen species (RNS), such as 1 O 2 , H 2 O 2 , • OH, and • NO. Importantly, triM nanozyme exhibits the significant neutral environment preference for removing the • OH, 1 O 2 , and • NO free radical, indicating its highly catalytic selectivity. The density functional theory (DFT) calculations reveal that triM nanozyme can capture electrons very easily and provides more attraction to reactive oxygen and nitrogen species (RONS) radicals in the neutral environment. In vitro experiments show that triM nanozyme can improve the viability of injured neural cell. In the LPS-induced brain injury model, the superoxide dismutase (SOD) activity and lipid peroxidation can be greatly recovered after triM nanozyme treatment. Moreover, the triM nanozyme treatment can significantly improve the survival rate, neuroinflammation, and reference memory of injured mice. Present work provides a feasible route for improving selectivity of nanozyme in the physiological environment as well as exploring potential applications in brain science.

Aging is a complex phenomenon that has detrimental effects on tissue homeostasis. The skeletal muscle is one of the earliest tissues to be affected and to manifest age-related changes such as functional impairment and the loss of mass. Common to these alterations and to most of tissues during aging is the disruption of the proteostasis network by detrimental changes in the ubiquitin-proteasomal system (UPS) and the autophagy-lysosomal system (ALS). In fact, during aging the accumulation of protein aggregates, a process mainly driven by increased levels of oxidative stress, has been observed, clearly demonstrating UPS and ALS dysregulation. Since the UPS and ALS are the two most important pathways for the removal of misfolded and aggregated proteins and also of damaged organelles, we provide here an overview on the current knowledge regarding the connection between the loss of proteostasis and skeletal muscle functional impairment and also how redox regulation can play a role during aging. Therefore, this review serves for a better understanding of skeletal muscle aging in regard to the loss of proteostasis and how redox regulation can impact its function and maintenance., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: This study was conducted in order to investigate the effects of reactive oxygen species (ROS) levels on the seminal plasma (SP) metabolite milieu and sperm dysfunction., Methods: Semen specimens of 151 normozoospermic men were analyzed for ROS by chemiluminescence and classified according to seminal ROS levels [in relative light units (RLU)/s/10 6 sperm]: group 1 (n = 39): low (ROS < 20), group 2 (n = 38): mild (20 ≤ ROS < 40), group 3 (n = 31): moderate (40 ≤ ROS < 60), and group 4 (n = 43): high (ROS ≥ 60). A comprehensive analysis of SP and semen parameters, including conventional semen characteristics, measurement of total antioxidant capacity (TAC), sperm DNA fragmentation index (DFI), chromatin maturation index (CMI), H19-Igf2 methylation status, and untargeted seminal metabolic profiling using nuclear magnetic resonance spectroscopy (1H-NMR), was carried out., Result(s): The methylation status of H19 and Igf2 was significantly different in specimens with high ROS (P < 0.005). Metabolic fingerprinting of these SP samples showed upregulation of trimethylamine N-oxide (P < 0.001) and downregulations of tryptophan (P < 0.05) and tyrosine/tyrosol (P < 0.01). High ROS significantly reduced total sperm motility (P < 0.05), sperm concentration (P < 0.001), and seminal TAC (P < 0.001) but increased CMI and DFI (P < 0.005). ROS levels have a positive correlation with Igf2 methylation (r = 0.19, P < 0.05), DFI (r = 0.40, P < 0.001), CMI (r = 0.39, P < 0.001), and trimethylamine N-oxide (r = 0.45, P < 0.05) and a negative correlation with H19 methylation (r = - 0.20, P < 0.05), tryptophan (r = - 0.45, P < 0.05), sperm motility (r = - 0.20, P < 0.05), sperm viability (r = - 0.23, P < 0.01), and sperm concentration (r = - 0.30, P < 0.001)., Conclusion(s): Results showed significant correlation between ROS levels and H19-Igf2 gene methylation as well as semen parameters. These findings are critical to identify idiopathic male infertility and its management through assisted reproduction technology (ART).

Most of the key radionuclides in the nuclear fuel cycle, such as actinides, possess a combination of heavy metal chemotoxicity and radiotoxicity and therefore represent a severe threat to the ecological environment and public safety. The radiotoxicity originates from direct radiation-induced organ damage and indirect damage, mostly through radiation-induced reactive oxygen species (ROS). Although effective chelating agents that can accelerate the excretion of actinides, such as uranium, have been developed in the past several decades, very few of them can reduce radiation-induced damage from internal contamination. In fact, the strategy of simultaneous removal of actinides and their induced-ROS in vivo has scarcely been considered. Here, we report a 3,2-hydroxypyridinone-grafted chitosan oligosaccharide nanoparticle (COS-HOPO) as a new type of decorporation agent that is effective for the removal of both uranium and ROS in vivo. The cytotoxicity and decorporation assays indicate that the marriage of chitosan oligosaccharide (COS) and hydroxypyridinone (HOPO) gives rise to a remarkable decrease in toxicity and promotion of the uranium removal capability from both kidneys and femurs. The decorporation efficacy can reach up to 43% in rat proximal tubular epithelial cells (NRK-52E), 44% in kidneys, and 32% in femurs. Moreover, the ROS levels of the cells treated with COS-HOPO are significantly lower than those of the control group, implying a promising radiation protection effect. The detoxification mechanism of COS-HOPO is closely related to both chelating U(VI)- and scavenging U(VI)-induced intracellular ROS.

