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Cryopreservation of Isolated Rat Hepatocytes: Effects of Iron-Mediated Oxidative Stress on Metabolic Activity
34, 150–156 (1997) CY961993
CRYOBIOLOGY ARTICLE NO.
Cryopreservation of Isolated Rat Hepatocytes: Effects of Iron-Mediated Oxidative Stress on Metabolic Activity PETER DE LOECKER,* BARRY J. FULLER,† VITALI A. KOPTELOV,‡ VALENTIN I. GRISCHENKO,‡ AND WILLIAM DE LOECKER* *Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium; †University Department of Surgery, Royal Free Hospital School of Medicine, London NW3 2QG, England; and ‡Institute for Problems of Cryobiology and Cryomedicine of the Ukraine Academy of Sciences, 310015 Kharkov, Ukraine In an attempt to quantitatively evaluate the destructive effects of free radicals on metabolism, freshly prepared and cryopreserved isolated rat hepatocytes were exposed to and incubated with Fe2/ compounds, reputedly inducing oxygen-derived free radicals (OFR) capable of attacking the lipid structures of cellular membranes. Malondialdehyde (MDA) formation was interpreted as an expression of free radical interaction with polyunsaturated lipids, and in vitro incubations were carried out during the period of constant MDA formation. Protein synthesizing activity was evaluated by incubating control hepatocytes and cells previously exposed to 100 mM of Fe2/, to 100 mM of Fe2/, and 100 mM of desferrioxamine and to 100 mM of desferrioxamine alone with 0.1 mCi of L-[U-14C]isoleucine and in the presence of these compounds. Membrane transport activity was similarly evaluated by following the cellular uptake of a-amino-[1-14C]isobutyric acid. Protein-synthesizing activity of freshly prepared and cryopreserved hepatocytes was not affected by Fe2/ treatment, nor by the additions of the iron chelator desferrioxamine. Amino acid transport, however, was inhibited by 100 mM of Fe2/, but was effectively neutralized by the simultaneous addition of 100 mM of desferrioxamine. Cryopreserved hepatocytes equally presented a significantly inhibited amino acid transport activity over the incubation period. The results suggest that the metabolic depression measured in thawed hepatocytes does not result to any large extent from iron-catalysed OFR effects. When OFR production was deliberately induced, the most significant early change was seen in transmembrane amino acid uptake in both fresh and cryopreserved cells. q 1997 Academic Press
Hepatocyte cryopreservation is of interest in areas of drug toxicity testing, cell transplantation, and development of artificial liver support (6). Current methods allow good recoveries of cells, but often metabolic activity measured immediately after thawing is depressed (8). One theory of freeze-induced damage in biological systems involves the production of harmful oxygen-derived free radicals (OFR), although this is not generally accepted (1, 9). Some studies have demonstrated OFR activity in populations of recovered cells after thawing, but this may have resulted from release of substances from the subpopulation of damaged cells rather than
Received July 5, 1996; accepted November 27, 1996.
the freezing process itself (3). Certainly it is known that hypoxia and in vitro manipulation of cells can cause release of catalytic iron (a transition metal intimately involved in biological OFR production), events which are an inevitable consequence of hepatocyte isolation and cryopreservation protocols (7, 14). The current studies were undertaken to investigate the possible role of iron-induced OFR in depressing hepatocyte metabolism postcryopreservation by using the iron-chelating agent desferrioxamine to remove any catalytic iron before further incubation. Metabolism was measured by protein synthesis and transmembrane amino acid uptake. Further experiments were performed by deliberately inducing ironmediated OFR stress in fresh and cryopreserved hepatocytes to evaluate patterns of change in metabolism where OFR effects
150 0011-2240/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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could be freely demonstrated by increases in lipid peroxidation. MATERIALS AND METHODS
Isolation of Hepatocytes Hepatocytes were prepared from Wistar R rat livers as described in detail for previous experiments (4, 5). The liver was removed and attached to a closed-circuit perfusion system to obtain free hepatocytes by collagenase digestion of the parenchyma. The hepatocytes were suspended at a concentration of 5 1 106 viable cells/ml in Krebs–Ringer bicarbonate buffer (KR). In all experiments a 4-ml Lamino acid supplement (Vamin, Vitrum, Stockholm 12, Sweden) as well as the usual additions of antibiotics were added to 100 ml of KR. Viability of the cells was assessed by the trypan blue exclusion test before and after exposure and incubation with the different additions (4). Only preparations containing a minimum of 85% viable cells were used throughout these experiments.
Iron Stress Experiments
Cryopreservation After exposure to 1.8 M of dimethylsulfoxide for 30 min at 207C, the samples placed in polypropylene tubes were progressively cooled at 17C/min to 077C in an alcohol bath (Fryka-Therm-FT 8000, Copenhagen, Denmark) and after dissipation of latent crystallization heat further to 0387C. At that temperature the samples were placed for 30 min in the gas phase, followed by submersion into the liquid nitrogen, and preserved at 01967C for up to 15 days. After thawing in a waterbath at 377C, the cells were twice, diluted 101, and washed to remove the cryoprotectant. An original volume of fresh buffer containing the Fe2/ and desferrioxamine additions was added to the sedimented hepatocytes ready to be incubated. Incubation Procedures To evaluate protein synthesizing activity, incubation of 1.0 ml of the freshly prepared or cryopreserved hepatocytes took place for
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up to 60 min at 377C in KR containing 0.1 mCi of L-[U-14C]isoleucine (spec radioact 150 mCi/mmol; Amersham International, Amersham, Bucks, UK) as well as the appropriate Fe2/ and desferrioxamine additions. After incubation, protein synthesis was blocked by the addition of 5 ml of trichloroacetic acid (Merck, TCA 20%). This was followed by the usual washing and counting procedures (4). Amino acid transport activity was followed by incubating the fresh or the cryopreserved hepatocytes in the presence of 0.1 mCi of aamino-[1-14C]isobutyric acid (AIB, spec radioact 60 mCi/mmol; Amersham International) and with the appropriate Fe2/ and desferrioxamine additions. After the incubation period, the hepatocytes were diluted with 10 ml ice-cold fresh KR containing 30 mmol of nonradioactive AIB (Sigma) to arrest amino acid transport. After a low-speed centrifugation and three subsequent identical washes, the cells were further processed to assess the intracellular radioactivity (4).
