CuZn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in pancreatic islets and other tissues in the mouse.

Exogenous superoxide dismutase, catalase and scavengers of the hydroxyl radical protect pancreatic-islet cells against the toxic actions of alloxan in vitro [Grankvist et al. (1979) Biochem. J. 182, 17--25]. To test whether the extraordinary sensitivity of islet cells to alloxan is due to a deficiency of endogenous enzymes protecting against oxygen-reduction products, we assayed CuZn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in mouse islets and other tissues. To correct for any blood contamination, haemoglobin was also measured in the tissue samples. Pancreatic islets were found to belong to tissues with relatively little activity of the protective enzymes. However, the deviation from other tissues in this respect is probably not large enough to explain the especially great susceptibility of islet cells to alloxan.     

Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro.

Experiments with isolated pancreatic islets or dispersed islet cells from non-inbred ob/ob mice were performed to test the hypothesis that free radicals, notably OH., mediate the diabetogenic toxicity of alloxan. Accumulation of 86Rb+ by whole islets and exclusion of Trypan Blue by dispersed cells were used as previously validated criteria of islet-cell viability. Alloxan alone drastically inhibited the Rb+ accumulation and significantly decreased the frequency of cells excluding Trypan Blue. Enzymic scavengers of O2.- and H2O2 or non-enzymic scavengers of OH. or singlet oxygen were added to the incubation medium and tested for their ability to protect against these effects of alloxan. Superoxide dismutase, catalase, dimethyl sulphoxide, benzoate, and mannitol counteracted the effects of alloxan in both cytotoxicity assays. Significant protection of the Rb+-accumulating capacity was also afforded by butanol, caffeine, theophylline, NADH, NADPH and, to a small extent, NAD+. Urea has a poor affinity for OH. and did not protect against alloxan. No effect was obtained with the singlet-oxygen scavenger, histidine. Except for the protection by NADH and NADPH, which may be due to a direct reaction with alloxan in the medium, the results strongly support the hypothesis. beta-Cells may be particularly vulnerable to alloxan because their metabolic specialization facilitates reduction of the drug and perhaps of other substrates for O2.--yielding redox cycles.     

Protection by superoxide dismutase, catalase, and poly(ADP-ribose) synthetase inhibitors against alloxan- and streptozotocin-induced islet DNA strand breaks and against the inhibition of proinsulin synthesis

We have shown previously that alloxan and streptozotocin, two major diabetogenic agents, cause DNA strand breaks in rat pancreatic islets and stimulate nuclear poly(ADP-ribose) synthetase, thereby depleting intracellular NAD level and inhibiting proinsulin synthesis (Okamoto, H. (1981) Mol. Cell. Biochem. 37, 43-61; Yamamoto, H., Uchigata, Y., and Okamoto, H. (1981) Nature 294, 284-286). In the present study, superoxide dismutase and catalase, scavengers of radical oxygens, were found to protect against islet DNA strand breaks and inhibition of proinsulin synthesis induced by alloxan. The radical scavengers did not affect islet DNA strand breaks or inhibition of proinsulin synthesis induced by streptozotocin. On the other hand, compounds that inhibit islet nuclear poly(ADP-ribose) synthetase were found to protect against alloxan- as well as streptozotocin-induced inhibition of proinsulin synthesis. The poly(ADP-ribose) synthetase inhibitors were ineffective in protection against DNA strand breaks induced by the agents. These results may provide an important clue for elucidating the prevention of insulin-dependent diabetes as well as for understanding the cause of diabetes.     

The role of oxygen radicals in dye-mediated photodynamic effects in Escherichia coli B

