INT . J . RADIAT . BIOL .,
1977,
VOL .
32,
NO .
1, 89-101
Effect of salt solutions on radiosensitivity of mammalian cells II . Treatment with hypotonic solutions G . P . RAAPHORST and J . KRUUV Department of Physics, University of Waterloo, Waterloo, Ontario, Canada
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(Received 25 October 1976 ; accepted 16 March 1977)
Chinese hamster (V79) cells were treated with hypotonic NaCl, NaCl-ouabain, KCl, LiCI, NH 4 C1, NaNO3 or sucrose solutions and irradiated at various times during exposure to the solution . The extreme increase in the radiosensitivity of these cells, mainly characterized by changes in Do , could be attributed to increases in the cell water-content and possibly the decrease in total cell water structure . The various ions may exert their specific effects on radiosensitivity by influencing the above water-related factors .
1 . Introduction Radiation damage in biological systems is caused to a large extent by the action of ionizing radiation on cellular water, which results in the radiochemical production of free-radical species . The radicals can diffuse to and interact with biological macromolecules, resulting in sub-lethal or lethal damage in living organisms . It has been shown that up to 62 per cent of all death in cultured mammalian cells caused by ionizing radiation may be due to the OH radical (Reuvers, Greenstock, Borsa and Chapman 1973) . Thus changes in the water of biological systems may result in changes in their radiosensitivity . Nuclear magnetic resonance evidence has accumulated to indicate that the water of cells may exist in a structured state (Hazlewood, Nichols and Chamberlain 1969, Cope 1969, Abetsedarakaya, Miftakhutdinova and Fedotov 1968, Drost-Hansen 1971) and that the self-diffusion coefficient of such water is lower than that of bulk water (Chang, Rorschach, Nichols and Hazlewood 1973, Hansen 1971, Abetsedarakaya et al. 1968, Thompson, Kydon and Pintar 1976) . It was reported by Raaphorst, Kruuv and Pintar (1975) that the total cellular water structure and content may be altered by subjecting cells to various ionic solutions . Furthermore it was shown that when cells were subjected to a wide range of salt solutions, large changes were observed in the cellular radiosensitivity . Extreme increases in cellular radiosensitivity were observed for cells treated with hypotonic concentrations of various salt solutions (Raaphorst and Kruuv 1976, 1977 a) . Under these conditions the cell swells, thus acquiring more water . Hence, the structure of the total cell water decreases (Raaphorst et al. 1975) . Other investigators have shown that the cell-volume undergoes various changes as a function of the time that the cells are exposed to various hypotonic solutions and that these changes in volume are dependent on the types of ions present in the cell bathing solution (Roti Roti and Rothstein 1973 a, Doljanski, Ben-Sasson, Reich and Grover 1974, Shank, Rosenberg and Horowitz 1973) . In this paper, experiments are presented in which the relationships between the changes in cell water and radiosensitivity are examined when the cells are treated with various hypotonic solutions .
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G. P . Raaphorst and J . Kruuv
Materials and methods The cells used were originally derived from the V-79 S-171 line of Chinese hamster fibroblasts and the sub-line used was S-171-W1 . Tissue-culture methods for the S171-W2 sub-line have been described by Koch and Kruuv (1971) ; the same culture techniques were used in these experiments . Exponentially-growing cells were inoculated into plastic Petri dishes (60 mm) containing complete medium (Eagle's Basal Medium with Hanks' salts supplemented by 15 per cent foetal calf serum) and were incubated at 37 ° C for 22 hours for attachment . The medium was then removed and replaced by a salt solution equilibrated to room temperature (22 ° C) . In the last (x) minutes of the exposure to the salt, a dose of radiation, requiring (x) minutes to deliver, was administered to the ' experimental ' plates . Immediately after irradiation was completed, the salt solution was removed from each plate, which was then rinsed with 3 ml of medium at room temperature . Finally, complete medium was added to each plate and incubation for the colony-survival assay followed . For the window experiment (figure 3), cells were treated for 20 min with a salt solution and irradiated at various times before, during, or after exposure to the salt . For irradiation before exposure to salt, the plates (containing medium) were taken out of the incubator immediately before irradiation, after which they were kept at room temperature until they were exposed to salt at zero time . All other plates were removed from the incubator at zero time ; the medium was replaced by room-temperature salt solutions for 20 . 0 min, followed by a change to room-temperature medium . The plates were reincubated only after both exposure to salt and irradiation had been carried out . For each experiment toxicity controls (No . 1) were carried out in which the cells were treated with the salt solutions in the same manner as the experimental plates, except that irradiation was omitted . Another control (No . 