- Email: [email protected]
In vivo imaging of increased oxidative stress in the liver by electron spin resonance-computed tomography
Con A injection was elevated in mice of all ages, however plasma ALT level m mice of 3 weeks old was sigmficantly lower compared with other ages of mice, and plasma ALT level was getting higher in accordance with ages (mean + - SE, KU/L: 3 w 1062 + - 299; 5 w 3240 + - 744; 7 w 5970 + - 370; 10 w 5348 + - 248, n=4-5), Plasma IFN-% IL-2 and IL-4 levels 2 and 4 h after Con A injection were significantly reduced in mice of 3 weeks old compared w~th that in mice of 7 weeks of age., However plasma TNF-e~ and IDIO levels were not significant difierence between 3 and 7 weeks old. In mice of 3 weeks old, the pretreatmenl of lL-7 significantly increased plasma ALT level 8 h after Con A injection compared with vehmle treatment (vehicle 1452 + - 322; lk-7 3260 + - 483, n = 6 ) Plasma IFN-y, Ik-2 and ID4 levels 2 and 8 h after Cou A injection were also significantly increased by IL-7 pretreatment in mice of 3 weeks old, Conclusion:These findings suggested that Con A-induced liver mjury was lessened through inhibition of pminflammatory cytokines production and IL-7 exacerbated the liver injury and cytokines production in immature mice,
potential effects on cytokines, cellular signaling, and apoptosis. Because of the clinical synergism noted for alcoholic liver disease and chronic HCV intection, we developed an in vitro system whereby the proxidative properties of core protein could be studied in bepatocytes that overexpress the P450 enzyme CYP2E 1. NepG2 cells which express CYP2E 1 (E47), or control cells (C34), (a kind gift of Dr. A Cederhaum), were transiected with expression vectors containing HCV core sequence genotype lb, (nt 342-915). Cells expressing CYP2E1 and core as well as plasmid controls were cloned using antibiotic selection medium. Clonal cell lines expressing HCV core protein and CYP2E1 were stable lot extended passages and the long-term expression of CYP2E1 in antibiotic selection medium was not affected by" concomitant expression of HC%~ core protein. Compared to the E47 and C34 parental cell lines, cells expressing HCV core showed increased dichlorofluorescin (DCF) fluorescence and hydroethidinm (HE) staining, suggesting that core promotes higher levels of intracellular hydrogen peroxide and superoxide t~spectively E47 ceils that co-expressed core showed decreased cellular levels of glntathione and enhanced sensitivity to glutathtone depletion with bnthionine sultbximme (BSO) than E47 or C34 control cells as measured by cdlular viability studies. These results suggest that core protein expression can lead to an increase in cellular oxidant production and a decrease in cellular glutatbione levels, resulting in decreased hepatocyte viability. Consequently, HCV core may enhance the toxicity and hepatic injury' of CYP2E1 substrates such as ethanol when both disease processes are present in patients.
