Effects of female sex steroids on concanavalin A-mediated agglutination of hepatocytes from nonregenerating and regenerating rat liver and hepatic tumor marker enzymes

Effect of treatment of female rats with an oral contraceptive agent (OCA), Ovulen-50, for 7 weeks on agglutination of hepatocytes with concanavalin A (con A) and activities of certain tumor marker enzymes were examined to find out if OCA treatment is related to preneoplastic or neoplastic processes. Hepatocytes from regenerating and nonregenerating livers of control female rats showed negligible agglutination with Con A, whereas hepatocytes from non regenerating but not from the regenerating livers of female rats treated with a combination of 5 micrograms ethinyl estradiol and 100 micrograms ethynodiol diacetate showed agglutination. Of the tumor marker enzymes such as hepatic glucose 6-phosphatase, gamma-glutamyl transpeptidase (gamma-GT), and arginase examined in the liver, only gamma-glutamyl transpeptidase showed a significant increase in activity in the steroid-treated rats. Plasma alkaline phosphatase activity was also higher in the treated animals. However, the magnitude of the changes observed was relatively small and perhaps unrelated to the neoplastic process.     

Ontogeny of the enzymes related to γ-glutamyl cycle in the human fetal system

Measurements have been made of the enzymes associated with γ-glutamyl cycleviz, γ-glutamyl transpeptidase, γ-glutamyl cysteine synthetase, and 5-oxoprolinase in human fetal brain, liver and kidney over 12–36 weeks of gestation. γ-Glutamyl transpeptidase activity increases gradually with age. γ-Glutamyl cysteine synthetase and 5-oxoprolinase show biphasic pattern of development in human fetal brain. The data presented in this communication may indicate a relationship between γ-glutamyl cycle and amino acid transport.     

The Redox Status in Rats Treated with Flaxseed Oil and Lead-Induced Hepatotoxicity

Lead is a persistent environmental pollutant, and its toxicity continues to be a major health problem due to its interference with natural environment. In the present study, we have evaluated the effect of flaxseed oil on lead acetate-mediated hepatic oxidative stress and toxicity in rats. Lead acetate enhanced lipid peroxidation and nitric oxide production in both serum and liver with concomitant reduction in glutathione, catalase, superoxide dismutase, glutathione reductase, glutathione-S-transferase, and glutathione peroxidase activities, these findings were associated with DNA fragmentation. In addition, lead acetate caused liver injury as indicated by histopathological changed of the liver with an elevation in total bilirubin, serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transpeptidase, and alkaline phosphatase. Treatment of rats with flaxseed oil resulted in marked improvement in most of the studied parameters as well as histopathological features. On the basis of the above results it can hypothesized that flaxseed oil is a natural product can be protect against lead acetate-mediated hepatic cytotoxicity.     

Effect of phenobarbital and thyroxine on hepatocarcinogenesis initiated in mice by nitrosoethylurea and diethylnitrosamine

13-14-day old mice of ICR and CBA strains were given a single intraperitoneal injection of nitrosoethylurea (80 mg/kg) or diethylnitrosamine (50 mg/kg). 2 weeks later, they were given drinking water containing phenobarbital (1 g/L) or thyroxine (2 mg/L). The control mice were given only tap water. 29.4% of male and 42.1% of female ICR mice who had received nitrosoethylurea died of leukemia within 3-6 months after the carcinogen treatment. There was no case of leukemia in mice treated with diethylnitrosamine. Nitrosoethylurea induced 3-more often lung adenomas than diethylnitrosamine. Phenobarbital and thyroxine did not affect development of either leukemias or lung adenomas. By contrast, phenobarbital significantly elevated the number and size of hepatic lesions, whereas thyroxine markedly decreased them in all the experiments. The total and free thyroxine levels were significantly decreased in the blood of mice given phenobarbital and increased in mice given thyroxine. The data obtained indicate that thyroid hormones suppress tumor development in the mouse liver and that the promotion of hepatic tumoro-genesis by phenobarbital is presumably caused by the elimination of this suppressing effect of the thyroid hormones.     

Effect of lithium on hepatic drug-metabolizing enzymes of protein-deficient rats

Protein deficiency was produced by feeding synthetic 8%-protein diet. Lithium carbonate at the dose level of 1.1g/kg diet was administered to normal and protein-deficient rats for a period of one mo. A significant inhibition in the levels of cytochrome (cyt) P₄₅₀, cyt b₅, glutathione (GSH), glutathione S-transferase (GST) and glutathione peroxidase (GPx), but an increase in γ-glutamyl transpeptidase (γ-GT), was observed in low-protein LP-fed rats. Lithium treatment to normal rats caused no significant change in the activities of cyt P₄₅₀, cyt b₅, GST, and GSH levels, whereas there was elevation in the activities of γ-GT and GPx and suppression in glutathione reductase (GRd) activity. Lithium administration to LP-fed rats resulted in significant increases in the hepatic γ-GT and GPx activities.     

