Concentrations Of Oxygen Radicals Research Articles

Induction of p21 Mediated by Reactive Oxygen Species Formed during the Metabolism of Aziridinylbenzoquinones by HCT116 Cells

Aziridinylbenzoquinones are a group of antitumor agents that elicit cytotoxicity by generating either alkylating intermediates or reactive oxygen species. The mechanism of toxicity may not always, however, involve profound damage of cellular constituents, but may involve a cytostatic effect through interference with the cell cycle. In this context, we have examined the induction of the cell cycle inhibitor p21 (WAF1, CIP1, or sdi1), whose overexpression suppresses the growth of various tumor cells, in human tumor cells metabolizing 3,6-diaziridinyl-1,4-benzoquinone (DZQ) and its C2,C5-substituted derivatives: 2,5-bis-(carboethoxyamino) (AZQ) and 2, 5-bis-2(-hydroxyethylamino) (BZQ). Both DZQ and AZQ were effectively activated by HCT116 human colonic carcinoma cells; the activation of the former involved largely a dicoumarol-sensitive activity, whereas that of the latter appeared to be accomplished primarily by one-electron transfer reductases. BZQ was not a substrate for the dicoumarol-sensitive enzyme in HCT116 cells. Cellular activation of the first two quinones was associated with formation of oxygen-centered radicals as detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide. The redox transitions of DZQ involved hydroxyl radical formation and were strongly inhibited by catalase, whereas those of AZQ showed a strong superoxide anion component sensitive to superoxide dismutase. These signals were suppressed by N-acetylcysteine with concomitant production of a thiyl radical adduct. This suggests an effective electron transfer between the thiol and free radicals formed during the activation of these quinones. DZQ and AZQ induced significantly the expression of p21 in HCT116 cells, but a 10-fold higher concentration of AZQ was required to achieve the level of induction elicited by DZQ. BZQ had little effect on p21 expression. p21 induction at both mRNA and protein levels correlated with the inhibition of either cyclin-dependent kinase activity or cell proliferation. p21 induction elicited by the above quinones was inhibited by N-acetylcysteine, whereas the non-sulfur analog, N-acetylalanine, was without effect. Catalase and superoxide dismutase did not effect p21 induction by aziridinylbenzoquinones in HCT116 cells, thus suggesting that extracellular sources of oxygen radicals generated by plasma membrane reductases have no influence in the expression of this gene. Hydrogen peroxide, a product of quinone redox cycling, elicited an increase of p21 mRNA levels in HCT116 and K562 human chronic myelogenous leukemia cells. The latter lacks p53, one of the activators of p21 transcription, thus suggesting that p21 expression can be accomplished in a p53-independent manner in these cells. This study suggests that p21 induction is mediated by an increase in the cellular steady-state concentration of oxygen radicals and that the greater effectiveness in p21 induction by DZQ may be related to its efficient metabolism by NAD(P)H:quinone oxidoreductase activity in HCT116 cells.

Radiation Protection and Radiation Recovery with Essential Metalloelement Chelates

Understanding essential metalloelement metabolism and its role in tissue maintenance and function, as well as the roles of essential metalloelement-dependent enzymes in responding to injury, offer a new approach to decreasing and/or treating radiation injury. This review presents the roles of some essential metalloelement-dependent enzymes in tissue maintenance and function, and their responses to radiation injury in accounting for radiation protection and recovery effects observed for nontoxic doses of essential metalloelement compounds. Effects of biochemicals including water undergoing bond radiolysis and the effects of free radicals derived from diatomic oxygen account for the acute and chronic aspects of radiation injury. Recognized biochemical roles of essential metalloelement-dependent enzymes and the observed pharmacological effects of small-molecular mass chelates predict the therapeutic usefulness of essential metalloelement complexes in decreasing and/or treatment of radiation injury. Copper chelates have radiation protection and radiation recovery activities and cause rapid recovery of immunocompetency and recovery from radiation-induced histopathology. Mice treated with Cu(II)2(3,5-diisopropylsalicylate)4[Cu (II)2(3,5-DIPS)4] had increased survival and corresponding increases in numbers of myeloid and multipotential progenitor cells early after irradiation and earlier recovery of immune reactivity. Examination of radiation-induced histopathology in spleen, bone marrow, thymus, and small intestine also revealed Cu(II)2(3,5-DIPS)4-mediated rapid recovery of radiation-induced histopathology. Most recently, Fe, Mn, and Zn complexes have also been found to prevent death in lethally irradiated mice. These pharmacological effects of essential metalloelement chelates can be understood as due to facilitation of de novo synthesis of essential metalloelement-dependent enzymes which have roles in preventing the accumulation of pathological concentrations of oxygen radicals or repairing biochemical damage caused by radiation-induced bond homolysis. Essential metalloelement chelates offer a physiological approach to prevention and/or treatment of radiation injury.

