Activity of antioxidant enzymes in the wound in patients with deep burns.

Analysis of wound discharge in children with deep burns over 3 weeks after the injury revealed gradual increase in catalase activity. The increase in activities of myeloperoxidase, glutathione-S-transferase, and catalase was maximum in patients with the most severe burns. Local complications were observed during the period of maximum myeloperoxidase activity, while the beginning of epithelialization was associated with its reduction. Analysis of wound impressions confirms long-term persistence of neutrophils in the wound.

Comparative study of cytokine content in the plasma and wound exudate from children with severe burns.

The content of 27 cytokines was measured in blood plasma from 19 children with severe uncomplicated burns (group 1) and complicated burns (septic toxemia, toxemia, and pneumonia; group 2). Before surgical treatment (day 4 (+/-2) after burn), significant differences were found in the concentrations of interleukin-1 receptor antagonist, interleukin-6, interleukin-8, interleukin-10, tumor necrosis factor-alpha, interferon-gamma, MCP-1, and granulocyte colony-stimulating factor. Cytokine concentration in group 2 patients was much higher than in group 1 patients and healthy children. The concentrations of interleukin-6, interleukin-8, and MCP-1 in group 1 patients significantly surpassed the normal level. Cytokine concentration in the plasma and wound exudates and myeloperoxidase activity in wound exudates from 4 patients of group 2 were measured over 18 days after burn. The inflammatory response was characterized by an increase in the content of interleukin-1beta, interleukin-8, MCP-1, tumor necrosis factor-alpha, MIP-1alpha, and granulocyte-macrophage colony-stimulating factor in the wound (as compared to that in the plasma). Activity of myeloperoxidase in all patients was shown to correlate with the amount of MIP-1alpha (r=0.47), tumor necrosis factor-alpha (r=0.47), and granulocyte-macrophage colony-stimulating factor (r=0.55, p<0.05). Interleukin-8 concentration was beyond the limits of calibration. No correlation was found between the concentration of any of 27 cytokines in blood plasma and exudate. Our results indicate that during active surgical treatment, the wound serves as the source of inflammatory cytokines. Cytokines play a role in the systemic response and increase the degree of local inflammation, which modulates the number and activity of wound neutrophils.

Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health.

Phenylpropanoids (PPs) belong to the largest group of secondary metabolites produced by plants, mainly, in response to biotic or abiotic stresses such as infections, wounding, UV irradiation, exposure to ozone, pollutants, and other hostile environmental conditions. It is thought that the molecular basis for the protective action of phenylpropanoids in plants is their antioxidant and free radical scavenging properties. These numerous phenolic compounds are major biologically active components of human diet, spices, aromas, wines, beer, essential oils, propolis, and traditional medicine. Last few years, much interest has been attracted to natural and synthetic phenylpropanoids for medicinal use as antioxidant, UV screens, anticancer, anti-virus, anti-inflammatory, wound healing, and antibacterial agents. They are of great interest for cosmetic and perfume industries as active natural ingredients. In the present review, the metabolic pathways of phenylpropanoid biosynthesis in plants and the mechanism of phenylpropanoid-mediated plant defense are described. Learning from plants, free radical-driven, molecular and cellular processes modulated by phenylpropanoids in human cell cultures in vitro and in the in vivo animal models of tumors, inflammation, and cellular damage are also reviewed.

Molecular mechanisms underlying wound healing and anti-inflammatory properties of naturally occurring biotechnologically produced phenylpropanoid glycosides.

Two phenylpropanoid glycosides, verbascoside (VB) and teupolioside (TP), produced biotechnologically by Syringa vulgaris and Ajuga reptans plant cell cultures, were studied in vitro and in vivo for their anti-inflammatory and wound healing activities. It was shown that TP- and VB-containing extracts significantly accelerated wound healing and possessed remarkable anti-inflammatory action in the excision wound model. These effects correlated with the inhibition of reactive oxygen species release from the whole blood leukocytes and with the ferrous ion chelating capacity. On the other hand, they don't correlate either with free radical scavenging or with the inhibition of lipid peroxidation in the cell-free systems. Furthermore, both VB- and TP-containing extracts were extremely effective inhibitors of chemokine and growth factor expression by cultured human keratinocytes treated with pro-inflammatory cytokines, TNF-alpha and interferon-gamma.

Release of active oxygen radicals by leukocytes of Fanconi anemia patients.

