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reperfusion on heat-shock protein 70 and 90 gene expression in rat liver: Relation to nutritional status
382A
ii01
AASLD ABSTRACTS EFFECT OF I S C H E M I A / R E P E R F U S I O N ON HEAT.SHOCK PROTEIN 70 AND 90 GENE EXPRESSION IN RAT LIVER: RELATION T O NUTRITIONAL STATUS. A Gnsbarrini, S De21i
1102
Esoeati*. S De Notariis. P Caranoni. S Loffredo*. A Abraham*, M Simoneini. A Cnlantoni. F Tmvisani. G Gasbardni'. M Buraardi. Universita' di Bologna and Universita' Cattoliea di Roma', Italy; Brown University*, Providence, USA.
EFFECTS OF HEPATOCYTE GROWTH FACTOR ON DIMETHYLNITROSOAMINE INDUCED LIVER INJURY IN RATS. K. Okabe, H. Suzuki, S. Suemori, T. Yamamoto,Y Mamata, N. Mat sumoto, H . M i z u n o , M . K o n o , M . S u g a , M . S u z u k i , Y K a t o , A.Sato,T.Meguro. Deparment of Internal Medicine and Research Laboratory. Yokohama City S e ~ u Hospital, St.Marianna University School of Medicine, Yokohama, Japan Hepatocyte growth factor ( H G F ) is a multifunctional cytokine with numerous activities in a variety o f cell lines.The aim of this study was to investigate the effects of an exogenous addition of H G F on expenmentaliy induced chronic liver injury. [ M e t h o d s ] Group 1:Control(normal rats), G r o u p 2 : D M N administrated group(rats received dimethylnitrosoamine (DMN;10 gg/kg) by intraperitoneal injection every other day, 3 times a w e e k , for 4 weeks) ,Group 3: D M N + H G F group; rats ,administrated with DMN, received an intravenous injection o f H G F (500 gg/kg) twice a d a y . Recombinant h u m a n H G F (5 amino acid residue-deleted type H G F ) was used in this study.Serum AST, ALT, totalprotein,fibrinogen, a 2plasmin inhibitor, normotest were determined andhistological chages were investigated. [ R e s u l t s ] In laboratory findings, the intravenous injection o f H G F i n c r e a s e d platelct count, total protein a n d fibrinogen, and suppressed the elevation o f A S T and ALT levels. D M N administration i n d u c e d h e m o r r h a g i c necrosis a n d H G F r e m a r k a b l y supressed this hemorrhagic necrosis and increased P C N A labeling index. Tabie 1.Effects of dIIGF in rats with DMN-induced liver injury DMN (n=4) DMN+dHGF(n=4) Control (n=8) PLT(xl04//z I) 9.3+--4.0 ** 65.3+-5.4 ## 72.7+-1 .S T. protein(g/dl) AST(mu/ml)
4.0+-0,15 ** 350+-74 **
ALT(mu/ml)
235+-45 **
Normotest(%) Fibrinogen(mg/dl) ar2-plasmininhibitor(%)
39+-1.3 ** 66+-16 * 67+-3
4.9+-0.17 ##
141±9.7 108+-5 75+-5.9 ## 243+-25 ## 94~+4.5"#
5.1-k 0,04 69+-3
38+-3 100+-8.5 163+-29 75+-7
Mean+-S.E. •* & *; Significantly different from Normal control (p<0.01&0.05) #-it & #; Significantlydifferent from DMN (p<0.01&0.05)
[ D i s c u s s i o n ] E x o g e n o u s addition o f H G F remarkably improved coagulation and fibrinolytic system and liver injury and these dam suggest that an exogenous addition o f H G F m a y be effective in suppressing aggravation o f liver injury.
