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Nitric oxide, an inhibition of lipid oxidation by lipoxygenase and cyclooxygenase
Inflammation
15.17
CHARACTERIZATION OF A NOVEL, CYTOTOXIC OXIDANT PRODUCED FROM THE INTERACTION BETWEEN SUPEROXIDE (02-) AND HYPOTHIOCYANOUS ACID (HOSCN). Matthew B. Grisham, Christine Volkmer and Tamaki Yamada. Department of Physiology, LSU Medical Center, Shreveport, LA. 71130. Saliva contains a potent antimicrobial system composed of salivary peroxidase (SPO), hydrogen peroxide (H202), and thiocyanate anion (SCN-). SPO catalyzes the oxidation of SCN- by H202 to yield the potent oxidant HOSCN. Because large numbers of activated neutrophils are present within the gingiva and the oral cavity during chronic gingival inflammation it is possible that some of the tissue injury associated with this condition may be mediated by secondary oxidants generated from the interaction between granulocyte-derived 02- and salivary HOSCN. Thus, the objective of this study was to characterize the oxidizing and cytotoxic properties of O2-, HOSCN and the secondary oxidants produced from the interaction between O 2- and HOSCN. We found that the addition of potassium 02- (KO2; 85 #M) to solutions containing HOSCN and catalase at pH 6.0 increased the number of oxidizing equivalents from the 57 +_ 3 #M originally present as HOSCN to 185 _+ 6 #M. Omission of either KO 2 or HOSCN eliminated the formation of the secondary oxidant. This novel oxidant did not degrade deoxyribose suggesting that hydroxyl radical was not produced. Furthermore this potent oxidizing agent exhibited unusual stability with greater than 50% of its oxidizing activity remaining after a 30 min at 37"C. Finally, we found that the addition of KO 2 to HOSCN in the presence of catalase enhanced the cytotoxicity of 50 #M HOSCN toward cultured epithelial cells from 6 +_ 1% to 66 + 4% (p<0.01). Our data suggest that granulocyte-derived O~- interacts with salivary HOSCN to yield a potent oxidizing agent that may mediate injury to the oral tissues.
15,19HYDROGEN PEROXIDE-INDUCED
HEPATOCYTE DAMAGE IN RAT HEPATOCYTE / KUPFFER CELL COCULTURE Regine Kahl, Herbert Desel, Gregor Latocha, and Andrea Kastien Department of Clinical Pharmacology, University of GOttingen, D-3400 G0ttingen, West Germany Cultured rat hepatocytes are remarkably resistent to exogenous hydrogen peroxide. H202 at 1 mM does not result in substantial pro£uciion of malondialdehyde (MDA) and release of lactate dehydrogenase (LDH). At i0 mM H202, MDA release amounts to 1-2 fmoles/cell and k s terminated within the first hour. LDH release only starts when H202 concentration has dropped below 0.i mM via an azide-sensitive elimination process with a half life of 2.5 min. At 180 min, 1-2 ~U/cell have been released.
In a hepatocyte / Kupffer c e l l coculture ( I : i ) . gross hepatocyte damage as determined by LDH release is not observed even a f t e r stimulation of the Kupffer cells with zymosan or phorbol myristate acetate (PMA). Under these conditions, 0.5-1 fmoles MDA/cell were released at 60 min. The toxicity of exogenous H202 is markedly enhanced by the presence of the Kupffer cells. In a typical duplicate experiment with i0 mM H202, MDA production in the coculture at 60 min was 6.5 fmoles/cell as compared to 1.5 fmoles/cell in the absence of Kupffer cells. The amount of LDH released after 180 min was also markedly increased. These data indicate that a Kupffer cell-derived compound sensitizes the hepatocyte to exogenously added H202 . The identity of the mediator(s) involved zs at present under investigation.