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To each of the experimental samples of hepatocytes 100 ml of a freshly prepared stock solution of FeSO4r7H2O (Merck) was added to obtain a final concentration of 1, 10, 100, or 1000 mM of Fe2/. The basic experiments however were carried out with an addition of 100 mM of Fe2/. To some experiments 100 mM of ascorbic acid and 100 mM of citric acid were added to further ensure iron to remain in its ferrous form. To neutralize the ironmediated generation of oxygen-derived free radicals, 100 mM of the iron chelator desferrioxamine mesylate (desferoxamine mesylate, Sigma) was added together with the Fe2/ supplement. To evaluate possible toxicity, 100 mM of desferrioxamine alone was added to separate groups. Samples of hepatocytes, from all control and treated groups, were taken, and the thiobarbituric acid-reactive compound malondialdehyde (MDA) as stable end-product of lipid peroxidation was determined according to the Yagi method (13). Reaction tubes contained 7% (w/v) sodium dodecyl sul-
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fate (0.2 ml), 0.1 M HCl (2.0 ml), 10% (w/v) phosphotungstic acid (0.3 ml), and 0.67% (w/ v) thiobarbituric acid (1.0 ml) (Merck) to which 0.1 ml of a tissue sample was added, this being the total content of an original experimental sample, hepatocytes, and supernatant and containing approx 0.5–1.0 mg/protein (12). After heating at 957C for 60 min, the tubes were cooled and 5 ml of butan-1-ol (Merck) was added. After mixing and centrifugation, the chromophore in the solvent phase was evaluated by spectrofluorometry (l ex. 515 nm; l em. 540–560 nm) (Aminco SPF500 Ratio spectrofluorimeter). Malondialdehyde (Merck) was used to obtain the standard curve. Statistical Evaluation Radioactive measurements were expressed as disintegrations per min (dpm) per 1 1 106 viable hepatocytes { SEM. The concentration of MDA found in the tissue samples was expressed as nmol MDA/mg protein { SEM. Statistical evaluation of significance was carried out by analysis of variance (ANOVA). Each result in a specific group of data was compared to the control values by the Dunnett’s test. Where appropriate, pairwise comparisons were made by the Tukey– Kramer test. Differences of P õ 0.01 were considered to be significant. RESULTS 2/
Effects of Free Fe Concentrations on Lipid Peroxidation in Hepatocytes Freshly prepared hepatocytes were exposed to different concentrations of Fe2/ from 0 to 1000 mM for 30 min followed by incubation at 377C for 60 min. MDA formation as a stable end product of lipid peroxidation was measured in samples of hepatocytes. The results demonstrated that at Fe2/ concentrations of 100 mM or more, maximal MDA formation was induced, amounting to 16.8 { 1.2 nmol/mg protein and significantly higher than the controls (P õ 0.01) (Fig. 1). With cryopreserved hepato-
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cytes, the MDA pattern obtained after identical exposure was not significantly different. Hereafter, the chosen concentration used for iron stress was 100 mM in most instances. Ascorbic acid and citric acid added complementary did not seem to modify the ferrous induced effects. Exposure of fresh or cryopreserved hepatocytes to a combination of 100 mM of Fe2/ and 100 mM of desferrioxamine or to 100 mM of desferrioxamine alone reduced MDA levels to control values. Effects of Free Fe2/ on AIB Uptake in Fresh and Cryopreserved Cells Following these initial studies, further experiments were performed to measure AIB uptake in fresh and cryopreserved hepatocytes. The AIB uptake in fresh hepatocytes after 60 min of incubation amounted to 4800 { 380 dpm/106 cells. Addition of 100 mM Fe2/ reduced AIB uptake in fresh hepatocytes by 15.7% (P õ 0.01) after 40 min and by 20.8% (P õ 0.01) after 60 min of incubation. When desferrioxamine (100 mM) was added to fresh hepatocytes, AIB uptake was similar to that of untreated control cells, and when both desferrioxamine and Fe2/ were added together at 100 mM, again AIB uptake was not significantly changed from the fresh control series (Fig. 2). After cryopreservation, AIB uptake in hepatocytes was already significantly lower than that in fresh cells (2206 { 367 dpm/106 cells). Addition of Fe2/ at 100 mM to induce oxidative stress further reduced AIB uptake in cryopreserved cells by 37.2–47.8% (P õ 0.01) compared to the value in cryopreserved cells without addition of Fe2/. When desferrioxamine alone (100 mM) or desferrioxamine combined with 100 mM Fe2/ was added to cryopreserved cells, AIB uptake at 60 min was not different from that seen in untreated cryopreserved cells (Fig. 2). Effects of Free Fe2/ Stress on 14C-AIB Uptake Additions of various concentrations of Fe2/ up to 1000 mM were made, and AIB uptake
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FIG. 1. Effect of Fe2/ concentrations on malondialdehyde (MDA) formation. Freshly prepared hepatocytes in KR (5 1 106 cells/ml) were exposed to different concentrations (1.0, 10, 100, and 1000 mM) of Fe2/ for 30 min, followed by incubation at 377C for 60 min, after which the MDA formed was evaluated by taking 0.1 ml of the sample (cells suspended in buffer) to react with sodium dodecyl sulfate reagent (Materials and Methods). MDA values were expressed as nmol/mg protein. Each value represents the mean of six experiments { SEM. P õ 0.01 (*) indicates significant difference from the controls.
