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Studies on the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay
8, 293-299
CRYOBIOLOGY,
Studies
III.
on the Mechanism of Freezing Damage to Mouse Using a Mitochondrial Enzyme Assay
Cryophyllaxis
with
Dimethyl WILLIAM
Biochemistru
Sulfoxide
and
Enzyme
Liver
Localization’
N. FISHBEI?;
Branch. Armed Washington,
Forces
Institute
of Pathology,
D. c. POSO5
Previous communications in this series (4, 5) have demonstrated that the succinate cytochrome c reductase complex, a multienzyme electron transport segment of mitochondria, which is suitable for multiple sample spectrophotometric assay, provides a graded response to freeze-thaw treat’ments of mouse liver slices, so that information can be obtained as to the mechanism of freezing damage in nucleated cells with rapidly obtained quantitative data. During slow freezing, the loss of enzyme activity was shown to coincide with the phase transition of the medium from liquid to solid state, and to be independent of other variables studied. In rapid freezing procedures a critical cooling rate was inferred, in the neighborhood of lOO”C/sec, beyond which a markedly greater loss in enzyme activity resulted. In these respects, therefore, the enzyme complex behaved in a manner analogous to t,hat of whole cell systems studied by much more tedious methods (9, 11, 12, 14). In t’his report we show that the analogy is maintained with regard to the protection of the enzyme complex against’ freezing damage by a permeant cryophyllactic agent, dimethyl sulfoxide.
In addition, the unusual stability of the enzyme complex in water has permit’ted the demonstration that dimethyl sulfoxide cryoprotection depends to a significant extent upon the presence of external solute. Finally evidence is presented that t,he enzyme complex under study is localized in the mit,orhondria of mouse liver. EXPERIMENTAL
PROCEDURE
All chemicals used in the study were purchased as reagent grade and used without, further purification. Liver slices measuring approximately 10 X 5 X 1 mm from freshly sacrificed male CAF,(BALB/c x A/) mice were placed in glass homogenizing tubes containing l-2 ml of appropriate chilled medium and cooled at l’C/ min from 4°C to a final nadir at -25°C in a Canalco slow freezing apparatus (Canal Industrial Corp., Bethesda, &Id.) as previously described (4). dfter thawing by immersion of the tube in a 37°C water bath, and replacement of the freeze-thaw medium by fresh, chilled 0.25 M sucrose, 10% homogenates were prepared lT--ith Teflon pasties against smooth glass walls at 4”C, and assayed for succinate cytochrome c reductase activity as described in our first report (4). Received February 19, 1971 ‘This investigation was supported in part by a Dilutions of the same homogenates were used for Kumber 3AO6211OA821, protein determinations by the method of Lowry rrstwch grant, Project from the Medical Research and Development et al. (8)) using bovine serum albumin as stundCommand, U.S. Army, Washington, D.C. 20012. In ard, a.nd the specific activities of the frozenconducting the research described in this report, thawed specimens were calculated as percentsgea the investigators adhered to the “Guide for Laboof the corresponding specific activit’ies ot’ unratory Animal Facilities and Care,” as promulfrozen cont,rol specimens. Buffer and salt solugated by t,he Committee on the Guide for Laboratory Animal Facilities and Care of the Institute tions were prepared in 50% dimethyl sulfoside of Laboratory Animal Resources, National Acadas well as in wat,er, so that appropriate volumetemy of Sciences-National Research Council. The ric mixing of the two solutions yielded various opinions or assertions contained herein are the prilevels of dimethyl sulfoxide without altering the vat,e views of the author and are not to be conconcentration of other constituents. strued as official or as reflecting the views of the For studies on the localization of the enzyme Department of the Army or the Department of complex, mitochondria were prepared at 4°C Defense. 293
294
FISHBEIN
from homogenates as follows: (average g forces are cited, maximum g forces were about 20% higher) after a lo-min centrifugation at 700g to remove nuclei and intact cells, the mitochondria were sedimented at 5500g for 10 min and were centrifuged twice more at 5500g for 15 min after two further resuspensions in 0.25 M sucrose. All fractions and washes were saved, as well as an aliquot of the initial homogenate, for activity, protein, and PNA determinations, the latter by the orcinol reaction (15). Mitochondrial swelling experiments, as evidenced by absorption changes, were performed on these isolates using a home-made fragilograph (2) incorporating a Coleman spectrophotometer wired to a pica-ammeter and recorder. A mitochondrial suspension was injected into the l-mm thick diffusion chamber formed by dialysis membrane stretched over a metal frame. This was placed in a large test tube containing 100 ml of external fluid and insert,ed horizontally into the light path of the photometer, so that t#he optical effect of mitochondria settling during the experimental period was minimized.
must elapse before freezing occurs when a l”C/ min cooling rate is employed, it was first necessary to determine whether or not dimethyl sulfoxide itself produced direct alteration in the succinate cytochrome c reductase activity, in the absence of freezing and thawing. The liver slices were therefore preincubat,ed in dimethyl sulfoxide containing medium at 4°C for various periods of time before beginning the freeze-thaw experiment. Four such experiments with two different concentrations of dimethyl sulfoxide are shown in Fig. 1. Although there was variation in recovery from one experiment to the next, it is clear that within any one experiment incubation of the slice with dimethyl sulfoxide at 4°C for periods up to 1 hr resulted in no greater loss in enzyme activity than if the freeze-thaw experiment was begun at once. It was concluded, therefore, that direct toxic effects of dimethyl sulfoxide on this enzyme complex were negligible under these conditions. The protection afforded by various concentrations of dimethyl sulfoxide incorporated into sucrose solutions against the freeze-thaw damage of succinate cytochrome c reductase is shown in Fig. 2. All concentrations investigated from 5 to 30% (v/v) provided significant protection (p < 0.05). Although 15% gave the highest mean recovery value, this was not significant in comparison to that afforded by other concentrations (p
RESULTS Cryoprotection with dimethyl sulfoxide was evaluated by incorporating the agent, in the concentrations noted, into the suspending medium prior to freezing. Since a lO-15-min period
0
SO E
0
0
0
lO%DMSO
t
20
0 0
*
I 15
0
I 30
I 60
I 45
Tim DMSO Preseti kfore Tissue Freezing
I 75
hid
FIG. 1. Lack of influence of preincubation of mouse liver slices with dimethyl sulfoxide (DMSO) in 0.25 M sucrose at 4°C on the recovery of succinate cytochrome c reductase activity after freeze-thaw. The percentage of DMSO is given on a volume/volume basis. The minimum time for freezing at a cooling rate of l”C/min was 10-15 min, and thawing was accomplished rapidly by immersion in a 37°C water bath. Four separate experiments are shown.