2) was carried out in which the cells were irradiated in whole medium without prior or subsequent salt treatment ; these cells were subjected to the same temperature profile as the salt treated cells . (Previous control experiments with cells in complete medium had shown that there was no significant difference in survival between cells irradiated immediately on removal from the 37 ° C incubator and cells subjected for up to 30 min to room temperature before irradiation .) This control is indicated on each figure by a cross . Ouabain was dissolved in medium or salt solutions at a concentration of 5 x 10 -4 M . Exponentially-growing cells were exposed to the solution of ouabain and medium for 12 hours at 37 ° C . After this time, the cells were released from the plates by treatment with trypsin, inoculated into new plates containing medium with 5 x 10 -4 M ouabain and put into the incubator for 2 . 5 hours for attachment . The treatment of cells with salt solutions containing ouabain was carried out as described above . The plating efficiency (PE) of the S-171-W1 cells in complete medium was above 80 per cent in all experiments . The results for controls Nos . 1 and 2 and the radiation survival results for the salt-treated cells (experimental cells) were normalized to the PE . The standard errors are indicated in the figures when greater than the actual graph point size . Two petri dishes were used for each radiation point ; three dishes were used for each toxicity point . All the solutions used were unbuffered, i .e . the salt or the sucrose was dissolved in distilled water . The pH at the time of irradiation was about 5 . 5
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due to CO 2 absorption . Monitoring of the pH has previously been described (Raaphorst and Kruuv 1976) . Growth curve experiments with salt-treated cells were carried out as follows . After a 2 . 5 hour incubation period at 37 ° C for attachment, the cells in the 60 mm Petri dishes were treated for 20 min with a salt solution at room temperature (22 ° C) . The salt solution was then replaced by whole medium at room temperature, and the plates were put into the 37°C incubator . This was considered zero time in the growth experiment . Controls were treated in an identical manner, except that the cells were treated for 20 min with medium at room temperature instead of salt solution . All chemicals used were reagent grade . The ouabain (Strophanthin G) was obtained from Calbiochem . Irradiation was carried out with a Radiation Machinery Corporation ' Gammator B', which has a caesium-137 source delivering an absorbed dose of 460 rad/min to the cells . 3.
Results The growth of cells is not significantly affected by a 20 min treatment at room temperature with a 0 . 05 or 0 . 10 M NaCl solution (figure 1) . The doublingtime for salt-treated cells and control cells was the same (about 10 hours) . It is possible that the salt treatment has caused a small initial decrease in the number of cells that were counted .
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a w
w a
N J W U
Figure 1 . Growth curves of cells given 20 . 0 min equilibration at room temperature in solutions of 0 . 05 M (triangles) or 0 . 1 M NaCl (squares) . The circles represent growth of cells that were treated with whole medium for 20 . 0 min at room temperature . Zero time on the abscissa represents the end of the solution equilibration times . Complete radiation survival curves of cells treated with various hypotonic NaCl or KCl solutions are shown in figure 2 . The survival-curve parameters are listed in table 1 . In each case the radiation survival data were corrected for the salt toxicity (i .e . control No . 1) . The toxicity effect of the treatment with 0 . 01 M NaCl or KCl solutions reduced the cell survival to 30 . 9 and 8 . 3 per cent, respectively . The cell toxicity survival after treatment with 0 . 04 M, or the more concentrated solutions of NaCl or KCl, was greater than 80 per cent .
G . P . Raaphorst and j . Kruuv
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Figure 2 . Survival curves of cells treated with various NaCl (closed symbols) or KCI (open symbols) solutions for 20 . 0 min at room temperature and irradiated in the last minutes of exposure to salt . The survival curve of control cells, irradiated in medium, is represented by the closed circles ; cells exposed to 0 . 01 and 0 . 04 M salt solutions are signified by diamonds and squares, respectively . The open and closed triangles represent results with cells exposed to 0 . 15 M KCl and 0 . 116 M NaCl, respectively .
From table 1 it can be observed that the changes in cell survival are characterized mainly by changes in Do of the survival curve, while n does not change significantly. In all cases the treatment with hypotonic solutions caused an increase in cellular radiosensitivity . The cells treated with 0 . 01 or 0 . 04 M KCl solutions have a lower survival level than the cells treated with the corresponding concentrations of NaCl . Description
Control 0 . 010 M 0 . 040 M 0 . 116 M 0 . 010 M 0 . 040 M 0 . 150 M Table 1 .
NaCl NaCl NaCl KCl KCl KCl
Do (rad)
Da (rad)
n
124 48 72 112 45 58 113
540 210 280 440 180 280 450
60 60 50 50 60 70 60
D,, salt/ Do control
0 . 39 0 . 58 0 . 90 0 . 36 0 . 46 0 . 90
Parameters from the NaCl and KCI survival curves .