was assessed. It can be seen that Fe2/ concentrations up to 10 mM had no effect on AIB uptake. However, when Fe2/ was increased to 100 or 1000 mM, a significant reduction in AIB uptake was seen. After 40 min of incubation, inhibition by Fe2/ (1000 mM) reduced AIB uptake by 22.5% (P õ 0.01) compared to the Fe2/ (100 mM)-treated samples. After 60 min of incubation, Fe2/ (1000 mM) further reduced AIB uptake by 18.4% (P õ 0.01) compared to the samples treated with Fe2/ (100 mM) (Fig. 3).
fresh hepatocytes. Similarly, although untreated cryopreserved hepatocytes showed a generally reduced incorporation of [14C]isoleucine compared to fresh cells, addition of Fe2/ alone, desferrioxamine alone, or the two agents combined did not alter significantly the incorporation of [14C]isoleucine into the proteins in cryopreserved hepatocytes (Fig. 4). Throughout these experiments, viability assessed by the trypan blue exclusion test was not affected by the addition of Fe2/, desferrioxamine, or a combination of both.
Effects of Cryopreservation and Fe2/ Stress on Incorporation of [14C]Isoleucine into Proteins
DISCUSSION
The incorporation of [14C]isoleucine by fresh control hepatocytes amounted to 1133 { 52 dpm/106 cells after 60 min of incubation. After cryopreservation, this incorporation decreased by 78%. When fresh hepatocytes were deliberately exposed to iron-induced stress by incubating with 100 mM Fe2/, 14C incorporation was not different from control values. Addition of desferrioxamine (100 mM) alone or in the presence of Fe2/ (100 mM) also did not change the incorporation of [14C]isoleucine in
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The results of the present study on cryopreserved hepatocyte function confirm those of earlier reports where complex metabolic activities were depressed following thawing (4, 11). This reduction in metabolism has been attributed to a variety of causes, including damage to the plasma membrane causing loss of intracellular homeostasis and ultrastructural alterations in mitochondria, resulting in poor energy production (4). In theory, both of these changes could also promote damage by OFR, particularly if traces of transition metal catalysts such as low-molecular-weight iron com-
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FIG. 2. Effects of Fe2/ and desferrioxamine on the aamino-[1-14C]isobutyric acid uptake by fresh and cryopreserved hepatocytes. Fresh hepatocytes (l) were incubated in KR (5 1 106 viable cells/ml) in the presence of 0.1 mCi of a-amino-[1-14C]isobutyric acid at 377C for up to 60 min. Other samples were first exposed for 30 min at 207C to 100 mCi of Fe2/ (s), 100 mM of Fe2/, and 100 mM of desferrioxamine (.) or to 100 mM of desferrioxamine alone (,) before being incubated in the buffer containing the respective additions. Hepatocytes to be cryopreserved in KR (5 1 106 viable cells/ml) were exposed for 30 min to 1.8 M of Me2SO before being frozen and cryopreserved at 01967C. After thawing and washing, the cells were resuspended in fresh KR containing the labeled amino acid analogue. The control series (m) and the experimental series after exposure for 30 min at 207C to 100 mM of Fe2/ (n), to 100 mM of Fe2/, and 100 mM of desferrioxamine (j) or to 100 mM of desferrioxamine alone (h) were incubated for up to 60 min at 377C in the presence of the mentioned additions. To avoid overlapping on the graph, some data from different experimental groups are slightly moved from their exact time values of 20, 40, and 60 min, respectively. Uptake values are expressed as disintegrations per min (dpm) per 1 1 106 originally viable cells. Each value represents the mean of six experiments { SEM. P õ 0.01 (*) indicates significant difference from corresponding controls.
plexes were released from damaged cells. Such oxidative stress has been monitored in other cell types after thawing, and treatment of thawed cells with the iron chelator desferrioxamine has enhanced recovery (2, 3). In the present study, treatment of thawed cells with desferrioxamine at concentrations of 100 mM produced no measurable improvement in ei-
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ther uptake of amino acid analogue AIB or incorporation of radiolabeled isoleucine into protein. Similar concentrations of desferrioxamine have been shown to inhibit iron-catalyzed OFR reactions in cell or tissue systems exposed to hypoxia and/or hypothermia, and under those conditions improvements in some indices of function were recorded (10, 14). This leads us to believe that the reduced metabolism reported in hepatocytes after cryopreservation is unlikely to result to any significant degree from iron-catalyzed OFR interactions. In the experiments where OFR production was deliberately stimulated by adding free iron salts, different responses were noted in the two different metabolic pathways. Iron at concentrations of 100 mM or more produced
FIG. 3. Effects of different concentrations of Fe2/ on the a-amino-[1-14C]isobutyric acid uptake by fresh hepatocytes. Freshly prepared hepatocytes (5 1 106 viable cells/ml) were exposed to different concentrations of Fe2/ (FeSO4 .7H2O) for 30 min at 207C, before being incubated at 377C for 60 min in the presence of 0.1 mCi of a-amino[1-14C]isobutyric acid. The KR buffer with the control cells (l) did not contain any additions. Other samples contained 1.0 mM of Fe2/ (s), 10 mM of Fe2/ (j), 100 mM of Fe2/ (h), and 1000 mM of Fe2/ (m). After incubation, the cells were processed as for Fig. 2. Each value represents the mean of six experiments { SEM. P õ 0.01 (*) indicates significance between groups treated with Fe2/, 100 and 1000 mM, respectively, after 40 and 60 min of incubation.