$893 Glutathion Peroxidase Gene Knockout Accelerates Liver Regeneration After 70% Partml Hepateetomy Nobutaka lwakuma, Nobuya lshibashi, Kazuya Momosaki, 8hogo Yoshida, Atsnshi Kaibara, Yousuke Oka, Kazuo Sh~rouzu Rationale: Reactive oxygen species and antioxidant enzymes have been implicated m control mechanisms of cellular growth and prolitemtion. There is an accumulating evidence that supplementation of antioxidant as well as antioxidatu enzymes gene transter reduce liver injury, msuhs improvement of liver regeneration after partial hepateetomy, however, no evidence can be seen in the literature using selective antioxidant enzymes gene knockout model for this study to date. Here, we investigate the influence of gene knockout ot intracellular glutathione peroxidase(GPxl)on liver regeneration Method: C57BU6 background normal mice (Wild group) and GPxl knockout mice (KO group) were underwent 70% bepatectomy(PH). 1,3,6,24,48,72 and 96 hours alter PH, mice were sacrificed, remnant liver and blood were harvested. Serum levels of AST, ALl', IL-6 and TNF-,x, rate of liver regeneration (RLR), BrdU labeling indices were measured Data was analyzed by ANOVA followed by Fisber's PLSD method. Results: Them was a significant increase in RLR and BrdU labeling indices at 48 and 72hours after PH in KO group ( 4 8 h r s : 3 . 7 _+ 1.3, 72hrs:5.3 • 0.7)compared with Wild group(48hrs:3.2 7_+0.5, 72hrs:4 3 +- 0.4)(p<0.05) The values of AST and ALT in KO group (48hrs:AST/ALT = 76.0• 3.8/29.1 -+ 1.7 SF unitlrnl) were significantly lower than in Wild group (48hrs:AST/ALT = 197.5 • 49.2/293.6 -+ 104.1 SF unit/ml) (p<0.05). Serum lL-6 and TNF-cr were rapidly induced aider PH in KO group(3hrs:lL-6 = 5051 +_8.8 pg/ml, 6hrs:TNF-c~ = 387.5"+ 6.3 pg/ml), while Wild group showed slower induction and slguifieant lower levels of cytukines(3hrs:lL-6 = 126.3 "+42.9 pgknl, 6hrs:TNF-c~ = 137.9 • 569 pg/ml)(p
$896 A New Anti Radical Agent, Edaravone Markedly Protects Rats from Acute Liver Injury Nobuhim Nakamoto, Hidetsugu Saito, Shmichiro Tad.a, Kumi Kitamura, Satoshi Kurita, Yoshimasa Saito, Naok: Kumagat, Kanji Tsuchimoto, Hiromasa lshii (background and aim} Reactive oxygen radicals play an important role it: various forms of liver injury, suggesting that inhibition of radical formation is important in the regulation of the liver injury. Edaravone is a newly synthesized free radical scavenger and has been already clinically used in the prevention of brain damage, but its eftect on liver injury has not been studied.We exanrined its effect on carbon tetrachloride (CCl4)-induced acute liver damage. {Methods) The clinical dose of edaravone (3mg/kg) was intravenously administrated immediately after and 3 hours following intraperimneal administration of CCl4 in rats. Rats were sacrificed 24 horn's after CC14 injection, and serum levels of ALT, TB, and LDH were measured. Histological examination, TUNEL assay, and :mmunohistochemical staining with a monoclonal antibody specific for 4-HNE and 8-OHdG were perfomed. Serum levels of TNF-c~, IL-6 and 1L-10 were measured by ELLS& and the transcnption level of these inflamraatory cytokines was semi-quantified by RT-PCR. (Results) After the administration ot CCl4, serum ALT and LDH levels were significantly elevated at 6, 12, and 24 hours timedependently- in the absence of edamvone. Edaravone treatment, however, attenuated the increase (at 24 hours: ALT, 1 6 3 0 . 6 + 6 0 6 . 9 IU/dl vs 119.4"+ 113.5 IU/dl; LDH, 5068.0"+2956.5 IU/dI vs 369.7 • IU/dl). Histological examination of the liver by H&E and oil red O staining showed a marked reduction of steatosis in the CCL + edaravone group compared with the CCL alone group. Significant inhibition of apoptosis was demonstrated by TUNEL methnd in the CCl4 +edaravone rats. Serum levds of 1L-6, TNF
$894 Liver Regeneration Is Delayed After Partial Hepatectomy in Mice Deficient For Uneoupfing Protein-2 Masayoshi Horimoto, Peter Fulop, Jack R. Wands, Gyorgy Barry BACKGROUND. Liver regeneration is a remarkable process with complex regulation. Hepatocyte pmlfli~mtion reqmres large amounts of metabolic" energy" to match increased macromolecular synthesis. These mechanisms are m part modulated by cellular redox-sensuive factors. Mituchondnal uncoupling protein-2 (UCP2) is an inner membrane protein that sbows increased expression during liver regeneraUon ~bllowmg partial hepatectomy (PH). UCP2 ntediates proton leak, regulates the ATP/ADP ratio, and negatively" affects mitocbondrial superox~de