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Purpose: To verify the influence of the software version of the dose control system on the equivalent dose and effective dose calculation values., Methods: We performed chest CT and liver to pelvic CT imaging with a human phantom placed on a CT bed. After the imaging was completed, the radiation dose structured reports (RDSRs) generated by the equipment were transferred to several dose management systems with different software versions for equivalent dose and effective dose calculations., Results: The equivalent and effective doses calculated from the same RDSR differed depending on the software version of the dose management system with a difference of approximately 2 times for the effective dose and up to 50 times for the equivalent dose., Conclusion: It is considered that the voxel phantom geometry and dose calculation algorithm may differ depending on the software version of the dose management system. Careful attention should be paid in handling the dose calculation values because the exposure explanations and risk assessments using the equivalent doses, and effective dose calculated by the dose management systems may be overestimated.

Branched-chain amino acids (BCAA) reverse malnutrition and l-carnitine leads to the reduction of hyperammonemia and muscle cramps in cirrhotic patients. BCAA and l-carnitine are involved in glucose and fatty acid metabolism, however their mechanistic activity in cirrhotic liver is not fully understood. We aim to define the molecular mechanism(s) and combined effects of BCAA and l-carnitine using a cirrhotic rat model. Rats were administered carbon tetrachloride for 10 weeks to induce cirrhosis. During the last 6 weeks of administration, cirrhotic rats received BCAA, l-carnitine or a combination of BCAA and l-carnitine daily via gavage. We found that BCAA and l-carnitine treatments significantly improved hepatocellular function associated with reduced triglyceride level, lipid deposition and adipophilin expression, in cirrhotic liver. Lipidomic analysis revealed dynamic changes in hepatic lipid composition by BCAA and l-carnitine administrations. BCAA and l-carnitine globally increased molecular species of phosphatidylcholine. Liver triacylglycerol and phosphatidylcholine hydroperoxides were significantly decreased by BCAA and l-carnitine. Furthermore, serum and liver ATP levels were significantly increased in all treatments, which were attributed to the elevation of mature cardiolipins and mitochondrial component gene expressions. Finally, BCAA and l-carnitine dramatically reduced hepatocellular death. In conclusion, BCAA and l-carnitine treatments attenuate hepatocellular damage through the reduction of lipid peroxides and the overall maintenance of mitochondrial integrity within the cirrhotic liver. These effectiveness of BCAA and l-carnitine support the therapeutic strategies in human chronic liver diseases., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Protein S is a vitamin K-dependent glycoprotein produced mainly in the liver with anticoagulant, anti-inflammatory, immune-modulatory, and antiapoptotic properties. Protein S exacerbates acute liver injury by prolonging the survival of liver immune cells. However, the effect of protein S on chronic liver injury and fibrosis is unknown. Here, we investigated whether human protein S can affect chronic liver injury and fibrosis. Liver injury/fibrosis was induced by carbon tetrachloride injection in mice overexpressing human protein S and in wild-type mice. Human protein S transgenic mice receiving carbon tetrachloride showed significantly higher circulating levels of liver transaminases, increased liver expression of inflammatory cytokines, significantly more extended liver fibrosis, and areas with DNA breakage after chronic injury compared with wild-type mice. Wild-type mice infused with exogenous human protein S exhibited exacerbated liver injury and increased number of hepatic stellate cells compared with untreated mice. Human protein S inhibited apoptosis and increased Akt pathway activation in hepatic stellate cells. The antiapoptotic activity of protein S may play a role in chronic liver injury and subsequent liver fibrosis., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to assess the significance and degree of correlation between the intake of fish oil, magnesium (Mg), and zinc (Zn) and metabolic parameters., Methods: Correlation coefficients among nutrient intake and physical and laboratory parameters were determined using Spearman's rho (ρ) test or a multiple regression model among Japanese individuals (male:female, 37:66; median age, 55 y) who completed a semiquantitative food questionnaire and underwent testing for diabetes. Individuals with diabetes were excluded., Results: Spearman's test revealed several weak but significant correlations between intake of fish oil including ω-3 polyunsaturated fatty acids (PUFAs) and various metabolic parameters. The test showed that Zn intake in women significantly correlated with reduced systolic blood pressure (SBP), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GPT), and homeostasis model assessment-insulin resistance (HOMA-IR). Multivariate analysis revealed that intake of fish oil, eicosapentaenoic acid (EPA), and Zn was significantly associated with increased serum levels of high-density lipoprotein cholesterol (HDL-C; fish oil versus HDL-C, P = 0.0438; 95% confidence interval [CI], 0.0055-0.3724; EPA versus HDL-C, P = 0.0439; 95% CI, 0.0053-0.3724; Zn versus HDL-C, P = 0.0041; 95% CI, 0.0890-0.4609). Multivariate analysis revealed that ω-3 PUFAs were associated with decreased serum ALT levels (P = 0.0240; 95% CI, -5.000 to -0.0367) and that Zn correlated with SBP (P = 0.0239; 95% CI, -0.5149 to -0.0377) in women., Conclusion: Intake of fish oil, Mg, and Zn was associated with some metabolic parameters. Abundant intake of fish oil including ω-3 PUFAs and Zn can exert antiarteriosclerotic effects through increasing serum levels of HDL-C. ω-3 PUFAs can reduce liver inflammation and Zn can reduce SBP in women., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Alcohol consumption is a major cause of liver injury but the mechanisms are not completely understood. Protein S (PS) is an anticoagulant glycoprotein with multiple functions. The role of PS in liver injury is unknown., Objectives: This study investigated the role of PS in acute alcoholic hepatitis., Methods: A mouse overexpressing human PS (hPS-TG) was generated in which acute hepatitis was induced by intraperitoneal injection of ethanol., Results: The levels of serum liver enzymes and liver tissue inflammatory cytokines and the degree of hepatic steatosis were significantly increased in hPS-TG mice treated with ethanol compared with ethanol-treated wild type (WT) mice. Cell expansion, activation and inhibition of apoptosis were significantly augmented in natural killer T (NKT) cells from hPS-TG mice compared with WT mice. Liver mononuclear cells from hPS-TG mice express higher levels of inflammatory cytokines than those from WT mice after stimulation with a specific stimulant of NKT cells in vitro. In a co-culture system of hepatocytes and NKT cells, the effects of PS on ethanol-mediated cell injury were suppressed by a CD1d neutralizing antibody. Alcoholic liver injury was significantly improved in mice pre-treated with PS siRNA and anti-protein S antibody compared with control mice. Patients with alcoholic hepatitis showed significantly increased plasma PS levels and enhanced liver expression of PS and CD1d compared with controls., Conclusions: The results of this study suggest that PS exacerbates acute alcoholic hepatitis by inhibiting apoptosis of activated NKT cells., (© 2014 International Society on Thrombosis and Haemostasis.)

Transarterial chemoembolization (TACE) and radiofrequency ablation (RFA) are effective treatments for hepatocellular carcinoma (HCC). However, the extent of treatment depends on hepatic functional reserve. L-Carnitine is a vitamin-like substance and several reports have described the usefulness of L-carnitine supplementation in cases of cirrhosis, with confirmed effectiveness against refractory hepatic encephalopathy. On the other hand, we have previously reported that in patients who underwent TACE or RFA, administration of branched-chain amino acids (BCAAs) pre-intervention significantly reduced inflammatory reactions. We first determined serum levels of total, free, and acyl-carnitine before and at 7 d after performing TACE in 10 HCC patients. We administered levocarnitine (L-carnitine chloride, a biologically active form of carnitine) at 900 mg/d to 69 consecutive HCC patients hospitalized to undergo TACE and/or RFA, and compared changes in blood test values with those in 119 consecutive patients not administered this drug. Sixty-seven patients had a history of using BCAAs at the time of admission. We found that after 7 d of TACE, serum levels of total and acyl-carnitine are significantly decreased. On comparing the four groups, the carnitine+BCAA, carnitine-alone, and BCAA-alone groups showed significantly higher values for changes in NH3 when compared with the non-dosed group. The decrease in albumin (Alb) was significantly suppressed in the carnitine+BCAA and BCAA-alone groups. We also conducted the same examinations in a subset of patients classified as Child-Pugh class A, and noted the same trends. Administration of levocarnitine and/or BCAAs during invasive treatments reduced blood NH3 concentrations and suppressed decreases in Alb.