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FIG. 4. Effects of Fe2/ and desferrioxamine on the incorporation of L-[U-14C]isoleucine into the proteins of fresh and cryopreserved hepatocytes. Freshly prepared hepatocytes were incubated in KR (5 1 106 viable cells/ml) in the presence of 0.1 mCi of L-[U-14C]isoleucine at 377C for up to 60 min (l). Other samples were first exposed for 30 min at 207C to 100 mM of Fe2/ (s), 100 mM of Fe2/, and 100 mM of desferrioxamine (.) or to 100 mM of desferrioxamine alone (,), before being incubated in the buffer containing the mentioned additions. The hepatocytes to be cryopreserved in KR (5 1 106 viable cells/ ml) were exposed for 30 min to 1.8 M of Me2SO. After cryopreservation at 01967C and thawing, the samples were washed and resuspended in fresh KR containing the labeled amino acid and were incubated as for the freshly prepared series. The control cryopreserved (m) hepatocytes contained no other additions, while the experimental groups were incubated after being exposed to and in the presence of 100 mM of Fe2/ (n), 100 mM of Fe2/, and 100 mM of desferrioxamine (j) or 100 mM of desferrioxamine alone (h). Incorporation values are expressed as disintegrations per min (dpm) per 1 1 106 originally viable cells. Each value represents the mean of six experiments { SEM.
significant oxidative stress in exposed hepatocytes as indicated by increases in products of lipid peroxidation, and this is in line with effects seen using isolated membrane vesicles (10). By treating the hepatocytes simultaneously with an equal concentration of desferrioxamine, the burst of lipid peroxidation could be completely inhibited. In both fresh and cryopreserved cells, iron-induced OFR production reduced the uptake of radiolabeled AIB after incubation for 1 h. The reduction
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for cryopreserved cells after 1 h (45%) was greater than that in fresh suspensions of hepatocytes (21%), although AIB uptake was already significantly reduced in the thawed cells incubated without added iron salts. In both cases, desferrioxamine treatment prevented the iron-induced depression of AIB uptake. This may suggest that by causing lipid peroxidation in the phospholipid environment of the plasma membrane, externally added iron salts could affect the efficiency of the transport process itself, either by directly disrupting the conformation of the transporter protein or altering the fluidity of the phospholipid environment adjacent to the transporter. Similar inhibition of activity in proteins of mitochondrial energy-transducing complexes has been reported after stimulating OFR production with exogenous iron salts (15). In contrast, addition of iron salts did not affect the rate of incorporation of 14C-labeled isoleucine into protein by either fresh or cryopreserved hepatocytes under the selected conditions of incubation. This may be explained by the early effects of iron-induced OFR damage being limited to the external plasma membrane, avoiding damage to ribosomal and mitochondrial functions necessary for peptide synthesis. Since the data on AIB uptake suggested a reduction (but not complete inhibition) of amino acid transport, we can only assume that under the conditions of incubation, transport of 14C-labeled isoleucine was not a limiting factor for protein synthesis. This may be a reasonable assumption because high concentrations of amino acids were supplied in the supplement added to the incubation medium. Most likely a more prolonged exposure to iron-induced OFR would eventually reduce protein synthesis activity as more products of denatured lipids and proteins accumulate within the cells and the available transported amino acids become depleted. In summary, the OFR theory of injury during cryopreservation of hepatocytes does not seem to be a significant factor in early cell function following thawing unless high concentrations of free radicals are induced into the system.
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The authors are indebted to the Belgian National Foundation for Medical Research and the INTAS of EC for a research grant. They express their appreciation to Mrs. F. De Wever for her excellent technical assistance.
8.
9. REFERENCES 1. Ashwood-Smith, M. J. Genetic damage is not produced by normal cryopreservation procedures involving either glycerol or dimethyl sulfoxide: A cautionary note, however, on the possible effects of dimethyl sulfoxide. Cryobiology 22, 427–433 (1985). 2. Benson, E. E., and Lynch, P. T. Enhancement of postthaw recovery in cryopreserved rice cells treated with desferrioxamine. Cryo-Letters 16, 317 (1995). 3. Benson, E. E., and Noronha-Dutra, A. A. Chemiluminescence in cryopreserved plant tissue cultures: The possible involvement of singlet oxygen in cryoinjury. Cryo-Letters 9, 120–131 (1988). 4. De Loecker, R., Fuller, B. J., Gruwez, J., and De Loecker, W. The effects of cryopreservation on membrane integrity, membrane transport and protein synthesis in rat hepatocytes. Cryobiology 27, 143–152 (1990). 5. De Loecker, P., Fuller, B. J., Koptelov, V. A., and De Loecker, W. Metabolic activity of freshly prepared and cryopreserved hepatocytes in monolayer culture. Cryobiology 30, 12–18 (1993). 6. Fuller, B. J., and De Loecker, W. Cryopreservation of isolated hepatocytes. Transplantology 5, 147– 150 (1994). 7. Fuller, B. J., Gower, J. D., and Green, C. J. Free radi-
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10.