Photosensitive dyes representative of the thiazines, xanthenes, acridines, and phenazines mediated phototoxicity in Escherichia coli B. The observed phototoxicity was sensitizer-, light-, and oxygen-dependent and is therefore a photodynamic effect. Hydroxyl radical scavengers conferred protection against the photodynamic action of all of the representative dyes. The extent of protection was dependent on the concentration of scavenger and on the in vitro reactivity of the scavenger with the hydroxyl radical. Exogenous superoxide dismutase and catalase partially protected the cells against the dye-mediated phototoxicity, and prior induction of intracellular superoxide dismutase and catalase by growth in glucose minimal medium containing manganese and paraquat substantially protected E. coli B against the photodynamic action of all of the dyes examined. Combinations of protective treatments against the phototoxicity of all four classes of dyes, including superoxide dismutase and catalase preinduction and addition of extracellular superoxide dismutase and catalase or the addition of hydroxyl radical scavengers, provided nearly complete protection against the oxygen-dependent component of dye-mediated lethality. E. coli B grown in glucose minimal medium containing manganese and photosensitive dyes induced manganese superoxide dismutase. The extent of induction was correlated with the dyes' ability to photooxidize NADH in vitro. Thus, oxygen radicals are primarily responsible for the oxygen-dependent toxicity of the photosensitive dyes examined, and one adaptive response of E. coli B to a dye-mediated oxidative threat is to induce superoxide dismutase.     

Relative importance of cellular uptake and reactive oxygen species for the toxicity of alloxan and dialuric acid to insulin-producing cells

The diabetogenic agent alloxan is selectively accumulated in insulin-producing cells through uptake via the GLUT2 glucose transporter in the plasma membrane. In the presence of intracellular thiols, especially glutathione, alloxan generates "reactive oxygen species" (ROS) in a cyclic reaction between this substance and its reduction product, dialuric acid. The cytotoxic action of alloxan is initiated by free radicals formed in this redox reaction. Autoxidation of dialuric acid generates superoxide radicals (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), and finally hydroxyl radicals ((*)OH). Thus, while superoxide dismutase (SOD) only reduced the toxicity, catalase, in particular in the presence of SOD, provided complete protection of insulin-producing cells against the cytotoxic action of alloxan and dialuric acid due to H(2)O(2) destruction and the prevention of hydroxyl radical ((*)OH) formation, indicating that it is the hydroxyl radical ((*)OH) which is the ROS ultimately responsible for cell death. After selective accumulation in pancreatic beta cells, which are weakly protected against oxidative stress, the cytotoxic glucose analogue alloxan destroys these insulin-producing cells and causes a state of insulin-dependent diabetes mellitus through ROS-mediated toxicity in rodents and in other animal species, which express this glucose transporter isoform in their beta cells.     

Diethyldithiocarbamate, but not disulfiram, inhibits alloxan-induced dye accumulation of isolated mouse islet beta-cells

The diabetogenic action of alloxan on pancreatic beta-cells is thought to be mediated by hydroxyl radicals. The initial attack of the radicals is probably at the plasma membrane level. Diethyldithiocarbamate (DDTC) and its dimer disulfiram (Antabuse) have recently been shown to protect against damage by free radical generating agents. The ability of DDTC and disulfiram to inhibit alloxan-induced dye accumulation of isolated ob/ob mice islet beta-cells was therefore studied. Evans blue was used as an indicator of plasma membrane permeability. DDTC (100 microM 1 mM) but not disulfiram (100 microM 1 mM) inhibited alloxan-induced dye uptake of beta-cells. The effect of DDTC on oxygen consumption in a mixture of reduced glutathione (GSH), alloxan and FeSO4 was studied with a Clark-type oxygen electrode. DDTC (20, 100 microM) had no effect on the oxygen consumption of this mixture. It is suggested that the DDTC inhibition of alloxan-induced dye uptake of isolated beta-cells takes place at a step beyond the generation of free radicals.     

In vitro and in vivo protective effect of Ganoderma lucidum polysaccharides on alloxan-induced pancreatic islets damage

This study was undertaken to investigate the protective effect against alloxan-induced pancreatic islets damage by Ganoderma lucidum Polysaccharides (Gl-PS) isolated from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. In vitro, alloxan caused dose-dependent toxicity on the isolated pancreatic islets. Pre-treatment of islets with Gl-PS for 12 h and 24 h significantly reversed alloxan-induced islets viability loss. Gl-PS was also found to inhibit the free radicals production induced by alloxan in the isolated pancreatic islets using confocal microscopy. Gl-PS dose-dependently increased serum insulin and reduced serum glucose levels when pretreated intragastrically for 10 days in alloxan-induced diabetic mice. It was found that the pancreas homogenates had higher lipid peroxidation products in alloxan-treated mice than in the Gl-PS-treated animals. Aldehyde fuchsin staining revealed that alloxan caused nearly all the beta cells disappearing from the pancreatic islets, while Gl-PS partly protected the beta cells from necrosis. Alloxan (60 mg/kg) induced NF-kappa B activation in the pancreas at 30 min after injection, pretreatment with Gl-PS inhibited alloxan-induced activation of NF-kappa B. These results suggest that Gl-PS was useful in protecting against alloxan-induced pancreatic islets damage in vitro and in vivo; one of the mechanisms is through its scavenging ability to protect the pancreatic islets from free radicals-damage induced by alloxan.     