production It is therefore conceivable to suppose that UCP2 has a significant influence on liver regeneration. AIM. We asked whether the absence of UCP2 changes the kinetms of liver regeneration fofiowmg PH. METHODS. Eight4o-twelve weeks old female wild-t~q~e (WT) and UCP2-A mice were subiected to P H The weight of remnant livers was measured at dittemnt time points up to 5 days tollowing PH. Cell proli|eration was measured by BrdU incorporation. Apoptosis rate was assessed by "I15NEL assay. Oxidative stress in liver tissue was characterized by TBARS production. RESULTS. The renmant liver size, expressed as a liver weight/body weight ratio, revealed a significant delay in liver regeneration of UCP2-/- mice as compared to 'v:~ (day 2, 20.8+/-1.3 vs. 246+/-1.8 rag/g, p
$897 In vivo Imaging of Increased Oxidative Stress in the Liver by Electron Spin Resonance-Computed Tomography Junitsu ho, Hitoshi Togashi, Tom Adachi, Kazuhiko Sugahara, Sumio Kawata Background: Although magnetic resonauce imaging, which relies on imaging signals from protons, is being used for clinical purposes, the practical relevance of electron spin resonance (ESR) imaging which relies on imaging free radicals, has not yet been established. The aim of this study" is to investigate whether increased hepatic oxidative stress would be visualized m living aninrals before the onset of obvious liver injury, Materials and Methods: Acute hepatic injury was induced m mice by priming with heat-kiUed C. parvum tbnowed by injection of a low dose of lipopolysacchafide (LPS). Low fl:equency band ESR-CT with carbamoyl-PROX'lL (CP) was used to visualize hepatic oxidative stress, Results: Biochemical and histological investigations performed 3 h after injection of LPS revealed no obvious injury to the liver. Conversely, significant hepatic oxidative stress could be detected at this time. The kinetic clearance of CP after intravenous administration was delayed significantly in mice that had received LPS, due to impairment of the reduction system by hepatic oxidative stress ESR-CT of the murine abdomen revealed a high intensity area of CP, which consisted mainly of the liver and enlarged spleen. Time
$895 HCV Core Protein Promotes Increased Oxidative Stress and Glutathione Depletion in HepG2 Cells that Overexpress P450 microsomal enzyme CYP2E1 Feng Wen, Maber u Abdalla, Costiea Aloman, lman Ahtnad, Jinhua Xiang, Michael L McCormick, K Brown, J. Walewski, A. Branch, Douglas R. Spitz, Bradley Britigan, Warren N. 5chmidt Chronic hepatitis C virus (HCV) iniection causes hepatic oxidative stress and likely influences the redox imbalances caused by" hepatic toxins such as ethanol Recent evidence suggests that the H O / nudeocapsid core protein itself can promote oxidative stress, suggesting
AASLD
Abstracts
A-722
potential effects on cytokines, cellular signaling, and apoptosis. Because of the clinical synergism noted for alcoholic liver disease and chronic HCV intection, we developed an in vitro system whereby the proxidative properties of core protein could be studied in bepatocytes that overexpress the P450 enzyme CYP2E 1. NepG2 cells which express CYP2E 1 (E47), or control cells (C34), (a kind gift of Dr. A Cederhaum), were transiected with expression vectors containing HCV core sequence genotype lb, (nt 342-915). Cells expressing CYP2E1 and core as well as plasmid controls were cloned using antibiotic selection medium. Clonal cell lines expressing HCV core protein and CYP2E1 were stable lot extended passages and the long-term expression of CYP2E1 in antibiotic selection medium was not affected by" concomitant expression of HC%~ core protein. Compared to the E47 and C34 parental cell lines, cells expressing HCV core showed increased dichlorofluorescin (DCF) fluorescence and hydroethidinm (HE) staining, suggesting that core promotes higher levels of intracellular hydrogen peroxide and superoxide t~spectively E47 ceils that co-expressed core showed decreased cellular levels of glntathione and enhanced sensitivity to glutathtone depletion with bnthionine sultbximme (BSO) than E47 or C34 control cells as measured by cdlular viability studies. These results suggest that core protein expression can lead to an increase in cellular oxidant production and a decrease in cellular glutatbione levels, resulting in decreased hepatocyte viability. Consequently, HCV core may enhance the toxicity and hepatic injury' of CYP2E1 substrates such as ethanol when both disease processes are present in patients.
