CRYOBIOLOGY
28, 1-7 (1991)
Effect of Polyethylene
Glycol on Lipid Peroxidation Rat Hepatocytes’
J. E. MACK, J. A. KERR, P. K. VREUGDENHIL, J. H. SOUTHARD
in Cold-Stored
F. 0. BELZER,
AND
Department of Surgery, University of Wisconsin, Madison, Wisconsin 53792 A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause per-oxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondiaIdehyde, MDA). Hepatocytes were stored in the UW solution 2 glutathione (GSH) or k polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37°C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 k 0.73 1 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 -+ 0.564 nmohmg protein. When freshly isolated hepatocytes were incubated (37°C) in the presence of iron (160 CLM)MDA formation was maximally stimulated (3.314 f 0.941 nmoVmg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmoYmg protein at 24 h and 0.752 nmol/mg protein at 48 h). In the absence of PEG, iron-stimulated MDA formation was nearly maximal at all times of storage. These results show that lipid peroxidation is stimulated by cold storage of hepatocytes. Inclusion of PEG in the storage medium suppressed lipid peroxidation suggesting that PEG is accumulated, in a time-dependent manner, by hepatocytes (either into the plasma membrane or into the cell cytosol) and either scavenges oxygen free radicals or alters the availability of lipids to these radicals. PEG may be a useful additive t0 organ preSerVatiOn SOhtiOnS. 0 1991 Academic Press, Inc.
The complexities inherent in determining stored in an organ preservation solution the mechanisms of cell injury due to drugs, (VW solution) or in a tissue culture metoxins, hypoxia, ischemia, or preservation dium. It was shown that for successful in whole organs have lead a number of in- preservation of hepatocytes, polyethylene vestigators to develop a simpler system us- glycol (3-5 g%) was necessary to suppress ing isolated cells. We (13, 14), as well as hypothermic-induced cell swelling. others (IO, 27), have used isolated hepatoOne mechanism thought to cause loss of cytes to determine how hypothermic pres- organ viability is the generation of oxygen ervation conditions affect cell metabolism free radicals (7, 16). Agents or conditions and viability. We have shown that hepato- that stimulate oxygen free radical generacyte viability can be maintained (based tion often lead to lipid peroxidation and the upon LDH release) for up to 3 days when production of malondialdehyde (MDA). Fuller et al. (4, 5) have shown increased Received January 6, 1989; accepted February 15, MDA formation in tissues following preservation and have implicated an iron1990. mediated mechanism for lipid peroxidation. ’ Supported by NIH Grant 35143. 1 OOll-2240191$3.00 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
2
MACK
ET AL.
ml when used. In some experiments glutathione was omitted. The following groups were studied: Group 1, UW solution containing PEG ? GSH; Group 2, UW solution minus PEG; and Group 3, UW solution minus both PEG and GSH. For the determination of lipid peroxidation thiobarbiturate (malondialdehyde) reactive material was determined by the method of Rush et al. (22). After cold storage, hepatocytes (or freshly prepared hepatocytes) were sedimented (77Og, 5 min) and the storage medium was discarded. The cells were resuspended in a physiologically balanced salt solution (Weinberg et al. (30) solution A) at 5-6 mg protein/ml and an aliquot of this suspension was added to an equal volume of trichloroacetic acid (10%) to precipitate the protein. Protein was removed by centrifugation (14,000 rpm, 2 min: Eppendorf microcentrifugal) and MDA concentration determined in the suMATERIALS AND METHODS pernate fluid. MDA was also determined afHepatocytes were prepared from rats ter normothermic incubation in Weinberg’s (Sprague-Dawley, 20 to 250 g) by the solution A at 37°C under an atmosphere of method of Seglen (23) as described by oxygen (100%) for 30 and 60 min. At the Marsh et al. (14). Only preparations that end of incubation an aliquot of cells was showed less than 10% uptake of trypan blue added to an equal volume of TCA and MDA were used in these studies. Immediately af- determined in the protein-free supernate ter preparation hepatocytes were sus- fluid. Iron-stimulated MDA formation was pended in the preservation solution at 5°C determined as described by Hogberg et al. which had been equilibrated with nitrogen (9) by adding aliquots of a stock solution (100%). Hepatocytes were stored in sealed containing ADP (100 mM) and ferric chlotest tubes at 5°C at a concentration of 5-6 ride (2 mM) to obtain a final concentration mg protein/ml (Biuret method) without of iron of 20 to 320 @Y. The concentration shaking to simulate simple cold storage of of MDA was determined by measuring the absorbance at 535 nm against a blank conorgans (i.e., cold ischemia). The storage (preservation) solution was taining all reagents minus the supernate similar to the UW solution (28) with hy- fluid. The absorbance was compared to that droxyethyl starch and contained: 100 mM obtained with a known concentration of lactobionate (K), 30 mM rafftnose, 5 mM 1,1,3,3-tetraethoxypropane and the results magnesium sulfate, 5 mit4 adenosine, 1 mM were expressed as nmol of MDA/mg proallopurinol , 3 mM glutathione , neutralized tein. with sodium and potassium hydroxides to Experimental results were obtained from give a final concentration of 30 * 5 mM = at least four separate preparations of hepaNa, and 120 ? 5 mM = K, with an osmo- tocytes, MDA assays were performed in at lality of 300 mOsm/liter. Polyethylene gly- least duplicates, and the means were calcuco1(PEG, 8000 mol wt) was added at 5 g/100 lated. The results presented are the means These studies, as well as others (1,31), suggest that suppression of the generation of oxygen free radicals, or effectively scavenging cytotoxic products of oxygen metabolism, may improve the quality of organ preservation. The University of Wisconsin (UW) solution has been shown to effectively preserve the liver (ll), kidney (21), and pancreas (28). This solution contains a number of agents that could act as antioxidants: allopurinol, glutathione (GSH), raffinose, and hydroxyethyl starch. Additionally, PEG used in hepatocyte cold-storage studies (13, 14) could also act as an antioxidant (2). In this study we have determined how preservation of hepatocytes in the UW solution affects lipid peroxidation and how the omission of two potential antioxidants (PEG and GSH) affected the extent of MDA formation.