Heparins as essential antioxidants

Chronically inflamed tissues contain significant concentrations of oxygen radicals, possibly generated by transition metal species such as Fe(II) and Cu(I). In addition, the pH of such tissues may well be lower than that of normal counterpart tissues. In this communication, the effect of variation of pH on the antioxidant activity of caeruloplasmin (a copper-containing acute-phase protein possessing important ferroxidase and other antioxidant activities) and of heparin are reported. Heparin, unlike caeruloplasmin, retains antioxidant activity at low pH. Heparins possess a variety of metal cation-binding chemical groups allowing them to bind damaging Cu(I) and Fe(II) ions and consequently prevent their redox involvement in oxygen radical-generating Fenton reactions. In conclusion, heparins may function as endogenous antioxidants and sequestration and/or oxidation by them, of ions such as Fe(II), may contribute to their effectiveness.

Lyell's syndrome: treatment with Centoxin and selenium

Lyell's syndrome (LS) (toxic epidermal necrolysis) is also known as “scalded skin syndrome.” When caused by staphylococci, it has a favorable prognosis. However, as a drug reaction, primarily to sulfonamides, anticonvulsives, and antirheumatic agents, it often takes a fatal course. On the basis of a case report, the authors present a new therapeutic concept aimed at influencing the pathobiochemical changes underlying LS. After taking a single dose of a sulfonamide preparation for a mild urinary tract infection, a 14-year-old girl developed LS with skin changes covering almost the whole body surface. Within 12 h the child showed signs of sepsis with imminent organ failure, leuco- and thrombocytopenia, and symptoms of beginning enteroparalysis. Wholeblood chemiluminescence analyses revealed massive formation of reactive oxygen species. Parallel to this, a drastic increase in PMN-elastase and strong activation of the kallikrein-kinin system were found. Blood concentrations of glutathione peroxidase and reduced glutathione were extremely low. The therapeutic regimen was intended to prevent both endotoxinemia and overproduction of toxic oxygen metabolites: the girl received 100 μg monoclonal antibody against endotoxin (Centoxin) in combination with sodium selenite (Selenase). By day 2 her circulatory condition had stabilized and the blood concentration of oxygen radicals decreased. Neither liver nor kidney function showed pathological changes. Although on day 2 maximum activation of the kallikreinkinin system was observed, the expected drop in blood pressure did not occur. The blood concentration of elastate fell markedly, indicating that the release of elastase from neutrophil granulocytes, stimulated by endotoxin and complement, could be prevented. Both blood and plasma activities of glutathione peroxidase increased sharply as a consequence of the selenium therapy. On the 8th day of therapy all parameters had returned to normal (elastase, kallikrein-kinin, leucocytes, selenium). The whole-blood chemiliminescence response showed normal values and the skin changes had almost completely disappeared.

Role of nutrients in the cause and prevention of oxygen radical pathology.

Nutrients function in both the generation and catabolism of oxygen radicals, i.e., as oxidants and antioxidants. Experimental animals fed large excesses of prooxidant nutrients, such as polyunsaturated fatty acids and iron, develop pathologies that have been firmly linked to the formation of toxic concentrations of oxygen radicals in the tissues. Similar pathologies have been demonstrated to occur as a result of experimental deficiencies of some antioxidant nutrients, notably vitamin E and selenium. Clinical diseases resulting from dietary deficiencies of these nutrients also have been well documented in farm animals. There is evidence that pharmacological intakes of some antioxidants may reduce the risk of developing some clinical diseases associated with oxygen radical pathology in human subjects consuming a normal diet and ameliorate the tissue damage associated with others. It remains unclear, however, whether oxygen radical pathology occurs in humans as a result of inadequate or excessive intakes of nutrients from the general food supply.