DMSO CRYOPHYLLAXIS
OF SUCCINATE CYTOCHROME c REDKTASE
5
20 15 10 % DMSO PRESENT DURING FREEZING
25
‘-,!I5
30
Fro. 2. Dimethyl sulfoxide (DMSO) cryophyllaxis of succinate cytochrome c reductase activity after freeze-thaw of mouse liver slices. The mean recovery, standard error, and number of experiments is shown for each DMSO concentration (v/v) in 0.25M sucrose. > 0.1). Glycerol at similar concentrations gave a shown in Table 1. In sucrose, phosphate, and comparable degree of protection in the few ex- Tris solutions, dimethyl sulfoxide conferred a 3540% protective effect against freezing damperiments made. We had earlier found that freeze-thaw damage age, whereas in water the protective effect was to succinate cytochrome c reductase in liver virtually nil. The significance of these findings slices was independent of the suspending me- will be discussedbelow. Lest confusion arise, it is important to point dium used, whether this was 0.25 M sucrose, 0.15 N phosphate buffer, 0.12 M Tris-HCl buffer, or out that the succinate cytochrome c reductase most strikingly, simply water, the loss of activ- assay is not equivalent to the assay of succinate ity in all cases being about 45% (4). This unu- dehydrogenase, although the latter enzyme is the sual situation permitted the evaluation of di- first member of the sequence measured by the methyl sulfoxide protection in these diverse reductase assay. Although t’he cytochrome c remedia, and the results of such an experiment are ductase method was introduced aa an assay for
296
FISHBEIN TABLE
1
INFLUENCE OF THE SUSPENDING MEDIUM ON DIMETHYLSULFOXIDE (DMSO) CRYOPHYLLAXIS CINATE CYTOCHROME c REDUCTASE (SCcR) IN ~-MM THICK MOUSE LIVER SLICES~ Medium
No.
expts.
Y. Protection with 2.1 M DMSO M&Ill
SE
PCS% (1)
(1) Wat.er (2) 0.25 M Sucrose (3) 0.10 M Phosphat’e
5 5 5
8.4 39.4 32.4
5.5 5.1 6.1
++ +
(4) 0.17 M Tris-HCl
5
33.8
7.4
+
(+)
FOR Suc-
or 1% (++)
(2)
(3)
(4)
++
+ -
+ -
-
-
a Cooling at l”C/min to -25°C was performed on separate slices in each medium with and without 2.1 M DMSO (15’%, v/v). After rapid thawing in a 37°C water bath, the loss (L) of SCcR activity in the tissue immersed in medium only (Lm) and medium supplemented with DMSO (L,) was determined by comparison with a nonfrozen control. The percentage protection afforded by DMSO was calculated as lOO[(L, - L,)/L,], and denotes the added protection due to DMSO, above that provided by each medium alone, which was about 507, in each case. The probability matrix at the right permits rapid comparison of any two numbered groups with regard to a significant difference between their means at t,he 5y0 (+) or 1% (++) level.
succinate dehydrogenase (1)) the two assays are strikingly different in their response to physical damage (5) . The localization of the reductase enzyme complex was evaluated by centrifugal isolation of mitochondria from the homogenates. All fractions were assayed and corrected to initial volume so as to permit the determination of yields of the enzyme, protein, and nucleic acid as compared to the original homogenate. This is important to assesswhether any activation or inhibition of the enzyme occurs during fractionation, which would interfere with the interpretation of activity changes due to physical damage. The fractionation procedure was designed primarily for purity in the mitochondrial fraction. No special effort was made to obtain nuclear fractions free of mit,ochondria, and phase cont,rast and elect,ron microscopy revealed a large number of mitochondria as well as intact, cells in this fraction (gentle homogenization with Teflon pestles against smooth glass was used in this work).