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.     

Decreased Hepatic 5-HT<Subscript>1A</Subscript> Receptors During Liver Regeneration and Neoplasia in Rats

In the present study we investigated the role of 5-hydroxytryptamine (5-HT) and 5-HT_1A receptor during liver regeneration after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in male Wistar rats. 5-HT content was significantly increased during liver regeneration after PH and NDEA induced hepatocellular carcinoma. Scatchard analysis using 8-OH-DPAT, a 5-HT_1A specific agonist showed a decreased receptor during liver regeneration after PH and NDEA induced hepatocellular carcinoma. 5-HT when added alone to primary hepatocyte culture did not increase DNA synthesis but was able to increase the EGF mediated DNA synthesis and inhibit the TGFβ1 mediated DNA synthesis suppression in vitro. This confirmed the co-mitogenic activity of 5-HT. 8-OH-DPAT at a concentration of 10⁻⁴ M inhibited the basal and EGF-mediated DNA synthesis in primary hepatocyte cultures. It also suppressed the TGFβ1-mediated DNA synthesis suppression. This clearly showed that activated 5-HT_1A receptor inhibited hepatocyte DNA synthesis. Our results suggest that decreased hepatic 5-HT_1A receptor function during hepatocyte regeneration and neoplasia has clinical significance in the control of cell proliferation.     

FORMATION OF γ-GLUTAMYLHISTAMINE FROM HISTAMINE IN RAT BRAIN

Abstract– γ-Glutamyl amides of histamine, serotonin and dopamine were formed from these amines by the transfer of the γ-glutamyl moiety from γ-glutamyl peptides in the presence of γ-glutamyl transpeptidase. [^I4C]Histamine was injected intraventricular into rats, and the formation of γ-glutamyl-[¹⁴C] histamine in the brain was confirmed by purification and identification with the authentic compound. The radioactivity was highest 30 min after the injection. The possible significance of γ-glutamyl amides in nerve transmission is discussed.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.     

Expression of hepatic calcium-binding protein regucalcin mRNA is decreased by phenobarbital administration in rats

The effect of phenobarbital on the expression of calcium-binding protein regucalcin mRNA in rat liver was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb of open reading frame). Phenobarbital (4, 8 and 12 mg/ 100 g body weight) was intraperitoneally administered to rats 3 times with 24 h intervals, and the animals were sacrificed by bleeding at 24 h after the last administration. The hepatic regucalcin mRNA levels were markedly reduced by phenobarbital administration. This decrease was about 50% of control level with the 12 mg/100 g dose. Moreover, the hepatic regucalcin concentration was significantly decreased by the administration of phenobarbital (12 mg/100 g), although the serum regucalcin concentration was not altered appreciably. Meanwhile, serum transaminases (GOT and GPT) activities were not increased by the administration of phenobarbital (4 and 12 mg/100 g). The present study demonstrates that the expression of hepatic regucalcin mRNA is decreased by phenobarbital administration in rats, suggesting that regucalcin does not have a role in drug metabolism related to phenobarbital.     

Rate of regeneration of two arms in the field and its effect on body components in Luidia clathrata (Echinodermata: Asteroidea)

The incidence of arm regeneration, effects of arm regeneration on non-regenerating body components and rate of arm regeneration were examined under field conditions. Approximately 60% of Luidia clathrata found in the upper portion of Tampa Bay, Florida were regenerating one or more arms. Individuals with two arms amputated at the disk edge took 380 days to regenerate completely. Both arms of an individual regenerated at the same rate. Length of regenerating arms initially increased faster than dry mass. No difference was found between non-regenerating and regenerating individuals for the dry mass of the body wall, pyloric caeca, and gonad of a non-regenerating arm. The absolute masses of the gonad and pyloric caeca were low through out the year possibly indicating a low food environment. Based on the rate of regeneration, arm loss just prior to the reproductive season may reduce the reproductive potential of this species.     

