- Email: [email protected]
Prostaglandin E2 synthesis affected by nitric oxide in macrophages
112
Poster Session 2C. Nitric Oxide
LPS (10 IlglmJ) repressed CYP2Bl mRNA after 3 hours. 'The effect was maximalafter 12 hours and persisted at 24 hours. When a nitric oxide synthase inhibitor was added to the culture media (30 to 300 IlM) . no difference was noted. To determine the efficiency of the inhibitorswe measured NO production. which was blocked when an inhibitor was in the culture medium. We concluded that NO was not involved in the repression of CYP2BI mRNA by LPS in rat hepatocytes,
I
P2C74
1 STUDIES ON THE EFFECT OF CARBON DISULFIDE ON NITRIC OXIDE AND LIPID PEROXIDATION
Zhang Wen-chang " Tu Li-qing. Department of Occupational Health. Fujian Medical University Fuzhou 350004. P.R. China Studies on the effect of CS2 on the rat nitric oxide/endothelium-derived relaxing factor (NOIEDRF) and it, mechanism were carried out, The results showed that serum and cardiac NO were reducedand were significantly lower in Csj-exposed rats (320.160 mg/kg) than in the controls, and serum LPO (lipid peroxidation) higher in rats exposed to CS2than in controls (p < O.O\), but changes cardiac NOS (nitric oxide synthases) activities had not been found (p > 0.05). The results of stepwise regression analysis (serum NO (y). CSz dose (x l ). serum LPO (x2), cardiac NOS (x S) and cardiac NO (x4) showed that there was a close correlation between serum ~O and LPO or the dose of CS2. When F critical value was 5, the serum LPO became the only factor in the stepwise regression equation (R = -0.6665, p < 0.01). We also found that the serum LPO content was significantly reduced, and NO level was significantly increased when some anti-oxidation substances (Sc, Zn, Vit E and Vit C) were taken by rats which were exposed to CS2 at the same time. So, it was suggested that the lipid peroxidation resulted from CS2 would accelerate the oxidation and decomposition of NO. and the NO level in the body was reduced which resulted in the toxic effect on rnycardiovascular system.
IP2C7S1
STREPTOMYCES ANULATUS INDUCED PRODUCTION OF NITRIC OXIDE AND CYTOTOXICITY IN HUMAN ALVEOLAR TYPE 11 EPITHEUAL CELLS (A549)
M.-R. Hirvoncn .1 ,J. Jussila1 , M. Ruotsalainen 1 • K. Savolainerr", A. Nevalainen 1 , J Division ofEnvironmental Health. National Public Healrh Institute. P.D. Box 95. FIN-7070I Kuopio; 2Department of Pharmacology and Toxicology, University of Kuopio, P.D. Box 1627. FIN-702// Kuopio, Finland Dampness and mold growth in buildings cause spore generation into indoor air. which is associated with respiratory tract disorders. Specific agents or cellular mechanisms of diseases have not yet been identified. A likely contributingfactor in the etiological mechanism of these respiratory symptoms could be inflammatory response towards specific organic materials in the microbes. NO plays an important role as an immune defense molecule. A variety of cells in the airways can be stimulated to produce large amounts of NO for long periods of time through stimulation of inducible NO synthase (iNOS). The ability of Streptomyces anulatus, gram positive bacteria which was isolated from moldy buildings. and lipopolysaccharide (LPS) or interferon gamma (IFN) to induce NO production and to cause cytotoxicity in human alveolar type II epithelial cells (A549) were studied. Here were report that Streptomyces anulatus or IFN induce a significant dose-, and time dependent increase in l'O-production and decrease in the cell viability of A549 cells. The effects induced by Streptomyces anulatus alone. were further increased by IFN. LPS was not able to induce NO-production in these cells or to cause cytotoxicity. The present results suggests that Streptomyces anulatus, initiates a cascade of events in human alveolar A549 cells leading to cytotoxicity and production of NO, which is believed to
he an important inflammatory mediator in the pathogenesisof many respiratory diseases.
IP2C76!