Uric acid is the final product of purine metabolism in humans and is considered to be quantitatively the main antioxidant in plasma. In vitro studies showed that the oxidation of uric acid by peroxidases, in presence of superoxide, generates urate free radical and urate hydroperoxide. Urate hydroperoxide is a strong oxidant and might be a relevant intermediate in inflammatory conditions. However, the identification of urate hydroperoxide in cells and biological samples has been a challenge due to its high reactivity. By using mass spectrometry, we undoubtedly demonstrated the formation of urate hydroperoxide and its corresponding alcohol, hydroxyisourate during the respiratory burst in peripheral blood neutrophils and in human leukemic cells differentiated in neutrophils (dHL-60). The respiratory burst was induced by phorbol myristate acetate (PMA) and greatly increased oxygen consumption and superoxide production. Both oxygen consumption and superoxide production were further augmented by incubation with uric acid. Conversely, uric acid significantly decreased the levels of HOCl, probably because of the competition with chloride by the catalysis of myeloperoxidase. In spite of the decrease in HOCl, the overall oxidative status, measured by GSH/GSSG ratio, was augmented in the presence of uric acid. In summary, the present results support the formation of urate hydroperoxide, a novel oxidant in neutrophils oxidative burst. Urate hydroperoxide is a strong oxidant and alters the redox balance toward a pro-oxidative environment. The production of urate hydroperoxide in inflammatory conditions could explain, at least in part, the harmful effect associated to uric acid., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are by-products of aerobic metabolism, and excessive production can result in oxidative stress and cell damage. In addition, ROS function as cellular messengers, working as redox regulators in a multitude of biological processes. Understanding ROS signalling and stress responses requires methods for precise imaging and quantification to monitor local, subcellular and global ROS dynamics with high selectivity, sensitivity and spatiotemporal resolution. In this review, we summarize the present knowledge for in vivo plant ROS imaging and detection, using both chemical probes and fluorescent protein-based biosensors. Certain characteristics of plant tissues, for example high background autofluorescence in photosynthetic organs and the multitude of endogenous antioxidants, can interfere with ROS and redox potential detection, making imaging extra challenging. Novel methods and techniques to measure in vivo plant ROS and redox changes with better selectivity, accuracy, and spatiotemporal resolution are therefore desirable to fully acknowledge the remarkably complex plant ROS signalling networks., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cerebral toxoplasmosis is characterized by activation of brain resident cells and recruitment of specific immune cell subsets from the periphery to the central nervous system (CNS). Our studies revealed that the rapidly invaded Ly6G + neutrophil granulocytes are an early non-lymphoid source of interferon-gamma (IFN-γ), the cytokine known to be the major mediator of host resistance to Toxoplasma gondii ( T. gondii ). Upon selective depletion of Ly6G + neutrophils, we detected reduced IFN-γ production and increased parasite burden in the CNS. Ablation of Ly6G + cells resulted in diminished recruitment of Ly6C hi monocytes into the CNS, indicating a pronounced interplay. Additionally, we identified infiltrated Ly6G + neutrophils to be a heterogeneous population. The Ly6G + CD62-L hi CXCR4 + subset released cathelicidin-related antimicrobial peptide (CRAMP), which can promote monocyte dynamics. On the other hand, the Ly6G + CD62-L lo CXCR4 + subset produced IFN-γ to establish early inflammatory response. Collectively, our findings revealed that the recruited Ly6G + CXCR4 + neutrophil granulocytes display a heterogeneity in the CNS with a repertoire of effector functions crucial in parasite control and immune regulation upon experimental cerebral toxoplasmosis.

Superoxide dismutases (SOD) are a group of enzymes that catalyze the dismutation of superoxide (O 2 - ) radicals into molecular oxygen (O 2 ) and H 2 O 2 as a first line of defense against oxidative stress. Here, we show that glycine-functionalized copper(ii) hydroxide nanoparticles (Gly-Cu(OH) 2 NPs) are functional SOD mimics, whereas bulk Cu(OH) 2 is insoluble in water and catalytically inactive. In contrast, Gly-Cu(OH) 2 NPs form water-dispersible mesocrystals with a SOD-like activity that is larger than that of their natural CuZn enzyme counterpart. Based on this finding, we devised an application where Gly-Cu(OH) 2 NPs were incorporated into cigarette filters. Cigarette smoke contains high concentrations of toxic reactive oxygen species (ROS, >10 16 molecules per puff) including superoxide and reactive nitrogen species which lead to the development of chronic and degenerative diseases via oxidative damage and subsequent cell death. Embedded in cigarette filters Gly-Cu(OH) 2 NPs efficiently removed ROS from smoke, thereby protecting lung cancer cell lines from cytotoxic effects. Their stability, ease of production and versatility make them a powerful tool for a wide range of applications in environmental chemistry, biotechnology and medicine.

There is a growing interest in the physiological roles of reactive oxygen species (ROS) as essential components of molecular mechanisms regulating key cellular processes, including proliferation, differentiation and apoptosis. This interest has fostered the development of new molecular tools to localize and quantify ROS production in cultured cells and in whole living organisms. An equally important but often neglected aspect in the study of ROS biology is the development of accurate procedures to introduce a ROS source in the biological system under study. At present, this experimental requirement is solved in most cases by an external and systemic administration of ROS, usually hydrogen peroxide. We have previously shown that a photodynamic treatment based on the endogenous photosensitizer protoporphyrin IX and further irradiation of the target with adequate light source can be used to transiently switch on an in situ ROS production in human cultured keratinocytes and in mouse skin in vivo. Using this approach we reported that qualitatively low levels of ROS can activate cell proliferation in cultured cells and promote a transient and reversible hyperproliferative response in the skin, particularly, in the hair follicle stem cell niche, promoting physiological responses like acceleration of hair growth and supporting the notion that a local and transient ROS production can regulate stem cell function and tissue homeostasis in a whole organism. Our principal aim here is to provide a detailed description of this experimental methodology as a useful tool to investigate physiological roles for ROS in vivo in different experimental systems., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) are by-products of photosynthesis and respiration in plant tissues. Abiotic and biotic stressors also induce the production and temporary accumulation of ROS in plants, including hydrogen peroxide (H 2 O 2 ), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H 2 O 2 acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H 2 O 2 levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H 2 O 2 estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H 2 O 2 in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg -1 FW range. Commonly used additives for H 2 O 2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H 2 O 2 directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Over the past ten years, alternative methods for the rapid screening of PSs have been developed. In the present work, a study was undertaken to correlate the phototoxicity of plant extracts on either prokaryotic or eukaryotic cells, with the total oxidation status (TOS) as well as with their ability to produce 1 O 2 . Results demonstrated that the extracts containing PSs that were active either on eukaryotic cells or bacteria increased their TOS after illumination, and that there was a certain degree of positive correlation between the extract phototoxic efficacy and TOS levels. The production of 1 O 2 by the illuminated extracts was indirectly measured by the use of the fluorescence of "singlet oxygen sensor green", which is a method that has proved highly sensitive for such measurement. 1 O 2 was detectable only upon illumination of the most active extracts. In addition, the oxidation of tryptophan and was employed as a method capable of measuring ROS generated by both type I and II ROS reactions. However, it turned out to be not sensitive enough to detect the species generated by plant extracts. Results demonstrated that the TOS method, initially developed to measure the oxidant status in plasma, can be readily applied to plant extracts. Unlike the method used to detect 1 O 2 , the method employed for the detection of TOS proved to be accurate, since all the extracts that displayed a high phototoxic activity on either prokaryotic or eukaryotic cells, presented high TOS levels after illumination., (Copyright © 2016 Elsevier Inc. All rights reserved.)