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.     

Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B.

Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c. The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase. Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol. This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline. Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. The addition of superoxide dismutase and catalase to the incubation medium partially protected E. coli B against the light-dependent lethality. Preinduction of intracellular superoxide dismutase and catalase substantially protected E. coli B against the phototoxicity of tetracyclines. Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality. Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity. The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical. These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E. coli B.     

The involvement of superoxide and iron ions in the NADPH-dependent lipid peroxidation in human placental mitochondria

Incubation of human term placental mitochondria with Fe2+ and a NADPH-generating system initiated high levels of lipid peroxidation, as measured by the production of malondialdehyde. Malondialdehyde formation was accompanied by a corresponding decrease of the unsaturated fatty acid content. This NADPH-dependent lipid peroxidation was strongly inhibited by superoxide dismutase and singlet oxygen scavengers, markedly stimulated by paraquat, but was not affected by hydroxyl radical scavengers. Catalase enhanced the production of malondialdehyde by placental mitochondria. The effects of catalase and hydroxyl radical scavengers suggest that the initiation of NADPH-dependent lipid peroxidation is not dependent upon the hydroxyl radical produced via an iron-catalyzed Fenton reaction. These studies provide evidence that hydrogen peroxide strongly inhibits NADPH-dependent mitochondrial lipid peroxidation. The inhibitory effect of superoxide dismutase and stimulatory effect of paraquat, which was abolished by the addition of superoxide dismutase, suggests that superoxide may promote NADPH-dependent lipid peroxidation in human placental mitochondria.     

Comparison of the ability of ferric complexes to catalyze microsomal chemiluminescence, lipid peroxidation, and hydroxyl radical generation

The interaction of microsomes with iron and NADPH to generate active oxygen radicals was determined by assaying for low level chemiluminescence. The ability of several ferric complexes to catalyze light emission was compared to their effect on microsomal lipid peroxidation or hydroxyl radical generation. In the absence of added iron, microsomal light emission was very low; chemiluminescence could be enhanced by several cycles of freeze-thawing of the microsomes. The addition of ferric ammonium sulfate, ferric-citrate, or ferric-ADP produced an increase in chemiluminescence, whereas ferric-EDTA or -diethylenetriaminepentaacetic acid (detapac) were inhibitory. The same response to these ferric complexes was found when assaying for malondialdehyde as an index of microsomal lipid peroxidation. In contrast, hydroxyl radical generation, assessed as oxidation of chemical scavengers, was significantly enhanced in the presence of ferric-EDTA and -detapac and only weakly elevated by the other ferric complexes. Ferric-desferrioxamine was essentially inert in catalyzing any of these reactions. Chemiluminescence and lipid peroxidation were not affected by superoxide dismutase, catalase, or competitive hydroxyl radical scavengers whereas hydroxyl radical production was decreased by the latter two but not by superoxide dismutase. Chemiluminescence was decreased by the antioxidants propylgallate or glutathione and by inhibiting NADPH-cytochrome P-450 reductase with copper, but was not inhibited by metyrapone or carbon monoxide. The similar pattern exhibited by ferric complexes on microsomal light emission and lipid peroxidation, and the same response of both processes to radical scavenging agents, suggests a close association between chemiluminescence and lipid peroxidation, whereas both processes can be readily dissociated from free hydroxyl radical generation by microsomes.     

