Hepatic Cell Injury Research Articles

Evaluation of purine nucleoside phosphorylase release as a measure of hepatic endothelial cell injury

With emerging data that endothelial cell (EC) injury is the limiting factor in liver preservation and hepatic function, a simple and reliable biochemical technique for monitoring EC injury is needed. Measurement of purine nucleoside phosphorylase (PNP) release into the circulation from perfused liver has been proposed as such a method. However, our experiments with perfused rat liver did not display a clear or direct relationship between PNP release and endothelial cell injury. Therefore, we re-examined the suitability of using PNP as a measure of nonparenchymal injury by measuring its distribution in purified populations of hepatocytes, ECs, and Kupffer cells (KCs) and correlating cell injury and enzyme release in short-term cultures at 37 degrees C of each cell type. Purified cells were incubated (4 x 10(6) cells/mL) in oxygen or nitrogen saturated. Wisconsin solution or Krebs buffer for 6 hours, with cell viability and PNP release assayed every 2 hours. ECs had the lowest specific activity (27 +/- 9 U/mg protein; mean +/- standard error of the mean [SEM]) compared with both hepatocytes (115 +/- 15) and KCs (66 +/- 18). Despite a decrement in EC and KC viability over time in each incubation solution, there was poor correlation between time of incubation and PNP release (r = .01 to .22), and between cell viability and PNP release (r = .01 to .16). In contrast, PNP release from incubated hepatocytes correlated with the length of incubation (r = .57 to .78) as well as cell injury (r = .63 to .77) in all four test solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

A Case Study on Cyclosporine A in a Patient with Acute Hepatitis.

The relationship between the pharmacokinetics of Cyclosporine A (CyA) and the hepatic function was studied in a 15-year-old woman with hepatitis. At the early stage of treatment, the values of glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and total serum bilirubin (t-Bil) were higher than the normal range, suggesting hepatic cell injury and impaired bile formation and/or duct. The blood concentration ratio of CyA metabolites (Met) to CyA was markedly decreased from 1.92 to 0.74 in accordance with the clinical improvement in liver function. In addition a significant positive correlation was demonstrated between the Met/ CyA ratio and GOT (r=0.621, p<0.05), GPT (r=0.678, p<0.02) and t-Bil (r=0.824, p<0.001), while no significant correlation were found in the concentration-to-dosage ratio of CyA and Met. These results suggest the Met/CyA ratio could be a useful indicator for evaluate the clinical response and/or hepatic function. We would like to report this as a typical case that presented on apparent correlation of Met/CyA with liver functions. These results also indicate that the Met level as well as the unchanged CyA level should be evaluated in the hepatitis patient.

Effects of paraquat on Mg2+-ATPase activity in rat liver

Intraperitoneal injection of paraquat (70 mg/kg) elicited a significant decrease of 20% in activity of Mg(2+)-ATPase in hepatic mitochondria, which is always surrounded by a high concentration of oxygen, in rats. The decrease of mitochondrial Mg(2+)-ATPase activity was completely abolished by pretreatment with vitamin E, which is a scavenger of oxygen radicals. On the other hand, paraquat administration did not change the Mg(2+)-ATPase activity in endoplasmic reticulum, which exists in an anaerobic condition in living cells. When liver microsomes were incubated with 1 mM paraquat under aerobic conditions, the Mg(2+)-ATPase activity was decreased by 42%. The decrease of Mg(2+)-ATPase activity was completely eliminated by pretreatment with vitamin E. Furthermore, lipid peroxidation in microsomes was tremendously increased by the addition of 1 mM paraquat under aerobic conditions. The increase of lipid peroxidation was completely abolished by preadministration of vitamin E in rats. The results suggest that the inhibition of Mg(2+)-ATPase activity induced by paraquat may be mediated by active oxygen, which is produced by the reaction of paraquat radicals; molecular oxygen may be involved in the induction of hepatic cell injury.

The relationship between LeY antigen and the therapeutic efficacy of interferon in chronic hepatitis C.