PEG EFFECT ON COLD-STORED HEPATOCYTES
3
(+-SD) obtained by combining the results from each hepatocyte preparation. Statistical analysis was done using the Student t test.
more MDA on rewarming than hepatocytes stored in the UW solution containing PEG (Group 1) after 48,72, and 96 h of storage (P < 0.05 at 48 and 72 h but not at 96 h). The omission of both PEG and GSH (Group 3) RESULTS resulted in an even greater production of The concentration of MDA in freshly iso- MDA on rewarming when compared to lated hepatocytes averaged 0.335 + 0.055 Group 2 (no PEG). However, a significant nmol/mg protein. There was no significant difference between Group 3 and Group 2 increase in MDA during a 120-min incuba- was obtained only after 48 h of storage and tion of hepatocytes in an atmosphere of ox- rewarming. After 96 h of storage and reygen (refer to Fig. 2, bottom trace, 0 FM warming the MDA concentrations were ADP-Fe). When hepatocytes were stored 1.152 + 0.276 nmol/mg in Group 2 vs 1.618 for up to 96 h at 5°C under an atmosphere of + 0.731 nmol/mg protein in Group 3. nitrogen there was also practically no inThese results show that PEG suppresses crease in MDA compared to freshly iso- MDA formation in cold-stored hepatocytes. PEG was equally effective in the lated hepatocytes. When stored in the UW solution contain- presence or absence of GSH (Group 1). ing PEG (Group 1 + GSH) a significant in- However, GSH did appear to have some crease in MDA occurred (Fig. l), but only antioxidant properties because the omisafter 96 h of storage (0.827 k 0.564 nmol/mg sion of both PEG and GSH (Group 3) from protein vs 0.355 + 0.055 nmol/mg protein, the UW solution resulted in a greater proP < 0.05). The concentration of MDA in duction of MDA than in hepatocytes stored hepatocytes rewarmed after 24, 48, and 72 only in the absence of PEG (Group 2). To determine how iron (ADP-Fe) would h of storage (Group 1) was not significantly different than that in freshly isolated hepa- effect lipid peroxidation in cold-stored hepatocytes, we determined the concentratocytes rewarmed for 120 min. Hepatocytes stored in the UW solution tion of ADP-Fe to maximally stimulate (not containing PEG, Group 2) produced MDA formation in freshly isolated hepatocytes (Fig. 2). In this set of experiments,
0
48
24 Preservation
72
96
Time (hr)
FIG. 1. Effects of cold storage of rat hepatocytes on MDA formation after rewarming. Rat hepatocytes were cold-stored for up to 96 h as described under Materials and Methods. At the end of preservation the cells were incubated at 37°C in a physiologically balanced salt solution for 60 min and the concentration of MDA was determined. Results shown are means (*SD) obtained from at least four separate hepatocyte preparations. *P < 0.05 compared to Group 1.
0
30
60
TIME (MN)
FIG. 2. Effects of iron on MDA formation in freshly isolated hepatocytes. Iron (ADP-Fe) was added to freshly isolated hepatocytes suspended in Weinberg’s solution A and incubated for up to 60 min at 37°C in the presence of oxygen. MDA concentrations were determined as described under Materials and Methods. *P < 0.05 versus 160 @4.
4
MACK ET AL.
freshly isolated hepatocytes contained 0.208 ? 0.028 nmol MDA/mg protein. Maximal MDA production was obtained within a 30-min incubation. The maximal concentration of MDA (a lo-fold increase) was produced by incubation in the presence of 160 Q4 ADP-Fe (2.193 f 0.223 nmol/mg protein, not significantly different from 320 00 The results in Fig. 3 show how iron affected lipid peroxidation in hepatocytes after cold storage for up to 96 h. In this set of experiments the addition of iron (160 PM) to freshly isolated hepatocytes also increased MDA by lo-fold (0.355 ? 0.055 to 3.314 + 0.941 nmoYmg protein). When hepatocytes were cold-stored in UW solution containing PEG 2 GSH (Group l), there was a decrease in the capability of iron to stimulate lipid peroxidation that was dependent upon the time of storage. After 48 h of storage the iron-induced formation of MDA was reduced from 3.314 to 0.752 + 0.293 nmol/mg protein and MDA remained relatively low after rewarming hepatocytes cold-stored for 72 and 96 h. When PEG was omitted from the UW solution (Group 2), there was an initial decrease in the ironstimulated production of MDA after 24 h of storage but after 48 to 96 h of storage the
OJ 0
24
48 Preservation
72 lime
1 96
(hr)
FIG. 3. Effects of iron on MDA production in rat hepatocyte cold-stored for up to 96 h after rewarming. Methods are as described in the legend to Fig. 1 and under Materials and Methods. Iron (ADP-Fe) was added (160 pJ4) at the beginning of normothermic incubation, after the indicated preservation times. *P < 0.05 versus Group 1.
