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The relation of free radicals ot organic and inorganic semiconductors with special reference to silicon
140
15.5
Inflammation ROLE OF NEUTROPHIL-DERIVED OXIDANTS IN THE PATHOPHYSIOLOGY OF INTESTINAL INFLAMMATION. Matthew B. Grisham, Hiroshi Tamai, D. Nell Granger and Timothy S. Gaginella. Department of Physiology, LSU Medical Center, Shreveport, La 71130 and Searle Research and Development, Skokie, IL 60077, USA. There is a growing body of evidence to suggest that much of the pathophysiology (diarrhea, mucosal injury) associated with inflammatory bowel disease may be mediated by extravasated neutrophils. Activated neutrophils release large quantities of relatively stable oxidants including hydrogen peroxide (H202) , hypochlorous acid (HOC1),monochloramine (NH2C1),and taurine monoehloramine (TauNHCI). The objective of this study was to assess the ability of neutrophilderived oxidants to enhance intestinal mucosal permeability in vivo and stimulate CI- secretion in vitro. Perfusion of the intestinal lumen with 100-500 #M H202, HOCI, and NHzCI produced dose-dependent increases in mucosal permeability ranging from 3-12 fold as measured by the blood-to-lumen clearance of SlCr-EDTA. TauNHC1 did not significantly enhance mucosal permeability. We found that the HOC1 and NH~Cl-induced increases in permeability were related to their cytotoxic activities toward intestinal epithelial cells in vitro whereas H202-mediated increases involved noncytotoxic mechanisms. Furthermore, we found that noncytotoxic concentrations of H~O 2 and NH2CI (l 0-100 #M) dramatically enhanced intestinal C1- secretion as measured by increases in short circuit current. HOC1 and TauNHCI were ineffective in this system. Subsequent experiments demonstrated that some of the H202 and NH2Cl-induced secretory response was inhibited by tetrodotoxin or piroxicam suggesting that the nervous system and prostaglandins respectively were involved in oxidant-mediated electrolyte transport. Taken together our data suggest that certain neutrophil-derived oxidants may play an important role in the pathogenesis of inflammationinduced diarrhea and mucosal barrier dysfunction.
15.7
K I N E T I C S OF S U P E R O X I D E F O R M A T I O N B Y STIMULATED NEUTROPHILS A m r a m Samuni*, C. Murali Krishna, Christopher D.V. Black, John Cook and Angelo Russo * M o l e c u l a r B i o l o g y , School of M e d i c i n e , H e b r e w University, Jerusalem, Israel, and Radiation Oncology Branch, NCI, NIH, Bethesda, MD. 20892, USA. The kinetics of radical formation by stimulated neutrophils is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminolamplified chemiluminescence surveillance of superoxide and H 2 0 2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such finding. The kinetics of superoxide production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin-trapping combined with e l e c t r o n spin r e s o n a n c e ( E P R ) d e t e c t i o n , a n d ferricytochrome c reduction. The effects of pH and [02] on luminol-amplified CL were determined using hypoxanthine / xanthine oxidase to produce superoxide in cell-free systems. T h e present results show that stimulated neutrophils continued to generate superoxide for several hours and that the failure o f the CL assay to report superoxide and H202 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged superoxide fluxes does not terminate radical production, and that the use of luminol-amplified CL is not a valid assay for continuous monitoring of superoxide or H202 generated by stimulated neutrophils or cell-free systems.
OXIDATIVE DAMAGE TO THE KEY METABOLIC ENZYME G3PDH OCCURS/n v/tro AFTER EXPOSURE TO H202 AND IN AN ANIMAL MODEL OF ACUTE INFLAMMATORY ARTHRITIS.
15.6
Mark S. Baker & Dennis A. Lowther John Curtin School of Medical Research, Canberra & Monash University, Melbourne, Australia.
The production of reactive oxidants (02-, H202, OCI- & HO-) by polymorphonuclear neutrophils (PMNLs) is suspected as being one cause of cellular and tissue damage during acute inflammation. Exposure of articular cartilage to H202 in vitro inhibits proteoglycan (PG) synthesis in a fashion which could be markedly increased by inhibition of either catalase (azide, 3-AT) or glutathione metabofising systems (BCNU). The kinetics of suppression of PG synthesis by H202 suggested that injury occurred at the level of general protein synthesis. Exposure to H202 caused an immediate (<2 min) dose dependent decrease in cartilage ATP levels, that was identified as being due specifically to the oxidative inactivation of glyceraldehyde-3phosphate dehydrogenase (G-3-PDH). Articular cartilage biosynthetic pathways mostly use ATP generated by glycolysis, since PG synthesis was inhibited by the glycolytic inhibitor 2-deoxyglucose (65%), but to a much lesser degree (10%) by the uncoupler, 2,4-dinitrophenol. Most recently we have found that there is a loss of the relatively specific labelling of G3PDH by 3H-IAA in rabbit articular cartilage during carrageenin-induced arthritis, corresponding with the maximal influx of PMNL cells into the arthritic synovial fluid and the substantial inhibition of PG core protein synthesis. We suggest that inflammatory cartilage injury by "activated" PMNLs and macrophages occurs through oxidation of the sensitive thiol (-SH) residue at the active center of G3PDH, with subsequent reduction in the rate of glycolytic ATP synthesis, required for DNA, protein, proteoglycan and hyaluronic acid synthesis.