143
SUPEROXIDE ANION GENERATION IN POLYMORPHONUCLEAR LEUKOCYTES OBTAINED FROM RATS WITH GALACTOSAMINE-INDUCED HEPATIC INJURY Yuji Hino, Ryukichi Kumashiro, Michio Sata, and Kyuichi Tanikawa Second Department of Internal Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830 Japan
15.18
Superoxide anions have been known to induce damages in cultured vascular endothelial cells. Injury to the endothelia is an important process leading to liver cell necrosis. In our present study, the effect of superoxide anions generated by polymorphonuclear leukocytes (PMNs) obtained from galactosamine-treated rats on sinusoidal endothelial cells was discussed. Wistar male rats (body weight: 200 g) received intraperitoneally a dose of i000 mg/kg D-galactosamine HCI. PMNs were isolated from peripheral blood by the dextran sedimentation technique and percoll gradient centrifugation at 24 hr after galactosamine administration. The generation of superoxide anions in PMNs stimulated by phorbor miristate acetate (PMA) was determined by reduction of cytochrome C. PMNs in galactosamine-treated rats indicated a remarkable ten fold generation of superoxide anion, as compared with those in untreated control rats. As the galactosamine-treated rats were characterized with hepatic dysfunctions, it is suggested that the free radical generation will be helpful in explaining the pathogenesis of severe acute hepatic injury including dysfunction of sinusoidal endothelial cells.
NITRIC OXIDE, AN INHIBITOR OF LIPID OXIDATION BY LIPOXYGENASE AND CYCLOOXYGENASE loseph Kanner, Rina Granit and Bezalel A k i r i Department of Food Science, ARO, The Volcani I n s t i t u t e , PO Box 6, Bet Dagan 50250, ISRAEL, Recent discovery has shown that n i t r i c oxide is an important mammalian metabolite. The main function of NO. seems to be the stimulation of the soluble guanylate cyclase to elevate cGF~ p. N i t r i c oxide is thought to activate soluble guanylate cyclase through an interaction with iron atom in the heme of the enzyme. The system activates a v a r i e t y of biological actions which include endothelium-dependent relaxation, vascular tone, platel e t aEEregation and adhesion. Our study demonstrated that n i t r i c oxide i n h i b i t s lipoxygenase, cyclooxygenase and hemoglobin toward l i p i d oxidation, which was determined by coupled oxidation of linoleate-~-carotene and accumulation of conjugated dienes. I t is assumed that n i t r i c oxide m i g h t act as a modulator of the arachidonic acid cascade and by this to affect many physiological and pathological reactions.
15.20
15.17
CHARACTERIZATION OF A NOVEL, CYTOTOXIC OXIDANT PRODUCED FROM THE INTERACTION BETWEEN SUPEROXIDE (02-) AND HYPOTHIOCYANOUS ACID (HOSCN). Matthew B. Grisham, Christine Volkmer and Tamaki Yamada. Department of Physiology, LSU Medical Center, Shreveport, LA. 71130. Saliva contains a potent antimicrobial system composed of salivary peroxidase (SPO), hydrogen peroxide (H202), and thiocyanate anion (SCN-). SPO catalyzes the oxidation of SCN- by H202 to yield the potent oxidant HOSCN. Because large numbers of activated neutrophils are present within the gingiva and the oral cavity during chronic gingival inflammation it is possible that some of the tissue injury associated with this condition may be mediated by secondary oxidants generated from the interaction between granulocyte-derived 02- and salivary HOSCN. Thus, the objective of this study was to characterize the oxidizing and cytotoxic properties of O2-, HOSCN and the secondary oxidants produced from the interaction between O 2- and HOSCN. We found that the addition of potassium 02- (KO2; 85 #M) to solutions containing HOSCN and catalase at pH 6.0 increased the number of oxidizing equivalents from the 57 +_ 3 #M originally present as HOSCN to 185 _+ 6 #M. Omission of either KO 2 or HOSCN eliminated the formation of the secondary oxidant. This novel oxidant did not degrade deoxyribose suggesting that hydroxyl radical was not produced. Furthermore this potent oxidizing agent exhibited unusual stability with greater than 50% of its oxidizing activity remaining after a 30 min at 37"C. Finally, we found that the addition of KO 2 to HOSCN in the presence of catalase enhanced the cytotoxicity of 50 #M HOSCN toward cultured epithelial cells from 6 +_ 1% to 66 + 4% (p<0.01). Our data suggest that granulocyte-derived O~- interacts with salivary HOSCN to yield a potent oxidizing agent that may mediate injury to the oral tissues.
