Presence Of Hepatocytes Research Articles

A MODERATE FORM OF HYPERHOMOCYSTEINEMIA IS ACCOMPANIED BY POLYPLOIDIZATION OF HEPATOCYTES

The aim of research is to find out the effect of hyperhomocysteinemia on the regenerative process of liver cells. The histological structure of the liver was studied in Rattus norvegicus Berk white rats (n=25) (control group - 10 animals, comparison group with moderate hyperhomocysteinemia - 15 animals). On histological sections stained with hematoxylin and eosin, the following were calculated: the number of nuclei, the proportion of binuclear cells, the area and the diameter of the nucleus; the nuclear cytoplasmic index was calculated. With the use of immunohistochemical stain by antibodies to detect the expression of the Ki-67 marker (rabbit IgG, 1:200; Cell Marque Corporation, USA), the number and intensity of expression of Ki-67-positive cells in the field of view of the microscope were determined. In the liver of animals with moderate hyperhomocysteinemia, the presence of two processes was revealed at the same time. It is reactive-dystrophic (the presence of periportal leukocyte-lymphocyte infiltrates, the appearance of cells in a state of dystrophy and necrosis) and regenerative (an increase in the core area from 52,51±4,5 to 56,68±5,58 µm, nuclear-cytoplasmic ratio, an increase in the number and intensity of Ki-67+cells expression). The presence of hepatocytes with very large nuclei (polyploid), which make up 12,5 % of the entire population is characteristic of homocysteine-induced liver pathology. Hyperhomocysteinemia, along with a decrease in the number and dystrophy of individual hepatocytes, leads to an increase in the diameter and area of the cell nucleus, an increase in the intensity of proliferation, the appearance of polyploid nuclei, which increases the regenerative potential of the liver and provides a crucial role in the homeostasis of the gland. The findings require further research to determine the "critical point" of transition, which will allow modulation of liver tissue function.

Liver Involvement by Perforated Peptic Ulcer: A Systematic Review.

Background and Objective:Liver penetration by a confined perforation of peptic ulcer is a rare but severe event. Its clinical and pathological features are unclear.Methods:In total, 41 qualified English publications were identified using the PubMed database and one in-house case.Results:Among the 42 patients, 20 patients had liver involvement by a perforated duodenal ulcer and 22 by a gastric ulcer. Among the 23 cases of known ulcer histology, 2 ulcers were malignant and were adenocarcinomas in the gastric remnant and the remaining 21 ulcers were confirmed as histologically benign (for frequency of malignancy in duodenal versus gastric ulcers, p = 0.48). The presence of hepatocytes was the clue of diagnosis for 19 cases. The median ages of the patients were 64.5 years (95% Confidence Intervals [CI] 53.40–71.90) for duodenal ulcer and 65.5 years (95% CI: 59.23–70.95) for gastric ulcer, respectively. The male to female ratio was 1.5:1 for duodenal ulcers and 2:1 for gastric ulcers. Patients with liver involvement of a perforated gastric ulcer were more likely to have a larger ulcer (median largest dimension, 4.75 cm versus 2.5 cm, p = 0.014). Female patients with liver involvement of a gastric ulcer were older than male patients (median age 72 versus 60 years, p = 0.045). There were no differences in gender, region (Asia, Europe, America versus others), use of non-steroidal anti-inflammatory drugs (n = 15), H. Pylori positivity (n = 10), possible history of peptic ulcer disease (n = 19) or mortality (n = 32) between duodenal and gastric ulcers.Conclusions:Careful histologic examination, clinicopathological correlation, and immunohistochemistry are critical to establish the diagnosis and avoid misdiagnosing liver involvement as malignancy.

