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Glutathione Content during the Rinsing and Rewarming Process of Rat Hepatocytes Preserved in University of Wisconsin Solution
Cryobiology 40, 270 –276 (2000) doi:10.1006/cryo.2000.2242, available online at http://www.idealibrary.com on
BRIEF COMMUNICATION Glutathione Content during the Rinsing and Rewarming Process of Rat Hepatocytes Preserved in University of Wisconsin Solution Marı´a E. Mamprin,* Edgardo E. Guibert,† and Joaquı´n V. Rodriguez* ,1 *Farmacologı´a, Departamento de Ciencias Fisiolo´gicas, and †Biologı´a Molecular, Departamento de Ciencias Biolo´gicas, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Suipacha, 531-2000 Rosario, Argentina 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. © 2000 Academic Press Key Words: UW solution; glutathione; methionine; hepatocyte; rinse solution.
and as a reductant of peroxides (7). The cold storage of hepatocytes in UW solution causes a reduction of GSH content due to increased catabolic processes and this sensitizes cells to various forms of oxidative injury (10). The extent of reoxygenation injury is related to the composition of the rewarming solution (6) and we have shown that after 96 h of cold storage, the viability of hepatocytes is dependent on the pH of that solution (5). Several authors (7, 10) have demonstrated that the cellular concentration of GSH decreases during cold storage of rat hepatocytes when GSH is not added to UW solution. The addition of GSH to UW solution increases the intracellular content of GSH and improves the viability of cells. We have previously shown (7) that hepatocytes are permeable to GSH during hypothermic storage in UW solution. Preliminary experiments carried out in our laboratory revealed that depletion of GSH can occur when the hepatocytes are transferred from UW solution to the rewarming solution. Because of this,
Hepatocyte suspensions provide a valuable experimental model for studies to determine how hypothermic storage in University of Wisconsin (UW) solution affects liver cell metabolism and viability. Hepatocytes perform numerous functions, including synthesis and secretion of plasma proteins and energy production involving carbohydrate, lipid, and amino acid metabolism (5). Hepatocytes may also be considered for use in their own right for cellular transplantation or in bioartificial liver-assist devices. During preservation at low temperatures (⬃4°C) metabolites are depleted and a timedependent injury develops. Glutathione (GSH) has important protective and metabolic functions; in particular, it has a role in the detoxification of electrophilic compounds and epoxides Received January 4, 2000, accepted February 28, 2000. E.E.G. and J.V.R. are members of the National Council of Research, Argentina. This study was funded by PID 202-UNR. 1 To whom correspondence should be addressed. E-mail: [email protected].
0011-2240/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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it has been necessary to develop methods that help the cells to maintain their GSH content following preservation. Several compounds were considered potentially interesting—N-acetylcysteine, GSH monoethyl ester, and some amino acids (4). In this study we have examined how different solution compositions affect the GSH content and the cellular viability of hepatocytes cold stored in UW solution up to 72 h. Hepatocyte isolation. Adult male Wistar rats weighing 250 –300 g were fed ad libitum and received care in compliance with international regulations. They were anesthetized with sodium thiopental (70 mg/kg bw, ip). The cells were isolated from fed animals using collagenase according to the method described previously (5). Cell viability was tested by the exclusion of 0.4% trypan blue stain (TBE) in phosphate-buffered saline. Over 500 million cells were obtained per liver and preparations with a TBE greater than 90% were considered suitable for the experiments. Hepatocyte preservation. Isolated hepatocytes were rinsed twice and resuspended in freshly prepared UW solution at 4°C. The composition of the modified UW solution (7) was 100 mM lactobionic acid, 25 mM K 2HPO 4, 5 mM MgSO 4, 30 mM raffinose, 2.5 mM adenosine, 3 mM GSH, 1 mM allopurinol, 5% polyethylene glycol, 15 mM glycine, 0.25 mg/ml streptomycin, and 10 UI/ml penicillin G; pH 7.40. The solution was bubbled with 100% N 2 for 15 min at 4°C before use. Hepatocytes (75 ⫻ 10 6 cells in 30 ml UW solution) were allowed to settle to the bottom of the 50-ml screw cup polycarbonate tubes and were left undisturbed at 4°C for up to 72 h. One of the stored suspensions was removed on each day and was used to estimate the time course changes of the viability assessed by lactate dehydrogenase (LDH) release, GSH content, and morphology. Viability measurement—LDH release. The LDH activity in the cell suspension (total activity) and in the supernatant (extracellular LDH) was determined as described previously (5). Results were expressed as the percentage of total enzyme activity in the extracellular medium.
