bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Nitric Oxide on Iron–Mediated Oxidative Stress in Primary Rat Hepatocyte Culture
Authors: Sergent O. , Griffon B. , Morel I. , Chevanne M. , Dubos M. , Cillard P. , Cillard J.
Published: July 2004

An iron–mediated oxidative stress caused by an increase of the intracellular pool of low molecular weight complex of iron (LMWC) can be observed with iron overloading or ethanol metabolism. The aim of this study was to determine whether nitric oxide (NO) behaved as a pro–oxidant or an antioxidant in such an iron–mediated oxidative stress in rat hepatocytes. The cells were set up in primary cultures and incubated with lipopolysaccharide (LPS) and γ–interferon (IFN) for 18 hours to induce NO synthase and to trigger NO production. Then 20 μmol/L iron or 50 mmol/L ethanol were added. Oxidative stress was evaluated by measuring lipoperoxidation using two markers: malondialdehyde (MDA) and conjugated dienes. Simultaneously, NO production was followed by the quantitation of nitrites in the culture medium, dinitrosyl iron complexes (DNICs) and mononitrosyl iron complexes (MNICs) in intact hepatocytes. DNIC and MNIC, evaluated by electron paramagnetic resonance (EPR), corresponded to NO bound to iron–containing molecules and to free NO, respectively. In cultures preincubated with LPS and IFN before iron or ethanol addition, a net decrease of lipid peroxidation induced by either NO, iron, or ethanol was noted. Moreover, an elevation of iron–bound NO and a decrease of free NO were observed in these cultures compared with the cultures incubated with only LPS and IFN. These data support the idea that there is a relationship between the changes of NO pool and the inhibition of oxidative stress. In addition, using NG–monomethyl–l–arginine (l–NMMA), a NO synthase inhibitor, NO was shown to be involved in the inhibition of oxidative stress induced by iron or ethanol. Addition of the chelator of LMWC iron, deferiprone, was followed by the inhibition of the increase of iron–bound NO and the reincrease of lipid peroxidation extent, which was as high as in cultures incubated only with LPS and IFN. Thus LMWC iron appeared to be involved also in the inhibition of oxidative stress induced by NO. All the results favor the conclusion that NO acts as an antioxidant in iron–mediated oxidative stress in rat hepatocytes. NO reacted with LMWC iron to form inactive iron complexes unable to induce oxidative stress in rat hepatocytes. Thus NO played a critical role in protecting the liver from oxidative stress.
Mechanisms of Alcohol Impairment of Recovery From Mechanically Denuded Areas Made Within Cultured Rat Hepatocytes
Authors: Anderson R. J. , Sponsel H. T. , Brown S. E. , Breckon R. , Ray C. , Simon F. R. , Guzelian P. S.
Published: July 2004

The effect of ethanol on the ability of a denuded hepatocyte cell surface to recover is unknown. We therefore determined the effect of ethanol on the rate of renewal of mechanical wounds made in near–confluent monolayers of primary cultures of rat hepatocytes. We found that ethanol exerted a dose–dependent effect to impair rat hepatocyte recovery and that at least 12 hours of exposure to ethanol was required to induce this impairment. The effect of ethanol to impair recovery of denuded areas of epithelium was not seen in two established renal tubular epithelial cell lines. Ethanol impairment of rat hepatocyte recovery could be blocked by an alcohol dehydrogenase inhibitor (4–methyl pyrazole) and potentiated by an inhibitor of aldehyde dehydrogenase (pargyline). The effect of ethanol to inhibit rat hepatocyte recovery of denuded areas was not associated with any change in hepatocyte cell surface expression of the β1 integrin subunit as determined by flow cytometry. These results suggest that acetaldehyde is responsible for ethanol inhibition of hepatocyte recovery from mechanical injury and that this impairment occurs independent of cell surface expression of the β1 integrin subunit.
Carnitine Metabolism in Patients With Chronic Liver Disease
Authors: Krähenbühl S. , Reichen J.
Published: July 2004

Carnitine metabolism was studied in 79 patients with chronic liver disease, including 22 patients with noncirrhotic liver disease and 57 patients with different types of cirrhosis (22 patients with hepatitis B– or C–associated cirrhosis, 15 patients with alcohol–induced cirrhosis, 15 patients with primary biliary cirrhosis [PBC], and 5 patients with cryptogenic cirrhosis), and compared with 28 control subjects. In comparison with control subjects, patients with noncirrhotic liver disease showed no change in the plasma carnitine pool, whereas patients with cirrhosis had a 29% increase in the long–chain acylcarnitine concentration. Analysis of subgroups of patients with cirrhosis showed that patients with alcohol–induced cirrhosis had an increase in the total plasma carnitine concentration (67.8 ± 29.5 vs. 55.2 ± 9.9 μmol/L in control subjects), resulting from increases in both the short–chain and long–chain acylcarnitine concentration. In this group of patients, the acylcarnitine concentrations showed a close correlation with the total carnitine concentration, and the total carnitine concentration with the serum bilirubin concentration. Urinary excretion of carnitine was not different between patients with noncirrhotic or cirrhotic liver disease and control patients. However, patients with PBC showed an increased urinary excretion of total carnitine (52.5 ± 40.0 vs. 28.0 ± 16.7 micromol carnitine/μmol creatinine), resulting from an increase in the fractional excretion of both free carnitine and short–chain acylcarnitine. The current studies show that patients with cirrhosis are normally not carnitine deficient. Patients with alcohol–induced cirrhosis have increased plasma carnitine concentrations, which may result from increased carnitine biosynthesis because of increased skeletal muscle protein turnover. The increase in the fractional carnitine excretion in patients with primary biliary cirrhosis may result from competition of bile acids and/or bilirubin with tubular carnitine reabsorption and/or from a reduced activity of the carnitine transporter located in the proximal tubule.
The Synthesis Rates of Total Liver Protein and Plasma Albumin Determined Simultaneously In Vivo In Humans
Authors: Barle H. , Nyberg B. , Essén P. , Andersson K. , McNurlan M. A. , Wernerman J. , Garlick P. J.
Published: July 2004

