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A4—Reactive oxygen species and mutagenesis
8
10th Russian Seminar on Oxygen Radicals
that peripheral erythrocytes of patients with hemolytic anemias or hemolytic states can be lysed by oxygen radicals. Some authors have emphasized that free radicals appear to be leukocytes killers in drug- and radiation-induced agranulocytosis. On the other hand, it is suggested that the molecular basis of hereditary diseases with the features of aplastic anemia such as Fanconi anemia, Bloom's syndrome, and Down's syndrome is the change of cellular superoxide dismutase (SOD) activity. We are especially interested in the development of analytical assays that can be used in small blood or bone marrow punctate samples obtained from children to monitor the free radical formation and utilization. In general, we have studied children affected by different hereditary aplastic anemias such as Fanconi and Diamond-Blackfan anemias, acquired aplastic anemias (idiopathic or irradiation-induced anemias), acute lympho- and myelo-leukemias, thalassemia, and other disorders connected with unstable hemoglobin. The next very important point is to study and to develop the antioxidants and chelators as drugs for treating such patients. It was found that some of these diseases were characterized by a sharply increased level of oxygen radical production by peripheral and bone marrow granulocytes (Fanconi and irradiation-induced anemias, myeloleukemia, thalassemia), which can be corrected by the long-term administration of rutin (bioflavonoid exhibiting the properties of an antioxidant as well as an iron chelator), of a mixture containing vitamin E and/3-carotene, and other oral and parenteral chelators. For example, after treatment with rutin of several Fanconi anemia patients for 2 or more years, significant improvement of their conditions, an increase in DNA stability, and the enhancement of the amount of colony-forming bone marrow cells in culture were observed. In contrast to Fanconi anemia, thalassemia leukocytes exhibited a reduced level of oxygen radical production. Nonetheless, the treatment ofthalassemia patients with oral and parenteral chelators normalized not only iron content but also the level of oxygen radical production. We also suppose that an increase in oxygen radical production by leukocytes of children irradiated during therapeutic treatment, or by accident, is one of the most likely risk factors for leukemia development. Therefore, we propose to use antioxidant vitamin administration in order to reduce the risk of future malignancies.
A3--Free Radical Lipoperoxidation During Atherosclerosis V. Z. Lankin Russia Cardiology Research Centre, Academy of Medical Science, Moscow, Russia Feeding of cholesterol (Ch) to rabbits and mini-pigs is followed by a decrease in the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSHpx), cytosolic antioxidative enzymes of liver. Simultaneously, an increase in the level of hydroxyphospholipids and a decrease in the 7aCh hydroxilase activity of liver microsomes was observed.
In patients with postinfarct cardiosclerosis, we found a sharp increase in the blood levels of primary and seconda~ products of lipoperoxidation, but the activity of blood GSHpx was drastically lowered. In our study, which was carried out in collaboration with our colleagues from Humboldt University in Germany (FEBS Lett., 1991) we investigated the lipid composition of human aortas with atherosclerotic lesions obtained at autopsy 1-3 h after death by a high performance liquid chromatography (HPLC) method. The major component of oxygenated Ch esters in the region ot aorta atherosclerotic plaques was hydroperoxyl-derivative of Ch linoleate, a major lipid component of these specimens. We found that unsaturated Ch esters oxygenated with a high rate during the incubation with mammalian lipoxygenase (LOX) (reticulocyte LOX) in vitro. Structure of the Ch-oxygenated products points at the possibility of their lipoxygenase origin. In the human aortas we observed a significant.decrease in the activity of SOD and GSHpx in the area of atherosclerotic lesions. Our findings enable us to explain a known fact of diminished prostacyclin production in the atherosclerotic aorta by accumulation of lipoperoxides inhibiting prostacyclin synthetase. Thus, intensified lipoperoxidation in atherosclerosis may be a direct cause of thromboses, which are the most frequent and severe event in this disease. Our observations are consistent with the view that antioxidative drugs may exhibit a good effect during treatment of atherosclerosis. In blood of patients with cardiosclerosis, we observed a drastic increase in the activity GSHpx during the 3 months of treatment with the antioxidant probucol. In our experiments, feeding oxidized Ch to rabbits led to rapid development of hypercholesterolemia and lipoidosis in liver and aorta, in contrast to the findings obtained with the group of animals fed by nonoxidized Ch. These findings support our assumption that not only Ch itself but its oxidation products formed in the tissues in situ or given with food, are likely to contribute substantially to atherogenesis. It seems unavoidable to conclude that experimental data obtained in our experiments indicates that atherosclerosis is a classical example of free radical pathology.