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-3 510
5
10 30
-10 0 TIME
40
20 IN
60
MIN
Figure 3 . Survival of cells exposed for 20 . 0 min to 0. 10 M NaCl solutions and irradiated with a dose of 1380 rad before this exposure time (negative time on the abscissa), during this exposure (0-20 . 0 min on the abscissa) or after the salt exposure (20 . 0-50 . 0 min on the abscissa) . All salt, radiation and medium exposures were carried out at room temperature . The cross represents the control irradiated in whole medium . The horizontal bars represent the time-span of radiation exposure (3 . 0 min) . The experiment depicted in figure 3 (termed a ` window' experiment) shows the radiation survival of cells irradiated either before, during, or after, a 20 min exposure to a 0 . 10 M NaCl solution . The horizontal bars on the data points represent the time required (3 min) to administer the 1380 rad dose of radiation . Large changes in cell survival occur if irradiation is carried out immediately before or after the addition of the NaCl solution . The survival ratio (SR), i .e . the ratio of the experimental point to control No . 2 (cross), is 0 . 208 for cells irradiated immediately before the addition of the salt and 0 . 014 if irradiation is carried out immediately after the salt addition . Likewise, there is an abrupt increase in the SR if cells are irradiated immediately after the salt solution is removed (0 . 317) compared with immediately before the salt is removed (0 . 137) . Also as the cells are irradiated at later times in the period of exposure to salt (between 0 and 20 min on figure 3), the cell survival increases from a SR of 0 . 014 All points have a significantly lower survival at 0-3 min to 0 . 137 at 17-20 min . level than control No . 2 . Figures 4 to 6 display the survival of cells treated with hypotonic salt or sucrose solutions for various times before administering a dose of 1380 rad in the last three minutes of the exposure time . In the case of KCl the radiation dose was 1012 rad and was given in the last 2. 2 min of exposure to solution. The curves of the irradiated cells (closed symbols ; left axis) have been normalized to the toxicity control (open symbols ; right axis) . R .B .
H
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0
0 Q
L 10"
5 10 0 20
40 TIME
60 IN MIN
80
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Figure 4 . Isodose survival curves (closed symbols, left axis) of cells irradiated with a 1380 rad dose in the last 3 . 0 min of various exposure times to 0 . 10 M sucrose (circles) or 0 . 13 M LiCl (squares) solutions as indicated on the abscissa . All pre-irradiation solution exposure times from 0 to 80 . 0 min were carried out at room temperature . The open symbols (right axis) represent the toxicity effect on cells equilibrated in 0 . 10 M sucrose (circles) or 0 . 13 M LiCl (squares) solutions for the various times indicated on the abscissa . The cross (left axis) represents the survival of cells irradiated in whole medium .
For both the 0 . 13 M LiCl and 0 . 10 M sucrose curves in figure 4, the cell survival is the lowest when irradiation is carried out immediately after the addition of the solution (i .e . SRs of 0 . 021 and 0 . 122 for the sucrose and LiC1 curve, respectively) . However, if the length of exposure to solution before irradiation is increased, survival increases . Initially the increase is very rapid, but with longer pre-exposure to solution, the curves appear to approach asymptotically a constant value with SR of 1 . 040 and 0 . 556 at 80 min for sucrose and LiCl, respectively . The curve of cells treated with 0 . 10 M NaCl solutions for various times (figure 5) displays the same characteristics as the LiCl or sucrose curves of figure 4 . The SRs for irradiation immediately after the addition of salt and at 80 min after are 0 . 041 and 0 . 413, respectively . However, the curve of cells treated with ouabain and 0 . 10 M NaCl solutions shows a slight decrease in cell survival as the length of exposure to solution before irradiation is increased, the SRs being 0 . 037 and 0 . 023 for irradiation carried out at 0 and 80 min exposure times, respectively . The open triangle represents the survival of cells treated with ouabain in an identical manner to the experimental cells, except that treatment
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Figure 5 . Isodose survival curves (closed symbols, left axis) of cells irradiated with a 1380 rad dose in the last 3 . 0 min of various exposure times to 0 . 10 M NaCl (circles) or 0 . 10 M NaCl-ouabain (squares) solutions as indicated on the abscissa . All pre-irradiation solution exposure times from 0 to 80 . 0 min were carried out at room temperature . The open symbols (right axis) represent the toxicity effect on cells equilibrated in 0 . 10 M NaCl or 0 . 10 M NaCl-ouabain solutions for the various times indicated on the abscissa . The closed and open triangles (left axis) represent the survival of cells irradiated in untreated whole medium and survival of cells pretreated for 12 hours in medium-plus-ouabain solutions at 37°C before irradiation, respectively .
with a salt solution containing ouabain was omitted ; thus, the cells were irradiated in whole medium containing ouabain . It should be noted that ouabain treatment by itself does radiosensitize cells ; the SR is 0 . 12 at the 1380 rad dose level . Figure 6 shows that the radiation survival of cells decreases as the 0 . 12 M KCl exposure to solution before radiation is increased ; the SRs are 0 . 016 and 0 . 00035 for the 0 and 80 min exposure times, respectively. Even after 80 min, the curve appears to be still decreasing . This effect is opposite to the results obtained for cells treated with hypotonic LiCl, NaCl or sucrose, but similar to those with cells treated with 0 . 10 M NaCl plus ouabain solutions . Control No . 2 for the KC1 experiment, which is not shown on figure 6, had a fraction survival level of 6 . 66 x 10-2 . The radiation survival curve of cells treated with 0 . 12 M NH 4C1 does not show as large an increase with time as cells treated with hypotonic NaCl, LiCl or sucrose solutions . Also the initial drop in survival is not very large compared with the LiC1, NaCl, KCl or sucrose results . The SRs at the 0 and 40 min are
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16,
0-1
1
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16,
a
5
1
1 1
10 0
20
40 TIME
IN
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Figure 6 . The survival of cells irradiated with a dose of 1012 rad in the last 2 . 2 min of various exposure times to 0 . 12 M KCl solutions (closed circles, left axis) or with a dose of 1380 rad in the last 3 . 0 min of various exposure times to 0 . 12 M NH 4C1 solutions (closed squares, left axis) . All solution exposures were carried out at room temperature . The open symbols (right axis) represent the toxicity effect on cells exposed for various times to solutions of 0 . 12 M KCl (circles) or 0 . 12 M NH 4 C1 (squares) . The fraction survival of the radiation control (not shown) of cells irradiated in whole medium with a 1012 rad dose was 6 . 66 x 10 -2 . The cross (left ordinate) represents the survival of cells irradiated in whole medium with a dose of 1380 rad .