At low levels, reactive oxygen species (ROS) can act as signaling molecules within cells. When ROS production greatly exceeds the capacity of endogenous antioxidant systems, or antioxidant levels are reduced, ROS levels increase further. The latter is associated with induction of oxidative stress and associated signal transduction and characterized by ROS-induced changes in cellular redox homeostasis and/or damaging effects on biomolecules (e.g. DNA, proteins and lipids). Given the complex mechanisms involved in ROS production and removal, in combination with the lack of reporter molecules that are truly specific for a particular type of ROS, quantification of (sub)cellular ROS levels is a challenging task. In this chapter we describe two strategies to measure ROS: one approach to assess general oxidant levels using the chemical reporter CM-H 2 DCFDA (5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate), and a second approach allowing more specific analysis of cytosolic hydrogen peroxide (H 2 O 2 ) levels using protein-based sensors (HyPer and SypHer)., (Copyright © 2016. Published by Elsevier Inc.)

The critical involvement of reactive oxygen species (ROS) in both physiological and pathological processes in cell biology makes their detection and assessment a fundamental topic in biomedical research. Established methodologies to study ROS in cell biology take advantage of oxidation reactions between the ROS and a reduced probe. After reacting the probe reveals the presence of ROS either by the appearance of colour (chromogenic reaction) or fluorescence (fluorogenic reaction). However current methodologies rarely allow for a site-specific detection of ROS production. Here we propose a colorimetric reaction driven by the oxidation of 3,3'-diaminobenzidine (DAB) by photodynamically-produced ROS that allows for fine detection of the ROS production site. The introduced methodology is fast, easy to implement and permits cellular resolution at the submicrometric level. Although the basic protocol is proved in a photodynamic model of ROS generation, the principle is applicable to many different scenarios of intracellular ROS production. As a consequence this proposed methodology should greatly complement other techniques aiming at establishing a precise subcellular localization of ROS generation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The paper presents an image processing method for the quantitative assessment of ROS accumulation areas in leaves stained with DAB or NBT for H 2 O 2 and O 2 - detection, respectively. Three types of images determined by the combination of staining method and background color are considered. The method is based on the principle of supervised machine learning with manually labeled image patterns used for training. The method's algorithm is developed as a JavaScript macro in the public domain Fiji (ImageJ) environment. It allows to select the stained regions of ROS-mediated histochemical reactions, subsequently fractionated according to the weak, medium and intense staining intensity and thus ROS accumulation. It also evaluates total leaf blade area. The precision of ROS accumulation area detection is validated by the Dice Similarity Coefficient in the case of manual patterns. The proposed framework reduces the computation complexity, once prepared, requires less image processing expertise than the competitive methods and represents a routine quantitative imaging assay for a general histochemical image classification., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP targeted to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The photodynamic mechanism sensitized by N,N-dimethyl-2-[4-(3-N,N,N-trimethylammoniopropoxy)phenyl]fulleropyrrolidinium (DPC 60 2+ ) was investigated in Staphylococcus aureus cells. Different experimental conditions were used to detect reactive oxygen species (ROS) in S. aureus cell suspensions. First, a photoinactivation of 4 log decrease of S. aureus viability was chosen using 0.5μM DPC 60 2+ and 15min irradiation. An anoxic atmosphere indicated that oxygen was required for an effective photoinactivation. Also, photoprotection was found in the presence of sodium azide, whereas the photocytotoxicity induced by DPC 60 2+ increased in D 2 O. The addition of diazabicyclo[2.2.2]octane or d-mannitol produced a reduction in the S. aureus photokilling. Moreover, singlet molecular oxygen, O 2 ( 1 Δ g ), was detected by the reaction with 9,10-dimethylanthracene into the S. aureus cells. A decrease in the photoinactivation of S. aureus was observed in the presence of β-nicotinamide adenine dinucleotide reduced form, which was dependent on the NADH concentration. Therefore, under aerobic condition the photocytotoxicity activity induced by DPC 60 2+ was mediated by mainly a contribution of type II process. Moreover, photoinactivation of S. aureus was possible with DPC 60 2+ in the presence of azide anions under anoxic condition. However, these conditions were not effective to photoinactivate Escherichia coli. On the other hand, the addition of potassium iodide produced an increase in the photokilling of bacteria, depending on the KI concentration and irradiation times. The formation of reactive iodine species may be contributing to inactivate S. aureus cells photoinduced by DPC 60 2+ ., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Singlet molecular oxygen, O 2 (a 1 Δ g ), is a Reactive Oxygen Species, ROS, that acts as a signaling and/or perturbing agent in mammalian cells, influencing processes that range from cell proliferation to cell death. Although the importance of O 2 (a 1 Δ g ) in this regard is acknowledged, an understanding of the targets and mechanisms of O 2 (a 1 Δ g ) action is inadequate. Thus, methods that better facilitate studies of O 2 (a 1 Δ g ) in mammalian cells are highly desired. This is particularly important because, as a consequence of its chemistry in a cell, O 2 (a 1 Δ g ) can spawn the generation of other ROS (e.g., the hydroxyl radical) that, in turn, can have a unique influence on cell behavior and function. Therefore, exerting better control and specificity in O 2 (a 1 Δ g ) experiments ultimately reduces the number of variables in general studies to unravel the details of ROS-dependent cell dynamics. In this article, we summarize our recent efforts to produce O 2 (a 1 Δ g ) with increased control and selectivity in microscope-based single-cell experiments. The topics addressed include (1) two-photon excitation of a photosensitizer using a focused laser to create a spatially-localized volume of O 2 (a 1 Δ g ) with sub-cellular dimensions, (2) protein-encapsulated photosensitizers that can be localized in a specific cellular domain using genetic engineering, and (3) direct excitation of dissolved oxygen in sensitizer-free experiments to selectively produce O 2 (a 1 Δ g ) at the expense of other ROS. We also comment on our recent efforts to monitor O 2 (a 1 Δ g ) in cells and to monitor the cell's response to O 2 (a 1 Δ g )., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Photodynamic therapy (PDT) is a clinical modality of photochemotherapy based on the accumulation of a photosensitizer in target cells and subsequent irradiation of the tissue with light of adequate wavelength promoting reactive oxygen species (ROS) formation and cell death. PDT is used in several medical specialties as an organ-specific therapy for different entities. In this review we focus on the current dermatological procedure of PDT. In the most widely used PDT protocol in dermatology, ROS production occurs by accumulation of the endogenous photosensitizer protoporphyrin IX after treatment with the metabolic precursors 5-methylaminolevulinic acid (MAL) or 5-aminolevulinic acid (ALA). To date, current approved dermatological indications of PDT include actinic keratoses (AK), basal cell carcinoma (BCC) and in situ squamous cell carcinoma (SCC) also known as Bowen disease (BD). With regards to AKs, PDT can also treat the cancerization field carrying an oncogenic risk. In addition, an increasing number of pathologies, such as other skin cancers, infectious, inflammatory or pilosebaceous diseases are being considered as potentially treatable entities with PDT. Besides the known therapeutic properties of PDT, there is a modality used for skin rejuvenation and aesthetic purposes defined as photodynamic photorejuvenation. This technique enables the remodelling of collagen, which in turn prevents and treats photoaging stygmata. Finally we explore a new potential treatment field for PDT determined by the activation of follicular bulge stem cells caused by in situ ROS formation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Reactive oxygen species (ROS) play a critical role in cell signaling and disease pathogenesis. Despite their biological importance, assessment of ROS often involves measurement of indirect byproducts or measurement of ROS from excised tissue. Herein, we describe a microdialysis technique that utilizes the Amplex Ultrared assay to directly measure hydrogen peroxide (H 2 O 2 ) and superoxide in tissue of living, anesthetized rats and mice. We demonstrate the application of this methodology in the penis, adipose tissue, skeletal muscle, kidney, and liver. We provide data demonstrating the impact of important methodological considerations such as membrane length, perfusion rate, and time-dependence upon probe insertion. In this report, we provide a complete list of equipment, troubleshooting tips, and suggestions for implementing this technique in a new system. The data herein demonstrate the feasibility of measuring both in vivo H 2 O 2 and superoxide in the extracellular environment of various rodent tissues, providing a technique with potential application to a vast array of disease states which are subject to oxidative stress., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Significance: Due to its large families of redox-active enzymes, genetic amenability, and complete transparency, the nematode Caenorhabditis elegans has the potential to become an important model for the in vivo study of redox biology., Recent Advances: The recent development of several genetically encoded ratiometric reactive oxygen species (ROS) and redox sensors has revolutionized the quantification and precise localization of ROS and redox signals in living organisms. Only few exploratory studies have applied these sensors in C. elegans and undoubtedly much remains to be discovered in this model. As a follow-up to our recent findings that the C. elegans somatic gonad uses superoxide and hydrogen peroxide (H2O2) signals to communicate with the germline, we here analyze the patterns of H2O2 inside the C. elegans germline., Critical Issues: Despite the advantages of genetically encoded ROS and redox sensors over classic chemical sensors, still several general as well as C. elegans-specific issues need to be addressed. The major concerns for the application of these sensors in C. elegans are (i) decreased vitality of some reporter strains, (ii) interference of autofluorescent compartments with the sensor signal, and (iii) the use of immobilization methods that do not influence the worm's redox physiology., Future Directions: We propose that several of the current issues may be solved by designing reporter strains carrying single copies of codon-optimized sensors. Preferably, these sensors should have their emission wavelengths in the red region, where autofluorescence is absent. Worm analysis could be optimized using four-dimensional ratiometric fluorescence microscopy of worms immobilized in microfluidic chips. Antioxid. Redox Signal. 25, 577-592.

