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Carbon monoxide acts via inhibition of cytochrome oxidase and generation of mitochondrial reactive oxygen species
228
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
not responsive to change using either tool. Functional independence was correlated with SF-12 and SF-36 PCS. PTSD, depression, gender, and social support were correlated with SF-12 and SF-36 MCS. Conclusions: The SF-12 can be used to assess quality of life in trauma patients. The lack of responsiveness to change of the MCS suggests other methods may be necessary to fully evaluate mental QOL. Combining the SF-12 with measures to assess psychosocial variables in this population should be further investigated. QOL measure
1 month*
6 month*
Population norms
SF-36 SF-12 SF-36 SF-12
31.8 ⫾ 0.7 32.3 ⫾ 0.7 47.5 ⫾ 0.9 47.2 ⫾ 0.8
41.4 ⫾ 1.1 41.0 ⫾ 1.1 47.6 ⫾ 1.1 47.5 ⫾ 1.0
50.0 ⫾ 0.2 50.0 ⫾ 0.2 50.0 ⫾ 0.2 50.0 ⫾ 0.2
PCS PCS MCS MCS
*p ⬍ 0.05 compared to population norms, SF-12 and SF-36 PCS and MCS.
TRAUMA/CRITICAL CARE IV-Apoptosis and Oxidative stress 178. CARBON MONOXIDE ACTS VIA INHIBITION OF CYTOCHROME OXIDASE AND GENERATION OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES. R. Vallabhaneni1, J. Rao1, L. Otterbein2, T. Billiar1, B. S. Zuckerbraun1; 1University of Pittsburgh, Pittsburgh, PA, 2Harvard University, Boston, MA. Introduction. Critical illness and multisystem organ failure can result when the inflammatory stress response to trauma and injury is excessive and sustained after severe trauma. The stress response also involves the compensatory upregulation of anti-inflammatory genes. One such gene is heme oxygense-1. Many of the antiinflammatory properties of HO-1 have been attributed to its byproducts of heme catabolism, specifically on carbon monoxide. Although CO has been shown to be cytoprotective, the mechanisms of action of CO are not well known. The purpose of these studies was to test the hypothesis that CO limits the inflammatory response via inhibition of cytochrome oxidase and generation of mitochondrial reactive oxygen species. Methods. RAW 264.7 cells were utilized for all experiments. Mutant 0 cells were generated from wild-type RAW cells by incubation with ethidium bromide (25ng/mL) for 2 weeks. This inhibits replication of mitochondrial DNA, and critical subunits of the electron transport chain (ETC). Cells were treated with CO 250 ppm throughout the duration of all experiments. Cytochrome oxidase activity was determined from intact cells. Antimycin A was utilized as an inhibitor of complex III of the ETC. Elaboration of reactive oxygen species (ROS) was determined by DCF fluorescence. TNF-␣ production was measured in LPS (1g/mL) treated RAW cells by ELISA. Statistics were determined using one-way ANOVA. Results. CO was confirmed to significantly inhibit intact cell cytochrome oxidase activity (P⬍0.01). CO increased ROS generation in a time-dependent fashion as determined by DCF fluorescence. Changes could be detected within minutes and were 2.3⫾0.14 fold increased over control at 30 minutes (P⬍0.05). Antimycin A alone modestly increased ROS production compared to controls (1.26⫾0.13 fold) but diminished CO-induced DCF fluorescence to 