Although the metabolism of liver–derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin

in vivo

were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. l–[2H5]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 ± 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC–MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 ± 3.1 %/d (mean ± SD), and that of albumin was 5.9 ±1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 ± 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human

in vivo

model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein.
Predictive Value of Intraoperative Biopsies and Liver Function Tests for Preservation Injury in Orthotopic Liver Transplantation
Authors: Gaffey M. J. , Boyd J. C. , Traweek S. T. , Ali M. A. , Rezeig M. , Caldwell S. H. , Iezzoni J. C. , McCullough C. , Stevenson W. C. , Khuroo S. , Nezamuddin N. , Ishitani M. B. , Pruett T. L.
Published: July 2004

Eighty liver allografts were studied to determine the predictive value of intraoperative biopsies and postoperative liver function tests for the development of preservation injury (PI). Peak transaminase (aspartate transaminase [AST] and alanine transaminase [ALT]) and prothrombin time (PT) values achieved by each patient during postoperative days (POD) 1 through 7 were determined. PI in day 0 preperfusion biopsies (0Pre) (obtained immediately before implantation) and postperfusion biopsies (0Post) (obtained immediately after revascularization) was categorized by histological criteria as present or absent. PI in biopsies taken during POD 2 through 14 was histologically graded as either moderate–to–severe, mild, or absent. Of the 80 allografts, 8 were omitted because of primary nonfunction or postoperative complications. 0Pre and 0Post biopsies were available on 25 of 72 (35%) and 69 of 72 (96%) allografts, respectively. Only 2 (8%) of the 0Pre biopsies showed histological PI compared with 48 (70%) of the 0Post biopsies. Fifty–nine patients were biopsied between POD 2 through 14. Of these, 15, 28, and 16 patients developed moderate–to–severe, mild, or no evidence of PI, respectively. The presence of PI in the 0Post biopsy strongly correlated with the development of PI during POD 2 through 14 (

P

< .0005). Peak AST and ALT values in patients with moderate–to–severe PI on POD 2 through 14 were significantly elevated compared with those patients with either mild (

P

= .01 and .03) or no PI (

P

= .02 and .006). Because of extensive overlap in AST and ALT values between the three groups, however, transaminase values were not useful in predicting the presence or absence of PI in the individual case. The development of PI during POD 2 through 14 correlated with advanced donor age (

P

= .06) but was unassociated with 0Pre biopsy findings, cold ischemia time, or peak PT values. We conclude that the 0Post biopsy is a valuable tool for the prediction of subsequent PI in the early postoperative period. In contrast, 0Pre biopsy findings and peak AST and ALT values are not useful in the assessment of PI.
Graft and Systemic Disease in Long–Term Survivors of Liver Transplantation
Authors: Slapak G. I. , Saxena R. , Portmann B. , Gane E. , Devlin J. , Calne R. , Williams R.
Published: July 2004

In this clinicopathological study, we have examined the prevalence of biochemical and histological graft abnormalities in 116 patients who have lived for longer than five years since orthotopic liver transplantation (mean, 8.4 years [range, 5.2–19.5 years]). In each patient, the protocol biopsy was carried out in addition to full clinical, biochemical, and immunological evaluation. The renal function was preserved in most patients (mean creatinine, 86 micromol/L [0.95 mg%]) with only 35% showing any degree of impairment, and only one patient with pretransplant renal failure required dialysis. Forty one (35%) of the patients had normal biochemical liver function tests (LFTs); 19 of these patients had histologic abnormalities. Of the remaining 75 patients with abnormal LFTs, 65 were found to have histological abnormalities on liver biopsy. Some of the 84 patients with an abnormal histology had more than one finding but were placed into 6 categories according to the main change as follows: chronic hepatitis in 27; primary biliary cirrhosis–like changes in 8; cholangitis in 15; structural anomalies in 26; chronic rejection in 2; mild acute cellular rejection in 1; and miscellaneous in 5. The recurrence of B, C, or autoimmune hepatitis accounted for 14 cases of chronic hepatitis, whereas, in 13 cases, no cause could be detected. The latter cases included 8 patients who had liver transplants for primary biliary cirrhosis (PBC), leaving suboptimally treated rejection or disease recurrence as possible causes. Characteristic histological features of PBC were observed in 8 additional cases that had liver transplantations for this disease. Of the 26 cases with structural anomalies, various types of occlusive vasculopathy were detected during pre– or posttransplantation in 12 cases. All but one of these were maintained on azathioprine, the only possible etiological factor in the 14 other cases, 5 of whom showed stabilization or clinical and histological improvement following withdrawal of the drug.