decisive increase in specific activity occurred in the mitochondrial fraction, where it reached 3-5 times the specific activity of the homogenate on a protein basis. This specific activity increase is the maximum that can be expected for any enzyme in mitochondria, since they contain about one quarter of the protein of the liver cell. The much higher specific activity relative to PNA reflects the low level of nuclear and microsomal contamination in the mitochondrial fraction. (3) There is clearly no succinate cytochrome c re-
Finally,
higher, we may infer that no significant
PNA was determined
by the orcinol re-
ductase activity
in ribosomes, endoplasmic retic-
ulum, or soluble protein since less than 3% of the activity is found in these fractions, and the specific activity is E-20 times lower than t,hat of the homogenate. (4) The nuclear fraction contains a large amount of enzyme activity, about as much as is present in the mitochondrial fraction, although its specific activity is only 1.5 times that of the homogenate. Since we know that this fraction contains a significant number of mitochondria with specific activities 2-3 times contri-
action, which sums the RNA and the DNA con- bution to the total enzyme activity is being protribution. The result,s of four such fractionation vided by a separate nuclear enzyme complex. experiments are shown in Table 2, which may be The mitochondrial isolates prepared above summarized with the following observations: were used to test for penetrance by dimethyl (1) 95% of the total succinate cytochrome c sulfoxide by a straightforward osmotic argureductase activity of the homogenate, as well as ment, using increase in light transmission at 600 85% of the tot,al protein and PNA, was re- nm as the index of mitochondrial swelling. The covered in the va,rious fractions, so that activa- mitochondrial suspension in sucrose was injected tion or inhibition of the enzyme during the fractionation procedure is negligible. (2) The only
into the dialysis compartment
of a fragilograph,
the large surrounding volume being occupied by
DMSO CRTOPHYLLAXIS
OF SUCCINSTE TABLE
LOC.~LIZATION
AND
YIELD
OF
SUCCINATE
TIONATION
Mousr,
“97
c REDUCTASE
2
CYTOCHROME OF
CYTOCHROME
c REDUCTME LIVER
(SCcR) RY
CENTKIFUGAL
FR.~c-
HOMOGENSTES”
-~
% Total content SCcR
H A B C A+B+C
activity
100 47.1 (32.4-62.8) 45.6 (38.1-58.2) 2.62 (1.94-3.25) 95.3 (82.9-102.9)
Protein
100 27.2 (20.5-33.9) 12.1 (11.7-12.5) 44.5 (42.3-46.8) 83.9 (75.4-92.4)
Relative PNA
100 28.4 (24.7-32.0) 3.2 (2.8-3.8) 52.8 (50.3-55.7) 84.4 (78. I-90.7)
specific
activity
on basis
Protein
1.00 1.72 (1.58-1.85) 3.75 (3.26-4.66) 0.060 (0.042-o. 077) 1.14 (1.10-1.24)
of:
P?r’A
1.00 1.75 (1.01-2.54) 14.35 (12.3-15.fij 0.049 (0.039%0.059j 1.14 (0.92-1.31)
Q See the text for procedures and discussion. The data for each of four experiments was calculated on the basis of the corresponding homogenate value as lOO%, and corrected to total original volume. H = homogenate, A = nuclear fraction, B = mitochondrial fractiou, C = ribosome, endoplasmic ret,iciilum, and soluble protein fraction. The mean values and range are shown for the four experiments; pentose nucleic acid (PNA) values include both DNA and RNA. In a perfect experimeut, the values for A + B + C would equal the homogenate values. water, 0.25 M sucrose, or 0.25 41 dimethyl sulfoxide in water. With water in the outer compartment, the sucrose concentration of the mitochondrinl suspension falls gradually by diffusion, and the mitochondria, facing a hypotonic solution, absorb water and swell, accompanied by an increase in light t.ransmission. With sucrose in the out,er compartment,, the concentration of solutc wit,hin and without the dialysis membrane will not change on diffusion, and the mitochondria should swell little or not at all in the same time period. With 0.25 31 dimethyl sulfoxide in the outer compartment the reasoning is as follows: If it does not penetrate the mitochondrial membranes, it will act as an external solute and the osmotic pressure inside the dialysis membrane will not change as the dimethyl sulfoxide exchanges for sucrose by diffusion; t’he light transmission will then follow the pattern seen with sucrose in the external compartment. On the other hand, if the mitochondria are permeable t’o dimethyl sulfoxide, then t,he swelling and thus the increase in light transmission should follow the pattern seen when water is used in the external compartment. A final control involves 0.25 M dimethyl sulfoxide plus 0.25 M sucrose in the estrrnal compartment; if simple osmotic considerations apply and there is no facilitation of sucrose uptake by dimethyl sulfoxide, then the external tonicity maintained by sucrose should
be sufficient to prevent mitochondrial swelling despite the uptake of dimethyl sulfoxide. The results are shown in Fig. 3, which makes it clear that the mitochondria are fully permeable to dimethyl sulfoxide. We do not interpret the slightly greater initial increase in transmiasion in the presence of dimethyl sulfoxide to indicate that it penet,rates faster than water;
Y/4
OMSO IN M/4 SUCROSE
MINUTES
FIG. 3. Permeability of mouse liver mitochondria to dimethyl sulfoxide and water as evidenced by increased light transmission due to osmotic swelling in a fragilograph. A suspension of mitochondria in 0.25 M sucrose was injected into the small inner dialysis compartment. The captions indicate the solution placed in the large outer cornpart,ment. See the text for discussion.