The release of oxygen radicals by blood and bone marrow leukocytes of patients with Fanconi anemia (FA) has been studied. It was found that the nonstimulated FA leukocytes and those stimulated by concanavalin A, SiO2, latex, and opsonized zymosan produced enhanced levels of luminol- and lucigenin-dependent chemiluminescence (CL) in comparison with normal leukocytes. At the same time, the ratio of the intensity of lucigenin-dependent CL to that of luminol-dependent CL was significantly smaller for FA leukocytes than for normal leukocytes. From these findings and from the effects of antioxidative enzymes and free radical scavengers on CL, it was concluded that FA leukocytes release enhanced amounts of oxygen radicals and that these free radicals contain enhanced amounts of hydroxyl or hydroxyl-like radicals more active than superoxide ion. It was proposed that elevated reactivity of the oxygen radicals released by FA leukocytes may be a major factor in the development of Fanconi anemia; this proposal is supported by the first positive results of treatment of FA patients with rutin (a nontoxic natural free radical scavenger and chelator).

[Examination of the local antioxidative system of the eye in experimental corneal burn injury and the prospects for pharmacological correction of its parameters].

The present paper deals with the study of the efficiency of oral use of the antioxidative drug Immugen (a complex of alpha-tocopherol, oubichinone, selenium aspartate, methionine, and soyabean phospholipids) on a rabbit model of severe alkaline-induced corneal burn. The investigations have indicated that addition of Immugen to the rabbit feed exerts a significant positive effect on the parameters of the local antioxidative system of the eye and causes an increase in the activity of superoxide dismutase and catalase, and, on day 14, in antioxidative activity. The early experimental periods were marked by a slight rise in the frequency of deep corneal ulcerations. Moreover, the long-term clinical effect of use of Immugen appears as a significant increase in the area of the transparency-preserving affected cornea. The findings suggest that the antioxidants can show their optimal effect in the complex therapy for burn processes, including the use of proteinase inhibitors.

Autoxidation of dopamine: a comparison of luminescent and spectrophotometric detection in basic solutions.

Oxidation products of catecholamines, particularly dopamine, could play an important role in the physiology and pathology of the nervous system. This study has therefore characterised autoxidation of dopamine monitored in a variety of systems. Lucigenin-dependent chemiluminescence and reduction of cytochrome c were exploited to register generation of the byproduct superoxide anion, whereas the quinone product was detected by a direct spectrophotometric measurement. Accumulation of hydrogen peroxide was followed as an increase in luminol-dependent chemiluminescence. In all cases, basic solutions were used to initiate the oxidation of dopamine. The results obtained could be interpreted as specific reactions at the particular stages of the autoxidation process: the luminol-dependent chemiluminescence system detected accumulation of hydrogen peroxide during dopamine oxidation, whereas the lucigenin-dependent chemiluminescence indicated generation of superoxide anion. Furthermore, cytochrome c reduction, observed during dopamine oxidation, was probably attributed to a direct interaction with dopamine semiquinone. In addition, the effects of superoxide dismutase, catalase, and peroxidase were examined in each of the systems: Each enzyme exhibited a different effect in each system used. The possible reaction mechanisms leading to different action of enzymes affecting reactive oxygen species are discussed. The methods described here of monitoring dopamine autoxidation could thus be used in parallel to detect the effects of different preparations on various stages of the dopamine autoxidation process.

A possible interaction between acetylcholinesterase and dopamine molecules during autoxidation of the amine.

Acetylcholinesterase has an action in the central nervous system, independent of hydrolysis of acetylcholine. This study explored the possible interaction between the two molecules: the effects of acetylcholinesterase on the autoxidation of the catecholamine were tested, and, in turn, modification of the catalytic activity of the enzyme by products of dopamine oxidation were studied. Acetylcholinesterase selectively inhibited the speed of quinone production from dopamine as well as accumulation of hydrogen peroxide, whilst the rate of generation of superoxide was increased. Analysis of absorption spectra revealed the formation of a new product, which appeared after mixing acetylcholinesterase and dopamine in neutral pH. In all cases, butyrylcholinesterase was ineffective. Incubation of acetylcholinesterase in the presence of dopamine resulted in a significant decrease in the catalytic activity of the enzyme. The effects of application of preparations modifying autoxidation of dopamine (SOD, catalase, peroxidase) suggested that inactivation of the enzyme occurred as a result of the direct interaction of a quinone and/or semiquinone oxidation product with enzyme, as opposed to any effects of reactive oxygen species. Because acetylcholinesterase and dopamine are co-released from the neurons degenerating in Parkinson's disease, a direct chemical interaction between these two molecules could have significance both for the normal functioning of the substantia nigra and for related pathological states.