De–novo

malignancy was seen in 3.4% of cases, causing mortality in only one patient to date. This study demonstrates an unexpectedly high prevalence of histological abnormalities that is found often in the presence of normal biochemical liver–function in liver grafts of long– term survivors. Although graft loss at this time is rare, protocol liver biopsies at 5 years, and thereafter at 2–year intervals, may allow for earlier beneficial therapeutic intervention.
Presence of Antibodies to the Hepatitis B Surface Antigen Is Associated With An Excess Risk for Hepatocellular Carcinoma Among Non–Asians in Los Angeles County, California
Authors: Yu M. C. , Yuan J. , Ross R. K. , Govindarajan S.
Published: July 2004

Hepatocellular carcinoma (HCC) exhibits a more than 50–fold variation in incidence worldwide. High–risk regions include East Asia and sub–Saharan Africa, while non–Asians in the United States constitute a low–risk population. We assessed 111 cases of histologically confirmed HCC and 128 community control subjects among non–Asians of Los Angeles County for the presence in serum of hepatitis B surface antigen (HBsAg), antibodies to HBsAg (anti–HBs), antibodies to the hepatitis B core antigen (anti–HBc), HBV DNA, and antibodies to the hepatitis C virus (anti–HCV). Anti–HCV positivity was significantly associated with a 12.6–fold increase in HCC risk (95% confidence limits = 4.7, 33.6). As expected, the presence of serum HBsAg and the presence of anti–HBc in the absence of anti–HBs were both positively associated with the risk of HCC. But most interestingly, among our study subjects, the presence of anti–HBs in the absence of HBsAg and HBV DNA (indicative of a resolved infection) was significantly related to a 4.7–fold increased risk for HCC (95% confidence limits = 2.2, 9.4). Overall, any serological evidence of prior HBV exposure was associated with a 9.4–fold elevation in HCC risk (95% confidence limits = 4.7, 18.7). The data also demonstrate a synergistic effect of HBV and HCV infections on the risk of HCC. We estimate that about 55% of HCC cases occurring in non–Asians of Los Angeles can be attributed to infection by the hepatitis B and/or C viruses.
Chronic Viral Hepatitis Induced by Hepatitis C But Not Hepatitis B Virus Infection Correlates With Increased Liver Angiogenesis
Authors: Mazzanti R. , Messerini L. , Monsacchi L. , Buzzelli G. , Zignego A. L. , Foschi M. , Monti M. , Laffi G. , Morbidelli L. , Fantappié O. , Omer Bartoloni Saint F. , Ziche M.
Published: July 2004

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections lead to cirrhosis and increase the risk for the development of hepatocellular carcinoma (HCC). Angiogenesis is an essential step in oncogenesis and contributes to tumor progression in adult organs; however, to what extent angiogenesis occurs in the liver during chronic viral hepatitis has not been studied. Ninety–nine matched patients affected by chronic hepatitis due to either HBV or HCV were studied together with 13 controls (5 patients were affected by familial hyperbilirubinemia with normal liver histology; 6 patients with stage II primary biliary cirrhosis; and 2 patients with pseudo inflammatory tumor). Microvessel density was assessed in liver biopsies by immunostaining using two different antibodies against endothelial cell antigens, QB–END/10 and Factor VIII. In addition, the liver homogenates and sera of HCV– or HBV–positive patients and controls were tested for their capacity to stimulate the migration and proliferation of freshly isolated human endothelial cells in vitro. Evidence of angiogenesis was significantly more frequent in HCV–positive patients compared with HBV–infected subjects or controls (74% vs. 39% vs. 8%) (χ2 = 20.78;

P

< .0001) (HCV+ vs. HBV+ vs. controls). The degree of microvessel density was also higher in HCV– than in HBV–positive patients or controls (χ2 = 12.28;

P

< .005). In addition, HCV–positive sera and liver homogenates stimulated a higher migration and proliferation of human endothelial cells

in vitro

compared with HBV–positive or control sera and liver homogenates. These observations indicate that angiogenesis is particularly linked to HCV infection, suggesting a possible contribution to HCV–related liver oncogenesis.
Nitric Oxide Donors Stimulate Bile Flow and Glutathione Disulfide Excretion Independent of Guanosine 3′,5′–Cyclic Monophosphate in the Isolated Perfused Rat Liver
Authors: Trauner M , Nathanson M H , Mennone A , Rydberg S A , Boyer J L
Published: July 2004

ABSTRACT

Nitric oxide (NO) modulates several metabolic functions in hepatocytes, but the role of NO in bile secretion has not been clearly defined. In the present study, we examined the effects of NO on bile flow and biliary HCO3
- and glutathione excretion in the isolated perfused rat liver and assessed the role of guanosine 3′,5′–cyclic monophosphate (cGMP) in mediating these effects. The NO donors sodium nitroprusside (SNP) and S–nitroso–acetyl–penicillamine stimulated bile flow and increased both HCO3
- and glutathione excretion. Increases in bile flow were linearly related to increases in biliary glutathione concentration and output (