298
FISHBEIN
the data are simply not that accurate. However, the patterns are distinctive enough from that of dimethyl sulfoxide in sucrose to indicate that the osmotic arguments are fulfilled. Phase microscopy of samples before and after the swelling experiment verified that the increase in light transmission truly measured mitochondrial swelling, which involved a 2-4-fold increase in diameters of individual organelles as estimated under oil immersion. DISCUSSION This report completes the evidence indicating that a relatively simple enzyme sequence, consisting of but five or six enzymes of well-categorized behavior, and without any elaborate and mysterious energy-transducing concomitants, can nevertheless evince, in a graded manner, the characteristic phenomena of freeze-thaw damage exhibited by intact cells. Thus the damage is produced by the phase change that accompanies freezing, a critical cooling rate is observed with rapid freezing techniques, and a distinct cryophyllaxis is exhibited by dimethyl sulfoxide. In theoretic terms, this argues, therefore, that mechanisms of freeze-thaw damage, if they are to be generally valid, must be formulated on a molecular rather than a cellular basis, and be able to explain the disruption of a few small molecules as well as that of the cell membrane or nucleus. Thus, for example, the mechanical disruption of cells by internal ice crystals can hardly be of significance in explaining the damage to a five to six molecule sequence, and hence is not a satisfactory general mechanism for freeze-thaw damage. On the other hand, the hypertonicity theory of Lovelock (6, 7)) and the dehydration theory of Meryman (13) and Mazur (lo), provide etiologic hypotheses which may satisfactorily apply to an individual macromolecule as well as to that remarkable concatenation of these which we call a cell. On the pathogenetic level, a general hypothesis has been advanced that macromolecular dissociation followed by absent or abnormal reassociation underlies the appearance of freeze-thaw damage in biologic systems. The site of damage would then be determined by the particular types of macromolecules involved (3). On the practical level, the availability of a simply assayed enzyme complex as an indicator of freezing damage permits the ready accumulation of quantitative data on the nature of freez-
ing damage, which is otherwise very tedious to obt’ain on intact cells, and almost impossible to obtain on tissue slices. The limitations of this method rela,te to the absence of any a priori evidence to indicate that the enzyme damage will regularly correlate with other aspects of cellular damage. For the present this has not been a problem, since we can be certain that the cells are damaged if any element of them is damaged. If, however, under other conditions the enzyme is fully protected, this is no guarantee that other parameters of cell function and structure would also remain intact. For this we must be able to correlate parameters of structural damage with functional damage, obviously not a feasible operation in any quantitative way in tissue slices. -4 quantitative structural evaluation would be feasible, however, with mitochondrial isolates, and since the enzyme complex is mitochondrial in origin, this is the logical approach for subsequent work. Dimethyl sulfoxide cryophyllaxis, although important as a parameter of the response of succinate cytochrome c reductase to freeze-thaw, is hardly a new observation. What is unusual, however, is the clear demonstration that this cryoprotective action was abolished when no external solute was present in the medium. Certainly: then, some minimal external osmotic pressure is required to withdraw sufficient water from the cell during freezing, for the internal dimethyl sulfoxide to exert its protective effect. This is compatible with the simplistic view of permeant cryophyllactics as antifreeze agents, assuming that lowering the amount of intracellular water relative to that of dimethyl sulfoxide will lower the intracellular freezing point. However, in indicating that exchange of dimethyl sulfoxide for water is important, rather than simply the intracellular addition of dimethyl sulfoxide, the data also raise the possibility that exchange at specific sites, rather than a simple nonspecific exchange, may also be important. A more critical investigation of this possibility is currently in progress. SUMMARY Dimethyl sulfoxide, at concentrations of 5-30% (v/v) protected succinate cytochrome c reductase from loss in activity on cooling l-mm thick mouse liver slices to -25°C at l”C/min. The cryophyllactic agent had no direct deleterious effects on the enzyme complex under the
DMSO CRYOPHYLLAXIS
OF SUCCINATE
conditions used. iZlthough equally effective in 0.25 M sucrose, 0.15 M phosphate, and Cl.12 M Tris media, dimethyl sulfoxide exhibited no cryoprotective activity in pure aqueous solutions,
which themselves result in the same degree of freeze-thaw
damage as do buffered media. The
inference is that intracellular dimethyl
sulfoxide
accumulation of
is not of itself a sufficient
condition for cryophyllaxis, but that exchange of the compound for water is required. Succinate cytochrome c reductase was found to be localized in liver mitochondria, and these were found to be fully permea.ble to dimethyl
sulfoxide. The
enzyme complex fulfills the requirements for a graded response to freeze-thaw damage exhibiting the major features characteristic of cryogenic damage in intact cells, while providing a simply quant,ified facile assay for the evaluation of cryogenic damage to tissue slices. REFERENCES 1. Cooperstein, S. J.. Lazarow, A., and Kurfess,
N. J. A microspectrophotometric method for 2.
3.
4.
5.
the determination of succinic dehydrogenase. J. Biol. Chem. 186, 129-139 (1950). Danon, D. A rapid micro method for recording red cell osmotic fragility by continuous decrease of salt concentration. J. Clin. Pathol. 16,377-382 (1963). Fishbein, W. N., and Griffin, J. L. A pathogenetic theory of freeze-thaw injury to biologic systems-protomer dissociation and dys-association. Cryobiology 6,261-262 (1969). Fishbein, W. N., and Stowell, R. E. Studies on the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay I. Temporal localization of the injury phase 4, 283-289 during slow freezing. Cryobiology (1968). Fishbein, W. N., and Stowell, R. E. Studies on
CYTOCHROME
c REDUCTASE
299
the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay II. Comparison of slow and rapid cooling rates. Cryobiology 6,227-234 (1969). 6. Lovelock, J. E. The haemolysis of human red blood-cells by freezing and thawing. B&him. Biophys. Acta lo,414426 (1953). 7. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Bioclrim. Biophys. iicta 11, 28-36 (1953). 8. Lowry, 0. H., Rosebrough, K. J., Farr, A. I,., and Randall, R. J. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275 (1951). 9. Luyet. B. J.. Rapatz. G. I,.. and Gehenio. I’. M. On the mode of action of rapid cooling in the preservation of erythrocytes in frozen blood. Biodynamics 9, 95-124 (1963). 10. Mazur. P. Causes of injury in frozen and thawed cells. Fed. Amer. Sot. Exp. Biol. Proc. 24, Suppl. 15, S175-S182 (1965). 11. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). 12. Mazur, P., and Schmidt, J. J. Interactions of cooling velocity, temperature, and warming velocity on the survival of frozen and thawed 5, 1-17 (1968). yeast. Cryobiology 13. Meryman, H. T. The relationship between dchydration and freezing injury in the human erythrocyte. “Proceedings of the International Conference on Low Temperature Science.” Vol. 2, pp. 231-244. University of Hokkaido, Japan, 1967. 14. Rapatz, G., Sullivan, J. J., and Luyet. B. Preservation of erythrocytes in frozen blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature. Cryobiology 5, 18-25 (1968). 15. Schneider, W. C. Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol.
3,680-682
(1957).