Gene cloning and protein expression of γ-glutamyltranspeptidases from <Emphasis Type="Italic">Thermus thermophilus</Emphasis> and <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis>: comparison of molecular and structural properties with mesophilic counterparts

γ-Glutamyltranspeptidase (γ-GT) is an ubiquitous enzyme that catalyzes the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. γ-GTs from extremophiles, bacteria adapted to live in hostile environments, were selected as model systems to study the molecular underpinnings of their adaptation to extreme conditions and to find out special properties of potential biotechnological interest. Here, we report the cloning, expression and purification of two members of γ-GT family from two different extremophilic species, Thermus thermophilus (TtGT) and Deinococcus radiodurans (DrGT); the first is an aerobic eubacterium, growing at high temperatures (50–82°C), the second is a polyextremophile, as it tolerates radiations, cold, dehydration, vacuum, and acid. TtGT and DrGT were both synthesized as precursor proteins of 59–60 kDa, undergoing an intramolecular auto-cleavage to yield two subunits of 40 and 19–20 kDa, respectively. However, like the γ-GT from Geobacillus thermodenitrificans, but differently from the other characterized bacterial and eukaryotic γ-GTs, the two new extremophilic enzymes displayed γ-glutamyl hydrolase, but not transpeptidase activity in the 37–50°C temperature range, pH 8.0. The comparison of sequences and structural models of these two proteins with experimental-determined structures of other known mesophilic γ-GTs suggests that the extremophilic members of this protein family have found a common strategy to adapt to different hostile environments. Moreover, a phylogenetic analysis suggests that γ-GTs displaying only γ-glutamyl hydrolase activity could represent the progenitors of the bacterial and eukaryotic counterparts.     

Four waves of hepatocyte proliferation linked with three waves of hepatic fat accumulation during partial hepatectomy-induced liver regeneration

Partial hepatectomy (PH) triggers hepatocyte proliferation-mediated liver repair and is widely used to study the mechanisms governing liver regeneration in mice. However, the dynamics of the hepatocyte proliferative response to PH remain unclear. We found that PH-induced mouse liver regrowth was driven by four consecutive waves of hepatocyte replication. The first wave exhibited the highest magnitude followed by two moderate waves and one minor wave. Underlying this continuous hepatocyte replication was persistent activation of cell cycle components throughout the period of liver regeneration. Hepatocyte mitotic activity in the first three proliferative cycles showed a circadian rhythm manifested by three corresponding mitosis peaks, which were always observed at Zeitgeber time 0. The Bmal1-Clock/Wee1/Cdc2 pathway has been proposed by others to govern the circadian rhythm of hepatocyte mitosis during liver regeneration. However, we did not observe the correlations in the expression or phosphorylation of these proteins in regenerating livers. Notably, Bmal1 protein displayed frequent changes in hepatic distribution and cellular localization as the liver regrowth progressed. Further, three waves of hepatic fat accumulation occurred during hepatic regeneration. The first started before and lasted through the first round of hepatocyte proliferation, whereas the second and third occurred concomitantly with the second and third mitotic peaks, respectively. CONCLUSION: PH-induced liver regeneration consists of four continuous waves of hepatocyte proliferation coupled with three waves of hepatic fat accumulation. Bmal1, Wee1, and Cdc2 may not form a pathway regulating the circadian rhythm of hepatocyte mitosis during liver regeneration.     

PARTIAL PURIFICATION AND KINETICS OF γ-GLUTAMYL TRANSPEPTIDASE FROM BOVINE CHOROID PLEXUS

Abstract— γ-Glutamyl transpeptidase from bovine choroid plexus has been shown to be a membrane-bound enzyme. Partial purification of the enzyme has been accomplished using detergent extraction and ammonium sulfate fractionation. Important determinants of enzymatic activity with acceptor substrates included chain length, stereoisomerism, and amino acid composition of the acceptors. L-Methionine was the best amino acid substrate and its corresponding peptides L-methionylmethionine and L-methionyl-L-serine were also good γ-glutamyl acceptors. L-Alanine and glycine were poor acceptor substrates; whereas, some peptides containing these amino acids were excellent substrates. Glycylglycine was significantly more effective as a γ-glutamyl acceptor than glycine, triglycine, or tetraglycine. L-Alanylglycine was a superior acceptor to glycine, L-alanine, or L-alanylglycylglycine, while the D-isomer of alanylglycine was only minimally effective as an acceptor substrate. In general glycyl peptides were the best acceptor substrates examined. Our findings that γ-glutamyl transpeptidase could catalyze the transfer of γ-glutamyl groups to glycylglycyl-L-alanine and L-alanylglycylglycine are of special interest, since few examples of tripeptide acceptors for the enzyme have been found. It is suggested that γ-glutamyl transpeptidase might play a role in the inactivation and/or transport of biologically active peptides.     