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Glutathione assay. Hepatocytes (2.5 ⫻ 10 6 cell/ml) were separated from the incubation medium by centrifugation (13000g, 30 s) and the cell pellet was deproteinized by addition of 450 l of cold 3% HClO 4. All of the procedures were performed on ice. After centrifugation, the protein-free supernatant was neutralized with K 2CO 3. After removal of insoluble potassium perchlorate by spinning down (13000g, 30 s), total glutathione—GSH plus GSSG— concentration (GSH t) was determined by the enzymecoupled spectrophotometric assay of Tietze (9). It was shown in previous experiments (7) that the GSH concentration seen in the acid-extracted hepatocytes was not an artifact caused by GSH trapped in the extracellular water of the cell pellet. Hepatocyte rinsing. After 72 h of cold storage, the hepatocytes were gently resuspended to form an homogeneous suspension. An aliquot of cells was taken to determine GSH content and LDH release. After that, the hepatocyte suspension was sedimented (50g, 3 min), the supernatant was removed, and the cells were resuspended in warm Krebs–Henseleit solution (KHR). This procedure, called “hepatocyte rinsing,” was repeated twice to facilitate the removal of preservation solution. In all cases the ratio between the volume of rinse solution and the pelleted cell volume was 4:1. After each rinse, an aliquot of cell suspension was removed and centrifuged (50g, 3 min) and the GSH content was determined on the sedimented cells. The KHR composition was 114 mM NaCl, 25 mM NaHCO 3, 4.8 mM KCl, 1.2 mM KH 2PO 4, 1.2 mM MgSO 4, 1.2 mM CaCl 2, 10 mM hepes, 5 mM fructose, 5 mM glucose, 2.5 mM adenosine, 1 mM allopurinol, 3 mM glycine, and 1% BSA; pH 7.20. To obtain the time course of cellular GSH content during this procedure, we studied four experimental groups; each group was rinsed twice with one of the following solutions: (a) KHR (control); (b) KHR ⫹ 80 M GSH; (c) KHR ⫹ 1 mM methionine; (d) KHR ⫹ 3 mM GSH, and (e) KHR ⫹ 3 mM GSH ⫹ 1 mM methionine. Hepatocyte rewarming. The hepatocytes rinsed in (d) solution (the best rinsing condi-
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FIG. 1. The effect of addition of GSH and methionine to the rinse solution on intracellular GSH t concentration in rat hepatocytes that were cold preserved for up to 72 h. Cells were rinsed twice without GSH (group a); with 80 M GSH (group b); 3 mM GSH (group d); 1 mM methionine (group c); and 3 mM GSH ⫹ 1 mM methionine (group e). Data are given as means ⫾ SD for hepatocytes prepared from four rats. *P ⬍ 0.05 indicates that the GSH t of hepatocytes is statistically different from UW 72 h, groups c, d, and e. **P ⬍ 0.05 indicates that the GSH t of hepatocytes is statistically different from UW 72 h, groups d and e.
tions, see below) were incubated in one of the following solutions: (I) KHR (control); (II) KHR ⫹ 80 M GSH; (III) KHR ⫹ 3 mM GSH; (IV) KHR ⫹ 80 M GSH ⫹ 1 mM methionine, and (V) KHR ⫹ 1 mM methionine. In every case cells were incubated (120 min, 37°C, 1 ⫻ 10 6 cells/ml) under carbogen atmosphere in a Dubnoff metabolic shaker, and an aliquot was removed to determine the GSH content and LDH release at 0, 30, 60, and 120 min. Statistical analysis. Results are presented as means ⫾ SD and the number of preparations analyzed was four or more, as indicated in each figure. Statistical significance of the differences between values was assessed by multifactor analysis of variance followed by Scheffe’s multiple range test. If P was less than 0.05 it was considered statistically significant (Statgraphics, Statistical graphics System, U.S.A.). Cell viability during cold storage in UW solution. Viability of suspensions of hepatocytes stored in modified UW solution was assessed by LDH release from the cells into the cold storage solution. At the start of the experiments (time ⫽
0), the LDH release was 0.45 ⫾ 0.18% of total LDH. Hepatocyte viability was well maintained during cold storage of cells suspensions for up 72 h (LDH released at 24 h: 1.03 ⫾ 0.22, at 48 h: 2.21 ⫾ 0.45%, and after 72 h of preservation: 3.33 ⫾ 0.55%; n ⫽ 12). Light microscopy showed that the cells had a normal rounded shape without blebs. Judged by performance following rewarming, we consider that 72 h of cold storage is the limiting preservation time; at this stage the release of LDH from the cells was around 3.50 ⫾ 0.51% and this correlated with good morphology. LDH release at 96 h of cold storage was 6.90 ⫾ 0.62% and the cells tended to form blebs. Evolution of cellular glutathione content during rinsing. Figure 1 shows the cellular GSHt concentration (nmol GSH/10 6 cell) after 72 h of preservation and rinsing twice in the KHR solution. The hepatocytes rinsed without GSH (group a) and with 80 M GSH (group b) showed a major depletion of GSH. When cells were rinsed once in KHR (group a), the GSH t level diminished by 77.33 ⫾ 4.14% following
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the first rinse and by 83.81 ⫾ 4.33% following the second. For group b, the GSH t level diminished by 78.33 ⫾ 3.55% for the first rinse and by 83.81 ⫾ 6.34% for the second. In both experimental conditions the cells lost GSH by efflux into the extracellular medium. The GSH bidirectional transport system could be a factor in this loss. This carrier-mediated process, which was characterized by Kaplowitz and colleagues (3), will operate as a net efflux pump during the rinsing procedure because: (a) the cells had been loaded with GSH during cold storage in UW, and (b) there is a concentration gradient of GSH between the cells and the rinsing solution. When the hepatocytes were rinsed in 1 mM methionine (group c), the GSH t level was statistically different (P ⬍ 0.05) with respect to groups a and b. In this case, the GSH t level diminished by 44.83 ⫾ 6.02% on the first rinse and by 65.37 ⫾ 8.21% on the second. On the other hand, GSH levels were statistically higher (P ⬍ 0.05) in groups d and e when compared with the other groups, but no difference was observed between them. For group d, the GSH t level diminished by only 1.86 ⫾ 0.12% during the first rinse and by 16.33 ⫾ 2.34% during the second; in group e, GSH t levels increased by 10.18 ⫾ 1.84% following the first rinse and diminished by 10.33 ⫾ 2.40% after the second. Two different concentrations of GSH were used in the KHR solution (3 mM and 80 M). The first concentration was chosen because it is the same as the concentration added to the UW solution, and the second is the concentration of GSH in the plasma of our rats (78.42 ⫾ 1.45 M; n ⫽ 6). We also included 1 mM methionine, since it has been reported that this amino acid inhibits the efflux of GSH in isolated hepatocytes (1). In both situations the GSH level recorded after the second rinse was similar to the GSH content of freshly isolated hepatocytes (see Fig. 1, dotted line, 55.6 ⫾ 4.6 nmol GSH/ 10 6 cells; n ⫽ 4). During cold storage in UW solution, the hepatocytes accumulate GSH (7), and when the cells are rinsed in a solution containing 3 mM GSH there is no decrease in GSH level; this is probably due to the absence
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FIG. 2. Evolution of cellular GSH content during the rewarming procedure. The cells were cold preserved up to 72 h, rinsed twice in KHR ⫹ 3 mM GSH, and incubated up to 120 min in KHR (group I) and KHR ⫹ 80 M GSH (group II); 3 mM GSH (group III); 80 M GSH ⫹ 1 mM methionine (group IV); and 1 mM methionine (group V). Data are given as means ⫾ SD for hepatocytes prepared from four rats. The asterisk indicates that the GSH t of hepatocytes from groups IV and V groups is statistically different from the other groups at P ⬍ 0.05.