Long-term supplementation with branched-chain amino acids (BCAA) is associated with prolonged survival and decreased frequency of development of hepatocellular carcinoma (HCC) in patients with liver cirrhosis. However, the pharmaceutical mechanism underlying this association is still unclear. We investigated whether continuous BCAA supplementation increases survival rate of rats exposed to a fibrogenic agent and influences the iron accumulation, oxidative stress, fibrosis, and gluconeogenesis in the liver. Further, the effects of BCAA on gluconeogenesis in cultured cells were also investigated. A significant improvement in cumulative survival was observed in BCAA-supplemented rats with advanced cirrhosis compared to untreated rats with cirrhosis (P<0.05). The prolonged survival due to BCAA supplementation was associated with reduction of iron contents, reactive oxygen species production and attenuated fibrosis in the liver. In addition, BCAA ameliorated glucose metabolism by forkhead box protein O1 pathway in the liver. BCAA prolongs survival in cirrhotic rats and this was likely the consequences of reduced iron accumulation, oxidative stress and fibrosis and improved glucose metabolism in the liver.

Nonalcoholic fatty liver disease (NAFLD) is a major causative agent of chronic liver disease worldwide, but the actual mechanisms responsible for liver injury remain unclear. NAFLD includes a spectrum of clinical entities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH) with possible evolution to cirrhosis and hepatocellular carcinoma. Iron is considered a putative element that interacts with oxygen radicals in inducing liver damage/fibrosis and insulin resistance. The role of hepatic iron in the progression of NASH remains controversial, but in some patients, iron may have a role in the pathogenesis of NASH. Though genetic factors, insulin resistance, dysregulation of iron-regulatory molecules, erythrophagocytosis by Kupffer cells may be responsible for hepatic iron accumulation in NASH, exact mechanisms involved in iron overload remain to be clarified. Iron reduction therapy such as phlebotomy or iron-restricted diet may be promising in patients with NAFLD/NASH to reduce hepatic injury as well as insulin resistance. Larger controlled trials of longer duration are warranted to assess the long-term clinical benefit of phlebotomy and/or iron-restricted diet in NAFLD/NASH.

Aims: In this study, we investigated the effect of synthetic triglyceride containing an arachidonic acid branch (8A8) on lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) in macrophages, and LPS-induced liver injury in the rat., Main Methods: RAW264.7 macrophages were co-incubated with 8A8 and LPS (100ng/ml), and TNF-alpha mRNA/protein levels, nuclear factor (NF)-kappaB DNA binding activity, expression of inducible-type NO synthase (NOS2), and NO(2) production were measured. Male Wistar rats were given a single intraperitoneal injection of 8A8 prior to an intravenous injection of LPS (5mg/kg), and liver histology, apoptotic cell death, serum TNF-alpha levels, and hepatic TNF-alpha mRNA were then evaluated., Key Findings: LPS-induced increases in TNF-alpha production in RAW264.7 macrophages were blunted by 8A8 in a dose-dependent manner, with 40% inhibition at 100ppm. Further, 8A8 dose-dependently prevented LPS-induced increases in TNF-alpha mRNA levels, as well as NF-kappaB DNA binding activities, in RAW264.7 macrophages. LPS-induction of NOS2 and NO(2) release from these cells was also decreased by 8A8 in a dose-dependent manner. In vivo, LPS-induced liver injury, including hepatocyte apoptosis, was largely prevented when 8A8 (100microl/kg) was given 30min prior to LPS. Indeed, 8A8 blunted increases in both serum TNF-alpha and hepatic TNF-alpha mRNA levels significantly., Significance: LPS-induced liver injury was prevented by 8A8 most likely through the inhibition of TNF-alpha and NO production from hepatic macrophages, suggesting a potential usefulness of 8A8 as an immuno-modulating nutrient for prevention/treatment of endotoxin-related organ injuries including alcoholic liver disease and non-alcoholic steatohepatitis (NASH).

Although the oxidative stress frequently occurs in patients with chronic hepatitis C, its role in future hepatocellular carcinoma (HCC) development is unknown. Hepatic 8-hydroxydeoxyguanosine (8-OHdG) was quantified using liver biopsy samples from 118 naïve patients who underwent liver biopsy from 1995 to 2001. The predictability of 8-OHdG for future HCC development and its relations to epidemiologic, biochemical and histological baseline characteristics were evaluated. During the follow-up period (mean was 6.7+/-3.3 years), HCC was identified in 36 patients (30.5%). Univariate analysis revealed that 16 variables, including 8-OHdG counts (65.2+/-20.2 vs 40.0+/-23.5 cells per 10(5) microm2, P<0.0001), were significantly different between patients with and without HCC. Cox proportional hazard analysis showed that the hepatic 8-OHdG (P=0.0058) and fibrosis (P=0.0181) were independent predicting factors of HCC. Remarkably, 8-OHdG levels were positively correlated with body and hepatic iron storage markers (vs ferritin, P<0.0001 vs hepatic iron score, P<0.0001). This study showed that oxidative DNA damage is associated with increased risk for HCC and hepatic 8-OHdG levels are useful as markers to identify the extreme high-risk subgroup. The strong correlation between hepatic DNA damage and iron overload suggests that the iron content may be a strong mediator of oxidative stress and iron reduction may reduce HCC incidence in patients with chronic hepatitis C.

Altered gene expression in liver sinusoidal endothelial cells (SEC) is associated with a variety of aspects of liver pathophysiology. It is, therefore, possible to envision a new therapeutic strategy for treatment of intractable liver diseases and achievement of graft-specific immunotolerance through modulation of SEC functions by genetic engineering. The SEC possesses unique hyaluronan receptors that recognize and internalize hyaluronic acid (HA). This characteristic was used in the development of a system for targeting foreign DNA to SEC. A gene carrier system was prepared by coupling HA oligomers to poly L-lysine (PLL) in a 1:1 weight ratio by reductive amination reaction. The resulting copolymer (PLL-g-HA) was mixed with various amounts of DNA in 154 mM NaCl. Inter-polyelectrolyte complex formation between PLL-g-HA and DNA exhibited minimal self-aggregation, explaining the highly soluble nature of the complex. Complex formation between PLL-g-HA and DNA was further assessed with a gel retardation assay. The titration point representing the minimum proportion of PLL-g-HA required to retard the DNA completely occurred at a 1:1 copolymer (based on PLL) to DNA charge ratio. Following intravenous injection of (32)P-labeled pSV beta-Gal plasmid complexed to PLL-g-HA in Wistar rats, >90% of the injected counts were shown to be taken up by the liver. Further, it was shown that the PLL-g-HA/DNA complex was distributed exclusively in the SEC. At 72 h after injection of 90 mug of pSV beta-Gal in a PLL-g-HA-complexed form, a large number of SEC expressing beta-galactosidase were detected. So, the PLL-g-HA/DNA system permits targeted delivery of exogenous nucleotide agents selectively to the liver SEC, providing a novel strategy for manipulation of SEC functions.

In alcoholic liver disease, ethanol-induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol-induced injury. To elucidate the role of sphingosine 1-phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 mumol/L S1P attenuated ethanol-induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P-induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, N(G)-nitro-L-arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca(2+)-sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury.