Significance: Reactive oxygen species (ROS) fulfill numerous roles in biology ranging from signal transduction to the induction of cell death. To advance our understanding of these sometimes contradictory roles, quantitative, specific, and sensitive ROS measurements are required., Recent Advances: Several organic or genetically encoded probes were successfully developed for ROS detection., Critical Issues: In some cases, ROS production occurs in a harsh environment such as low pH or high concentration of proteases. However, the ROS sensor may be sensitive to such environmental conditions and therefore becomes inaccurate. While the sensitivity of many ROS sensors to pH is known, many other environmental conditions remain unexplored. This article illustrates the interference between ROS sensors and their environment using the phagosome as an example. In the phagosome, pH changes, high concentration of ROS, and the presence of many proteases generate a hostile and rapidly changing environment., Future Directions: Difficulties due to cell movement and continuous formation of new phagosomes can be reduced by ratio measurements, if appropriate dyes are identified. For detection in live cells and subcellular locations, fluorescent proteins (FPs) offer several advantages and are used to create biosensors for ROS. Some FPs are directly sensitive to certain ROS as shown here. Although this may compromise their use in an environment with high levels of ROS, it can also be exploited for ROS measurement directly with the FPs themselves. For all types of ROS detection, we suggest a set of basic guidelines for testing the environmental sensitivity of an ROS sensor. Antioxid. Redox Signal. 25, 564-576.

