- Email: [email protected]
Anti-Inflammatory Action of Glutamine in Acute Liver Failure Induced by Thioacetamide in Rats
POSTER PRESENTATIONS time period ( p 0.024), predominantly due to an increase in the percentage of patients surviving with transplantation – see graph. In patients meeting the Kings College Hospital Poor Prognostic Criteria (KCC), spontaneous survival increased from 0% in 1992–1996 to 31.2% in 2007–2011 ( p < 0.001). Overall survival has also increased in this group of patients, from 28.6% to 66.6%.
Conclusions: There has been a decrease in the total number of admission to SLTU over the past 20 years, with no change in number of admissions with POD. Spontaneous survival rates have not improved over time but have improved significantly in the group meeting KCC. Overall survival rates have increased due to more patients surviving with transplantation. THU-408 INHIBITION OF CONNEXIN HEMICHANNELS ALLEVIATES ACETAMINOPHEN-INDUCED HEPATOTOXICITY IN MOUSE M. Maes1, S.C. Yanguas1, J. Willebrords1, T.C. da Silva2, M. Lebosky3, I.V. Pereira2, H. Jaeschke3, B. Cogliati2, M. Vinken1. 1Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium; 2Department of Pathology, University of Sao Paulo, Sao Paulo, Brazil; 3Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States E-mail: [email protected] Background and Aims: Traditionally, connexin hemichannels are considered as the structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in extracellular signaling. In contrast to gap junctions, which mediate intercellular communication, connexin hemichannels seem to be mainly activated in pathological processes, including cell death and inflammation. In the present study, the involvement of connexin hemichannels composed of connexin32 and connexin43, the predominant connexin species expressed in hepatic tissue, in liver toxicity induced by acetaminophen was investigated. These experiments were based on the use of TAT-Gap24 and TAT-Gap19, claimed to be specific inhibitors of connexin32 and connexin43 hemichannels, respectively. Methods: In order to confirm channel specificity of both inhibitors, cultures of primary hepatocytes were exposed to TAT-Gap24 and TAT-Gap19, followed by measurement of gap junction and connexin hemichannel activity. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 and/or TATGap19, and sampling at 0, 3, 6, 24 and 48 hours. Evaluation of the effect of connexin hemichannel inhibition was based on a number of clinically relevant read-outs, including acetaminophen-protein adduct formation, measurement of aminotransferase activity and histopathological examination of liver tissue. Inflammation was judged on hepatic and serum quantities of cytokines, while oxidative stress was monitored by measuring liver amounts of oxidized and reduced glutathione.
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Results: In vitro testing confirmed suppression of connexin32 and connexin43 hemichannel activity by TAT-Gap24 and TAT-Gap19, respectively. Treatment of acetaminophen-overdosed animals with TAT-Gap19 did not show major improvement of liver insults. In contrast, a signification reduction in liver damage was observed upon treatment with TAT-Gap24. Most importantly, simultaneous treatment of acetaminophen-overdosed mice with TAT-Gap24 and TAT-Gap19 revealed a synergistic protective effect against acetaminophen-induced liver injury. Conclusions: Inhibition of overall connexin hemichannel activity might represent a therapeutic strategy in the treatment of acetaminophen-induced acute liver toxicity. THU-409 ANTI-INFLAMMATORY ACTION OF GLUTAMINE IN ACUTE LIVER FAILURE INDUCED BY THIOACETAMIDE IN RATS E.G. Schemitt1, J.R. Colares2, R.M. Hartmann1, F. Licks1, M. Do Couto Soares1, J. De Oliveira Salvi2, C.A. Marroni3, N.A.P. Marroni4. 1 Universidade Federal do Rio Grande do Sul, Porto Alegre; 2Universidade Luterana do Brasil, Canoas; 3Postgraduate Program in Liver Diseases, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre; 4 Postgraduate Program in Cell and Molecular Biology Applied to Health, Universidade Luterana do Brasil, Canoas, Brazil E-mail: [email protected] Background and Aims: Severe acute liver failure (SALF) is a syndrome which leads to functional impairment of the liver. The thioacetamide (TAA) is a xenobiotic that causes liver damage. Glutamine (G) is an amino acid involved in the synthesis of glutathione. The aim of this study was to assess the hepatotoxic effect of thioacetamide and the anti-inflammatory action of glutamine. Methods: CEUA project/HCPA: 12-0116. 