CRYOBIOLOGY,
Studies
III.
on the Mechanism of Freezing Damage to Mouse Using a Mitochondrial Enzyme Assay
Cryophyllaxis
with
Dimethyl WILLIAM
Biochemistru
Sulfoxide
and
Enzyme
Liver
Localization’
N. FISHBEI?;
Branch. Armed Washington,
Forces
Institute
of Pathology,
D. c. POSO5
Previous communications in this series (4, 5) have demonstrated that the succinate cytochrome c reductase complex, a multienzyme electron transport segment of mitochondria, which is suitable for multiple sample spectrophotometric assay, provides a graded response to freeze-thaw treat’ments of mouse liver slices, so that information can be obtained as to the mechanism of freezing damage in nucleated cells with rapidly obtained quantitative data. During slow freezing, the loss of enzyme activity was shown to coincide with the phase transition of the medium from liquid to solid state, and to be independent of other variables studied. In rapid freezing procedures a critical cooling rate was inferred, in the neighborhood of lOO”C/sec, beyond which a markedly greater loss in enzyme activity resulted. In these respects, therefore, the enzyme complex behaved in a manner analogous to t,hat of whole cell systems studied by much more tedious methods (9, 11, 12, 14). In t’his report we show that the analogy is maintained with regard to the protection of the enzyme complex against’ freezing damage by a permeant cryophyllactic agent, dimethyl sulfoxide.
In addition, the unusual stability of the enzyme complex in water has permit’ted the demonstration that dimethyl sulfoxide cryoprotection depends to a significant extent upon the presence of external solute. Finally evidence is presented that t,he enzyme complex under study is localized in the mit,orhondria of mouse liver. EXPERIMENTAL
PROCEDURE
All chemicals used in the study were purchased as reagent grade and used without, further purification. Liver slices measuring approximately 10 X 5 X 1 mm from freshly sacrificed male CAF,(BALB/c x A/) mice were placed in glass homogenizing tubes containing l-2 ml of appropriate chilled medium and cooled at l’C/ min from 4°C to a final nadir at -25°C in a Canalco slow freezing apparatus (Canal Industrial Corp., Bethesda, &Id.) as previously described (4). dfter thawing by immersion of the tube in a 37°C water bath, and replacement of the freeze-thaw medium by fresh, chilled 0.25 M sucrose, 10% homogenates were prepared lT--ith Teflon pasties against smooth glass walls at 4”C, and assayed for succinate cytochrome c reductase activity as described in our first report (4). Received February 19, 1971 ‘This investigation was supported in part by a Dilutions of the same homogenates were used for Kumber 3AO6211OA821, protein determinations by the method of Lowry rrstwch grant, Project from the Medical Research and Development et al. (8)) using bovine serum albumin as stundCommand, U.S. Army, Washington, D.C. 20012. In ard, a.nd the specific activities of the frozenconducting the research described in this report, thawed specimens were calculated as percentsgea the investigators adhered to the “Guide for Laboof the corresponding specific activit’ies ot’ unratory Animal Facilities and Care,” as promulfrozen cont,rol specimens. Buffer and salt solugated by t,he Committee on the Guide for Laboratory Animal Facilities and Care of the Institute tions were prepared in 50% dimethyl sulfoside of Laboratory Animal Resources, National Acadas well as in wat,er, so that appropriate volumetemy of Sciences-National Research Council. The ric mixing of the two solutions yielded various opinions or assertions contained herein are the prilevels of dimethyl sulfoxide without altering the vat,e views of the author and are not to be conconcentration of other constituents. strued as official or as reflecting the views of the For studies on the localization of the enzyme Department of the Army or the Department of complex, mitochondria were prepared at 4°C Defense. 293
294
FISHBEIN
from homogenates as follows: (average g forces are cited, maximum g forces were about 20% higher) after a lo-min centrifugation at 700g to remove nuclei and intact cells, the mitochondria were sedimented at 5500g for 10 min and were centrifuged twice more at 5500g for 15 min after two further resuspensions in 0.25 M sucrose. All fractions and washes were saved, as well as an aliquot of the initial homogenate, for activity, protein, and PNA determinations, the latter by the orcinol reaction (15). Mitochondrial swelling experiments, as evidenced by absorption changes, were performed on these isolates using a home-made fragilograph (2) incorporating a Coleman spectrophotometer wired to a pica-ammeter and recorder. A mitochondrial suspension was injected into the l-mm thick diffusion chamber formed by dialysis membrane stretched over a metal frame. This was placed in a large test tube containing 100 ml of external fluid and insert,ed horizontally into the light path of the photometer, so that t#he optical effect of mitochondria settling during the experimental period was minimized.
must elapse before freezing occurs when a l”C/ min cooling rate is employed, it was first necessary to determine whether or not dimethyl sulfoxide itself produced direct alteration in the succinate cytochrome c reductase activity, in the absence of freezing and thawing. The liver slices were therefore preincubat,ed in dimethyl sulfoxide containing medium at 4°C for various periods of time before beginning the freeze-thaw experiment. Four such experiments with two different concentrations of dimethyl sulfoxide are shown in Fig. 1. Although there was variation in recovery from one experiment to the next, it is clear that within any one experiment incubation of the slice with dimethyl sulfoxide at 4°C for periods up to 1 hr resulted in no greater loss in enzyme activity than if the freeze-thaw experiment was begun at once. It was concluded, therefore, that direct toxic effects of dimethyl sulfoxide on this enzyme complex were negligible under these conditions. The protection afforded by various concentrations of dimethyl sulfoxide incorporated into sucrose solutions against the freeze-thaw damage of succinate cytochrome c reductase is shown in Fig. 2. All concentrations investigated from 5 to 30% (v/v) provided significant protection (p < 0.05). Although 15% gave the highest mean recovery value, this was not significant in comparison to that afforded by other concentrations (p
RESULTS Cryoprotection with dimethyl sulfoxide was evaluated by incorporating the agent, in the concentrations noted, into the suspending medium prior to freezing. Since a lO-15-min period
0
SO E
0
0
0
lO%DMSO
t
20
0 0
*
I 15
0
I 30
I 60
I 45
Tim DMSO Preseti kfore Tissue Freezing
I 75
hid
FIG. 1. Lack of influence of preincubation of mouse liver slices with dimethyl sulfoxide (DMSO) in 0.25 M sucrose at 4°C on the recovery of succinate cytochrome c reductase activity after freeze-thaw. The percentage of DMSO is given on a volume/volume basis. The minimum time for freezing at a cooling rate of l”C/min was 10-15 min, and thawing was accomplished rapidly by immersion in a 37°C water bath. Four separate experiments are shown.
