THE EFFECT OF ROENTGEN RAYS ON THE COLLOIDAL PROPERTIES OF ERYTHROCYTES’ HELEN QUINCY WOODARD Memorial Hospital, New York City, New York Received June 99, 1957 INTRODUCTION
In a recent address Failla (2) advanced a theory of the action of penetrating radiation on living material. The theory is based on the observation that both the cells of irradiated tissue and their nuclei swell following exposure to Roentgen rays or to the alpha, beta, or gamma rays of radium. It is known that radiation of these types ionizes matter, producing “radio ions.” These are LLatoms, molecules, or aggregates which either have lost or have acquired electrons.” They are distinct from ordinary chemical ions, and usually recombine rapidly by exchange of an electron. They may, however, regroup themselves with the formation of new chemical substances. If this latter process takes place inside a living cell, and if the new substances have a smaller molecular weight than the parent substances, the osmotic pressure inside the cell will increase. If the intercellular fluid, such as blood or lymph, is affected to a less extent than the cell contents, or if it is replaced with unchanged fluid by the circulation, the cells will swell. Direct injury to the cell membrane would intensify this effect by weakening the resistance of the membrane to the increased internal pressure. It is difficult to test this theory with cells imbedded in a slowly permeable stroma, such as those in the intact organism or in tissue culture. Of isolated cells the most readily available are mammalian erythrocytes. These are undesirable because of their well-known high radioresistance. On the other hand, since they are non-nucleated, they are free from the complications which might be introduced by the presence of a second osmotic system within the first. Accordingly, suspensions of washed sheep erythrocytes were chosen for the present study. Since it was found that hemolysis played an important part in the phenomena observed, it will be convenient here to review the mechanism of this process. When erythrocytes are placed in hypotonic solutions they swell in accordance with osmotic laws (4,7). There is a t first no I
Presented at the Fourteenth Colloid Symposium, held a t Minneapolis, Minnesota, June 10-12, 1937. 47
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change in the permeability or electrical properties of the membrane (3). Khen the membrane has been stretched to a critical degree, however, it suddenly becomes completely permeable to hemoglobin, about 90 per cent of which then escapes into the surrounding medium in less than 1 sec. (9). The electrical properties of the membrane change at the moment of hemolysis, but the membrane persists unruptured as a “ghost.” If the hemolyzed ghosts are then placed in hypertonic solutions they shrink like normal erythrocytes and regain most of their former osmotic and electrical resistance (9). The permeability of the membrane in hypotonic hemolysis is nearly constant regardless of the tonicity of the solution used to bring about the hemolysis (9). Khen hemolysis is brought about by a lysin such as saponin, instead of by exposure to hypotonic solutions, the primary effect is on the membrane rather than on the contents of the cell. As in hypotonic hemolysis, the cell hemolyzes completely or not a t all. However, after hemolysis by saponin the cell membrane is irreparably damaged, does not recover its former properties, and may disappear altogether (3). .Uthough the hemolysis of the individual erythrocyte is an “all or none” process, the hemolysis of any given sample of blood is not, because the red cell population is a heterogeneous one showing all degrees of resistance. Hence it is possible by appropriate treatment t o hemolyze any fraction of a sample of cells from zero to 100 per cent. Since hemoglobin solutions obey Beer’s law (I), the per cent of hemolysis is readily determined by meann of photoelectric readings on the supernatant liquid obtained by centrifuging a partly henioly2ed suspension. METHOD
Preliminary work was done on heparinized blood in order to avoid possible osmotic effects from a small molecule anticoagulant such as oxalate. Heparin did not proye a satisfactory anticoagulant for large volumes, hoiTever, so, as no difference was detected in the behavior of washed cells from oxalated and heparinized samples, oxalated blood was used in most of the work. Sheep blood containing 2 mg. of potassium oxalate per cubic centimeter was obtained in 300-cc. lots fresh from the slaughter house. It {vas centrifuged at once, the plasma pipetted off, and the cells decanted from the potassium oxalate precipitate and washed three times with 0.9 per cent sodium chloride solution. The cells were then suspended in such a quantity of 0.9 per cent sodium chloride solution that, when packed, they would occupy about 50 per cent of the volume of the suspension. The suspension was kept in the ice box until used. Before being irradiated the suspension was placed in 1-cc. hematocrit tube.. closed with vaccine stoppers and centrifuged at a force of 1000 X gravity. Forty-five minutes was required for the packed cells to coni? to