Evidence of direct toxic effects of free radicals on the myocardium

The hypothesis that oxygen-derived free radicals do indeed play a role in myocardial ischemic and reperfusion injury has received a lot of support. Experimental results have shown that free radical scavengers can protect against certain aspects of myocardial ischemic injury and that on reperfusion the heart approaches a level that is more normal than those hearts not receiving additional scavenging agents. Superoxide dismutase, catalase, glutathione peroxidase, hydroxyl radical scavengers and iron chelators such as desferrioxamine have proven successful in providing an increased level of recovery. These results indicate, as would be expected, that superoxide, hydrogen peroxide and hydroxyl radicals may all, at some point, either contribute to the injury or be important in generating a subsequent radical which causes damage. In addition, solutions capable of generating free radicals have been shown to cause damage to myocardial cells and the vascular endothelium that is similar to the damage observed during myocardial ischemic and reperfusion injury. Alterations in function, structure, flow, and membrane biochemistry have been documented and compared to ischemic injury. The continued investigation of the role of free radicals in ischemic injury is warranted in the hope of further elucidating the mechanisms involved in free radical injury, the sources of their generation, and in defining a treatment that will provide significant protection against this particular aspect of ischemic damage.     

Autoxidation reactions of hemoglobin A free from other red cell components: a minimal mechanism

Rates of autoxidation reactions are determined for normal human hemoglobin A preparations which are extensively purified to remove all other redox active red cell components. The effects of superoxide dismutase, catalase, and hydroxyl radical scavengers on the reaction provide evidence for superoxide formation as the rate determing step followed by fast reactions that involve peroxide and hydroxyl radical. These results support a minimum overall mechanism for heme iron(II) oxidation and dioxygen reduction to water. Side reactions also occur that result in the modification and precipitation of the protein moiety; catalase and hydroxyl radical scavengers reduce the extent of the side reactions. These studies provide insight into the basis of oxidant stress in the red cell.     

Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase

Yeast glutathione reductase catalyzes an NADPH-dependent reduction of the herbicide paraquat in vitro. The single-electron reduced paraquat radical reacts with O2 to generate the superoxide radical, O2.-. Hydroxyl radicals (OH.) can also be detected in this assay system by their reaction with phenol to form diphenols, as assayed quantitatively by a highly specific and sensitive method employing gas-liquid chromatography. Formation of hydroxyl radicals can be virtually completely suppressed by catalase and partially suppressed by superoxide dismutase. The role of hydroxyl radicals and superoxide in paraquat toxicity in vivo is discussed.     

Bleomycin-dependent damage to the bases in DNA is a minor side reaction.

The antitumor antibiotic bleomycin degrades DNA in the presence of ferric ions and H2O2 or in the presence of ferric ions, oxygen, and ascorbic acid. When DNA degradation is measured as formation of base propenals by the thiobarbituric acid assay, it is not inhibited by superoxide dismutase and scavengers of the hydroxyl radical or by catalase (except that catalase inhibits in the bleomycin/ferric ion/H2O2 system by removing H2O2). Using the technique of gas chromatography/mass spectrometry with selected-ion monitoring, we show that DNA degradation is accompanied by formation of small amounts of modified DNA bases. The products formed are identical with those generated when hydroxyl radicals react with DNA bases. Base modification is significantly inhibited by catalase and partially inhibited by scavengers of the hydroxyl radical and by superoxide dismutase. We suggest that the bleomycin-oxo-iron ion complex that cleaves the DNA to form base propenals can decompose in a minor side reaction to generate hydroxyl radical, which accounts for the base modification in DNA. However, hydroxyl radical makes no detectable contribution to the base propenal formation.     

Hydroxyl radical-mediated, cytochrome P-450-dependent metabolic activation of benzene in microsomes and reconstituted enzyme systems from rabbit liver