Direct measurement of myocardial free radical generation in an in vivo model: Effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase

Objectives. The purpose of this study was to determine whether postischemic reperfusion of the heart in living rabbits induces a burst of oxygen free radical generation that can be attenuated by recombinant human superoxide dismutase administered at the moment of reflow.Background. This phenomenon was previously demonstrated in crystalloid perfused, globally ischemic rabbit hearts.Methods. Thirty-two open chest rabbits were assigned to one of four groups of eight animals each: Group I (control animals), no coronary artery occlusion; Group II, 30 min of circumflex marginal coronary artery occlusion without reperfusion; Group III, 30 min of coronary occlusion followed by 60 s of reperfusion, and Group IV, 30 min of coronary occlusion followed by treatment with recombinant human superoxide dismutase (a 20-mg/kg body weight bolus 90 s before reperfusion and a 0.17-mg/kg infusion during 60 s of reperfusion). Full thickness biopsy specimens taken from the ischemic region were then rapidly freeze clamped and electron paramagnetic resonance spectroscopy was performed at 77 °K.Results. Three radical signals similar to those previously identified in the isolated, crystalloid perfused rabbit heart were observed: an isotropic signal with g = 2.004 suggestive of a semiquinone, an anisotropic signal with gI= 2.033 and gI= 2.005 suggestive of an oxygen-centered alkyl peroxy radical, and a triplet with g = 2.000 and aN= 24 G suggestive of a nitrogencentered radical. In addition, a fourth signal consistent with an iron-sulfur center was seen. The oxygen-centered free radical concentration during normal perfusion (Group I) was 1.8 ± 0.8 μmol compared with 4.4 ± 0.9 μmol after 30 min of regional ischemia without reperfusion (Group II) and 13.0 ± 2.5 μmol after 60 s of reperfusion (Group III) (p < 0.05 among all three groups). In contrast, superoxide dismutase treated-rabbits (Group IV) demonstrated a peak oxygen radical concentration of only 5.9 ± 1.2 μmol (p < 0.05 vs. Group III).Conclusions. This study demonstrates that reperfusion after regional myocardial ischemia in the intact rabbit is associated with a burst of oxygen-centered free radicals. The magnitude of this burst is greater than that seen after a comparable duration of global ischemia in the isolated, buffer-perfused rabbit heart preparation and is significantly reduced by superoxide dismutase administration begun just before reflow.

Essential Metalloelement Metabolism and Radiation Protection and Recovery

Understanding the metabolism of essential metalloelements and their role in tissue maintenance and function as well as the roles of essential metalloelement-dependent enzymes in responding to injury offers a new approach to preventing and/or treating radiation injury. This review presents the roles of some essential metalloelement-dependent enzymes in the maintenance and function of tissues and their responses to radiation injury and gives an account of the observed effects of nontoxic doses of essential metalloelement compounds on protection against radiation damage and its recovery. The radiolysis of chemical bonds and free radicals derived from oxygen accounts for the acute and chronic aspects of radiation injury. The recognized biochemical roles of essential metalloelements and their observed pharmacological effects predict the therapeutic usefulness of essential metalloelement complexes in the prevention and/or treatment of radiation injury. Copper complexes have radiation protection and radiation recovery activities and cause rapid recovery of immunocompetence and radiation-induced damage to cells and tissues. Recently, iron, manganese, and zinc complexes have also been found to prevent death in lethally irradiated mice. These pharmacological effects of essential metalloelement complexes can be understood to be due to facilitation of de novo synthesis of essential metalloelement-dependent enzymes which have roles in preventing the accumulation of pathological concentrations of oxygen radicals or repairing damage caused by radiation-induced bond homolysis. Essential metalloelement complexes offer a physiological approach to prevention and/or treatment of radiation injury.

Nitroblue tetrazolium reduction and bone resorption by osteoclasts in vitro inhibited by a manganese-based superoxide dismutase mimic.

Oxygen-derived free radicals are produced by osteoclasts. Oxygen radical formation occurs at the osteoclast/bone surface interface. This location next to bone implies that oxygen radicals, including but not limited to superoxide, are needed for bone resorption. Compounds that scavenge superoxide are being developed as pharmaceutical agents to inhibit the damaging effects of oxygen radical formation on tissues. One such scavenger is the Desferal-manganese complex (DMnC). DMnC reduced the amount of formazan staining produced by the interaction of oxygen radicals with nitroblue tetrazolium (NBT) in both individual mouse calvarial osteoclasts in tissue explants and isolated osteoclasts. As a result of the reduced concentrations of oxygen radicals, DMnC inhibited bone resorption by calvarial explants and isolated osteoclasts. Superoxide dismutase (SOD) inhibited NBT reduction and bone resorption by isolated osteoclasts but to a lesser degree than DMnC. Inhibition of bone resorption in the isolated osteoclast system increased in parallel to the concentration of DMnC in cultures. Desferal without Mn had no effect on bone resorption by isolated osteoclasts. These results support the hypothesis that osteoclasts produce oxygen radicals as part of the process of bone resorption.