THE RELATION OF FREE RADICALS TO ORGANIC AND INORGANIC SEMICONDUCTORS WITH SPECIAL REFERENCE TO SILICON Faik Atroshi, Jouko Parantainen, and Tuomas Westermarck Research Laboratories, Medica Pharmaceutical Co.Ltd, Box 325, 00101, Helsinki; Department of Pharmacology and Toxicology, College of Veterinary Medicine, Box 6, Helsinki 55, Finland Silicon (Si) is the major element used to regulate electron flow in electronic (semiconduction), and silicon together with siliconized structures may have static electric charges. Si compounds may have proporties of electrolytes, which regulate conductivity by definition. Negative charges have been measured in Si-complexes formed in living tissue. Si has marked effects on free radical formation and lipid peroxidation, the essence of which is to add electrons to oxygen. More important than silicon as such, however, may be the principles by which trace elements and some other factors (e.g. redox state and pH) regulate electric conductivity in organic structures. In detoxifying of hydrogen peroxide by glutathione peroxidase, selenium may be needed as an reductant: to add electrons to the oxygen radicals: electrons are added to oxygen. While the process may be initiated by Si, Se (GSH-Px) may be needed in the later phase. We have correlated the changes of trace elements and antioxidants to conduction capacity of milk, and studied the mechanisms also of blood and inflamed tissue, which are new findings.
15.8
15.5
Inflammation ROLE OF NEUTROPHIL-DERIVED OXIDANTS IN THE PATHOPHYSIOLOGY OF INTESTINAL INFLAMMATION. Matthew B. Grisham, Hiroshi Tamai, D. Nell Granger and Timothy S. Gaginella. Department of Physiology, LSU Medical Center, Shreveport, La 71130 and Searle Research and Development, Skokie, IL 60077, USA. There is a growing body of evidence to suggest that much of the pathophysiology (diarrhea, mucosal injury) associated with inflammatory bowel disease may be mediated by extravasated neutrophils. Activated neutrophils release large quantities of relatively stable oxidants including hydrogen peroxide (H202) , hypochlorous acid (HOC1),monochloramine (NH2C1),and taurine monoehloramine (TauNHCI). The objective of this study was to assess the ability of neutrophilderived oxidants to enhance intestinal mucosal permeability in vivo and stimulate CI- secretion in vitro. Perfusion of the intestinal lumen with 100-500 #M H202, HOCI, and NHzCI produced dose-dependent increases in mucosal permeability ranging from 3-12 fold as measured by the blood-to-lumen clearance of SlCr-EDTA. TauNHC1 did not significantly enhance mucosal permeability. We found that the HOC1 and NH~Cl-induced increases in permeability were related to their cytotoxic activities toward intestinal epithelial cells in vitro whereas H202-mediated increases involved noncytotoxic mechanisms. Furthermore, we found that noncytotoxic concentrations of H~O 2 and NH2CI (l 0-100 #M) dramatically enhanced intestinal C1- secretion as measured by increases in short circuit current. HOC1 and TauNHCI were ineffective in this system. Subsequent experiments demonstrated that some of the H202 and NH2Cl-induced secretory response was inhibited by tetrodotoxin or piroxicam suggesting that the nervous system and prostaglandins respectively were involved in oxidant-mediated electrolyte transport. Taken together our data suggest that certain neutrophil-derived oxidants may play an important role in the pathogenesis of inflammationinduced diarrhea and mucosal barrier dysfunction.