ARECA NUT EXTRACTS STIMULATE DNA STRAND BREAKS BY GENERATION OF NITRIC OXIDE
T.y. Liu ", C.W.Chi. Veterans General Hospital-Taipei. Taipei, Taiwan. Republic of China International Agency for Research on Cancer (!ARC) clearly stated that chewingbetel quid links to the development of oral cancer. Areca nut is the majorcomponentof betelquid. Previously, we havedemonstrated that areca nut extract (ANE) can generate reactive oxygen species (ROS) and cause oxidative DNA damage through hydrogen peroxide formation and Fenton-type reaction, In this study, we have investigated the possibility that nitric oxide is also involved in the ANE-mediated DNAdamages. By using alkaline single cell gel electrophoresis (SCGE), we have shown that Al'E-induced DNA strand breaks dose-dependently in bovine endothelial aortic cells (BEAC). S-methyl-L-thiocitrulline and Nw-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthase, could suppress the ANE-induced DNA strand breaks. Increase of nitroryrosine, a stable product of nitric oxide, was also detected in ANE-treated cells. These results suggest that ANE treatment may generate nitric oxide to damage DNA and consequently may be important in the pathogenesis of oral cancer.
[P2C'7!] NO PRODUCTION IN A UNDIFFERENTIATED AND DIFFERENTIATED HUMAN INTESTINAL CELL LINE
A.L. Vignoli, F. Zucco v" , A. Stammati, R.C. Srivastava". Istituto Superiore di Sanaa, Roma; J Istituto di Tecnologie Biomediche. CNR. Roma, Italy: 2 Industrial Toxicology Research Centre, Lucknow; India Nitric oxide (NO) may play an important role in intestinal inflammation by acting on the barrier integrity. thus reducing its selective permeability. Few studies have been performed in vitro on human intestinal cell lines. An inducible NO synthase has been shown in HT29 and OLD-I cells following proinflammatory cytokine exposure. On Caco-2 cells, able to spontaneously differentiate in vitro to small intestinal enterocytes, only indirect evidence has been obtained of NO production. By the use of different biochemical methods. we have been able to showthat Caco-2cells are producing ~O following PMA + yll'F exposure. In undifferentiated cells (7 days of culture) the production is very low, while at the 21'1 day of culture. when the cells are completely differentiated. a consisted production is detected. This effect is inhibited by nitro-D-arginine methyl ester (D-NAME). but N-monomethyl-L-arginine (NMLA) is more effective, suggesting that an inducible NO synthase (NOS) is involved. Indeed. the NOS activity has been detected by 3H-argininclcitrulline conversion method. Further studies concerning thc induction of the enzyme are in progress,as well as its correlation with the cytoskeletal modification and barrier integrity.
[P2Ctii] PROSTAGLANDIN E
2 SYNTHESIS AFFECTED BY NITRIC OXIDE IN MACROPHAGES
Cecilia Guastadisegni .1. Maria Balduzzi'", Luisa Minghetti2 • Alessia Nicolini", Elisabetta Polazzi2 . JLaboratory of EnvironmentalHygiene; 2Neurobiology Section. Laboratory of Pathophysiology Istituto Superiore di Sanita; .I Biomedical and Toxicological Department. ENEA. Rome. Italy Prostaglandins (PGs). the arachidonic acid (AA) metabolites of
Poster Session 2C. Nitric Oxide
the cyclooxygenase (COX) pathway, and Nitric oxide (NO) play major roles in inflammation and increasing evidence indicates the existence of a cross talk between the pathways leading to their synthesis. In a previous study (Guastadisegni et al. FEBS Letters 413 (1997) 314-3 18), we showed that the macrophage-like RAW 264.7 cells stimulated with S-nitroso-N-acetylpenicillamine (SNAP) a compound which releases NO, PGE2 synthesis was increased as a result of the increased AA release, which counteracted the decreasing effect on COX-2 expression. On the other hand after stimulation with LPS, SNAP decreased PGE2 due to the slight increase in AA release. Recently NONOates have been recognised a good source of NO due to their well defined half-life. We studied the effect of NO derived from PAPNNO (tl/2 15 min) and DETAINO (tl/2 20 hr) in RAW 264.7 cells. We found that both NONOates (500 JLM) markedly increased the basal and the LPS-stimulated PGE2 production. Basal PGE2 productionwas 5 fold and 6 fold increased after 24 hr incubation with PAPAINO and DETNNO respectively. PAPNNO and DETAINO exposure was accompanied by a 2 fold and 3 fold increase respectively of the LPS-stimulated PGE2 release. The AA release was only slightly increased but interestingly DETAINO released AA (22% and 30% over basal and LPS.induced respectively) after 2 hr incubation while PAPAINO (15% and 12% over basal and LPS-induced respectively) after 5 hr incubation. At variance with SNAP, the massive increase in basal and LPS-stimulated PGE2 production is very unlikely to be mediated by an increased AA release, while western blot analysis of RAW 264.7 proteins gave indication of an increased expression of COX-2.