A4--Reactive Oxygen Species and Mutagenesis A. D. Durnev, N. V. Guseva, and S. B. Seredenin Research Institute of Pharmacology, Moscow, Russia Many observations indicate that the mutagenic effect of oxidative stress imposed by different mechanisms is mediated by reactive oxygen species. On the other hand, oxidative stress may be developed in mammalians under the action of chemical drugs or emotional impacts. By the use of the chemiluminescence method, it was shown that the drugs bleomycin and dioxidine; the alkylating agents photrin, trenimon, thio-TEPA; and the industrial pollutants chrysotil-asbcstos fibers and zeolite particles, stimulated the production of reactive oxygen species by phagocytosis in a concentration-dependent manner. The same concentrations of these agents induced an increase in the level of chromosomal aberrations in the cultures of whole human blood. Superoxide dismutase but not catalase
10th Russian Seminar on Oxygen Radicals diminished the clastogenic effect ofbleomycin, catalase and not superoxide dismutase diminished cytogenetic activity of dioxidine and trenimon and eliminated clastogenic effect of zeolite particles. Both enzymes prevent an increase in the amount of aberrant cells by photrin and chrysotile-asbestos fibers, but did not affect the clastogenicity ofthio-TEPA. In in vitro (cultured whole human blood) and in vivo (mice C57BL/6) experiments, mutagenic effects of chemical and corpuscular xenobiotics (thio-TEPA and trenimon were not studied) were dose-dependently weakened by the use of antioxidative drugs (1.4-benzodiazepine and 2-mercaptobenzimidasole derivatives, some flavonoids, and in some cases ascorbic acid). Exposition of inbred mice C57B1/6 and BALB/c in the open field (experimental model of emotional stress) causes a 4-5 fold increase of the level of chromosomal aberrations in bone marrow cells of C57B1/6 mice, but not in the cells of BALB/c mice. Moreover, the products of lipid peroxidation were registered in C57B1/6 mice in higher concentrations and for longer periods of time than in BALB/c mice. The data obtained suggest that oxygen reactive species play a significant role in mutagenic action of studied xenobiotics (except ofthio-TEPA) and that the mutagenic effect of emotional stress is mediated by the induction of lipid peroxidation processes.
A5--Active Oxygen and Plant Resistance to Infective Diseases A. A. Aver'yanov Research Institute ofPhytopathology, p/o B. Vyazemi, Moscow Region, Russia
Participation of active oxygen in antiinfective reactions of animals (namely in phagocytosis) is well established. Higher plants possess similar antioxidant defense systems. In the case of plants, this was stated by Pogosyan I and was supported experimentally in the study ofgossypol. This phytoalexin of cotton plants inhibits the development of causative fungus of wilt disease; the effect is completely due to superoxide generation. 2 Other phenols of plant origin, for example, caffeic, chlorogenic, and ferulic acids also generate superoxide in model systems and demonstrate fungitoxicity, which is dependent of O2"- and "OH) It was also proposed that many naturally occurring plant photosensitizers generated singlet oxygen, protecting tissues against microbial invasion. In the whole plants, inoculation leads to a fast rise in the chemically assayed superoxide formation, which is usually higher in resistant varieties than in susceptible ones. That was observed in potato inoculated with blight fungus, in tobacco inoculated with tobacco mosaic virus (TMV) and bacteria Pseudomonas syringae, in tomato infested with nematode Meloidogyne incognita, and in rice inoculated with blast disease. We showed that in rice leaf excretions active oxygen is formed in the amount toxic for the parasite fungus Pyricularia oryzae. 4 This fact may be an early indication of varietal immunity. The resistance induced by environmental conditions (intensive light or high temperature)
9