1.5⫾0.17 fold controls (P⬍0.05 compared to CO alone). CO did not increase ROS generation in 0 cells. LPS increased TNF-␣ production 12.3⫾0.5 fold compared to controls (P⬍0.01). Antimycin A alone or with LPS had no significant effect. CO treatment diminished LPS-induced TNF-␣ production to 5.1⫾0.9 fold that of controls (P⬍0.01 compared to LPS alone). However, antimycin A abrogated the effects of CO resulting in a 9.7⫾1.3 fold increase compared to controls (P⬍0.01 compared to CO⫹LPS group). Furthermore, inhibition of upstream ETC by antimycin A reversed CO induced phosphorylation of p38 and JNK. Conclusions. These results suggest that the antiinflammatory effects of CO occur via inhibition of cytochrome oxidase
and subsequent mitochondrial generation of ROS from upstream components of the ETC chain. CO may prove to be a useful clinical adjunct to decreasing inflammation, yet further investigation is needed to characterize these mechanisms. 179. NITRIC OXIDE (NO) DECREASES LIVER DAMAGE AND CASPASE-3 ACTIVITY DURING CHOLESTATIC LIVER INJURY. Bucher B, Jeyabalan G, Kaizu T, Feng X, Murase N, Geller D; University of Pittsburgh Background: Bile acid-induced apoptosis is an important mediator of cellular injury in cholestatic liver disease. The iNOS gene is known to be activated during cholestatic liver diseases, however, the role of NO in this setting is unknown. Therefore, the purpose of this work was to study the effects of nitric oxide (NO) on bile salt glycochenodeoxycholic acid (GCDC)-induced liver injury in rat hepatocyte cultures (HCs), as well as in vivo in a model of cholestatic liver injury. Methods: For in vitro hepatocellular injury, rat hepatocytes (HC) were treated with GCDC (200 M). The NO donor SNAP was used as a source of exogenous NO. Adenoviral iNOS (AdiNOS) transduction was used to generate endogenous NO synthesis, and HC were transfected with AdiNOS or AdY5 (empty Ad vector) at an MOI⫽3 prior to GCDC treatment. Caspase-3 activity was measured from the protein extracts 6 hrs after GCDC treatment. For the in vivo model of cholestatic liver injury, Lewis rats were pretreated with either AdiNOS or AdLacZ 4 days prior to complete bile duct ligation (BDL). Serum LFTs were measured 24 hrs after BDL. Results: GCDC induced significant caspase-3 activity in HC (Table). AdY5 or AdiNOS alone had no effect on basal caspase-3 activation (data not shown). The NO donor SNAP decreased caspase-3 activation in a dose-dependent manner. Oxidized (inactive) SNAP had no effect (data not shown). AdiNOS delivery (but not AdY5) significantly decreased caspase-3 activation, and this was reversed by the iNOS inhibitor L-NIO. In vivo, bile duct ligation produced elevated serum AST (2,190⫾165) and ALT (1,367⫾14) levels after one day, and the hepatic injury was significantly decreased by pre-treatment with AdiNOS (989⫾246ⴱ and 846⫾193ⴱ, respectively), ⴱ p⬍0.05 compared to AdLacZ control. Conclusion: Bile salts (GCDC) caused significant hepatocellular apoptosis in vitro, and this was reversed by endogenous or exogenous NO. Further, endogenous NO synthesis with AdiNOS transduction decreased cholestatic liver injury in vivo, suggesting a possible beneficial role for NO in cholestatic liver conditions.