The activation of Kupffer cells represents a central mechanism of liver injury involving the production of TNF-alpha. It is known that glycine prevents LPS-induced production of TNF-alpha in isolated Kupffer cells. In this study, the possibility that glycine analogues might affect Kupffer cells was investigated. As a result, aminomethanesulfonic acid (AMS) inhibited the production of TNF-alpha in LPS-stimulated Kupffer cells. Furthermore, LPS treatment caused a transient increase in intracellular calcium ([Ca(2+)](i)) which was blunted by AMS. Thus, the addition of AMS is protective against the LPS-induced increase [Ca(2+)](i) and subsequent production of TNF-alpha. Moreover, in vivo studies demonstrated that pretreatment of mice with AMS increased the rate of survival after injection with LPS/d-gal and reduced the TNF-alpha serum level and the mRNA level in the liver. These results indicate that intake of AMS attenuates the LPS-induced hepatotoxicity resulting from activation of Kupffer cells.

Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). To develop a system for targeting foreign DNA to SECs, comb-type polycations having HA side chains were prepared by coupling HA to poly(L-lysine) (PLL). The HA-grafted-PLL copolymer (PLL-g-HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains. To determine whether the PLL-g-HA/DNA complexes were recognized by SEC HA receptors in vivo, we injected Wistar rats i.v. via the tail vein with PLL-g-HA complexed to a beta-galactosidase expression plasmid (pSV beta-Gal) labeled with 32P. One hour postinjection, >90% of the injected radioactivity remained in the liver. Administration of the PLL-g-HA complexed to an FITC-labeled DNA revealed that the carrier-DNA complex was distributed exclusively in SECs. A large number of SECs expressing beta-galactosidase was detected along the sinusoidal lining after transfection with PLL-g-HA/pSV beta-Gal. Moreover, PLL-g-HA effectively stabilized DNA triplex formation. In conclusion, the new PLL-g-HA/DNA carrier system permits targeted transfer of exogenous genes selectively to the SECs.

Altered gene expression of liver sinusoidal endothelial cells (SECs) is associated with impaired immune response. Here we report that the decoy technique effectively suppresses TNFalpha-induced ICAM-1 expression in SEC. An NF-kappaB decoy (NF-kappaB31: 5(')-TGGGGACTTTCCAGTTTCTGGAAAGTCCCCA-3), which contains a consensus sequence for NF-kappaB, was complexed to PLL-g-HA [hyaluronate-grafted poly(L-lysine) copolymer] that permits transfer of exogenous DNA selectively to the SEC. The PLL-g-HA/NF-kappaB31 complex was added to the culture media of LSE cells, a human SEC-derived cell line. Then, cells were stimulated with TNFalpha (5ng/mL). PLL-g-HA/NF-kappaB31, but not control oligodeoxynucleotides having a reverse or scrambled sequence, inhibited the intranuclear localization of NF-kappaB induced by TNFalpha, with almost complete inhibition at 2.5microg/mL as DNA. NF-kappaB31 attenuated the increase in ICAM-1 mRNA as well as protein levels in LSE cells. The decoy technique in combination with PLL-g-HA may provide a novel strategy for manipulation of SEC functions.

Background & Aims: Sensitization of Kupffer cells (KCs) to lipopolysaccharide (LPS) and overproduction of tumor necrosis factor (TNF) alpha are critical for progression of alcoholic liver injury. Thalidomide has been shown to suppress TNF-alpha production from macrophages. Accordingly, the purpose of this study was to determine whether thalidomide could prevent alcohol-induced liver injury., Methods: Rats were given ethanol (5 g/kg body wt) and thalidomide (5 mg/kg) once every 24 hours intragastrically. To assess the sensitization of Kupffer cells, LPS (5 mg/kg intravenously) was administered and liver histology was evaluated 24 hours later. KCs were isolated after 4 weeks of ethanol treatment and intracellular Ca2+ ([Ca2+]i) was measured using fura-2, whereas TNF-alpha was evaluated by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. CD14 was determined by Western and fluorescence staining., Results: Treatment with ethanol for 8 weeks caused marked steatosis, necrosis, and inflammation in the liver. These pathologic parameters were diminished markedly by treatment with thalidomide. In the 4-week ethanol group, the LPS-induced liver damage was aggravated and KCs were sensitized to LPS. Coadministration of thalidomide with ethanol prevented the KC sensitization completely. Furthermore, thalidomide abolished the LPS-induced increase in CD14 expression and [Ca2+]i elevation in KCs. Gut permeability was increased about 10-fold after 4 weeks of ethanol exposure, which was not affected by thalidomide. Moreover, thalidomide reduced the LPS-induced TNF-alpha production by KCs by decreasing TNF-alpha messenger RNA., Conclusions: These results collectively indicate that thalidomide prevents alcoholic liver injury through suppression of TNF-alpha production and abolishment of KC sensitization.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hidekazu Tsukamoto and Yoshiyuki Takei. The presentations were (1) Tribute to Professor Rajendar K. Chawla, by Craig J. McClain; (2) Dysregulated TNF signaling in alcoholic liver disease, by Craig J. McClain, S. Joshi-Barve, D. Hill, J Schmidt, I. Deaciuc, and S. Barve; (3) The role of mitochondria in ethanol-mediated sensitization of the liver, by Anna Colell, Carmen Garcia-Ruiz, Neil Kaplowitz, and Jose C. Fernandez-Checa; (4) A peroxisome proliferator (bezafibrate) can prevent superoxide anion release into hepatic sinusoid after acute ethanol administration, by Hirokazu Yokoyama, Yukishige Okamura, Yuji Nakamura, and Hiromasa Ishii; (5) S-adenosylmethionine affects tumor necrosis factor-alpha gene expression in macrophages, by Rajendar K. Chawla, S. Barve, S. Joshi-Barve, W. Watson, W. Nelson, and C. McClain; (6) Iron, retinoic acid and hepatic macrophage TNFalpha gene expression in ALD, by Hidekazu Tsukamoto, Min Lin, Mitsuru Ohata, and Kenta Motomura; and (7) Role of Kupffer cells and gut-derived endotoxin in alcoholic liver injury, by N. Enomoto, K. Ikejima, T. Kitamura, H. Oide, Y. Takei, M. Hirose, B. U. Bradford, C. A. Rivera, H. Kono, S. Peter, S. Yamashina, A. Konno, M. Ishikawa, H. Shimizu, N. Sato, and R. Thurman.

Apoptosis of sinusoidal endothelial cells (SECs) is one of the initial events in the development of ischemia-reperfusion injury of the liver. Glycine has been shown to diminish ischemia-reperfusion injury in the liver and improve graft survival in the rat liver transplantation model. Here, we investigated the effect of glycine on apoptosis of primary cultured rat SECs induced by vascular endothelial growth factor (VEGF) deprivation. Isolated rat SECs were cultured in EBM-2 medium supplemented with 10% fetal bovine serum (FBS) and growth factors including 20 ng/mL VEGF for 3 days. SECs at 3 days of culture showed spindle-like shapes; however, cells started shrinking and detaching from dishes by VEGF deprivation. Apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated d-uridine triphosphate (dUTP)-biotin nick end labeling (TUNEL) staining in these conditions. Control SECs contained only a few percent of TUNEL-positive cells; however, they started increasing 4 hours after VEGF deprivation, and the percentage of TUNEL-positive cells reached about 50% at 8 hours and almost 100% at 16 hours after VEGF deprivation. Interestingly, this increase in TUNEL-positive cells after VEGF deprivation was prevented significantly when glycine (1-10 mmol/L) was added to the medium, the levels being around 60% of VEGF deprivation without glycine. Furthermore, strychnine (1 micromol/L), a glycine receptor antagonist, inhibited this effect of glycine, suggesting the possible involvement of the glycine receptor/chloride channel in the mechanism. Moreover, Bcl-2 protein levels in SECs were decreased 8 hours after VEGF deprivation, which was prevented almost completely by glycine. It is concluded that glycine prevents apoptosis of primary cultured SECs under VEGF deprivation.