P

< .0001), which were almost entirely caused by glutathione disulfide, whereas the excretion of reduced glutathione remained unchanged. NO donors increased cGMP concentrations in bile and perfusate, and the membrane–permeant cGMP analogue dibutyryl cGMP was also found to stimulate bile flow and HCO3
- excretion. However, in contrast to the NO donors, dibutyryl cGMP did not increase glutathione excretion. Furthermore, the NO donors failed to stimulate bile flow in mutant TR–rats in which the canalicular transport of glutathione and glutathione conjugates is deficient, although dibutyryl cGMP increased bile flow and HCO3
- excretion in the mutant rats as in normals. These findings indicate that exogenous sources of NO increase bile acid–independent bile flow by stimulating glutathione disulfide excretion, effects that are independent of cGMP.
Expression of the Rat Liver Na+/Taurocholate Cotransporter Is Regulated In Vivo By Retention of Biliary Constituents But Not Their Depletion
Authors: Gartung C , Schuele S , Schlosser S F , Boyer J L
Published: July 2004

ABSTRACT

Expression and function of the hepatic Na+/taurocholate cotransporter (

ntcp

) are down–regulated in several models of experimental cholestasis. To test whether retention and/or depletion of biliary constituents are involved in

ntcp

regulation,

ntcp

expression was quantified in several animal models with altered levels of these constituents. In choledochocaval fistula rats (CCF) (retention model),

ntcp

mRNA expression specifically declined after 1 and 3 days by 76 ± 4% (

P

< .005) and 31 ± 9% (

P

< .05), respectively, returning to control levels by 7 days. However, protein expression as assessed by Western blotting remained unchanged for up to 7 days of CCF. In rats with bile fistulas (depletion model) for 0.5, 1, 2, 4, and 7 days, both

ntcp

protein and mRNA expression remained unaltered. Infusion of either taurocholate or taurochenodeoxycholate for 12 hours also did not effect

ntcp

mRNA expression in intact animals, probably because of its inability to increase serum and intrahepatic bile acid levels. In rats with selective bile duct ligation (SBDL),

ntcp

mRNA levels were down–regulated by 40 ± 10% (

P

< .05) only after 12 and 24 hours in ligated lobes, and mRNA levels returned to control values in these lobes after 2 and 4 days.

ntcp

mRNA expression remained unchanged in the nonobstructed lobes at any time. When data from CCF and SBDL rats were combined, serum bile acids correlated linearly with

ntcp

mRNA (r = .62,

P

< .0005) over a 0 to 110–micromol/L range. Our results indicate that

ntcp

is constitutively expressed and remains uneffected by either depletion or increased flux of biliary constituents. However, retention of biliary constituents results in rapid down–regulation of

ntcp

mRNA, consistent with the concept that hepatocytes may be protected from bile acid toxicity during cholestasis by this mechanism.
Improvement in Cholestasis–Associated Fatigue With A Serotonin Receptor Agonist Using A Novel Rat Model of Fatigue Assessment
Authors: Swain M G , Maric M
Published: July 2004

ABSTRACT

Fatigue is a common complaint in patients with liver disease; however, the etiology of fatigue is poorly understood and no therapeutic options are available to treat it. Altered central neurotransmission, especially serotonergic, appears to play a role in the genesis of fatigue. In this study, we describe a rat model of fatigue assessment using a swim tank, and we used this model to document the degree of fatigue in rat models of cholestasis caused by bile duct resection (BDR) and of hepatitis caused by carbon tetrachloride (CCl4) administration. Fatigue was quantitated as the time spent floating and struggling over a 15–minute period after placement in the swim tank, and an overall activity score was calculated. Using this technique, BDR rats exhibited significantly enhanced floating times and an overall reduction in activity score compared with noncholestatic controls (

P

≤ .01). On the other hand, CCl4–treated rats showed a marked variability in floating and struggling times and activity scores such that, overall, CCl4–treated rats were not significantly different from normal controls. Therefore, we used BDR and noncholestatic control rats to examine the effects of a serotonin (5HT1A) receptor agonist (LY293284) on cholestasis–associated fatigue. BDR rats treated with LY293284 (0.3 mg/kg subcutaneously 24, 5, and 1 hour before placement in the swim tank) showed marked reductions in floating times and an increase in overall activity scores compared with BDR controls (

P

≤ .001). LY293284 was without effect in noncholestatic animals. These results suggest that fatigue can be quantitated in rat models of liver disease and that 5HT1A receptor agonists may provide a useful therapeutic tool in the treatment of cholestatic liver disease– associated fatigue.
Regional Cerebral Edema and Chloride Space in Galactosamine–Induced Liver Failure in Rats
Authors: Gove C D , Hughes R D , Ede R J , Williams R
Published: July 2004

ABSTRACT

The pathogenesis of cerebral edema, which is a major complication of fulminant hepatic failure, is poorly understood. In previous studies, increased regional brain water content was observed in rats at an early stage of acute liver failure caused by galactosamine. At a later stage when the animals had developed deep coma, brain water content was reduced, possibly as a result of generalized dehydration. In the present investigation, we have determined brain water content at a late stage of liver failure, 48 hours after galactosamine, in animals that had been maintained in fluid balance by continuous intraperitoneal infusion of glucose solution. In these animals, brain water content, determined from the ratio of wet to dry weight, showed a greater increase than that observed previously at the early stage (hindbrain region [cerebellum, pons, and brain stem] increased by 4.2%; forebrain region increased by 1.4% compared with controls). Regional analysis of brain water, using a tissue–specific gravity method, showed a significant increase in cerebellar gray matter water content. Analysis of chloride space showed the extra fluid to be mainly extracellular in the hindbrain region, but not in the forebrain region. Ultrastructural examination of capillaries in gray matter from cerebellum and cerebrum showed no evidence of gross disruption of the tight junctions. Swelling of the astroglial foot processes was observed in the cerebellar gray matter. These results suggest that both vasogenic and cytotoxic mechanisms of edema formation occur in the brain during liver failure.
Prenatal Alcohol Exposure Affects Galactosyltransferase Activity and Glycoconjugates in the Golgi Apparatus of Fetal Rat Hepatocytes
Authors: Renau-Piqueras J , Guasch R , Azorín I , Seguí J , Guerri C
Published: July 2004