LYSOSOMAL ENZYMES AS EARLY AND QUANTITATIVE M A R K E R S OF H E P A T I C I S C H E M I A / R E P E R F U S I O N I N J U R Y . w Liu. o Schdb. J Pu~mire. D Jackson. KA Znckur. DE Fry, and RH Glew. Dpts of Surgery and Biochemistry, University of New Mexico, Albuquerque Due to considerable delay in response serum lactate dchydrogeaase (LDH), gmnmaglutamyltransferase (GGT). and aspartatu aminotransferasc (AST), do not satisfy to early assess hepatic injury/failure. Lysosomai evzymes, however, appe,~ to be more eligible and this study was performed to investigate their properties as early indicators and prognostic factors in isehemia-reperfusioninjury of the fiver. In ten male swine (25 to 35 kg) following 30, 45, and 90 rain of acute hepatic ischemia, the release of eight lysosomal enzymes and lipid hydroperoxide production were determined and compared to pre- and post-operativelevels of LDH, GGT and AST. The release of ali lysosomal enzymes was dose-depandant on the length of the iachemic p e r i o d Isc~mlJ ~rlod (45 ~ 90 mln) ~1 ~-Gluzoslde~" eso (e,g., g-galactosidsse mean of f o l d increase: 30 min 1.9-fold; 45 rain [ 8.3-fold; 90 rain 22.8-fold, p< 0.002) and peaked within the In'st three hours. 5 Lipid p e r o x i d e production followed timeoff l e p e r f u s i o n the same pattern. AST,LDH and GGT scarcely increasedinitially andpeaked24 hours after restonnghepatic blood flow. In the case of irreversible damage (90 rain ischemia) the lysosomal enzymes showed a continuous increase till the animals expired in liver failure (6 to 12 In's) whereas in reversible damage the lysosomai enzymes started dropping after 2-3 hrs and reached normal values within 24 hours. We concinde that 8-galactusidaseand g-glucosidase appear to be more sensitive and earlier markers for 1. quanffyingliver injury and 2. distinguishing reversible and irreversible hepatic injury than AST, LDH, or GGT.
Heat shock proteins (HSP) are inUacelhtlasproteins associated with a generalized response of the cell to stress conditions. Anion superoxide ganemtion is one of the factor that determine HSP gane expression in post-ischemic condition. Our group recently showed that the production of an/on superoxide induced by reoxyganation in hepatocytes isolated from fasted rats is greater than in cells obtained from fed animals. Aim of the study: m assess the levels of messanger RNA (mRNA) for HSP genes 70 and 90 in liver from fed or 24 h-fasted rats in baseline condition and during a period of 60 min ischemia/120 rain repeffusinn. Reduced glutathione (GSH) was evaluated to assess antioxidant status of the tissue. Methods: liver ischemia was induced by piaeing mic~'ovascularclamps around the appropriale branches of the portal vein and hepatic m'tery of the left lateral and median lobe. Northern blot analysis of total RNA extracted from liver tissue was performed. Specific P32 labelled eDNA probes for HSP 70, HSP 90, albumin and reference Riboprobe were utilized. Densitometry analysis of antoradiograms was performed to obtain semiquantitative dala. GSH was assessed by spettrophotumetric methods. Results: in baseline condition, liver from fasted animals presented significant differences compared to organ from fed rats: mRNA for HSP 70 and 90 were increased 2-3 fold and GSH was decreased by 40 % (0.72±0.1 vs 1.26±0.1 nM/mg; p<0.01). After 60 min ischemia, liver from starved rats presanted a 2-3 fold decrease in HSP 70 and 90 mRNA, whiie HSP gent expression did not change significantely in liver from fed animals, GSH decreased 55 % (to 0.32±0.1 riM/rag) in fasted liver and 25 % (to 0.94±0.1 nMlmg) in fed organ. Upon 120 rain repeffnsion, HSP 70 and 90 mRNA rose 4-5 folds only in fasted animals, whiie a slight decrement in gene expression was observed in the fed group; GSH concantration returned to 65 and 85 % of baseline value in liver from fasted and fed rats, respectively (to 0.47±0.1 and to 1.07±0.1 nM/mg). Conclusion: a prolonged period of fasting determines by itseif a significant reduction in liver antioxidant status and an induction of HSP gane expression. Moreover, only in the fasted group the repeffusion phase that follows a period of isehcmia was characterized by a m~ked rise in HSP mRNA. The reduced antioxidant status and the greater generation of anion superoxide determined by fasting may be the mechanisms underlying this phenomena.
1103
HEPATOLOGYOctober 1995
m
1104
.