0116 M
0. 0775 M
Salt Volume KCl NaCl CaCl2 LiCI Na 2 SO 4
2290 1550 1110 1600 1400
SR
µ3 1 .8 9.1 8.0 6 .0 7.0
x x x x x
10-2 10 -2 10 -1 10 -2 10 -'
I Table 2
Volume p 3 2660 1580 1190 1680 1580
SR 1.0 3.8 3.0 6.4 1 .4
x x x x x
10-3 10-3 10-1 10 -3 10 -1
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0 . 43 and 0-78, respectively ; no further increase is observed at times between 40 and 80 min . The cross represents control No . 2 for the NH 4 C1 curve only . Results for cells treated with hypotonic (0 . 10 M) NaNO 3 solutions (not shown) are similar to the results observed for NH 4 C1 treatment in that the radiosensitivity does not decrease rapidly after the initial salt solution treatment and that the total decrease is not very large (i .e . SRs range from 0 . 072 to 0 . 152 at 0 and 80 min, respectively) . The data presented in table 2 are from the cell-volume curves of Raaphorst et al. (1975) and from the radiation survival data of Raaphorst and Kruuv (1976, 1977 a) . The numbers were obtained by either direct measurement or interpolation from the curves for cell-volume or radiation survival in the various solutions . Generally, the SR of cells increases as the cell volume decreases in the various salt solutions . However, if SR is plotted versus cell-volume (semilog), the slope of this plot is different for each salt solution . That is, even though radiosensitivity is generally directly related to cell-volume, there are ion-specific effects . 4.
Discussion The data show that the treatment of cells with hypotonic NaCl solutions for 20 min has no effect on the long-term growth of the cells . Roti Roti and Rothstein (1973 b) have shown that mammalian cells could grow indefinitely in hypotonic media with osmolalities as low as 185 mosm (equivalent to 0 . 101 M NaCl solution) and that when the hypotonic medium was removed, normal growth was resumed . Furthermore, it was shown that cells, exposed for 15 min to 60 mosm medium (equivalent to 0 . 031 M NaCl solution), undergo an initial decrease in cell survival but resume normal growth when returned to isotonic medium . Our results with cells treated for 20 min with hypotonic NaCl solutions at 0 . 05 M are similar . The treatment of cells with hypotonic solutions causes substantial increases in cellular radiosensitivity, as can be observed from the large changes in D o of the survival-curve data . These changes in radiosensitivity may be associated with changes in the amount and structure of cellular water . Raaphorst and Kruuv (1977 b) have shown that when cells are subjected to hypotonic solutions the water-content of the cell increases and the total water structure of the cell decreases, as measured by N .M .R . The data in table 2 generally support the hypothesis that, in hypotonic solutions, the radiation survival is related to the cell-volume . That is, cells with a smaller volume, such as those treated with CaCl2 , are less radiosensitive than cells treated with hypotonic KCl solutions, which results in a much larger volume . However, as pointed out previously, it is also possible that the various ions may have differing effects on cellular radiosensitivity over and above their effects on cell-volume . It has been shown for several different cell-types that in hypotonic solutions single cells rapidly swell, then equilibrate in size with time, eventually shrinking to a volume smaller than expected from the Boyle-van't Hoff relationship by losing cellular potassium (Roti Roti and Rothstein 1973 a, Doljanski et al. 1974) . This final shrinking phase was reported to be complete in 10 to 30 min (Doljanski et al . 1974, Hendil and Hoffman 1974, Shank et al. 1973, Roti Roti and Rothstein 1973 a, Rosenberg, Shank and Gregg 1972) . It should be pointed out that some
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of the results that are referenced above were derived from experiments carried out at 25°C, while others were done at 37 . 5°C ; however, the cell swelling and shrinking dynamics during hypotonic treatment were not greatly different at these different temperatures . Thus the cell may lose about 47 per cent of its K` equilibrating in 189 mosmolar K+-free medium for 16 min (Doljanski et al . 1974) . No substantial protein loss occurs during this process . It should be mentioned that the dynamics of the cell-volume adaption are such that the swelling phase of the cell requires 2-3 min at 25 ° C and that no K+ is lost from the cell during this phase . It can be seen from figures 4 and 5 that the radiosensitivity of the hypotonic sucrose, LiCI or NaCl treated cells has almost recovered to the control level after 30 min in the salt solution . This time correlates well with the time required for the cells to shrink to a new equilibrium phase due to a loss of potassium and water . When investigators exposed cells to hypotonic phosphate-buffered potassium solutions, the cells continued to swell beyond the largest size observed for the initial swelling phase when cells had been subjected to hypotonic phosphatebuffered saline solutions . This swelling phase was completed in 40 min and no subsequent shrinking phase was observed (Doljanski et al . 