presence of iron-stimulated MDA production (significant at P > 0.05 versus Group 1 at 48-96 h). Iron-stimulated MDA production was maximally obtained after all periods of storage when both PEG and GSH (Group 3) were omitted from the UW solution (P < 0.05 versus Group 1 at 24-72 h). These results show that the storage of hepatocytes in the presence of PEG suppresses iron-stimulated MDA formation. DISCUSSION
The UW solution has been shown to be an effective flushout solution for the preservation of the liver and is currently used clinically (25) with preservation times of up to 35 h. The mechanism of action of the UW solution is not clearly understood and we have recently begun to use isolated hepatocytes as an experimental model to determine how hypothermic preservation affects cellular metabolism. In previous studies (13, 14) we used a preservation model for hepatocytes that simulated continuous perfusion of the liver: hepatocytes were coldstored (S’C) in the presence of oxygen with continuous shaking. In these studies we demonstrated that viability of hepatocytes was dependent upon suppressing hypothermic-induced cell swelling. We showed that even in the presence of impermeants (lactobionic acid, raffinose, and sucrose) hepatocytes became swollen when stored at 5°C. This is unlike whole livers (26) or liver slices (29) which do not gain water when stored in solutions containing these impermeants. This suggested that the process of digestion of the liver tissue with collagenase changed the permeability properties of the plasma membrane of hepatocytes. Therefore, to study the mechanisms of hypothermic-induced cell death in the absence of cell swelling, another agent was required. We found that PEG suppressed hypothermic-induced cell swelling in hepatocytes stored for up to 5 days (14) and have, consequently, included PEG in our studied of isolated hepatocytes. However,
PEG
EFFECT
ON
COLD-STORED
HEPATOCYTES
5
brane bound lipids to initiators of lipid peroxidation by changing membrane structure we have investigated the effects of PEG on or by scavenging initiators before they lipid peroxidation in cold-stored hepato- reach the lipids, similar to the mechanism proposed for the antioxidant properties of cytes . PEG is an effective antioxidant and sup- vitamin E (20). Glutathione is the major thiol in tissue pressed lipid peroxidation after rewarming hepatocytes cold-stored in the UW solution and has an one of its function the reduction for up to 96 h. The effectiveness of PEG of hydrogen peroxide and lipid peroxides was seen in hepatocytes rewarmed in the (for review see (17)). In the presence of hyabsence and presence of iron. The effec- drogen peroxide, GSH is oxidized to GSSG tiveness of PEG as an antioxidant increased (glutathione peroxidase) to form oxygen with time of storage of hepatocytes in the and water. Thus, the reduction in the celUW solution. This was clearly demon- lular concentration of hydrogen peroxide strated when iron-stimulated lipid peroxi- reduces the concentration of hydroxyl raddation was determined. After 24 h of stor- icals formed by the iron-catalyzed HaberWeiss reaction (6). In this study the conage in PEG there was an approximately 50% decrease in iron-stimulated MDA pro- centration of MDA produced by coldduction. After 48 h of storage there was a stored hepatocytes was independent of the presence of GSH when the cells were complete suppression of iron-stimulated MDA production. There are a number of stored in the presence of PEG. However, possible explanations for the time-depen- when stored without PEG the presence of dent effect of PEG. One is that cold storage GSH decreased the amount of MDA proof hepatocytes reduced the capability of the duced in rewarming hepatocytes. The recells to produce the initiators of lipid per- sults show that the omission of both PEG oxidation (superoxide anion, hydroxyl rad- and GSH from the UW solution caused a icals). However, MDA was produced (plus greater increase in MDA formation than or minus iron) in hepatocytes cold-stored in when just the PEG was omitted. Others the absence of PEG and therefore, cold have also shown suppression of lipid perstorage does not appear to affect the gener- oxidation by GSH (8, 15) in models other ation of initiators of lipid peroxidation. A than cold storage. There are a number of studies that sugsecond explanation is that PEG scavenges gest that oxygen free radicals are a factor in initiators of lipid peroxidation (hydroxyl radicals, 19) and to be effective a critical injury to organs following preservation (7, concentration of PEG must be obtained in 16) and other forms of organ injury (12,24). the hepatocytes. In the cold it appears that How these cytotoxic products of oxygen it takes about 48 h for the effective concen- metabolism cause cell death is not clear but tration of PEG to accumulate in hepato- may be related to peroxidation of memcytes. The exact location of PEG in hepa- brane lipids and changes in membrane tocytes is not clear and it could be either in properties (permeability and enzyme activthe cell cytosol or in the cell membranes. ity). One concept in the development of the PEG has been shown to bind to phospho- UW solution was the addition of agents that lipids and accumulate in cell membranes would suppress oxygen free radical injury (3); thus, this is a possible sight for the ac- in preserved organs. In addition to GSH the tion of PEG in suppressing lipid peroxida- UW solution contains allopurinol. Allopution. The presence of PEG in cell mem- rinol is an inhibitor of xanthine oxidase which has been suggested to be a source of branes couuld suppress lipid peroxidation by either altering the availability of mem- superoxide anions during reperfusion of PEG may have other effects on the cell than
just the suppression of cell swelling. Thus,
6
MACK
ischemic or injured organs (16). In this study the UW solution contained allopurinol. However, lipid peroxidation was evident even in the presence of allopurinol, suggesting that xanthine oxidase may not be the only source of superoxide anions in cold-stored livers (hepatocytes) which has been previously suggested (18). The effectiveness of PEG in suppressing lipid peroxidation in this study suggests that PEG might be a useful agent in organ preservation solutions. Wicomb and Collins (32) have recently shown that the UW solution containing PEG is effective for a 24-h preservation of the rabbit heart. This may be due to the effectiveness of PEG as an inhibitor of lipid peroxidation. The usefulness of PEG in the preservation of other organs, however, is not known but these results suggest that PEG may be beneficial in organ preservation. REFERENCES 1. Atalla S. L., Toledo-Pereyra, L. G., MacKenzie, G. H., and Cedema, J. P. Influence of oxygenderived free radical scavengers on ischemic livers. Transplantation 40, 584-590 (1985). 2. Bennett, J. F., Bry, W. I., Collins, G. M., and Halasz, N. A. The effects of oxygen free radicals on the preserved kidney. Cryobiology 24, 261269 (1987). 3. Boni, L. T., Hah, J. S., Hui, S. H., Mukhetjee, P., Ho, J. T., and Jung, C. Y. Aggregation and fusion of unilamellar vesicles by polyethylene glycol. Biochim. Biophys. Acta 775, 409-418 (1984). 4. Fuller, B. J., Lunec, J., Healing, G., Simpkin, S., and Green, C. J. Reduction of susceptibility to lipid peroxidation by desferrioxamine in rabbit kidneys subjected to 24-hour cold ischemia and 43, 604-606 reperfusion. Transplantation (1987). 5. Green, C. J., Healing, G., Lunec, J., Fuller, B. J., and Simpkin, S. Evidence of free-radical-induced damage in rabbit kidneys after simple hypothermic preservation and autotransplantation. Transplantation 41, 161-165 (1986). 6. Halliwell, B., and Gutteridge, J. M. C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1-14 (1984). 7. Hemandez, L. A., and Granger, D. N. Role of antioxidants in organ preservation and transplantation. Crit. Care Med. 16, 543-549 (1988).