The expression of LeY antigen (Fuc alpha 1-->2Gal beta 1-->4 [Fuc alpha-->3] GlcNAc beta 1-->R), recognized by the monoclonal antibody BM-1, was studied in peripheral blood T-lymphocyte subpopulations in patients with viral hepatitis, and in liver tissue of patients with acute viral hepatitis. The relationship between the expression of LeY antigen on peripheral blood T-lymphocytes and the effects of interferon (IFN) therapy for chronic hepatitis type C were also evaluated. LeY antigen is not markedly expressed in B or T-lymphocytes of healthy individuals. However, it was strongly expressed in CD8 and CD4 T-lymphocytes in patients with viral hepatitis. In the acute phase of acute viral hepatitis, the expression of LeY antigen was more markedly expressed on peripheral CD8 T-lymphocytes than on CD4 T-lymphocytes. In chronic hepatitis type B and type C, it was significantly expressed more often on CD4 T-lymphocytes. In the liver tissues of patients with acute viral hepatitis, LeY antigen was expressed on hepatocytes and infiltrating lymphocytes. IFN therapy for chronic active hepatitis type C proved most effective when LeY antigen was more markedly expressed on the patient's CD4 and CD8 peripheral blood T-lymphocytes before treatment. Further studies are needed to clarify the relationship between the mechanisms of hepatic cell injury and LeY antigen.

Cadmium-induced hepatic endothelial cell injury in inbred strains of mice

Susceptibility to cadmium (Cd) hepatotoxicity differs among inbred strains of mice. For example, C3H/HeJ mice are sensitive to Cd-induced hepatotoxicity, whereas DBA/2J micre are resistant. The mechanism of genetic predisposition to Cd hepatotoxicity is unknown. A contemporary theory for acute target organ intoxication maintains that Cd initially damages vascular endothelium and parenchymal cell injury is a secondary event that results from localized ischemia. In the present study, the hypothesis that hepatic endothelial cells (EC) of C3H mice are more susceptible to Cd toxicity than those of DBA mice was tested. Hepatic parenchymal and endothelial cells were grown separately on monolayer cultures for 22 h and subsequently treated with various concentrations of Cd. Hepatocellular toxicity was assessed by lactate dehydrogenase leakeage and intracellular K + loss, whereas endothelial cell injury was assessed by trypan blue exclusion and the inhibition of protein synthesis. The susceptibility of hepatocytes to the cytotoxic effects of Cd was identical between strains. In contrast, the vulnerability of EC to Cd intoxication was strain-dependent. When exposed to 2.5–10.0 μM Cd, EC of Cd-sensitive mice were more susceptible to the cytotoxic effects of Cd than those of Cd-resistant mice. Basal metallothionein (MT) levels as well as Cd uptake into EC were similar in the two strains. Following Cd exposure, EC of Cd-sensitive mice accumulated similar amounts of MT as EC of Cd-resistant mice. These observations suggest that the microvasculature in livers of inbred mice is the target tissue responsible for strain-dependent susceptibility to Cd-induced liver injury. The mechanisms that account for this genetic variation in endothelial cell response to Cd are unknown, but do not appear to be related to the cellular disposition of Cd nor to a defect in the metabolism of MT.

Prevention of paracetamol-induced liver injury by fructose

Hepatic cell injury was studied in an in vitro system using rat liver slices incubated in two stages. During the first 2 hr slices were exposed to 10 mM paracetamol, this was absent during the subsequent 4hr of incubation. Cell damage was quantified at the end by measuring leakage of lactic dehydrogenase, increase in water content and potassium loss. Treatment of slices with 20 mM fructose in the second period of incubation prevented paracetamol-induced damage. The effect of fructose was not modified by the continued presence of paracetamol in the second incubation period. The inhibition of glycolysis either with 1 mM NaF or 10 μM iodoacetate blocked the effect of fructose. The protective effect afforded by fructose was not duplicated by the addition of lactate. All these findings strongly suggest an increase in intracellular ATP levels as the most probable explanation for the protective effect of fructose, and point to fructose as a potentially useful therapeutic tool for protection of the liver late in paracetamol intoxication.

The enhancement of liver injury by lipopolysaccharide in an experimental drug-induced allergic hepatitis model

When guinea pigs sensitized with trinitrophenylated (TNPed) liver protein 1 (LP1) were intravenously injected with TNPed isolated hepatocytes, remarkable hepatic cell injury was induced 24 hours later. In this experimental model, drug-induced allergic liver injury was induced by using trinitrobenzen sulfonic acid (TNBS) as the hapten and LP1 as the carrier. When these sensitized guinea pigs were intravenously injected with 10 micrograms of lipopolysaccharide (LPS) along with TNPed isolated hepatocytes, serum AST and ALT levels were remarkably higher than those of the guinea pigs not injected with LPS. Hepatic cell necrosis was also more extensive, and some bleeding was observed. Although none of the guinea pigs died even after 24 hours, when 50 micrograms of LPS was injected, many of the guinea pigs started to die and the survival rate was 5% at 24 hours. These results suggested that LPS enhanced liver injury in this experimental drug-induced allergic liver injury model.