and the physiological status of plants also could be due to similarly expressed oxygen activation. Several fungicides against blast disease act as inducers of oxygen activation in plants. They are tricyclazol, phtalide, probenazol, 5,6 and newly designed metal-organic complex. 7 As oxygen activation is one of the factors of disease resistance, antioxidative substances of a host and a parasite may stimulate disease susceptibility. Thus, several sugars, which are OH" scavengers effectively protect fungal spores from the toxicity of leaf excretions and artificially generated hydroxyls. 8 We have found that the pigment melanin typical for P. oryzae has antioxidant and cell-protective properties towards any form of active oxygen. 9 Pigment mutations in fungus or the changes in melanin biosynthesis cause correlative changes in pathogenicity of fungus spores and their tolerance to oxidative stress. Therefore, antioxidative capacity of a parasite apparently determines its pathogenicity. For example, tricyclazol and phtalide inhibited melanin pigmentation and sensitized the fungus to damage in leafdiffusate and O2"--generating systems. Mechanism of the superoxide generation associated with disease resistance is unclear and supposedly is relevant to the reactions of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase or peroxidase. The process is induced by some elicitors of plant defense responses. 9 Besides direct toxic effects, active oxygen species is presumably involved in diverse antiinfective reactions such as hypersensitivity and synthesis of ethylene, phytoalexins, and lignin. REFERENCES
1. Pogosyan S. I. et al. (1978) Doklady Biophysics (Proceedings of the Academy of Sciences of the USSR) 239:57-60. 2. Aver'yanov A. A. et al. (1980) Biol. Sci. (In Russian). N8: 77-79. 3. Aver'yanov A. A., Lapikova V. P. (1985) Biology Bulletin (Izvestiya Akademii Nauk SSSR, Seriya Biologicheskaya). N4:352-357. 4. Aver'yanov A. A. et al. (1987a) Soviet Plant Physiology.34:301306. 5. Nikolaev O. N. et al. (1990) Soviet Plant Physiology. 37.'124130. 6. Nikolaev O. N. et al. (1991a) Soviet Plant Physiology. 1991. 38:375-381. 7. Nikolaev O. N. et al. (1991b) Doklady Botanical Sciences (Proceedings of the Academy of Sciences of the USSR). 316: 26-28. 8. Aver'yanov A. A., Lapikova V. P. (1989) Biochemistry-USSR. 54:1344-1349. 9. Aver'yanov A. A. et al. (1987b) Biochemistry-USSR. 52: 13341341.
A6--Free Radical Oxidative Reactions in Lipids of Plant Membranes M. N. Merzlyak Department of Cell Physiology and Immunology, Faculty of Biology, Moscow State University, Moscow, Russia
At present, many lines of evidence suggest that lipid peroxidation mediated by activated oxygen species is an important (and probably even a universal) mechanism of membrane damage operating in plants under stress conditions
10th Russian Seminar on Oxygen Radicals
that peripheral erythrocytes of patients with hemolytic anemias or hemolytic states can be lysed by oxygen radicals. Some authors have emphasized that free radicals appear to be leukocytes killers in drug- and radiation-induced agranulocytosis. On the other hand, it is suggested that the molecular basis of hereditary diseases with the features of aplastic anemia such as Fanconi anemia, Bloom's syndrome, and Down's syndrome is the change of cellular superoxide dismutase (SOD) activity. We are especially interested in the development of analytical assays that can be used in small blood or bone marrow punctate samples obtained from children to monitor the free radical formation and utilization. In general, we have studied children affected by different hereditary aplastic anemias such as Fanconi and Diamond-Blackfan anemias, acquired aplastic anemias (idiopathic or irradiation-induced anemias), acute lympho- and myelo-leukemias, thalassemia, and other disorders connected with unstable hemoglobin. The next very important point is to study and to develop the antioxidants and chelators as drugs for treating such patients. It was found that some of these diseases were characterized by a sharply increased level of oxygen radical production by peripheral and bone marrow granulocytes (Fanconi and irradiation-induced anemias, myeloleukemia, thalassemia), which can be corrected by the long-term administration of rutin (bioflavonoid exhibiting the properties of an antioxidant as well as an iron chelator), of a mixture containing vitamin E and/3-carotene, and other oral and parenteral chelators. For example, after treatment with rutin of several Fanconi anemia patients for 2 or more years, significant improvement of their conditions, an increase in DNA stability, and the enhancement of the amount of colony-forming bone marrow cells in culture were observed. In contrast to Fanconi anemia, thalassemia leukocytes exhibited a reduced level of oxygen radical production. Nonetheless, the treatment ofthalassemia patients with oral and parenteral chelators normalized not only iron content but also the level of oxygen radical production. We also suppose that an increase in oxygen radical production by leukocytes of children irradiated during therapeutic treatment, or by accident, is one of the most likely risk factors for leukemia development. Therefore, we propose to use antioxidant vitamin administration in order to reduce the risk of future malignancies.