ET AL.
8. Hogberg, J., and Kristoferson, A. A correlation between glutathione levels and cellular damage in isolated hepatocytes. Eur. J. Biochem. 74, 77-82 (1977). 9. Hogberg J., Orrenius, S., and Larson, R. E. Lipid peroxidation in isolated hepatocytes. Eur. J. Biochem. 50, 592-602 (1975). 10. Innes, G. K., Fuller, J. J., and Hobbs, K. E. Lipid peroxidation in hepatocyte cell cultures: Modulation by free radical scavengers and iron. In Vitro Cell Dev. Biol. 24, 126-132 (1988). 11. Jamieson, N. V., Sundberg, R., Lindell, S., Southard, J. H., and Belzer, F. 0. Preservation of the canine liver for 24-t8 hours using simple cold storage with UW solution. Transplantation 46, 517-525 (1988). 12. Jennische, E. Possible influence of glutathione on postischemic liver injury. Acta Pathol. Microbiol. Immunol. Stand. 92, 55-64 (1984). 13. Marsh, D. C., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes II. Importance of Ca and amino acids. Cryobiology 27, l-8 (1990). 14. Marsh, D. C., Lindell, S. L., Fox, L. E., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes. I. Role of cell swelling. Cryobiology 26, 524-534 (1989). 15. Masaki, N., Kyle, M. E., and Farber, J. L. TertButyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids. Arch. Biothem. Biophys. 269, 390-399 (1989). 16. McCord, J. M. Oxygen-derived free radicals in postischemica tissue injury. N. Engl. J. Med. 312, 159-165 (1985). 17. Meister, A. Glutathione metabolism and its selective modification. J. Biol. Chem. 263, 17,20517,208(1988). 18. Metzger, J., Dore, S. P., and Lauterburg, B. H. Oxidant stress during reperfusion of ischemic liver: No evidence for a role of xanthine oxidase. Hepatology 8, 58CL584 (1988). 19. Miller, J. S., and Comwell, D. G. The role of cryoprotective agents and hydroxyl radical scavengers. Cryobiology 15, 585-588 (1978). 20. Pascoe, G. A., and Reed, D. J. Cell calcium, vitamin E, and the thiol redox system in cytotoxicity. Free Radicals. Biol. Med. 6, 209-224 (1989). 21. Ploeg, R. J., Goossens, D., McAnulty, J. F., Southard, J. H., and Belzer, F. 0. Successful 72-hour cold storage of dog kidneys with UW solution. Transplantation 46, 191-196 (1988). 22. Rush, G. F., Gorski, J. R., Ripple, M. G., Sowinski, J., Bugelski, P., and Hewitt, W. R. Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes. Toxicol. Appl. Pharmacol. 78, 473-483 (1985).
PEG
EFFECT
ON
COLD-STORED
23. Seglen, P. 0. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 30-83 (1976). 24. Southom, P. A., and Powis, G. Free radicals in medicine. II. Involvement in human disease. Mayo Clin. hoc. 63, 390X18 (1988). 25. Todo, S., Nery, J., Yanaga, K., Podesta, L., Gordon, R. D., and Starzl, T. E. Extended preservation of human liver grafts with UW solution. .I. Amer. Med. Assoc. 261, 711-714 (1989). 26. Todo, S., Podesta, L., Ueda, Y., Inventarza, O., Casavilla, A., Oks, A., Okuda, K., Malesnik, M., Venkataramanan, R., and Starzy, T. E. Comparison of UW with other solutions for liver preservation in dogs. C/in. Transplant. 3, 253-259 (1989). 27. Umeshiata, K., Monden, J., Fujimori, T., Sekai, H., Gotoh, M., Okamura, J., and Mori, T. Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation. Cryobiology 25, 102-109 (1988).
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7
28. Wahlberg, J. A., Love, R., Landegaard, L., Southard, J. H., and Belzer, F. 0. 72-hour preservation of the canine pancreas. Transpluntation 43, 5-10 (1987). 29. Wahlberg, J. A., Southard, J. H., and Belzer, F. 0. Development of a cold storage solution for pancreas preservation. Cryobiology 23,477487 (1986). 30. Weinberg, J. M. Oxygen deprivation-induced injury to isolated rabbit kidney tubules. J. C/in. Invest. 76, 1193-1208 (1985). 31. Wickens, D. G., Li, M. K., Atkins, G., Fuller, B. J., Hobbs, K. E., and Dormandy, T. L. Free radicals in hypothermic rat heart preservation: prevention of damage by mannitol and desferrioxame. Free Radicals Res. Commun. 4, 189-195 (1987). 32. Wicomb, W. N., and Collins, Cl. M. 24-hour rabbit heart storage with UW solution. Transplantation 48, 6-9 (1989).
