Evaluation of the protection exerted by Pisum sativum Ferredoxin-NADP(H) Reductase against injury induced by hypothermia on Cos-7 cells.

Hypothermia is employed as a method to diminish metabolism rates and preserve tissues and cells. However, low temperatures constitute a stress that produces biochemical changes whose extension depends on the duration and degree of cold exposure and is manifested when physiological temperature is restored. For many cellular types, cold induces an oxidative stress that is dependent on the elevation of intracellular iron, damages macromolecules, and is prevented by the addition of iron chelators. Pisum sativum Ferredoxin-NADP(H) Reductase (FNR) has been implicated in protection from injury mediated by intracellular iron increase and successfully used to reduce oxidative damage on bacterial, plant and mammalian systems. In this work, FNR was expressed in Cos-7 cells; then, they were submitted to cold incubation and iron overload to ascertain whether this enzyme was capable of diminishing the harm produced by these challenges. Contrary to expected, FNR was not protective and even exacerbated the damage under certain circumstances. It was also found that the injury induced by hypothermia in Cos-7 cells presented both iron-dependent and iron-independent components of damage when cells were actively dividing but only iron-independent component when cells were in an arrested state. This is in agreement with previous findings which showed that iron-dependent damage is also an energy-dependent process.

Expression and distribution of aquaporin 8 in rat hepatocytes cold stored 72 hours in modified University of Wisconsin and bes-gluconate-sucrose solutions. Study of their correlation with water content.

Since few data are availble on the genetic responses to low temperatures, we investigated if cold storage of hepatocytes (0 degree C, mUW or BGS solutions, 72 h) can affect gene expression and/or cellular localization of AQP8 and their correlation with water movements. Cold preserved hepatocytes showed a significant decrease in water content (P less than 0.05) but were able to regulate their volume when they returned to physiological conditions. These changes were not related to modulation in the expression and the pattern of distribution of AQP8 suggesting that other mechanisms are involved. The study of the quantitative changes in the expression of genes coding for liver specific proteins in cold preserved hepatic cells is of interest in order to develop new preservation methods or solutions that could contribute to maintain the utility of these cells when destined to be applied in clinical models.

Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation.

UNLABELLED: There is increasing evidence that carbon monoxide (CO), a signaling molecule generated during the degradation of heme by heme oxygenase-1 (HO-1) in biological systems, has a variety of cytoprotective actions, including anti-hypoxic effects at low temperatures. However, during liver cold preservation, a direct effect needs to be established. Here, we designed a study to analyze the role of CO, delivered via a carbon monoxide-releasing molecule (CO-RM) in the maintenance of liver function, and integrity in rats during cold ischemia/reperfusion (CI/R) injury. We used an isolated normothermic perfused liver system (INPL) following a clinically relevant model of ex vivo 48 h cold ischemia stored in a modified University of Wisconsin (UW) solution, to determine the specific effects of CO in a rat model. CO was generated from 50 microM tricarbonylchloro ruthenium-glycinato (CORM-3), a water-soluble transition metal carbonyl that exerts pharmacological activities via the liberation of controlled amounts of CO in biological systems. The physiological effects of CORM-3 were confirmed by the parallel use of a specific inactive compound (iCORM-3), which does not liberate CO in the cellular environment. CORM-3 addition was found to prevent the injury caused by cold storage by improving significantly the perfusion flow during reperfusion (by almost 90%), and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold preserved in UW alone. Also, CORM-3 supplementation preserved good metabolic capacity as indicated by hepatic oxygen consumption, glycogen content, and release of lactate dehydrogenase. Liver histology was also partially preserved by CORM-3 treatment. CONCLUSIONS: These findings suggest that CO-RM could be utilized as adjuvant therapeutics in UW solutions to limit the injury sustained by donor livers during cold storage prior to transplantation, as has been similarly proposed for the heart, and kidney.

Glutathione synthesis during the rewarming of rat hepatocytes preserved in the University of Wisconsin solution.