1974) . This agrees well with the data of table 2, where the volume of cells subjected to KCl solutions for 15 min is much larger than that of cells subjected to NaCl solutions for 15 min . Similarly, the large increase in the radiosensitivity of mammalian cells, as the times of exposure to hypotonic KCl solution are increased, correlates well with the continuing increase in cell-volume and the prevention of the loss of potassium and water by the presence of potassium ions in the external bathing solution (Doljanski et al. 1974) . Thus, it appears that the dynamics of the radiosensitivity of cells subjected to hypotonic solutions is closely related to the changes in cell-volume and potassium content ; that is, the initial rapid cell-volume increase due to an increase in cell water-content after hypotonic treatment is accompanied by an increase in radiosensitivity in all experiments (NaCl, KCl, NaCl plus ouabain, NH 4 C1, LiCl and sucrose). Following this, the cells either continue to swell and increase in radiosensitivity if K+ is plentiful in the bathing solution or shrink by losing K+ and water with a concomitant decrease in radiosensitivity if no K+ is present in the external solution . However, the above experiments do not unequivocally prove whether the change in the cell radiosensitivity during this last phase is caused by changes in the amount of intracellular water or K± or both . It has been reported (Lehninger 1975) that NH 4 + can be substituted for K+ in maintaining the N+- K+ ATPase activity in erythrocytes . However, the changes in radiosensitivity of cells exposed to NH 4 Cl solutions are not the same as those observed during KCI treatment . Results of cells treated with hypotonic (0 . 10 M) NaNO 3 indicate that the anion NO3 can produce effects similar to the cation NH 4 +, since the results obtained with hypotonic NaCl solutions (figure 5) are quite different from those with either NaNO 3 or NH 4 CI solutions (figure 6 and § 3) . It has been shown that the cross-linking in DNA irradiated in vitro can be increased by increasing the water-content of the DNA (Alexander 1962) and that bacterial spores become more radiosensitive when their water-content is increased (Iwasaki, Tallentire, Kimler and Powers 1974) . Likewise, in
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hypotonically-treated cells, the amount of water present may be instrumental in the increase in the radiosensitivity . In vivo experiments, injecting distilled water into tumours, have shown that the cells swell and that tumour regression after irradiation is enhanced (Sugiura 1940) . Similarly, in our experiments during the initial rapid swelling phase in hypotonic solutions (i .e . when there is no loss of K+ taking place), whether the external bathing solution consists of hypotonic sucrose, NaCl, LiC1 or NaNO3 there was a dramatic increase in radiosensitivity with respect to the control in the first 3 . 0 min of exposure to salt, and the same applied to KCI solutions . It is only after this initial event that the dynamics of radiosensitivity show ionic specificity . Thus, it seems likely that the rapid influx of water is responsible for the initial increase in radiosensitivity of hypotonically treated cells . The data of the window experiment (figure 3) of cells subjected to 0 . 1 M NaCl solutions support the results of the hypotonic solution experiments discussed above . There is a large initial drop in survival when the cells are subjected to the hypotonic solution and irradiated immediately, but as the time between initial exposure to salt and irradiation is increased, the radiosensitivity of the cells decreases, i .e . the latter temporally corresponds to the cell shrinking phase . The radiosensitivity of cells decreases when the irradiation is carried out immediately after the removal of the salt solution and the addition of the complete medium . It has been shown that when hypotonically-adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K+ content (Roti Roti and Rothstein 1973 a) . Thus, the initial shrinking phase is accompanied by a decrease in radiosensitivity and then, as the cells swell and gain K+, the radiosensitivity increases again . Thus, it appears that a gain in K+ and water in the cell corresponds to an increase in radiosensitivity, which agrees with the shrinking phase of the experiment depicted in figure 5, where a loss of cell water and K+ was accompanied by a decrease in radiosensitivity . When cells are restored to whole medium, they may equilibrate back to their normal size after 30 min (Roti Roti and Rothstein 1973 a) ; however, a large increase in radiosensitivity persists (figure 3), but the reasons are not clear . The decrease in survival as the time of irradiation before