15.7
K I N E T I C S OF S U P E R O X I D E F O R M A T I O N B Y STIMULATED NEUTROPHILS A m r a m Samuni*, C. Murali Krishna, Christopher D.V. Black, John Cook and Angelo Russo * M o l e c u l a r B i o l o g y , School of M e d i c i n e , H e b r e w University, Jerusalem, Israel, and Radiation Oncology Branch, NCI, NIH, Bethesda, MD. 20892, USA. The kinetics of radical formation by stimulated neutrophils is the subject of extensive ongoing research. Most previous studies of neutrophil activation indicate that there is a transient production of reactive oxygen species. Luminolamplified chemiluminescence surveillance of superoxide and H 2 0 2 supported these general findings. Yet, recent studies showed that production of reactive oxygen species by stimulated neutrophils is not transient but persistent; however, luminol-dependent methods do not corroborate such finding. The kinetics of superoxide production by human neutrophils were studied using luminol-amplified chemiluminescence (CL), spin-trapping combined with e l e c t r o n spin r e s o n a n c e ( E P R ) d e t e c t i o n , a n d ferricytochrome c reduction. The effects of pH and [02] on luminol-amplified CL were determined using hypoxanthine / xanthine oxidase to produce superoxide in cell-free systems. T h e present results show that stimulated neutrophils continued to generate superoxide for several hours and that the failure o f the CL assay to report superoxide and H202 formation results from some luminol reaction product which interferes with the light reaction. Our results show that the cells are operative for long periods indicating that cell exposure to prolonged superoxide fluxes does not terminate radical production, and that the use of luminol-amplified CL is not a valid assay for continuous monitoring of superoxide or H202 generated by stimulated neutrophils or cell-free systems.
OXIDATIVE DAMAGE TO THE KEY METABOLIC ENZYME G3PDH OCCURS/n v/tro AFTER EXPOSURE TO H202 AND IN AN ANIMAL MODEL OF ACUTE INFLAMMATORY ARTHRITIS.
15.6
Mark S. Baker & Dennis A. Lowther John Curtin School of Medical Research, Canberra & Monash University, Melbourne, Australia.
The production of reactive oxidants (02-, H202, OCI- & HO-) by polymorphonuclear neutrophils (PMNLs) is suspected as being one cause of cellular and tissue damage during acute inflammation. Exposure of articular cartilage to H202 in vitro inhibits proteoglycan (PG) synthesis in a fashion which could be markedly increased by inhibition of either catalase (azide, 3-AT) or glutathione metabofising systems (BCNU). The kinetics of suppression of PG synthesis by H202 suggested that injury occurred at the level of general protein synthesis. Exposure to H202 caused an immediate (<2 min) dose dependent decrease in cartilage ATP levels, that was identified as being due specifically to the oxidative inactivation of glyceraldehyde-3phosphate dehydrogenase (G-3-PDH). Articular cartilage biosynthetic pathways mostly use ATP generated by glycolysis, since PG synthesis was inhibited by the glycolytic inhibitor 2-deoxyglucose (65%), but to a much lesser degree (10%) by the uncoupler, 2,4-dinitrophenol. Most recently we have found that there is a loss of the relatively specific labelling of G3PDH by 3H-IAA in rabbit articular cartilage during carrageenin-induced arthritis, corresponding with the maximal influx of PMNL cells into the arthritic synovial fluid and the substantial inhibition of PG core protein synthesis. We suggest that inflammatory cartilage injury by "activated" PMNLs and macrophages occurs through oxidation of the sensitive thiol (-SH) residue at the active center of G3PDH, with subsequent reduction in the rate of glycolytic ATP synthesis, required for DNA, protein, proteoglycan and hyaluronic acid synthesis.
THE RELATION OF FREE RADICALS TO ORGANIC AND INORGANIC SEMICONDUCTORS WITH SPECIAL REFERENCE TO SILICON Faik Atroshi, Jouko Parantainen, and Tuomas Westermarck Research Laboratories, Medica Pharmaceutical Co.Ltd, Box 325, 00101, Helsinki; Department of Pharmacology and Toxicology, College of Veterinary Medicine, Box 6, Helsinki 55, Finland Silicon (Si) is the major element used to regulate electron flow in electronic (semiconduction), and silicon together with siliconized structures may have static electric charges. Si compounds may have proporties of electrolytes, which regulate conductivity by definition. Negative charges have been measured in Si-complexes formed in living tissue. Si has marked effects on free radical formation and lipid peroxidation, the essence of which is to add electrons to oxygen. More important than silicon as such, however, may be the principles by which trace elements and some other factors (e.g. redox state and pH) regulate electric conductivity in organic structures. In detoxifying of hydrogen peroxide by glutathione peroxidase, selenium may be needed as an reductant: to add electrons to the oxygen radicals: electrons are added to oxygen. While the process may be initiated by Si, Se (GSH-Px) may be needed in the later phase. We have correlated the changes of trace elements and antioxidants to conduction capacity of milk, and studied the mechanisms also of blood and inflamed tissue, which are new findings.
15.8