DMSO CRYOPHYLLAXIS
OF SUCCINATE CYTOCHROME c REDKTASE
5
20 15 10 % DMSO PRESENT DURING FREEZING
25
‘-,!I5
30
Fro. 2. Dimethyl sulfoxide (DMSO) cryophyllaxis of succinate cytochrome c reductase activity after freeze-thaw of mouse liver slices. The mean recovery, standard error, and number of experiments is shown for each DMSO concentration (v/v) in 0.25M sucrose. > 0.1). Glycerol at similar concentrations gave a shown in Table 1. In sucrose, phosphate, and comparable degree of protection in the few ex- Tris solutions, dimethyl sulfoxide conferred a 3540% protective effect against freezing damperiments made. We had earlier found that freeze-thaw damage age, whereas in water the protective effect was to succinate cytochrome c reductase in liver virtually nil. The significance of these findings slices was independent of the suspending me- will be discussedbelow. Lest confusion arise, it is important to point dium used, whether this was 0.25 M sucrose, 0.15 N phosphate buffer, 0.12 M Tris-HCl buffer, or out that the succinate cytochrome c reductase most strikingly, simply water, the loss of activ- assay is not equivalent to the assay of succinate ity in all cases being about 45% (4). This unu- dehydrogenase, although the latter enzyme is the sual situation permitted the evaluation of di- first member of the sequence measured by the methyl sulfoxide protection in these diverse reductase assay. Although t’he cytochrome c remedia, and the results of such an experiment are ductase method was introduced aa an assay for
296
FISHBEIN TABLE
1
INFLUENCE OF THE SUSPENDING MEDIUM ON DIMETHYLSULFOXIDE (DMSO) CRYOPHYLLAXIS CINATE CYTOCHROME c REDUCTASE (SCcR) IN ~-MM THICK MOUSE LIVER SLICES~ Medium
No.
expts.
Y. Protection with 2.1 M DMSO M&Ill
SE
PCS% (1)
(1) Wat.er (2) 0.25 M Sucrose (3) 0.10 M Phosphat’e
5 5 5
8.4 39.4 32.4
5.5 5.1 6.1
++ +
(4) 0.17 M Tris-HCl
5
33.8
7.4
+
(+)
FOR Suc-
or 1% (++)
(2)
(3)
(4)
++
+ -
+ -
-
-
a Cooling at l”C/min to -25°C was performed on separate slices in each medium with and without 2.1 M DMSO (15’%, v/v). After rapid thawing in a 37°C water bath, the loss (L) of SCcR activity in the tissue immersed in medium only (Lm) and medium supplemented with DMSO (L,) was determined by comparison with a nonfrozen control. The percentage protection afforded by DMSO was calculated as lOO[(L, - L,)/L,], and denotes the added protection due to DMSO, above that provided by each medium alone, which was about 507, in each case. The probability matrix at the right permits rapid comparison of any two numbered groups with regard to a significant difference between their means at t,he 5y0 (+) or 1% (++) level.
succinate dehydrogenase (1)) the two assays are strikingly different in their response to physical damage (5) . The localization of the reductase enzyme complex was evaluated by centrifugal isolation of mitochondria from the homogenates. All fractions were assayed and corrected to initial volume so as to permit the determination of yields of the enzyme, protein, and nucleic acid as compared to the original homogenate. This is important to assesswhether any activation or inhibition of the enzyme occurs during fractionation, which would interfere with the interpretation of activity changes due to physical damage. The fractionation procedure was designed primarily for purity in the mitochondrial fraction. No special effort was made to obtain nuclear fractions free of mit,ochondria, and phase cont,rast and elect,ron microscopy revealed a large number of mitochondria as well as intact, cells in this fraction (gentle homogenization with Teflon pestles against smooth glass was used in this work).