IP2C79\
THE ROLEOF TUMORNECROSIS FACTOR-a AND NITRICOXIDE IN ACUTELIVERINJURYINDUCED BY CARBONTETRACHLORIDE
L.A. Morio *, D.L. Laskin. Joint Graduate Program in Toxicology, Rutgers University and UMDNJ-R oben Wood Johnson Medical School, Piscataway. New Jersey, USA Our laboratory has been investigating the role of macrophages in chemically-induced hepatotoxicity. In a number of different experimental models, hepatotoxicity is prevented or reduced by pretreatment of animals with the macrophage inhibitor. gadolinium chloride. These findings demonstrate that macrophagcs playa critical role in the pathogenesis of liver injury. Macrophages arc known to release a numberof differentinflammatory mediators with cytotoxicpotential. ln the present studies we analyzed the role of two macrophagederived mediators, tumor necrosis factor-a (TNF-a) and nitric oxide, in liver inj ury induced by carbon tetrachloride (CCLj). Treatment of mice with CC4 (0.3 ml/kg, i.p.) resulted in a time dependent induction of centrilobular hepatic necrosis. This was correlated with a dramatic increase in serum transaminase levels. Recent studies have shown that CCI4 treatment of animals also induces TNF-a and nitric oxide production in the liver. To study the role of these mediators in hepatotoxicity, we used knockout mice lacking the gene for the 55 kDa TNF-a receptor or inducible nitric oxide synthase (iNOS). We found that CC4 was significantly less effectivein inducing hepatotoxicity in mice lacking the p55 TNF-a receptor. These data are consistent with the observation that pretreatment of animals with soluble TNF-a receptor or anti-TNF-a antibody decreased CC4-induced liver injury. In contrast, CC4-induced liver injury was markedly increased in knockout mice lacking the gene for iNOS suggesting a hepatoprotective role of nitric oxide. Taken together, these data suggest that macrophage-derived TN F-a and nitric oxide are important regulators of CC4 -induced liver injury (Supported by NIH GM343I0 and the Burroughs Wellcome Fund).
IP2CBO I
113
MICE LACKINGINDUCIBLE NITRIC OXIDESYNTHASE ARE PROTECTED FROMACETAMINOPHEN-INDUCED HEPATD-TOXICITY
J.D. Laskin *. en. Gardner, D.E. Heck, H. Chiu, S.K. Durham, D.L. Laskin. Rutgers University and UMDNJ-Roben Wood Johnson Medical School, Piscataway. NJ and Bristol Myers Squibb Pharmaceutical Research Institute, Prin ceton. NJ. USA
Nitric oxide (NO) is a highly reactive inflammatory mediator implicated in chemically-induced tissue injury. In the present srudies we analyzed the role of NO in the hepatotoxicity of acetaminophen (AA) using knockout mice lacking the gene for inducible nitric oxide synthase (NOSII). Treatment of wild type control mice with AA (100-300 mglkg, ip) resulted in a dose and time dependent induction of centrilobular necrosis. This was associated with a marked increase in serum ALT and AST levels. AA was significantly less effective in inducing hepatotoxicity in mice lacking NOSH. These data demonstrate that NO produced via NOSH contributes to liver injury. To assess the contribution of parenchymal cell-derived NO to AA-induced toxicity, we quantified hepatocyte NO production. AA administration resulted in a time dependent increase in NO production and NOSII expression by hepatocytes. Pretreatment of animals with the NOSII inhibitor aminoguanidine for 3 days (100 mg/kg, sc, I x /day) completely abrogated hepatotoxicity. Hepatocytes isolated from animals pretreated with aminoguanidine also produced significantly less NO than cells from animals treated with AA alone. This was associated with decreased NOSH protein and mRNA. These results suggest that aminoguanidine exerts multiple sites of inhibitory action on NO production. This is supported by our finding that a single injectionof arninoguanidine immediately before AA was significantly less effective in blocking hepatotoxicity. Taken together these data demonstrate that NO is a critical mediator of hepatotoxicity. Moreover, hepatocytes appear to be a major source of NO in the liver during the pathogenesis of AA induced liver injury (Supported by NIH GM343I0 and the Burroughs Wellcome Fund).