28, 1-7 (1991)
Effect of Polyethylene
Glycol on Lipid Peroxidation Rat Hepatocytes’
J. E. MACK, J. A. KERR, P. K. VREUGDENHIL, J. H. SOUTHARD
in Cold-Stored
F. 0. BELZER,
AND
Department of Surgery, University of Wisconsin, Madison, Wisconsin 53792 A mechanism suggested to cause injury to preserved organs is the generation of oxygen free radicals either during the cold-storage period or after transplantation (reperfusion). Oxygen free radicals can cause per-oxidation of lipids and alter the structural and functional properties of the cell membranes. Methods to suppress generation of oxygen free radicals of suppression of lipid peroxidation may lead to improved methods of organ preservation. In this study we determined how cold storage of rat hepatocytes affected lipid peroxidation by measuring thiobarbituric acid reactive products (malondiaIdehyde, MDA). Hepatocytes were stored in the UW solution 2 glutathione (GSH) or k polyethylene glycol (PEG) for up to 96 h and rewarmed (resuspended in a physiologically balanced saline solution and incubated at 37°C under an atmosphere of oxygen) after each day of storage. Hepatocytes rewarmed after storage in the UW solution not containing PEG or GSH showed a nearly linear increase in MDA production with time of storage and contained 1.618 k 0.73 1 nmol MDA/mg protein after 96 h. When the storage solution contained PEG and GSH there was no significant increase in MDA production after up to 72 h of storage and at 96 h MDA was 0.827 -+ 0.564 nmohmg protein. When freshly isolated hepatocytes were incubated (37°C) in the presence of iron (160 CLM)MDA formation was maximally stimulated (3.314 f 0.941 nmoVmg protein). When hepatocytes were stored in the presence of PEG there was a decrease in the capability of iron to maximally stimulate lipid peroxidation. The decrease in iron-stimulated MDA production was dependent upon the time of storage in PEG (1.773 nmoYmg protein at 24 h and 0.752 nmol/mg protein at 48 h). In the absence of PEG, iron-stimulated MDA formation was nearly maximal at all times of storage. These results show that lipid peroxidation is stimulated by cold storage of hepatocytes. Inclusion of PEG in the storage medium suppressed lipid peroxidation suggesting that PEG is accumulated, in a time-dependent manner, by hepatocytes (either into the plasma membrane or into the cell cytosol) and either scavenges oxygen free radicals or alters the availability of lipids to these radicals. PEG may be a useful additive t0 organ preSerVatiOn SOhtiOnS. 0 1991 Academic Press, Inc.
The complexities inherent in determining stored in an organ preservation solution the mechanisms of cell injury due to drugs, (VW solution) or in a tissue culture metoxins, hypoxia, ischemia, or preservation dium. It was shown that for successful in whole organs have lead a number of in- preservation of hepatocytes, polyethylene vestigators to develop a simpler system us- glycol (3-5 g%) was necessary to suppress ing isolated cells. We (13, 14), as well as hypothermic-induced cell swelling. others (IO, 27), have used isolated hepatoOne mechanism thought to cause loss of cytes to determine how hypothermic pres- organ viability is the generation of oxygen ervation conditions affect cell metabolism free radicals (7, 16). Agents or conditions and viability. We have shown that hepato- that stimulate oxygen free radical generacyte viability can be maintained (based tion often lead to lipid peroxidation and the upon LDH release) for up to 3 days when production of malondialdehyde (MDA). Fuller et al. (4, 5) have shown increased Received January 6, 1989; accepted February 15, MDA formation in tissues following preservation and have implicated an iron1990. mediated mechanism for lipid peroxidation. ’ Supported by NIH Grant 35143. 1 OOll-2240191$3.00 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.
2
MACK
ET AL.
ml when used. In some experiments glutathione was omitted. The following groups were studied: Group 1, UW solution containing PEG ? GSH; Group 2, UW solution minus PEG; and Group 3, UW solution minus both PEG and GSH. For the determination of lipid peroxidation thiobarbiturate (malondialdehyde) reactive material was determined by the method of Rush et al. (22). After cold storage, hepatocytes (or freshly prepared hepatocytes) were sedimented (77Og, 5 min) and the storage medium was discarded. The cells were resuspended in a physiologically balanced salt solution (Weinberg et al. (30) solution A) at 5-6 mg protein/ml and an aliquot of this suspension was added to an equal volume of trichloroacetic acid (10%) to precipitate the protein. Protein was removed by centrifugation (14,000 rpm, 2 min: Eppendorf microcentrifugal) and MDA concentration determined in the suMATERIALS AND METHODS pernate fluid. MDA was also determined afHepatocytes were prepared from rats ter normothermic incubation in Weinberg’s (Sprague-Dawley, 20 to 250 g) by the solution A at 37°C under an atmosphere of method of Seglen (23) as described by oxygen (100%) for 30 and 60 min. At the Marsh et al. (14). Only preparations that end of incubation an aliquot of cells was showed less than 10% uptake of trypan blue added to an equal volume of TCA and MDA were used in these studies. Immediately af- determined in the protein-free supernate ter preparation hepatocytes were sus- fluid. Iron-stimulated MDA formation was pended in the preservation solution at 5°C determined as described by Hogberg et al. which had been equilibrated with nitrogen (9) by adding aliquots of a stock solution (100%). Hepatocytes were stored in sealed containing ADP (100 mM) and ferric chlotest tubes at 5°C at a concentration of 5-6 ride (2 mM) to obtain a final concentration mg protein/ml (Biuret method) without of iron of 20 to 320 @Y. The concentration shaking to simulate simple cold storage of of MDA was determined by measuring the absorbance at 535 nm against a blank conorgans (i.e., cold ischemia). The storage (preservation) solution was taining all reagents minus the supernate similar to the UW solution (28) with hy- fluid. The absorbance was compared to that droxyethyl starch and contained: 100 mM obtained with a known concentration of lactobionate (K), 30 mM rafftnose, 5 mM 1,1,3,3-tetraethoxypropane and the results magnesium sulfate, 5 mit4 adenosine, 1 mM were expressed as nmol of MDA/mg proallopurinol , 3 mM glutathione , neutralized tein. with sodium and potassium hydroxides to Experimental results were obtained from give a final concentration of 30 * 5 mM = at least four separate preparations of hepaNa, and 120 ? 5 mM = K, with an osmo- tocytes, MDA assays were performed in at lality of 300 mOsm/liter. Polyethylene gly- least duplicates, and the means were calcuco1(PEG, 8000 mol wt) was added at 5 g/100 lated. The results presented are the means These studies, as well as others (1,31), suggest that suppression of the generation of oxygen free radicals, or effectively scavenging cytotoxic products of oxygen metabolism, may improve the quality of organ preservation. The University of Wisconsin (UW) solution has been shown to effectively preserve the liver (ll), kidney (21), and pancreas (28). This solution contains a number of agents that could act as antioxidants: allopurinol, glutathione (GSH), raffinose, and hydroxyethyl starch. Additionally, PEG used in hepatocyte cold-storage studies (13, 14) could also act as an antioxidant (2). In this study we have determined how preservation of hepatocytes in the UW solution affects lipid peroxidation and how the omission of two potential antioxidants (PEG and GSH) affected the extent of MDA formation.
