Despite numerous theories, the etiology and pathogenesis of primary varicose veins remain unclear. The etiology of chronic venous diseases (CVDs) known as chronic venous insufficiency (CVI) is related to leukocyte trapping. Leukocyte trapping involves trapping of white cells in vessel walls followed by their activation and translocation outside the vessel. Release of reactive oxygen species (ROS) from trapped white cells has been documented. Superoxide dismutase (SOD) directly inhibits the generation of free radicals and compounds that are produced during oxidation by ROS, such as malonyldialdehyde (MDA). The aim of this study was to determine the involvement of free radicals in the etiology of venous changes. The following material was used for the study: fragments of sufficient or insufficient venous system and varices from 31 patients diagnosed with chronic venous disease in the 2nd or 3rd degree, according to clinical state, etiology, anatomy and pathophysiology (CEAP), which were qualified for surgical procedure. The levels of oxidative stress markers strongly correlated with lesions observed by USG in insufficient and varicose veins. In both a higher concentration of MDA was observed, which is a sign of lipid peroxidation. Antioxidative mechanisms, SOD activity and total antioxidative power expressed as FRAP were inversely proportional to MDA concentration. In insufficient and varicose veins both FRAP and SOD activities were significantly lower than in normal veins. The severity of clinical changes was inversely dependent on the efficiency of scavenging of ROS, which additionally proves the participation of free radicals in pathogenesis of CVDs.
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The aim of the study was an evaluation of changes in protein level and activity of SOD isoenzymes, and the participation of AP-1 and NF-κB in subsequent stages of colorectal cancer development. Studies were conducted on 65 colorectal cancers. Controls were unchanged colon regions. Activity of SOD isoenzymes, lipid peroxidation level (TBARS), and protein level of SOD1, SOD2, AP-1 and NF-κB were determined. We found that the protein level and activity of SOD isoenzymes and protein level of AP-1 and NF-κB change in subsequent stages of clinical advancement of colorectal cancer, according to UICC (I-IV), and in grades of tumor cells differentiation (G1-G3). These results indicate adaptation of colorectal cancer cells to oxidative stress, and show that the observed changes of SOD activity and protein level depend on gradual progression of colorectal cancer, and suggest an impairment of processes regulated by AP-1 and NF-κB which are critical for tumor progression (proliferation, differentiation and apoptosis).
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Colorectal cancer (CRC) is a serious medical and economical problem of our times. It is the most common gastrointestinal cancer in the world. In Poland, the treatment and detection of CRC are poorly developed and the pathogenesis is still unclear. One hypothesis suggests a role of reactive oxygen species (ROS) in the pathogenesis of CRC. Experimental studies in recent years confirm the participation of ROS in the initiation and promotion of CRC.The aim of the study was to examine the effect of the following coordination compounds coordination compounds: dinitrate (V) tetra(3,4,5-trimethyl-N1-pyrazole-κN2) copper(II), dichloro di(3,4,5-trimethyl-N1-pyrazole-κN2) copper(II), dinitrate (V) di(1,4,5-trimethyl-N1-pyrazole-κN2) copper(II), dichloro di(1,3,4,5-tetramethyl-N1-pyrazole-κN2) copper(II) on the activity of antioxidant enzymes superoxide dismutase (SOD, ZnCu-SOD) and catalase (CAT) in a group of patients with colorectal cancer (CRC) and in the control group consisting of patients with minor gastrointestinal complaints.Material and methods. The study was conducted in 20 patients diagnosed with colorectal cancer at the age of 66.5±10.2 years (10 men and 10 women) versus the control group of 20 people (10 men and 10 women) aged 57.89±17.10 years without cancer lesions in the biological material - hemolysate prepared in a proportion of 1ml of water per 1 ml of blood. CAT activity was measured by the Beers method (1952), while SOD activity was measured by the Misra and Fridovich method (1972).Results. We found that patients with CRC showed a statistically significant decrease of SOD and CAT activity (CAT - 12,75±1.97 U/g Hb, SOD - 1111.52±155.52 U/g Hb) in comparison with the control group (CAT - 19.65±2,17 U/g Hb, SOD - 2046.26±507.22 U/g Hb). Simultaneously, we observed that the investigated coordination compounds of Cu(II) significantly increased the antioxidant activity of CAT and SOD in patients with CRC (mean: CAT 25.23±4.86 U/g Hb, SOD - 3075.96±940.20 U/g Hb).Conclusions. Patients with colorectal cancer are characterized by reduced activity of antioxidant enzymes catalase and superoxide dismutase which suggests impaired antioxidant barrier. Therefore, coordination compounds of Cu (II), which enhance the activity of CAT and SOD, may prove useful in the prevention and treatment of colorectal cancer.
