Salomon L. Abrahamse

Evaluation of rat liver apoptotic and necrotic cell death after cold storage using Uw, Htk, and celsior

Background. The benefit of Celsior in liver graft preservation is controversial. In the isolated perfused rat liver model, we compared the effects of Celsior, University of Wisconsin (UW), and histidine-tryptophan-ketoglutarate (HTK) preservation solutions on liver cell death. Methods. Rat livers were stored at 4°C for 0, 8, 16, or 24 hr in either Celsior, UW, or HTK and reperfused for 90 min (37°C). Bile secretion and perfusate levels of liver enzymes and histone-associated DNA fragments were measured. Apoptosis and oncotic necrosis were analyzed in biopsies by DNA gel electrophoresis, hematoxylin and eosin histology, and enzyme histochemistry for lactate dehydrogenase (LDH) and 5′-nucleotidase (5′-NT). Results. Perfusate flow rate through the liver during perfusion did not significantly differ among preservation solutions. Bile secretion was best preserved in UW livers after 16-hr (versus HTK livers) and 24-hr storage (versus HTK and Celsior livers). Enzyme leakage from UW livers was lower compared with HTK livers after 8-hr storage (serum glutamic oxaloacetic transaminase [SGOT], LDH) and with Celsior and HTK livers after 16-hr (SGOT, LDH) and 24-hr storage (SGOT, serum glutamic pyruvic transaminase, LDH, purine nucleoside phosphorylase). In situ LDH and 5′-NT activities were best preserved in UW livers (up to 24 hr), whereas enzyme activities declined remarkably in HTK livers (after 8 hr) and Celsior livers (after 16 hr of cold storage). Although perfusate DNA fragment levels were repeatedly lowest from Celsior livers, apoptotic DNA laddering and the number of fragmented nuclei in hematoxylin and eosin sections was not different among livers after 8, 16, or 24 hr of storage. Conclusions. Celsior and UW are equally effective in preventing rat liver cell death after 0–16 hr of cold preservation as compared with the less effective HTK solution. After 24-hr cold storage, rat livers were best preserved in UW. Furthermore, there was no significant difference in mode of cell death (apoptosis or oncotic necrosis) after storage in any of the three solutions.

Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW, HTK, and Celsior solutions.

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4°C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not enhanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.

Treatment of acute liver failure in pigs reduces hepatocyte function in a bioartificial liver support system.

Several different types of bioartificial liver (BAL) support systems have been developed to bridge patients suffering from acute liver failure (ALF) to transplantation or liver regeneration. In this study we assessed the effects of ALF plasma on hepatocyte function in the BAL system that has been developed in our center. Pigs (40–60 kg) were anaesthetised and a total hepatectomy was performed. Cells were isolated from the resected livers and were transferred to the bioreactor of the BAL system. Twenty hours after cell isolation, hepatocytes in the BAL were tested for cell viability and functional activity by using a recirculating test medium in which assessment of LDH leakage, ammonia clearance, urea synthesis, 7-ethoxycoumarin O-deethylase (ECOD) activity and pseudocholine esterase production was performed. Subsequently, two groups were studied. In one group (I, n=5), the cell-loaded bioreactor was used to treat the donor pig, rendered anhepatic, for 24 hours. In the second group (II, n=5) the bioreactor was cultured for 24 h and served as a control. After 24 hours treatment or culturing, the cell viability count and functional activity tests were repeated. The results show that hepatocytes in the BAL remained viable after 24 h treatment of anhepatic pigs, as shown by the LDH release and pseudocholine esterase production. However, metabolic functions such as ammonia clearance, ECOD and urea synthesis were reduced after 24 h exposure of hepatocytes to autologous ALF plasma, whereas these functions were unaltered after 24 h culturing of the cells in the bioreactor.

Enhancement of ovalbumin-induced antibody production and mucosal mast cell response by mercury.

Food contaminants may contribute to the recent increased incidence of food allergies. We have investigated this hypothesis experimentally. It was our objective to determine whether toxicity to the intestinal tissue by orally applied mercury (Hg) could modulate the immune response to food allergens. Effective mechanisms were studied with functional immunological and toxicological parameters. Brown Norway rats were immunized intraperitoneally by ovalbumin (OVA). Before oral challenge with OVA, immunized and non-immunized animals were exposed to HgCl2. Immunological responses were measured by enzyme-linked immunosorbent assays [anti-OVA-IgE and-IgG, rat mast cell protease II (RMCPII), interferon-gamma, interleukin-4, lymphocyte proliferation] and by flow cytometry (lymphocyte subpopulations). Toxicity of Hg to the intestinal barrier was determined by measuring viability, DNA damage and induction of glutathione S-transferase in isolated intestinal epithelial cells and lymph node cells, and by measuring permeability, short-circuit current and tissue conductance of the intact intestinal epithelium. A single high oral dose of HgCl2 enhanced the serum concentrations of anti-OVA-IgE and IgG (P < 0.05) and of RMCPII (P < 0.05) in immunized rats. The treatment resulted in a higher number of CD4/CD25+ T cells in the lymph nodes (P < 0.05). The multiple application of low HgCl2 doses (5 x 0.2 mg/kg body weight) only resulted in an elevated RMCPII serum concentration (P < 0.05). Neither treatment schedules impaired proliferation and cytokine production of lymphocytes. In non-immunized rats only minor immunological changes were observed. Oral HgCl2 induced genotoxic damage in lymph node cells and in jejunal epithelial cells (P < 0.05). Moreover, HgCl2 increased the permeability of intestinal epithelial tissue and of Caco-2 monolayers and was genotoxic and cytotoxic to isolated intestinal epithelial cells in vitro. In conclusion, these studies indicate that the food contaminant Hg can stimulate the immune response to OVA in immunized rats. One possible mechanism could be the toxicity of Hg to the intestinal epithelial and the lymph node cells. Whether humans with allergies respond to high oral doses of Hg in a similar way needs to be investigated in further studies.