decisive increase in specific activity occurred in the mitochondrial fraction, where it reached 3-5 times the specific activity of the homogenate on a protein basis. This specific activity increase is the maximum that can be expected for any enzyme in mitochondria, since they contain about one quarter of the protein of the liver cell. The much higher specific activity relative to PNA reflects the low level of nuclear and microsomal contamination in the mitochondrial fraction. (3) There is clearly no succinate cytochrome c re-
Finally,
higher, we may infer that no significant
PNA was determined
by the orcinol re-
ductase activity
in ribosomes, endoplasmic retic-
ulum, or soluble protein since less than 3% of the activity is found in these fractions, and the specific activity is E-20 times lower than t,hat of the homogenate. (4) The nuclear fraction contains a large amount of enzyme activity, about as much as is present in the mitochondrial fraction, although its specific activity is only 1.5 times that of the homogenate. Since we know that this fraction contains a significant number of mitochondria with specific activities 2-3 times contri-
action, which sums the RNA and the DNA con- bution to the total enzyme activity is being protribution. The result,s of four such fractionation vided by a separate nuclear enzyme complex. experiments are shown in Table 2, which may be The mitochondrial isolates prepared above summarized with the following observations: were used to test for penetrance by dimethyl (1) 95% of the total succinate cytochrome c sulfoxide by a straightforward osmotic argureductase activity of the homogenate, as well as ment, using increase in light transmission at 600 85% of the tot,al protein and PNA, was re- nm as the index of mitochondrial swelling. The covered in the va,rious fractions, so that activa- mitochondrial suspension in sucrose was injected tion or inhibition of the enzyme during the fractionation procedure is negligible. (2) The only
into the dialysis compartment
of a fragilograph,
the large surrounding volume being occupied by
DMSO CRTOPHYLLAXIS
OF SUCCINSTE TABLE
LOC.~LIZATION
AND
YIELD
OF
SUCCINATE
TIONATION
Mousr,
“97
c REDUCTASE
2
CYTOCHROME OF
CYTOCHROME
c REDUCTME LIVER
(SCcR) RY
CENTKIFUGAL
FR.~c-
HOMOGENSTES”
-~
% Total content SCcR
H A B C A+B+C
activity
100 47.1 (32.4-62.8) 45.6 (38.1-58.2) 2.62 (1.94-3.25) 95.3 (82.9-102.9)
Protein
100 27.2 (20.5-33.9) 12.1 (11.7-12.5) 44.5 (42.3-46.8) 83.9 (75.4-92.4)
Relative PNA
100 28.4 (24.7-32.0) 3.2 (2.8-3.8) 52.8 (50.3-55.7) 84.4 (78. I-90.7)
specific
activity
on basis
Protein
1.00 1.72 (1.58-1.85) 3.75 (3.26-4.66) 0.060 (0.042-o. 077) 1.14 (1.10-1.24)
of:
P?r’A
1.00 1.75 (1.01-2.54) 14.35 (12.3-15.fij 0.049 (0.039%0.059j 1.14 (0.92-1.31)
Q See the text for procedures and discussion. The data for each of four experiments was calculated on the basis of the corresponding homogenate value as lOO%, and corrected to total original volume. H = homogenate, A = nuclear fraction, B = mitochondrial fractiou, C = ribosome, endoplasmic ret,iciilum, and soluble protein fraction. The mean values and range are shown for the four experiments; pentose nucleic acid (PNA) values include both DNA and RNA. In a perfect experimeut, the values for A + B + C would equal the homogenate values. water, 0.25 M sucrose, or 0.25 41 dimethyl sulfoxide in water. With water in the outer compartment, the sucrose concentration of the mitochondrinl suspension falls gradually by diffusion, and the mitochondria, facing a hypotonic solution, absorb water and swell, accompanied by an increase in light t.ransmission. With sucrose in the out,er compartment,, the concentration of solutc wit,hin and without the dialysis membrane will not change on diffusion, and the mitochondria should swell little or not at all in the same time period. With 0.25 31 dimethyl sulfoxide in the outer compartment the reasoning is as follows: If it does not penetrate the mitochondrial membranes, it will act as an external solute and the osmotic pressure inside the dialysis membrane will not change as the dimethyl sulfoxide exchanges for sucrose by diffusion; t’he light transmission will then follow the pattern seen with sucrose in the external compartment. On the other hand, if the mitochondria are permeable t’o dimethyl sulfoxide, then t,he swelling and thus the increase in light transmission should follow the pattern seen when water is used in the external compartment. A final control involves 0.25 M dimethyl sulfoxide plus 0.25 M sucrose in the estrrnal compartment; if simple osmotic considerations apply and there is no facilitation of sucrose uptake by dimethyl sulfoxide, then the external tonicity maintained by sucrose should
be sufficient to prevent mitochondrial swelling despite the uptake of dimethyl sulfoxide. The results are shown in Fig. 3, which makes it clear that the mitochondria are fully permeable to dimethyl sulfoxide. We do not interpret the slightly greater initial increase in transmiasion in the presence of dimethyl sulfoxide to indicate that it penet,rates faster than water;
Y/4
OMSO IN M/4 SUCROSE
MINUTES
FIG. 3. Permeability of mouse liver mitochondria to dimethyl sulfoxide and water as evidenced by increased light transmission due to osmotic swelling in a fragilograph. A suspension of mitochondria in 0.25 M sucrose was injected into the small inner dialysis compartment. The captions indicate the solution placed in the large outer cornpart,ment. See the text for discussion.