ABSTRACT

Prenatal exposure to alcohol affects the morphological, structural, and functional features of the Golgi apparatus (GA), thus altering the glycosylation process in fetal hepatocytes. To elucidate the cellular mechanisms underlying these alterations, we have studied the effect of alcohol exposure

in utero

on the activity of liver galactosyltransferase, an enzyme involved in the glycosylation process, and on the hepatic glycoprotein sugar composition. For this, livers from 21–day–old fetuses obtained from control and ethanol–fed rats were used. Galactosyltransferase (GT) activity was determined in isolated GA cis and trans fractions. Colloidal gold–labeled lectin cytochemistry was used to analyze sugar residues in hepatocytes at the subcellular level. Finally, the integrity of the GA after alcohol treatment was assessed by electron microscopy and by evaluating the distribution of the Golgi β–COP, a protein involved in vesicular trafficking. Prenatal alcohol exposure induces a significant increase in both liver weight and total protein content in the trans Golgi. Moreover, this treatment decreases the activity of galactosyltransferase, increases α–l–Fuc residues, and reduces the number of α–Man, GlcNAc(β1,4,GlcNAc)1,2, GalNAc α1,3GalNAc, α–GalNAc, and a–Gal residues. Alcohol exposure also causes the Golgi cisternae to disappear in about 30% of the hepatocytes, and reduces 75% the number of anti–Golgi β–COP protein binding sites. Our results suggest that the decrease in galactosyltransferase activity, the alterations in the oligosaccharide chain composition, and the reduction in the amount of Golgi β–COP protein could be involved in the alterations in the glycosylation process, as well as in the accumulation of hepatic proteins observed after prenatal alcohol exposure. These alterations could contribute, therefore, to the alcohol–induced injury in the developing liver.
Kupffer Cells Generate Superoxide Anions and Modulate Reperfusion Injury in Rat Livers After Cold Preservation
Authors: Shibuya H , Ohkohchi N , Seya K , Satomi S
Published: July 2004

ABSTRACT

This study was designed to determine the source of reactive oxygen species (ROS) and whether Kupffer cells modulate the injury of perfused rat liver after cold preservation. The livers of male Lewis rats pretreated with schizophyllan glucan (SPG) (10 mg/kg, SPG group) or gadolinium chloride (5 mg/kg; Gd group) and untreated rats (control group) were preserved in University of Wisconsin solution for 0, 12, and 24 hours at 4 degrees C and then perfused for 60 minutes with oxygenated Krebs–Henseleit bicarbonate buffer. Real–time chemiluminescence (CL) of the liver during perfusion was measured using a sensitive photomultiplier, and reperfusion injury was assessed by measuring lipid peroxidation and lactate dehydrogenase release. CL intensity reached a peak within 5 minutes of reperfusion, and superoxide dismutase completely inhibited this CL in all groups. In the control group, the total CL intensity was high after 24 hours of preservation. It significantly (

P

< .05 vs. control group) increased in the SPG group, while it decreased in the Gd group after 12 and 24 hours of preservation. The total CL intensity decreased by 70% (when diphenyliodonium chloride (100 micromol/L; an NADPH oxidase inhibitor) was added to the perfusate before preservation of untreated rats. Lipid peroxidation and lactate dehydrogenase release significantly (

P

< .05) deteriorated in the SPG group, while they ameliorated in the Gd group after 24 hours of preservation. These results demonstrate that Kupffer cells primarily generate superoxide anions and modulate the organ injury in the initial phase of reperfusion after cold preservation.
Oxidative Stress–Mediated Apoptosis of Hepatocytes Exposed to Acute Ethanol Intoxication
Authors: Kurose I , Higuchi H , Miura S , Saito H , Watanabe N , Hokari R , Hirokawa M , Takaishi M , Zeki S , Nakamura T , Ebinuma H , Kato S , Ishii H
Published: July 2004

ABSTRACT

The present study was designed to investigate whether acute ethanol intoxication increases the production of active oxidants, and subsequently promotes apoptosis of hepatocytes. Hepatocytes were isolated from male Wistar rats, and cultured in the presence or absence of ethanol. The fluorescence

in situ

nick end labeling method and an enzyme–linked immunosorbent assay (ELISA) system to quantify fragmented DNA were used to estimate apoptotic change in hepatocytes. Nuclear morphological alterations and membrane barrier dysfunction of hepatocytes were assessed by staining with Hoechst 33342 and propidium iodide (PI). Intracellular glutathione level was determined as the fluorescence of monochlorobimane (MCLB), which forms conjugate with glutathione to become fluorescent. Ethanol (100 mmol/L) increased the amount of fragmented DNA and the number of apoptotic hepatocytes

in vivo

as well as

in vitro

. These ethanol–induced alterations in hepatocytes were attenuated by simultaneous incubation with either 4–methylpyrazole, an inhibitor of alcohol dehydrogenase, or dimethylthiourea, an intracellular oxidant scavenger. Diethyl maleic acid (DMA), a glutathione depletor, enhanced the induction of apoptotic change, and decreased membrane barrier function in ethanol–treated hepatocytes, whereas ethanol