of a concentration gradient between the rinsing solution and the cells. Because no differences in GSH levels were found between the rinsing solution containing 3 mM GSH (d) and the solution that contained 3 mM GSH ⫹ 1 mM methionine (e), we chose the rinse solution (d) to carry out the rewarming step. Evolution of cellular glutathione content during the rewarming step. Figure 2 shows the time course of cellular GSH t content during 120 min of rewarming. Each group of cells was preserved for up to 72 h in UW, rinsed twice in KHR ⫹ 3 mM GSH (solution d), and resuspended in the rewarming solution. The hepatocytes from groups (I) and (II) showed a major decrease in GSH t content during the rewarming step and the initial value of GSH (time 0) was four times lower than in the other groups. The time 0 represents 5 min, the time required to
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FIG. 3. Time course of LDH release (viability) of rat hepatocytes rewarmed in KHR with different substrates. The cells were cold preserved, rinsed twice in KHR ⫹ 3 mM GSH, and incubated up to 120 min in KHR (group I); and KHR ⫹ 80 M GSH (group II); 3 mM GSH (group III); 80 M GSH ⫹ 1 mM methionine (group IV), and 1 mM methionine (group V). Data are given as means ⫾ SD for hepatocytes prepared from four rats. The asterisk indicates that the LDH release of hepatocytes from groups IV and V groups is statistically different from the other groups at P ⬍ 0.05.
centrifuge the cells from the rinsing solution and resuspend them in the rewarming solution. For groups I and II, the rewarming solution did not contain GSH (I) or contained 80 M GSH (II). GSH t content (nmol GSH/10 6 cells) for the cells rewarmed in KHR solution (group I) was 12.25 ⫾ 3.40 at time 0; 3.63 ⫾ 1.11 at 60 min; and 0.17 ⫾ 0.14 after 120 min of reoxygenation. When the hepatocytes were rewarmed in the presence of 80 M GSH (group II), the GSH t content was 11.75 ⫾ 2.06 at zero time; 3.35 ⫾ 1.10 at 60 min; and 0.14 ⫾ 0.08 after 120 min. The lower values of GSH obtained in groups I and II could be caused by the loss of GSH due to efflux from the cells to the medium (1) as well as to the catabolism produced during the 120 min of rewarming. When the hepatocytes were incubated with 3 mM GSH (group III), the GSH t level was statistically different (P ⬍ 0.05) with respect to groups (I) and (II). In this case the GSH t level at time zero was 51.43 ⫾ 5.40; 42.65 ⫾ 4.89 at 60 min; and 17.88 ⫾ 4.92 after 120 min of reoxygenation. However, the GSH levels were statistically higher (P ⬍ 0.05) in groups (IV) and (V) than in the other groups, but no difference was observed between them. The GSH t content of
hepatocytes from group IV, which had been incubated in the presence of 80 M GSH ⫹ 1 mM methionine was 54.75 ⫾ 4.86 at zero time; 36.25 ⫾ 7.04 at 60 min; and 31.75 ⫾ 4.92 after 120 min of reoxygenation. When the hepatocytes were rewarmed in the presence of 1 mM methionine (group V) the GSH t content was 55.75 ⫾ 4.44 at zero time; 37.25 ⫾ 4.57 at 60 min; and 33.75 ⫾ 5.26 at 120 min. The time course of cellular GSH in freshly isolated hepatocytes incubated in KHR without GSH or methionine was 55.6 ⫾ 4.6 at time zero and 30.25 ⫾ 4.1 nmol GSH/10 6 cells (n ⫽ 4) after 120 min (see Fig. 2, dotted line). These values correlate with those obtained when preserved hepatocytes were incubated with solutions IV and V. Evolution of LDH release during the rewarming step. In this study we used LDH release to assess hepatocyte injury because it is a good measure of membrane integrity for cells in suspension (6). The LDH released from hepatocytes resuspended at 37°C and rewarmed in the different solutions up to 120 min are shown in Fig. 3. The data from groups IV and V show statistical differences (P ⬍ 0.05) after 120 min of rewarming. LDH release was lower for the
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cells incubated in presence of 80 M GSH ⫹ 1 mM methionine (group IV) and 1 mM methionine (group V) with respect to cells incubated in the other solutions (9.67 ⫾ 0.76% for group IV, 10.33 ⫾ 1.53% for group V, 18.11 ⫾ 2.30% for group I, 16.33 ⫾ 2.15% for group II, and 15.67 ⫾ 3.31% for group III). Furthermore, the amount of LDH released by freshly isolated hepatocytes after 120 min of rewarming in KHR were similar to the values obtained for groups IV and V (see Fig. 3, dotted line 11.33 ⫾ 2.15%; n ⫽ 6). This result indicates the superior performance of cells rewarmed in solutions IV and V rather than solutions I, II, and III. We observed the loss of cellular GSH during the rewarming step in all groups, and hepatocytes