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cal damage and organ preservation: Fact or fiction? Cryobiology 25, 377–393 (1988). Fuller, B. J., Morris, G. J., Nutt, L., and Attenburrow, V. D. Functional recovery of isolated rat hepatocytes upon thawing from 01967C. Cryo-Letters 1, 139–146 (1980). Heckley, R. J., and Quay, J. Adventitious chemistry at reduced water activities: Free radicals and polyhydroxy agents. Cryobiology 20, 613–624 (1983). Horton, R., Rice-Evans, C., and Fuller, B. J. The effects of iron-mediated oxidative stress in isolated renal cortical brush border membrane vesicles at normothermic and hypothermic temperatures. Free Rad. Res. Commun. 5, 267–275 (1989). Innes, G. K., Fuller, B. J., and Hobbs, K. E. F. Functional testing of hepatocytes following their recovery from cryopreservation. Cryobiology 25, 23– 30 (1988). Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951). Ochkawa, H., Ohishi, N., and Yagi, K. Assay for lipid peroxides in animal tissue by thiobarbituric acid reactions. Anal. Biochem. 95, 351–355 (1979). Paller, M. S., and Hedlund, B. E. Extracellular iron chelators protect kidney cells from hypoxia/reoxygenation. Free Rad. Biol. Med. 17, 597–603 (1994). Tru¨mper, L., Hoffman, B., Wiswedel, I., and Augustin, W. Impairment of the respiratory chain in the b–c1 region as early functional event during Fe2//ascorbate induced peroxidation in rat liver mitochondria. Biomed. Biochim. Acta 47, 933– 939 (1988).
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Cryopreservation of Isolated Rat Hepatocytes: Effects of Iron-Mediated Oxidative Stress on Metabolic Activity PETER DE LOECKER,* BARRY J. FULLER,† VITALI A. KOPTELOV,‡ VALENTIN I. GRISCHENKO,‡ AND WILLIAM DE LOECKER* *Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit te Leuven, B-3000 Leuven, Belgium; †University Department of Surgery, Royal Free Hospital School of Medicine, London NW3 2QG, England; and ‡Institute for Problems of Cryobiology and Cryomedicine of the Ukraine Academy of Sciences, 310015 Kharkov, Ukraine In an attempt to quantitatively evaluate the destructive effects of free radicals on metabolism, freshly prepared and cryopreserved isolated rat hepatocytes were exposed to and incubated with Fe2/ compounds, reputedly inducing oxygen-derived free radicals (OFR) capable of attacking the lipid structures of cellular membranes. Malondialdehyde (MDA) formation was interpreted as an expression of free radical interaction with polyunsaturated lipids, and in vitro incubations were carried out during the period of constant MDA formation. Protein synthesizing activity was evaluated by incubating control hepatocytes and cells previously exposed to 100 mM of Fe2/, to 100 mM of Fe2/, and 100 mM of desferrioxamine and to 100 mM of desferrioxamine alone with 0.1 mCi of L-[U-14C]isoleucine and in the presence of these compounds. Membrane transport activity was similarly evaluated by following the cellular uptake of a-amino-[1-14C]isobutyric acid. Protein-synthesizing activity of freshly prepared and cryopreserved hepatocytes was not affected by Fe2/ treatment, nor by the additions of the iron chelator desferrioxamine. Amino acid transport, however, was inhibited by 100 mM of Fe2/, but was effectively neutralized by the simultaneous addition of 100 mM of desferrioxamine. Cryopreserved hepatocytes equally presented a significantly inhibited amino acid transport activity over the incubation period. The results suggest that the metabolic depression measured in thawed hepatocytes does not result to any large extent from iron-catalysed OFR effects. When OFR production was deliberately induced, the most significant early change was seen in transmembrane amino acid uptake in both fresh and cryopreserved cells. q 1997 Academic Press
Hepatocyte cryopreservation is of interest in areas of drug toxicity testing, cell transplantation, and development of artificial liver support (6). Current methods allow good recoveries of cells, but often metabolic activity measured immediately after thawing is depressed (8). One theory of freeze-induced damage in biological systems involves the production of harmful oxygen-derived free radicals (OFR), although this is not generally accepted (1, 9). Some studies have demonstrated OFR activity in populations of recovered cells after thawing, but this may have resulted from release of substances from the subpopulation of damaged cells rather than
Received July 5, 1996; accepted November 27, 1996.
the freezing process itself (3). Certainly it is known that hypoxia and in vitro manipulation of cells can cause release of catalytic iron (a transition metal intimately involved in biological OFR production), events which are an inevitable consequence of hepatocyte isolation and cryopreservation protocols (7, 14). The current studies were undertaken to investigate the possible role of iron-induced OFR in depressing hepatocyte metabolism postcryopreservation by using the iron-chelating agent desferrioxamine to remove any catalytic iron before further incubation. Metabolism was measured by protein synthesis and transmembrane amino acid uptake. Further experiments were performed by deliberately inducing ironmediated OFR stress in fresh and cryopreserved hepatocytes to evaluate patterns of change in metabolism where OFR effects
150 0011-2240/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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could be freely demonstrated by increases in lipid peroxidation. MATERIALS AND METHODS
Isolation of Hepatocytes Hepatocytes were prepared from Wistar R rat livers as described in detail for previous experiments (4, 5). The liver was removed and attached to a closed-circuit perfusion system to obtain free hepatocytes by collagenase digestion of the parenchyma. The hepatocytes were suspended at a concentration of 5 1 106 viable cells/ml in Krebs–Ringer bicarbonate buffer (KR). In all experiments a 4-ml Lamino acid supplement (Vamin, Vitrum, Stockholm 12, Sweden) as well as the usual additions of antibiotics were added to 100 ml of KR. Viability of the cells was assessed by the trypan blue exclusion test before and after exposure and incubation with the different additions (4). Only preparations containing a minimum of 85% viable cells were used throughout these experiments.