salt treatment is decreased may be related to repair of potentially-lethal damage occuring at room temperature (Winans, Dewey and Dettor 1972) . The cells were irradiated and then held at room temperature until exposed to the salt solution at room temperature . Thus, the longer the interval between radiation and salt treatment, the greater the time in which repair of potentially-lethal damage could occur . The studies with ouabain-treated cells exposed to hypotonic NaCl-ouabain solutions show a similar behaviour in cell radiosensitivity as a function of the duration of exposure to salt as the KCI-treated cells . Roti Roti and Rothstein (1973 a) and Doljanski et al. (1974) have shown that cells treated with ouabain and exposed to hypotonic solutions containing ouabain undergo the initial swelling phase but not a subsequent shrinking phase, nor do they continue to swell to the same extent as cells treated with hypotonic phosphate-buffered potassium chloride (PBK) solutions . The survival of the ouabain-treated cells in the 0 . 10 M NaCl-ouabain solution undergoes a large initial drop corresponding to the initial cell swelling phase (figure 5), but then the small subsequent decrease in survival corresponds to the phase in which no large subsequent shrinking or
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swelling phase occurs . This seems to support the argument that the cellular radiosensitivity of hypotonic cells is related to the amount of water in the cells . Treatment with ouabain with no subsequent exposure to salt also increases the radiosensitivity of cells (figure 5) . It has been reported that treatment of cells with ouabain in growth-medium may increase the cell-volume by 10 per cent (Rosenberg et al. 1972) ; however, others have reported slight decreases (Roti Roti and Rothstein 1973 a, McGann, Kruuv, Frim and Frey 1974) . Ouabain is also known to alter many other cell characteristics, some of which are the electrolyte balance, growth-rate and rate of macromolecular synthesis (McDonald et al . 1972) . Thus, it is possible that, during ouabain treatment and ouabainwith-0 . 10 M-NaCl treatment, some of the changes in cellular radiosensitivity were due to the above-mentioned factors as well as to changes in the watercontent of the cell . However, if we limit our discussion to the relative importance of the K+ ion versus water-content in influencing radiosensitivity of hypotonic cells, it should be pointed out that cells pre-treated with ouabain in growth medium may lose two-thirds of their K+, while gaining a somewhat smaller amount of Na+ (Roti Roti and Rothstein 1973 a, McDonald et al. 1972) . When these low K+-high Na+ cells are exposed to hypotonic medium containing ouabain, they suffer a further loss in K+ and gain in Na+ . The K+ content of these cells may be only 12 per cent of that of control cells (Roti Roti and Rothstein 1973 a) . Since the loss of the K+ takes place as a function of time concomitant with the gain of the Na+ ion (McDonald et al. 1972), and since the cell-volume and the radiosensitivity (figure 5) are constant over this time, we conclude that the radiosensitivity of hypotonic cells is related to water-content . Thus, in summary, the increase in radiosensitivity of hypotonically-treated cells may be caused by the increase in the cellular water content with a concomitant decrease in cellular water structure and possibly a change in the ionic composition of the cellular protoplasm. More water in the cell would allow an increase in OH° production by radiation ; more ' free water ' would allow faster diffusion rates of these OH° radicals to their target sites. ACKNOWLEDGMENT
This work was supported by grants from the National Research Council of Canada . Les cellules (V79) d'hamsters chinois furent traitees avec des solutions de NaCl hypertoniques, NaCl-ouabain, KCl, LiC1, NH 4 C1, NaNO 3 ou des solutions de sucrose . Elles furent irradiees a differentes reprises pendant 1'exposition a la solution . L'augmentation extreme de la radiosensibilite de ces cellules, marquee surtout de changements de Do , pourrait resulter des augmentations du contenu de Peau cellulaire et peut-titre de la diminution de la structure de Peau cellulaire . Des ions peuvent exercer leurs effets specifiques sur la radiosensibilite en influencant les facteurs ci-dessus relatifs a Peau . Chinesische Hamsterzellen (V79) wurden mit hypotonischen NaCL-, NaCl-Ouabin-, KCl-, LiCI-, NH4 C1-, NaNO 3 -, oder Saccharose-Losungen behandelt and zu verschiedenen Zeiten wahrend der Behandlung bestrahlt . Die extreme Erhohung der Strahlenempfindlichkeit dieser Zellen, besonders charakterisiert durch Anderungen von D o , konnte auf einen Anstieg des Zellwassergehalts and moglicherweise auf eine Abnahme der Zellwasserstruktur zuruckgefuhrt werden . Die verschiedenen Ionen konnten vielleicht ihre spezifischen Wirkungen auf die Strahlenempfindlichkeit durch Einwirkung auf die obengenannten Faktoren ausiiben .