180. UP-REGULATION OF THE PRO-DEATH PROTEIN FADD REQUIRES CASPASE-8 ACTIVATION AND BID CLEAVAGE IN HEPATOCYTES. R. Vallabhaneni, Y. Wang, X. Zhang, Y. Yuan, X. Peng, X. Yin, T. Billiar; University of Pittsburgh, Pittsburgh, PA. Introduction: Fulminant hepatic failure is associated with excessive hepatocyte (HC) apoptosis. However, the mechanisms that govern apoptosis are only partially understood. We have previously shown that levels of Fas-Associated Death Domain Protein (FADD), an essential component of the Death Inducible Signaling Complex (DISC), increase by over 20 fold in HC undergoing apoptosis in vitro. We postulate that HC FADD up-regulation is an important feed forward apoptotic signaling mechanism in hepatocytes and that it requires the initial activation of apoptotic signaling pathways. We tested this hypothesis by examining the involvement of two of the more proximal steps of apoptotic signaling in HC, caspase-8 activa-
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
not responsive to change using either tool. Functional independence was correlated with SF-12 and SF-36 PCS. PTSD, depression, gender, and social support were correlated with SF-12 and SF-36 MCS. Conclusions: The SF-12 can be used to assess quality of life in trauma patients. The lack of responsiveness to change of the MCS suggests other methods may be necessary to fully evaluate mental QOL. Combining the SF-12 with measures to assess psychosocial variables in this population should be further investigated. QOL measure
1 month*
6 month*
Population norms
SF-36 SF-12 SF-36 SF-12
31.8 ⫾ 0.7 32.3 ⫾ 0.7 47.5 ⫾ 0.9 47.2 ⫾ 0.8
41.4 ⫾ 1.1 41.0 ⫾ 1.1 47.6 ⫾ 1.1 47.5 ⫾ 1.0
50.0 ⫾ 0.2 50.0 ⫾ 0.2 50.0 ⫾ 0.2 50.0 ⫾ 0.2
PCS PCS MCS MCS
*p ⬍ 0.05 compared to population norms, SF-12 and SF-36 PCS and MCS.
TRAUMA/CRITICAL CARE IV-Apoptosis and Oxidative stress 178. CARBON MONOXIDE ACTS VIA INHIBITION OF CYTOCHROME OXIDASE AND GENERATION OF MITOCHONDRIAL REACTIVE OXYGEN SPECIES. R. Vallabhaneni1, J. Rao1, L. Otterbein2, T. Billiar1, B. S. Zuckerbraun1; 1University of Pittsburgh, Pittsburgh, PA, 2Harvard University, Boston, MA. Introduction. Critical illness and multisystem organ failure can result when the inflammatory stress response to trauma and injury is excessive and sustained after severe trauma. The stress response also involves the compensatory upregulation of anti-inflammatory genes. One such gene is heme oxygense-1. Many of the antiinflammatory properties of HO-1 have been attributed to its byproducts of heme catabolism, specifically on carbon monoxide. Although CO has been shown to be cytoprotective, the mechanisms of action of CO are not well known. The purpose of these studies was to test the hypothesis that CO limits the inflammatory response via inhibition of cytochrome oxidase and generation of mitochondrial reactive oxygen species. Methods. RAW 264.7 cells were utilized for all experiments. Mutant 0 cells were generated from wild-type RAW cells by incubation with ethidium bromide (25ng/mL) for 2 weeks. This inhibits replication of mitochondrial DNA, and critical subunits of the electron transport chain (ETC). Cells were treated with CO 250 ppm throughout the duration of all experiments. Cytochrome oxidase activity was determined from intact cells. Antimycin A was utilized as an inhibitor of complex III of the ETC. Elaboration of reactive oxygen species (ROS) was determined by DCF fluorescence. TNF-␣ production was measured in LPS (1g/mL) treated RAW cells by ELISA. Statistics were determined using one-way ANOVA. Results. CO was confirmed to significantly inhibit intact cell cytochrome oxidase activity (P⬍0.01). CO increased ROS generation in a time-dependent fashion as determined by DCF fluorescence. Changes could be detected within minutes and were 2.3⫾0.14 fold increased over control at 30 minutes (P⬍0.05). Antimycin A alone modestly increased ROS production compared to controls (1.26⫾0.13 fold) but diminished CO-induced DCF fluorescence to 1.5⫾0.17 fold controls (P⬍0.05 compared to CO alone). CO did not increase ROS generation in 0 