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A biosynthetic human insulin precursor displayed enhanced susceptibility to deamidation at one particular site. The present study was undertaken to monitor progress of precursor deamidation at successive manufacturing stages. MALDI-TOF/TOF MS in combination with controlled endoproteinase Glu-C and endoproteinase Asp-N proteolysis was used for rapid and unambiguous determination of deamidated residue within the investigated structure. Close inspection of isotopic distribution patterns of peptides resulting from enzymatic digestion enabled determination of distinct precursor forms occurring during the production process. Asn, Asp, isoAsp and succinimide derivatives of the amino acid at position 26 were unambiguously identified. These modifications are related to the leader peptide of a precursor encompassing amino acid sequence corresponding to that of superoxide dismutase [Cu-Zn] (SOD1 1, EC=1.15.1.1). Monitoring of precursor deamidation process at successive manufacturing stages revealed that the protein folding stage was sufficient for a prominent replacement of asparagine by aspartic and isoaspartic acid and the deamidated human insulin precursor constituted the main manufactured product. Conversion proceeded through a succinimide intermediate. Significant deamidation is associated with the presence of SNG motif and confirms results achieved previously on model peptides. Our findings highlight an essential role of the specific amino acid sequence on accelerated rate of protein deamidation. To our knowledge, this is the first time that such a dramatic change in the relative abundance of Asp and isoAsp resulting from protein deamidation process is reported.
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Mutants of Saccharomyces cerevisiae devoid of Cu,Zn-superoxide dismutase are hypersensitive to a range of oxidants, hyperbaric oxygen and hyperosmotic media, show lysine and methionine auxotrophy when grown under the atmosphere of air and have a shortened replicative life span when compared to the wild-type strain. Ascorbate and other antioxidants can ameliorate these defects, which may be a basis of simple tests sensing the presence of antioxidants. In particular, tests of growth on solid medium (colony formation) in the absence of methionine and/or lysine, or in the presence of 0.8 M NaCl can be useful for detection and semiquantitative estimation of compounds of antioxidant properties. Hypoxic atmosphere was found to increase the sensitivity of detection of antioxidants. The test of abolishment of lysine auxotrophy showed a concentration dependence of the antioxidant effects of cysteine and N-acetylcysteine which, however, lost their protective action at high concentration, in contrast to glutathione which was effective also at higher concentrations.
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Cadmium (Cd), similarly to other heavy metals, inhibits plant growth. We have recently showed that Cd2+ either stimulates (1-4 μM) or inhibits (ł 6 μM) growth of soybean (Glycine max L.) cells in suspension culture (Sobkowiak & Deckert, 2003, Plant Physiol Biochem. 41: 767-72). Here, soybean cell suspension cultures were treated with various concentrations of Cd2+ (1-10 μM) and the following enzymes were analyzed by native electrophoresis: superoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APOX). We found a significant correlation between the cadmium-induced changes of soybean cell culture growth and the isoenzyme pattern of the antioxidant enzymes. The results suggest that inhibition of growth and modification of antioxidant defense reactions appear in soybean cells when Cd2+ concentration in culture medium increases only slightly, from 4 to 6 μM.
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Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (rs ~0.27; p=0.004 and rs =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in diminishing the effects of alcohol- and smoking-related oxidative stress.