rewarmed in an amino-acid-free medium (KHR) after cold storage were unable to regenerate GSH; their GSH content decreased. This depletion leads to changes in many metabolic processes and particularly in the accessibility of GSH for detoxification reactions. These phenomena will then enhance the susceptibility of cells to additional stresses, such as those involved in hepatocellular transplantation or the bioartificial liver. When we added 1 mM methionine or 1 mM methionine with 80 M GSH to the rewarming solution the depletion of GSH after 120 min of reoxygenation was smaller and cellular viability was better maintained. Indeed, the GSH t content of these cells was similar to values found when fresh hepatocytes were rewarmed in KHR for 120 min. The depletion of GSH below a critical level during the rewarming step is associated with an increased LDH release, indicating that cellular viability is being affected. Another solution, the “Carolina rinse,” was originally developed to inhibit reperfusion injury (8). It contains some of the substances we used in our rewarming solution—GSH, adenosine, fructose, allopurinol. This solution was shown to decrease damage during the preservation of rodent livers in an orthotopic liver transplantation model and in clinical liver transplantation even with short ischemia times. It is reasonable to think that the protective effect observed following the addition of 1 mM methionine to the rewarming solution is medi-
ated through an inhibition of GSH efflux from the isolated hepatocytes (1). The mechanism of such an effect is not clear and has not been studied in this work, but Kaplowitz and colleagues (2) has suggested an inhibition of a carrier-mediated GSH transport process. Krebs et al. (4) have shown that the addition of methionine to the incubation medium maintains GSH concentration in rat hepatocytes at concentrations from 0.2 to 1 mM. Since the normal concentration of methionine in the rat plasma is about 0.04 mM and in the liver is about 0.1 mM, it might be expected that the ability of 1 mM methionine to maintain GSH may be due to a gradual biosynthesis of cysteine from methionine via the cystathionine pathway, which is needed for the resynthesis of GSH. In conclusion, the addition of GSH to the rinsing solution and of methionine to the rewarming solution improves the viability of hepatocytes that were stored in the cold for up to 72 h. This knowledge can contribute to the development of new strategies to maintain functional and viable hepatocytes after cold preservation, to applications in hepatocellular transplantation, and in extrahepatic bioartificial liver support devices. REFERENCES 1. Aw, T. K., Ookhtens, M., and Kaplowitz, N. Inhibition of glutathione efflux from isolated rat hepatocytes by methionine. J. Biol. Chem. 15, 9355–9358 (1984). 2. Aw, T. Y, Ookhtens, M., Clement, R., and Kaplowitz, N. Kinetics of glutathione efflux from isolated rat hepatocytes. Am. J. Physiol. (Gastrointest. Liver Physiol. 250) 13, G236 –G243 (1986). 3. Garcı´a-Ruiz, C., Ferna´ndez Checa, J. C., and Kaplowitz, N. Bi-directional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles. J. Biol. Chem. 267, 22256 –22264 (1992). 4. Krebs, H. A., Hems, R., and Vin˜a, J. Regulation of the hepatic concentration of reduced glutathione. In “Functions of Glutathione in Liver and Kidney” (H. Sies and A. Wendel, Eds), Springer-Verlag, New York, 1978. 5. Mamprin, M. E., Rodriguez, J. V., and Guibert, E. E. The importance of pH in resuspension media on viability of hepatocytes preserved in University of Wisconsin solution. Cell Transplant. 4, 269 –274 (1995).
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6. Marsh, D. C., Hjelmhaug, J. A., Vreugdenhil, P. K., Kerr, J. A., Rice, M. J., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes. III. Effects of resuspension media on viability after up to 7 days of storage. Hepatology 13, 500 –508 (1991). 7. Rodriguez, J. V., Mamprin, M., Mediavilla, M., and Guibert, E. Glutathione (GSH) movements during cold preservation of rat hepatocytes. Cryobiology 36, 236 –244 (1998). 8. Sanchez-Urdazpal, L., Gores, G. J., Lemasters, J. J., Thurman, R. G., Steers, J. L., Wahistrom, H. E.,
Hay, E. I., Porayko, M. K., Wiesner, R. H., and Krom, R. A. Carolina Rinse Solution decreases liver injury during clinical liver transplantation. Transplant. Proc. 25, 1574 –1575 (1993). 9. Tietze, F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522 (1969). 10. Vreugdenhil, P. K., Belzer, F. O., and Southard, J. H. Effect of cold storage on tissue and cellular glutathione. Cryobiology 28, 143–149 (1991).