The protective effects of Sho-saiko-to in an experimental, immunologically-induced allergic hepatic cell injury.

The protective effects of Sho-saiko-to in an experimental, immunologically-induced allergic hepatic cell injury.

水素ガスクリアランス法による肝組織血流量測定 実験的ならびに臨床的研究

In this study, the author intended to examine the validity of the inhaled hydrogen gas clearance method (i-H2) for determination of the hepatic blood flow (HBF), and also to show some applicabilities of the method in experimental animals and patients with liver diseases. Simultaneous determinations of HBF by i-H2 and electromagnetic flowmetry in rabbits revealed an excellent correlation between the values obtained by the two methods. Moreover, HBF in rabbits measured by i-H2 varied in parallel with that by thermocouple flowmetry or laser Doppler velocimetry after administration of norepinephrine, propranolol or glucagon. In carbon tetrachloride-treated rats, HBF measured by i-H2 correlated better with the severity of damage in the sinusoidal structure than the severity of hepatic cell injury or the serum levels of transaminases. HBF as determined by i-H2 was significantly decreased in acute hepatitis (AH), chronic inactive hepatitis (CIH), chronic active hepatitis (CAH), liver cirrhosis (LC) and fatty liver. Reduced HBF in AH returned to normal during recovery of the disease. The ratio of HBF in tumor/normal tissue was greater than 1.0 for hepatocellular carcinoma in contrast to the ratio of less than 1.0 for metastatic liver carcinoma. Propranolol caused a decrease in HBF by 31%, and vasopressin by 39% in patients with CIH or LC. In contrast, glucagon induced its increase by 65%, 35% and 17%, respectively, in patients with CIH, AH and LC.

Multiple tissues express alpha 1-antitrypsin in transgenic mice and man.

Hepatocytes are considered to be the predominant source of alpha 1-antitrypsin (AAT), the major antiprotease in human plasma. The development of emphysema in the hereditary PiZ AAT deficiency state suggests that inhibition of leukocyte elastase in the lung is a major function of this protein. In addition, patients with AAT deficiency are at increased risk for developing cholestasis in infancy and chronic liver disease as adults. The mechanism for hepatic cell injury, however, is not understood. Transgenic mice that express the normal human AAT gene demonstrate abundant AAT in hepatocytes and specific cell types of numerous nonhepatic tissues. Immunoperoxidase techniques have previously disclosed AAT in many of the cell types seen in transgenic mice; however, the issue of local synthesis vs. endocytosis in these cell types has remained unresolved. In this study, AAT mRNA was seen in a variety of tissues in the transgenic mouse. Immunoelectron microscopy of renal tubular and small intestinal epithelial cells in the transgenic mice demonstrated AAT within the cisternae of the rough endoplasmic reticulum, as in hepatocytes. These findings support the possibility of local synthesis in the various cell types. The results suggest that in addition to maintaining tissue integrity in the lung, the protease/antiprotease balance may have physiological functions in other organs as well.

Hepatocyte injury during post-operative sepsis: activated neutrophils as potential mediators.

We evaluated the concept that the vascular entrance of both bacterial and nonbacterial particulate material could lead to hepatic parenchymal cell injury, either due to postphagocytic Kupffer cell activity or the margination of activated leukocytes in the liver. Injection of denatured, collagen-coated particles as well as heat-killed bacteria were used as particulate challenges. Hepatic parenchymal cell injury in vivo during postoperative sepsis was evaluated by plasma aspartate aminotransferase (AST) and ornithine carbamyl transferase (OCT) enzyme levels over 3-72 h. AST and OCT levels elevated following either laparotomy plus cecal ligation (mild sepsis) or laparotomy plus cecal ligation with puncture (severe sepsis), with the peak level at 24 h. In addition, the direct intravenous injection of either nonbacterial foreign particles or heat-killed Pseudomonas aeruginosa into normal rats also produced a dose-dependent elevation of AST and OCT. The plasma level of either AST or OCT actually increased 350-400% after injection of the non-bacterial particles. A similar dose related elevation in enzymes followed the intravenous injection of heat-killed Pseudomonas. To differentiate the potential contribution of activated hepatic Kupffer cells versus activated marginated neutrophils to the in vivo hepatic injury, we determined the release of the hepatic specific enzyme OCT by cultured hepatic parenchymal cells when they were exposed to isolated Kupffer cells or isolated PMNs that were activated by exposure to dead bacteria. Bacteria alone when added to cultured hepatocytes did not induce significant OCT release. In contrast, activated PMNs but not Kupffer cells induced a significant (p less than 0.05) release of OCT from parenchymal cells into the culture media. Thus, in vivo transient hepatic parenchymal cell injury with post-operative sepsis may be mediated by the margination of activated PMNs in the liver.

