Scavenging of reactive oxygen species by melatonin

Monocytes adherent to implanted biomaterials differentiate into macrophages while synthesizing large amounts of degradative enzymes, including cholesterol esterase (CE), which previously has been shown to degrade poly(urethane)s. Human peripheral blood monocytes were cultured on tissue culture grade polystyrene (PS), and two model poly(urethane)s were synthesized from (1) polycaprolactone (PCL) and (2) polytetramethylene oxide (PTMO), both with 2,4-toluene diisocyanate (TDI) and ethylene diamine (ED). The increase in CE and total protein per cell were measured on days 8 and 28 in culture and normalized to the DNA content per cell. At day 8 there consistently were fewer cells remaining on the PTMO-based polymer than on the PCL-based polymer or the PS (p < 0.05). When comparing day 28 to day 8, there was more CE activity and protein per cell on all materials. However, there was a disproportionate synthesis of CE per mg of total protein on PS and TDI/PCL/ED whereas on PTMO there was not. Significantly, there was more protein and CE per cell on PTMO than on PS or TDI/PCL/ED (p < 0.05). This in vitro model system of the chronic phase of inflammation has shown that it is possible to culture monocytes for a month and assess the material surface itself as a potent activator of the differentiation into macrophages without secondary stimulation. Since CE has been shown to degrade poly(ether and ester)-based poly(urethane)s, the differential production of this enzyme relative to the total protein on different surfaces may impact on the potential long-term biostability of an implanted material.     

Induction of reactive oxygen species in neurons by haloperidol

Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT-22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT-22 cells are equally sensitive to HP with an IC50 of 35 microM in the primary neurons and 45 microM in HT-22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as beta-estradiol and vitamin E, lowers the level of ROS and protects HT-22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca2+, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.     

Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species

The effects of pentobarbital on electroencephalogram (EEG) and auditory brain-stem response (ABR) were evaluated in 13 Japanese white male rabbits, divided into two groups, 7 and 6, respectively. After baseline evoked responses were obtained, pentobarbital was infused intravenously at 60 mg/kg/h in both groups. EEGs and ABR were recorded with 15 min intervals. When the blood concentration of pentobarbital reached therapeutic levels (2.0-5.0 mg/dL), cortical and hippocampal EEGs became isoelectric. Although the appearance of ABR waves was significantly delayed, each wave was clearly observed in spite of isoelectric EEG levels. The rabbits in one group were killed at that time, and their brains were removed to determine the concentration of pentobarbital in the brain tissue. In another group, pentobarbital was additionally infused at the rate of 120 mg/kg/h. Although the waves (II-IV) of the ABR gradually disappeared with increasing dosage, wave I was present until just prior to cardiac arrest. It is considered that the persistency of ABR at high doses of barbiturates is characteristic of patients in deep barbiturate coma. Therefore, at the diagnosis of brain death, there is no necessity to consider the half-life of the barbiturate, even if an excessive amount of barbiturate remains in the brain. In this study, pentobarbital concentration in the brain was nearly equal to the concentration in the blood. However, it is estimated that a large amount of barbiturate is accumulated in the brain of a patient after brain death because the blood flow in the brain is stagnant.     

Cyclophosphamide-induced generation of reactive oxygen species. Comparison with morphological changes in type II alveolar epithelial cells and lung capillaries

Immunosenescence involves modifications of humoral and cellular immunity. Here we report the analysis of human leukocyte antigen (HLA) expression on T lymphocytes, B lymphocytes and monocytes of 58 healthy subjects aged 23-95 years old. Using a double staining immunofluorescence and flow cytometry analysis, we have determined the percentages of cells expressing HLA class-I and HLA-DR antigens. The number of antigenic sites expressed per cell were evaluated for HLA-ABCw, HLA-A, HLA-B, HLA-DR locus with a flow cytometry quantification technique. With advancing age, we observed: (i) a significant decrease of the percentage of T cells and B cells expressing HLA-A products; (ii) a decrease of the number of HLA class-I antigenic sites expressed per cell on the three populations tested, predominantly on B cells and in a locus-dependent fashion; (iii) a decrease of the number of HLA-DR molecules expressed per T cell, although the percentage of T cells expressing DR products was increased; (iv) a significant diminution of the percentage of B cells expressing HLA-DR molecules, without changes of the number of HLA-DR antigenic sites per cells. These changes in HLA expression with increasing age could contribute to the decreased level of immunologic responsiveness observed with ageing and contribute to the modification of antigen recognition.     