$893 Glutathion Peroxidase Gene Knockout Accelerates Liver Regeneration After 70% Partml Hepateetomy Nobutaka lwakuma, Nobuya lshibashi, Kazuya Momosaki, 8hogo Yoshida, Atsnshi Kaibara, Yousuke Oka, Kazuo Sh~rouzu Rationale: Reactive oxygen species and antioxidant enzymes have been implicated m control mechanisms of cellular growth and prolitemtion. There is an accumulating evidence that supplementation of antioxidant as well as antioxidatu enzymes gene transter reduce liver injury, msuhs improvement of liver regeneration after partial hepateetomy, however, no evidence can be seen in the literature using selective antioxidant enzymes gene knockout model for this study to date. Here, we investigate the influence of gene knockout ot intracellular glutathione peroxidase(GPxl)on liver regeneration Method: C57BU6 background normal mice (Wild group) and GPxl knockout mice (KO group) were underwent 70% bepatectomy(PH). 1,3,6,24,48,72 and 96 hours alter PH, mice were sacrificed, remnant liver and blood were harvested. Serum levels of AST, ALl', IL-6 and TNF-,x, rate of liver regeneration (RLR), BrdU labeling indices were measured Data was analyzed by ANOVA followed by Fisber's PLSD method. Results: Them was a significant increase in RLR and BrdU labeling indices at 48 and 72hours after PH in KO group ( 4 8 h r s : 3 . 7 _+ 1.3, 72hrs:5.3 • 0.7)compared with Wild group(48hrs:3.2 7_+0.5, 72hrs:4 3 +- 0.4)(p<0.05) The values of AST and ALT in KO group (48hrs:AST/ALT = 76.0• 3.8/29.1 -+ 1.7 SF unitlrnl) were significantly lower than in Wild group (48hrs:AST/ALT = 197.5 • 49.2/293.6 -+ 104.1 SF unit/ml) (p<0.05). Serum lL-6 and TNF-cr were rapidly induced aider PH in KO group(3hrs:lL-6 = 5051 +_8.8 pg/ml, 6hrs:TNF-c~ = 387.5"+ 6.3 pg/ml), while Wild group showed slower induction and slguifieant lower levels of cytukines(3hrs:lL-6 = 126.3 "+42.9 pgknl, 6hrs:TNF-c~ = 137.9 • 569 pg/ml)(p
$896 A New Anti Radical Agent, Edaravone Markedly Protects Rats from Acute Liver Injury Nobuhim Nakamoto, Hidetsugu Saito, Shmichiro Tad.a, Kumi Kitamura, Satoshi Kurita, Yoshimasa Saito, Naok: Kumagat, Kanji Tsuchimoto, Hiromasa lshii (background and aim} Reactive oxygen radicals play an important role it: various forms of liver injury, suggesting that inhibition of radical formation is important in the regulation of the liver injury. Edaravone is a newly synthesized free radical scavenger and has been already clinically used in the prevention of brain damage, but its eftect on liver injury has not been studied.We exanrined its effect on carbon tetrachloride (CCl4)-induced acute liver damage. {Methods) The clinical dose of edaravone (3mg/kg) was intravenously administrated immediately after and 3 hours following intraperimneal administration of CCl4 in rats. Rats were sacrificed 24 horn's after CC14 injection, and serum levels of ALT, TB, and LDH were measured. Histological examination, TUNEL assay, and :mmunohistochemical staining with a monoclonal antibody specific for 4-HNE and 8-OHdG were perfomed. Serum levels of TNF-c~, IL-6 and 1L-10 were measured by ELLS& and the transcnption level of these inflamraatory cytokines was semi-quantified by RT-PCR. (Results) After the administration ot CCl4, serum ALT and LDH levels were significantly elevated at 6, 12, and 24 hours timedependently- in the absence of edamvone. Edaravone treatment, however, attenuated the increase (at 24 hours: ALT, 1 6 3 0 . 6 + 6 0 6 . 9 IU/dl vs 119.4"+ 113.5 IU/dl; LDH, 5068.0"+2956.5 IU/dI vs 369.7 • IU/dl). Histological examination of the liver by H&E and oil red O staining showed a marked reduction of steatosis in the CCL + edaravone group compared with the CCL alone group. Significant inhibition of apoptosis was demonstrated by TUNEL methnd in the CCl4 +edaravone rats. Serum levds of 1L-6, TNF