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49
constant volume at this centrifugal force. The sodium chloride solution mas then removed, the tubes re-stoppered, placed in a cardboard box, and
irradiated. Sodium chloride solution of the desired concentration was then added, the cells redispersed by shaking, and the tubes kept in a horizontal position in the ice box for seventeen to twenty hours. They were then re-centrifuged, and the cell volume read with a precision of about f 0 . 5 per cent. The per cent hemolysis was determined by photoelectric readings on the supernatant liquid in the hematocrit tubes. Reference values for 100 per cent hemolysis were obtained by centrifuging tubes of stock suspension, removing the saline, and adding distilled water. Owing to the high opacity of the completely hemolyzed preparations there was a possible error of 10 per cent in these photoelectric readings. The error in reading the partially hemolyzed preparations was proportionately less, so that, although all the determinations in a given experiment were subject to the possible error in the 100 per cent value, they were consistent among themselves to a higher precision than that of the 100 per cent standard. As Roentgen irradiation colors the glass of the hematocrit tubes, blank readings were always made on tubes of the same color as the experimental ones. Several investigators (5, 6) have shown that hemolysis is profoundly influenced by temperature. It was impossible in the present work to maintain a constant temperature throughout the course of an experiment. Hence great care was exercised to avoid temperature differences between irradiated and control tubes during manipulation. The Roentgen rays were obtained from a mechanically rectified machine with water-cooled tube having glass walls 4 mm. thick. It was operated a t 200 kv. and 30 ma.; distance from target to hematocrit = 40 cm.; 1 mm. cardboard; intensity = 270 r. per miiiute; filter = 1 mm. glass dose = 16,200roentgens. This dose is much greater than any which would be tolerated by the intact organism, but previous experience by the author and many others has shown that material irradiated in vitro is usually highly radioresistant.
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RESULTS
Preliminary work indicated that, when whole sheep blood was irradiated, or when centrifuged cells were irradiated alone and unirradiated plasma added, the irradiated cells swelled slightly. This is in accord with Failla’s theory. When plasma was irradiated and then added to unirradiated cells, no significant change in the volume of the cells a a s found. This may indicate either that there is a differential effect on the cell proteins or that changes produced by irradiation in the less concentrated plasma proteins are too small to produce psmotic effects greater than the experi-
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mental error. It was apparent at once, however, that much larger effects could be produced by equilibrating the irradiated cells with sodium chloride solutions. This is to be expected from the Donnan relations. Hence most of the work was done with cells equilibrated with salt solutions. As the effects of irradiation on fresh and aged blood differed markedly, the changes which took place in the control suspensions with time will first be outlined. Figure 1 shows changes taking place in two stock suspensions of washed cells which were kept in 0.9 per cent sodium chloride solution in the ice box. The volume of the cells as shown by the hem-
FIG. 1 FIG.2 FIG. 1. Changes in stock suspensions with time. Abscissae = age in days. Ordinates: A = absolute cell volume from hematocrit readings; B = per cent hemolysis; C = specific cell volume; D = photoelectric readings on completely hemolyzed
samples. FIG.2. Changes with time in the absolute cell volume of unirradiated cells equilibrated tvith fresh sodium chldride solutions. Abscissae 5 age in days of cells when fresh solutions were added. Ordinates = absolute cell volume from hematocrit readings. Curves in A, cells in 1.8 per cent sodium chloride; curves in B, cells in 0.9 per cent sodium chloride; curves in C, cells in 0.6 per cent sodium chloride.