Importance of reactive oxygen species in rheumatoid arthritis

Free radical oxidation--peroxidation products, superoxide dismutase (SOD) activity--and nonproteic thiols were measured in blood from 10 normal subjects and 10 patients with rheumatoid arthritis (RA). Peroxidation products and SOD activity have been found significantly elevated, while blood nonproteic thiols have been found significantly lower in RA patients, as compared to normal controls. Also, plasmatic concentration of ceruloplasmin has been found significantly higher in RA patients than in controls.     

The role of reactive oxygen species in atherosclerosis

Reactive oxygen species (ROS) are probably not only unintended, toxic side-products of oxygen metabolism in mammalian cells, they also have several important physiologic functions including antimicrobial killing, regulation of cellular proliferation and growth, and regulation of vascular tone. ROS are generated within the vessel wall by several mechanisms, including a vascular type of a NAD(P)H oxidase. ROS formation can be stimulated by mechanical stress, environmental factors, the peptide angiotensin II, cytokines, native low-density lipoproteins (LDL), and in the presence of catalytic metal ions. Their ability to modify LDL, react with endothelial-derived nitric oxide subsequently forming peroxynitrite, and amplify the expression of various genes important for leukocyte recruitment within the arterial wall are the basis of the oxidant injury theory of atherosclerosis. In animal studies, antioxidant therapy (probucol, butylated hydroxytoluene, N', N'-diphenylenediamide, vitamin E, superoxide dismutase) have been successfully used to prevent fatty streak formation, and to restore impaired nitric oxide-dependent vasorelaxation. In man, antioxidant therapy (e.g., supplementation with vitamin E) clearly increased the resistance of LDL to oxidative modification. Case-controlled and prospective clinical studies suggest a relation between baseline antioxidant plasma levels and/or antioxidant supplementation and risk of cardiovascular events. In one secondary prevention trial (randomized, blinded, placebo-controlled), vitamin E supplementation reduced significantly the risk for non-fatal myocardial infarctions. Before general recommendations can be made, results of further large-scale trials should be awaited.     

Involvement of reactive oxygen species in kidney damage

There is considerable evidence suggesting that reactive oxygen species (ROS) are implicated in the pathogenesis of ischemic, toxic, and immunologically-mediated renal injury. In experimental renal ischemia, ROS sources include the electron transport chain, oxidant enzymes (xanthine oxidase), phagocytes, and auto-oxidation of epinephrine. ROS cause lipid peroxidation of cell and organelle membranes and, hence, disruption of the structural integrity and capacity for cell transport and energy production, especially in the proximal tubule segment. In experimental immune glomerulonephritis, ROS are generated by both infiltrating blood-borne cells (polymorphonuclear leukocytes and monocytes) and resident glomerular cells, mainly mesangial cells. Their formation results in morphologic lesions and in modifications of glomerular permeability to proteins through activation of proteases and reduction of proteoglycan synthesis. Additionally, they promote a reduction in glomerular blood flow and glomerular filtration rate through liberation of vasoconstrictory bioactive lipids (prostaglandins, thromboxane, and platelet activating factor) and, possibly, inactivation of relaxing nitric oxide. Further studies are needed to address the role of ROS in human glomerular diseases.     

Degradation of glycosaminoglycans by reactive oxygen species derived from stimulated polymorphonuclear leukocytes

The effect of reactive oxygen species (ROS), generated by in vitro stimulation of isolated PMN upon the main GAG components of mineralised and non-mineralised connective tissues was investigated. PMN were isolated from whole blood and the production of the ROS superoxide (O2.-) and hydroxyl radicals (.OH) was stimulated by the addition of phorbol myristyl acetate (PMA) and PMA/FeCl3-EDTA chelate respectively and their production assessed over a 24 h period. The glycosaminoglycans (GAG), hyaluronan, chondroitin 4-sulphate and dermatan sulphate, were exposed to the ROS fluxes, incubated at 37 degrees C for 1 h and 24 h. GAG fragmentation was examined by gel exclusion chromatography and modification to hexuronic acid and hexosamine residues determined. Stimulation of PMN with PMA resulted in a burst of O2.- production for 1 h, which was sustained at a reduced level for 24 h. Fragmentation of GAG was observed for all GAG examined. Modification to the GAG was evident, with hyaluronan being more susceptible to loss of GAG residues than sulphated GAG. Modification of sugar residues increased with the incubation time and loss of the hexuronic acid residues was greater than loss of hexosamine residues. Addition of FeCl3-EDTA chelate, which led to the generation of .OH and was sustained over the 24 h period, demonstrated similar trends of GAG modification although increased degradation and loss of hexosamine and hexuronic acid were observed. GAG chains are constituents of PGs and their modification is likely to affect the function of these macromolecules and be of importance in considering the pathogenesis of inflammatory diseases, including periodontal diseases.     