Background: Hepatic stellate cells have been reported to play important roles in the regulation of hepatic microcirculation via cell contraction. Increase in intracellular calcium concentration is required to induce cell contraction. We have already reported the existence of L-type voltage-operated Ca2+ channels (VOCC), such as smooth muscle cells. On the other hand, alcohol has been known to disturb hepatic microcirculation. In this study, we evaluated the effect of acute and chronic treatment of alcohol on VOCC in rat hepatic stellate cells., Methods: Stellate cells isolated from rats were cultured with or without 100 mM ethanol for up to 14 days. VOCC were detected by the patch clamp technique. Cells cultured for 14 days without ethanol were exposed to ethanol to investigate calcium current during membrane depolarization. alpha-Smooth muscle actin (alpha-SMA) was stained by indirect immunofluorescence., Results: In the control model, VOCC were recognized in cells cultured for more than 7 days. Detection of VOCC increased from 9% on day 7 to 55% on day 14. On the other hand, VOCC in cells treated chronically with 100 mM ethanol appeared earlier than in the control and the incidences were significantly higher than those of the control accompanied with an early activation of cells. In contrast, simultaneous exposure to ethanol during the membrane depolarization inhibited Ca2+ current., Conclusions: The expression of Ca2+ channels in stellate cells were up-regulated by the chronic treatment of alcohol accompanied with the transformation to myofibroblast-like phenotype. However, alcohol itself inhibited Ca2+ current.

Gap junctional intercellular communication (GJIC) between hepatocytes is important for the maintenance of differentiated liver functions. Taurine is known to be cytoprotective, and is used clinically to improve liver functions. We evaluated the effect of taurine on GJIC in hepatocyte doublets under oxidative stress. Hepatocyte doublets were isolated from female Wistar rats, using a collagenase perfusion technique, and cultured in Leibovitz-15 medium containing fetal bovine serum (10%). H2O2 (2 mM) and/or taurine (0.1-1 mM) were added 2 h after inoculation, and the culture was incubated for 3 h. Fluorescent dye (Lucifer Yellow CH) coupling between adjacent cells was evaluated by microinjection. The distribution and quantity of connexin 32 (Cx32) in hepatocytes were detected using indirect immunofluorescence analysis and Western blotting. Steady state mRNA levels of Cx32 were detected by Northern blotting. The percentage of dye coupling 5 h after inoculation was 88 +/- 6.3% in the control. however, this was decreased to almost half the control value by H2O2. Taurine prevented the decrease caused by H2O2 in a dose-dependent manner. Immunofluorescence analysis for Cx32 demonstrated numerous punctate fluorescent spots along the intercellular plasma membrane in controls, which were significantly decreased by H2O2. Taurine prevented the decrease of Cx32. Western blot analysis also showed the decrease of Cx32 protein levels by H2O2 treatment, which decrease was prevented by taurine. Interestingly, H2O2 and/or taurine treatments did not affect Cx32 mRNA levels. Our findings indicated that H2O2 treatment decreased GJIC between hepatocytes, most likely due to augmenting the degradation of Cx32 proteins, whereas taurine prevented this process. This effect of taurine is beneficial for the preservation of differentiated functions in the liver under oxidative stress.

To evaluate the role of nitric oxide (NO) in hepatic microcirculation and liver injury during endotoxemia, we studied O2 transport in the hepatic microcirculation of endotoxin-infused rats. Rats were continuously infused with Escherichia coli lipopolysaccharide (LPS) (0.8 mg/kg/h) for 7 hours. LPS increased the plasma levels of NO2- + NO3- and aspartate transaminase (AST), and decreased the bile flow rate and hepatic adenosine triphosphate (ATP) level. Hepatic microcirculation was evaluated by two methods: reflectance spectrophotometry showed a decrease in the oxygenation of hemoglobin (Hb) in the liver, and dual-spot microspectroscopy indicated that LPS administration decreased blood velocity, the oxygenation of Hb, and O2 release from sinusoids to hepatocytes. The observed decreases in the O2 transport parameters were prominent in pericentral sinusoids. All of these phenomena were further aggravated by the administration of N(w)-nitro-L-arginine methyl ester (L-NAME) (5 mg/kg/h) plus LPS, and by aminoguanidine (AMG) (5 mg/kg/h) plus LPS, and these could be reversed by the concomitant administration of L-arginine (L-Arg) (100 mg/kg/h). These results suggest that deterioration of hepatic oxygen transport and liver function induced by endotoxin can be ameliorated by NO.

The critical injury causing graft failure after prolonged liver storage involves reperfusion-induced killing of sinusoidal endothelial cells and activation of Kupffer cells. Treatment of stored livers with Carolina rinse solution (CRS) prevents endothelial cell killing, reduces Kupffer cell activation, and improves graft survival. Accordingly, our aim was to evaluate the components of CRS and other agents for protection against reperfusion injury to rat livers stored 24 hr in University of Wisconsin solution. CRS virtually abolished endothelial cell killing, prevented denudation of the sinusoidal lining, and decreased structural changes in Kupffer cells indicative of activation. The only component of CRS preventing endothelial cell killing was acidic pH of 6.5. However, when pH was subsequently increased to 7.4, antioxidants (allopurinol, deferoxamine mesylate, and glutathione), vasodilators (adenosine and nicardipine), and possibly energy substrates (fructose, glucose, and insulin) partially blocked pH-dependent cell killing (pH paradox). Na+/H+ exchange inhibition, protease inhibition, and Ca(2+)-free buffer did not decrease reperfusion injury, but the amino acid glycine protected strongly. Strychnine, which binds to glycine receptors in the central nervous system, protected equally well. Protection by glycine and CRS was synergistic, virtually.

Long-term storage of liver grafts results in increased adhesion of leukocytes onto the sinusoidal walls. This eventually leads to posttransplant graft damage through disturbances of hepatic microcirculation. Intracellular adhesion molecule-1 (ICAM-1) is known to be involved in attachment of leukocytes. This study was designed to examine whether ICAM-1 participated in the pathogenesis of posttransplant liver injury. Inbred Lewis rats were used as both donors and recipients to avoid immunoreactivity. Donor livers were stored for either 1 or 6 hr in ice-cold Euro-Collins solution and subsequently implanted. Expression of ICAM-1 was examined immunohistochemically. In some rats that received livers stored for 6 hr, the intact IgG (1.0 mg/kg) or the F(ab')2 fragment (0.5 mg/kg) of an anti-ICAM-1 mAb (1A29) was administered via the tail vein immediately after reperfusion of portal blood. In the group receiving livers stored for 6 hr, ICAM-1 began to be expressed on the sinusoidal endothelial cells as early as 15 min after reperfusion of the portal blood. Strong ICAM-1 expression was observed from 2 hr up to 24 hr after reperfusion. In contrast, expression of ICAM-1 was not evident at any time point after surgery in the 1-hr storage group as well as in untransplanted, normal livers. Serum alanine aminotransferase (ALT) levels were significantly higher in the 6-hr storage group compared with those of the 1-hr storage group (1-hr: 171 +/- 9 IU/L; 6-hr: 825 +/- 109 IU/L, P < 0.05; mean +/- SEM) 24 hr after transplantation. Serum ALT levels were markedly reduced by treatment with the F(ab')2 fragment of 1A29 (247 +/- 34 IU/L, P < 0.05 vs. 6-hr storage group). This was associated with reduced accumulation of leukocytes in the liver. In marked contrast, treatment with the intact IgG of 1A29 increased serum ALT levels dramatically (5297 +/- 634 IU/L, P < 0.05 vs. 6-hr storage group) and reduced serum complement. Histological examination revealed focal hepatocellular necrosis 24 hr after surgery in the 6-hr storage group. Treatment with the F(ab')2 fragment decreased the liver damage; in marked contrast, treatment with the intact IgG strikingly aggravated the injury, as characterized by massive necrosis throughout the liver. Liver damage caused by the intact IgG might be related to activation of the complement system by the Fc portion of the antibody. Taken together, these results indicate that ICAM-1 is involved in the mechanism of postoperative liver injury following liver transplantation.

