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Endotoxin-induced mitochondrial injury in the ileum is attenuated by a free radical spin trap
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ACUTE AND CHRONIC STRESS-INDUCED GASTROINTESTINAL INJURY IN RATS,AND PROTECTION BY A NOVEL IH636 GRAPE SEED PROANTHOCYANIDIN EXTRACT (GSPE). l&&g&j, C.B. Williams,M. Milnes, J. Balm&i,X:Ye, D. Bagchi, S.J. Stohs, Creighton Univ. Health Sciences Center, Omaha, NE 68 178. Reactive oxygen species (ROS) are implicated in the pathogenesis of stress-induced gastrointestinal(GI) injury. In the present study, we have investigated the effects of acute stress (AS) and chronic stress (CS) on the enhanced production of superoxide anion (SA) as determined by cvtochrome c reduction assav (CCR). and correlated the enhanced -a ~~ production if SA with in&eased lipid peroxidation (LP), DNA fragmentation (DF), and membrane microviscosity (MV), indices of oxidative tissue injury, in gastric mucosa (GM) and intestinal mucosa (IM), and determined the protective effects of GSPE against mucosal injury. AS was induced by water-immersion for 90 min, while CS was induced by water-immersion for 15 mm/day for 15 consecutive days. Half of the animals exposed to AS were pretreated orally with 100 mg GSPEkg/day for 15 consecutive days. Similarly, half of the animals exposed to CS were pretreated orally with 100mg GSPE/kg/day for 15 consecutive days. AS produced maximal injury to both GM and IA4 as compared to CS. AS and CS increased CCR by 12.2- and 4.8-fold, respectively, in the GM, and l2.1- and 4.6-fold in the IM. AS increased LP, DF and MV by’3.3-, 4. I- and 11&fold, respectively, in the GM, and 4.4-, 5.2- and 16.6fold in IM. GSPE decreased AS-induced LP, DF and MV by 15% 12% and 13%, respectively, in the GM, and by 13%, 14% and 16%, respectively, in the IM. CS increased LP, DF and MV by 2.9-, 3.3- and 6.3- fold resoectivelv. in the GM. and 3.3-. 4.2- and 9.3-fold. respectively,in the IM: GSPE &creased CS-induced LP, DF and MV by 23%, 2 1% and 25%, respectively, in the. GM, and by 26%, 26% and 25%, respectively, in the IM. These results demonstrate that AS and CS stress can induce GI oxidative stress and mucosal injury through enhanced production of ROS, and that GSPE provides significantprotectionagainst GI oxidative stress and mucosal injury by scavenging these ROS.
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ENDOTOXIN-INDUCED MITOCHONDRIAL INJURY IN THE ILEUM IS ATTENUATED BY A FREE RADICAL SPIN TRAP
We have previously shown that intravenous endotoxin (U’S) causes severe ileal mitochondrlal injury which correlates with altered oxygen metabolism in the ileum. Purthermore, LPSinduced mitochondrial inlurv has been shown to be associated with the prevalence of r&c&e oxygen species (ROS) in the ileum. A potent scavenger of RGS, alpha-phenyl-N-tert-butylnltrone (PBN), has been st&n to improve s&vlvaj in septic animals. Hypothesis: LPS-induced ileal mitochondrlal injury is attenuated by PIlN. Methods: We assessed ileal mltochondrlal ultrastructure at 2 hours in &in situc/I> autoperfused feline heal preparations in 3 groups: Control (n=S), LPS-treated (n=8,3.0 mg/kg IV) and LPS (3.0 &kg, IV) following PBN (100 mg/kg, IVl pretreatment (n=3). Electron micrographs were made to assess rrdtocbondrlal ultrast~cture and scored (scale of O-5)based on the cell injury staging system described by Trump et al (Scan. Elec. Micrcscupy Ih437-62; 1980). Results: LPS treatment was associated with significant mltochondrlal injury relative to Controls (35 f 0.2 cI>vs 0.7 f 0.2, p < 0.001). Pretreatment with PBN conferred significant protection against LPSinduced ileal mltochondrlal injury (1.7 f 0.3, p < 0.01). Conclusions: LPSinduced ndtochondrlal lnktrv in the lIeal mucosa is attenuated, but not completely preventad, by pretreatment with a I
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potent free radical scavenger (PBN). These results suggest that ROS play an important role in &e development of mltocho&ial injury in the ileum following LPS and may explain the pathogen&s of altered oxygen metabolismobserved in sepsis. Funded by: ALA Grant RGO3&Nand OSU Seed Grant 221860.