L~
iNTERACTIONS OF CYCLOSPORIN A A N D ITS DERIVATIVE IMM-125 WITH RAT HEPATOCYTES A Wolf. C Tronclolonbura. L. Aicher. U $¢hrgmm, A Fahr. G Fdcker. 5andoz Pharma AG, Basle, Switzerland
Treatment with Cyclosporin A may be accompanied by hepatic side effects such as cholestasis. Therefore, interactions of cyclospodn A (CSA) and its novel derivative D-Serin,-cyclosporin (IMM-125) with hepatocytes were investigated in freshly isolated rat liver cells, primary hepatocyte cultures and isolated perfused rat liver. Na'-dependent taurocholate uptake into freshly isolated rat liver cells was appearantly competitively inhibited to a similar extent by CSA and tMM-U25. Uptake of both cyclosporins, however, was not Na'dependent nor saturable, nor could it be inhibited by taurocholate and cholate, indicating that inhibition of bile salt uptake by cyclosporins occurs by an indirect interaction rather than by direct competition for an uptake system. This finding was supported by experiments with artificial lipid membranes showing dose dependent changes in membrane fluidity after incubation with both cyclosporins. Incubation of primary hepatocyte cultures with 10 IJM CSA or IMM-125 resulted in a moderate increase of LDH release. After incubation with 25 and 50 [aM of each cyclosporin the release of LDH was higher in the presence of IMM-125 than in the presence of CSA, This finding was confirmed in isolated rat livers, when perfusion with 25 tJM and 50 tJM IMM-125 resulted in a significantly earlier release of LDH than perfusion with CSA. The release of the transaminases GOT and GPT was not significantly different from controls for both cyclosporins, Bile flow was immediately impaired after addition of 50 IJM of each cyciosporin into the perfusate. It diminished to about 55 % of controls in the presence of CSA and to 35 % of controls in the presence of IMM- 125. In order to see whether intracellular events are involved in hepatic cyctosporin side effects the molar ratio of cellular reduced and oxidized glutathione (GSH/GSSG) was determined in primary hepatocyte cultures, tt was significantly lower than in controls already at concentrations 2 1 }JM of both cyclosporins with a more pronounced effect of IMM-125. The data provide evidence that IMM-125 might exert higher hepatic side effects than CSA and that oxidative stress might be part of the mechanisms by which cyclosporins cause liver toxicity.
ii01
AASLD ABSTRACTS EFFECT OF I S C H E M I A / R E P E R F U S I O N ON HEAT.SHOCK PROTEIN 70 AND 90 GENE EXPRESSION IN RAT LIVER: RELATION T O NUTRITIONAL STATUS. A Gnsbarrini, S De21i
1102
Esoeati*. S De Notariis. P Caranoni. S Loffredo*. A Abraham*, M Simoneini. A Cnlantoni. F Tmvisani. G Gasbardni'. M Buraardi. Universita' di Bologna and Universita' Cattoliea di Roma', Italy; Brown University*, Providence, USA.
EFFECTS OF HEPATOCYTE GROWTH FACTOR ON DIMETHYLNITROSOAMINE INDUCED LIVER INJURY IN RATS. K. Okabe, H. Suzuki, S. Suemori, T. Yamamoto,Y Mamata, N. Mat sumoto, H . M i z u n o , M . K o n o , M . S u g a , M . S u z u k i , Y K a t o , A.Sato,T.Meguro. Deparment of Internal Medicine and Research Laboratory. Yokohama City S e ~ u Hospital, St.Marianna University School of Medicine, Yokohama, Japan Hepatocyte growth factor ( H G F ) is a multifunctional cytokine with numerous activities in a variety o f cell lines.The aim of this study was to investigate the effects of an exogenous addition of H G F on expenmentaliy induced chronic liver injury. [ M e t h o d s ] Group 1:Control(normal rats), G r o u p 2 : D M N administrated