Kupffer cells (KC) become activated in response to lipopolysaccharide (LPS) and produce a variety of mediators. Among them, TNF alpha is known to injure the liver. Here we report that TNF alpha mediates apoptosis in KC and sinusoidal endothelial cells. After stimulation for 24 h with LPS (0-10 micrograms/mL), apoptosis in KC detected by TUNEL TdT-mediated dUTP-biotin nick end labelling (TUNEL) increased in a concentration-dependent manner (0 micrograms/mL, 12 +/- 4%; 0.1 microgram/mL, 36 +/- 11%; 1.0 micrograms/mL, 65 +/- 9%; 10 micrograms/mL, 78 +/- 15%). In contrast, co-incubation of endothelial cells with LPS-stimulated KC resulted in a marked increase in TUNEL-positive endothelial cells. TNF alpha antibody blocked apoptosis in both KC and endothelial cells. Apoptosis was observed in cells adjacent to or in contact with KC. Reducing transmembrane TNF alpha expressed on KC also led to a decrease in endothelial cell apoptosis, suggesting that transmembrane TNF alpha is implicated in the cell-to-cell contact mechanism of induction of apoptosis. Thus, TNF alpha mediates apoptosis in KC and endothelial cells.

The purpose of this study was to clarify the role of endogenous nitric oxide in ethanol-induced perturbation of microcirculation and hepatic injury in perfused rat liver. Infusion of ethanol into the portal vein at 25 and 100 mmol/L increased portal pressure, which is an indicator of hepatic vasoconstriction, in a concentration-dependent fashion. Portal pressure started to rise immediately after ethanol load, then decreased gradually and remained at higher than basal levels throughout the period of ethanol infusion. Release of lactate dehydrogenase into the effluent perfusate began to increase after 30 min of ethanol infusion and continued to increase during the 60-min period of ethanol infusion. The lactate dehydrogenase level in the effluent perfusate at 60 min was dependent on the ethanol concentration (0 mmol/L, 8 +/- 3 IU/L; 25 mmol/L, 16 +/- 2 IU/L; 100 mmol/L, 52 +/- 6 IU/L). Simultaneous infusion of NG-monomethyl-L-arginine, a nitric oxide synthesis inhibitor, enhanced significantly the ethanol-induced increase in portal pressure by 100% to 400% and increased lactate dehydrogenase release by 40% to 80%. The effect of NG-monomethyl-L-arginine on the ethanol-induced increase in portal pressure was completely reversed by the co-infusion of an excess dose of L-arginine. Change in portal pressure averaged over 60 min of ethanol infusion correlated with levels of lactate dehydrogenase release 60 min after the initiation of ethanol infusion (r = 0.77, p < 0.01). In conclusion, inhibition of the action of endogenous nitric oxide was associated with an increase in hepatic vasoconstriction and hepatocellular damage.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of an organoselenium compound, ebselen [2-phenyl-1,2-benzisoselenazol-3-(2H)-one], on ethanol-induced liver damage through perturbation of microcirculation was investigated in perfused livers from fed rats. Infusion of ethanol at concentrations > or = 25 mM into the portal vein increased portal pressure in a concentration-dependent manner. Release of lactate dehydrogenase (LDH) into the effluent perfusate was minimal at 30 min; thereafter LDH release began to increase gradually until the end of the experiment (60 min after the onset of ethanol infusion) and was dependent on ethanol concentration. Simultaneous infusion of ebselen at a concentration of 10 or 30 microM with ethanol reduced significantly this ethanol-induced increase in portal pressure by 50 to 75% (P < .05) and LDH release by 70% (P < .05). When endothelin-1 or phenylephrine was infused into the liver, portal pressure was increased, reaching maximal levels (50 +/- 17 and 46 +/- 7 mm of H2O, respectively) and then decreasing gradually. Ebselen reduced the maximal increase in portal pressure induced by endothelin-1 (18 +/- 2 mm of H2O) by 64% (P < .05). In addition, ebselen decreased the maximal levels of portal pressure induced by phenylephrine (8 +/- 1 mm of H2O) by 83% (P < .05). These data indicate that ebselen has a vasodilative effect on constriction of hepatic vasculature and diminishes ethanol-induced hepatic damage by offsetting ethanol-induced increase in portal pressure. Thus, ebselen may prove useful for treatment of alcoholic liver injury via improvement of microcirculatory disturbances.

Nuclear protein transport was inhibited in permeabilized HeLa cells which had been prepared after culture with pertussis toxin, suggesting that the pertussis toxin-sensitive protein(s) might be involved in the nuclear protein transport. To investigate the mechanism whereby pertussis toxin inhibited the nuclear protein transport, an accumulation of proteins containing a nuclear localization signal sequence (NLS) into isolated rat liver nuclei was investigated. The NLS-containing protein accumulation required ATP and cytosolic proteins, and was temperature- and wheat germ agglutinin-sensitive as had been observed in permeabilized cells. Non-hydrolyzable GTP analogues, such as guanosine 5'-(gamma-thio)triphosphate and guanosine 5'-(beta, gamma-methylene)triphosphate, but not ATP analogues, inhibited the NLS-containing protein accumulation in the isolated nuclei. The NLS-containing protein accumulation was also inhibited by prior treatment of the nuclei with pertussis toxin plus NAD, and the effect of pertussis toxin was blocked when guanosine 5'-(gamma-thio)triphosphate was simultaneously added during the pretreatment with pertussis toxin. The inhibition induced by pertussis toxin and the blockage by guanosine 5'-(gamma-thio)triphosphate were well correlated to ADP-ribosylation of 40-kDa protein in nuclear fraction. These results suggested that the nuclear pertussis toxin-sensitive GTP-binding protein is involved in a pathway of nuclear protein transport.

Endothelin-1 (ET-1), a vasoactive peptide, causes a significant rise in portal vein pressure, which is most likely a result of severe vasoconstriction in the liver. In this study, the effect of ET-1 on sinusoidal vasoconstriction in the liver after ischemia and reperfusion was directly investigated using intravital microscopy. In anesthetized female Sprague Dawley rats (200-250 g) ischemia of the median and left liver lobes was induced for 90 min by temporary ligation of the left pedicle. After declamping and a 90-min reperfusion period, the livers were exposed for intravital microscopy. Using a Nikon MM-11 fluorescence microscope (545 nm, 330x), a CCD camera (Cohu FK 6990), and a SVHS video recording unit, the hepatic microcirculation was directly investigated. Besides sham groups, two ischemia groups were studied, receiving ET-1 antiserum (anti-ET-1; 0.5 ml; Peptide Inst., Osaka, Japan) or NaCl 0.9% (0.5 ml) 5 min prior to reperfusion of the liver (n = 6/group). Following a transient drop in the mean arterial blood pressure in the anti-ET-1-treated groups, comparable systemic hemodynamic conditions among the four groups were noted during intravital microscopic assessment at the end of the 90-min reperfusion period. Reduction in the sinusoidal diameters during postischemic reperfusion (7.7 +/- 0.5 microm) was prevented by anti-ET-1 treatment (9.6 +/- 0.25 microm; P < 0.01; mean + SEM) back to control values (9.6 +/- 0.32 microm), while most of other microcirculatory parameters did not show significant differences. The results supported further the role of ET-1 in dysregulation of the sinusoidal vascular tone in the liver, e.g., after ischemia and reperfusion.