PEG EFFECT ON COLD-STORED HEPATOCYTES
3
(+-SD) obtained by combining the results from each hepatocyte preparation. Statistical analysis was done using the Student t test.
more MDA on rewarming than hepatocytes stored in the UW solution containing PEG (Group 1) after 48,72, and 96 h of storage (P < 0.05 at 48 and 72 h but not at 96 h). The omission of both PEG and GSH (Group 3) RESULTS resulted in an even greater production of The concentration of MDA in freshly iso- MDA on rewarming when compared to lated hepatocytes averaged 0.335 + 0.055 Group 2 (no PEG). However, a significant nmol/mg protein. There was no significant difference between Group 3 and Group 2 increase in MDA during a 120-min incuba- was obtained only after 48 h of storage and tion of hepatocytes in an atmosphere of ox- rewarming. After 96 h of storage and reygen (refer to Fig. 2, bottom trace, 0 FM warming the MDA concentrations were ADP-Fe). When hepatocytes were stored 1.152 + 0.276 nmol/mg in Group 2 vs 1.618 for up to 96 h at 5°C under an atmosphere of + 0.731 nmol/mg protein in Group 3. nitrogen there was also practically no inThese results show that PEG suppresses crease in MDA compared to freshly iso- MDA formation in cold-stored hepatocytes. PEG was equally effective in the lated hepatocytes. When stored in the UW solution contain- presence or absence of GSH (Group 1). ing PEG (Group 1 + GSH) a significant in- However, GSH did appear to have some crease in MDA occurred (Fig. l), but only antioxidant properties because the omisafter 96 h of storage (0.827 k 0.564 nmol/mg sion of both PEG and GSH (Group 3) from protein vs 0.355 + 0.055 nmol/mg protein, the UW solution resulted in a greater proP < 0.05). The concentration of MDA in duction of MDA than in hepatocytes stored hepatocytes rewarmed after 24, 48, and 72 only in the absence of PEG (Group 2). To determine how iron (ADP-Fe) would h of storage (Group 1) was not significantly different than that in freshly isolated hepa- effect lipid peroxidation in cold-stored hepatocytes, we determined the concentratocytes rewarmed for 120 min. Hepatocytes stored in the UW solution tion of ADP-Fe to maximally stimulate (not containing PEG, Group 2) produced MDA formation in freshly isolated hepatocytes (Fig. 2). In this set of experiments,
0
48
24 Preservation
72
96
Time (hr)
FIG. 1. Effects of cold storage of rat hepatocytes on MDA formation after rewarming. Rat hepatocytes were cold-stored for up to 96 h as described under Materials and Methods. At the end of preservation the cells were incubated at 37°C in a physiologically balanced salt solution for 60 min and the concentration of MDA was determined. Results shown are means (*SD) obtained from at least four separate hepatocyte preparations. *P < 0.05 compared to Group 1.
0
30
60
TIME (MN)
FIG. 2. Effects of iron on MDA formation in freshly isolated hepatocytes. Iron (ADP-Fe) was added to freshly isolated hepatocytes suspended in Weinberg’s solution A and incubated for up to 60 min at 37°C in the presence of oxygen. MDA concentrations were determined as described under Materials and Methods. *P < 0.05 versus 160 @4.
4
MACK ET AL.
freshly isolated hepatocytes contained 0.208 ? 0.028 nmol MDA/mg protein. Maximal MDA production was obtained within a 30-min incubation. The maximal concentration of MDA (a lo-fold increase) was produced by incubation in the presence of 160 Q4 ADP-Fe (2.193 f 0.223 nmol/mg protein, not significantly different from 320 00 The results in Fig. 3 show how iron affected lipid peroxidation in hepatocytes after cold storage for up to 96 h. In this set of experiments the addition of iron (160 PM) to freshly isolated hepatocytes also increased MDA by lo-fold (0.355 ? 0.055 to 3.314 + 0.941 nmoYmg protein). When hepatocytes were cold-stored in UW solution containing PEG 2 GSH (Group l), there was a decrease in the capability of iron to stimulate lipid peroxidation that was dependent upon the time of storage. After 48 h of storage the iron-induced formation of MDA was reduced from 3.314 to 0.752 + 0.293 nmol/mg protein and MDA remained relatively low after rewarming hepatocytes cold-stored for 72 and 96 h. When PEG was omitted from the UW solution (Group 2), there was an initial decrease in the ironstimulated production of MDA after 24 h of storage but after 48 to 96 h of storage the
OJ 0
24
48 Preservation
72 lime
1 96
(hr)
FIG. 3. Effects of iron on MDA production in rat hepatocyte cold-stored for up to 96 h after rewarming. Methods are as described in the legend to Fig. 1 and under Materials and Methods. Iron (ADP-Fe) was added (160 pJ4) at the beginning of normothermic incubation, after the indicated preservation times. *P < 0.05 versus Group 1.