Lindane-Induced Oxidative Stress. I. Time Course of Changes in Hepatic Microsomal Parameters, Antioxidant Enzymes, Lipid Peroxidative Indices and Morphological Characteristics

1. Lindane (60 mg/kg) administered orally to rats increased liver cytochrome P-450 content and superoxide radical (O2-.) generation 24 h after treatment, while formation of thiobarbituric acid reactants and NADPH/ADP-supported microsomal chemiluminescence were significantly increased 4 h after treatment. 2. Hepatic superoxide dismutase (SOD) and catalase decreased 6 h after lindane treatment and SOD/O2-. ratio progressively decreased during 4 to 24 h after lindane treatment. 3. Morphological evidence of hepatic cell injury after lindane treatment was seen at all times studied, and appeared to increase with time. 4. Lindane administration results in time-dependent oxidative stress in liver which involves an early component (4-6 h) related to the reductive metabolism of lindane, and a late component (24 h) associated with the induction of cytochrome P-450; the biochemical changes correlated with the observed morphological lesions.

Effects of dietary fat on alcohol-pyrazole hepatitis in rats: the pathogenetic role of the nonalcohol dehydrogenase pathway in alcohol-induced hepatic cell injury.

Rats were fed with two different alcohol-containing (36% of total calories) liquid diets of high fat and low fat (35% and 15% of total calories) with or without 2 mM of pyrazole for 12 weeks. At the 12th week, the serum glutamic oxaloacetic transaminase level was significantly elevated in the alcohol-pyrazole high fat group, but not in the low fat group. Ballooning and necrotic changes of the hepatocytes in the centrolobular area were more prominent in the alcohol-pyrazole high fat group than in the low fat group and alcohol alone groups, indicating that high fat diet accelerates the development of alcohol-pyrazole hepatitis. In the alcohol-pyrazole high fat group, a decrease of hepatic microtubules content and an accumulation of hepatic export proteins in the hepatocytes were found. The protein accumulation was prominent only in the ballooned hepatocytes. Hepatic acetaldehyde levels were significantly higher in the alcohol-pyrazole high fat group than in the alcohol-pyrazole low fat group. These results suggest that the accelerated ethanol metabolism in the nonalcohol dehydrogenase pathway by a high fat diet may play an important role in the development of hepatocytic injuries, by impairing the microtubular function of the hepatocytes.

The phosphorylation of liver ribosomal protein S6 during the development of acute hepatic cell injury induced by D-galactosamine

The administration of D-galactosamine-HCl (GalN) to rats initiates a sequence of metabolic events in the liver leading to a morphological and biochemical hepatitis-like liver injury [l] . The formation of UDPgalactosamine metabolites plays a pathogenetic key role since they reduce strongly the hepatocellular pool of uridine phosphates and UDP-sugars by a trap mechanism [2]. As a consequence, macromolecular synthesis, e.g., of RNA [3] and protein [4-91, is inhibited. It is presently unknown whether the impairment of protein biosynthesis is due to a direct effect on translation or to a secondary response to inhibition of RNA synthesis [2]. A direct influence on the translation machinery is suggested by experiments which indicate that the inhibition of protein synthesis by GalN can be reversed by uridine in the presence of actinomycin D [9]. However from present knowledge on the molecular mechanism of polypeptide synthesis, in particular on the role of uridine nucleotides, a specific action of UTP deficiency on translation is hardly to explain. Therefore it is conceivable that the effect of GalN on cellular protein synthesis is a direct one but mediated by a common, nonspecific pathway. Postsynthetic modifications of ribosomal proteins have been suggested as a regulatory mechanism of protein biosynthesis [lo] . Recently presented evidence

False-Positive Infectious Mononucleosis Spot Test in Pancreatic Carcinoma

Two patients with pancreatic carcinoma were found to have positive differential spot tests for mononucleosis (Monospot tests) early in the course of their disease. The Monospot test is considered highly specific for infectious mononucleosis with false-positive reactions reported only in acute leukemia, lymphoma, and infectious hepatitis. Liver biopsies in both patients showed no hepatic cell injury or mononuclear infiltrate, thus excluding the possibility of coexisting infectious hepatitis.