28 rats were divided into 4 groups: control (CO), Glutamine (G), Thioacetamide (TAA), Glutamine + thioacetamide (TAA + G). TAA doses were administered (400 mg/kg ip) with an interval of eight hours. Glutamine (25 mg/kg ip) was administered 30 minutes after the last dose of TAA. 24 hours after the beginning of the experiment, the animals were anesthetized and killed. Blood samples were collected to assess the levels of AST, ALT and AP. The liver was removed for analysis interleukins (Assay kits using Luminex® technology by Invitrogen™) and immunohistochemical evaluation of NF-kB, TNF-α and iNOS. Statistical analysis was ANOVA + Student-Newman-Keuls test (mean ± SE) being significant p < 0.05. Results: There was an increase in AST, ALT and AP levels in TAA group (598.8 ± 39.4, 298.4 ± 9.1 and 58.7 ± 2.6 U/L, respectively) relative to groups CO (42.0 ± 9.2, 24.2 ± 2.5 and 15.4 ± 0.9 U/L, respectively) and G (48.5 ± 5.5, 29.8 ± 3.6 and 16.8 ± 1 U/L, respectively) and a decrease in the TAA + G group (249.7 ± 24.9, 54.5 ± 5.1 and 19.6 ± 1.2 U/L respectively) ( p < 0.001). IL-1β increased in TAA group (465.9 ± 15.9 pg/mL) relative to groups CO (61.1 ± 1.8 pg/mL) and G (62.1 ± 1.3 pg/mL) and decreased in the group TAA + G (104 ± 8.5 pg/mL) ( p < 0.001). IL-6 increased in TAA group (359.4 ± 12.6 pg/mL) relative to groups CO (73.2 ± 0.9 pg/mL) and G (78.0 ± 0.8 pg/mL) and decreased in the group TAA + G (155.9 ± 6.9 pg/mL) ( p < 0.01). An increase in IL-10 was observed in the TAA group (58.2 ± 4.5 pg/mL) when compared to the CO group (8.6 ± 0.9 pg/mL) and G (13.2 ± 1.2 pg/mL) and a decrease in TAA + G group (21.3 ± 3.1 pg/mL) ( p < 0.01). Immunohistochemical expression of NF-kB, TNF-α and iNOS in animals exposed to TAA showed the highest staining for all proteins when compared to the control groups. Glutamine treatment led to decreased expression of the protein in the significantly TAA + G group. Conclusions: TAA causes alterations in biochemical and inflammatory parameters. Glutamine has been shown to have antiinflammatory effects against liver damage generated by this experimental model thioacetamide.
Journal of Hepatology 2016 vol. 64 | S213–S424
Conclusions: There has been a decrease in the total number of admission to SLTU over the past 20 years, with no change in number of admissions with POD. Spontaneous survival rates have not improved over time but have improved significantly in the group meeting KCC. Overall survival rates have increased due to more patients surviving with transplantation. THU-408 INHIBITION OF CONNEXIN HEMICHANNELS ALLEVIATES ACETAMINOPHEN-INDUCED HEPATOTOXICITY IN MOUSE M. Maes1, S.C. Yanguas1, J. Willebrords1, T.C. da Silva2, M. Lebosky3, I.V. Pereira2, H. Jaeschke3, B. Cogliati2, M. Vinken1. 1Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium; 2Department of Pathology, University of Sao Paulo, Sao Paulo, Brazil; 3Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, United States E-mail: [email protected] Background and Aims: Traditionally, connexin hemichannels are considered as the structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in extracellular signaling. In contrast to gap junctions, which mediate intercellular communication, connexin hemichannels seem to be mainly activated in pathological processes, including cell death and inflammation. In the present study, the involvement of connexin hemichannels composed of connexin32 and connexin43, the predominant connexin species expressed in hepatic tissue, in liver toxicity induced by acetaminophen was investigated. These experiments were based on the use of TAT-Gap24 and TAT-Gap19, claimed to be specific inhibitors of connexin32 and connexin43 hemichannels, respectively. Methods: In order to confirm channel specificity of both inhibitors, cultures of primary hepatocytes were exposed to TAT-Gap24 and TAT-Gap19, followed by measurement of gap junction and connexin hemichannel activity. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 and/or TATGap19, and sampling at 0, 3, 6, 24 and 48 hours. Evaluation of the effect of connexin hemichannel inhibition was based on a number of clinically relevant read-outs, including acetaminophen-protein adduct formation, measurement of aminotransferase activity and histopathological examination of liver tissue. Inflammation was judged on hepatic and serum quantities of cytokines, while oxidative stress was monitored by measuring liver amounts of oxidized and reduced glutathione.