Oxygen Atom Induced Deposition of Silicon Dioxide

ABSTRACTSilicon dioxide was deposited from tetraethylorthosilicate (TEOS) and remote microwave oxygen plasma on a heated silicon substrate in a cold-wall reactor. The deposition rate and film quality were examined as functions of substrate temperature, total pressure, absorbed plasma power and O2:TEOS flow ratio. The deposition reaction exhibited an activation energy of approximately 10 kJ/mol for substrate temperatures in the range of 323–623 K. The deposition rate reached a maximum with increasing total pressure. The rate was found to be a near-linear function of the absorbed microwave power. At fixedabsorbed power the rate reached a maximum with increasing O2:TEOS flow ratio. A one-dimensional mathematical model was developed to predict the oxygen radical concentration at the exit of the afterglow region of the oxygen discharge. Comparisons of the predicted oxygen radical concentrations with the deposition rates at corresponding deposition conditions supports the view that the overall SiO2 deposition reaction is largely controlled by the concentration of oxygen radicals. The average refractive index ofthe deposited films was 1.466 ± 0.011. Fourier transform infra-red (FTIR) transmission spectra showed significant concentrations of hydroxyls in the deposited films.

Treatment with deferoxamine during ischemia improves functional and metabolic recovery and reduces reperfusion-induced oxygen radical generation in rabbit hearts.

Iron may play a central role in oxygen radical generation during myocardial ischemia and after reperfusion. Because conditions during ischemia may also liberate iron, we hypothesized that administration of the iron chelator deferoxamine during ischemia would result in improved functional and metabolic recovery after postischemic reperfusion. Isolated, perfused rabbit hearts were studied by phosphorus-31 nuclear magnetic resonance spectroscopy. The hearts received one of three treatments: deferoxamine at the onset of 30 minutes of global ischemia (n = 9), deferoxamine as a bolus followed by a continuous 15-minute infusion begun at reflow (n = 9), or standard perfusate (n = 7). Hearts treated with deferoxamine during ischemia showed better recovery of developed pressure than did control hearts (63.2 +/- 7.5% versus 41.2 +/- 2.9% of baseline) (p = 0.02) and better recovery of myocardial phosphocreatine content (92.4 +/- 10.3% versus 68.2 +/- 4.5% of baseline, p less than 0.05). These functional and metabolic benefits were comparable to those obtained with deferoxamine treatment during early reperfusion. In 15 additional hearts, intraischemic treatment with deferoxamine resulted in no reduction in oxygen radical concentrations as measured on frozen tissue by electron paramagnetic resonance spectroscopy at end ischemia, but the treatment eliminated the reperfusion-induced increase of free radical generation observed in control hearts (2.9 +/- 0.01 versus 7.0 +/- 0.07 microM, p less than 0.001). The magnitude of reduction was similar to that when deferoxamine was given at the onset of reflow (2.4 +/- 0.02 microM, p less than 0.001 versus control). These results demonstrate improved functional and metabolic recovery of myocardium treated with deferoxamine during ischemia, accompanied by a reduction in reperfusion-induced oxygen free-radical generation to the same degree as reflow treatment, confirming the importance of iron in the pathogenesis of myocardial reperfusion injury.

The effect of UV radiation and sun blockers on free radical defence in human and guinea pig epidermis

Defence against oxidative damage by UV-generated free radicals in both guinea pig and human skin has been found to be mediated by the ubiquitous thioprotein, thioredoxin reductase. Human keratinocytes contain approximately 5% thioredoxin reductase in their total acidic protein fraction and also express membrane-associated enzyme activity in cells cultured in synthetic medium. The thioredoxin reductase/thioredoxin system has been shown to reduce superoxide anion radicals through hydrogen peroxide to water. However, both UVA and UVB radiation, below the minimal erythemal dose, generate a sufficiently high concentration of oxygen radicals to deactivate thioredoxin reductase considerably. In albino guinea pigs, enzyme deactivation was up to 70% for UVA and 66% for UVB (n = 10 animals/protocol). The application of sun blockers SPF4, SPF8 and SPF15 to albino guinea pig skin offered no significant protection for the deactivation of thioredoxin reductase by either UVA or UVB radiation. In the human population (n = 15), thioredoxin reductase was deactivated by 54% with UVA and 36% with UVB radiation, although the degree of enzyme inhibition depended on skin phototype (I-VI, Fitzpatrick Classification). SPF24 offered considerable protection for thioredoxin reductase against both UVA and UVB for skin types I and II. However, SPF24 yielded no significant protection with UVA for skin types III-VI, and enhanced the enzyme inhibition with UVB additively. These results indicate that UVB photo-oxidation of oxybenzone (the UVA filter in SPF24) may deactivate thioredoxin reductase in more pigmented members of the population by Michael addition of oxybenzone semiquinone to the thiolate active site of this enzyme.