atocrit readings remains approximately constant over a period of several days. The degree of hemolysis, on the other hand, increases rather suddenly after a variable period of time which, for the samples shown, was about six days. If the specific cell volume is calculated by the formula, specific cell volume
=
absolute cell volume - per cent hemolysis
100
it is seen that the volume of the remaining intact cells increases. At the same time the light absorption of the hemoglobin in samples completely hemolyzed by the addition of distilled water increases, as is shon-n by the
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COLLOIDAL PROPERTIES OF ERYTHROCYTES
drop in the curves for the photoelectric cell readings. The dotted portions
of the curves indicate regions in which the photoelectric readings on the completely hemolyzed samples were so small that calculations of per cent hemolysis were subject to large errors. These changes suggest that a spontaneous change takes place in the cell contents which results in an increased intracellular osmotic pressure. This causes the cells to swell, and brings about the hemolysis of those in which the process is most pronounced. I n the samples illustrated, the relative rates of swelling and hemolysiswere such that the totalvolume of the unhemolyzed cellsremained approximately constant until near the end of the period of observation. I
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FIG.3 FIQ.4 FIG.3. Changes with time in the per cent hemolysis of unirradiated cells equilibrated with fresh sodium chloride solutions. Abscissae = age in days of cells when iresh solutions were added. Ordinates = per cent hemolysis. Curves i n A, cells in 1.8 per cent sodium chloride; curves in B, cells in 0.9 per cent sodium chloride; curves in C, cells in 0.6 per cent sodium chloride. FIG.4. Changes with time in the specific cell volume of unirradiated cells equilibrated with fresh sodium chloride solutions. Abscissae = age in days of cells when fresh solutions were added. Ordinates = specific cell volume. Curves in A, cells in 1.8 per cent cent sodium chloride; curves in B, cells in 0.9 per cent sodium chloride.
When samples of such suspensions are removed from time to time, centrifuged, the old saline removed, and the cells equilibrated for eighteen hours in the ice box with hypertonic, isotonic, and hypotonic sodium chloride solutions, curves like those in figures 2, 3, and 4 are obtained. The absolute cell volume, per cent hemolysis, and specific cell volume are plotted against days of age of the cells when placed in fresh saline. Curves No. 2 and 3 are for the same suspensions as those illustrated in figure 1, and the cell volumes are corrected for losses from hemolysis in the stock suspension. Data were not complete on the hemolysis, which was small, in the stock suspension for curve No. 1, and the cell volumes for this prepara-
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tion are uncorrected. In these preparations the cells which were sholvn by calculation of the specific cell volume in the stock suspension to be somewhat swollen were unable to withstand the further swelling caused by hypotonic solutions, and hemolyzed so extensively that the absolute volume of the intact cells fell rapidly. The specific volume of the cells in hypotonic saline was found to rise very rapidly with increasing age of the cells, but the experimental error of both hematocrit and photoelectric readings is so large in this range that calculations of specific cell volume are without quantitative significance, and are not shown in the chart. On the other hand, if cells of the same lot were placed in isotonic saline, they underwent some additional increase in volume, but this was not sufficient to cause much hemolysis. Cells placed in hypertonic saline, while they shrank somewhat less than they did when fresh, nevertheless diminished somemhat in volume and hemolyzed only very slightly. If the preparations were observed still longer, a marked increase in the light absorption of hemoglobin was found to take place in sodium chloride solutions of all concentrations, the absolute cell volume decreased, and hemolysis finally became complete. Numerous other samples of erythrocytes were found to behave in the same way as those illustrated, although the changes outlined took place a t different ages in the different samples If the changes induced by irradiation are due to a change in the state of aggregation of the cell contents, it is to be expected that the ef'irct of irradiation of fresh and old cells will be different. This is found to be the case, as is shown in figures 5, G , and 7. The percentage change in absolute and specific cell volumes and the absolute change in per cent hemolysis are plotted against age of blood sample a t the time of irradiation. It is seen that, when fresh cells are irradiated and then equilibrated with sodium chloride solutions of different concentrations, the hemolysis undergone