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Results: In vitro testing confirmed suppression of connexin32 and connexin43 hemichannel activity by TAT-Gap24 and TAT-Gap19, respectively. Treatment of acetaminophen-overdosed animals with TAT-Gap19 did not show major improvement of liver insults. In contrast, a signification reduction in liver damage was observed upon treatment with TAT-Gap24. Most importantly, simultaneous treatment of acetaminophen-overdosed mice with TAT-Gap24 and TAT-Gap19 revealed a synergistic protective effect against acetaminophen-induced liver injury. Conclusions: Inhibition of overall connexin hemichannel activity might represent a therapeutic strategy in the treatment of acetaminophen-induced acute liver toxicity. THU-409 ANTI-INFLAMMATORY ACTION OF GLUTAMINE IN ACUTE LIVER FAILURE INDUCED BY THIOACETAMIDE IN RATS E.G. Schemitt1, J.R. Colares2, R.M. Hartmann1, F. Licks1, M. Do Couto Soares1, J. De Oliveira Salvi2, C.A. Marroni3, N.A.P. Marroni4. 1 Universidade Federal do Rio Grande do Sul, Porto Alegre; 2Universidade Luterana do Brasil, Canoas; 3Postgraduate Program in Liver Diseases, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre; 4 Postgraduate Program in Cell and Molecular Biology Applied to Health, Universidade Luterana do Brasil, Canoas, Brazil E-mail: [email protected] Background and Aims: Severe acute liver failure (SALF) is a syndrome which leads to functional impairment of the liver. The thioacetamide (TAA) is a xenobiotic that causes liver damage. Glutamine (G) is an amino acid involved in the synthesis of glutathione. The aim of this study was to assess the hepatotoxic effect of thioacetamide and the anti-inflammatory action of glutamine. Methods: CEUA project/HCPA: 12-0116. 28 rats were divided into 4 groups: control (CO), Glutamine (G), Thioacetamide (TAA), Glutamine + thioacetamide (TAA + G). TAA doses were administered (400 mg/kg ip) with an interval of eight hours. Glutamine (25 mg/kg ip) was administered 30 minutes after the last dose of TAA. 24 hours after the beginning of the experiment, the animals were anesthetized and killed. Blood samples were collected to assess the levels of AST, ALT and AP. The liver was removed for analysis interleukins (Assay kits using Luminex® technology by Invitrogen™) and immunohistochemical evaluation of NF-kB, TNF-α and iNOS. Statistical analysis was ANOVA + Student-Newman-Keuls test (mean ± SE) being significant p < 0.05. Results: There was an increase in AST, ALT and AP levels in TAA group (598.8 ± 39.4, 298.4 ± 9.1 and 58.7 ± 2.6 U/L, respectively) relative to groups CO (42.0 ± 9.2, 24.2 ± 2.5 and 15.4 ± 0.9 U/L, respectively) and G (48.5 ± 5.5, 29.8 ± 3.6 and 16.8 ± 1 U/L, respectively) and a decrease in the TAA + G group (249.7 ± 24.9, 54.5 ± 5.1 and 19.6 ± 1.2 U/L respectively) ( p < 0.001). IL-1β increased in TAA group (465.9 ± 15.9 pg/mL) relative to groups CO (61.1 ± 1.8 pg/mL) and G (62.1 ± 1.3 pg/mL) and decreased in the group TAA + G (104 ± 8.5 pg/mL) ( p < 0.001). IL-6 increased in TAA group (359.4 ± 12.6 pg/mL) relative to groups CO (73.2 ± 0.9 pg/mL) and G (78.0 ± 0.8 pg/mL) and decreased in the group TAA + G (155.9 ± 6.9 pg/mL) ( p < 0.01). An increase in IL-10 was observed in the TAA group (58.2 ± 4.5 pg/mL) when compared to the CO group (8.6 ± 0.9 pg/mL) and G (13.2 ± 1.2 pg/mL) and a decrease in TAA + G group (21.3 ± 3.1 pg/mL) ( p < 0.01). Immunohistochemical expression of NF-kB, TNF-α and iNOS in animals exposed to TAA showed the highest staining for all proteins when compared to the control groups. Glutamine treatment led to decreased expression of the protein in the significantly TAA + G group. Conclusions: TAA causes alterations in biochemical and inflammatory parameters. Glutamine has been shown to have antiinflammatory effects against liver damage generated by this experimental model thioacetamide.
Journal of Hepatology 2016 vol. 64 | S213–S424