Avaluation of morphofunctional condition of rats organism for study of toxicity of the preparation tilmicosine basis

The article presents the study results of the acute and subacute toxicity of the veterinary drug “Tylmozyn 25” (solution for oral administration) based on tilmicosin. Intra-gastric administration of “Tylmozyn 25” to white mice at a dose of 25000 mg/kg of body weight caused the death of 100% of the animals, a dose of 15000 mg/kg of body weight caused the death of 66% of the white mice. The average time of death was 2 and 5 hours correspondingly. While determining the toxicity of “Tylmozyn 25” in white rats, we did not spot the death of any studied animal at any administered dose (5000, 15000, 25000 mg/kg of body weight). Based on the result of our study, we conclude that the veterinary drug Tylmozyn 25 belongs to the fourth of toxicity class – low toxic substances. LD50 of Tylmozyn 25 in white mice is 14167 mg/kg, while in white rats LD50 is higher than 25000 mg/kg. Testing on white rats intra-gastric drug “Tylmozyn 25” during for 14 days, both in therapeutic (80 mg/kg of body weight) and 10-fold doses (800 mg/kg of body weight) did not cause animal death, but caused a decrease in body weight, a significant decrease in the coefficients of weight of the liver and spleen and a tendency to increase the coefficients of weight of the heart and lungs compared with the animals of the control group. Animals which got the drug at a dose of 800 mg/kg of body weight showed erythrocytosis, leukopenia, increased enzymatic activity of AST, ALT, and LDH, the content of total protein against to decrease urea and creatinine, which may indicate impaired liver, kidney function and hematopoietic organs. The macroscopic and microscopic structure of the internal organs of the experimental rats is preserved. Rats receiving a tenfold therapeutic dose of the drug for 14 days, histologically revealed the most granular protein dystrophy in the liver and kidneys, which was manifested by discomplexation of the lamellae, presence of hepatocytes with uneven granular cytoplasm, slightly colored cytoplasm, hypertrophied nuclei, renal convoluted tubules and narrowing of their lumen, compaction of the mesh of the renal corpuscle. In the myocardium, the branching, swelling of the muscle fibers, swelling of the stroma with cell infiltrates, mainly of the lympho-histiocytic series, was observed, which indicated the development of serosa myocarditis. Structural changes in the liver, kidneys and heart were confirmed by biochemical parameters of the enzymatic activity of the serum of rats of this group.

Histostructure of the internal organs and skin of rats for the long-term effect of the drug “VetMikoDerm”

The investigation of the skin toxicity of the liniment “VetMikoDerm” for prolonged use was performed on young and healthy laboratory rats with intact skin, body weight 200–220 g. The investigated environment was applied to a clean, non-woolly area (dorsal/lateral surface) of at least 10% of the total surface area. The spotted thistle oil was applied to the pre-prepared area of the skin of the animals of the control group, and the other two experimental groups of animals got the studied drug at the doses of 50 (I) and 500 mg/kg (II), respectively. The drug was applied to the skin daily for 28 days. At the end of the experiment, after the decapitation of the rats conducted under the light etheric anesthesia, a complete pathoanatomical dissection was performed, the coefficients of the mass of the internal organs were determined, samples of liver, kidney and skin tissues were taken for their histological examination. According to the results it was established that the liver and kidneys histology, and skin of rats of the 1st experimental group were similar to those that were of the control animals. At the same time, receiving the maximum dose (10 times the therapeutic dose) of the drug “VetMikoDerm”, the majority of rats of the 2nd group showed a discomplication of the lamellar structure of the liver lobules, hepatocytes were placed in separate groups, sinusoidal capillaries were expanded. The presence of hepatocytes with heterogeneous, granular and weakly colored cytoplasm was observed in the central parts of the lobules, the nuclei of individual hepatocytes were increased, indicating the development of granular protein degeneration of the parenchyma. Histologically, under these conditions, foci of granular dystrophy of the epithelium of the vorticular and direct renal tubules with the expansion of their lumen were found in the structure of kidney of rats. The kernels of individual nephropyelitis had signs of karyopicnosis and cariorexis. The histological structure of skin of rats in all experimental groups was of the same type and consisted of the epidermis, dermis and hypodermis. Animals of the 2nd experimental group which repeatedly and for a long time were applied a “VetMikoDerm” lineulation in a dose that exceeded its optimal amount by 10 times showed the epidermal thickening, the presence of small-mesenchymal polymorphocytic infiltration, microcirculatory corneal disorder and sebaceous gland hyperplasia glands in the dermis that indicated the development of a compensatory-adaptive reaction in places of application of a 10-fold dose of the investigational medicinal product.

Characterization and spatial relationships of the hepatic vascular-biliary tracts, and their associated pancreocytes and macrophages, in the model fish guppy (Poecilia reticulata): A study of serial sections by light microscopy.