by the irradiated cells is always greater than that of the controls. Because of the pronounced hemolysis of irradiated rells suspended in hypotonic saline the absolute volume of the remaining rells is less than that of the controls, while that of the less hemolyzed irradiated cplls in isotonic saline undergoes little change, and that of irradiated cells in hypertonic saline remains greater than that of the controls. The specific volume of all the cells irradiated when fresh is greater than that of the controls. As the cells age, however, these relationships change, SO that in the oldest prepnrations the hemolysis of the irradiated cells is less than that of the controls. Thi.; reversal of radiosensitivity takes place at approximately the same age ns the most marked increase in the light absorption of the hemoglobin. It appears therefore that irradiation of fresh parked sheep erythrocytes results in the splitting of the hemoglobin t o rompounds of smaller molecular -\vpigl-lt. This causes the cells to swell relatively to the controls when subsequently placed in sodium chloride solutions, and to hemolyze when
COLLOIDAL PROPERTIES O F ERYTHROCYTES
53
the swelling is great enough. This is in accord with the theory of Failla. As the cells age, however, a spontaneous change takes place in the cell
FIG.5 FIG. 6 FIG.5. Changes in radiosensitivity with time. Abscissae = age in days of cells when irradiated. Ordinates = absolute volume of irradiated cells/absolute volume of control cells. Curves in A, cells in 1.8 per cent sodium chloride; curves in B, cells in 0.9 per cent sodium chloride; curves in C, cells in 0.6 per cent sodium chloride. FIG.6. Changes in radiosensitivity with time. Abscissae = age in days of cells when irradiated. Ordinates = per cent hemolysis of irradiated cells minus per cent hemolysis of control cells. Curves in A, cells in 1.8 per cent sodium chloride; curves in B, cells in 0.9 per cent sodium chloride; curves in C, cells in 0.6 per cent sodium chloride.
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FIG.7. Changes in radiosensitivity with time. Abscissae = age in days of cells when irradiated. Ordinates = specific volume of irradiated cells/specific volume of control cells. Curves in A, cells in 1.8 per cent sodium chloride; curves in B, cells in 0.9 per cent sodium chloride.
contents which causes the cells to swell. Irradiation of these swollen cells reduces their volume, probably through coagulation of the altered hemoglobin. No evidence is a t hand a t present as to the nature of the
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spontaneous change in the hemoglobin which results in a reversal of the radiation effect. Preliminary work in which the catalase of the cells was inactivated with sodium chlorate indicates that methemoglobin formation may play a part. No mention has been made of the possible effect of radiation on the cell membrane. The data are more in harmony with the theory that the major portion of the irradiation effect is on the cell contents. It is hoped that direct evidence on this question may be obtained by a study of the radiosensitivity of cells whose membranes have been weakened by a lysin. Since the sensitivity of cells which have been irradiated while packed and almost free from saline and then suspended in saline of different concentrations is the same within the experimental error as the sensitivity of these cells when they have been suspended in the corresponding saline solutions just prior to irradiation, it is obvious that the effect of the radiation is on the cells themselves rather than on the medium. SUMMARY
The effect of 200 kv. Roentgen radiation on the osmotic properties of sheep erythrocytes has been studied. The volume and the susceptibility to. hemolysis of erythrocytes which were irradiated when fresh was increased by irradiation. This effect was reversed when the cells were irradiated after they had been kept in physiological saline for several days at low temperatures. This is interpreted as meaning that the hemoglobin of fresh cells is split to compounds of smaller molecules by Roentgen radiation, while that of aged cells is coagulated. The author wishes to express her thanks to Dr. G. Failla for suggesting the problem, and to Mr. John Sachs for technical assistance. REFERENCES
(1) DRABKIN, D. L., AND AUSTIN,J. H.: J. Biol. Chem. 112, 105-15 (1935). (2) FAILLA,G.: Address at the meeting of the American Association for the Advancement of Science, December, 1936. (3) FRICKE, H.,AND CURTIS,H. J . : J. Gen. Physiol. 18,821-36 (1934-35). (4) JACOBS, M. H.:Biol. Bull. 62, 17&94 (1932). (5) JACOBS, M. H., GLASSMAN, H. N., AND PARPART, A. K.: J. Cellular Comp. Physiol. 8, 403-17 (1936). (6) JACOBS, M.H.,AND PARPART, A. K.: Biol. Bull. 60, 95-119 (1931). (7) JACOBS, M.H., AND PARPART, A. K.: Biol. Bull. 65, 224-34 (1932). (8) PONDER, E.: Proc. SOC.Exptl. Biol. Med. 33, 630-3 (1936). (9) PONDER, E., AND MARSLAND, D . : J. Gen. Physiol. 19, 3544 (1935).