group(rats received dimethylnitrosoamine (DMN;10 gg/kg) by intraperitoneal injection every other day, 3 times a w e e k , for 4 weeks) ,Group 3: D M N + H G F group; rats ,administrated with DMN, received an intravenous injection o f H G F (500 gg/kg) twice a d a y . Recombinant h u m a n H G F (5 amino acid residue-deleted type H G F ) was used in this study.Serum AST, ALT, totalprotein,fibrinogen, a 2plasmin inhibitor, normotest were determined andhistological chages were investigated. [ R e s u l t s ] In laboratory findings, the intravenous injection o f H G F i n c r e a s e d platelct count, total protein a n d fibrinogen, and suppressed the elevation o f A S T and ALT levels. D M N administration i n d u c e d h e m o r r h a g i c necrosis a n d H G F r e m a r k a b l y supressed this hemorrhagic necrosis and increased P C N A labeling index. Tabie 1.Effects of dIIGF in rats with DMN-induced liver injury DMN (n=4) DMN+dHGF(n=4) Control (n=8) PLT(xl04//z I) 9.3+--4.0 ** 65.3+-5.4 ## 72.7+-1 .S T. protein(g/dl) AST(mu/ml)
4.0+-0,15 ** 350+-74 **
ALT(mu/ml)
235+-45 **
Normotest(%) Fibrinogen(mg/dl) ar2-plasmininhibitor(%)
39+-1.3 ** 66+-16 * 67+-3
4.9+-0.17 ##
141±9.7 108+-5 75+-5.9 ## 243+-25 ## 94~+4.5"#
5.1-k 0,04 69+-3
38+-3 100+-8.5 163+-29 75+-7
Mean+-S.E. •* & *; Significantly different from Normal control (p<0.01&0.05) #-it & #; Significantlydifferent from DMN (p<0.01&0.05)
[ D i s c u s s i o n ] E x o g e n o u s addition o f H G F remarkably improved coagulation and fibrinolytic system and liver injury and these dam suggest that an exogenous addition o f H G F m a y be effective in suppressing aggravation o f liver injury.
LYSOSOMAL ENZYMES AS EARLY AND QUANTITATIVE M A R K E R S OF H E P A T I C I S C H E M I A / R E P E R F U S I O N I N J U R Y . w Liu. o Schdb. J Pu~mire. D Jackson. KA Znckur. DE Fry, and RH Glew. Dpts of Surgery and Biochemistry, University of New Mexico, Albuquerque Due to considerable delay in response serum lactate dchydrogeaase (LDH), gmnmaglutamyltransferase (GGT). and aspartatu aminotransferasc (AST), do not satisfy to early assess hepatic injury/failure. Lysosomai evzymes, however, appe,~ to be more eligible and this study was performed to investigate their properties as early indicators and prognostic factors in isehemia-reperfusioninjury of the fiver. In ten male swine (25 to 35 kg) following 30, 45, and 90 rain of acute hepatic ischemia, the release of eight lysosomal enzymes and lipid hydroperoxide production were determined and compared to pre- and post-operativelevels of LDH, GGT and AST. The release of ali lysosomal enzymes was dose-depandant on the length of the iachemic p e r i o d Isc~mlJ ~rlod (45 ~ 90 mln) ~1 ~-Gluzoslde~" eso (e,g., g-galactosidsse mean of f o l d increase: 30 min 1.9-fold; 45 rain [ 8.3-fold; 90 rain 22.8-fold, p< 0.002) and peaked within the In'st three hours. 5 Lipid p e r o x i d e production followed timeoff l e p e r f u s i o n the same pattern. AST,LDH and GGT scarcely increasedinitially andpeaked24 hours after restonnghepatic blood flow. In the case of irreversible damage (90 rain ischemia) the lysosomal enzymes showed a continuous increase till the animals expired in liver failure (6 to 12 In's) whereas in reversible damage the lysosomai enzymes started dropping after 2-3 hrs and reached normal values within 24 hours. We concinde that 8-galactusidaseand g-glucosidase appear to be more sensitive and earlier markers for 1. quanffyingliver injury and 2. distinguishing reversible and irreversible hepatic injury than AST, LDH, or GGT.