BRIEF COMMUNICATION Glutathione Content during the Rinsing and Rewarming Process of Rat Hepatocytes Preserved in University of Wisconsin Solution Marı´a E. Mamprin,* Edgardo E. Guibert,† and Joaquı´n V. Rodriguez* ,1 *Farmacologı´a, Departamento de Ciencias Fisiolo´gicas, and †Biologı´a Molecular, Departamento de Ciencias Biolo´gicas, Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario, Suipacha, 531-2000 Rosario, Argentina 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. © 2000 Academic Press Key Words: UW solution; glutathione; methionine; hepatocyte; rinse solution.
and as a reductant of peroxides (7). The cold storage of hepatocytes in UW solution causes a reduction of GSH content due to increased catabolic processes and this sensitizes cells to various forms of oxidative injury (10). The extent of reoxygenation injury is related to the composition of the rewarming solution (6) and we have shown that after 96 h of cold storage, the viability of hepatocytes is dependent on the pH of that solution (5). Several authors (7, 10) have demonstrated that the cellular concentration of GSH decreases during cold storage of rat hepatocytes when GSH is not added to UW solution. The addition of GSH to UW solution increases the intracellular content of GSH and improves the viability of cells. We have previously shown (7) that hepatocytes are permeable to GSH during hypothermic storage in UW solution. Preliminary experiments carried out in our laboratory revealed that depletion of GSH can occur when the hepatocytes are transferred from UW solution to the rewarming solution. Because of this,
Hepatocyte suspensions provide a valuable experimental model for studies to determine how hypothermic storage in University of Wisconsin (UW) solution affects liver cell metabolism and viability. Hepatocytes perform numerous functions, including synthesis and secretion of plasma proteins and energy production involving carbohydrate, lipid, and amino acid metabolism (5). Hepatocytes may also be considered for use in their own right for cellular transplantation or in bioartificial liver-assist devices. During preservation at low temperatures (⬃4°C) metabolites are depleted and a timedependent injury develops. Glutathione (GSH) has important protective and metabolic functions; in particular, it has a role in the detoxification of electrophilic compounds and epoxides Received January 4, 2000, accepted February 28, 2000. E.E.G. and J.V.R. are members of the National Council of Research, Argentina. This study was funded by PID 202-UNR. 1 To whom correspondence should be addressed. E-mail: [email protected].
0011-2240/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
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it has been necessary to develop methods that help the cells to maintain their GSH content following preservation. Several compounds were considered potentially interesting—N-acetylcysteine, GSH monoethyl ester, and some amino acids (4). In this study we have examined how different solution compositions affect the GSH content and the cellular viability of hepatocytes cold stored in UW solution up to 72 h. Hepatocyte isolation. Adult male Wistar rats weighing 250 –300 g were fed ad libitum and received care in compliance with international regulations. They were anesthetized with sodium thiopental (70 mg/kg bw, ip). The cells were isolated from fed animals using collagenase according to the method described previously (5). Cell viability was tested by the exclusion of 0.4% trypan blue stain (TBE) in phosphate-buffered saline. Over 500 million cells were obtained per liver and preparations with a TBE greater than 90% were considered suitable for the experiments. Hepatocyte preservation. Isolated hepatocytes were rinsed twice and resuspended in freshly prepared UW solution at 4°C. The composition of the modified UW solution (7) was 100 mM lactobionic acid, 25 mM K 2HPO 4, 5 mM MgSO 4, 30 mM raffinose, 2.5 mM adenosine, 3 mM GSH, 1 mM allopurinol, 5% polyethylene glycol, 15 mM glycine, 0.25 mg/ml streptomycin, and 10 UI/ml penicillin G; pH 7.40. The solution was bubbled with 100% N 2 for 15 min at 4°C before use. Hepatocytes (75 ⫻ 10 6 cells in 30 ml UW solution) were allowed to settle to the bottom of the 50-ml screw cup polycarbonate tubes and were left undisturbed at 4°C for up to 72 h. One of the stored suspensions was removed on each day and was used to estimate the time course changes of the viability assessed by lactate dehydrogenase (LDH) release, GSH content, and morphology. Viability measurement—LDH release. The LDH activity in the cell suspension (total activity) and in the supernatant (extracellular LDH) was determined as described previously (5). Results were expressed as the percentage of total enzyme activity in the extracellular medium.