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REFERENCES ABETSEDARAKAYA, L. A ., MIFTAKHUTDINOVA, F . G ., and FEDOTOV, V . D ., 1968, Biophysics, 13, 750 . ALEXANDER, A ., 1962, Ann . N .Y. Acad . Sci., 24, 966 . CHANG, D . C ., RORSCHACH, H . E ., NICHOLS, B . L ., and HAZLEWOOD, C . F ., 1973, Ann . N.Y. Acad. Sci., 204, 434 . COPE, F. W ., 1969, Biophys . Y ., 9, 303 . DOLJANSKI, F., BEN-SASSON, S ., REICH, M ., and GROVER, N . B ., 1974, ,. cell. Physiol ., 84, 215 . DROST-HANSEN, W ., 1971, Chemistry of the Cell Interface (New York : Academic Press) . HANSEN, J . R ., 1971, Biochem . biophys . Acta, 230, 482 . HAZLEWOOD, C . F ., NICHOLS, B . L ., and CHAMBERLAIN, N . F ., 1969, Nature, Lond., 222, 747 . HENDIL, K . B ., and HOFFMAN, E . K ., 1974, Y. cell . Physiol ., 84,115 . IWASAKI, T., TALLENTIRE, A ., KIMLER, B . F ., and POWERS, E . L ., 1974, Radiat . Res ., 57, 306 . KOCH, C. J ., and KRuuv, J ., 1971, Radiat . Res ., 48, 74 . LEHNINGER, A . L., 1975, Biochemistry (New York : Worth) . MCDONALD, T . F ., SACHS, H . G ., ORR, C. W . M ., and EBERT, J . D ., 1972, Expl Cell Res ., 74,201 . McGANN, L . E ., KRUUV, J ., FRIM, J ., and FREY, H . E ., 1974, Cryobiology, 11, 332 . RAAPHORST, G . P ., 1976, Ph .D . Thesis, University of Waterloo, Canada . RAAPHORST, G . P ., and KRUUV, J ., 1976, Int. Y. Radiat . Biol ., 29, 493 ; 1977 a, Ibid ., 32, 71 ; 1977 b, Contemporary Biophysics, Vol . 1 (New York : Marcel Dekker) (in the press) . RAAPHORST, G . P ., KRUUV, J ., and PINTAR, M . M ., 1975, Biophys. Y., 15, 391 . REUVERS, A . P ., GREENSTOCK, C . L ., BORSA, J ., and CHAPMAN, J . D ., 1973, Int . -7. Radiat . Biol., 24, 533 . ROSENBERG, H . M ., SHANK, B . B ., and GREGG, E . C ., 1972, Y. cell. Physiol., 80, 23 . ROTI ROTI, L. W ., and ROTHSTEIN, A ., 1973 a, Expl Cell Res ., 79, 295 ; 1973 b, Ibid ., 79, 311 . SHANK, B . B ., ROSENBERG, H . M ., and HOROWITZ, C ., 1973, Y. cell. Physiol ., 82, 257 . SUGIURA, K., 1940, Am . Y. Roentgen ., 43, 533 . THOMPSON, R . T ., KYDON, D . W ., and PINTAR M . M ., 1976, Bull . Am . phys. Soc ., 21, 58 . WINANS, L . E ., DEWEY, W . C ., and DETTOR, C . M ., 1972, Radiat. Res ., 52, 333 .
1977,
VOL .
32,
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Effect of salt solutions on radiosensitivity of mammalian cells II . Treatment with hypotonic solutions G . P . RAAPHORST and J . KRUUV Department of Physics, University of Waterloo, Waterloo, Ontario, Canada
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(Received 25 October 1976 ; accepted 16 March 1977)
Chinese hamster (V79) cells were treated with hypotonic NaCl, NaCl-ouabain, KCl, LiCI, NH 4 C1, NaNO3 or sucrose solutions and irradiated at various times during exposure to the solution . The extreme increase in the radiosensitivity of these cells, mainly characterized by changes in Do , could be attributed to increases in the cell water-content and possibly the decrease in total cell water structure . The various ions may exert their specific effects on radiosensitivity by influencing the above water-related factors .
1 . Introduction Radiation damage in biological systems is caused to a large extent by the action of ionizing radiation on cellular water, which results in the radiochemical production of free-radical species . The radicals can diffuse to and interact with biological macromolecules, resulting in sub-lethal or lethal damage in living organisms . It has been shown that up to 62 per cent of all death in cultured mammalian cells caused by ionizing radiation may be due to the OH radical (Reuvers, Greenstock, Borsa and Chapman 1973) . Thus changes in the water of biological systems may result in changes in their radiosensitivity . Nuclear magnetic resonance evidence has accumulated to indicate that the water of cells may exist in a structured state (Hazlewood, Nichols and Chamberlain 1969, Cope 1969, Abetsedarakaya, Miftakhutdinova and Fedotov 1968, Drost-Hansen 1971) and that the self-diffusion coefficient of such water is lower than that of bulk water (Chang, Rorschach, Nichols and Hazlewood 1973, Hansen 1971, Abetsedarakaya et al. 1968, Thompson, Kydon and Pintar 1976) . It was reported by Raaphorst, Kruuv and Pintar (1975) that the total cellular water structure and content may be altered by subjecting cells to various ionic solutions . Furthermore it was shown that when cells were subjected to a wide range of salt solutions, large changes were observed in the cellular radiosensitivity . Extreme increases in cellular radiosensitivity were observed for cells treated with hypotonic concentrations of various salt solutions (Raaphorst and Kruuv 1976, 1977 a) . Under these conditions the cell swells, thus acquiring more water . Hence, the structure of the total cell water decreases (Raaphorst et al. 1975) . Other investigators have shown that the cell-volume undergoes various changes as a function of the time that the cells are exposed to various hypotonic solutions and that these changes in volume are dependent on the types of ions present in the cell bathing solution (Roti Roti and Rothstein 1973 a, Doljanski, Ben-Sasson, Reich and Grover 1974, Shank, Rosenberg and Horowitz 1973) . In this paper, experiments are presented in which the relationships between the changes in cell water and radiosensitivity are examined when the cells are treated with various hypotonic solutions .