IP2CB1 I
THE ROLEOF NITRIC OXIDE ON LEAD NEPHROTOXICITY IN PERFUSED RATKJDNEY
M. Ghazi-Khansari *, S. Ala. M. Essalat, A.R. Dchpour. Departm ent of Pharmacology. School ofMedicine. Tehran Unive rsity of Medical Sciences. P. O. Box /3/45-784. Tehran. I.R., Iran Lead nephrotoxicity has been shown by a useful early markerof renal injury namely urinary N-acetyl-tl-D-glucosaminidase (NAG). In this study the effect of lead acetate on nephrotoxicity and its correlation with the nitric oxide (NO) system by detecting the NAG release in perfused rat kidney was investigated. Lead acetate (20 and 50 JLgldl) caused time and dose-dependent increase in enzymuria. The effect of concurrent perfusion with lead and L-arginine (L-Arg) or L-NG-nitro argininemethylester (L-NAME) [precursor and inhibitor of NO synthase respectively] in the perfusion fluid was also studied by measuring NAG activity in the perfusate kidney rat. L-Arg (2 roM ) has significantly decreased the lead-induced NAG release (p < 0.00 I), and L-NAME (0.1 mM) has significantly increased the lead-induced enzyme release in the time-dependent manner (p < 0.001). Moreover, histological studies using light microscope shows that some of the epithelial cells of the proximal compoluted tubules are degenerated or necrotic and desquamated into the lumens in rat treated with lead acetate. This changes occurs at 50 ILgidl of lead acetate and increases by addition of L-NAME to lead acetate. This may suggested that lead may interfere with NO system in rat kidney.
Poster Session 2C. Nitric Oxide
LPS (10 IlglmJ) repressed CYP2Bl mRNA after 3 hours. 'The effect was maximalafter 12 hours and persisted at 24 hours. When a nitric oxide synthase inhibitor was added to the culture media (30 to 300 IlM) . no difference was noted. To determine the efficiency of the inhibitorswe measured NO production. which was blocked when an inhibitor was in the culture medium. We concluded that NO was not involved in the repression of CYP2BI mRNA by LPS in rat hepatocytes,
I
P2C74
1 STUDIES ON THE EFFECT OF CARBON DISULFIDE ON NITRIC OXIDE AND LIPID PEROXIDATION
Zhang Wen-chang " Tu Li-qing. Department of Occupational Health. Fujian Medical University Fuzhou 350004. P.R. China Studies on the effect of CS2 on the rat nitric oxide/endothelium-derived relaxing factor (NOIEDRF) and it, mechanism were carried out, The results showed that serum and cardiac NO were reducedand were significantly lower in Csj-exposed rats (320.160 mg/kg) than in the controls, and serum LPO (lipid peroxidation) higher in rats exposed to CS2than in controls (p < O.O\), but changes cardiac NOS (nitric oxide synthases) activities had not been found (p > 0.05). The results of stepwise regression analysis (serum NO (y). CSz dose (x l ). serum LPO (x2), cardiac NOS (x S) and cardiac NO (x4) showed that there was a close correlation between serum ~O and LPO or the dose of CS2. When F critical value was 5, the serum LPO became the only factor in the stepwise regression equation (R = -0.6665, p < 0.01). We also found that the serum LPO content was significantly reduced, and NO level was significantly increased when some anti-oxidation substances (Sc, Zn, Vit E and Vit C) were taken by rats which were exposed to CS2 at the same time. So, it was suggested that the lipid peroxidation resulted from CS2 would accelerate the oxidation and decomposition of NO. and the NO level in the body was reduced which resulted in the toxic effect on rnycardiovascular system.