The guppy is a tropical fish that has been used as an experimental model organism in science. It is a species well adapted to the natural environment and that can support adverse environmental conditions, and so, at occasions, its presence can be indicative of environmental disturbances. Moreover, as the liver is very important when studying fish diseases, the knowledge of normal microanatomy is essential to assess histological changes, e.g., related to environmental change or toxic pollutants. The target organ of this histological study is the liver. The main objective is to contribute to the identification of anatomical and structural variations of this organ in different teleost species. We studied the distribution and spatial organization of the different types of blood vessels and biliary ducts and the relationships between them are established. For this, each liver was totally sectioned and the serial sections inspected in detail. The guppy liver presented intra-hepatic pancreatic tissue and so reported its association with the vascular and biliary elements. We observed that the input of afferent vessels (i.e., bringing blood into the liver) occur not only in the hilum but pierce and enter the organ at various points. Within the liver, venous vessels and bile ducts are seen, isolated or associated as venous-arteriolar tracts (VAT), and venous-biliary- arteriolar tracts (VBAT). Sometimes, pancreocytes appear within the liver surrounding isolated veins, forming venous tract with pancreatic acini (VT-P), or dual associations with afferent vessels, forming venous-arteriolar tracts with pancreatic acini (VAT-P). Intrahepatic pancreatic ducts were tiny and rare, putting in question the functional role of the acini. Contrary to other fish species, we did not spot isolated arterioles and associations between these and biliary ducts (BAT).We found aggregates of macrophages, namely associated with afferent and efferent (i.e., draining blood out) venous vessels; the latter fact not commonly reported in other fish species. There was a reduced arterialization of the organ (as arterioles were extremely rare), contrasting with an over predominance of a random distribution of the venous vascularization. The guppy differs to some extent from other previously studied models, highlighting the importance of making this kind of study to offer specific frameworks that can explain specific physiological processes or avoid misinterpretations; for instance about gene expression, as the whole liver specific expression will reflect the presence of hepatocytes and pancreocytes as well.

Histological changes of liver tissue and serobiochemical relation in does with pregnancy ketosis

<p class="abstrak2">Histological changes of liver in does with pregnancy ketosis were characterized. Twenty pregnant does at day 80 of pregnancy were used for this experiment. A total of 10 does were fed by grass (Napier) and goat concentrate with water ad libitum. Those 10 goats considered as healthy pregnant goat, and another 10 goats showing clinical and subclinical signs of ketosis considered as unhealthy pregnant does. Liver biopsies were performed when clinical signs appeared. Beta-Hydroxybutyrate (BHBA), free fatty acid (FFA), and glucose were dosed. Histological preparation revealed similar incidence and intensity of mild liver steatosis with lower cellular vacuolation in hepatocyte presence in healthy late pregnant does. Almost all of the pregnant does with ketosis state (n=8/10) had large amount of small lipid droplets in almost every hepatocyte over the whole liver acinus with higher number of cellular vacuolation, and related with higher BHBA and FFA levels while low in glucose level.</p>

Isolation of Shewanella putrefaciens from Goldfish (Carassius auratus auratus)

In the present study, the etiological agent that caused mortality in goldfish at an aquarium company located in Bursa in northwestern Turkey was identified. Diseased fish were lethargic, displaying a loss of appetite, ascites, and exophthalmia. Internal examination revealed that the kidney was surrounded by exudate and that the spleen and liver were pale. Microbiological examination showed that the kidney, spleen, and liver were infected by gramnegative, cytochrome oxidase, catalase, and H2S positive rod-shaped bacteria identified as Shewanella putrefaciens. Identification of the bacteria was confirmed by 16S rRNA gene sequencing. Histopathological examination of the kidney, spleen, and liver revealed necrosis in kidney tubules and the presence of hepatocytes and hemosiderin in melanomacrophages. According to the disk diffusion method, isolates were sensitive to enrofloxacin (5 µg), florfenicol (30 µg), gentamycin (120 µg), and sulfamehoxazole+ trimethoprim (25 µg).