A3--Free Radical Lipoperoxidation During Atherosclerosis V. Z. Lankin Russia Cardiology Research Centre, Academy of Medical Science, Moscow, Russia Feeding of cholesterol (Ch) to rabbits and mini-pigs is followed by a decrease in the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSHpx), cytosolic antioxidative enzymes of liver. Simultaneously, an increase in the level of hydroxyphospholipids and a decrease in the 7aCh hydroxilase activity of liver microsomes was observed.
In patients with postinfarct cardiosclerosis, we found a sharp increase in the blood levels of primary and seconda~ products of lipoperoxidation, but the activity of blood GSHpx was drastically lowered. In our study, which was carried out in collaboration with our colleagues from Humboldt University in Germany (FEBS Lett., 1991) we investigated the lipid composition of human aortas with atherosclerotic lesions obtained at autopsy 1-3 h after death by a high performance liquid chromatography (HPLC) method. The major component of oxygenated Ch esters in the region ot aorta atherosclerotic plaques was hydroperoxyl-derivative of Ch linoleate, a major lipid component of these specimens. We found that unsaturated Ch esters oxygenated with a high rate during the incubation with mammalian lipoxygenase (LOX) (reticulocyte LOX) in vitro. Structure of the Ch-oxygenated products points at the possibility of their lipoxygenase origin. In the human aortas we observed a significant.decrease in the activity of SOD and GSHpx in the area of atherosclerotic lesions. Our findings enable us to explain a known fact of diminished prostacyclin production in the atherosclerotic aorta by accumulation of lipoperoxides inhibiting prostacyclin synthetase. Thus, intensified lipoperoxidation in atherosclerosis may be a direct cause of thromboses, which are the most frequent and severe event in this disease. Our observations are consistent with the view that antioxidative drugs may exhibit a good effect during treatment of atherosclerosis. In blood of patients with cardiosclerosis, we observed a drastic increase in the activity GSHpx during the 3 months of treatment with the antioxidant probucol. In our experiments, feeding oxidized Ch to rabbits led to rapid development of hypercholesterolemia and lipoidosis in liver and aorta, in contrast to the findings obtained with the group of animals fed by nonoxidized Ch. These findings support our assumption that not only Ch itself but its oxidation products formed in the tissues in situ or given with food, are likely to contribute substantially to atherogenesis. It seems unavoidable to conclude that experimental data obtained in our experiments indicates that atherosclerosis is a classical example of free radical pathology.
A4--Reactive Oxygen Species and Mutagenesis A. D. Durnev, N. V. Guseva, and S. B. Seredenin Research Institute of Pharmacology, Moscow, Russia Many observations indicate that the mutagenic effect of oxidative stress imposed by different mechanisms is mediated by reactive oxygen species. On the other hand, oxidative stress may be developed in mammalians under the action of chemical drugs or emotional impacts. By the use of the chemiluminescence method, it was shown that the drugs bleomycin and dioxidine; the alkylating agents photrin, trenimon, thio-TEPA; and the industrial pollutants chrysotil-asbcstos fibers and zeolite particles, stimulated the production of reactive oxygen species by phagocytosis in a concentration-dependent manner. The same concentrations of these agents induced an increase in the level of chromosomal aberrations in the cultures of whole human blood. Superoxide dismutase but not catalase
10th Russian Seminar on Oxygen Radicals diminished the clastogenic effect ofbleomycin, catalase and not superoxide dismutase diminished cytogenetic activity of dioxidine and trenimon and eliminated clastogenic effect of zeolite particles. Both enzymes prevent an increase in the amount of aberrant cells by photrin and chrysotile-asbestos fibers, but did not affect the clastogenicity ofthio-TEPA. In in vitro (cultured whole human blood) and in vivo (mice C57BL/6) experiments, mutagenic effects of chemical and corpuscular xenobiotics (thio-TEPA and trenimon were not studied) were dose-dependently weakened by the use of antioxidative drugs (1.4-benzodiazepine and 2-mercaptobenzimidasole derivatives, some flavonoids, and in some cases ascorbic acid). Exposition of inbred mice C57B1/6 and BALB/c in the open field (experimental model of emotional stress) causes a 4-5 fold increase of the level of chromosomal aberrations in bone marrow cells of C57B1/6 mice, but not in the cells of BALB/c mice. Moreover, the products of lipid peroxidation were registered in C57B1/6 mice in higher concentrations and for longer periods of time than in BALB/c mice. The data obtained suggest that oxygen reactive species play a significant role in mutagenic action of studied xenobiotics (except ofthio-TEPA) and that the mutagenic effect of emotional stress is mediated by the induction of lipid peroxidation processes.