Heat shock proteins (HSP) are inUacelhtlasproteins associated with a generalized response of the cell to stress conditions. Anion superoxide ganemtion is one of the factor that determine HSP gane expression in post-ischemic condition. Our group recently showed that the production of an/on superoxide induced by reoxyganation in hepatocytes isolated from fasted rats is greater than in cells obtained from fed animals. Aim of the study: m assess the levels of messanger RNA (mRNA) for HSP genes 70 and 90 in liver from fed or 24 h-fasted rats in baseline condition and during a period of 60 min ischemia/120 rain repeffusinn. Reduced glutathione (GSH) was evaluated to assess antioxidant status of the tissue. Methods: liver ischemia was induced by piaeing mic~'ovascularclamps around the appropriale branches of the portal vein and hepatic m'tery of the left lateral and median lobe. Northern blot analysis of total RNA extracted from liver tissue was performed. Specific P32 labelled eDNA probes for HSP 70, HSP 90, albumin and reference Riboprobe were utilized. Densitometry analysis of antoradiograms was performed to obtain semiquantitative dala. GSH was assessed by spettrophotumetric methods. Results: in baseline condition, liver from fasted animals presented significant differences compared to organ from fed rats: mRNA for HSP 70 and 90 were increased 2-3 fold and GSH was decreased by 40 % (0.72±0.1 vs 1.26±0.1 nM/mg; p<0.01). After 60 min ischemia, liver from starved rats presanted a 2-3 fold decrease in HSP 70 and 90 mRNA, whiie HSP gent expression did not change significantely in liver from fed animals, GSH decreased 55 % (to 0.32±0.1 riM/rag) in fasted liver and 25 % (to 0.94±0.1 nMlmg) in fed organ. Upon 120 rain repeffnsion, HSP 70 and 90 mRNA rose 4-5 folds only in fasted animals, whiie a slight decrement in gene expression was observed in the fed group; GSH concantration returned to 65 and 85 % of baseline value in liver from fasted and fed rats, respectively (to 0.47±0.1 and to 1.07±0.1 nM/mg). Conclusion: a prolonged period of fasting determines by itseif a significant reduction in liver antioxidant status and an induction of HSP gane expression. Moreover, only in the fasted group the repeffusion phase that follows a period of isehcmia was characterized by a m~ked rise in HSP mRNA. The reduced antioxidant status and the greater generation of anion superoxide determined by fasting may be the mechanisms underlying this phenomena.
1103
HEPATOLOGYOctober 1995
m
1104
.
L~
iNTERACTIONS OF CYCLOSPORIN A A N D ITS DERIVATIVE IMM-125 WITH RAT HEPATOCYTES A Wolf. C Tronclolonbura. L. Aicher. U $¢hrgmm, A Fahr. G Fdcker. 5andoz Pharma AG, Basle, Switzerland
Treatment with Cyclosporin A may be accompanied by hepatic side effects such as cholestasis. Therefore, interactions of cyclospodn A (CSA) and its novel derivative D-Serin,-cyclosporin (IMM-125) with hepatocytes were investigated in freshly isolated rat liver cells, primary hepatocyte cultures and isolated perfused rat liver. Na'-dependent taurocholate uptake into freshly isolated rat liver cells was appearantly competitively inhibited to a similar extent by CSA and tMM-U25. Uptake of both cyclosporins, however, was not Na'dependent nor saturable, nor could it be inhibited by taurocholate and cholate, indicating that inhibition of bile salt uptake by cyclosporins occurs by an indirect interaction rather than by direct competition for an uptake system. This finding was supported by experiments with artificial lipid membranes showing dose dependent changes in membrane fluidity after incubation with both cyclosporins. Incubation of primary hepatocyte cultures with 10 IJM CSA or IMM-125 resulted in a moderate increase of LDH release. After incubation with 25 and 50 [aM of each cyclosporin the release of LDH was higher in the presence of IMM-125 than in the presence of CSA, This finding was confirmed in isolated rat livers, when perfusion with 25 tJM and 50 tJM IMM-125 resulted in a significantly earlier release of LDH than perfusion with CSA. The release of the transaminases GOT and GPT was not significantly different from controls for both cyclosporins, Bile flow was immediately impaired after addition of 50 IJM of each cyciosporin into the perfusate. It diminished to about 55 % of controls in the presence of CSA and to 35 % of controls in the presence of IMM- 125. In order to see whether intracellular events are involved in hepatic cyctosporin side effects the molar ratio of cellular reduced and oxidized glutathione (GSH/GSSG) was determined in primary hepatocyte cultures, tt was significantly lower than in controls already at concentrations 2 1 }JM of both cyclosporins with a more pronounced effect of IMM-125. The data provide evidence that IMM-125 might exert higher hepatic side effects than CSA and that oxidative stress might be part of the mechanisms by which cyclosporins cause liver toxicity.