Effects of two mesenchymal cell populations on hepatocytes and lymphocytes

The inflammatory response to liver injury plays an important role in the onset of liver fibrosis, which may ultimately lead to liver failure. The attenuation of inflammation and hepatocyte rescue are, therefore, of the utmost importance for recovery. Mesenchymal stromal cells (MSCs) from adult bone marrow have been shown to rescue hepatocyte function. Here we explore a more plentiful source of neonatal MSCs: human umbilical cord perivascular cells (HUCPVCs). We cocultured HUCPVCs or bone marrow-derived mesenchymal stromal cells (BM-MSCs) with rat hepatocytes or human peripheral blood mononuclear cells in order to identify their effects on hepatocyte functionality and the proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells (phaPBMCs). The expression of hepatotrophic factors by both types of MSCs in the presence of hepatocytes and the functional implications of blocking putative MSC anti-inflammatory factors were compared. Both types of MSCs improved albumin secretion, ureagenesis, hepatospecific gene expression, cytochrome P450 (CYP) activity, and functional hepatocyte mass maintenance. However, although HUCPVCs had an improved effect on the maintenance of ureagenesis, BM-MSCs had a strong effect on hepatocyte CYP activity. Additionally, each MSC type differentially expressed putative hepatotrophic factors, whereas phaPBMC proliferation was significantly decreased. Indoleamine 2,3-dioxygenase (IDO) was the main immunosuppressive mechanism used by both types of MSCs, but HUCPVCs exhibited higher expression of programmed death 1 ligands. However, the functional significance of the difference in anti-inflammatory factor expression still remains to be elucidated. Thus, both MSC types can serve as hepatocyte stromal cells and mitigate inflammation with IDO, but they present differences in the manner in which they affect hepatocytes and in the expression of both hepatotrophic and anti-inflammatory factors.

Presence of primary human hepatocytes inhibits expansion of virus-specific CD8+ T cells in vitro

Background: A hallmark of chronic infections with hepatotropic viruses such as HBV and HCV is the notoriously weak antiviral immune response by CD8 T cells. It is believed that the liver-specific environment can induce tolerance either by induction of dysfunctional CD8 T cells or by premature death of activated CD8 T cells. Recent reports from mouse models indicate that hepatocytes can act as antigen-presenting cells. Direct antigen presentation by virally infected hepatocytes thus might inhibit the CD8 T cell response. Methods: Primary hepatocytes were isolated from resected liver fragments of patients undergoing surgery and cultured in the presence of PBMCs with a memory CD8 T cell response to HLA-A2 restricted Flu- or CMV-epitopes. After 10 days the frequency of specific CD8 T cells and production of cytokines was determined. Results: The frequency of virus-specific CD8+ T cells after 10 days expansion is dramatically reduced in the presence of hepatocytes. 16h of co-culture of PBMCs with hepatocytes were sufficient for this suppressive effect. Expansion of CD8 T cells in the presence of hepatocytes was almost completely restored in transwell experiments indicating that the suppressive effect is cell-contact dependent. However, the effect was not dependent of antigen-specific contact between the virus-specific TCR on CD8+ T cells and the HLA/peptide complex on hepatocytes as the suppressive effect was observed in the presence of hepatocytes from both HLA-A2-positive and –negative donors. Analysis of the cytokine-profile from culture supernatants revealed 3-fold increased concentrations of TGFbeta in the presence of hepatocytes compared to their absence. Conclusions: Presence of primary human hepatocytes has a strong suppressive effect on expansion of virus-specific memory CD8 T cells from PBMC in vitro. The role of different cellular subsets and cytokines is currently investigated.

Regulation of osteopontin and related proteins in rat CC531 colorectal cancer cells