Effect of rutin and its copper complex on superoxide formation and lipid peroxidation in rat liver microsomes.

Two free radical scavengers, bioflavonoid rutin and the copper-rutin complex Cu(Rut)Cl2, inhibited lucigenin-amplified chemiluminescence and lipid peroxidation in rat liver microsomes, Cu(Rut)Cl2 being a 5-9 times more efficient inhibitor than rutin. The enhanced inhibitory activity of Cu(Rut)Cl2 was due to the presence of the additional superoxide-dismutating center (Cu), as follows from the comparison of its effects on microsomal chemiluminescence and cytochrome c reduction by xanthine oxidase. Similar effects of both inhibitors on superoxide production and lipid peroxidation as well as the elevated activity of Cu(Rut)Cl2 indicate an important role of superoxide ion in the initiation of microsomal lipid peroxidation.

Cytoskeleton alterations of erythrocytes from patients with Fanconi's anemia.

Fanconi's anemia (FA) is a very rare genetically heterogeneous disease which has been hypothesized to be defective in the detoxification of reactive oxygen species. In this work we report the results obtained by morphometric and biochemical analyses on the red blood cells (RBCs) from FA patients. With respect to RBCs from healthy donors the following changes have been detected: (i) a variety of ultrastructural alterations, mainly surface blebbing typical of acanthocytes and stomatocytes; (ii) a significant quantitative increase of these altered forms; (iii) modifications of spectrin cytoskeleton network; (iv) an altered redox balance, e.g. a decreased catalase activity and significant variations in the GSSG/GSH ratio. We hypothesize that remodeling of the redox state occurring in FA patients results in cytoskeleton-associated alterations of red blood cell integrity and function.

Membrane fluorescent probes for revealing lymphocyte population heterogeneity. II. T-and B-lymphocytes of human peripheral blood.

Lymphocytes of human peripheral blood were stained with a fluorescent probe 3-methoxybenzanthrone (MBA) and their fluorescence intensities measured using a cytofluorimeter. The lymphocytes are shown to be heterogeneous in fluorescence intensity. The fluorescence intensity of B-cells is 2-3 times higher than that of T-cells. Some data suggest that null-cells have an intermediate fluorescence intensity. Thus, with MBA it is possible to distinguish T- and B-lymphocytes in man.

Membrane fluorescent probes for the demonstration of lymphocyte population heterogeneity. I T. and B lymphocytes of mice and rats.

Non-fixed lymphocytes of rats and mice were stained with the membrane fluorescent probe, 3-methoxybenzanthrone. The probe is capable of binding preferentially with lymphocyte membranes and fluoresces in the green spectral region. Microfluorometry of single cells showed that lymphocytes differ in all lymphoid organs and these may be a 3-10-fold variation in fluorescence intensity. Lymphocytes can be divided into two groups according to fluorescence intensity: "bright" and "dim". The proportions of "bright" and "dim" cells were determined in rats and mice for various lymphoid organs in the normal state, after thymectomy and cyclophosphamide treatment, and also after lymphocyte separation on a nylon wool column. In all cases the proportion of bright lymphocytes corresponded to the B-cell content, and the proportion of dim lymphocytes corresponded to the content of T-cells.

Different antioxidant activities of bioflavonoid rutin in normal and iron-overloading rats.

The effects of rutin on liver microsomes, peritoneal macrophages, and blood neutrophils isolated from iron-overloading (IOL) and normal rats were studied. The formation of 2-thiobarbituric acid-reactive products and the level of lucigenin-amplified chemiluminescence (CL) were determined in liver microsomes. Oxygen radical production by phagocytes was measured by luminol- and lucigenin-amplified CL and superoxide dismutase-sensitive cytochrome c reduction. These ex vivo findings were compared with the in vitro effects of rutin on cellular free processes. It was found that rutin administration sharply suppressed free radical production in liver microsomes and by phagocytes of IOL animals and only slightly affected these processes in normal rats. The selective inhibitory effect of rutin under pathologic conditions induced by iron overload is thought to be due to the formation of inactive iron-rutin complexes which are unable to catalyse the conversion of superoxide ion into reactive hydroxyl radicals, a process responsible for the free radical-mediated toxic effects of iron overload. These findings may account for the favourable effects of the treatment of pathologies associated with iron overload with rutin.

Enhancement of antioxidant and anti-inflammatory activities of bioflavonoid rutin by complexation with transition metals.