Free radicals, particularly reactive oxygen species (ROS), are involved in various pathologies, injuries related to radiation, ischemia-reperfusion or ageing. Unfortunately, it is virtually impossible to directly detect free radicals in vivo, but the redox status of the whole organism or particular organ can be studied in vivo by using magnetic resonance techniques (EPR and MRI) and paramagnetic stable free radicals - nitroxides. Here we review results obtained in vivo following the pharmacokinetics of nitroxides on experimental animals (and a few in humans) under various conditions. The focus was on conditions where the redox status has been altered by induced diseases or harmful agents, clearly demonstrating that various EPR/MRI/nitroxide combinations can reliably detect metabolically induced changes in the redox status of organs. These findings can improve our understanding of oxidative stress and provide a basis for studying the effectiveness of interventions aimed to modulate oxidative stress. Also, we anticipate that the in vivo EPR/MRI approach in studying the redox status can play a vital role in the clinical management of various pathologies in the years to come providing the development of adequate equipment and probes., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Herein, we report a highly sensitive electrocatalytic sensor-cell construct that can electrochemically communicate with the internal environment of immune cells (e.g., macrophages) via the selective monitoring of a particular reactive oxygen species (ROS), hydrogen peroxide. The sensor, which is based on vertically aligned single-walled carbon nanotubes functionalized with an osmium electrocatalyst, enabled the unprecedented detection of a local intracellular "pulse" of ROS on a short second time scale in response to bacterial endotoxin (lipopolysaccharide-LPS) stimulation. Our studies have shown that this initial pulse of ROS is dependent on NADPH oxidase (NOX) and toll like receptor 4 (TLR4). The results suggest that bacteria can induce a rapid intracellular pulse of ROS in macrophages that initiates the classical innate immune response of these cells to infection.

Monitoring of reactive oxygen species like superoxide anion (O2(∙-)) turns to be of increasing significance considering their potential damages to organism. In the present work, we fabricated a novel O2(∙-) electrochemical sensor through immobilizing superoxide dismutase (SOD) onto a Pt-Pd/MWCNTs hybrid modified electrode surface. The Pt-Pd/MWCNTs hybrid was synthesized via a facile one-step alcohol-reduction process, and well characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction. The immobilization of SOD was accomplished using a simple drop-casting method, and the performance of the assembled enzyme-based sensor for O2(∙-) detection was systematically investigated by several electrochemcial techniques. Thanks to the specific biocatalysis of SOD towards O2(∙-) and the Pt-Pd/MWCNTs - promoted fast electron transfer at the fabricated interface, the developed biosensor exhibits a fast, selective and linear amperometric response upon O2(∙-) in the concentration scope of 40-1550 μM (R(2)=0.9941), with a sensitivity of 0.601 mA cm(-2) mM(-1) and a detection limit of 0.71 μM (S/N=3). In addition, the favorable biocompatibility of this electrode interface endows the prepared biosensor with excellent long-term stability (a sensitivity loss of only 3% over a period of 30 days). It is promising that the proposed sensor will be utilized as an effective tool to quantitatively monitor the dynamic changes of O2(∙-) in biological systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract., Results: Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH.), superoxide anion (O2 .-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO-), singlet oxygen (1O2), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC50 of the extract showed significant difference only in singlet oxygen (1O2) and iron chelating activity when compared with the controls., Conclusions: The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.

Despite the growing use of carbon nanomaterials in commercial applications, very little is known about the fate of these nanomaterials once they are released into the environment. The carbon-carbon bonding of spherical sp(2) hybridized fullerene (C60) forms a strong and resilient material that resists biodegradation. Moreover, C60 is widely reported to be bactericidal. Here however, we observe the changing properties of fullerene nanoparticle aggregates aged in the presence of microbes. C60 aggregates were observed to decrease in size with aging, while hydroxylation and photosensitized reactivity measured by the production of reactive oxygen species (ROS) increased, suggesting that chemically and/or biologically-mediated activity is capable of partially transforming fullerene structure and reactivity in the environment. However, stable-isotope-labeling C60 aggregates incubated with microbial cultures from aged suspensions for 203 days did not produce significant labeled carbon dioxide, despite significant reduction in aggregate radius for biological samples. These results suggest that either the rate of biodegradation of these particles is too slow to quantify or that the biologically-enhanced transformation of these particles does not occur through microbial biodegradation to carbon dioxide., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Context: Most current knowledge of pancreatic islet pathophysiology in diabetes mellitus has come from animal models. Even though islets from humans are readily available, only a few come from diabetic donors. We had the uncommon opportunity to acquire islets from humans with type 2 diabetes and used it to perform a study not previously done with human or animal islets., Objectives: Oxidative stress has been proposed as a mechanism for impaired β-cell function in type 2 diabetes. Lipid peroxides caused by reactive oxygen species are damaging to body tissues. The objective was to determine whether lipid peroxide-protein adducts occur in pancreatic islets of humans with type 2 diabetes., Design: Immunoblots with two antibodies to hydroxynonenal and 2 other antibodies we generated against reactive small aliphatic compounds were used to detect lipid peroxide-protein adducts in islets of patients with type 2 diabetes and controls., Results: The antibodies reacted strongly to ≥5 islet proteins. The major hydroxynonenal adduct in the islets of type 2 diabetes patients was a 52-kDa protein seen with all 4 antibodies that was also seen in islets of nondiabetic humans, rat islets, and insulinoma cells and in mitochondria of various rat tissues. Nano-LC-MS/MS (liquid chromatography-tandem mass spectrometry) and MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) analysis identified the protein as the β-chain of the mitochondrial F-ATP synthase, an enzyme responsible for 95% of ATP formed in tissues., Conclusions: Lipid peroxide-protein adducts occur in β-cells in the nondiabetic state and in diabetes. Lipid peroxidation is thought to be damaging to tissues. Analogous to various other unhealthy characteristics, the presence in nondiabetic individuals of lipid peroxide-protein adducts does not necessarily indicate they are not detrimental.

Reactive oxygen and nitrogen species (ROS and RNS) produced by macrophages are essential for protecting a human body against bacteria and viruses. Micrometer-sized electrodes coated with Pt black have previously been used for selective and sensitive detection of ROS and RNS in biological systems. To determine ROS and RNS inside macrophages, one needs smaller (i.e., nanometer-sized) sensors. In this article, the methodologies have been extended to the fabrication and characterization of Pt/Pt black nanoelectrodes. Electrodes with the metal surface flush with glass insulator, most suitable for quantitative voltammetric experiments, were fabricated by electrodeposition of Pt black inside an etched nanocavity under the atomic force microscope control. Despite a nanometer-scale radius, the true surface area of Pt electrodes was sufficiently large to yield stable and reproducible responses to ROS and RNS in vitro. The prepared nanoprobes were used to penetrate cells and detect ROS and RNS inside macrophages. Weak and very short leaks of ROS/RNS from the vacuoles into the cytoplasm were detected, which a macrophage is equipped to clean within a couple of seconds, while higher intensity oxidative bursts due to the emptying of vacuoles outside persist on the time scale of tens of seconds.