per se

did not increase the number of PI–positive hepatocytes. Furthermore, combination of ethanol and DMA but not ethanol alone decreased the hepatocyte MCLB fluorescence. Taken together, the present study suggests that active oxidants produced during ethanol metabolism mediate fragmentation of DNA in hepatocytes, and that intracellular antioxidants such as glutathione play a critical role in the cytoprotective mechanisms of hepatocyte against lethal cell death, ie, apoptosis, induced by ethanol.
Membrane Potential of Hepatic Mitochondria After Acute Cocaine Administration in Rats—The Role of Mitochondrial Reduced Glutathione
Authors: Masini A , Gallesi D , Giovannini F , Trenti T , Ceccarelli D
Published: July 2004

ABSTRACT

Cocaine hepatotoxicity may be mediated by oxidative damage, possibly involving mitochondrial injury. The effect of an acute dose of cocaine in rats on the mitochondrial level of reduced glutathione, nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH), important determinants in cellular defense against oxidative stress, was investigated. Under these conditions, the extent of lipid peroxidation was assessed as thiobarbituric acid reactive substances formation and the energy transducing capability of the inner mitochondrial membrane was evaluated by membrane potential measurements. Female Wistar albino rats were given an acute 50 mg/kg intraperitoneal dose of cocaine and, 6 hours later, hepatic and mitochondrial biochemical analyses were made. Rats administered intraperitoneally, 7.5 hours before the sacrifice, a specific inhibitor of glutathione synthesis, l–buthionine–(S,R)–sulphoximine, either alone or in combination with cocaine, underwent in parallel the same determinations. Cocaine intoxication did not impair mitochondrial functions, although a significant increase of lipid peroxidation occurred. By contrast the combination of l–buthionine–(S,R)–sulphoximine with cocaine induced a severe derangement of mitochondrial functional efficiency, a large depletion of reduced glutathione, and a further enhancement of lipid peroxidation. The mitochondrial functional anomalies were largely restored by the use of cyclosporin A, ethyleneglycotetraacetic acid (EGTA) and glutathione methylmonoester. A nonspecific calcium dependent inner membrane permeabilty transition (pore opening) accounted for the partial loss of mitochondrial coupled functions at a period of cocaine intoxication when no cell damage occurred. The level of mitochondrial glutathione played a critical role in protecting inner membrane functional integrity against cocaine–induced oxidative stress.
Characterization of A Swelling–Activated Anion Conductance in Homozygous Typing Cell Hepatoma Cells
Authors: Bodily K , Wang Y , Roman R , Sostman A , Fitz J G
Published: July 2004

ABSTRACT

Liver cell volume and intracellular ion concentrations are maintained within a narrow physiologic range by regulated changes in membrane ion permeability. These studies of homozygous HTC hepatoma cells, a model liver cell line, evaluate the relationship between cell volume and membrane ion permeability, and assess the possibility that cell swelling allows the efflux of the intracellular osmolite taurine through the opening of a conductive pathway. Cell swelling induced by exposure to hypotonic solutions (203 mOsm) caused a rapid increase in cell volume, followed by recovery toward basal values. Volume recovery was inhibited by Cl- depletion or by exposure to the putative Cl- channel blocker 5–nitro–2–(3–phenylpropyl–amino) benzoic acid (NPPB) (25 μmol/L). Swelling increased the efflux rates of 36Cl (181% ± 15%,

P

< .01) and 125I (310% ± 21%,

P

< .01). In whole cell patch clamp recordings, cell swelling induced by 1) exposure to hypotonic solution or 2) intracellular perfusion with hypertonic sucrose–containing solutions activated an anion–selective current which was outwardly rectified and showed time–dependent inactivation at depolarizing potentials. The current density at –80 mV increased proportionally with increases in the transmembrane osmotic gradient from basal values of –1 pA/pF to maximal values of 70 pA/pF with 100 mmol/L sucrose in the pipette. Basal taurine permeability was low, but cell swelling increased the efflux of [1,2–3H]taurine to 1,587% ± 172% of basal levels (

P

< .05). Intracellular perfusion with hypertonic solutions activated currents carried by anionic taurine, with an estimated taurine/Cl- permeability ratio of .88 ± .17 for whole cell currents. These studies demonstrate that the HTC membrane anion permeability is closely coupled to changes in cell volume, and that the recovery from swelling depends upon activation of anion–selective conductance pathways permeable to both Cl- and taurine.
The Anti–Inflammatory Drug Sodium Salicylate Inhibits Nitric Oxide Formation Induced by Interleukin–1β At A Translational Step, But Not At A Transcriptional Step, in Hepatocytes
Authors: Sakitani K , Kitade H , Inoue K , Kamiyama Y , Nishizawa M , Okumura T , Ito S
Published: July 2004