The antioxidant and anti-inflammatory activities of two transition metal complexes of bioflavonoid rutin, Fe(rut)Cl(3) and Cu(rut)Cl(2), were studied. It was found that Cu(rut)Cl(2) was a highly efficient in vitro and ex vivo free radical scavenger that sharply decreased (by 2-30 times compared to the parent rutin): oxygen radical production by xanthine oxidase, rat liver microsomes, and rat peritoneal macrophages; the formation of thiobarbituric acid-reactive products in microsomal lipid peroxidation; and the generation of oxygen radicals by broncho-alveolar cells from bleomycin-treated rats. The copper-rutin complex was also a superior inhibitor of inflammatory and fibrotic processes (characterized by such parameters as macrophage/neutrophil ratio, wet lung weight, total protein content, and hydroxyproline concentration) in the bleomycin-treated rats. The antioxidant activity of Fe(rut)Cl(3) was much lower and in some cases approached that of rutin. Fe(rut)Cl(3) also stimulated to some degree spontaneous oxygen radical production by macrophages. We suggested that the superior antioxidant and anti-inflammatory activity of the copper-rutin complex is a consequence of its acquiring the additional superoxide-dismuting copper center. The inhibitory activity of Fe(rut)Cl(3) was lower, probably due to the partial reduction into Fe(rut)Cl(2) in the presence of biological reductants; however, similarly to the copper-rutin complex, this complex efficiently suppressed lung edema.

Psoralen-photosensitized damage of rat peritoneal exudate cells.

Psoralen-sensitized photodamage (PUVA) of rat peritoneal exudate cells was investigated. Quartz-activated luminol-dependent chemiluminescence (ChL) was registered and the amount of trypan-positive cells was determined. Irradiation of peritoneal exudate cells in the presence of psoralen resulted in a dose-dependent monotonous inhibition of ChL. The reciprocity law of irradiation intensity and duration of irradiation was not valid for the observed inhibition of ChL: the inhibition increased with higher intensity. When psoralen previously photooxidized in ethanol (POP) was added to peritoneal exudate cell suspension, a double-phase response depending on psoralen irradiation dose was obtained: ChL activation was observed at low doses of UVA, ChL inhibition at high doses. Chemiluminescence inhibition correlated well with the increase in the number of trypan-positive cells. It may be supposed that the observed effects of PUVA or POP treatment are caused by cell cytoplasmic membrane damage.

The role of oxidative stress in developmental and reproductive toxicity of tamoxifen.

The antiestrogen tamoxifen (TAM) is widely used as a drug against breast cancer and is currently being tested as a chemopreventive agent. However, a number of studies showed genotoxic and carcinogenic effects of TAM. These effects are thought to be related to oxygen radical overproduction which occurs during TAM metabolic activation. There is no evidence, thus far, on TAM toxicity to embryos and gametes. The present study was designed to elucidate the mechanisms of TAM-induced developmental, reproductive and cytogenetic toxicity towards sea urchin (SU) embryos with regard to the possibility of TAM-initiated oxidative stress. Embryo cultures from SU were subjected to long-term (throughout embryogenesis) or short-term (two hours) incubation with TAM at concentrations from 10(-8) to 10(-5) M. The experiments on TAM-induced toxicity to gametes were carried out with SU sperm, or unfertilized eggs, suspended in TAM (10(-8) to 10(-6) M). To assess the effects of TAM to embryos or to gametes, developmental defects, embryonic mortality, fertilization success, and cytogenetic abnormalities were scored. Oxidative damage to DNA and lipids was detected by measurements of 8OHdG levels and lipid peroxidation, respectively. Reactive oxygen species (ROS) production by eggs and embryos was recorded by luminol-dependent chemiluminescence (LDCL) and cytochrome c reduction methods. The changes in activities of SU superoxide dismutase (SOD) and catalase were also evaluated. TAM exerted: a) early embryonic mortality to exposed embryos and to the offspring of exposed eggs; b) developmental defects to the offspring of exposed sperm; c) decrease in sperm fertilization success, and d) cytogenetic effects in the offspring of exposed sperm or eggs. These morphological effects corresponded to the state of oxidative stress in SU embryos (increased oxidative damage to DNA and lipids and induction of antioxidant enzymes). Since TAM did increase significantly ROS production by embryos, it is suggested that TAM may be metabolically activated by SU embryonic oxidases and peroxidases, which in turn could be induced by TAM. The present study provides further support to the utilization of the SU system as a useful model to help elucidate mechanisms of chemical teratogenesis and carcinogenesis.

