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).

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.

[The mutagenic action of the dust of natural zeolites and chrysotile asbestos]. / Issledovanie mutagennogo deistviia pyli prirodnykh tseolitov i khrizotil-asbesta.

The cell chemiluminescence method was used to demonstrate the ability of asbest and zeolite dusts from 8 deposits of the USSR to induce generation of free oxygen radicals in the phagocytosing cells suspension. It has been found that asbest and zeolite (0.01 and 0.05 mg/ml) increase levels of cells with chromosome aberrations in human cell cultures. The cytogenetic effect of asbest was inhibited by superoxide dismutase (50 mg/ml). The damaging effect of zeolite was decreased by the pharmacological drug bemithyl (0.007-0.07 mM) and completely eliminated by catalase (20 mg/ml). The results obtained indicate that mutagenic effect of dust particles of asbest and zeolite is mediated by oxygen radicals.

[Determination of phospholipase A2 activity in liver mitochondria using fluorescent dye 1,8-anilinonaphthalene sulfonate]. / Opredelenie aktivnosti fosfolipazy A2 v mitokhondriiakh pecheni s pomoshch'iu fliuorestsentnogo krasitelia 1,8-anilinonaftalin sul'fonata.

Quantitative estimation of free fatty acids was carried out in liver mitochondria of normal and thyroidectomized rabbits by means of two procedures--spectrophotometry using rhodamine 6Zh and a fluorescent method with 1,8-aniline naphthalene sulfonate /ANS/ as a dye. The use of ANS enabled to estimate quantitatively the free fatty acids beginning from 25 nmol in a sample /with eggor of about 17%/ by visual evaluation of the spot areas. As compared with the rhodamine procedure, use of ANS permitted to increase 5-fold the sensitivity of free fatty acid estimation, to elevate 6-fold the accuracy of the analysis, to overcome the step of fatty acid elution from chromatogramms and to decrease the time required for analysis. The data obtained suggest that the hyperthyroid state in rabbits was accompanied by an increase in the phospholipase A2 activity in liver mitochondria.

[Chemiluminescent determination of superoxide dismutase activity]. / Khemiliuminestsentnoe opredelenie aktivnosti superoksiddismutazy.

A specific chemiluminescent method for the assessment of superoxide dismutase (SOD) activity in human blood has been developed. Intensive lucinogen-dependent chemiluminescence was observed during adrenalin oxidation in alkaline medium, and the level of this chemiluminescence essentially decreased in the presence of SOD. 50% inhibition of chemiluminescence was observed in the presence of 25 ng/ml of the enzyme, this indicating a high sensitivity of the method as against a similar spectrophotometric technique. SOD activities in donor blood samples were measured. The described method is offered for clinical application.

[Effect of endogenous lipid peroxides on respiration in rat liver mitochondria]. / Deivtvie Zndogennykh Perekisei Lipidou Na Dykhanie Mitokhondrii Pecheni Krys

Effect of Fe2+- and ascorbate-induced lipid peroxidation on mitochondrial respiration in 3 and 4 states (according to Chance) is studied. Peroxidation was shown to result in an increase of the oxygen uptake rate in 4th state, and in the KCl-containing medium (but not in sucrose medium) it caused a decrease of the oxygen uptake rate in the phosphorylating state, which partially reversed when cytochrom c was added. These effects were observed only after the development of peroxidation (after "slow" flash of chemiluminescence), and the value of and effect correlated with the content of peroxidation products unsaturated fatty acids. No considerable differences in the damaging effect of peroxidation were observed under incubation of mitochondria in KCl, NaCl and choline containing media.

Free radical generation during interaction of chrysotile asbestos with natural compounds.