presence of iron-stimulated MDA production (significant at P > 0.05 versus Group 1 at 48-96 h). Iron-stimulated MDA production was maximally obtained after all periods of storage when both PEG and GSH (Group 3) were omitted from the UW solution (P < 0.05 versus Group 1 at 24-72 h). These results show that the storage of hepatocytes in the presence of PEG suppresses iron-stimulated MDA formation. DISCUSSION
The UW solution has been shown to be an effective flushout solution for the preservation of the liver and is currently used clinically (25) with preservation times of up to 35 h. The mechanism of action of the UW solution is not clearly understood and we have recently begun to use isolated hepatocytes as an experimental model to determine how hypothermic preservation affects cellular metabolism. In previous studies (13, 14) we used a preservation model for hepatocytes that simulated continuous perfusion of the liver: hepatocytes were coldstored (S’C) in the presence of oxygen with continuous shaking. In these studies we demonstrated that viability of hepatocytes was dependent upon suppressing hypothermic-induced cell swelling. We showed that even in the presence of impermeants (lactobionic acid, raffinose, and sucrose) hepatocytes became swollen when stored at 5°C. This is unlike whole livers (26) or liver slices (29) which do not gain water when stored in solutions containing these impermeants. This suggested that the process of digestion of the liver tissue with collagenase changed the permeability properties of the plasma membrane of hepatocytes. Therefore, to study the mechanisms of hypothermic-induced cell death in the absence of cell swelling, another agent was required. We found that PEG suppressed hypothermic-induced cell swelling in hepatocytes stored for up to 5 days (14) and have, consequently, included PEG in our studied of isolated hepatocytes. However,
PEG
EFFECT
ON
COLD-STORED
HEPATOCYTES
5
brane bound lipids to initiators of lipid peroxidation by changing membrane structure we have investigated the effects of PEG on or by scavenging initiators before they lipid peroxidation in cold-stored hepato- reach the lipids, similar to the mechanism proposed for the antioxidant properties of cytes . PEG is an effective antioxidant and sup- vitamin E (20). Glutathione is the major thiol in tissue pressed lipid peroxidation after rewarming hepatocytes cold-stored in the UW solution and has an one of its function the reduction for up to 96 h. The effectiveness of PEG of hydrogen peroxide and lipid peroxides was seen in hepatocytes rewarmed in the (for review see (17)). In the presence of hyabsence and presence of iron. The effec- drogen peroxide, GSH is oxidized to GSSG tiveness of PEG as an antioxidant increased (glutathione peroxidase) to form oxygen with time of storage of hepatocytes in the and water. Thus, the reduction in the celUW solution. This was clearly demon- lular concentration of hydrogen peroxide strated when iron-stimulated lipid peroxi- reduces the concentration of hydroxyl raddation was determined. After 24 h of stor- icals formed by the iron-catalyzed HaberWeiss reaction (6). In this study the conage in PEG there was an approximately 50% decrease in iron-stimulated MDA pro- centration of MDA produced by coldduction. After 48 h of storage there was a stored hepatocytes was independent of the presence of GSH when the cells were complete suppression of iron-stimulated MDA production. There are a number of stored in the presence of PEG. However, possible explanations for the time-depen- when stored without PEG the presence of dent effect of PEG. One is that cold storage GSH decreased the amount of MDA proof hepatocytes reduced the capability of the duced in rewarming hepatocytes. The recells to produce the initiators of lipid per- sults show that the omission of both PEG oxidation (superoxide anion, hydroxyl rad- and GSH from the UW solution caused a icals). However, MDA was produced (plus greater increase in MDA formation than or minus iron) in hepatocytes cold-stored in when just the PEG was omitted. Others the absence of PEG and therefore, cold have also shown suppression of lipid perstorage does not appear to affect the gener- oxidation by GSH (8, 15) in models other ation of initiators of lipid peroxidation. A than cold storage. There are a number of studies that sugsecond explanation is that PEG scavenges gest that oxygen free radicals are a factor in initiators of lipid peroxidation (hydroxyl radicals, 19) and to be effective a critical injury to organs following preservation (7, concentration of PEG must be obtained in 16) and other forms of organ injury (12,24). the hepatocytes. In the cold it appears that How these cytotoxic products of oxygen it takes about 48 h for the effective concen- metabolism cause cell death is not clear but tration of PEG to accumulate in hepato- may be related to peroxidation of memcytes. The exact location of PEG in hepa- brane lipids and changes in membrane tocytes is not clear and it could be either in properties (permeability and enzyme activthe cell cytosol or in the cell membranes. ity). One concept in the development of the PEG has been shown to bind to phospho- UW solution was the addition of agents that lipids and accumulate in cell membranes would suppress oxygen free radical injury (3); thus, this is a possible sight for the ac- in preserved organs. In addition to GSH the tion of PEG in suppressing lipid peroxida- UW solution contains allopurinol. Allopution. The presence of PEG in cell mem- rinol is an inhibitor of xanthine oxidase which has been suggested to be a source of branes couuld suppress lipid peroxidation by either altering the availability of mem- superoxide anions during reperfusion of PEG may have other effects on the cell than
just the suppression of cell swelling. Thus,
6
MACK
ischemic or injured organs (16). In this study the UW solution contained allopurinol. However, lipid peroxidation was evident even in the presence of allopurinol, suggesting that xanthine oxidase may not be the only source of superoxide anions in cold-stored livers (hepatocytes) which has been previously suggested (18). The effectiveness of PEG in suppressing lipid peroxidation in this study suggests that PEG might be a useful agent in organ preservation solutions. Wicomb and Collins (32) have recently shown that the UW solution containing PEG is effective for a 24-h preservation of the rabbit heart. This may be due to the effectiveness of PEG as an inhibitor of lipid peroxidation. The usefulness of PEG in the preservation of other organs, however, is not known but these results suggest that PEG may be beneficial in organ preservation. REFERENCES 1. Atalla S. L., Toledo-Pereyra, L. G., MacKenzie, G. H., and Cedema, J. P. Influence of oxygenderived free radical scavengers on ischemic livers. Transplantation 40, 584-590 (1985). 2. Bennett, J. F., Bry, W. I., Collins, G. M., and Halasz, N. A. The effects of oxygen free radicals on the preserved kidney. Cryobiology 24, 261269 (1987). 3. Boni, L. T., Hah, J. S., Hui, S. H., Mukhetjee, P., Ho, J. T., and Jung, C. Y. Aggregation and fusion of unilamellar vesicles by polyethylene glycol. Biochim. Biophys. Acta 775, 409-418 (1984). 4. Fuller, B. J., Lunec, J., Healing, G., Simpkin, S., and Green, C. J. Reduction of susceptibility to lipid peroxidation by desferrioxamine in rabbit kidneys subjected to 24-hour cold ischemia and 43, 604-606 reperfusion. Transplantation (1987). 5. Green, C. J., Healing, G., Lunec, J., Fuller, B. J., and Simpkin, S. Evidence of free-radical-induced damage in rabbit kidneys after simple hypothermic preservation and autotransplantation. Transplantation 41, 161-165 (1986). 6. Halliwell, B., and Gutteridge, J. M. C. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem. J. 219, 1-14 (1984). 7. Hemandez, L. A., and Granger, D. N. Role of antioxidants in organ preservation and transplantation. Crit. Care Med. 16, 543-549 (1988).