cells. LPS increased TNF-␣ production 12.3⫾0.5 fold compared to controls (P⬍0.01). Antimycin A alone or with LPS had no significant effect. CO treatment diminished LPS-induced TNF-␣ production to 5.1⫾0.9 fold that of controls (P⬍0.01 compared to LPS alone). However, antimycin A abrogated the effects of CO resulting in a 9.7⫾1.3 fold increase compared to controls (P⬍0.01 compared to CO⫹LPS group). Furthermore, inhibition of upstream ETC by antimycin A reversed CO induced phosphorylation of p38 and JNK. Conclusions. These results suggest that the antiinflammatory effects of CO occur via inhibition of cytochrome oxidase
and subsequent mitochondrial generation of ROS from upstream components of the ETC chain. CO may prove to be a useful clinical adjunct to decreasing inflammation, yet further investigation is needed to characterize these mechanisms. 179. NITRIC OXIDE (NO) DECREASES LIVER DAMAGE AND CASPASE-3 ACTIVITY DURING CHOLESTATIC LIVER INJURY. Bucher B, Jeyabalan G, Kaizu T, Feng X, Murase N, Geller D; University of Pittsburgh Background: Bile acid-induced apoptosis is an important mediator of cellular injury in cholestatic liver disease. The iNOS gene is known to be activated during cholestatic liver diseases, however, the role of NO in this setting is unknown. Therefore, the purpose of this work was to study the effects of nitric oxide (NO) on bile salt glycochenodeoxycholic acid (GCDC)-induced liver injury in rat hepatocyte cultures (HCs), as well as in vivo in a model of cholestatic liver injury. Methods: For in vitro hepatocellular injury, rat hepatocytes (HC) were treated with GCDC (200 M). The NO donor SNAP was used as a source of exogenous NO. Adenoviral iNOS (AdiNOS) transduction was used to generate endogenous NO synthesis, and HC were transfected with AdiNOS or AdY5 (empty Ad vector) at an MOI⫽3 prior to GCDC treatment. Caspase-3 activity was measured from the protein extracts 6 hrs after GCDC treatment. For the in vivo model of cholestatic liver injury, Lewis rats were pretreated with either AdiNOS or AdLacZ 4 days prior to complete bile duct ligation (BDL). Serum LFTs were measured 24 hrs after BDL. Results: GCDC induced significant caspase-3 activity in HC (Table). AdY5 or AdiNOS alone had no effect on basal caspase-3 activation (data not shown). The NO donor SNAP decreased caspase-3 activation in a dose-dependent manner. Oxidized (inactive) SNAP had no effect (data not shown). AdiNOS delivery (but not AdY5) significantly decreased caspase-3 activation, and this was reversed by the iNOS inhibitor L-NIO. In vivo, bile duct ligation produced elevated serum AST (2,190⫾165) and ALT (1,367⫾14) levels after one day, and the hepatic injury was significantly decreased by pre-treatment with AdiNOS (989⫾246ⴱ and 846⫾193ⴱ, respectively), ⴱ p⬍0.05 compared to AdLacZ control. Conclusion: Bile salts (GCDC) caused significant hepatocellular apoptosis in vitro, and this was reversed by endogenous or exogenous NO. Further, endogenous NO synthesis with AdiNOS transduction decreased cholestatic liver injury in vivo, suggesting a possible beneficial role for NO in cholestatic liver conditions.
180. UP-REGULATION OF THE PRO-DEATH PROTEIN FADD REQUIRES CASPASE-8 ACTIVATION AND BID CLEAVAGE IN HEPATOCYTES. R. Vallabhaneni, Y. Wang, X. Zhang, Y. Yuan, X. Peng, X. Yin, T. Billiar; University of Pittsburgh, Pittsburgh, PA. Introduction: Fulminant hepatic failure is associated with excessive hepatocyte (HC) apoptosis. However, the mechanisms that govern apoptosis are only partially understood. We have previously shown that levels of Fas-Associated Death Domain Protein (FADD), an essential component of the Death Inducible Signaling Complex (DISC), increase by over 20 fold in HC undergoing apoptosis in vitro. We postulate that HC FADD up-regulation is an important feed forward apoptotic signaling mechanism in hepatocytes and that it requires the initial activation of apoptotic signaling pathways. We tested this hypothesis by examining the involvement of two of the more proximal steps of apoptotic signaling in HC, caspase-8 activa-