IP2C7S1
STREPTOMYCES ANULATUS INDUCED PRODUCTION OF NITRIC OXIDE AND CYTOTOXICITY IN HUMAN ALVEOLAR TYPE 11 EPITHEUAL CELLS (A549)
M.-R. Hirvoncn .1 ,J. Jussila1 , M. Ruotsalainen 1 • K. Savolainerr", A. Nevalainen 1 , J Division ofEnvironmental Health. National Public Healrh Institute. P.D. Box 95. FIN-7070I Kuopio; 2Department of Pharmacology and Toxicology, University of Kuopio, P.D. Box 1627. FIN-702// Kuopio, Finland Dampness and mold growth in buildings cause spore generation into indoor air. which is associated with respiratory tract disorders. Specific agents or cellular mechanisms of diseases have not yet been identified. A likely contributingfactor in the etiological mechanism of these respiratory symptoms could be inflammatory response towards specific organic materials in the microbes. NO plays an important role as an immune defense molecule. A variety of cells in the airways can be stimulated to produce large amounts of NO for long periods of time through stimulation of inducible NO synthase (iNOS). The ability of Streptomyces anulatus, gram positive bacteria which was isolated from moldy buildings. and lipopolysaccharide (LPS) or interferon gamma (IFN) to induce NO production and to cause cytotoxicity in human alveolar type II epithelial cells (A549) were studied. Here were report that Streptomyces anulatus or IFN induce a significant dose-, and time dependent increase in l'O-production and decrease in the cell viability of A549 cells. The effects induced by Streptomyces anulatus alone. were further increased by IFN. LPS was not able to induce NO-production in these cells or to cause cytotoxicity. The present results suggests that Streptomyces anulatus, initiates a cascade of events in human alveolar A549 cells leading to cytotoxicity and production of NO, which is believed to
he an important inflammatory mediator in the pathogenesisof many respiratory diseases.
IP2C76!
ARECA NUT EXTRACTS STIMULATE DNA STRAND BREAKS BY GENERATION OF NITRIC OXIDE
T.y. Liu ", C.W.Chi. Veterans General Hospital-Taipei. Taipei, Taiwan. Republic of China International Agency for Research on Cancer (!ARC) clearly stated that chewingbetel quid links to the development of oral cancer. Areca nut is the majorcomponentof betelquid. Previously, we havedemonstrated that areca nut extract (ANE) can generate reactive oxygen species (ROS) and cause oxidative DNA damage through hydrogen peroxide formation and Fenton-type reaction, In this study, we have investigated the possibility that nitric oxide is also involved in the ANE-mediated DNAdamages. By using alkaline single cell gel electrophoresis (SCGE), we have shown that Al'E-induced DNA strand breaks dose-dependently in bovine endothelial aortic cells (BEAC). S-methyl-L-thiocitrulline and Nw-nitro-L-arginine methyl ester, inhibitors of nitric oxide synthase, could suppress the ANE-induced DNA strand breaks. Increase of nitroryrosine, a stable product of nitric oxide, was also detected in ANE-treated cells. These results suggest that ANE treatment may generate nitric oxide to damage DNA and consequently may be important in the pathogenesis of oral cancer.
[P2C'7!] NO PRODUCTION IN A UNDIFFERENTIATED AND DIFFERENTIATED HUMAN INTESTINAL CELL LINE
A.L. Vignoli, F. Zucco v" , A. Stammati, R.C. Srivastava". Istituto Superiore di Sanaa, Roma; J Istituto di Tecnologie Biomediche. CNR. Roma, Italy: 2 Industrial Toxicology Research Centre, Lucknow; India Nitric oxide (NO) may play an important role in intestinal inflammation by acting on the barrier integrity. thus reducing its selective permeability. Few studies have been performed in vitro on human intestinal cell lines. An inducible NO synthase has been shown in HT29 and OLD-I cells following proinflammatory cytokine exposure. On Caco-2 cells, able to spontaneously differentiate in vitro to small intestinal enterocytes, only indirect evidence has been obtained of NO production. By the use of different biochemical methods. we have been able to showthat Caco-2cells are producing ~O following PMA + yll'F exposure. In undifferentiated cells (7 days of culture) the production is very low, while at the 21'1 day of culture. when the cells are completely differentiated. a consisted production is detected. This effect is inhibited by nitro-D-arginine methyl ester (D-NAME). but N-monomethyl-L-arginine (NMLA) is more effective, suggesting that an inducible NO synthase (NOS) is involved. Indeed. the NOS activity has been detected by 3H-argininclcitrulline conversion method. Further studies concerning thc induction of the enzyme are in progress,as well as its correlation with the cytoskeletal modification and barrier integrity.