Application of confocal laser scanning microscopy to detect oxidative stress in human colon cells.

Introduction Excess of intracellular reactive oxygen species in relation to antioxidative systems results in an oxidative environment which may modulate gene expression or damage cellular molecules. These events are expected to greatly contribute to processes of carcinogenesis. Only few studies are available on the oxidative/reductive conditions in the colon, an important tumour target tissue. It was the objective of this work to further develop methods to assess intracellular oxidative stress within human colon cells as a tool to study such associations in nutritional toxicology. Methods We have measured H2O2-induced oxidative stress in different colon cell lines, in freshly isolated human colon crypts, and, for comparative purposes, in NIH3T3 mouse embryo fibroblasts. Detection was performed by loading the cells with the fluorigenic peroxide-sensitive dye 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (diacetoxymethyl ester), followed by in vitro treatment with H2O2 and fluorescence detection with confocal laser scanning microscopy (CLSM). Using the microgel electrophoresis (“Comet”) Assay, we also examined HT29 stem and clone 19A cells and freshly isolated primary colon cells for their relative sensitivity toward H2O2-induced DNA damage and for steady-state levels of endogenous oxidative DNA damage. Results A dose-response relationship was found for the H2O2-induced dye decomposition in NIH3T3 cells (7.8–125 μM H2O2) whereas no effect occurred in the human colon tumour cell lines HT29 stem and HT29 clone 19A (62–1000 μM H2O2). Fluorescence was significantly increased at 62 μM H2O2 in the human colon adenocarcinoma cell line Caco-2. In isolated human colon crypts, the lower crypt cells (targets of colon cancer) were more sensitive towards H2O2 than the more differentiated upper crypt cells. In contrast to the CLSM results, oxidative DNA damage was detected in both cell lines using the Comet Assay. Endogenous oxidative DNA damage was highest in HT29 clone 19A, followed by the primary colon cells and HT29 stem cells. Conclusions Oxidative stress in colon cells leads to damage of macromolecules which is sensitively detected in the Comet Assay. The lacking response of the CLSM-approach in colon tumour cells is probably due to intrinsic modes of protective activities of these cells. In general, however, the CLSM method is a sensitive technique to detect very low concentrations of H2O2-induced oxidative stress in NIH3T3 cells. Moreover, by using colon crypts it provides the unique possibility of assessing cell specific levels of oxidative stress in explanted human tissues. Our results demonstrate that the actual target cells of colon cancer induction are indeed susceptible to the oxidative activity of H2O2.

Blood coagulation in anhepatic pigs: effects of treatment with the AMC‐bioartificial liver

Summary.  The function of a newly devised bioartificial liver (AMC‐BAL) based on viable, freshly isolated porcine hepatocytes has been evaluated in anhepatic pigs. The aim of this study was to assess the contribution of BAL treatment on blood coagulation parameters. Pigs were anesthetized and a total hepatectomy was performed (n = 15). The infrahepatic caval vein and the portal vein were connected to the subdiaphragmatic caval vein using a three‐way prosthesis. Animals received standard intensive care (control, n= 5), treatment with an empty BAL (device control, n= 5) or with a cell‐loaded BAL (BAL‐treatment, n= 5) for a period of 24 h starting 24 h after hepatectomy. Coagulation parameters studied concerned prothrombin time (PT), platelet count, the procoagulant system (factors (F)II, FV, FVII, FVIII and fibrinogen), anticoagulant system (AT III), fibrinolytic system (t‐PA, PAI‐1) as well as markers of coagulation factor activation (TAT complexes, prothrombin fragment F1 + 2). FII, FV, FVII, AT III and fibrinogen rapidly decreased after total hepatectomy in pigs in accordance with the anhepatic state of the animals. FVIII levels were not influenced by the hepatectomy. A mild drop in platelet count was seen in all groups. Treatment of anhepatic pigs with the cell‐loaded BAL did not restore PT or clotting factor levels. TAT and F1 + 2 complexes, however, were significantly increased in this group. Levels of t‐PA and PAI‐1 were not influenced by cell‐loaded BAL treatment. Treatment of anhepatic pigs with the AMC‐BAL based on freshly isolated porcine hepatocytes does not result in an improved coagulation state due to extensive consumption of clotting factors. However, increased levels of TAT complexes and prothrombin fragments F1 + 2 during treatment of anhepatic pigs indicate synthesis and direct activation of coagulation factors, leading to thrombin generation. This demonstrates that this bioartificial liver is capable of synthesizing coagulation factors.

In vitro function of porcine carotid arteries preserved in UW, HTK and Celsior solutions

We compared the efficacy of histidine‐tryptophan‐ketoglutarate (HTK) and University of Wisconsin (UW) solution with Celsior solution using hypothermically‐preserved porcine carotid arteries and studied the importance of different components of these solutions by preserving carotid arteries in modified HTK solutions.