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Glutathione assay. Hepatocytes (2.5 ⫻ 10 6 cell/ml) were separated from the incubation medium by centrifugation (13000g, 30 s) and the cell pellet was deproteinized by addition of 450 l of cold 3% HClO 4. All of the procedures were performed on ice. After centrifugation, the protein-free supernatant was neutralized with K 2CO 3. After removal of insoluble potassium perchlorate by spinning down (13000g, 30 s), total glutathione—GSH plus GSSG— concentration (GSH t) was determined by the enzymecoupled spectrophotometric assay of Tietze (9). It was shown in previous experiments (7) that the GSH concentration seen in the acid-extracted hepatocytes was not an artifact caused by GSH trapped in the extracellular water of the cell pellet. Hepatocyte rinsing. After 72 h of cold storage, the hepatocytes were gently resuspended to form an homogeneous suspension. An aliquot of cells was taken to determine GSH content and LDH release. After that, the hepatocyte suspension was sedimented (50g, 3 min), the supernatant was removed, and the cells were resuspended in warm Krebs–Henseleit solution (KHR). This procedure, called “hepatocyte rinsing,” was repeated twice to facilitate the removal of preservation solution. In all cases the ratio between the volume of rinse solution and the pelleted cell volume was 4:1. After each rinse, an aliquot of cell suspension was removed and centrifuged (50g, 3 min) and the GSH content was determined on the sedimented cells. The KHR composition was 114 mM NaCl, 25 mM NaHCO 3, 4.8 mM KCl, 1.2 mM KH 2PO 4, 1.2 mM MgSO 4, 1.2 mM CaCl 2, 10 mM hepes, 5 mM fructose, 5 mM glucose, 2.5 mM adenosine, 1 mM allopurinol, 3 mM glycine, and 1% BSA; pH 7.20. To obtain the time course of cellular GSH content during this procedure, we studied four experimental groups; each group was rinsed twice with one of the following solutions: (a) KHR (control); (b) KHR ⫹ 80 M GSH; (c) KHR ⫹ 1 mM methionine; (d) KHR ⫹ 3 mM GSH, and (e) KHR ⫹ 3 mM GSH ⫹ 1 mM methionine. Hepatocyte rewarming. The hepatocytes rinsed in (d) solution (the best rinsing condi-
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FIG. 1. The effect of addition of GSH and methionine to the rinse solution on intracellular GSH t concentration in rat hepatocytes that were cold preserved for up to 72 h. Cells were rinsed twice without GSH (group a); with 80 M GSH (group b); 3 mM GSH (group d); 1 mM methionine (group c); and 3 mM GSH ⫹ 1 mM methionine (group e). Data are given as means ⫾ SD for hepatocytes prepared from four rats. *P ⬍ 0.05 indicates that the GSH t of hepatocytes is statistically different from UW 72 h, groups c, d, and e. **P ⬍ 0.05 indicates that the GSH t of hepatocytes is statistically different from UW 72 h, groups d and e.
tions, see below) were incubated in one of the following solutions: (I) KHR (control); (II) KHR ⫹ 80 M GSH; (III) KHR ⫹ 3 mM GSH; (IV) KHR ⫹ 80 M GSH ⫹ 1 mM methionine, and (V) KHR ⫹ 1 mM methionine. In every case cells were incubated (120 min, 37°C, 1 ⫻ 10 6 cells/ml) under carbogen atmosphere in a Dubnoff metabolic shaker, and an aliquot was removed to determine the GSH content and LDH release at 0, 30, 60, and 120 min. Statistical analysis. Results are presented as means ⫾ SD and the number of preparations analyzed was four or more, as indicated in each figure. Statistical significance of the differences between values was assessed by multifactor analysis of variance followed by Scheffe’s multiple range test. If P was less than 0.05 it was considered statistically significant (Statgraphics, Statistical graphics System, U.S.A.). Cell viability during cold storage in UW solution. Viability of suspensions of hepatocytes stored in modified UW solution was assessed by LDH release from the cells into the cold storage solution. At the start of the experiments (time ⫽
0), the LDH release was 0.45 ⫾ 0.18% of total LDH. Hepatocyte viability was well maintained during cold storage of cells suspensions for up 72 h (LDH released at 24 h: 1.03 ⫾ 0.22, at 48 h: 2.21 ⫾ 0.45%, and after 72 h of preservation: 3.33 ⫾ 0.55%; n ⫽ 12). Light microscopy showed that the cells had a normal rounded shape without blebs. Judged by performance following rewarming, we consider that 72 h of cold storage is the limiting preservation time; at this stage the release of LDH from the cells was around 3.50 ⫾ 0.51% and this correlated with good morphology. LDH release at 96 h of cold storage was 6.90 ⫾ 0.62% and the cells tended to form blebs. Evolution of cellular glutathione content during rinsing. Figure 1 shows the cellular GSHt concentration (nmol GSH/10 6 cell) after 72 h of preservation and rinsing twice in the KHR solution. The hepatocytes rinsed without GSH (group a) and with 80 M GSH (group b) showed a major depletion of GSH. When cells were rinsed once in KHR (group a), the GSH t level diminished by 77.33 ⫾ 4.14% following
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the first rinse and by 83.81 ⫾ 4.33% following the second. For group b, the GSH t level diminished by 78.33 ⫾ 3.55% for the first rinse and by 83.81 ⫾ 6.34% for the second. In both experimental conditions the cells lost GSH by efflux into the extracellular medium. The GSH bidirectional transport system could be a factor in this loss. This carrier-mediated process, which was characterized by Kaplowitz and colleagues (3), will operate as a net efflux pump during the rinsing procedure because: (a) the cells had been loaded with GSH during cold storage in UW, and (b) there is a concentration gradient of GSH between the cells and the rinsing solution. When the hepatocytes were rinsed in 1 mM methionine (group c), the GSH t level was statistically different (P ⬍ 0.05) with respect to groups a and b. In this case, the GSH t level diminished by 44.83 ⫾ 6.02% on the first rinse and by 65.37 ⫾ 8.21% on the second. On the other hand, GSH levels were statistically higher (P ⬍ 0.05) in groups d and e when compared with the other groups, but no difference was observed between them. For group d, the GSH t level diminished by only 1.86 ⫾ 0.12% during the first rinse and by 16.33 ⫾ 2.34% during the second; in group e, GSH t levels increased by 10.18 ⫾ 1.84% following the first rinse and diminished by 10.33 ⫾ 2.40% after the second. Two different concentrations of GSH were used in the KHR solution (3 mM and 80 M). The first concentration was chosen because it is the same as the concentration added to the UW solution, and the second is the concentration of GSH in the plasma of our rats (78.42 ⫾ 1.45 M; n ⫽ 6). We also included 1 mM methionine, since it has been reported that this amino acid inhibits the efflux of GSH in isolated hepatocytes (1). In both situations the GSH level recorded after the second rinse was similar to the GSH content of freshly isolated hepatocytes (see Fig. 1, dotted line, 55.6 ⫾ 4.6 nmol GSH/ 10 6 cells; n ⫽ 4). During cold storage in UW solution, the hepatocytes accumulate GSH (7), and when the cells are rinsed in a solution containing 3 mM GSH there is no decrease in GSH level; this is probably due to the absence
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FIG. 2. Evolution of cellular GSH content during the rewarming procedure. The cells were cold preserved up to 72 h, rinsed twice in KHR ⫹ 3 mM GSH, and incubated up to 120 min in KHR (group I) and KHR ⫹ 80 M GSH (group II); 3 mM GSH (group III); 80 M GSH ⫹ 1 mM methionine (group IV); and 1 mM methionine (group V). Data are given as means ⫾ SD for hepatocytes prepared from four rats. The asterisk indicates that the GSH t of hepatocytes from groups IV and V groups is statistically different from the other groups at P ⬍ 0.05.