[P2Ctii] PROSTAGLANDIN E
2 SYNTHESIS AFFECTED BY NITRIC OXIDE IN MACROPHAGES
Cecilia Guastadisegni .1. Maria Balduzzi'", Luisa Minghetti2 • Alessia Nicolini", Elisabetta Polazzi2 . JLaboratory of EnvironmentalHygiene; 2Neurobiology Section. Laboratory of Pathophysiology Istituto Superiore di Sanita; .I Biomedical and Toxicological Department. ENEA. Rome. Italy Prostaglandins (PGs). the arachidonic acid (AA) metabolites of
Poster Session 2C. Nitric Oxide
the cyclooxygenase (COX) pathway, and Nitric oxide (NO) play major roles in inflammation and increasing evidence indicates the existence of a cross talk between the pathways leading to their synthesis. In a previous study (Guastadisegni et al. FEBS Letters 413 (1997) 314-3 18), we showed that the macrophage-like RAW 264.7 cells stimulated with S-nitroso-N-acetylpenicillamine (SNAP) a compound which releases NO, PGE2 synthesis was increased as a result of the increased AA release, which counteracted the decreasing effect on COX-2 expression. On the other hand after stimulation with LPS, SNAP decreased PGE2 due to the slight increase in AA release. Recently NONOates have been recognised a good source of NO due to their well defined half-life. We studied the effect of NO derived from PAPNNO (tl/2 15 min) and DETAINO (tl/2 20 hr) in RAW 264.7 cells. We found that both NONOates (500 JLM) markedly increased the basal and the LPS-stimulated PGE2 production. Basal PGE2 productionwas 5 fold and 6 fold increased after 24 hr incubation with PAPAINO and DETNNO respectively. PAPNNO and DETAINO exposure was accompanied by a 2 fold and 3 fold increase respectively of the LPS-stimulated PGE2 release. The AA release was only slightly increased but interestingly DETAINO released AA (22% and 30% over basal and LPS.induced respectively) after 2 hr incubation while PAPAINO (15% and 12% over basal and LPS-induced respectively) after 5 hr incubation. At variance with SNAP, the massive increase in basal and LPS-stimulated PGE2 production is very unlikely to be mediated by an increased AA release, while western blot analysis of RAW 264.7 proteins gave indication of an increased expression of COX-2.
IP2C79\
THE ROLEOF TUMORNECROSIS FACTOR-a AND NITRICOXIDE IN ACUTELIVERINJURYINDUCED BY CARBONTETRACHLORIDE
L.A. Morio *, D.L. Laskin. Joint Graduate Program in Toxicology, Rutgers University and UMDNJ-R oben Wood Johnson Medical School, Piscataway. New Jersey, USA Our laboratory has been investigating the role of macrophages in chemically-induced hepatotoxicity. In a number of different experimental models, hepatotoxicity is prevented or reduced by pretreatment of animals with the macrophage inhibitor. gadolinium chloride. These findings demonstrate that macrophagcs playa critical role in the pathogenesis of liver injury. Macrophages arc known to release a numberof differentinflammatory mediators with cytotoxicpotential. ln the present studies we analyzed the role of two macrophagederived mediators, tumor necrosis factor-a (TNF-a) and nitric oxide, in liver inj ury induced by carbon tetrachloride (CCLj). Treatment of mice with CC4 (0.3 ml/kg, i.p.) resulted in a time dependent induction of centrilobular hepatic necrosis. This was correlated with a dramatic increase in serum transaminase levels. Recent studies have shown that CCI4 treatment of animals also induces TNF-a and nitric oxide production in the liver. To study the role of these mediators in hepatotoxicity, we used knockout mice lacking the gene for the 55 kDa TNF-a receptor or inducible nitric oxide synthase (iNOS). We found that CC4 was significantly less effectivein inducing hepatotoxicity in mice lacking the p55 TNF-a receptor. These data are consistent with the observation that pretreatment of animals with soluble TNF-a receptor or anti-TNF-a antibody decreased CC4-induced liver injury. In contrast, CC4-induced liver injury was markedly increased in knockout mice lacking the gene for iNOS suggesting a hepatoprotective role of nitric oxide. Taken together, these data suggest that macrophage-derived TN F-a and nitric oxide are important regulators of CC4 -induced liver injury (Supported by NIH GM343I0 and the Burroughs Wellcome Fund).