INTRODUCTION: Reactive oxygen species (ROS), which cause increased degradation of nitric oxide and defects in endothelial function, play an essential role in the pathogenesis of increased vascular contractility. Reactive oxygen species are produced in the vascular bed by endothelial cells, smooth muscle cells and fibroblasts. The destructive effect of ROS is of great importance to the pathogenesis of many diseases such as atheromatosis, ischemia, heart fail-ure, cerebral insufficiency and respiratory failure. MATERIAL AND METHODS: A total of 36 patients (31 females, 5 males; mean age 47 7 years) with connective tissue diseases (systemic lupus erythematosus – 23 patients, systemic sclerosis – 13 patients) and 40 (37 females, 3 males) healthy volunteers (mean age 50 2 years) of both sexes were enrolled in the study. This study evaluated the total antioxidant capacity in serum, using the method of reducing radical DPPH and radical ABTS•+. RESULTS: The blood plasma of the patients had lower antioxidant activities compared to the blood plasma of the healthy controls. CONCLUSIONS: This study shows the differences in the antioxidant activity in healthy volunteers and patients with connective tissue diseases. These parameters can be monitored during treatment by modulating oxidative metabolism and by stimulating antioxidant activity.
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WSTĘP: Reaktywne formy tlenu zwiększają degradację tlenku azotu i upośledzają funkcję śródbłonka, odgrywają ponadto istotną rolę w patogenezie zwiększonej kurczliwości naczyń. Produkowane są przez komórki śródbłonka łożyska naczyniowego, komórki mięśni gładkich oraz fibroblasty. Destrukcyjny wpływ reaktywnych form tlenu ma duże znaczenie w patogenezie wielu chorób, takich jak: miażdżyca, niedokrwienie, niewydolność serca, niewydolność naczyń mózgowych oraz niewydolność oddechowa. MATERIAŁ I METODY: W grupie badanej było 36 pacjentów (31 kobiet i 5 mężczyzn; średnia wieku 47 ± 7 lat) z chorobą tkanki łącznej (23 osoby z toczniem rumieniowatym układowym oraz 13 osób z twardziną układową), w grupie kontrolnej 40 zdrowych ochotników (37 kobiet i 3 mężczyzn; średnia wieku 50 2 lata). W pracy oceniono całkowitą zdolność antyoksydacyjną w surowicy, wykorzystując metodę redukcji rodnika DPPH i rodnika ABTS•+. WYNIKI: U pacjentów z chorobą układową tkanki łącznej aktywność antyoksydacyjna osocza jest mniejsza w porównaniu z grupą zdrowych ochotników. WNIOSKI: W pracy wykazano różnice w aktywności antyoksydacyjnej między zdrowymi ochotnikami a pacjentami z chorobą tkanki łącznej. Parametry te mogą być monitorowane podczas terapii przez modulowanie metabolizmu oksydacyjnego i stymulację aktywności antyoksydantów.
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Activities of superoxide dismutase, catalase and glutathione peroxidase in erythrocytes of cystic fibrosis children were studied in order to estimate the severity of their deficiency. Our results point to increased susceptibility of erythrocytes of cystic fibrosis subjects to oxidative injury and indicate that the antioxidant status of patients should be carefully monitored.
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A correlation is known to exist in yeast and other organisms between the cellular resistance to stress and the life span. The aim of this study was to examine whether stress treatment does affect the generative life span of yeast cells. Both heat shock (38°C, 30 min) and osmotic stress (0.3 M NaCl, 1 h) applied cyclically were found to increase the mean and maximum life span of Saccharomyces cerevisiae. Both effects were more pronounced in superoxide dismutase-deficient yeast strains (up to 50% prolongation of mean life span and up to 30% prolongation of maximum life span) than in their wild-type counterparts. These data point to the importance of the antioxidant barrier in the stress-induced prolongation of yeast life span.
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Previously, a stable cell suspension of cucumber tolerant to 100 µM CdCl2 was obtained (Gzyl & Gwóźdź, 2005, Plant Cell Tissue Organ Cult 80: 59-67). In this study, the relationship between the activity of antioxidant enzymes and cadmium tolerance of cucumber cells was analyzed. A cadmium-sensitive and the cadmium-tolerant cell lines were exposed to 100 µM and 200 µM CdCl2 and the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APOX) and guaiacol peroxidase (POX) were determined. In the sensitive cell line, a decrease of total activity of SOD and POX was observed, whereas the activity of CAT and APOX significantly increased in metal-supplemented medium. By contrast, in the tolerant cells, the total activity of antioxidant enzymes decreased (SOD, CAT) or was maintained at approximately the same level (APOX, POX). Moreover, a different pattern of isoenzyme activity was observed in the tolerant and sensitive cells. These results suggest that an enhanced activity of antioxidant enzymes is not directly involved in the increased tolerance to cadmium of the selected cucumber cell line.