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DIFFERENCES IN PENETRATION AND INTRACELLULAR LOCALIZATION BETWEEN CuZn SOD AND h4n SOD
Huang’. ’ Institut Sante & Developpement, Universite Paris VI, France * Lab. d’Analyse d’Images, Hopital Saint Louis, Paris, France Ingrid Emerit’, Paul0 Filipe”, Jany Vassy*,Vanda
Superoxide dismutase (SOD) rapidly (5’) binds to the surface of cells with slow uptake over the following hours.Membrane binding is most important for monocytes compared to neutrophils, lymphocytes and erythrocytes, as shown by flow cytometry. Using high resolution confocal laser scamring microscopy after covalent conjugation of fluorescent isothiocyanate (FITC) to the native enzyme, a typical diffise punctuate fluorescent pattern was seen in the cytoplasm of monocytes a&r Ih incubation with bovine as well as with human recombinant CuZnSOD. After 3 hs, the fluorescence had accumulated in the perinuclear area, giving the impression of a barrier for fbrther progression. Indeed, no fluorescence could be detected in the nucleus. In contrast hereto, MnSOD from E. coli showed rare spotty agglomerations in the cytoplasm without perinuclear accumulation. Also the number of labelled cells with the same incubation time and concentration was significantly lower for MnSOD than for the CuZnSODs. In agreement herewith, phorbohnyristate-stimulated 02- release by washed monocytes was not signifkantly reduced a&r preincubation with MnSOD, but inhibited by 40 % with CuZnSOD. The inhibitory effect of CuZn SODS was evident after an incubation period of 5’ only, when confocal laser microscopy located FITC-SOD on the membrane, but not yet in the cytoplasm.
OXYGEN
OXIDATIVE STRESS AND LOW LEVEL RADIATION Ingrid Emerit”, Urij Antipkine*,Natascha Zybina”,Amina Alaoui”, Paul0 Filipe“,Galina Katashkova”,Vanda Huang”, Marc Gentilin? ’ Institut Sante & Developpement, Univ. Paris VI,* Institute of Pediatrics, Kiev, ” Center for Radiation Medicine, StPetersburg. The plasma of persons, irradiated as a consequence of the Chernobyl accident, contains clastogenic factors (CF), which could be identified as lipid peroxidation products and cytokines with chromosome-damaging properties. The results of the CFtest were compared to other biomarkers of oxidative stress in 40 workers and 64 children exposed as the consequence of the Chernobyl accident. Clastogenic scores were in the abnormal range for 95 % of the workers (11 .Oltl.24 aberrations compared to O.S+l .O with normal plasma, p
activity of the plasma, increased 02- production by neutrophils, decreased plasma thiol or erythrocyte glutathione (GSH) and increased malondialdehyde levels. Glutathione peroxidase was decreased and erythrocyte SOD increased.
’ 9
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ACUTE AND CHRONIC STRESS-INDUCED GASTROINTESTINAL INJURY IN RATS,AND PROTECTION BY A NOVEL IH636 GRAPE SEED PROANTHOCYANIDIN EXTRACT (GSPE). l&&g&j, C.B. Williams,M. Milnes, J. Balm&i,X:Ye, D. Bagchi, S.J. Stohs, Creighton Univ. Health Sciences Center, Omaha, NE 68 178. Reactive oxygen species (ROS) are implicated in the pathogenesis of stress-induced gastrointestinal(GI) injury. In the present study, we have investigated the effects of acute stress (AS) and chronic stress (CS) on the enhanced production of superoxide anion (SA) as determined by cvtochrome c reduction assav (CCR). and correlated the enhanced -a ~~ production if SA with in&eased lipid peroxidation (LP), DNA fragmentation (DF), and membrane microviscosity (MV), indices of oxidative tissue injury, in gastric mucosa (GM) and intestinal mucosa (IM), and determined the protective effects of GSPE against mucosal injury. AS was induced by water-immersion for 90 min, while CS was induced by water-immersion for 15 mm/day for 15 consecutive days. Half of the animals exposed to AS were pretreated orally with 100 mg GSPEkg/day for 15 consecutive days. Similarly, half of the animals exposed to CS were pretreated orally with 100mg GSPE/kg/day for 15 consecutive days. AS produced maximal injury to both GM and IA4 as compared to CS. AS and CS increased CCR by 12.2- and 4.8-fold, respectively, in the GM, and l2.1- and 4.6-fold in the IM. AS increased LP, DF and MV by’3.3-, 4. I- and 11&fold, respectively, in the GM, and 4.4-, 5.2- and 16.6fold in IM. GSPE decreased AS-induced LP, DF and MV by 15% 12% and 13%, respectively, in the GM, and by 13%, 14% and 16%, respectively, in the IM. CS increased LP, DF and MV by 2.9-, 3.3- and 6.3- fold resoectivelv. in the GM. and 3.3-. 4.2- and 9.3-fold. respectively,in the IM: GSPE &creased CS-induced LP, DF and MV by 23%, 2 1% and 25%, respectively, in the. GM, and by 26%, 26% and 25%, respectively, in the IM. These results demonstrate that AS and CS stress can induce GI oxidative stress and mucosal injury through enhanced production of ROS, and that GSPE provides significantprotectionagainst GI oxidative stress and mucosal injury by scavenging these ROS.