Biomass combustion is used in heating and electric power generation in many areas of the world. Airborne particulate matter (PM) is released when biomass is brought to a facility, stored, and combusted. Occupational exposure to airborne PM within biomass-fueled facilities may lead to health problems. In March and August of 2006, airborne PM was collected from a biomass-fueled facility located in Denmark. In addition, source-specific PM was generated from straw and wood pellets using a rotating drum. The PM was analyzed for polycyclic aromatic hydrocarbons (PAHs), metals, microbial components, mutagenic activity, and ability to generate highly reactive oxygen species (hROS) in cell-free aqueous suspensions. PM collected from the boiler room and the biomass storage hall had higher levels of mutagenic activity, PAHs and metals, and a higher hROS generating potential than the source specific PM. The mutagenic activity was generally more potent without S9 activation, and on the metabolically enhanced strain YG1041, relative to TA98. Significant correlations were found between mutagenicity on YG1041 (without S9) and PAH concentration and mutagenicity on YG1041 (with S9) and hROS generating ability. PM collected in March was more toxic than PM collected in August. Overall, airborne PM collected from the facility, especially that from the boiler room, were more toxic than PM generated from straw and wood chips. The results suggest that exposure to combustion PM in a biomass-fueled facility, which likely includes PM from biomass combustion as well as internal combustion vehicles, may contribute to an elevated risk of adverse health effects., (2010 Wiley-Liss, Inc.)

Purpose: Cyclic GMP phosphodiesterase (PDE) is the light-regulated effector enzyme of vertebrate rods. Upon photo-activation of rhodopsin followed by activation of transducin/GTP, PDE rapidly hydrolyzes 3', 5'-cyclic GMP (cGMP) to 5'-GMP, which results in closure of cGMP-dependent ion channels and generation of a nerve signal. In the rod photoreceptors, PDE is entirely localized within the rod outer segment (ROS), a specialized compartment consisting of thousands of disc stacks. This study investigated the effects of light on the subcellular localization of PDE in ROS., Methods: Adult rats were either dark- or light-adapted for various durations before eyes were isolated and processed for transmission electron microscopy. Immunogold electron microscopy was performed with antibodies against PDE. Lateral displacement of PDE on ROS disc membrane was analyzed from electron micrographs. PDE enzymatic activities were measured with thin layer chromatography., Results: Light exposure induced translocation of PDE away from the edges of the dark-adapted disc membranes adjacent to the ROS plasma membrane. In dark-adapted ROS, a substantial portion (19+/-2%) of total PDE was localized near the edges of the disc membranes. Within 1 min of light exposure in the presence of GTP, over half of such PDE molecules (10+/-1% of total PDE) had moved away from the edges of the discs toward disc center. This light induced translocation of PDE was GTP dependent, as the effect was abolished when hydrolysis-resistant GTPgammaS was used in place of GTP. The percentage of PDE found near the disc edge corresponds to the fraction of PDE activity relative to maximal PDE activity revealed by limited trypsin proteolysis., Conclusions: These results suggest that light and GTP modulates lateral displacement of PDE, which might contribute to light-induced reduction of rod photoreceptor sensitivity.

Purpose: Vulnerable plaques are characterized by a myxoid matrix, necrotic lipidic core, reactive oxygen species, and high levels of microorganisms. Aerobic microbes such as Chlamydophila pneumoniae and Mycoplasma pneumoniae usually do not survive in oxidative stress media. Archaea are anaerobic microbes with powerful anti-oxidative enzymes that allow detoxification of free radicals whose presence might favor the survival of aerobic microorganisms. We searched for archaeal organisms in vulnerable plaques, and possible associations with myxoid matrix, chlamydia, and mycoplasma bodies., Methods: Twenty-nine tissue samples from 13 coronary artherectomies from large excentric ostial or bifurcational lesions were studied using optical and electron microscopy. Infectious agents compatible with archaea, chlamydia, and mycoplasma were semiquantified using electron micrographs and correlated with the amounts of fibromuscular tissue, myxoid matrix, and foam cells, as determined from semi-thin sections. Six of the cases were also submitted to polymerase chain reaction with archaeal primers., Results: All 13 specimens showed archaeal-compatible structures and chlamydial and mycoplasmal bodies in at least 1 sample. There was a positive correlation between extent of the of myxoid matrix and archaeal bodies (r = 0.44, P = 0.02); between archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and between chlamydial bodies and foam cells (r = 0.42; P = 0.03). The PCR test was positive for archaeal DNA in 4 of the 6 fragments., Discussion: DNA and forms suggestive of archaea are present in vulnerable plaques and may have a fundamental role in the proliferation of mycoplasma and chlamydia. This seems to be the first description of apparently pathogenic archaea in human internal organ lesions.

Respiration-deficient (petite) mutation is caused by hereditary impairment in mitochondrial functions. Yeasts have been grouped into "petite-positive" and "petite-negative" yeasts. Candida albicans has been regarded as a member of the petite-negative yeasts in which the respiration deficiency cannot be easily induced. We have succeeded in inducing the petite mutation in C. albicans by culturing in the presence of a chemical mutagen, acriflavine, at an elevated temperature. In the present review, we describe the cell biology of C. albicans petite mutants on the basis of experiments performed by our research group: namely, on respiratory activity and cytochrome composition, fine structures of cells and mitochondria, mitochondrial DNA structure, pathogenicity, oxidative stress sensitivity, generation of reactive oxygen species (ROS) and the roles of ROS in antifungal actions. We discuss also the usefulness of petite mutants in Candida research.