Iron Stress Experiments
Cryopreservation After exposure to 1.8 M of dimethylsulfoxide for 30 min at 207C, the samples placed in polypropylene tubes were progressively cooled at 17C/min to 077C in an alcohol bath (Fryka-Therm-FT 8000, Copenhagen, Denmark) and after dissipation of latent crystallization heat further to 0387C. At that temperature the samples were placed for 30 min in the gas phase, followed by submersion into the liquid nitrogen, and preserved at 01967C for up to 15 days. After thawing in a waterbath at 377C, the cells were twice, diluted 101, and washed to remove the cryoprotectant. An original volume of fresh buffer containing the Fe2/ and desferrioxamine additions was added to the sedimented hepatocytes ready to be incubated. Incubation Procedures To evaluate protein synthesizing activity, incubation of 1.0 ml of the freshly prepared or cryopreserved hepatocytes took place for
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up to 60 min at 377C in KR containing 0.1 mCi of L-[U-14C]isoleucine (spec radioact 150 mCi/mmol; Amersham International, Amersham, Bucks, UK) as well as the appropriate Fe2/ and desferrioxamine additions. After incubation, protein synthesis was blocked by the addition of 5 ml of trichloroacetic acid (Merck, TCA 20%). This was followed by the usual washing and counting procedures (4). Amino acid transport activity was followed by incubating the fresh or the cryopreserved hepatocytes in the presence of 0.1 mCi of aamino-[1-14C]isobutyric acid (AIB, spec radioact 60 mCi/mmol; Amersham International) and with the appropriate Fe2/ and desferrioxamine additions. After the incubation period, the hepatocytes were diluted with 10 ml ice-cold fresh KR containing 30 mmol of nonradioactive AIB (Sigma) to arrest amino acid transport. After a low-speed centrifugation and three subsequent identical washes, the cells were further processed to assess the intracellular radioactivity (4).
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To each of the experimental samples of hepatocytes 100 ml of a freshly prepared stock solution of FeSO4r7H2O (Merck) was added to obtain a final concentration of 1, 10, 100, or 1000 mM of Fe2/. The basic experiments however were carried out with an addition of 100 mM of Fe2/. To some experiments 100 mM of ascorbic acid and 100 mM of citric acid were added to further ensure iron to remain in its ferrous form. To neutralize the ironmediated generation of oxygen-derived free radicals, 100 mM of the iron chelator desferrioxamine mesylate (desferoxamine mesylate, Sigma) was added together with the Fe2/ supplement. To evaluate possible toxicity, 100 mM of desferrioxamine alone was added to separate groups. Samples of hepatocytes, from all control and treated groups, were taken, and the thiobarbituric acid-reactive compound malondialdehyde (MDA) as stable end-product of lipid peroxidation was determined according to the Yagi method (13). Reaction tubes contained 7% (w/v) sodium dodecyl sul-
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fate (0.2 ml), 0.1 M HCl (2.0 ml), 10% (w/v) phosphotungstic acid (0.3 ml), and 0.67% (w/ v) thiobarbituric acid (1.0 ml) (Merck) to which 0.1 ml of a tissue sample was added, this being the total content of an original experimental sample, hepatocytes, and supernatant and containing approx 0.5–1.0 mg/protein (12). After heating at 957C for 60 min, the tubes were cooled and 5 ml of butan-1-ol (Merck) was added. After mixing and centrifugation, the chromophore in the solvent phase was evaluated by spectrofluorometry (l ex. 515 nm; l em. 540–560 nm) (Aminco SPF500 Ratio spectrofluorimeter). Malondialdehyde (Merck) was used to obtain the standard curve. Statistical Evaluation Radioactive measurements were expressed as disintegrations per min (dpm) per 1 1 106 viable hepatocytes { SEM. The concentration of MDA found in the tissue samples was expressed as nmol MDA/mg protein { SEM. Statistical evaluation of significance was carried out by analysis of variance (ANOVA). Each result in a specific group of data was compared to the control values by the Dunnett’s test. Where appropriate, pairwise comparisons were made by the Tukey– Kramer test. Differences of P õ 0.01 were considered to be significant. RESULTS 2/
Effects of Free Fe Concentrations on Lipid Peroxidation in Hepatocytes Freshly prepared hepatocytes were exposed to different concentrations of Fe2/ from 0 to 1000 mM for 30 min followed by incubation at 377C for 60 min. MDA formation as a stable end product of lipid peroxidation was measured in samples of hepatocytes. The results demonstrated that at Fe2/ concentrations of 100 mM or more, maximal MDA formation was induced, amounting to 16.8 { 1.2 nmol/mg protein and significantly higher than the controls (P õ 0.01) (Fig. 1). With cryopreserved hepato-