Colorectal cancer accounts for 11% of all cancers and is the second most frequent cause of cancer-related death, with the majority of deaths attributable to hepatic metastasis. The main aim of the study was to investigate changes which occur in CC531 rat colon adenocarcinoma cells and are instrumental to the metastatic phenotype after homing to the liver. RT-PCR and Western blotting were used to detect the expression of certain proteins, transcription factors and enzymes, which are intimately linked to colorectal metastasis. These included osteopontin (OPN), bone sialoprotein ll (BSP ll), Runx2, Hoxc8, matrix metalloprotease-7 (MMP-7) and matrix-metalloprotease-9 (MMP-9). Subsequently, in order to detect the role of the hepatocytes in these changes seen in tumor cells, two models of co-culturing hepatocytes with CC531 cells were established. OPN, Runx2 and MMP-7 were found to be highly expressed in CC531 metastases explanted from the liver, but showed subsequent down-regulation and/or disappearance in cell culture. The inverse regulation of Hoxc8, OPN and Runx2 suggests that these genes may be regulated in a feed-back loop manner. MMP-9 mRNA and active MMP-7 protein were expressed in tumor cells themselves. The presence of hepatocytes was insufficient to induce induction of OPN and Runx2 in tumor cells in vitro, so was the addition of OPN or TGF-beta1. Whereas TGF-beta1 induced over-expression of OPN and Runx2 in hepatocytes, it did not exert the same effect on hepatocytes co-cultured with CC531 cells, indicating that this response was abrogated by CC531 cells.

New approach to study of T cellular immunity development: Parallel investigation of lymphoid organ formation and changes in immune proteasome amount in rat early ontogenesis

The expression pattern and distribution of proteasome immune subunits LMP7 and LMP2 in the developing rat spleen and liver as well as the periarterial lymphoid sheath formation were investigated. LMP7 and LMP2 were detected by immunoblotting in the spleen on the 21st embryonic day and during the first postnatal days in equal amounts. Their levels increased by the 8th and 18th postnatal days. Double immunofluorescent labeling the spleen cells revealed LMP7 and LMP2 in T and B lymphocytes localized in the red pulp in embryogenesis. Few T lymphocytes were discovered in periarterial zones on the 8th postnatal day. T lymphocytes filled these zones and formed lymphoid sheaths by the 18–19th day. In the liver, LMP7 and LMP2 were revealed by the 17–19th postnatal day. Immunofluorescent analysis showed their presence in hepatocytes at this period. The data suggest that T cell-mediated immune response in relation to hepatocytes is possible beginning from 18th to 19th postnatal day.

In situ identification of dimethyl diselenide in hepatocytes treated with methylseleninic acid by membrane inlet mass spectrometry

The advantages and drawbacks of membrane inlet mass spectrometry (MIMS) for identification of volatile selenium compounds were investigated. Hepatocytes were incubated with methylseleninic acid (MeSeA) directly in the MIMS reaction cell and dimethyl diselenide (DMeDSe) was identified as the major volatile metabolite. This compound was also identified as the major reaction product after incubation of glutathione (GSH) with MeSeA without the presence of hepatocytes. One advantage of MIMS is the possibility of in situ monitoring of volatile metabolites with minimum risk of sample loss and without the need for trapping devices. Another advantage is the possibility of concurrent monitoring of cell viability by simultaneous detection of oxygen consumption and CO2 production. Although ideal for screening, the technique suffers from a relative high detection limit (in the 1 µM range) and lack of robustness. This paper presents for the first time in situMS data on the formation of DMeDSe.

Binding of plasminogen to hepatocytes isolated from injured mice liver and nonparenchymal cell-dependent proliferation of hepatocytes

Plasmin is an essential enzyme located in the pericellular microenvironment of liver cells during liver regeneration. Previously, we reported that liver regeneration ability was significantly increased in alpha2-antiplasmin gene knockout mice as compared to wild-type mice, but it was significantly decreased in plasminogen knockout mice, or Plg/alpha2-antiplasmin gene knockout mice. The present study aimed to demonstrate direct interaction between plasminogen and mouse hepatocytes in the process of liver regeneration. Using the isolated hepatocytes from mice we analyzed following subjects: binding capacity of plasminogen to hepatocytes, plasminogen activation in the presence of hepatocytes, and proliferation ability of hepatocytes cocultured with liver nonparenchymal cells. The isolated hepatocytes from plasminogen wild-type mice bound to immobilized plasminogen. The mouse hepatocytes enhanced plasminogen activation, and impaired the inhibitory effect of alpha2-antiplasmin. The proliferation ability of hepatocytes after liver injury was studied. In plasminogen wild-type and plasminogen knockout mice, the hepatocytes cocultured with nonparenchymal cells, which were obtained from mice without CCl4 injection, showed similar proliferation abilities. On the contrary, the proliferation ability of hepatocytes cocultured with nonparenchymal cells, which were obtained from CCl4-treated plasminogen knockout mice, was significantly impaired as compared to wild-type mice. These results indicate that the plasminogen-plasmin system on the surface of mouse hepatocytes plays an important role in liver regeneration.