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8. Hogberg, J., and Kristoferson, A. A correlation between glutathione levels and cellular damage in isolated hepatocytes. Eur. J. Biochem. 74, 77-82 (1977). 9. Hogberg J., Orrenius, S., and Larson, R. E. Lipid peroxidation in isolated hepatocytes. Eur. J. Biochem. 50, 592-602 (1975). 10. Innes, G. K., Fuller, J. J., and Hobbs, K. E. Lipid peroxidation in hepatocyte cell cultures: Modulation by free radical scavengers and iron. In Vitro Cell Dev. Biol. 24, 126-132 (1988). 11. Jamieson, N. V., Sundberg, R., Lindell, S., Southard, J. H., and Belzer, F. 0. Preservation of the canine liver for 24-t8 hours using simple cold storage with UW solution. Transplantation 46, 517-525 (1988). 12. Jennische, E. Possible influence of glutathione on postischemic liver injury. Acta Pathol. Microbiol. Immunol. Stand. 92, 55-64 (1984). 13. Marsh, D. C., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes II. Importance of Ca and amino acids. Cryobiology 27, l-8 (1990). 14. Marsh, D. C., Lindell, S. L., Fox, L. E., Belzer, F. O., and Southard, J. H. Hypothermic preservation of hepatocytes. I. Role of cell swelling. Cryobiology 26, 524-534 (1989). 15. Masaki, N., Kyle, M. E., and Farber, J. L. TertButyl hydroperoxide kills cultured hepatocytes by peroxidizing membrane lipids. Arch. Biothem. Biophys. 269, 390-399 (1989). 16. McCord, J. M. Oxygen-derived free radicals in postischemica tissue injury. N. Engl. J. Med. 312, 159-165 (1985). 17. Meister, A. Glutathione metabolism and its selective modification. J. Biol. Chem. 263, 17,20517,208(1988). 18. Metzger, J., Dore, S. P., and Lauterburg, B. H. Oxidant stress during reperfusion of ischemic liver: No evidence for a role of xanthine oxidase. Hepatology 8, 58CL584 (1988). 19. Miller, J. S., and Comwell, D. G. The role of cryoprotective agents and hydroxyl radical scavengers. Cryobiology 15, 585-588 (1978). 20. Pascoe, G. A., and Reed, D. J. Cell calcium, vitamin E, and the thiol redox system in cytotoxicity. Free Radicals. Biol. Med. 6, 209-224 (1989). 21. Ploeg, R. J., Goossens, D., McAnulty, J. F., Southard, J. H., and Belzer, F. 0. Successful 72-hour cold storage of dog kidneys with UW solution. Transplantation 46, 191-196 (1988). 22. Rush, G. F., Gorski, J. R., Ripple, M. G., Sowinski, J., Bugelski, P., and Hewitt, W. R. Organic hydroperoxide-induced lipid peroxidation and cell death in isolated hepatocytes. Toxicol. Appl. Pharmacol. 78, 473-483 (1985).
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23. Seglen, P. 0. Preparation of isolated rat liver cells. Methods Cell Biol. 13, 30-83 (1976). 24. Southom, P. A., and Powis, G. Free radicals in medicine. II. Involvement in human disease. Mayo Clin. hoc. 63, 390X18 (1988). 25. Todo, S., Nery, J., Yanaga, K., Podesta, L., Gordon, R. D., and Starzl, T. E. Extended preservation of human liver grafts with UW solution. .I. Amer. Med. Assoc. 261, 711-714 (1989). 26. Todo, S., Podesta, L., Ueda, Y., Inventarza, O., Casavilla, A., Oks, A., Okuda, K., Malesnik, M., Venkataramanan, R., and Starzy, T. E. Comparison of UW with other solutions for liver preservation in dogs. C/in. Transplant. 3, 253-259 (1989). 27. Umeshiata, K., Monden, J., Fujimori, T., Sekai, H., Gotoh, M., Okamura, J., and Mori, T. Extracellular calcium protects cultured rat hepatocytes from injury caused by hypothermic preservation. Cryobiology 25, 102-109 (1988).
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28. Wahlberg, J. A., Love, R., Landegaard, L., Southard, J. H., and Belzer, F. 0. 72-hour preservation of the canine pancreas. Transpluntation 43, 5-10 (1987). 29. Wahlberg, J. A., Southard, J. H., and Belzer, F. 0. Development of a cold storage solution for pancreas preservation. Cryobiology 23,477487 (1986). 30. Weinberg, J. M. Oxygen deprivation-induced injury to isolated rabbit kidney tubules. J. C/in. Invest. 76, 1193-1208 (1985). 31. Wickens, D. G., Li, M. K., Atkins, G., Fuller, B. J., Hobbs, K. E., and Dormandy, T. L. Free radicals in hypothermic rat heart preservation: prevention of damage by mannitol and desferrioxame. Free Radicals Res. Commun. 4, 189-195 (1987). 32. Wicomb, W. N., and Collins, Cl. M. 24-hour rabbit heart storage with UW solution. Transplantation 48, 6-9 (1989).