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cytes, the MDA pattern obtained after identical exposure was not significantly different. Hereafter, the chosen concentration used for iron stress was 100 mM in most instances. Ascorbic acid and citric acid added complementary did not seem to modify the ferrous induced effects. Exposure of fresh or cryopreserved hepatocytes to a combination of 100 mM of Fe2/ and 100 mM of desferrioxamine or to 100 mM of desferrioxamine alone reduced MDA levels to control values. Effects of Free Fe2/ on AIB Uptake in Fresh and Cryopreserved Cells Following these initial studies, further experiments were performed to measure AIB uptake in fresh and cryopreserved hepatocytes. The AIB uptake in fresh hepatocytes after 60 min of incubation amounted to 4800 { 380 dpm/106 cells. Addition of 100 mM Fe2/ reduced AIB uptake in fresh hepatocytes by 15.7% (P õ 0.01) after 40 min and by 20.8% (P õ 0.01) after 60 min of incubation. When desferrioxamine (100 mM) was added to fresh hepatocytes, AIB uptake was similar to that of untreated control cells, and when both desferrioxamine and Fe2/ were added together at 100 mM, again AIB uptake was not significantly changed from the fresh control series (Fig. 2). After cryopreservation, AIB uptake in hepatocytes was already significantly lower than that in fresh cells (2206 { 367 dpm/106 cells). Addition of Fe2/ at 100 mM to induce oxidative stress further reduced AIB uptake in cryopreserved cells by 37.2–47.8% (P õ 0.01) compared to the value in cryopreserved cells without addition of Fe2/. When desferrioxamine alone (100 mM) or desferrioxamine combined with 100 mM Fe2/ was added to cryopreserved cells, AIB uptake at 60 min was not different from that seen in untreated cryopreserved cells (Fig. 2). Effects of Free Fe2/ Stress on 14C-AIB Uptake Additions of various concentrations of Fe2/ up to 1000 mM were made, and AIB uptake
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FIG. 1. Effect of Fe2/ concentrations on malondialdehyde (MDA) formation. Freshly prepared hepatocytes in KR (5 1 106 cells/ml) were exposed to different concentrations (1.0, 10, 100, and 1000 mM) of Fe2/ for 30 min, followed by incubation at 377C for 60 min, after which the MDA formed was evaluated by taking 0.1 ml of the sample (cells suspended in buffer) to react with sodium dodecyl sulfate reagent (Materials and Methods). MDA values were expressed as nmol/mg protein. Each value represents the mean of six experiments { SEM. P õ 0.01 (*) indicates significant difference from the controls.
was assessed. It can be seen that Fe2/ concentrations up to 10 mM had no effect on AIB uptake. However, when Fe2/ was increased to 100 or 1000 mM, a significant reduction in AIB uptake was seen. After 40 min of incubation, inhibition by Fe2/ (1000 mM) reduced AIB uptake by 22.5% (P õ 0.01) compared to the Fe2/ (100 mM)-treated samples. After 60 min of incubation, Fe2/ (1000 mM) further reduced AIB uptake by 18.4% (P õ 0.01) compared to the samples treated with Fe2/ (100 mM) (Fig. 3).
fresh hepatocytes. Similarly, although untreated cryopreserved hepatocytes showed a generally reduced incorporation of [14C]isoleucine compared to fresh cells, addition of Fe2/ alone, desferrioxamine alone, or the two agents combined did not alter significantly the incorporation of [14C]isoleucine into the proteins in cryopreserved hepatocytes (Fig. 4). Throughout these experiments, viability assessed by the trypan blue exclusion test was not affected by the addition of Fe2/, desferrioxamine, or a combination of both.
Effects of Cryopreservation and Fe2/ Stress on Incorporation of [14C]Isoleucine into Proteins
DISCUSSION
The incorporation of [14C]isoleucine by fresh control hepatocytes amounted to 1133 { 52 dpm/106 cells after 60 min of incubation. After cryopreservation, this incorporation decreased by 78%. When fresh hepatocytes were deliberately exposed to iron-induced stress by incubating with 100 mM Fe2/, 14C incorporation was not different from control values. Addition of desferrioxamine (100 mM) alone or in the presence of Fe2/ (100 mM) also did not change the incorporation of [14C]isoleucine in
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The results of the present study on cryopreserved hepatocyte function confirm those of earlier reports where complex metabolic activities were depressed following thawing (4, 11). This reduction in metabolism has been attributed to a variety of causes, including damage to the plasma membrane causing loss of intracellular homeostasis and ultrastructural alterations in mitochondria, resulting in poor energy production (4). In theory, both of these changes could also promote damage by OFR, particularly if traces of transition metal catalysts such as low-molecular-weight iron com-
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FIG. 2. Effects of Fe2/ and desferrioxamine on the aamino-[1-14C]isobutyric acid uptake by fresh and cryopreserved hepatocytes. Fresh hepatocytes (l) were incubated in KR (5 1 106 viable cells/ml) in the presence of 0.1 mCi of a-amino-[1-14C]isobutyric acid at 377C for up to 60 min. Other samples were first exposed for 30 min at 207C to 100 mCi of Fe2/ (s), 100 mM of Fe2/, and 100 mM of desferrioxamine (.) or to 100 mM of desferrioxamine alone (,) before being incubated in the buffer containing the respective additions. Hepatocytes to be cryopreserved in KR (5 1 106 viable cells/ml) were exposed for 30 min to 1.8 M of Me2SO before being frozen and cryopreserved at 01967C. After thawing and washing, the cells were resuspended in fresh KR containing the labeled amino acid analogue. The control series (m) and the experimental series after exposure for 30 min at 207C to 100 mM of Fe2/ (n), to 100 mM of Fe2/, and 100 mM of desferrioxamine (j) or to 100 mM of desferrioxamine alone (h) were incubated for up to 60 min at 377C in the presence of the mentioned additions. To avoid overlapping on the graph, some data from different experimental groups are slightly moved from their exact time values of 20, 40, and 60 min, respectively. Uptake values are expressed as disintegrations per min (dpm) per 1 1 106 originally viable cells. Each value represents the mean of six experiments { SEM. P õ 0.01 (*) indicates significant difference from corresponding controls.