IP2CBO I
113
MICE LACKINGINDUCIBLE NITRIC OXIDESYNTHASE ARE PROTECTED FROMACETAMINOPHEN-INDUCED HEPATD-TOXICITY
J.D. Laskin *. en. Gardner, D.E. Heck, H. Chiu, S.K. Durham, D.L. Laskin. Rutgers University and UMDNJ-Roben Wood Johnson Medical School, Piscataway. NJ and Bristol Myers Squibb Pharmaceutical Research Institute, Prin ceton. NJ. USA
Nitric oxide (NO) is a highly reactive inflammatory mediator implicated in chemically-induced tissue injury. In the present srudies we analyzed the role of NO in the hepatotoxicity of acetaminophen (AA) using knockout mice lacking the gene for inducible nitric oxide synthase (NOSII). Treatment of wild type control mice with AA (100-300 mglkg, ip) resulted in a dose and time dependent induction of centrilobular necrosis. This was associated with a marked increase in serum ALT and AST levels. AA was significantly less effective in inducing hepatotoxicity in mice lacking NOSH. These data demonstrate that NO produced via NOSH contributes to liver injury. To assess the contribution of parenchymal cell-derived NO to AA-induced toxicity, we quantified hepatocyte NO production. AA administration resulted in a time dependent increase in NO production and NOSII expression by hepatocytes. Pretreatment of animals with the NOSII inhibitor aminoguanidine for 3 days (100 mg/kg, sc, I x /day) completely abrogated hepatotoxicity. Hepatocytes isolated from animals pretreated with aminoguanidine also produced significantly less NO than cells from animals treated with AA alone. This was associated with decreased NOSH protein and mRNA. These results suggest that aminoguanidine exerts multiple sites of inhibitory action on NO production. This is supported by our finding that a single injectionof arninoguanidine immediately before AA was significantly less effective in blocking hepatotoxicity. Taken together these data demonstrate that NO is a critical mediator of hepatotoxicity. Moreover, hepatocytes appear to be a major source of NO in the liver during the pathogenesis of AA induced liver injury (Supported by NIH GM343I0 and the Burroughs Wellcome Fund).
IP2CB1 I
THE ROLEOF NITRIC OXIDE ON LEAD NEPHROTOXICITY IN PERFUSED RATKJDNEY
M. Ghazi-Khansari *, S. Ala. M. Essalat, A.R. Dchpour. Departm ent of Pharmacology. School ofMedicine. Tehran Unive rsity of Medical Sciences. P. O. Box /3/45-784. Tehran. I.R., Iran Lead nephrotoxicity has been shown by a useful early markerof renal injury namely urinary N-acetyl-tl-D-glucosaminidase (NAG). In this study the effect of lead acetate on nephrotoxicity and its correlation with the nitric oxide (NO) system by detecting the NAG release in perfused rat kidney was investigated. Lead acetate (20 and 50 JLgldl) caused time and dose-dependent increase in enzymuria. The effect of concurrent perfusion with lead and L-arginine (L-Arg) or L-NG-nitro argininemethylester (L-NAME) [precursor and inhibitor of NO synthase respectively] in the perfusion fluid was also studied by measuring NAG activity in the perfusate kidney rat. L-Arg (2 roM ) has significantly decreased the lead-induced NAG release (p < 0.00 I), and L-NAME (0.1 mM) has significantly increased the lead-induced enzyme release in the time-dependent manner (p < 0.001). Moreover, histological studies using light microscope shows that some of the epithelial cells of the proximal compoluted tubules are degenerated or necrotic and desquamated into the lumens in rat treated with lead acetate. This changes occurs at 50 ILgidl of lead acetate and increases by addition of L-NAME to lead acetate. This may suggested that lead may interfere with NO system in rat kidney.