Heparanase Enhances Early Hepatocyte Inclusion in the Recipient Liver after Transplantation in Partially Hepatectomized Rats

Hepatocyte transplantation is an emerging approach for the treatment of liver diseases. However, broad clinical application of this method has been limited by restricted source of cells and low efficiency of cell integration within the recipient liver. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, activity that affects cellular invasion associated with cancer metastasis and inflammation. This activity has a multifunctional effect on cell-cell interaction, cell adhesion, and angiogenesis. All these factors are important for successful integration of transplanted hepatocytes. Male donor hepatocytes pretreated with heparanase or untreated were transplanted into recipient female rat spleen following partial hepatectomy. Engraftment efficacy was evaluated by PCR for Y chromosome, histology and PCNA, and heparanase immunohistochemistry. In addition, proliferative activity of hepatocytes in vitro was determined by bromodeoxyuridine immunostaining. The number of heparanase-treated cells detected in the recipient liver was significantly increased three- to fivefold within 24-48 h posttransplantation and twofold at 14 days compared with untreated cells. The transplanted hepatocytes treated with heparanase were clearly seen inside portal vein radicles as cell aggregates up to 72 h posttransplantation. The number of portal radicles filled with heparanase-treated hepatocytes was increased compared to control early after transplantation. Heparanase treatment enhanced hepatocyte and sinusoidal endothelial cell proliferation in the liver, and hepatocyte proliferation within the spleen tissue. Preliminary in vitro studies with isolated hepatocytes treated with heparanase showed increased proliferative activity within 24-48 h of cell culture. These results suggest that preincubation of hepatocytes with heparanase increases the presence of hepatocytes within the recipient liver early following cell transplantation and stimulates both hepatocyte and sinusoidal endothelial cell proliferation.

Growth inhibitory actions of prothrombin on normal hepatocytes: Influence of matrix

Most hepatomas have a defect in prothrombin carboxylation, and can secrete under-carboxylated prothrombin or des-gamma-carboxy-prothrombin (DCP), the function of which is unknown. We considered that the prothrombin-DCP axis might also be involved in growth control. Hepatocytes and hepatoma cells were treated with prothrombin and DNA synthesis and cytoskeletal changes were studied. Prothrombin inhibited DNA synthesis in hepatocytes on fibronectin, but not collagen matrix. Hepatoma cell lines were not inhibited. We found that hepatoma cell matrix conferred resistance to hepatocytes. Prothrombin decreased fibronectin but not collagen amounts, but only in the presence of hepatocytes and not hepatoma cells, indicating that it has a differential action on matrix proteins. It also caused changes in cell shape and actin depolymerization. In vivo, there was a decrease in plasma prothrombin activity after a partial hepatectomy (PH), concomitant with the peak of DNA synthesis in the hepatocytes at 24h after PH. Injection of warfarin at the time of PH, further inhibited PT activity and enhanced this 24h peak of DNA synthesis. Furthermore, repeated injection of prothrombin lowered the peak DNA synthesis after PH. The data support the hypothesis that prothrombin can act as a hepatocyte growth inhibitor, likely at the level of fibronectin loss and result in cytoskeletal changes. Hepatomas resist this action, possibly due to their different matrix proteins. This represents a novel mechanism for growth regulation and provides a possible biological significance for the tumor marker DCP.

Nitric oxide inhibits T cell adhesion and migration by down-regulation of β1-integrin expression in immunologically liver-injured mice