plexes were released from damaged cells. Such oxidative stress has been monitored in other cell types after thawing, and treatment of thawed cells with the iron chelator desferrioxamine has enhanced recovery (2, 3). In the present study, treatment of thawed cells with desferrioxamine at concentrations of 100 mM produced no measurable improvement in ei-
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ther uptake of amino acid analogue AIB or incorporation of radiolabeled isoleucine into protein. Similar concentrations of desferrioxamine have been shown to inhibit iron-catalyzed OFR reactions in cell or tissue systems exposed to hypoxia and/or hypothermia, and under those conditions improvements in some indices of function were recorded (10, 14). This leads us to believe that the reduced metabolism reported in hepatocytes after cryopreservation is unlikely to result to any significant degree from iron-catalyzed OFR interactions. In the experiments where OFR production was deliberately stimulated by adding free iron salts, different responses were noted in the two different metabolic pathways. Iron at concentrations of 100 mM or more produced
FIG. 3. Effects of different concentrations of Fe2/ on the a-amino-[1-14C]isobutyric acid uptake by fresh hepatocytes. Freshly prepared hepatocytes (5 1 106 viable cells/ml) were exposed to different concentrations of Fe2/ (FeSO4 .7H2O) for 30 min at 207C, before being incubated at 377C for 60 min in the presence of 0.1 mCi of a-amino[1-14C]isobutyric acid. The KR buffer with the control cells (l) did not contain any additions. Other samples contained 1.0 mM of Fe2/ (s), 10 mM of Fe2/ (j), 100 mM of Fe2/ (h), and 1000 mM of Fe2/ (m). After incubation, the cells were processed as for Fig. 2. Each value represents the mean of six experiments { SEM. P õ 0.01 (*) indicates significance between groups treated with Fe2/, 100 and 1000 mM, respectively, after 40 and 60 min of incubation.
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FIG. 4. Effects of Fe2/ and desferrioxamine on the incorporation of L-[U-14C]isoleucine into the proteins of fresh and cryopreserved hepatocytes. Freshly prepared hepatocytes were incubated in KR (5 1 106 viable cells/ml) in the presence of 0.1 mCi of L-[U-14C]isoleucine at 377C for up to 60 min (l). Other samples were first exposed for 30 min at 207C to 100 mM of Fe2/ (s), 100 mM of Fe2/, and 100 mM of desferrioxamine (.) or to 100 mM of desferrioxamine alone (,), before being incubated in the buffer containing the mentioned additions. The hepatocytes to be cryopreserved in KR (5 1 106 viable cells/ ml) were exposed for 30 min to 1.8 M of Me2SO. After cryopreservation at 01967C and thawing, the samples were washed and resuspended in fresh KR containing the labeled amino acid and were incubated as for the freshly prepared series. The control cryopreserved (m) hepatocytes contained no other additions, while the experimental groups were incubated after being exposed to and in the presence of 100 mM of Fe2/ (n), 100 mM of Fe2/, and 100 mM of desferrioxamine (j) or 100 mM of desferrioxamine alone (h). Incorporation values are expressed as disintegrations per min (dpm) per 1 1 106 originally viable cells. Each value represents the mean of six experiments { SEM.
significant oxidative stress in exposed hepatocytes as indicated by increases in products of lipid peroxidation, and this is in line with effects seen using isolated membrane vesicles (10). By treating the hepatocytes simultaneously with an equal concentration of desferrioxamine, the burst of lipid peroxidation could be completely inhibited. In both fresh and cryopreserved cells, iron-induced OFR production reduced the uptake of radiolabeled AIB after incubation for 1 h. The reduction
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for cryopreserved cells after 1 h (45%) was greater than that in fresh suspensions of hepatocytes (21%), although AIB uptake was already significantly reduced in the thawed cells incubated without added iron salts. In both cases, desferrioxamine treatment prevented the iron-induced depression of AIB uptake. This may suggest that by causing lipid peroxidation in the phospholipid environment of the plasma membrane, externally added iron salts could affect the efficiency of the transport process itself, either by directly disrupting the conformation of the transporter protein or altering the fluidity of the phospholipid environment adjacent to the transporter. Similar inhibition of activity in proteins of mitochondrial energy-transducing complexes has been reported after stimulating OFR production with exogenous iron salts (15). In contrast, addition of iron salts did not affect the rate of incorporation of 14C-labeled isoleucine into protein by either fresh or cryopreserved hepatocytes under the selected conditions of incubation. This may be explained by the early effects of iron-induced OFR damage being limited to the external plasma membrane, avoiding damage to ribosomal and mitochondrial functions necessary for peptide synthesis. Since the data on AIB uptake suggested a reduction (but not complete inhibition) of amino acid transport, we can only assume that under the conditions of incubation, transport of 14C-labeled isoleucine was not a limiting factor for protein synthesis. This may be a reasonable assumption because high concentrations of amino acids were supplied in the supplement added to the incubation medium. Most likely a more prolonged exposure to iron-induced OFR would eventually reduce protein synthesis activity as more products of denatured lipids and proteins accumulate within the cells and the available transported amino acids become depleted. In summary, the OFR theory of injury during cryopreservation of hepatocytes does not seem to be a significant factor in early cell function following thawing unless high concentrations of free radicals are induced into the system.
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The authors are indebted to the Belgian National Foundation for Medical Research and the INTAS of EC for a research grant. They express their appreciation to Mrs. F. De Wever for her excellent technical assistance.
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