The mechanism of benzene oxygenation in liver microsomes and in reconstituted enzyme systems from rabbit liver was investigated. It was found that the NADPH-dependent transformation of benzene to water-soluble metabolites and to phenol catalyzed by cytochrome P-450 LM2 in membrane vesicles was inhibited by catalase, horseradish peroxidase, superoxide dismutase, and hydroxyl radical scavengers such as mannitol, dimethyl sulfoxide, and catechol, indicating the participation of hydrogen peroxide, superoxide anions, and hydroxyl radicals in the process. The cytochrome P-450 LM2-dependent, hydroxyl radical-mediated destruction of deoxyribose was inhibited concomitantly to the benzene oxidation. Also the microsomal benzene metabolism, which did not exhibit Michaelis-Menten kinetics, was effectively inhibited by six different hydroxyl radical scavengers. Biphenyl was formed in the reconstituted system, indicating the cytochrome P-450-dependent production of a hydroxycyclohexadienyl radical as a consequence of interactions between hydroxyl radicals and benzene. The formation of benzene metabolites covalently bound to protein was efficiently inhibited by radical scavengers but not by epoxide hydrolase. The results indicate that the microsomal cytochrome P-450-dependent oxidation of benzene is mediated by hydroxyl radicals formed in a modified Haber-Weiss reaction between hydrogen peroxide and superoxide anions and suggest that any cellular superoxide-generating system may be sufficient for the metabolic activation of benzene and structurally related compounds.     

Hydroxylation of aromatic compounds by reduced nicotinamide-adenine dinucleotide and phenazine methosulphate requires hydrogen peroxide and hydroxyl radicals, but not superoxide.

1. A mixture of NADH and phenazine methosulphate hydroxylates aromatic compounds at acidic pH values. 2. Hydroxylation is inhibited by catalase and by scavengers of the hydroxyl radical (-OH) but not by superoxide dismutase. 3. It is concluded that neither O2 leads to nor HO2- is sufficiently reactive to hydroxylate aromatic rings.     

Mitomycin C-induced deoxyribose degradation inhibited by superoxide dismutase. A reaction involving iron, hydroxyl and semiquinone radicals

Mitomycin C stimulates deoxyribose degradation with the release of thiobarbituric acid-reactive material under conditions of low oxygen concentration. This damage is inhibited by scavengers of the hydroxyl radical, iron chelators and the specific proteins catalase and superoxide dismutase. The reactive radical species appears to arise from a Fenton-type sequence in which iron is reduced by the mitomycin C semiquinone radical.     

Alloxan cytotoxicity in vitro. Inhibition of rubidium ion pumping in pancreatic beta-cells.

Exposing micro-dissected pancreatic islets of non-inbred ob/ob mice to 2-5 mM-alloxan for 10 min decreased the ability of the islets to accumulate Rb+. Rb+ accumulation in pieces of exocrine pancreas was unaffected by alloxan. When islets were treated with alloxan in the presence of 2-20 mM-D-glucose, the Rb+-accumulating ability was protected in a dose-dependent manner. The protective action of D-glucose was reproduced with 3-O-methyl-D-glucose but not with L-glucose or D-mannoheptulose; mannoheptulose prevented D-glucose from exerting its protective action. The inhibition of Rb+ accumulation was due to a decreased inward pumping, since alloxan did not affect Rb+ efflux from pre-loaded islets. The inhibitory effect of alloxan had a latency of about 1 min, as revealed by experiments with dispersed islet cells in suspension. Alloxan-treated islets showed only a marginal decrease in ATP and no change in glucose 6-phosphate concentration. Although alloxan slightly decreased the hydrolysis of ATP in a subcellular fraction enriched in plasma membranes, this effect could not be attributed to a ouabain-sensitive adenosine triphosphatase. The plasma membranes exhibited a K+-activated hydrolysis of p-nitrophenyl phosphate; this enzyme activity too was insensitive to alloxan. Glucose may protect the univalent-cation pump by preventing permeation of alloxan via a path coupled to the hexose-transport system. Inhibition of the pump may be fundamental to the induction of alloxan-diabetes.     

Prevention of doxorubicin-induced killing of MCF-7 human breast cancer cells by oxygen radical scavengers and iron chelating agents

This study investigated the effect of oxygen radical scavengers and iron chelating agents on the toxicity of doxorubicin for MCF-7 human breast cancer cells. Superoxide dismutase and catalase, but not the heat-inactivated enzymes, the hydroxyl radical scavenger N-acetylcysteine, and the organoselenium compound 2-phenyl-1-2-benzisoselenazol-3(2H)-one, which possesses glutathione peroxidase-like activity, significantly reduced or abolished tumor cell killing by doxorubicin. Similar protective activity was found only for those iron chelating agents capable of penetrating the tumor cell plasma membrane. These experiments suggest that an iron-dependent oxygen radical cascade contributes to the antineoplastic action of the anthracycline antibiotic doxorubicin.