ABSTRACT

Recent evidence suggests that nitric oxide (NO) mediates cellular injury under the pathological conditions such as endotoxemia in the liver of rats. Regulation of NO production is crucial for improving the hepatic dysfunction. We have previously reported that, in cultured rat hepatocytes, a single cytokine interleukin–1β (IL–1β) stimulated a release of nitrite, an oxidation product of NO, into culture medium dose– and time–dependently. The objective of this study was to investigate an ability of the anti–inflammatory drug NaSA to affect the production of NO in hepatocytes. IL–1β increased levels of inducible NO synthase (iNOS) messenger RNA (mRNA) with a maximal effect at 8 hours in primary cultures of rat hepatocytes. Nuclear factor–κB (NF–κB), that is an important nuclear factor protein in iNOS gene transcription in response to inflammatory mediators, also appeared in the nuclear fraction of hepatocytes 1 hour after addition of IL–1β. Sodium salicylate markedly inhibited the NO formation induced by IL–1β, but did not affect NF–κB activation and iNOS mRNA induction. Western blot analysis revealed that sodium salicylate (NaSA) blocked a step of iNOS protein synthesis. These findings indicate that NaSA may reduce hepatic injury by preventing the induction of NO formation in response to IL–1β at the posttranscriptional step.
Nicardipine As Antihypertensive Therapy in Liver Transplant Recipients: Results of Long–Term Use
Authors: Duvoux C , Cherqui D , Di Martino V , Métreau J , Salvat A , Lauzet J , Fagniez P , Dhumeaux D
Published: July 2004

ABSTRACT

Arterial hypertension is frequent in liver transplant recipients on cyclosporine A (CsA). Nicardipine is a calcium channel blocker (CCB) that has been shown to be efficient in controlling postoperative hypertension. However, its use has been limited in organ recipients because of its reported interaction with CsA metabolism. In this report, we studied the results of the long–term use of nicardipine after liver transplantation. Forty–nine consecutive liver transplant recipients with a follow–up longer than 2 years were studied. Immunosuppressive regimen was based on CsA and prednisone. Patients with immediate postoperative hypertension received intravenous nicardipine, secondarily switched to oral nicardipine (group 1, n = 27). Patients with delayed hypertension (i.e., >2 weeks posttransplant) received other antihypertensive drugs which did not interact with CsA metabolism. These patients and those without hypertension formed group 2 (n = 22). The two groups were similar for age, sex, body weight, and transplantation indications. Interaction of nicardipine with CsA metabolism was confirmed. Whereas cyclosporine blood levels were similar in both groups at any time during the study, the mean cyclosporine daily dose required to achieve such levels was 30% lower in group 1 compared with group 2 (

P

< .01). This resulted in a significant cost–containment. The use of nicardipine was not associated with an increased incidence of graft rejection or CsA toxicity episodes. The results in liver transplant recipients showed that nicardipine interacts with CsA metabolism, leading to a 30% reduction in CsA dose and does not increase the risk of CsA toxicity or graft rejection. Nicardipine can be used safely for the treatment of arterial hypertension after liver transplantation with a potential cost–containment.
Relation of Disease Activity During Chronic Hepatitis C Infection to Complexity of Hypervariable Region 1 Quasispecies
Authors: Yuki N , Hayashi N , Moribe T , Matsushita Y , Tabata T , Inoue T , Kanazawa Y , Ohkawa K , Kasahara A , Fusamoto H , Kamada T
Published: July 2004

ABSTRACT

We studied the heterogeneity in the E2/NS1 hypervariable region 1 of the hepatitis C virus (HCV) genome in relation to the natural course after infection. The subjects were composed of 38 chronic hepatitis C carriers who had been followed for 9 to 218 months after the onset of non–A, non–B (type C) hepatitis, being tested monthly for serum alanine aminotransferase levels. The complexity of the sequence heterogeneity was assessed by single–strand conformation polymorphism analysis. The quasispecies complexity had no relation to the route of infection, the time from infection and the duration of aminotransferase elevation after the onset. However, it had a significant relationship with the degree of aminotransferase elevation in the course of the disease. The quasispecies complexity was directly correlated with the first peak of serum aminotransferase at the onset (

r

= .48,

P

< .01) and the mean aminotransferase levels during the period of persistent aminotransferase elevation (

r

= .58,

P

< .01). Twenty–three of the 38 patients were further followed for 24 months with biweekly alanine transaminase (ALT) tests. Their aminotransferase levels remained within the normal range during follow–up, and no significant change was seen in the quasispecies complexity after this asymptomatic period. However among the 23 patients, the quasispecies complexity increased in six cases (26%) and decreased in five (22%). A significant direct relation was seen between changes in the quasispecies complexity and the mean aminotransferase levels during the asymptomatic period (

r

= .55,

P

= .01). These findings suggest that the development of the HCV quasispecies nature may be related to the severity of the hepatitis in the course of infection.
Detection of Type 2–Like T–Helper Cells in Hepatitis C Virus Infection: Implications for Hepatitis C Virus Chronicity
Authors: Tsai S , Liaw Y , Chen M , Huang C , Kuo G C
Published: July 2004

ABSTRACT

One striking clinical feature of hepatitis C virus (HCV) infection is that more than 50% of patients with acute hepatitis C will develop chronic infection. To investigate its possible mechanisms, we examined the activation of type 2–like T–helper (Th2–like) cells relating to the development of chronicity. Peripheral blood CD4+ T–cell proliferation and cytokine secretion in response to a panel of recombinant HCV antigens including core (C22), envelope 1 (E1), E2, nonstructural (NS) protein 4 (C100), fusion protein of NS3 and NS4 (C200), and NS5 were assayed in 17 patients with acute hepatitis C. All six patients with self–limited disease had a significant CD4+ T–cell proliferation to C22, E1, C100, C200, and NS5, running parallel with the antigen–stimulated secretion of interleukin (IL)–2 and interferon γ (IFN–γ), but not with interleukin (IL)–4 and IL–10, indicating predominant Th1 responses. Among the remaining 11 patients who developed chronicity, 6, 2, and 9 cases showed a specific CD4+ T–cell response to C22, C100, and C200, respectively, and the responses were significantly lower than those of cases with recovery in terms of stimulation index (SI) (