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The present study investigated the potential role of selected antioxidant enzymes: superoxide dismutase (SOD) and peroxidase (POX) as biochemical markers of the impact of tropospheric ozone. Experiments were carried out in ambient air conditions with two tobacco cultivars: Bel W3 and Bel B, which are sensitive and resistant to ozone, respectively. In this study, the degree of leaf injury of the sensitive cultivar was used as an indicator of the ozone level in correlation to the enzyme activity of both tobacco cultivars. In spite of low levels of tropospheric ozone during experimental season, the increase of antioxidant enzyme (SOD and POX) activity concomitant with the increase of ozone concentration was noticed in the sensitive cultivar as well as in the resistant one. This observation is especially important for the resistant tobacco, which does not exhibit any visual effects of ozone influence. Our results could be extrapolated to other plant species (i.e. Poaceae, Fabaceae, Solanaceae, Betulaceae, Salicaceae, Pinaceae), which do not reveal visible lesions in response to ozone stress.
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Lead, similar to other heavy metals and abiotic factors, causes many unfavorable changes at the subcellular and molecular levels in plant cells. An increased level of superoxide anion in Pisum sativum root cells treated with 1 mM Pb(NO3)2 evidenced oxidative stress conditions. We found increased activities of enzymatic components of the antioxidative system (catalase and superoxide dismutase) in the cytosol, mitochondrial and peroxisomal fractions isolated from root cells of Pisum sativum grown in modified Hoagland medium in the presence of lead ions (0.5 or 1 mM). Two isoenzyme forms of superoxide dismutase (Cu,Zn-SOD and Mn-SOD) found in different subcellular compartments of pea roots were more active in Pb-treated plants than in control. Increased amount of alternative oxidase accompanied by an increased activity of this enzyme was found in mitochondria isolated from lead-treated roots. These results show that plants storing excessive amounts of lead in roots defend themselves against the harmful oxidative stress caused by this heavy metal.
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Iron and copper toxicity has been presumed to involve the formation of hydroxyl radical (·OH) from H2O2 in the Fenton reaction. The aim of this study was to verify that Fe2+-O2 and Cu+-O2 chemistry is capable of generating ·OH in the quasi physiological environment of Krebs-Henseleit buffer (KH), and to compare the ability of the Fe2+-O2 system and of the Fenton system (Fe2+ + H2O2) to produce ·OH. The addition of Fe2+ and Cu+ (0-20 μM) to KH resulted in a concentration-dependent increase in ·OH formation, as measured by the salicylate method. While Fe3+ and Cu2+ (0-20 μM) did not result in ·OH formation, these ions mediated significant ·OH production in the presence of a number of reducing agents. The ·OH yield from the reaction mediated by Fe2+ was increased by exogenous Fe3+ and Cu2+ and was prevented by the deoxygenation of the buffer and reduced by superoxide dismutase, catalase, and desferrioxamine. Addition of 1 μM, 5 μM or 10 μM Fe2+ to a range of H2O2 concentrations (the Fenton system) resulted in a H2O2-concentration-dependent rise in ·OH formation. For each Fe2+ concentration tested, the ·OH yield doubled when the ratio [H2O2]:[Fe2+] was raised from zero to one. In conclusion: (i) Fe2+-O2 and Cu+-O2 chemistry is capable of promoting ·OH generation in the environment of oxygenated KH, in the absence of pre-existing superoxide and/or H2O2, and possibly through a mechanism initiated by the metal autoxidation; (ii) The process is enhanced by contaminating Fe3+ and Cu2+; (iii) In the presence of reducing agents also Fe3+ and Cu2+ promote the ·OH formation; (iv) Depending on the actual [H2O2]:[Fe2+] ratio, the efficiency of the Fe2+-O2 chemistry to generate ·OH is greater than or, at best, equal to that of the Fe2+-driven Fenton reaction.