A central role in the oxidative development of atherosclerotic lesions has been ascribed to the peroxidation of plasma low-density lipoprotein (LDL). Dietary supplementation with virgin olive oils increases the total plasma antioxidant status and the resistance of low-density lipoprotein to ex vivo oxidation. We have studied the effects of some dietary non-flavonoid phenols from Olea europaea L., both in purified form or in complex mixtures obtained by biotransformation of olive leaf extracts, on the LDL oxidation induced by Cu2+ ions. Cu2+-Induced LDL oxidation is inhibited by oleuropein and hydroxytyrosol in the initiation phase of the reaction at concentrations of phenols higher than that of Cu2+ ions. Interestingly, at lower concentration, both phenols anticipated the initiation process of LDL oxidation, thus exerting prooxidant capacities. Although similar effects are already described for flavonoids, such as quercetin, rutin, and apigenin, it is the first time that a prooxidant effect of dietary non-flavonoid phenols, such as oleuropein and hydroxytyrosol, on the LDL oxidation is reported. Our results show that a net effect of oleuropein and hydroxytyrosol on Cu2+-induced LDL peroxidation is determined by a balance of their pro- and antioxidant capacities. It is worth to underline that, during Cu2+-induced LDL oxidation in the presence of bioreactor eluates, we have evidence of a synergistic effect among phenolic compounds that enhance their antioxidant capacities so avoiding the prooxidant effects.

We investigated the role of glutathione (GSH) and antioxidant enzymes in menadione-resistance by using K300 cells (menadione-resistant cells) and parental P19 cells (menadione-sensitive cells). We found that acquisition of resistance was associated with elevations in glutathione content and DT-diaphorase activity. The activity of glutathione S-transferase (GST) was significantly decreased, while the activities of glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase in K300 cells were maintained at the same levels as compared to the parental P19 cells. Using reactive oxygen species (ROS)-sensitive fluorescence dye 2,7- dichlorodihydrofluorescein diacetate (DCFH/DA), we demonstrated that K300 cells are characterized by reduced cellular ROS as compared to the parental P19 cells during menadione's action. Menadione depleted glutathione to a small extent in the K300 cells, but a rapid depletion was observed in P19 cells. Pretreatment of K300 cells with dicumarol, a DT-diaphorase inhibitor, or buthionine sulfoximine (BSO), an inhibitor of gamma-glutamyl cysteine synthase, sensitized the cells to menadione. BSO treatment was less effective than dicumarol treatment in reversing menadione resistance in K300 cells. These results strongly support the belief that DT-diaphorase plays a central role in protecting cells against menadione-induced oxidative stress by decreasing the ROS formation.

Characterization of a mixed solution of sodium hypochlorite (NaClO) and hydrogen peroxide (H2O2), which have been used as root canal irrigants, was investigated using electron spin resonance spectroscopy combined with a spin-trapping technique. In the solution, two kinds of reactive oxygen species, the superoxide anion radical (O2-.) and the hydroxyl radical (.OH), were detected. The pH changes and dissolved oxygen values in the NaClO-H2O2 reaction mixture were investigated. The pH influenced the generation of O2-. and .OH, and O2 was generated from H2O2. It is suggested that root canal irrigation with NaClO and H2O2 induces biological effects, such as tissue irritation (from the chemical reactions of O2-. and .OH) and physical effects, such as O2 bubbling.

Aqueous extract of cigarette smoke (CS) contains some stable oxidants, which oxidize human plasma proteins, bovine serum albumin, amino acid homopolymers, and also cause extensive oxidative degradation of microsomal proteins. Similar observations are made when the aqueous extract of cigarette smoke is replaced by whole phase CS solution or whole phase cigarette smoke. CS-induced microsomal protein degradation is a two step process: (i) oxidation of proteins by the oxidants present in the CS and (ii) rapid proteolytic degradation of the oxidized proteins by proteases present in the microsomes. Using aqueous extract of CS equivalent to that produced from one-twentieth of a cigarette, the observed initial and postcigarette smoke treated values of different parameters of oxidative damage per milligram of microsomal proteins are respectively: 0.24 and 1.74 nmoles for carbonyl formation, 125.4 and 62.8 fluorescence units for tryptophan loss, 10.2 and 33.4 fluorescence units for bityrosine formation, and 58.3 and 12.2 nmoles for loss of protein thiols. When compared with sodium dodecyl sulphate polyacrylamide gel electrophoresis profiles of untreated microsomal proteins, the extent of microsomal protein degradation after treatment with whole phase CS solution or aqueous extract of CS is above 90%. Ascorbate (100 microM) almost completely prevents cigarette smoke-induced protein oxidation and thereby protects the microsomes from subsequent proteolytic degradation. Glutathione is partially effective, but other antioxidants including superoxide dismutase, catalase, vitamin E, probucol, beta-carotene, mannitol, thiourea, and histidine are ineffective. The gas phase cigarette smoke contains unstable reactive oxygen species such as superoxide (O2*-) and hydrogen peroxide (H2O2) that can cause substantial oxidation of pure protein like albumin but is unable to produce significant oxidative damage of microsomal proteins. Gas phase cigarette smoke-induced albumin oxidation is not only inhibited by ascorbate and glutathione but also by superoxide dismutase, catalase and mannitol. The stable oxidants in the cigarette smoke are not present in the tobacco and are apparently produced by the interaction of O2*-/H2O2/OH* of the gas phase with some components of the tar phase during/following the burning of tobacco.