Relationship between resazurin reduction test, reactive oxygen species generation, and gamma-glutamyltransferase

The production of reactive oxygen species (ROS) from spermatozoa has been measured by chemiluminescence in the two fractions of a Percoll gradient column (47 and 90%). Chemiluminescent signals were recorded in each fraction after addition of luminol and horse-radish peroxidase (basal state), and after stimulation with formyl-methionyl-leucyl-phenyl-alanine (FMLP) and phorbol ester (PMA). We found an inverse correlation between the reducing capacity of the semen as estimated by the resazurin test, and the production of ROS by the spermatozoa, especially after stimulation with PMA (r = 0.51, P < 0.001). gamma-glutamyltransferase activity of seminal plasma was inversely correlated with ROS (r = -0.47, P < 0.01) and the resazurin test result (r = -0.43, P < 0.001) suggesting a possible role of prostatic secretions in the protection of spermatozoa against oxygen radicals. The resazurin test has a positive predictive value of 92.5% at a criterion value of colour scale 4 to discriminate between normal and excessive ROS production by spermatozoa, with sensitivity 79% and specificity 74%. In addition, at criterion value of colour scale 3, the resazurin test could distinguish between samples with normal or low activity of gamma-glutamyltransferase, with sensitivity 57% and specificity 93%. It is concluded that the result of the resazurin test can be influenced by the oxidative stress of spermatozoa and by prostatic function.     

Inhibitory effect of local anaesthetics on reactive oxygen species production by human neutrophils

BACKGROUND: Reactive oxygen species (ROS) generated from neutrophils accumulated in various major organs are thought to play a pivotal role in the pathogenesis of host auto-injury. Lidocaine has been shown to reduce the injury. We investigated the effect of local anaesthetics (lidocaine, mepivacaine and bupivacaine) on ROS production by neutrophils using an in vitro system. METHODS: We measured the production of superoxide (ferricytochrome c method), hydrogen peroxide (H2O2: scopoletin fluorescence technique), and hydroxyl radical (OH.: ethylene gas method) by neutrophils isolated from human adult volunteers in the absence and presence of lidocaine (2-200 micrograms/mL), mepivacaine (3-300 micrograms/mL), and bupivacaine (3-300 micrograms/mL). We also measured the ROS generation in a cell-free (xanthine-xanthine oxidase) system. RESULTS: Lidocaine and mepivacaine at higher levels significantly decreased the production of ROS by neutrophils. However, these local anaesthetics at clinically relevant blood concentrations had no effect on the levels of ROS. Furthermore, lidocaine and mepivacaine failed to reduce ROS generated by the cell-free system. Bupivacaine did not decrease ROS generation by either generating system. CONCLUSION: In conclusion, in the present in vitro system, only concentrations of lidocaine and mepivacaine 100-fold higher than clinically feasible ones reduced ROS production by human neutrophils. However, the local anaesthetics at clinically relevant blood concentrations had no suppressive effect. Further studies using in vivo systems are required to elucidate the inhibitory effects of local anaesthetics on ROS generation in clinical settings.     

Protection against UVA damage and effects on neutrophil-derived reactive oxygen species by beta-carotene

OBJECTIVE: Phagocyte-derived reactive oxygen species (ROS) are involved in microbicidal activities as well as in tissue damage at sites of inflammation. Carotenoids play an important function in protecting cells from oxidant damage. We investigated the in vitro and in vivo effect of 13-cis and 9-cis-beta-carotene on human neutrophils. METHODS: Neutrophils from healthy donors in the presence of 0.25 mumol/L-1 mumol/l beta-carotene and from subjects under beta-carotene supplementation and UVA or UVA/B exposure were stimulated by opsonized zymosan and the generation of ROS was measured by electron spin resonance spectroscopy. RESULTS: Our in vitro results show different effects of the two isomers on stimulated neutrophils. 9-cis-beta-carotene did not produce any change, whereas 13-cis-beta-carotene significantly and concentration-dependent inhibited the ROS generation by stimulated neutrophils. Further, in a controlled study, we were able to demonstrate an in vivo protective effect of beta-carotene on neutrophils against UVA damage by beta-carotene supplemented subjects.     