298
FISHBEIN
the data are simply not that accurate. However, the patterns are distinctive enough from that of dimethyl sulfoxide in sucrose to indicate that the osmotic arguments are fulfilled. Phase microscopy of samples before and after the swelling experiment verified that the increase in light transmission truly measured mitochondrial swelling, which involved a 2-4-fold increase in diameters of individual organelles as estimated under oil immersion. DISCUSSION This report completes the evidence indicating that a relatively simple enzyme sequence, consisting of but five or six enzymes of well-categorized behavior, and without any elaborate and mysterious energy-transducing concomitants, can nevertheless evince, in a graded manner, the characteristic phenomena of freeze-thaw damage exhibited by intact cells. Thus the damage is produced by the phase change that accompanies freezing, a critical cooling rate is observed with rapid freezing techniques, and a distinct cryophyllaxis is exhibited by dimethyl sulfoxide. In theoretic terms, this argues, therefore, that mechanisms of freeze-thaw damage, if they are to be generally valid, must be formulated on a molecular rather than a cellular basis, and be able to explain the disruption of a few small molecules as well as that of the cell membrane or nucleus. Thus, for example, the mechanical disruption of cells by internal ice crystals can hardly be of significance in explaining the damage to a five to six molecule sequence, and hence is not a satisfactory general mechanism for freeze-thaw damage. On the other hand, the hypertonicity theory of Lovelock (6, 7)) and the dehydration theory of Meryman (13) and Mazur (lo), provide etiologic hypotheses which may satisfactorily apply to an individual macromolecule as well as to that remarkable concatenation of these which we call a cell. On the pathogenetic level, a general hypothesis has been advanced that macromolecular dissociation followed by absent or abnormal reassociation underlies the appearance of freeze-thaw damage in biologic systems. The site of damage would then be determined by the particular types of macromolecules involved (3). On the practical level, the availability of a simply assayed enzyme complex as an indicator of freezing damage permits the ready accumulation of quantitative data on the nature of freez-
ing damage, which is otherwise very tedious to obt’ain on intact cells, and almost impossible to obtain on tissue slices. The limitations of this method rela,te to the absence of any a priori evidence to indicate that the enzyme damage will regularly correlate with other aspects of cellular damage. For the present this has not been a problem, since we can be certain that the cells are damaged if any element of them is damaged. If, however, under other conditions the enzyme is fully protected, this is no guarantee that other parameters of cell function and structure would also remain intact. For this we must be able to correlate parameters of structural damage with functional damage, obviously not a feasible operation in any quantitative way in tissue slices. -4 quantitative structural evaluation would be feasible, however, with mitochondrial isolates, and since the enzyme complex is mitochondrial in origin, this is the logical approach for subsequent work. Dimethyl sulfoxide cryophyllaxis, although important as a parameter of the response of succinate cytochrome c reductase to freeze-thaw, is hardly a new observation. What is unusual, however, is the clear demonstration that this cryoprotective action was abolished when no external solute was present in the medium. Certainly: then, some minimal external osmotic pressure is required to withdraw sufficient water from the cell during freezing, for the internal dimethyl sulfoxide to exert its protective effect. This is compatible with the simplistic view of permeant cryophyllactics as antifreeze agents, assuming that lowering the amount of intracellular water relative to that of dimethyl sulfoxide will lower the intracellular freezing point. However, in indicating that exchange of dimethyl sulfoxide for water is important, rather than simply the intracellular addition of dimethyl sulfoxide, the data also raise the possibility that exchange at specific sites, rather than a simple nonspecific exchange, may also be important. A more critical investigation of this possibility is currently in progress. SUMMARY Dimethyl sulfoxide, at concentrations of 5-30% (v/v) protected succinate cytochrome c reductase from loss in activity on cooling l-mm thick mouse liver slices to -25°C at l”C/min. The cryophyllactic agent had no direct deleterious effects on the enzyme complex under the
DMSO CRYOPHYLLAXIS
OF SUCCINATE
conditions used. iZlthough equally effective in 0.25 M sucrose, 0.15 M phosphate, and Cl.12 M Tris media, dimethyl sulfoxide exhibited no cryoprotective activity in pure aqueous solutions,
which themselves result in the same degree of freeze-thaw
damage as do buffered media. The
inference is that intracellular dimethyl
sulfoxide
accumulation of
is not of itself a sufficient
condition for cryophyllaxis, but that exchange of the compound for water is required. Succinate cytochrome c reductase was found to be localized in liver mitochondria, and these were found to be fully permea.ble to dimethyl
sulfoxide. The
enzyme complex fulfills the requirements for a graded response to freeze-thaw damage exhibiting the major features characteristic of cryogenic damage in intact cells, while providing a simply quant,ified facile assay for the evaluation of cryogenic damage to tissue slices. REFERENCES 1. Cooperstein, S. J.. Lazarow, A., and Kurfess,
N. J. A microspectrophotometric method for 2.
3.
4.
5.
the determination of succinic dehydrogenase. J. Biol. Chem. 186, 129-139 (1950). Danon, D. A rapid micro method for recording red cell osmotic fragility by continuous decrease of salt concentration. J. Clin. Pathol. 16,377-382 (1963). Fishbein, W. N., and Griffin, J. L. A pathogenetic theory of freeze-thaw injury to biologic systems-protomer dissociation and dys-association. Cryobiology 6,261-262 (1969). Fishbein, W. N., and Stowell, R. E. Studies on the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay I. Temporal localization of the injury phase 4, 283-289 during slow freezing. Cryobiology (1968). Fishbein, W. N., and Stowell, R. E. Studies on
CYTOCHROME
c REDUCTASE
299
the mechanism of freezing damage to mouse liver using a mitochondrial enzyme assay II. Comparison of slow and rapid cooling rates. Cryobiology 6,227-234 (1969). 6. Lovelock, J. E. The haemolysis of human red blood-cells by freezing and thawing. B&him. Biophys. Acta lo,414426 (1953). 7. Lovelock, J. E. The mechanism of the protective action of glycerol against haemolysis by freezing and thawing. Bioclrim. Biophys. iicta 11, 28-36 (1953). 8. Lowry, 0. H., Rosebrough, K. J., Farr, A. I,., and Randall, R. J. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193, 265-275 (1951). 9. Luyet. B. J.. Rapatz. G. I,.. and Gehenio. I’. M. On the mode of action of rapid cooling in the preservation of erythrocytes in frozen blood. Biodynamics 9, 95-124 (1963). 10. Mazur. P. Causes of injury in frozen and thawed cells. Fed. Amer. Sot. Exp. Biol. Proc. 24, Suppl. 15, S175-S182 (1965). 11. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). 12. Mazur, P., and Schmidt, J. J. Interactions of cooling velocity, temperature, and warming velocity on the survival of frozen and thawed 5, 1-17 (1968). yeast. Cryobiology 13. Meryman, H. T. The relationship between dchydration and freezing injury in the human erythrocyte. “Proceedings of the International Conference on Low Temperature Science.” Vol. 2, pp. 231-244. University of Hokkaido, Japan, 1967. 14. Rapatz, G., Sullivan, J. J., and Luyet. B. Preservation of erythrocytes in frozen blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature. Cryobiology 5, 18-25 (1968). 15. Schneider, W. C. Determination of nucleic acids in tissues by pentose analysis. Methods Enzymol.
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(1957).