This study investigated the role of endogenous nitric oxide in the regulation of hepatic vascular tone in the presence of ethanol. In the perfused rat liver, upon the initiation of ethanol infusion into the liver, portal pressure was increased in a dose-dependent manner, reaching maximal levels in 2-5 min, then decreasing gradually. Simultaneous infusion of N(G)-monomethy 1-L-arginine, a nitric oxide synthesis inhibitor, enhanced this ethanol-induced increase in portal pressure. This enhancement was reversed by simultaneous infusion of a precursor of nitric oxide, L-arginine. These results suggest that endogenous nitric oxide acts as a vasodilator which reduces ethanol-induced vasoconstriction, thus improving the perturbation of hepatic microcirculation by ethanol.

The technique of generating 3-D images from CT and MR data has made progress. Manipulation of the images has become simpler, the processing time shorter, and the quality better. Clinical application of this technique has been performed for images of brain (5, 7, 8) and bone (1, 2, 6), but only a few studies have been done of abdominal organs (3, 4).

Portal circulation changes due to the progression of chronic liver disease and portal venous flow are also affected by pharmacotherapy. Thus, noninvasive measurement of effective portal venous flow (EPVF) is highly desirable. We evaluated EPVF under steady-state conditions using echo-Doppler flowmetry combined with per jejunal portal scintigraphy in 32 patients with chronic liver disease. After introduodenal administration of 37 MBq (1 mCi) of 123I-iodoamphetamine, scintigraphy of the pulmonary and hepatic regions was performed and a portosystemic shunt index (SI) calculated. EPVF was calculated as follows: EPVF = PVFx (1-SI/100). EPVF in chronic hepatitis, compensated cirrhosis and decompensated cirrhosis was 12.0 +/- 1.8 ml/min/kg, 10.3 +/- 1.6 ml/min/kg and 8.0 +/- 2.5 ml/min/kg, respectively. There were significant differences in EPVF between all groups, although PVF was similar in each group. EPVF correlated with liver function tests and was a better indicator of liver function than PVF. Measurement of EPVF may provide useful information in the management of patients with chronic liver disease.

This study was designed to investigate the mechanism for ethanol-induced hepatic vasoconstriction in isolated perfused rat liver. Upon initiation of ethanol infusion into the portal vein at concentrations ranging from 25 to 100 mM, portal pressure began to increase in a concentration-dependent manner and reached maximal levels in 2-5 min (initial phase), followed by a gradual decrease over the period of ethanol infusion (escape phenomenon). Endothelin-1 antiserum significantly inhibited this ethanol-induced hepatic vasoconstriction by 45-80%. Cessation of infusion of endothelin-1 antiserum was followed by a subsequent increase in portal pressure. On the other hand, when a nitric oxide synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA), was infused into the portal vein simultaneously with ethanol, the initial phase of the response of portal pressure to ethanol was not altered and the peak values of portal pressure remained unchanged. However, after the peak increase in portal pressure, the rate of decrease was less than in the absence of L-NMMA. Thus, L-NMMA diminished the escape phenomenon and sustained the vasoconstriction. This study supports the hypothesis that two endothelium-derived vasoactive factors, endothelin-1 and nitric oxide, regulate hepatic vascular tone in the presence of ethanol.

Adult rat hepatocytes attached on an asialoglycoprotein model polymer, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), formed anchored multilayer aggregates that had stable three-dimensional structure when epidermal growth factor (EGF) and insulin were added to the culture medium. The formation of multilayer aggregates depended on the concentrations of EGF and insulin added. Furthermore, the formation was synergistically accelerated by the presence of both hormones. Cells in the aggregates expressed a higher level of albumin secretion and lower proliferative ability than those in monolayer cultures on collagen. It seemed likely that the cells in multilayer aggregates experienced stable differentiated states resembling those in vivo through the formation of multilayer aggregates. The culture system described here has potential use for the study of the process of liver regeneration and the development of hepatic module systems such as a bioreactor and a hybrid artificial liver.

The role of microcirculation in the pathogenesis of alcoholic liver injury was investigated in isolated perfused livers from fed rats. Infusion of ethanol into the portal vein at concentrations ranging from 25 to 200 mmol/L increased portal pressure, which is an indicator of hepatic vasoconstriction, in a concentration-dependent fashion. Portal pressure started to rise immediately on initiation of ethanol load and remained at higher than basal levels throughout the period of ethanol infusion. Release of lactate dehydrogenase, an indicator of cell injury, into the effluent perfusate began to increase after 20 to 30 min of ethanol infusion and continued to increase until the end of the experiment (60 min after the initiation of ethanol infusion). The lactate dehydrogenase level in the effluent perfusate at 60 min was dependent on the ethanol concentration (0 mmol/L, 8 +/- 3 IU/L; 25 mmol/L, 22 +/- 3 IU/L; 50 mmol/L, 51 +/- 11 IU/L; 100 mmol/L, 60 +/- 7 IU/L; 200 mmol/L, 120 +/- 7 IU/L). Simultaneous infusion of sodium nitroprusside (100 mumol/L), a known vasodilator, inhibited significantly the ethanol-induced increases in portal pressure and lactate dehydrogenase release by abolishing hepatic vasoconstriction. In histological examinations focal hepatocellular necrosis, evidenced by trypan blue staining of cell nuclei, was detected predominantly in midzonal and pericentral areas of the liver lobule after 60 min of ethanol infusion. Change in portal pressure during 60 min of ethanol infusion correlated significantly with levels of lactate dehydrogenase after ethanol infusion (r = 0.82; p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

This study examines whether tumor necrosis factor and endotoxin are involved in the pathogenesis of primary nonfunction of graft and pulmonary complication after orthotopic liver transplantation. Livers from Lewis rats were stored for either 1 or 4 hr in ice-cold Euro-Collins solution (1-hr storage and 4-hr storage group, respectively). Subsequently, donor livers were implanted orthotopically. In some experiments, anti-tumor necrosis factor antibody was administered intravenously before and immediately after the surgery into animals that received livers stored for 4 hr. Blood samples for the measurement of tumor necrosis factor and endotoxin were collected by way of an indwelling catheter placed in the suprahepatic vena cava. Serum tumor necrosis factor was elevated at all time points studied postoperatively in rats of the 4-hr storage group; however, tumor necrosis factor was not detected in the serum in the 1-hr storage group. Endotoxin was also elevated significantly in the serum of the former group compared with levels in the serum of the latter group. The peak value of endotoxin occurred 1 hr earlier than that of tumor necrosis factor, suggesting that the rise in endotoxin stimulated release of tumor necrosis factor. The histological study of livers stored for 4 hr showed substantial hepatocellular degeneration 24 hr after surgery, whereas hepatocellular damage was minimal in the 1-hr storage group. Serum ALT levels 24 hr after the operation in the 1-hr and 4-hr storage groups were 169 +/- 46 IU/L and 374 +/- 41 IU/L (mean +/- S.E.M., p less than 0.05), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

We used a lactose-substituted polystyrene, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), as a substratum for adult rat hepatocytes in primary culture. Spherical-shaped hepatocytes attached on PVLA substratum formed stable multilayer aggregates anchored on substratum through the stimulation of epidermal growth factor (EGF). The cells required calcium ion essentially to form the aggregates. The formation of multilayer aggregates was inhibited by colchicine, but not by cytochalasin B. The inhibition was also observed by added PVLA molecules in the culture medium and by treating surfaces of PVLA-coated dishes with allo A lectin. It was suggested that adult rat hepatocytes attached on PVLA substratum required the specific interaction between asialoglycoprotein receptors on the cell surface and PVLA substratum to form anchored multilayer aggregates.