A5--Active Oxygen and Plant Resistance to Infective Diseases A. A. Aver'yanov Research Institute ofPhytopathology, p/o B. Vyazemi, Moscow Region, Russia
Participation of active oxygen in antiinfective reactions of animals (namely in phagocytosis) is well established. Higher plants possess similar antioxidant defense systems. In the case of plants, this was stated by Pogosyan I and was supported experimentally in the study ofgossypol. This phytoalexin of cotton plants inhibits the development of causative fungus of wilt disease; the effect is completely due to superoxide generation. 2 Other phenols of plant origin, for example, caffeic, chlorogenic, and ferulic acids also generate superoxide in model systems and demonstrate fungitoxicity, which is dependent of O2"- and "OH) It was also proposed that many naturally occurring plant photosensitizers generated singlet oxygen, protecting tissues against microbial invasion. In the whole plants, inoculation leads to a fast rise in the chemically assayed superoxide formation, which is usually higher in resistant varieties than in susceptible ones. That was observed in potato inoculated with blight fungus, in tobacco inoculated with tobacco mosaic virus (TMV) and bacteria Pseudomonas syringae, in tomato infested with nematode Meloidogyne incognita, and in rice inoculated with blast disease. We showed that in rice leaf excretions active oxygen is formed in the amount toxic for the parasite fungus Pyricularia oryzae. 4 This fact may be an early indication of varietal immunity. The resistance induced by environmental conditions (intensive light or high temperature)
9
and the physiological status of plants also could be due to similarly expressed oxygen activation. Several fungicides against blast disease act as inducers of oxygen activation in plants. They are tricyclazol, phtalide, probenazol, 5,6 and newly designed metal-organic complex. 7 As oxygen activation is one of the factors of disease resistance, antioxidative substances of a host and a parasite may stimulate disease susceptibility. Thus, several sugars, which are OH" scavengers effectively protect fungal spores from the toxicity of leaf excretions and artificially generated hydroxyls. 8 We have found that the pigment melanin typical for P. oryzae has antioxidant and cell-protective properties towards any form of active oxygen. 9 Pigment mutations in fungus or the changes in melanin biosynthesis cause correlative changes in pathogenicity of fungus spores and their tolerance to oxidative stress. Therefore, antioxidative capacity of a parasite apparently determines its pathogenicity. For example, tricyclazol and phtalide inhibited melanin pigmentation and sensitized the fungus to damage in leafdiffusate and O2"--generating systems. Mechanism of the superoxide generation associated with disease resistance is unclear and supposedly is relevant to the reactions of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase or peroxidase. The process is induced by some elicitors of plant defense responses. 9 Besides direct toxic effects, active oxygen species is presumably involved in diverse antiinfective reactions such as hypersensitivity and synthesis of ethylene, phytoalexins, and lignin. REFERENCES
1. Pogosyan S. I. et al. (1978) Doklady Biophysics (Proceedings of the Academy of Sciences of the USSR) 239:57-60. 2. Aver'yanov A. A. et al. (1980) Biol. Sci. (In Russian). N8: 77-79. 3. Aver'yanov A. A., Lapikova V. P. (1985) Biology Bulletin (Izvestiya Akademii Nauk SSSR, Seriya Biologicheskaya). N4:352-357. 4. Aver'yanov A. A. et al. (1987a) Soviet Plant Physiology.34:301306. 5. Nikolaev O. N. et al. (1990) Soviet Plant Physiology. 37.'124130. 6. Nikolaev O. N. et al. (1991a) Soviet Plant Physiology. 1991. 38:375-381. 7. Nikolaev O. N. et al. (1991b) Doklady Botanical Sciences (Proceedings of the Academy of Sciences of the USSR). 316: 26-28. 8. Aver'yanov A. A., Lapikova V. P. (1989) Biochemistry-USSR. 54:1344-1349. 9. Aver'yanov A. A. et al. (1987b) Biochemistry-USSR. 52: 13341341.
A6--Free Radical Oxidative Reactions in Lipids of Plant Membranes M. N. Merzlyak Department of Cell Physiology and Immunology, Faculty of Biology, Moscow State University, Moscow, Russia
At present, many lines of evidence suggest that lipid peroxidation mediated by activated oxygen species is an important (and probably even a universal) mechanism of membrane damage operating in plants under stress conditions