Walker 256 tumor cell degradation of extracellular matrices involves a latent gelatinase activated by reactive oxygen species

The invasion of blood vessel walls is a critical step in cancer metastasis, in which endothelial cells and their vascular basement membranes act as barriers to tumor cell passage. Here we report that Walker 256 carcinosarcoma (W256) cells degrade subendothelial matrices by a process involving both the generation of hydrogen peroxide and the secretion of a matrix metalloproteinase. As an assay of basement membrane degradation, [3H]proline-labeled subendothelial matrices were exposed to W256 cells in the presence or absence of the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). The release of [3H]proline, in the presence of 5 x 10(6) W256 cells, was increased from 49 +/- 2.5 to 64 +/- 2.2% by the addition of 10(-6) M fMLP. In the presence of fMLP-activated W256 cells, [3H]proline release was completely inhibited by the addition of 2000 units/ml catalase or by the metalloproteinase inhibitors 1,10-phenanthroline and EDTA at concentrations > or = 10 micrograms/ml. alpha 1-Antitrypsin or alpha 2-macroglobulin were without effect. Cell-free supernatants obtained from activated W256 cells were also able to promote basement membrane degradation. Electrophoresis of the cell-free supernatants from fMLP or PMA-activated W256 cells in gelatin-containing sodium dodecyl sulfate-polyacrylamide gels revealed a major band of gelatinolytic activity at 94 kDa. The 94-kDa band represented the activity of a latent gelatinase since incubation with 1 mM 4-aminophenylmercuric acetate (APMA; a known activator of latent metalloproteinases) resulted in the loss of gelatinolytic activity at 94 kDa and the appearance of five new bands of lower molecular weight (M(r) 86, 79, 74, 70, and 66 kDa). Two of these lower molecular weight bands (M(r) 86 and 66 kDa) were also detected in the absence of APMA, following 10-fold concentration of the cell-free supernatants. When the cell-free supernatants of phorbol myristate acetate-activated W256 cells (concentrated 10-fold) were incubated with increasing concentrations of hydrogen peroxide (35 to 70 mM), the band at 66 kDa demonstrated enhanced gelatinolytic activity. We suggest that W256 cells can secrete a latent metalloproteinase of molecular weight 94 kDa which, when activated by hydrogen peroxide, can degrade subendothelial matrices.     

Cytotoxicity-associated effects of reactive oxygen species on endothelin-1 secretion by pulmonary endothelial cells

In this study bovine pulmonary artery endothelial cells (BPAEC) were used as a model system to investigate the effects of the hypoxanthine-xanthine oxidase (HXXO) oxygen radical donor system on ET-1 secretion into pulmonary vasculature. Incubation of BPAEC with HXXO for 4 h caused a significant reduction in ET-1 secretion, which was significantly offset by allopurinol or catalase, but not by Cu/Zn superoxide dismutase (SOD). ET-1 secretion was also reduced by H2O2, and this effect was again significantly offset by catalase. XO alone also reduced ET-1 secretion, but to a significantly lesser degree than did HXXO, and this effect was not offset by allopurinol, catalase, or SOD. None of the oxidant treatments were associated with a loss of immunoreactive ET-1 from endothelial cell medium containing synthetic peptide. The HXXO- and H2O2-mediated reductions in ET-1 secretion were accompanied by evidence of reduced cell viability. This loss of viability was absent when cells were treated with HXXO + catalase, allopurinol, or mercaptopropionyl glycine, but not when SOD was present. We conclude that under conditions of oxidative stress, the pulmonary vascular endothelium responds by secreting less ET-1. This may be relevant to its vasodilator functions in the pulmonary vasculature, which would therefore be compromised when the endothelium is exposed to oxidant stress.     

Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment

Rat liver microsomes and, to a lesser extent, nuclei were previously shown to produce reactive oxygen species at elevated rates after chronic ethanol treatment. The ability of intact rat liver mitochondria to interact with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determined. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondrial lipid peroxidation. Rates were elevated two- to threefold with mitochondria from ethanol-fed rats with both reductants. Mitochondrial lipid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidants such as trolox. Mitochondrial generation of hydroxyl radical-like species (assayed by oxidation of chemical scavengers) was increased after chronic ethanol treatment, as was H2O2 production. Modifiers of mitochondrial metabolism such as rotenone, cyanide, or an uncoupling agent, had no effect on mitochondrial production of reactive oxygen intermediates. The membrane-impermeable thiol reagent, p-chloromercuribenzoate, was complete inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent production of oxygen radicals by rat liver mitochondria. The outer mitochondrial membrane fraction, prepared by digitonin fractionation, displayed increased rotenone-insensitive NADH-cytochrome c reductase activity after ethanol treatment and was more reactive in catalyzing scission of pBR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increase produced by chronic ethanol treatment are similar to those previously found with microsomes when NADH is the cofactor. Oxidation of ethanol by alcohol dehydrogenase generates NADH, and NADH-dependent production of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by ethanol.     

Breath ethane as a marker of reactive oxygen species during manipulation of diet and oxygen tension in rats

Breath ethane, O2 consumption, and CO2 production were analyzed in 24-mo-old female Fischer 344 rats that had been fed continuously ad libitum (AL) or restricted 30% of AL level (DR) diets since 6 wk of age. Rats were placed in a glass chamber that was first flushed with air, then with a gas mixture containing 12% O2. After equilibration, a sample of the outflow was collected in gas sampling bags for subsequent analyses of ethane and CO2. The O2 and CO2 levels were also directly monitored in the outflow of the chamber. O2 consumption and CO2 production increased for DR rats. Hypoxia decreased O2 consumption and CO2 production for the AL-fed and DR rats. These changes reflect changes in metabolic rate due to diet and PO2. A significant decrease in ethane generation was found in DR rats compared with AL-fed rats. Under normoxic conditions, breath ethane decreased from 2.20 to 1.61 pmol ethane/ml CO2. During hypoxia the levels of ethane generation increased, resulting in a DR-associated decrease in ethane from 2.60 to 1.90 pmol ethane/ml CO2. These results support the hypothesis that DR reduces the level of oxidative stress.     

Comparing two groups by simple graphs.

Considers that standard significance tests for testing whether 2 independent groups are equal in elevation or dispersion do not test the forms of those hypotheses which are frequently of most interest to behavioral scientists. More interesting forms of those hypotheses can be tested easily and quickly by drawing a simple graph. The same graph can also be used to perform several standard tests. In all these tests, the procedure is so simple that the chance of computational error is much smaller than with standard procedures. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Direct effects of intraperilymphatic reactive oxygen species generation on cochlear function

Reactive oxygen species (ROS) generation may play a role in ototoxicity, however, the specific effects of ROS generation upon cochlear function are unstudied. Therefore, guinea pig cochleas were instilled with artificial perilymph (AP), H2O2, or confirmed generating systems for the superoxide anion (O2-) or the hydroxyl radical (OH.), or with an ROS system plus its respective scavenger -catalase (CAT), superoxide dismutase (SOD) or deferoxamine (DEF). O2- generating system instillation led to significantly greater mean high frequency compound action potential (CAP) threshold shifts at 10 and 120 min post infusion than seen in AP control or SOD/O2- groups. H2O2 group CAP threshold shifts were significantly greater than control and CAT/H2O2 group values at 10 (16-30 kHz), and 120 min (above 12 kHz). OH generating system instillation led to significantly greater CAP threshold shifts at 10 (12-30 kHz) and 120 min (above 6 kHz) than seen in control or DEF/OH groups. No significant CAP differences were found between controls and scavenger/ROS groups. Mean 1.0 microV cochlear microphonic isopotential curve shift values did not systematically differ among groups. The rapid degradation of high frequency CAP threshold sensitivity seen here may provide insight into the portion of cochlear dysfunction which is ROS-mediated following noise, radiation or chemical exposures.     

The regulation of reactive oxygen species production during programmed cell death

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.     

Molecular mechanisms of cellular injury produced by neurotoxic amino acids that generate reactive oxygen species

There is now strong experimental evidence that the basic precursors for the synthesis of catechol(amine) and indolamine neurotransmitters, tyrosine and tryptophan can act as generators of ROS (reactive oxygen species): peroxides, superoxide and peroxyradicals. The consequences of free radicals formation from precursors during oxidative degradation process, their possible participation in electron transfer/addition reactions and chain processes involving cell antioxidant defense system were presented and discussed. Although the generation of neurotoxic ROS by tyrosine and tryptophan is accepted to occur in the presented model systems, doubts can exist as to the situation in vivo, which may be completely different and remain to be explored. The relevance of the present findings with regard to a variety of neurological diseases cannot be ignored.