In this study, we used isolated rat hepatocytes to investigate the effect of nucleoside content of the preserved cells on the ability to synthesize glutathione (GSH) during the rewarming process. We cold-stored hepatocytes in University of Wisconsin (UW) solution (72 h, 0 degrees C, N(2)) without nucleosides and with the addition of 5 mM adenosine or 10 mM ATP. After 72 h of cold storage, we determined the GSH synthesis rate and the ATP content of the cells. We found a GSH synthesis rate similar to that of freshly isolated hepatocytes only in the group of cells cold-stored with 10 mM ATP. When we tested the cellular ATP concentrations, we found that controls and preserved cells with 10 mM ATP showed a similar value of ATP during the rewarming step. Our results suggested that the incorporation of ATP in the UW solution increased the ATP content and the rate of GSH synthesis of cold-stored hepatocytes during rewarming.

Glutathione content during the rinsing and rewarming process of rat hepatocytes preserved in University of Wisconsin solution.

The addition of glutathione (GSH) to University of Wisconsin (UW) solution increases the intracellular content of GSH and decreases the release of lactate dehydrogenase used here as a measure of cell viability. However, we found a depletion of GSH when the cells were transferred from UW solution to the rewarming solution. This could sensitize the cells to various forms of oxidative injury. In this study we examined how different compositions of rinsing and rewarming solutions affected the GSH content and the viability of hepatocytes after 72 h of cold storage. For both the rinsing and the rewarming steps we used a Krebs-Henseleit solution with the addition of GSH, methionine, or both GSH and methionine. We found no loss of GSH when the hepatocytes were rinsed in the presence of 3 mM GSH. During the rewarming step we observed a loss of GSH in all of the study groups, but the cells that were incubated with 1 mM methionine showed a lesser depletion of GSH and improved viability. This finding may have valuable applications in hepatocellular transplantation and in the development of bioartificial liver support devices.

Hypothermic storage of periportal and perivenous rat hepatocytes.

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96 h-4 degrees C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37 degrees C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.

Glutathione movements during cold preservation of rat hepatocytes.

In this study we have examined the movements of glutathione (GSH) during cold preservation of rat hepatocytes in University of Wisconsin solution. During the preservation process at a low temperature (4 degrees C), with a high extracellular potassium concentration, an extracellular nondiffusible anion (lactobionate), and a Cl(-)-free medium, there is a depletion of metabolites and the development of a time-dependent injury. Also, there is a loss of GSH that is not compensated by transport or synthesis and is basically due to increased catabolic processes. This sensitizes the cells to different forms of oxidative injury, which can play a negative role during transplantation. The addition of GSH improves liver cell preservation but the mechanism is unclear. To elucidate this process we have isolated hepatocytes and preserved them under different conditions: with or without GSH: in the presence of DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione synthetase, and acivicine to inhibit the ectoactivity of cellular gammaglutamyl transpeptidase; or by obtaining hepatocytes from rats depleted of GSH by an injection of diethyl maleate. Under all these conditions we evaluated the GSH content of the cells during cold storage. We also report the time course of accumulation of [glycine-2-3H]GSH. Our results show that during hypothermic storage in University of Wisconsin solution, hepatocytes are permeable to GSH, and the mechanism involved is a rapid nonsaturable process, with linear dependence of the extracellular GSH concentration. This finding may have valuable applications in the improvement of the delivery of compounds to cells.

Importance of pH in resuspension media on viability of hepatocytes preserved in University of Wisconsin solution.

The effect of different pH of resuspension media on the viability of hepatocytes preserved (for 96 h at 4 degrees C) in University of Wisconsin solution (UW solution) was analyzed. After this cold resuspension media storage, we evaluated the rewarming step (incubation time 120 min at 37 degrees C) using different pH levels (6.80, 7.00, 7.20, and 7.40). Cell viability assessed by trypan blue exclusion (TBE) showed a significant difference (p < 0.05) for cells incubated at pH = 7.20. For instance, TBE expressed as percent of change was 78.1 +/- 1.4 compared with cells tested at other pH (pH = 6.80, TBE = 44.2 +/- 9.5; pH = 7.00, TBE = 66.5 +/- 1.1 and pH = 7.40, TBE = 62.0 +/- 1.4). We also evaluated the capacity of these cells both to maintain potassium content (0.509 +/- 0.230 microEq. K+/10(6) cells) and to synthesize urea (5.36 +/- 1.81 mumol Urea/10(6) cells). These results were compared with those obtained from freshly isolated non preserved hepatocytes (0.518 +/- 0.060 microEq. K+/10(6) cells and 5.91 +/- 0.43 mumol Urea/10(6) cells). The results show that viability is pH dependent and suggest that when resuspension media were used, the viability of hepatocytes was improved after 96 h of cold storage.