Alrp,a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Abstract

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of ‘Alrp homologous proteins’ in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

Intercellular communication in normal and regenerating rat liver: a quantitative analysis

We have compared intercellular communication in the regenerating and normal livers of weanling rats. The electrophysiological studies were conducted at the edge of the liver, and we have found that here as elsewhere in the liver there is a dramatic decrease in the number and size of gap junctions during regeneration. The area of hepatocyte membrane occupied by gap junctions is reduced 100-fold 29-35 h after hepatectomy. By combining observations made with the scanning electron microscope with our freeze fracture data we have estimated the number of "communicating interfaces" (areas of contact between hepatocytes that include at least one gap junction) formed by hepatocytes in normal and regenerating liver. In normal liver a hepatocyte forms gap junctions with every hepatocyte it contacts (approximately 6). In regenerating liver a hepatocyte forms detectable gap junctions with, on average, only one other hepatocyte. Intercellular spread of fluorescent dye and electric current is reduced in regenerating as compared with normal liver. The incidence of electric coupling is reduced from 100% of hepatocyte pairs tested in control liver to 92% in regenerating liver. Analysis of the spatial dependence of electronic potentials indicates a substantial increase in intercellular resistance in regenerating liver. A quantitative comparison of our morphological and physiological data is complicated by tortuous pattern of current flow and by inhomogeneities in the liver during regeneration. Nevertheless we believe that our results are consistent with the hypothesis that gap junctions are aggregates of channels between cell interiors.     

Alrp, a survival factor that controls the apoptotic process of regenerating liver after partial hepatectomy in rats

Augmenter of Liver Regeneration (Alrp) enhances, through unknown mechanism/s, hepatocyte proliferation only when administered to partially hepatectomized (PH) rats. Liver resection, besides stimulating hepatocyte proliferation, induces reactive oxygen species (ROS), triggering apoptosis. To clarify the role of Alrp in the process of liver regeneration, hepatocyte proliferation, apoptosis, ROS-induced parameters and morphological findings of regenerating liver were studied from PH rats Alrp-treated for 72 h after the surgery. The same parameters, evaluated on regenerating liver from albumin-treated PH rats, were used as control. The results demonstrated that Alrp administration induces the anti-apoptotic gene expression, inhibits hepatocyte apoptosis and reduces ROS-induced cell damage. These and similar data from in vitro studies and the presence of 'Alrp homologous proteins' in viruses as well as in mammals (i) allow to hypothesize that Alrp activity/ies may not be exclusive for regenerating liver and (ii) suggest the use of Alrp in the treatment of oxidative stress-related diseases.     

The first normal oral mucosa epithelium in which γ-glutamyl transpeptidase activity has been detected

Summary A portion of consistently -glutamyl transpeptidase-positive epithelium in the normal oral mucosa of rats is described. This is the first normal oral mucosa epithelium reported to express activity of the transpeptidase. This enzyme has been used as a marker of malignant transformation in tissues, such as epidermis and oral mucosa epithelium. Complementary studies of the enzyme-positive portion of oral mucosa and a neighbouring negative portion, suggest that, in this model, expression of -glutamyl transpeptidase is linked to a terminally-differentiated epithelium.     

Propolis protects CYP 2E1 enzymatic activity and oxidative stress induced by carbon tetrachloride

Induction of CYP 2E1 by carbon tetrachloride (CCl₄) is one of the central pathways by which CCl₄ generates oxidative stress in hepatocytes. Experimental liver injury was induced in rats by CCl₄ to determine toxicological actions on CYP 2E1 by microsomal drug metabolizing enzymes. In this report, ethanolic extract of propolis at a dose of 200 mg/kg (po) was used after 24 h of toxicant administration to validate its protective potential. Intraperitoneal injection of CCl₄ (1.5 ml/kg) induced hepatotoxicity after 24 h of its administration that was associated with elevated malonyldialdehyde (index of lipid peroxidation), lactate dehydrogenase and γ-glutamyl transpeptidase release (index of a cytotoxic effect). Hepatic microsomal drug metabolizing enzymes of CYP 2E1 showed sharp depletion as assessed by estimating aniline hydroxylase and amidopyrine N-demethylase activity after CCl₄ exposure. Toxic effect of CCl₄ was evident on CYP 2E1 activity by increased hexobarbitone induced sleep time and bromosulphalein retention. Propolis extract showed significant improvement in the activity of both enzymes and suppressed toxicant induced increase in sleep time and bromosulphalein retention. Choleretic activity of liver did not show any sign of toxicity after propolis treatment at a dose of 200 mg/kg (id). Histopathological evaluation of the liver revealed that propolis reduced the incidence of liver lesions including hepatocyte swelling and lymphocytic infiltrations induced by CCl₄. Electron microscopic observations also showed improvement in ultrastructure of liver and substantiated recovery in biochemical parameters. Protective activity of propolis at 200 mg/kg dose was statistically compared with positive control silymarin (50 mg/kg, po), a known hepatoprotective drug seems to be better in preventing hepatic CYP 2E1 activity deviated by CCl₄. These results lead us to speculate that propolis may play hepatoprotective role via improved CYP 2E1 activity and reduced oxidative stress in living system.