The effects of cortical tissue preparations (CTP) from human brain on the production of reactive oxygen species (ROS) has been investigated with several biochemical model reactions. As indicators for ROS, fragmentation of the methionine derivatives, alpha-keto-gamma-methylthiobutyric acid (KMB) or 1-amino-cyclopropane-1-carboxylic acid (ACC), yielding ethene have been used. With these systems we have shown that production of OH-radical-type oxidants by the xanthine oxidase (XOD)-system is strongly stimulated by CTP. This activity is due to intrinsic iron ions since ethene formation from KMB is stimulated by EDTA, inhibited by desferrioxamine (Desferal) and also visible with heat-denatured CTP. CTP by themselves have no XOD activity. 3-Hydroxykynurenine (3HK) is another possible substrate for XOD but produces H2O2 without XOD-catalysis, whereas allopurinol is not inhibiting. CTP contain measurable NAD(P)H oxidoreductase activity, producing OH- radical- type oxidants at the expense of NADPH and (to a lesser extent) NADH as electron donors, shown as redox-cycling of 2-methyl-5-hydroxy-1,4-naphthoquinone, plumbagin. Ethene formation from KMB is also driven by both morpholinosydnonimine (SIN) or ONOOH. The reaction driven by SIN is stimulated by CTP and inhibited by catalase, SOD and hemoglobin. Since ethene release from KMB driven by ONOOH is inhibited by CTP the mechanisms driving KMB fragmentation are different for SIN and ONOOH. Furthermore CTP contain approx. 4 U catalase activity per mg protein and very weak peroxidase (POD) activity shown as ACC fragmentation yielding ethene in the presence of both H2O2 and KBr or NaCl. Since ACC binds to CTP and both compounds, ACC and KMB are natural products, present in food (ACC) or synthesized from methionine in vivo (KMB), these compounds may represent protecting agents in systems where reactive oxygen species are formed. One might even speculate that the production of ethene at these membrane receptor sites may have biological functions, since ethene is known to possess anaesthetic activities.

Reactive oxygen species (ROS) in human semen, measured as luminol or lucigenin chemiluminescence (CL), originate from spermatozoa or leukocytes. To investigate the relative contribution of these cells to the overall CL signal, the leukocytes were separated by means of a magnetic separation system. Spermatograms according to WHO criteria were performed. The leukocytes were stained (o-toluidine) and counted in a counting chamber. A Berthold luminometer LB 953 was used to determine the luminol and lucigenin chemiluminescence at constant sperm concentrations of 10(7)/mL spermatozoa (MBSS as diluting agent). Then the leukocytes were separated by means of a magnetic cell separation system using different antibodies (MACS CD 14, 15, 16 or CD 45 RO microbeads). After these procedures, the mixture was filtered through a MACS column and the staining with o-toluidine was repeated. In the counting chamber no leukocytes could be found, indicating the complete separation of leukocytes. The CL counts in the spermatozoa fractions after separation of the leukocytes were significantly lower. The data show that leukocytes appear to be a major source for the CL in whole semen.

In 12 patients with primary hypogammaglobulinaemia we investigated the in vivo effect of one bolus injection (400 mg/kg) of intravenous immunoglobulin (IVIG) on lymphocyte subsets and monocytes in peripheral blood, on plasma levels of soluble factors reflecting monocyte and lymphocyte activity and on lymphocyte proliferation and generation of reactive oxygen species (ROS) from monocytes analysed in vitro. Several immunological changes were induced by IVIG infusion. First, there was a significant decrease in CD4+/CD8+ lymphocyte ratio in peripheral blood, reflecting a significant increase in circulating numbers of CD8+ lymphocytes. Second, although there was no significant change in plasma levels of soluble CD8 antigen, there was a significant decrease in soluble CD8 antigen/CD8+ lymphocyte ratio, suggesting a down-regulation of CD8+ lymphocyte activity. Third, there was a significant increase in plasma levels of neopterin, suggesting in vivo activation of monocytes/macrophages. Fourth, the was a down-modulation of mitogen-stimulated lymphocyte proliferation in vitro, and this down-regulation was significantly correlated with the increase in plasma neopterin levels. Finally, there was a significant decrease in zymosan-stimulated, but not in phorbol myristate acetate-stimulated, ROS generation from monocytes as evaluated by nitroblue tetrazolium reduction. The ability of IVIG administration in vivo to down-modulate lymphocyte proliferation and ROS generation from monocytes in patients with persistent immune activation may be relevant for the clinical effects of IVIG in a variety of immune-mediated disorders.

Humans exhibit an acute inflammatory response in the lungs after controlled laboratory exposure to ozone. The present study was designed to test whether biomarkers of inflammation are detectable in humans exposed to ozone and associated copollutants under natural conditions outdoors. Bronchoscopy with bronchoalveolar lavage (BAL) was carried out on 19 normal volunteer joggers from Governors Island, NY, who exercised in the afternoon during the 1992 summer (S1) season. Fifteen subjects were retested during the following, low ozone, winter season (W). The BAL protocol involved an initial instillation of 20 ml saline followed by four sequential 50-ml saline washes carried out in both the right middle lobe and the lingula. The eight 50-ml samples were pooled as the 'alveolar' sample. Analyses performed on the alveolar lavage samples included cell differentials, release of IL-8, TNF-alpha, and reactive oxygen species (ROS) by pooled cells, and levels of IL-8, protein, LDH, fibronectin, alpha1-antitrypsin (alpha1-AT), complement fragment 3a (C3a), and prostaglandin E2 (PGE2) in lavage fluids. Release of ROS by stimulated BAL cells was lower in S1 than in W (p = 0.03). In contrast, LDH levels in BAL fluids were 2-fold higher in S1 than in W (p = 0.02), as were IL-8 (p = 0.12) and PGE2 (p = 0.06). These results suggest a possible ongoing inflammatory response in the lungs of recreational joggers exposed to ozone and associated copollutants during the summer months.

Flow cytometry was used to study phagocytic function (uptake of fluorescein isothiocyanate-labeled bacteria) and release of reactive oxygen products (dihydrorhodamine 123 converted to rhodamine 123) following phagocytosis by neutrophil granulocytes of heparinized whole blood treated with adrenaline, noradrenaline, dopamine, dobutamine, or orciprenaline. Reduced neutrophil phagocytosis and reactive oxygen production were seen at 12 micrograms of adrenaline per liter (72% each compared with control values); at 120 micrograms of noradrenaline (72% each), dobutamine (83 and 80%, respectively), and orciprenaline (81 and 80%, respectively) per liter; and at 100 micrograms of dopamine per liter (66 and 70%) (P < 0.05 for all). At these dosages, neutrophil chemotaxis was reduced to < 50% of control values for all catecholamines. Treatment with catecholamines at lower dosages had no significant effect on phagocytosis or generation of reactive oxygen products or chemotaxis. The phagocytic capacity of granulocytes was related to the generation of reactive oxygen products (r = 0.789; P < 0.05). The results demonstrate that catecholamines have a suppressive effect on the response of phagocytic cells to bacterial pathogens at high therapeutic levels in blood.