Protective effect of glutathione (GSH) over glutathione monoethyl-ester (GSH-E) on cold preservation of isolated rat liver cells.

Hepatocyte suspensions provide a rapid method to determine how hypothermic storage affects liver cell metabolism and viability. We investigated whether reduced Glutathione (GSH) inclusion into a modified University of Wisconsin (UW) solution, has a protective effect over Glutathione derivatives, such as Glutathione-monoethylester (GSH-E), when suspensions of hepatocytes are cold stored for several days. Isolated rat liver cells were cold preserved 96 h in UW, UW plus 3 mM GSH and UW plus 3 mM GSH-E. During the cold storage, not significant changes in cell viability were observed, but the total Glutathione content was higher in systems with extracellular GSH over those with GSH-E or without. After cold storage, the liver cells were gently resuspended in Krebs-Henseleit-1% Albumin and used for 120 min of normothermic (37 degrees C) incubation. We evaluate the functional response of the cells measuring the exclusion of Trypan Blue (TBE). This response was clearly different in preserved cells in presence of GSH. These results indicate a protective role of extracellular Glutathione, due to an accumulation of it, rather than the derivative, for hepatic cell during the cold storage in UW solutions. And also, it is possible to extend experiments with hepatocytes from a single cell isolation over 4 or more consecutive days.

[Hypothermic preservation of the liver. Evaluation of Eurocollins solution using rats liver slices]. / Preservación hipotérmica del hígado. Evaluación de la solución Eurocollins utilizando cortes de hígado de rata.

In the present work, we analyze the effects produced by cold ischemia on the liver using Eurocollins solution (EC). This evaluates the function of slices obtained from preserved livers in EC (livers from adult female Wistar rats were used), which were perfused with Krebs-Henseleit solution (KH) for 10 min. and then with cold EC. These livers were stored at 4 degrees C in EC for 7, 24 and 48 hours. At the end of each period of preservation, the livers were sliced and they were incubated 1 hour at 37 degrees C in KH. The following parameters were determined: tissular water distribution and electrolytes content (K+ and Na+), LDH release at the incubation medium, thiobarbituric acid reactive substances (TBARS) and urea synthesis. The slices obtained from preserved livers (7 hours), showed an increase of tissular water, expressed by the expansion of the extracellular space, a progressive diminution of tissular K+ content and an increase in TBARS and the LDH release with the increment of the preservation time. No changes in ureogenesis rate were observed. These results suggest that the cold ischemia in EC, in periods that exceed 5 hours, cause, changes in the membrane permeability and severely affects the mechanism of regulation of the tissular volume and may compromise the functional viability of the organ to be transplanted.

Preservación hipotérmica del hígado. Evaluación de la solución Eurocollins utilizando cortes de hígado de rata. / [Hypothermic preservation of the liver. Evaluation of Eurocollins solution using rats liver slices]

In the present work, we analyze the effects produced by cold ischemia on the liver using Eurocollins solution (EC). This evaluates the function of slices obtained from preserved livers in EC (livers from adult female Wistar rats were used), which were perfused with Krebs-Henseleit solution (KH) for 10 min. and then with cold EC. These livers were stored at 4 degrees C in EC for 7, 24 and 48 hours. At the end of each period of preservation, the livers were sliced and they were incubated 1 hour at 37 degrees C in KH. The following parameters were determined: tissular water distribution and electrolytes content (K+ and Na+), LDH release at the incubation medium, thiobarbituric acid reactive substances (TBARS) and urea synthesis. The slices obtained from preserved livers (7 hours), showed an increase of tissular water, expressed by the expansion of the extracellular space, a progressive diminution of tissular K+ content and an increase in TBARS and the LDH release with the increment of the preservation time. No changes in ureogenesis rate were observed. These results suggest that the cold ischemia in EC, in periods that exceed 5 hours, cause, changes in the membrane permeability and severely affects the mechanism of regulation of the tissular volume and may compromise the functional viability of the organ to be transplanted.