15,19HYDROGEN PEROXIDE-INDUCED
HEPATOCYTE DAMAGE IN RAT HEPATOCYTE / KUPFFER CELL COCULTURE Regine Kahl, Herbert Desel, Gregor Latocha, and Andrea Kastien Department of Clinical Pharmacology, University of GOttingen, D-3400 G0ttingen, West Germany Cultured rat hepatocytes are remarkably resistent to exogenous hydrogen peroxide. H202 at 1 mM does not result in substantial pro£uciion of malondialdehyde (MDA) and release of lactate dehydrogenase (LDH). At i0 mM H202, MDA release amounts to 1-2 fmoles/cell and k s terminated within the first hour. LDH release only starts when H202 concentration has dropped below 0.i mM via an azide-sensitive elimination process with a half life of 2.5 min. At 180 min, 1-2 ~U/cell have been released.
In a hepatocyte / Kupffer c e l l coculture ( I : i ) . gross hepatocyte damage as determined by LDH release is not observed even a f t e r stimulation of the Kupffer cells with zymosan or phorbol myristate acetate (PMA). Under these conditions, 0.5-1 fmoles MDA/cell were released at 60 min. The toxicity of exogenous H202 is markedly enhanced by the presence of the Kupffer cells. In a typical duplicate experiment with i0 mM H202, MDA production in the coculture at 60 min was 6.5 fmoles/cell as compared to 1.5 fmoles/cell in the absence of Kupffer cells. The amount of LDH released after 180 min was also markedly increased. These data indicate that a Kupffer cell-derived compound sensitizes the hepatocyte to exogenously added H202 . The identity of the mediator(s) involved zs at present under investigation.
143
SUPEROXIDE ANION GENERATION IN POLYMORPHONUCLEAR LEUKOCYTES OBTAINED FROM RATS WITH GALACTOSAMINE-INDUCED HEPATIC INJURY Yuji Hino, Ryukichi Kumashiro, Michio Sata, and Kyuichi Tanikawa Second Department of Internal Medicine, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830 Japan
15.18
Superoxide anions have been known to induce damages in cultured vascular endothelial cells. Injury to the endothelia is an important process leading to liver cell necrosis. In our present study, the effect of superoxide anions generated by polymorphonuclear leukocytes (PMNs) obtained from galactosamine-treated rats on sinusoidal endothelial cells was discussed. Wistar male rats (body weight: 200 g) received intraperitoneally a dose of i000 mg/kg D-galactosamine HCI. PMNs were isolated from peripheral blood by the dextran sedimentation technique and percoll gradient centrifugation at 24 hr after galactosamine administration. The generation of superoxide anions in PMNs stimulated by phorbor miristate acetate (PMA) was determined by reduction of cytochrome C. PMNs in galactosamine-treated rats indicated a remarkable ten fold generation of superoxide anion, as compared with those in untreated control rats. As the galactosamine-treated rats were characterized with hepatic dysfunctions, it is suggested that the free radical generation will be helpful in explaining the pathogenesis of severe acute hepatic injury including dysfunction of sinusoidal endothelial cells.
NITRIC OXIDE, AN INHIBITOR OF LIPID OXIDATION BY LIPOXYGENASE AND CYCLOOXYGENASE loseph Kanner, Rina Granit and Bezalel A k i r i Department of Food Science, ARO, The Volcani I n s t i t u t e , PO Box 6, Bet Dagan 50250, ISRAEL, Recent discovery has shown that n i t r i c oxide is an important mammalian metabolite. The main function of NO. seems to be the stimulation of the soluble guanylate cyclase to elevate cGF~ p. N i t r i c oxide is thought to activate soluble guanylate cyclase through an interaction with iron atom in the heme of the enzyme. The system activates a v a r i e t y of biological actions which include endothelium-dependent relaxation, vascular tone, platel e t aEEregation and adhesion. Our study demonstrated that n i t r i c oxide i n h i b i t s lipoxygenase, cyclooxygenase and hemoglobin toward l i p i d oxidation, which was determined by coupled oxidation of linoleate-~-carotene and accumulation of conjugated dienes. I t is assumed that n i t r i c oxide m i g h t act as a modulator of the arachidonic acid cascade and by this to affect many physiological and pathological reactions.
15.20