$894 Liver Regeneration Is Delayed After Partial Hepatectomy in Mice Deficient For Uneoupfing Protein-2 Masayoshi Horimoto, Peter Fulop, Jack R. Wands, Gyorgy Barry BACKGROUND. Liver regeneration is a remarkable process with complex regulation. Hepatocyte pmlfli~mtion reqmres large amounts of metabolic" energy" to match increased macromolecular synthesis. These mechanisms are m part modulated by cellular redox-sensuive factors. Mituchondnal uncoupling protein-2 (UCP2) is an inner membrane protein that sbows increased expression during liver regeneraUon ~bllowmg partial hepatectomy (PH). UCP2 ntediates proton leak, regulates the ATP/ADP ratio, and negatively" affects mitocbondrial superox~de production It is therefore conceivable to suppose that UCP2 has a significant influence on liver regeneration. AIM. We asked whether the absence of UCP2 changes the kinetms of liver regeneration fofiowmg PH. METHODS. Eight4o-twelve weeks old female wild-t~q~e (WT) and UCP2-A mice were subiected to P H The weight of remnant livers was measured at dittemnt time points up to 5 days tollowing PH. Cell proli|eration was measured by BrdU incorporation. Apoptosis rate was assessed by "I15NEL assay. Oxidative stress in liver tissue was characterized by TBARS production. RESULTS. The renmant liver size, expressed as a liver weight/body weight ratio, revealed a significant delay in liver regeneration of UCP2-/- mice as compared to 'v:~ (day 2, 20.8+/-1.3 vs. 246+/-1.8 rag/g, p
$897 In vivo Imaging of Increased Oxidative Stress in the Liver by Electron Spin Resonance-Computed Tomography Junitsu ho, Hitoshi Togashi, Tom Adachi, Kazuhiko Sugahara, Sumio Kawata Background: Although magnetic resonauce imaging, which relies on imaging signals from protons, is being used for clinical purposes, the practical relevance of electron spin resonance (ESR) imaging which relies on imaging free radicals, has not yet been established. The aim of this study" is to investigate whether increased hepatic oxidative stress would be visualized m living aninrals before the onset of obvious liver injury, Materials and Methods: Acute hepatic injury was induced m mice by priming with heat-kiUed C. parvum tbnowed by injection of a low dose of lipopolysacchafide (LPS). Low fl:equency band ESR-CT with carbamoyl-PROX'lL (CP) was used to visualize hepatic oxidative stress, Results: Biochemical and histological investigations performed 3 h after injection of LPS revealed no obvious injury to the liver. Conversely, significant hepatic oxidative stress could be detected at this time. The kinetic clearance of CP after intravenous administration was delayed significantly in mice that had received LPS, due to impairment of the reduction system by hepatic oxidative stress ESR-CT of the murine abdomen revealed a high intensity area of CP, which consisted mainly of the liver and enlarged spleen. Time
$895 HCV Core Protein Promotes Increased Oxidative Stress and Glutathione Depletion in HepG2 Cells that Overexpress P450 microsomal enzyme CYP2E1 Feng Wen, Maber u Abdalla, Costiea Aloman, lman Ahtnad, Jinhua Xiang, Michael L McCormick, K Brown, J. Walewski, A. Branch, Douglas R. Spitz, Bradley Britigan, Warren N. 5chmidt Chronic hepatitis C virus (HCV) iniection causes hepatic oxidative stress and likely influences the redox imbalances caused by" hepatic toxins such as ethanol Recent evidence suggests that the H O / nudeocapsid core protein itself can promote oxidative stress, suggesting
AASLD
Abstracts
A-722