The Effect of Chronic Alcohol Ingestion on Free Radical Defense in the Miniature Pig

Cellular protection from free radical reactions was measured in hepatic tissue from controls and miniature pigs fed ethanol. The activities of copper-zinc superoxide dismutase (CuZnSOD) and glutathione peroxidase were lower and the activity of manganese superoxide dismutase (MnSOD) was higher in the pigs fed ethanol than in controls. Glutathione concentration was lower in the pigs fed ethanol than in controls. Ethanol consumption did not result in increased lipid peroxidation as assessed by thiobarbituric acid-reactive substances. The ethanol-induced effects on the activities of the superoxide dismutases may be a reflection of available metals and/or a response to increased hepatic concentration of oxygen radicals. Because the protection afforded by the glutathione system was altered, pigs fed ethanol may be at risk of peroxidative damage with continued ethanol exposure.

48 THE PARTICIPATION OF SELENIUM IN OXYGEN RADICAL PROTECTION OF THE HEART

Top of pageAbstract Selenium (Se) is an essential constituent of the peroxide metabolising enzyme glutathione peroxidase. Isolated perfused hearts from rats fed a Se-deficient diet were investigated. Glutathione peroxidase content in Se-deficient hearts was less than 5% of controls, and examination of ultrastructure revealed some areas with moderate cellular damage, mainly slight oedema of cells and mitochondrial changes. Se-deficient and control hearts were exposed for 11 min to a high and a low concentration of oxygen radicals generated by xanthine oxidase 0.0125 and 0.025 U/ml, and hypoxanthine 0.96 mM. With the low dose of oxygen radicals contractile function was gradually reduced, but in Se-deficient hearts the decrease was significantly more pronounced than in controls (32.7+6.5 vs 58.3±8.4% of initial values, mean±SEM). With the high dose of oxygen radicals contractility decreased abruptly in both groups, but a significantly lower tissue content of adenosine triphosphate was detected in the Se-deficient hearts. It is concluded that Se-deficiency reduces the tolerance of the heart to oxygen radicals.

RADIOMIMETIC TOXICITY OF OZONISED AIR

A simple way of detecting a morphological red cell effect of ozone in anlmals and in man was found, namely, the sphering of erythrocytes. Thus, the new red cell stants circulating as a flat disc and ends as a spherocyte, the change being promoted by exposure to ozone. It was also found that this change in shape of the suspended cells is greatiy speeded up by x irradiation in vitro, and that this radioinduced acceleration is sensitized by exposure of the animal to low ozone concentrations. It was shown that in the diluted blood of x- irradiated mice, rats, rabbits, and humans who breathed air containing 0.2 to 0.25 ppm of ozone (which can just be detected by smell) a sphering tendency of erythrocytes develops. A further direct demonstration of structural damage caused by ozone was found in the postmitotic nuclei of myocardial fibers of adult rabbits and mice that were exposed to 0.2 ppm of ozone during five hr per day for three weeks. They show rupture of the nuclear envelope and extrusion of nuclear contents which are never observed in the nuclei of normal fibers, but are very similar to nuclear irradiation effects in patients and in x-irradiatedmore » tumor cells. Inbred grey mice exposed to 0.1 or 0.2 ppm of ozone in air for 7 hr/day over three weeks had litters of normal size though the neonatal mortality in the first three weeks was 6.8 and 7.5%, against 1.6% in the controls. In the second litters of parents exposed to 0.1 ppm of ozone in air, this figure was 4.9%. In inbred C57 black mice exposed in the same way to 0.2 ppm of ozone the neonatal mortality in the first three weeks was 34%, against 9% in controls, It is concluded that exposure to low concentrations of ozone, such as produced by commercial ozonizers, is not innocuous. It is noted that health authorities are very much opposed to exposure to the smallest concentrations of oxygen radicals produced by iorizing radiation, whereas they are indifferent or they even encourage the use of an oxidant as powerful and as long-lived as ozone, which is copiously produced at home, in factories, and in hospitals, by so-called fresh air devices. (BBB)« less