Effects of bio-normalizer (a food supplementation) on free radical production by human blood neutrophils, erythrocytes, and rat peritoneal macrophages.

Bio-normalizer, a natural Japanese health food prepared by the fermentation of Carica papaya, exhibits therapeutic properties against various pathologies including tumors and immunodeficiency. To understand the mechanism of bio-normalizer's therapeutic effects, we studied its action on the production of active oxygen species in cell-free systems (the Fenton reaction, the xanthine-xanthine oxidase system, and the hydrogen peroxide-hypochloride or hydrogen peroxide-horseradish peroxidase systems) and by human blood neutrophils and erythrocytes and rat peritoneal macrophages. Bio-normalizer efficiently inhibited the formation of oxygen radicals in cell-free systems and partly decreased spontaneous and menadione-stimulated superoxide production by erythrocytes, but manifested both stimulatory and inhibitory effects on oxygen radical release by dormant and activated phagocytes (neutrophils and macrophages). We suggest that bio-normalizer is able to enhance the intracellular production of innocuous superoxide ion and, at the same time, to diminish the formation of reactive hydroxyl radicals, perhaps by the inactivation of ferrous ions, the catalysts of the superoxide-driven Fenton reaction. We also propose that the normalization of an organism's superoxide level is one of the molecular mechanisms of bio-normalizer activity.

Peculiarities of the clastogenic properties of chrysotile-asbestos fibers and zeolite particles.

It has been established that chrysotile-asbestos fibers and zeolite particles induce chromosome aberrations in human lymphocytes from whole blood cultures, peritoneal fluid cells and bone marrow cells of mice. It is shown that the level of cytogenetic response from the intraperitoneal administration of chrysotile-asbestos fibers and zeolite particles depends on the time of their exposure. Further, it is shown that SOD eliminates the cytogenetic effect of chrysotile-asbestos fibers, while catalase inhibits that of zeolite particles. Recommendations concerning testing for the mutagenic properties of mineral fibers and particles are given, and possible mechanisms of their damaging effects are discussed.

Oxygen radical-mediated mutagenic effect of asbestos on human lymphocytes: suppression by oxygen radical scavengers.

The mutagenic effect of chrysotile asbestos fibers and zeolite and latex particles on human lymphocytes in whole blood has been studied. It was concluded that their mutagenic activities were mediated by oxygen radicals because they were inhibited by antioxidant enzymes (SOD and catalase) and oxygen radical scavengers (rutin, ascorbic acid, and bemitil). It was proposed that oxygen radicals were released by phagocytes activated upon exposure to mineral dusts and fibers. The study of lucigenin- and luminol-amplified chemiluminescence of peritoneal macrophages stimulated by chrysotile fibers and zeolite and latex particles has shown that their mutagenic action is probably mediated by different oxygen species, namely, by the iron-oxygen complexes (perferryl ions) plus hydrogen peroxide, hydrogen peroxide, and superoxide ion, respectively. From the oxygen radical scavengers studied, rutin was the most effective inhibitor of the mutagenic effect of mineral fibers and dusts.

Congenital disorders sharing oxidative stress and cancer proneness as phenotypic hallmarks: prospects for joint research in pharmacology.

In spite of very distinct genotypic assets, a number of congenital conditions include oxidative stress as a phenotypic hallmark. These disorders include Fanconi's anaemia, ataxia telangiectasia, xeroderma pigmentosum and Bloom's syndrome, as well as two frequent congenital conditions: Down's syndrome and cystic fibrosis. Cancer proneness is a clinical feature shared by these disorders, while other manifestations include early ageing, neurological symptoms or congenital malformations. The onset of oxidative stress has been related to excess formation, or defective detoxification, of reactive oxygen species (ROS). This can arise from either the abnormal expression or inducibility of ROS-detoxifying enzymes, or by defective absorption of nutrient antioxidants. Resulting oxidative injury has been characterized through: (i) DNA, protein or lipid oxidative damage; (ii) excess ROS formation (in vitro and ex vivo); (iii) sensitivity to oxygen-related toxicity; (iv) improvement of cellular defects by either hypoxia or antioxidants; and (v) circumstantial evidence for in vivo oxidative stress (as e.g. clastogenic factors). Investigations conducted so far have been confined to individual disorders. Comparative studies of selected indicators for oxidative stress could provide further insights into the pathogenesis of each individual condition. Such a unified approach may have wide-ranging consequences for studies of ageing and cancer.