The effect of several hormones known to alter intracellular free Ca2+ on rates of O2 uptake in periportal and pericentral regions of the liver lobule was studied in the perfused liver. Regional O2 uptake was measured by stopping the flow and monitoring the decrease in O2 concentration. When perfusion was in the anterograde direction, basal rates of O2 uptake were two to three times higher in periportal than in pericentral regions, and phosphorylase alpha activity, which increases as a function of intracellular free Ca2+ levels, was higher in periportal regions. In contrast, when perfusion was in the retrograde direction, rates of O2 uptake were two to three times greater in pericentral regions. Infusion of epinephrine (0.1 microM) or angiotensin II (5 nM) increased the rate of O2 uptake nearly exclusively in downstream areas of the lobule where O2 tension was low. When perfusions were in the anterograde direction, epinephrine increased phosphorylase alpha activity significantly only in pericentral regions. Stimulation of O2 uptake by epinephrine was blocked by the alpha-adrenergic receptor blocker phentolamine (1 microM) but not by the beta-receptor blocker propranolol. Thus hormones that increase intracellular calcium stimulate O2 uptake predominantly in regions of the liver lobule where O2 tension is lowest, supporting the hypothesis that oxygen tension regulates O2 uptake in the liver via mechanisms involving intracellular free Ca2+.

The ADP-ribosyl moiety of NAD was transferred to a 40-kDa protein when rat liver nuclei were incubated with pertussis toxin. The 40-kDa substrate in the nuclei displayed unique properties as follows, some of which were apparently distinct from those observed with the toxin-substrate GTP-binding protein (Gi) in the liver plasma membranes. 1) The nuclear 40-kDa protein was recognized with antibodies reacting with the alpha-subunits (alpha i-1 and alpha i-2) of Gi, but not with anti-Go-alpha-subunit antibody. 2) The nuclear protein had a higher mobility than alpha-subunit of the plasma membrane-bound Gi upon electrophoresis with a urea/sodium dodecyl sulfate-containing polyacrylamide gel. 3) The nuclear protein was not extracted from the nuclei with 1% Triton X-100, whereas Gi was easily solubilized from the plasma membranes. 4) There was a beta gamma-subunit-like activity in the nuclei, which was assayed by an ability to support pertussis toxin-catalyzed ADP-ribosylation of a purified alpha-subunit of Gi. Moreover, a 36-kDa protein in the nuclei was recognized with antibody raised against purified beta-subunits of Gi. 5) Pertussis toxin-induced ADP-ribosylation of the nuclear protein was selectively inhibited by the addition of a nonhydrolyzable GTP analogue, and its inhibitory action was competitively blocked by the simultaneous addition of GDP or its analogues, as had been observed with plasma membrane-bound Gi. It thus appeared that a novel form of alpha beta gamma-trimeric GTP-binding protein serving as the substrate of pertussis toxin was present in rat liver nuclei. In order to examine a possible role of the nuclear GTP-binding protein, rats were injected with carbon tetrachloride, a necrosis inducer of hepatocytes. There was a marked increase in the nuclear substrate activity from 3-6 days after the injection, without a significant change in the activity of Gi in the plasma membranes. The time course of the increase corresponded with a recovering stage from the hepatocyte necrosis. These results suggested that the nuclear GTP-binding protein found in the present study might be involved at some stages in the hepatocyte growth.

The purpose of this study was to determine whether hepatic tissue oxygenation after ischemia-reperfusion procedures is an indicator for later liver injury. Partial ischemia in the liver was induced by ligating the left pedicles. Rats were divided into two groups according to duration of ischemia: group A (30-min ischemia) and group B (60-min ischemia). Indices of blood oxygenation and blood volume in regional hepatic tissue, serum ALT levels and histological appearance of livers were evaluated. Twenty-four hours after ischemia and reflow, all rats in group A were alive, whereas only 67% survived in group B. Blood-oxygenation index and blood-volume index in group A rats rebounded quickly after reperfusion. In group B, blood-oxygenation index and blood-volume index remained significantly lower than in group A after reperfusion. Serum ALT levels at 60 and 120 min after reperfusion in group B were significantly higher than those in group A. Blood-oxygenation index measured at 5 and 60 min of reperfusion showed significant correlation with serum ALT levels at 120 min of reperfusion. When the percentage recovery of blood-oxygenation index at 5 and 60 min after reperfusion was more than 75%, all rats survived. No obvious signs of hepatocellular degeneration were observed histologically 5 min after reperfusion; however, substantial hepatocellular degeneration had occurred at 120 min of reperfusion in groups A and B. These data indicate that a decline in hepatic tissue oxygenation during the early phase of reperfusion (even when no obvious hepatocellular degeneration has been observed) can be a predictor of subsequent liver injury and prognosis.

Liver scintigraphy was performed using a newly developed radiopharmaceutical, Tc-99m-DTPA-Galactosyl-Human-Serum-Albumin (Tc-99m-GSA), which binds specifically to the receptors on the hepatic cell surface, in 15 patients with chronic liver diseases. The scintigraphy was evaluated qualitatively and quantitatively, and the results were compared with those obtained from the Tc-99m-PMT or Tc-99m-sn-phytate scintigraphy, and the liver function tests. The Tc-99m-GSA scintigraphy showed clear liver images in chronic hepatitis. However, in liver cirrhosis, the liver images was not clear and the cardiac images still existed 40 minutes after administration of Tc-99m-GSA, suggesting that the image quality of the Tc-99m-GSA scintigrams may be inferior to that of Tc-99m-sn-phytate or Tc-99m-PMT in some cases of severe liver dysfunction. The time-activity curves of the heart and liver were analyzed by non-linear regression analysis. The clearance rate from plasma (Kd) were obtained from the time-activity curve of the heart, and the hepatic uptake rate (Ku), hepatic excretion rate (Ke) and peak time of hepatic uptake-excretion curve (PT) were obtained from the time-activity curve of the liver. Kd, Ku and PT values were more significantly decreased or prolonged in the patients with liver cirrhosis than those in the patients with chronic hepatitis. Kd, Ku and PT values had positive correlations with the result of the serum liver function tests, ICG-R15 and ICG-K. Ku and PT values had also correlations with the histological degrees of hepatic fibrosis.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to replace the liver function, the development of the system where natural hepatocytes are immobilized in a matrix polymer might be considered as the best way. Each hepatocyte has a number of specific receptors for several ligards. We noticed that the hepatocyte recognizes the structure of oligosaccharides via asialoglycoprotein receptors and synthesizes lactose-carrying styrene polymer (PVLA) as a asialoglycoprotein model. On PVLA-coated dish, the specific functions as well as attachment of hepatocyte were successfully maintained. Moreover, cultured hepatocytes on PVLA substratum started gradual movement, and then remarkably formed multilayer aggregations which had long-term survival. Hepatocytes in the aggregation exhibited better maintenance of specific hepatocyte-functions such as the synthesis of albumin and secretion of bile acid, and retained mitochondrial enzyme activity than those in the monolayer culture on collagen and fibronectin. Furthermore, we found that DNA synthesis in cultured hepatocytes was correlated to the cell-shape which could be controlled by the concentration of PVLA substratum. These results also suggested that the PVLA could potentiate the regulation of the differentiation and proliferation of hepatocytes.