of a concentration gradient between the rinsing solution and the cells. Because no differences in GSH levels were found between the rinsing solution containing 3 mM GSH (d) and the solution that contained 3 mM GSH ⫹ 1 mM methionine (e), we chose the rinse solution (d) to carry out the rewarming step. Evolution of cellular glutathione content during the rewarming step. Figure 2 shows the time course of cellular GSH t content during 120 min of rewarming. Each group of cells was preserved for up to 72 h in UW, rinsed twice in KHR ⫹ 3 mM GSH (solution d), and resuspended in the rewarming solution. The hepatocytes from groups (I) and (II) showed a major decrease in GSH t content during the rewarming step and the initial value of GSH (time 0) was four times lower than in the other groups. The time 0 represents 5 min, the time required to
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FIG. 3. Time course of LDH release (viability) of rat hepatocytes rewarmed in KHR with different substrates. The cells were cold preserved, rinsed twice in KHR ⫹ 3 mM GSH, and incubated up to 120 min in KHR (group I); and KHR ⫹ 80 M GSH (group II); 3 mM GSH (group III); 80 M GSH ⫹ 1 mM methionine (group IV), and 1 mM methionine (group V). Data are given as means ⫾ SD for hepatocytes prepared from four rats. The asterisk indicates that the LDH release of hepatocytes from groups IV and V groups is statistically different from the other groups at P ⬍ 0.05.
centrifuge the cells from the rinsing solution and resuspend them in the rewarming solution. For groups I and II, the rewarming solution did not contain GSH (I) or contained 80 M GSH (II). GSH t content (nmol GSH/10 6 cells) for the cells rewarmed in KHR solution (group I) was 12.25 ⫾ 3.40 at time 0; 3.63 ⫾ 1.11 at 60 min; and 0.17 ⫾ 0.14 after 120 min of reoxygenation. When the hepatocytes were rewarmed in the presence of 80 M GSH (group II), the GSH t content was 11.75 ⫾ 2.06 at zero time; 3.35 ⫾ 1.10 at 60 min; and 0.14 ⫾ 0.08 after 120 min. The lower values of GSH obtained in groups I and II could be caused by the loss of GSH due to efflux from the cells to the medium (1) as well as to the catabolism produced during the 120 min of rewarming. When the hepatocytes were incubated with 3 mM GSH (group III), the GSH t level was statistically different (P ⬍ 0.05) with respect to groups (I) and (II). In this case the GSH t level at time zero was 51.43 ⫾ 5.40; 42.65 ⫾ 4.89 at 60 min; and 17.88 ⫾ 4.92 after 120 min of reoxygenation. However, the GSH levels were statistically higher (P ⬍ 0.05) in groups (IV) and (V) than in the other groups, but no difference was observed between them. The GSH t content of
hepatocytes from group IV, which had been incubated in the presence of 80 M GSH ⫹ 1 mM methionine was 54.75 ⫾ 4.86 at zero time; 36.25 ⫾ 7.04 at 60 min; and 31.75 ⫾ 4.92 after 120 min of reoxygenation. When the hepatocytes were rewarmed in the presence of 1 mM methionine (group V) the GSH t content was 55.75 ⫾ 4.44 at zero time; 37.25 ⫾ 4.57 at 60 min; and 33.75 ⫾ 5.26 at 120 min. The time course of cellular GSH in freshly isolated hepatocytes incubated in KHR without GSH or methionine was 55.6 ⫾ 4.6 at time zero and 30.25 ⫾ 4.1 nmol GSH/10 6 cells (n ⫽ 4) after 120 min (see Fig. 2, dotted line). These values correlate with those obtained when preserved hepatocytes were incubated with solutions IV and V. Evolution of LDH release during the rewarming step. In this study we used LDH release to assess hepatocyte injury because it is a good measure of membrane integrity for cells in suspension (6). The LDH released from hepatocytes resuspended at 37°C and rewarmed in the different solutions up to 120 min are shown in Fig. 3. The data from groups IV and V show statistical differences (P ⬍ 0.05) after 120 min of rewarming. LDH release was lower for the
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cells incubated in presence of 80 M GSH ⫹ 1 mM methionine (group IV) and 1 mM methionine (group V) with respect to cells incubated in the other solutions (9.67 ⫾ 0.76% for group IV, 10.33 ⫾ 1.53% for group V, 18.11 ⫾ 2.30% for group I, 16.33 ⫾ 2.15% for group II, and 15.67 ⫾ 3.31% for group III). Furthermore, the amount of LDH released by freshly isolated hepatocytes after 120 min of rewarming in KHR were similar to the values obtained for groups IV and V (see Fig. 3, dotted line 11.33 ⫾ 2.15%; n ⫽ 6). This result indicates the superior performance of cells rewarmed in solutions IV and V rather than solutions I, II, and III. We observed the loss of cellular GSH during the rewarming step in all groups, and hepatocytes rewarmed in an amino-acid-free medium (KHR) after