The effect of ethanol and carbon tetrachloride administration on hepatic lipid-soluble antioxidant activity

The influence of ethanol or carbon tetrachloride administration on lipid soluble antioxidant activity of liver as well as mitochondria and microsomes was studied in a further evaluation of the hypothesis of the lipid peroxidation concept of ethanol-induced hepatic cell injury. While total liver lipid soluble antioxidant activity was not significantly modified in either ethanol or carbon tetrachloride-treated groups, the mitochondrial, but not microsomal, antioxidant concentration was significantly reduced 2 hours after the administration of ethanol. In contrast, microsomal lipid soluble antioxidant activity was significantly decreased in carbon tetrachloride-treated rats, while mitochondrial antioxidant activity remained unaltered. The selective changes in lipid soluble antioxidant activity correlate well with the subcellular sites of ethanol- and carbon tetrachloride-induced lesions and with previous demonstrations of lipoperoxidation. The intraperitoneal administration of the lipid soluble antioxidant DPPD, produced a three- to four-fold increase in total liver lipid antioxidant levels, as well as a comparable increase in brain tissue. The mitochondrial antioxidant levels were slightly increased while a profound enhancement occurred in the microsomal fraction in the DPPD-treated groups. The elevated mitochondrial and microsomal antioxidant levels in the DPPD-treated group were not significantly altered by ethanol or carbon tetrachloride administration. In an effort to establish the usefulness of antioxidant loss as an index of the peroxidation process, a comparative study was made employing the thiobarbituric acid procedure, i.e., malonaldehyde formation, conjugated diene analysis, and lipid soluble antioxidant activity determinations. The model was normal rat liver homogenates incubated either in the presence of air, nitrogen, oxygen, or in the presence of lipoxidase. Measurements were made at 0, 30, 60, 120 minutes of incubation. Malonaldehyde formation occurred in the following order, nitrogen < air < oxygen < lipoxidase. In all instances, the decrease in lipid soluble antioxidant concentration preceded the formation of malonaldehyde. In profound contrast to enhancement in malonaldehyde formation, or to the decrease in lipid-soluble antioxidant activity, the presence of conjugated dienes could not be detected in the liver homogenates incubated in nitrogen, air or oxygen atmospheres and only occurred in the presence of lipoxidase, denoting a relative degree of insensitivity and a possible limitation of the use of conjugated diene analysis to detect the peroxidation process. The present study further contributes to the concept that lipid antioxidants, such as DPPD exert their protective effect by their antioxidant activity exerted against an induced free radical attack at specific subcellular sites. While it remains to be established that the observed decline in antioxidant concentrations is sufficient to induce the mitochondrial or microsomal functional and ultrastructural aberrations which characterize acute ethanol or carbon tetrachloride administration, the present studies do extend the concept of lipid peroxidation in the pathogenesis of certain types of chemical induced hepatic injury and the possible role of antioxidants in the inhibition of injury.

Antioxidant maintenance of hepatic triglyceride secretory activity and hepatic function in carbon tetrachloride poisoned rats.

SummaryPrior administration of the lipid antioxidant, DPPD, to rats which subsequently received CC14 orally prevented the CCU-induced depression of plasma triglyc-eride concentration and maintained a normal hepatic release of triglyceride into the plasma compartment, as assessed by the post-Triton hypertriglyceridemic response. Administration of DPPD also prevented the CCl4-induced impairment in the hepatic removal of BSP. These findings, coupled with prior observation of inhibition of CCU-induced hepatic fatty infiltration, hepatic necrosis, and lethality, indicate that antioxidants can modify the degree and nature of hepatic cell injury resulting from exposure to certain toxic substances.

EFFECT OF THIOACETAMIDE-INDUCED SUBLETHAL HEPATIC CELL INJURY ON PROTEIN SYNTHESIS.

SummaryElectron microscopic and radiochemical studies were performed in mild sublethal hepatic cell injury (cloudy swelling) produced by small peroral doses of thioacetamide. Azurophilia, ribosomes and polyribosomes decreased or disappeared in injured cells at 5 1/2 hours. At 48 hours the cells in the outer portion of the lobule contained a marked increase in ribosomes and polyribosomes, while centrally located cells continued to show evidence of the earlier injury. At 5 1/2 hours after thioacetamide there was a decreased uptake of the label from administered C14-L-leucine in serum albumin and in total hepatic protein while it was increased in the hepatic RNA-protein. These findings were interpreted to indicate that incorporation of amino acid into hepatic RNA-protein is not measurably impaired by mild injury due to thioacetamide, but that synthesis or secretion of serum albumin is impaired. At 48 hours, amino acid incorporation in total hepatic protein was decreased in thioacetamide treated animals, but ...