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Zbadano wpływ słabego wolnozmiennego pola magnetycznego stosowanego w magnetostymulacji na aktywność dysmutazy ponadtlenkowej (SOD) w krwinkach czerwonych oraz na aktywność ceruloplazminy w surowicy krwi. Doświadczenie przeprowadzono na próbkach pełnej krwi ludzkiej i surowicy krwi pochodzącej od 12 zdrowych mężczyzn. Pełną krew i surowicę krwi, pochodzącą od tej samej osoby, rozdzielono na trzy próbki: jedną eksponowano na pole o indukcji maksymalnej 270 μT, drugą na 100 μT, trzecia nie była eksponowana (próba kontrolna). W próbkach krwinek czerwonych otrzymano zmniejszoną aktywność dysmutazy ponadtlenkowej po ekspozycji krwi pełnej na pole magnetostymulacyjne w porównaniu z kontrolą (p < 0,05). Otrzymana zmiana aktywności SOD może być wyjaśniona wpływem pola magnetycznego na stany spinowe wolnych rodników. W próbkach eksponowanych na pole magnetyczne zaobserwowano wzrost aktywności ceruloplazminy w surowicy krwi (p < 0,05), w porównaniu z wynikami grupy kontrolnej. Podwyższoną aktywność ceruloplazminy w surowicy krwi można tłumaczyć oddziaływaniem na nieskompensowane spiny magnetyczne paramagnetycznych jonów miedzi wchodzących w skład enzymu. Aktywności enzymów w próbkach poddanych działaniu pola o dwu różnych wartościach: 270 μT oraz 100 μT nie różniły się istotnie między sobą.
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The influence of the weak frequency magnetic field used in magnetostimulation on the activity of the superoxide dismutase (SOD) in the red cells of blood and the activity of the ceruloplasmin of the serum blood have been studied. The experiments were made on the samples of the full human blood and blood serum obtained from 12 healthy males - blood donors. The full blood and blood serums from the same donor were divided into three samples. One sample was exposed to the magnetic field with peak value 270 μT, second sample was exposed to the field of 100 μT. The third sample was not exposed and was used as a control. In samples of the red blood cells a statistically significant (p < 0.05) reduction of the superoxide dismutase (SOD) activity was observed after the exposure to the magnetic field compared with the control sample. The lowered superoxide dismutase (SOD) activity may indicate the reduction of the creation of the free radicals under the influence of magnetic field. Increased ceruloplasmine activity in the blood serum was observed (p < 0.05) compared with the results for the control group. The magnetic field acting on the uncompensated magnetic spins of the copper ion included in the enzyme may trigger a reaction with the participation of ceruloplasmine.
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Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37°C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O2-dependent. The formation of H2O2 as an intermediate and the thiol group in GSH seem to be important in this reaction.
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Parasites are designed by evolution to invade the host and survive in its organism until they are ready to reproduce. Parasites release a variety of molecules that help them to penetrate the defensive barriers and avoid the immune attack of the host. In this respect, particularly interesting are enzymes and their inhibitors secreted by the parasites. Serine-, aspartic-, cysteine-, and metalloproteinases are involved in tissue invasion and extracellular protein digestion. Helminths secrete inhibitors of these enzymes (serpins, aspins, and cystatins) to inhibit proteinases, both of the host and their own. Proteinases and their inhibitors, as well as helminth homologues of cytokines and molecules containing phosphorylcholine, influence the immune response of the host biasing it towards the anti-inflammatory Th2 type. Nucleotide-metabolizing enzymes and cholinesterase are secreted by worms to reduce inflammation and expel the parasites from the gastrointestinal tract. An intracellular metazoan parasite, Trichinella spiralis, secretes, among others, protein kinases and phosphatases, endonucleases, and DNA-binding proteins, which are all thought to interfere with the host cellular signals for muscle cell differentiation. Secretion of antioxidant enzymes is believed to protect the parasite from reactive oxygen species which arise from the infection-stimulated host phagocytes. Aside from superoxide dismutase, catalase (rarely found in helminths), and glutathione peroxidase (selenium-independent, thus having a poor activity with H2O2), peroxiredoxins are probably the major H2O2-detoxifying enzymes in helminths. Secretion of antioxidant enzymes is stage-specific and there are examples of regulation of their expression by the concentration of reactive oxygen species surrounding the parasite. The majority of parasite-secreted molecules are commonly found in free-living organisms, thus parasites have only adapted them to use in their way of life.
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