P

< .05) and of antigen–stimulated IL–2 and IFN–γ production. Importantly, IL–4 and IL–10 (Th2 responses) were detectable, and C22–specific Th2–like T–cell clones could be generated from patients with chronicity. The data suggested that activation of Th2 responses in acute hepatitis C patients may play a role in the development of chronicity.
Induction of Cytotoxic T–Cell Response Against Hepatitis C Virus Structural Antigens Using A Defective Recombinant Adenovirus
Authors: Bruña-Romero O , Lasarte J J , Wilkinson G , Grace K , Clarke B , Borrás-Cuesta F , Prieto J
Published: July 2004

ABSTRACT

A replication–defective recombinant adenovirus (RAd), RAdCMV–CE1, containing core and E1 genes of hepatitis C virus (HCV) was constructed. RAdCMV–CE1 was able to express core and E1 proteins both in mice and human cells. Immunization of BALB/c mice with RAdCMV–CE1 induced a specific cytotoxic T–cell response against the two HCV proteins. This response was characterized using a panel of 60 synthetic 14– or 15–mer overlapping peptides (10 amino–acid overlap) spanning the entire sequence of these proteins. Five main epitopes were found in the core protein, four of which had been previously described either in mice or humans. One single novel epitope was found in E1. Fine mapping of this E1 determinant, showed that octamer GHRMAWDM is the minimal epitope recognized by cytotoxic T lymphocytes (CTL). The cytotoxic T–cell response was

H–2

d restricted, lasted for at least 100 days, and was mediated by T cells with the classic CD4-CD8+ phenotype. This work demonstrates that replication–defective recombinant adenoviruses can efficiently express HCV proteins and are able to induce an

in vivo

cytotoxic T–cell response against a diversity of epitopes from HCV antigens. These vectors should be taken into consideration in the design of vaccines and also as a means to stimulate specific T–cell responses in chronic HCV carriers.
Acute Sporadic Non–A, Non–B, Non–C, Non–D, Non–E Hepatitis
Authors: Rochling F A , Jones W F , Chau K , DuCharme L , Mimms L T , Moore B , Scheffel J , Cuthbert J A , Thiele D L
Published: July 2004

ABSTRACT

Patients presenting with clinical and laboratory features consistent with a diagnosis of acute non–A, non–B hepatitis were evaluated for evidence of hepatitis C or hepatitis E infection and for evidence of severe or prolonged disease. Antibody to hepatitis C virus (anti–HCV) was detected in 75 of 108 (69%) patients, antibody to hepatitis E virus (anti–HEV) in three patients (3%), and neither antibody in 31 (29%) patients. One patient had both anti–HCV and anti–HEV. HCV RNA was not detected in sera from any of 20 patients with seronegative (non–ABCDE) hepatitis, but in all 10 patients with anti–HCV who were tested by polymerase chain reaction (PCR). Compared with patients with acute hepatitis C, those with non–ABCDE hepatitis had a lower incidence of parenteral risk factors (6% vs. 70%;

P

< .001), higher peak serum bilirubin levels (45% vs. 5% with peak levels > 15 mg/dL;

P

< .001), more prolonged jaundice (25% vs. 0% with peak bilirubin >5 weeks after onset;

P

< .01), more severe prothrombin time abnormalities (26% vs. 0% with >3 second prolongation;

P

< .001), more severe hypoalbuminemia (39% vs. 9% with albumin <3 g/dL;

P

< .01), and more frequent major clinical complications (13% vs. 0% with encephalopathy;

P

< .01; 10% vs. 0% with death or transplant;

P

= .024). Patients with acute non–ABCDE hepatitis were less likely to develop chronic hepatitis than those with acute hepatitis C (23% vs. 68%;

P

< .05). Thus, patients with acute non–ABCDE hepatitis are epidemiologically distinct from those with acute hepatitis C and have a significantly more severe acute illness.
The Hepatitis B Virus X Protein: the Quest for A Role in Viral Replication and Pathogenesis
Authors: Seeger C
Published: July 2004

ABSTRACT

Although the biological importance of hepatitis B virus X protein (HBX) in the life cycle of hepatitis B virus has been well established, the cellular and molecular basis of its function remains largely undefined. Despite the association of multiple activities with HBX, none of them appear to provide a unifying hypothesis regarding the true biological function of HBX. Identification and characterization of cellular targets of HBX remain an essential goal in the elucidation of the molecular mechanisms of HBX. Using the

Saccharomyces cerevisiae

two–hybrid system, we have identified and characterized a novel subunit of the proteasome complex (XAPC7) that interacts specifically with HBX. We also showed that HBX binds specifically to XAPC7

in vitro

. Mutagenesis studies have defined the domains of interaction to be critical for the function of HBX. Furthermore, overexpression of XAPC7 appeared to activate transcription by itself and antisense expression of XAPC7 was able to block transactivation by HBX. Therefore, the proteasome complex is possibly a functional target of HBX in cells.