Free radical generation was found after the addition of some natural biological substances (adrenaline, ascorbate, ubiquinone Q9, etc.) to a chrysotile asbestos suspension. This was detected by the chemiluminescent method with lucigenin as an indicator. The detailed study of the chemiluminescent reaction in the adrenaline-chrysotile system indicated that the reaction required hydroxyl ions, which arose in the chrysotile suspension, and was accompanied by a superoxide radical formation. At the same time, the radical production was very low in suspensions of amphybole asbestos, talc, and quartz, which could not alkalinize the water medium. On the basis of these results, it may be concluded that chrysotile has a unique ability to generate free radicals upon interaction with some biological molecules in a water medium. This fact may explain the great carcinogenicity of chrysotile. The injection of cigarette smoke solution into chrysotile (but not into amphibole asbestos or talc) suspension induced intensive chemiluminescence. This suggests that smoke aggravates the effect of chrysotile on human health by increasing free radical generation on the surface of the fibers.

Study of antioxidant properties of metal aspartates.

The effects of Zn, Mg, Cr, Cu, and Mn aspartates, their commercial formulation Inzolen, and the individual commercial medicine Unizinc, on oxygen radical production by enzymes [xanthine oxidase, horseradish peroxidase, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase] and phagocytic cells (human blood leukocytes) have been studied. The formation of oxygen radicals was measured by luminol- and lucigenin-amplified chemiluminescence and by the reduction of cytochrome c. All these compounds (excluding Cr aspartate) turn out to be inhibitors of oxygen radical formation in the systems studied (excluding horseradish peroxidase). Their inhibitory activities were a consequence of both the scavenging of free radicals and the inhibition of xanthine oxidase and NADPH oxidase activities. As expected, the most active free-radical scavengers were transition metal Cu and Mn aspartates, which mimicked the activities of copper-zinc and manganese dismutases. However, surprisingly non-transition metal Zn and Mg aspartates were also able to scavenge oxygen radicals. It was suggested that the scavenging activities of Zn and Mg aspartates may be explained by affecting the rate of spontaneous dismutation of the superoxide ion. In addition, it was found that Zn aspartate is an efficient inhibitor of the formation of the most reactive hydroxyl radicals. These antioxidant properties of Zn aspartate make it important in medicine for the prevention and treatment of free radical pathologies.

[Thyroid hormones and the electrical stability of rat liver mitochondrial membranes]. / Tireoidnye gormony i élektricheskaia stabil'nost' membran mitokhondri pecheni krys.

The electrical stability of mitochondrial membranes isolated from the liver of normal, hypothyroid, hyperthyroid (0.1 mg L-thyroxine per 100 g bw for 9 days) and thyrotoxic (4 mg L-thyroxine per 100 g bw for 6 days) rats was investigated. The electrical stability of mitochondrial membranes was estimated by the amount of potassium acetate required for electrical breakdown of the mitochondria. Of the four mitochondrial populations, the organelles of hyperthyroid and thyrotoxic animals had the most stable membranes, whereas the electrical stability of the mitochondria of normal and hypothyroid rats was approximately the same. It is assumed that the increased electrical stability of the mitochondria seen in hyperthyroid conditions is linked with the hormone-induced modifications in the composition of membrane phospholipids rather than with a direct action of excess thyroxine on the organelles.

[Chemoluminescence analysis of the functional state of blood phagocytes in silicosis and chronic dust-induced bronchitis]. / Khemoliuminestsentnyi analiz funktsional'nogo sostoianiia fagotsitov krovi pri silikoze i khronicheskom pylevom bronkhite.

Blood leukocyte function in patients with silicosis and chronic dust-induced bronchitis was examined by cellular chemiluminescence method for registration of free oxygen radical production. Significant differences in oxidation metabolism of blood neutrophils in the patients with these two nosologic entities were revealed. In silicosis blood phagocyte activation with silica particles in vitro activated production of active oxygen forms and chemiluminescent cells in all cases by 3 to 30 times. In chronic dust-induced bronchitis the production of active oxygen forms under the same conditions was either unchanged or enhanced but negligibly, the activation coefficient not surpassing 3. These data point to a new approach to the differential diagnosis between the above-mentioned conditions.