Our previous study has reported that nitric oxide (NO) exerts a protective role in immunologically liver-injured mice induced by delayed-type hypersensitivity to picryl chloride. To explore the mechanism of the protection, we have now examined the effect of NO on T cell adhesion and migration. First, we isolated hepatocytes and nonparenchymal cells from the liver-injured mice and separated the nonparenchymal cells into Kupffer cell-enriched and lymphocyte-enriched populations. When these hepatocytes or the fractions of nonparenchymal cells were co-cultured with spleen T cells of the liver-injured mice in a Transwell system, the adhesive potential of the T cells was significantly inhibited in the presence of hepatocytes or the Kupffer cell-enriched population but not the lymphocyte-enriched population of nonparenchymal cells. This effect was dependent on NO production. The NO synthase inhibitor NG-monomethyl-l-arginine (L-NMMA) could reverse this inhibition of cell adhesion and also decrease NO production. To confirm this effect of NO on T cells, we further examined the role of exogenous or endogenous NO on the adhesive activity of the Jurkat T cell line. As a result, the NO donor, S-nitroso-N-acetyl penicillamine (SNAP) caused a dose-dependent inhibition of the adhesion of Jurkat T cells. Furthermore, the binding ability of Jurkat T cells to collagen decreased gradually after co-incubation with macrophages stimulated by LPS+IFN-γ, an effect which correlated well with the increasing NO level in the medium. Such opposite changes in cell adhesion and in NO production were also markedly reversed by L-NMMA. Moreover, treatment with SNAP reduced adhesion, transmigration, matrix metalloproteinase-9 production and β1-integrin expression of spleen T cells of the liver-injured mice. Taken together, these findings suggest that NO can function as a down-regulator of T cell mobility, which might be one of the mechanisms by which NO exerts its protective effect in T cell-mediated liver injury.

Functional Characterization of Serum-Free Cultured Rat Hepatocytes for Downstream Transplantation Applications

Although ex vivo culture of hepatocytes is known to impair functionality, it may still be considered as desirable to propagate or manipulate them in culture prior to transplantation into the host liver. The aim of this study was to clarify whether rat hepatocytes cultured over different periods of time proliferate and retain their hepatocyte-specific functions following transplantation into the recipient liver. Rat hepatocytes were cultured under serum-free conditions in the presence of hepatocyte and epidermal growth factors. Cells derived from wild-type donor livers were transplanted into the livers of CD26-deficient rats. Cell proliferation and the expression of hepatocyte-specific markers were determined before and after transplantation. Cell number increased threefold over a culture period of 10 days. The expression of connexin 32 and phosphoenolpyruvate carboxykinase declined over time, indicating the loss of hepatocyte-specific functions. Hepatocytes cultured over 4 or 7 days and then transplanted proliferated in the host parenchyma. The transplanted cells expressed connexin 32, cytokeratin 18, and phosphoenolpyruvate carboxykinase, indicating the differentiated phenotype. The loss of hepatocyte-specific functions during culture may be restored after transplantation, suggesting that the proper physiological environment is required to maintain the differentiated phenotype.

Sinusoidal endothelial cells as a target for acetaminophen toxicity: Direct action versus requirement for hepatocyte activation in different mouse strains

Hepatic congestion occurs early in acetaminophen poisoning. This study examines whether acetaminophen is toxic to sinusoidal endothelial cells (SEC), which might lead to microcirculatory disruption. Acetaminophen toxicity was examined in vivo and in vitro in SEC and hepatocytes from C3H-HEN and Swiss Webster mice. In both strains, there was significantly more toxicity to SEC than to hepatocytes; in SEC from C3H-HEN mice, acetaminophen was directly toxic, but the presence of hepatocytes was required for toxicity to Swiss SEC. Acetaminophen, 750 mg/kg, by gavage caused toxicity with variability within and between strains, but all animals died between 3.5 and 6 hr with zone 3 hemorrhagic necrosis. Pretreatment of C3H-HEN SEC with aminobenzotriazole, a suicide inhibitor of P450, abolished toxicity. Baseline glutathione (GSH) levels were comparable, but a 12-hr incubation with acetaminophen decreased GSH by 60 and 8%, respectively, in C3H-HEN and Swiss SEC in single cell type culture. In co-culture, under conditions where Swiss SEC viability declined by 73%, hepatocyte viability and GSH only decreased by 21 and 20%, respectively. In conclusion, acetaminophen was toxic to SEC. It was directly toxic to SEC in one mouse strain and required hepatocyte activation in another strain. The lack of direct toxicity to Swiss SEC may be due to the lack of an activating P450 isozyme. Zone 3 hemorrhagic necrosis in vivo was comparable in both strains, despite differences in the pathways leading to SEC toxicity in vitro. We propose that toxicity to SEC may contribute to hepatic congestion in acetaminophen intoxication.