( 237
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ENDOTOXIN-INDUCED MITOCHONDRIAL INJURY IN THE ILEUM IS ATTENUATED BY A FREE RADICAL SPIN TRAP
We have previously shown that intravenous endotoxin (U’S) causes severe ileal mitochondrlal injury which correlates with altered oxygen metabolism in the ileum. Purthermore, LPSinduced mitochondrial inlurv has been shown to be associated with the prevalence of r&c&e oxygen species (ROS) in the ileum. A potent scavenger of RGS, alpha-phenyl-N-tert-butylnltrone (PBN), has been st&n to improve s&vlvaj in septic animals. Hypothesis: LPS-induced ileal mitochondrlal injury is attenuated by PIlN. Methods: We assessed ileal mltochondrlal ultrastructure at 2 hours in &in situc/I> autoperfused feline heal preparations in 3 groups: Control (n=S), LPS-treated (n=8,3.0 mg/kg IV) and LPS (3.0 &kg, IV) following PBN (100 mg/kg, IVl pretreatment (n=3). Electron micrographs were made to assess rrdtocbondrlal ultrast~cture and scored (scale of O-5)based on the cell injury staging system described by Trump et al (Scan. Elec. Micrcscupy Ih437-62; 1980). Results: LPS treatment was associated with significant mltochondrlal injury relative to Controls (35 f 0.2 cI>vs 0.7 f 0.2, p < 0.001). Pretreatment with PBN conferred significant protection against LPSinduced ileal mltochondrlal injury (1.7 f 0.3, p < 0.01). Conclusions: LPSinduced ndtochondrlal lnktrv in the lIeal mucosa is attenuated, but not completely preventad, by pretreatment with a I
.
potent free radical scavenger (PBN). These results suggest that ROS play an important role in &e development of mltocho&ial injury in the ileum following LPS and may explain the pathogen&s of altered oxygen metabolismobserved in sepsis. Funded by: ALA Grant RGO3&Nand OSU Seed Grant 221860.
1
1
DIFFERENCES IN PENETRATION AND INTRACELLULAR LOCALIZATION BETWEEN CuZn SOD AND h4n SOD
Huang’. ’ Institut Sante & Developpement, Universite Paris VI, France * Lab. d’Analyse d’Images, Hopital Saint Louis, Paris, France Ingrid Emerit’, Paul0 Filipe”, Jany Vassy*,Vanda
Superoxide dismutase (SOD) rapidly (5’) binds to the surface of cells with slow uptake over the following hours.Membrane binding is most important for monocytes compared to neutrophils, lymphocytes and erythrocytes, as shown by flow cytometry. Using high resolution confocal laser scamring microscopy after covalent conjugation of fluorescent isothiocyanate (FITC) to the native enzyme, a typical diffise punctuate fluorescent pattern was seen in the cytoplasm of monocytes a&r Ih incubation with bovine as well as with human recombinant CuZnSOD. After 3 hs, the fluorescence had accumulated in the perinuclear area, giving the impression of a barrier for fbrther progression. Indeed, no fluorescence could be detected in the nucleus. In contrast hereto, MnSOD from E. coli showed rare spotty agglomerations in the cytoplasm without perinuclear accumulation. Also the number of labelled cells with the same incubation time and concentration was significantly lower for MnSOD than for the CuZnSODs. In agreement herewith, phorbohnyristate-stimulated 02- release by washed monocytes was not signifkantly reduced a&r preincubation with MnSOD, but inhibited by 40 % with CuZnSOD. The inhibitory effect of CuZn SODS was evident after an incubation period of 5’ only, when confocal laser microscopy located FITC-SOD on the membrane, but not yet in the cytoplasm.
OXYGEN
OXIDATIVE STRESS AND LOW LEVEL RADIATION Ingrid Emerit”, Urij Antipkine*,Natascha Zybina”,Amina Alaoui”, Paul0 Filipe“,Galina Katashkova”,Vanda Huang”, Marc Gentilin? ’ Institut Sante & Developpement, Univ. Paris VI,* Institute of Pediatrics, Kiev, ” Center for Radiation Medicine, StPetersburg. The plasma of persons, irradiated as a consequence of the Chernobyl accident, contains clastogenic factors (CF), which could be identified as lipid peroxidation products and cytokines with chromosome-damaging properties. The results of the CFtest were compared to other biomarkers of oxidative stress in 40 workers and 64 children exposed as the consequence of the Chernobyl accident. Clastogenic scores were in the abnormal range for 95 % of the workers (11 .Oltl.24 aberrations compared to O.S+l .O with normal plasma, p
activity of the plasma, increased 02- production by neutrophils, decreased plasma thiol or erythrocyte glutathione (GSH) and increased malondialdehyde levels. Glutathione peroxidase was decreased and erythrocyte SOD increased.
’ 9
8
S83