cold storage were unable to regenerate GSH; their GSH content decreased. This depletion leads to changes in many metabolic processes and particularly in the accessibility of GSH for detoxification reactions. These phenomena will then enhance the susceptibility of cells to additional stresses, such as those involved in hepatocellular transplantation or the bioartificial liver. When we added 1 mM methionine or 1 mM methionine with 80 M GSH to the rewarming solution the depletion of GSH after 120 min of reoxygenation was smaller and cellular viability was better maintained. Indeed, the GSH t content of these cells was similar to values found when fresh hepatocytes were rewarmed in KHR for 120 min. The depletion of GSH below a critical level during the rewarming step is associated with an increased LDH release, indicating that cellular viability is being affected. Another solution, the “Carolina rinse,” was originally developed to inhibit reperfusion injury (8). It contains some of the substances we used in our rewarming solution—GSH, adenosine, fructose, allopurinol. This solution was shown to decrease damage during the preservation of rodent livers in an orthotopic liver transplantation model and in clinical liver transplantation even with short ischemia times. It is reasonable to think that the protective effect observed following the addition of 1 mM methionine to the rewarming solution is medi-
ated through an inhibition of GSH efflux from the isolated hepatocytes (1). The mechanism of such an effect is not clear and has not been studied in this work, but Kaplowitz and colleagues (2) has suggested an inhibition of a carrier-mediated GSH transport process. Krebs et al. (4) have shown that the addition of methionine to the incubation medium maintains GSH concentration in rat hepatocytes at concentrations from 0.2 to 1 mM. Since the normal concentration of methionine in the rat plasma is about 0.04 mM and in the liver is about 0.1 mM, it might be expected that the ability of 1 mM methionine to maintain GSH may be due to a gradual biosynthesis of cysteine from methionine via the cystathionine pathway, which is needed for the resynthesis of GSH. In conclusion, the addition of GSH to the rinsing solution and of methionine to the rewarming solution improves the viability of hepatocytes that were stored in the cold for up to 72 h. This knowledge can contribute to the development of new strategies to maintain functional and viable hepatocytes after cold preservation, to applications in hepatocellular transplantation, and in extrahepatic bioartificial liver support devices. REFERENCES 1. Aw, T. K., Ookhtens, M., and Kaplowitz, N. Inhibition of glutathione efflux from isolated rat hepatocytes by methionine. J. Biol. Chem. 15, 9355–9358 (1984). 2. Aw, T. Y, Ookhtens, M., Clement, R., and Kaplowitz, N. Kinetics of glutathione efflux from isolated rat hepatocytes. Am. J. Physiol. (Gastrointest. Liver Physiol. 250) 13, G236 –G243 (1986). 3. Garcı´a-Ruiz, C., Ferna´ndez Checa, J. C., and Kaplowitz, N. Bi-directional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles. J. Biol. Chem. 267, 22256 –22264 (1992). 4. Krebs, H. A., Hems, R., and Vin˜a, J. Regulation of the hepatic concentration of reduced glutathione. In “Functions of Glutathione in Liver and Kidney” (H. Sies and A. Wendel, Eds), Springer-Verlag, New York, 1978. 5. Mamprin, M. E., Rodriguez, J. V., and Guibert, E. E. The importance of pH in resuspension media on viability of hepatocytes preserved in University of Wisconsin solution. Cell Transplant. 4, 269 –274 (1995).
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6. Marsh, D. C., Hjelmhaug, J. A., Vreugdenhil, P. K., Kerr, J. A., Rice, M. J., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes. III. Effects of resuspension media on viability after up to 7 days of storage. Hepatology 13, 500 –508 (1991). 7. Rodriguez, J. V., Mamprin, M., Mediavilla, M., and Guibert, E. Glutathione (GSH) movements during cold preservation of rat hepatocytes. Cryobiology 36, 236 –244 (1998). 8. Sanchez-Urdazpal, L., Gores, G. J., Lemasters, J. J., Thurman, R. G., Steers, J. L., Wahistrom, H. E.,
Hay, E. I., Porayko, M. K., Wiesner, R. H., and Krom, R. A. Carolina Rinse Solution decreases liver injury during clinical liver transplantation. Transplant. Proc. 25, 1574 –1575 (1993). 9. Tietze, F. Enzymatic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Applications to mammalian blood and other tissues. Anal. Biochem. 27, 502–522 (1969). 10. Vreugdenhil, P. K., Belzer, F. O., and Southard, J. H. Effect of cold storage on tissue and cellular glutathione. Cryobiology 28, 143–149 (1991).