Denitrification of the genotoxicant 2-nitropropane : relationship to its mechanism of toxicity

1. The stabilities of the industrial chemical and constituent of cigarette smoke 2- nitropropane (2-NP) and its aci tautomer propane 2-nitronate (P2N) towards hepatic enzymes and proteins such as serum proteins and oxyhaemoglobin were investigated in vitro in biological (hepatocytes and subcellular liver fractions) and model systems (serum proteins, oxyhaemoglobin, methylene blue). 2. Denitrification of 2-NP, P2N and 2-deutero 2-nitropropane (2H-NP) occurred in murine hepatocytes significantly faster than in rat cells.For 2-NP the rates were 1271 ± 167 versus 820 ± 125 pmol nitrite × min−1 × 106 cells−1. 3. A similar observation was made in microsomes, where 2-NP denitrification was 1460 ± 110 (mouse) versus 480 ± 80 pmol nitrite × min−1 × mg protein−1 (rat). 4. The major NO2−-forming activity was found to be localized in the microsomal fraction. 5. Conversionof 2-NP into P2N, either chemically or enzymatically,was a prerequisite for rapid denitrification. 6. Serum proteins and oxyhaemoglobin proved to be capable of denitrifying P2N (198 ± 24 pmol nitrite × min−1 × mg protein−1 and 7·1 ± 1·0 nmol nitrite × min−1 × nmol HbO2−1 respectively), but were much less active towards 2-NP (24 ± 2 pmol nitrite × min−1 × mg protein−1 and none respectively). 7. Methylene blue decomposed 2-NP and P2N at rates of 11 ± 3 and 192 ± 4 pmol nitrite × min−1 × nmol methylene blue−1 respectively. The dye also enhanced NO2− formation from P2N and 2-NP in the presence of hepatocytes or serum proteins, with a concomitant enhancement of both 2-NP and P2N toxicity. 8. The results presented report species differences in the denitrification rate of 2-NP and highlight the crucial nitro-aci tautomerism of 2-NP as a pivotal determinant of 2-NP toxicity.

Catalysis of nitro-aci tautomerism of the genotoxicant 2-nitropropane by cytosol from rodent and human liver

2-Nitropropane (2-NP) is a genotoxicant and hepatocarcinogen in rodents. Conversion to propane 2-nitronate (P2N), the anion of the tautomeric aci form of 2-NP, seems to be a pivotal part of the mechanism by which 2-NP causes its toxicity. We tested the hypothesis that the tautomeric equilibration is influenced by enzymes in the liver, the target organ of 2-NP toxicity. Rat or mouse hepatocytes were incubated for 4 h with 2-NP, P2N or the 2-NP isotopomer 2-deutero 2-nitropropane ( 2H-2-NP), which equilibrates with P2N much more slowly than 2-NP. Tautomers were analysed by HPLC. The rates of conversion of 2-NP to P2N expressed as nmol P2N × (10 6 cells/ml) −1 × min −1 were 4.0 and 4.2 in the presence of hepatocytes from rats or mice, respectively, and 2.6 in the absence of cells. Production of 2-NP from P2N expressed as nmol 2-NP × (10 6 cells/ml) −1 × min −1 was increased from 6.1 in the absence of cells to 11.9 or 9.9 in the presence of hepatocytes from rats or mice, respectively. The rate of formation of P2N from 2H-2-NP as compared to 2-NP was characterised by a primary isotope effect of 3.4 and 3.8 in hepatocytes from rats and mice, respectively, contrasting with a value of 9.6 measured in medium omitting cells. When 2-NP was incubated with subfractions of rodent or human liver homogenate, production of P2N by cytosol was between 7.3 (mouse liver) and 28.1 times (human liver) higher than that observed in microsomes. Similarly generation of 2-NP from P2N by cytosol exceeded that in microsomes by a factor of two. Tautomerism in heat-inactivated cytosol, mitochondria or microsomes was not different from that in buffer only. The results suggest that the nitro-aci tautomerism of secondary nitroalkanes is catalysed by a hepatic enzyme which resides predominantly in the cytosol and may thus contribute to the generation of the toxic species via which 2-NP exerts its toxicity.