Temperature increases membrane fluidity and decreases vascular resistance in isolated organs. Therefore, these studies were designed to determine if a rinse with warm buffer could increase survival time in the rat model of orthotopic liver transplantation by improving hepatic microcirculation. Brief periods of warm ischemia (3-8 min) did not damage the liver as indexed by minimal release of LDH. Survival of rats for 30 days was greater than 90% in this model when livers were stored for 1 hr in Ringer's solution; yet grafts stored for 8 hr in Euro-Collins solution and rinsed with 20 ml of cold (0-4 degrees C) Ringer's solution survived postoperatively only around 3 days. However, livers stored for 8 hr in Euro-Collins and rinsed with 20 ml of warm (37 degrees C) Ringer's survived longer than 30 days (i.e., permanently). Serum transaminase levels reached peak values around 6000 U/L one day postoperatively in the cold-rinsed group, and liver injury assessed histologically was substantial. Under these conditions, pulmonary infiltration of inflammatory cells was observed in about 23% of lung tissue examined and was associated with massive bleeding. Following a warm rinse, however, maximal SGOT levels and injury to both liver and lung were reduced significantly by 80-90% 24 hr postoperatively. Moreover, the warm rinse improved hepatic microcirculation. It accelerated blood flow into the liver approximately two-fold, as indexed by the half-time of changes in hemoglobin reflectance from the liver surface, improved the distribution of colloidal carbon in the organ observed macroscopically, and decreased vascular resistance by over 50%. These data support the hypothesis that a brief rinse of liver grafts with warm buffer markedly improves the hepatic microcirculation, leading to dramatic improvement in graft survival. This work demonstrates clearly that a brief warm rinse may be useful clinically in liver transplantation.

The hypothesis was tested whether ingestion of ethanol might disturb the hepatic microcirculation with resulting hepatic hypoxia. Infusion of ethanol increased the portal pressure concentration-dependently in rat livers perfused with Krebs-Henseleit buffer at a constant flow rate (Emax = 11.5 cm H2O, EC50 = 90 mM). This increase in portal pressure was due to hepatic vasoconstriction, since it diminished in the presence of sodium nitroprusside, a direct acting vasodilator. The regional hepatic tissue hemoglobin concentration after perfusion with added erythrocyte suspension (hematocrit 1%), measured by tissue-reflectance spectrophotometry, was significantly diminished by the infusion of ethanol, indicating the impairment of the microcirculation of the superficial layer of the liver. When the absorption spectrum of the liver was examined by reflectance spectrophotometry, infusion of ethanol caused a parallel reduction of all the mitochondrial respiratory cytochromes in a concentration-dependent fashion, concomitant with the increase of portal pressure, indicating a marked reduction of oxygen concentration in superficial liver tissue. The reduction of the respiratory cytochromes was also associated with the decrease in oxygen consumption of the liver, indicating that the hepatic hypoxia was due to the reduction of oxygen delivery to hepatocytes rather than the increased oxygen consumption of the liver. The reduction of the respiratory cytochromes was correlated with the increase in portal pressure and was inhibited by sodium nitroprusside. These data indicate that the ethanol-induced hepatic vasoconstriction disturbs hepatic microcirculation, resulting in hepatic hypoxia and reduction of mitochondrial respiratory cytochromes.

The purpose of these experiments was to employ video microscopy to identify early pathological changes after orthotopic liver transplantation in the rat. Liver transplantation was performed using the cuff technique. Survival was greater than 90% when livers were stored for 1 hr in Ringer's solution prior to transplantation (survival conditions), whereas all rats died following storage of grafts for 4 hr in cold Euro-Collins solution (nonsurvival conditions). Postoperatively, each recipient animal was anesthetized, the abdomen was opened, and the liver was placed on the stage of an inverted fluorescence microscope equipped with a low-light ISIT video camera. Precautions were taken to prevent the liver surface from drying. The fluorescent dyes fluorescein sodium (1.0 mumol/kg), acridine orange (2.5 mumol/kg), and propidium iodide (1 mumol/kg) were injected intravenously to label hepatocytes, polymorphonuclear leukocytes, and the nuclei of irreversibly damaged cells, respectively. In livers from untransplanted rats, movement of labeled leukocytes through the hepatic sinusoids was smooth and rapid (velocity, 500-550 microns/sec) and margination (adhesion) of cells was minimal (less than 1%). After transplantation, however, velocity was diminished 2-3-fold, and margination was increased to 10% of leukocytes in survival groups and to 40% in nonsurvival groups 4 hr postoperatively. In the nonsurvival groups, irreversible cell death detected by propidium iodide fluorescence was minimal 15 min after transplantation--however, massive cell damage was detected 4 hr postoperatively. In addition, serum transaminases and necrosis were higher at 4 hr than 15 min postoperatively. Taken together, these data demonstrate that leukocyte margination increases following orthotopic liver transplantation and is followed rapidly by cell death. These events likely play a role in the mechanism of early graft failure following transplantation.

In the perfused rat liver, infusion of ethanol induced an initial increase in portal pressure which is an indicator of vasoconstriction and a subsequent increase in lactic dehydrogenase (LDH) release, which is an indicator of cell damage in a dose-dependent fashion. Simultaneous infusion of sodium nitroprusside, a potent vasodilator, (100 microM) inhibited the increases in portal pressure and LDH release. Focal hepatocellular necrosis evidenced by trypan-blue stained cell nuclei were localized in midzonal and pericentral area of the liver lobules at 60 min after ethanol load. These ethanol-induced microcirculatory disturbance might be involved in the pathogenesis of alcoholic liver disease.

Periportal and pericentral regions of the liver lobule were isolated from perfused rat liver using a micropunch and incubated in Krebs-Henseleit buffer (pH 7.6) containing 2% poly(ethylene glycol) in Eagle's basal medium, PMSF (50 micrograms/ml) and leupeptin (20 micrograms/ml) for 2 h at 25 degrees C under and O2/CO2 (95:5%) gas phase. Maximal rates of urea production from ammonium chloride were 96.4 +/- 8.7 and 32.8 +/- 5.4 mumol/g per h at 800 and 200 microM O2. Thus, urea synthesis was 2-3-times greater at high than low O2 tension in plugs from periportal and pericentral regions of the liver lobule.

Menadione bisulfite is a hepatotoxicant that damages periportal regions of the lobule in perfused liver in an oxygen-dependent manner. The effect of ethanol on menadione bisulfite toxicity was examined in perfused rat liver. Addition of menadione bisulfite (3 mM) alone to the perfusate increased oxygen uptake by 20-30 mumols/g/hr. Lactate dehydrogenase was released into the effluent after 60 min of perfusion and reached values around 100 units/g/hr. Under these conditions, trypan blue was taken up exclusively in periportal regions of the liver lobule; 44% of periportal cells were stained. In the presence of ethanol, maximal increases in oxygen uptake due to menadione bisulfite were much larger (about 90 mumols/g/hr), and lactate dehydrogenase release occurred earlier and reached higher maximal values (330 units/g/hr). Trypan blue staining was also more extensive; 90% of periportal cells were stained. The effect of ethanol on menadione bisulfite-induced oxygen uptake required metabolism via alcohol dehydrogenase (ADH), because ethanol increased oxygen uptake due to menadione bisulfite from 44 to 81 mumols/g/hr in deermice with ADH but had no effect in deermice lacking ADH. Other agents that increase NADH (xylitol and 2-ethyl-1-hexanol) also potentiated the stimulation of oxygen uptake due to menadione bisulfite, suggesting that ethanol was working by increasing the NADH redox state. Cyanide abolished the increase in oxygen uptake due to menadione bisulfite, both in the absence and in the presence of ethanol, supporting the hypothesis that the effect of ethanol on menadione bisulfite-mediated oxygen uptake involves the mitochondrial respiratory chain. Further, the stimulation of oxygen uptake by menadione bisulfite in isolated mitochondria was enhanced when matrix NADH was increased by addition of beta-hydroxybutyrate. These data indicate that ethanol potentiates oxygen uptake and toxicity due to menadione bisulfite most likely by generation of NADH for redox cycling of this model quinone.