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Materials and methods The cells used were originally derived from the V-79 S-171 line of Chinese hamster fibroblasts and the sub-line used was S-171-W1 . Tissue-culture methods for the S171-W2 sub-line have been described by Koch and Kruuv (1971) ; the same culture techniques were used in these experiments . Exponentially-growing cells were inoculated into plastic Petri dishes (60 mm) containing complete medium (Eagle's Basal Medium with Hanks' salts supplemented by 15 per cent foetal calf serum) and were incubated at 37 ° C for 22 hours for attachment . The medium was then removed and replaced by a salt solution equilibrated to room temperature (22 ° C) . In the last (x) minutes of the exposure to the salt, a dose of radiation, requiring (x) minutes to deliver, was administered to the ' experimental ' plates . Immediately after irradiation was completed, the salt solution was removed from each plate, which was then rinsed with 3 ml of medium at room temperature . Finally, complete medium was added to each plate and incubation for the colony-survival assay followed . For the window experiment (figure 3), cells were treated for 20 min with a salt solution and irradiated at various times before, during, or after exposure to the salt . For irradiation before exposure to salt, the plates (containing medium) were taken out of the incubator immediately before irradiation, after which they were kept at room temperature until they were exposed to salt at zero time . All other plates were removed from the incubator at zero time ; the medium was replaced by room-temperature salt solutions for 20 . 0 min, followed by a change to room-temperature medium . The plates were reincubated only after both exposure to salt and irradiation had been carried out . For each experiment toxicity controls (No . 1) were carried out in which the cells were treated with the salt solutions in the same manner as the experimental plates, except that irradiation was omitted . Another control (No . 2) was carried out in which the cells were irradiated in whole medium without prior or subsequent salt treatment ; these cells were subjected to the same temperature profile as the salt treated cells . (Previous control experiments with cells in complete medium had shown that there was no significant difference in survival between cells irradiated immediately on removal from the 37 ° C incubator and cells subjected for up to 30 min to room temperature before irradiation .) This control is indicated on each figure by a cross . Ouabain was dissolved in medium or salt solutions at a concentration of 5 x 10 -4 M . Exponentially-growing cells were exposed to the solution of ouabain and medium for 12 hours at 37 ° C . After this time, the cells were released from the plates by treatment with trypsin, inoculated into new plates containing medium with 5 x 10 -4 M ouabain and put into the incubator for 2 . 5 hours for attachment . The treatment of cells with salt solutions containing ouabain was carried out as described above . The plating efficiency (PE) of the S-171-W1 cells in complete medium was above 80 per cent in all experiments . The results for controls Nos . 1 and 2 and the radiation survival results for the salt-treated cells (experimental cells) were normalized to the PE . The standard errors are indicated in the figures when greater than the actual graph point size . Two petri dishes were used for each radiation point ; three dishes were used for each toxicity point . All the solutions used were unbuffered, i .e . the salt or the sucrose was dissolved in distilled water . The pH at the time of irradiation was about 5 . 5
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due to CO 2 absorption . Monitoring of the pH has previously been described (Raaphorst and Kruuv 1976) . Growth curve experiments with salt-treated cells were carried out as follows . After a 2 . 5 hour incubation period at 37 ° C for attachment, the cells in the 60 mm Petri dishes were treated for 20 min with a salt solution at room temperature (22 ° C) . The salt solution was then replaced by whole medium at room temperature, and the plates were put into the 37°C incubator . This was considered zero time in the growth experiment . Controls were treated in an identical manner, except that the cells were treated for 20 min with medium at room temperature instead of salt solution . All chemicals used were reagent grade . The ouabain (Strophanthin G) was obtained from Calbiochem . Irradiation was carried out with a Radiation Machinery Corporation ' Gammator B', which has a caesium-137 source delivering an absorbed dose of 460 rad/min to the cells . 3.
Results The growth of cells is not significantly affected by a 20 min treatment at room temperature with a 0 . 05 or 0 . 10 M NaCl solution (figure 1) . The doublingtime for salt-treated cells and control cells was the same (about 10 hours) . It is possible that the salt treatment has caused a small initial decrease in the number of cells that were counted .
W F Q
a w
w a
N J W U
Figure 1 . Growth curves of cells given 20 . 0 min equilibration at room temperature in solutions of 0 . 05 M (triangles) or 0 . 1 M NaCl (squares) . The circles represent growth of cells that were treated with whole medium for 20 . 0 min at room temperature . Zero time on the abscissa represents the end of the solution equilibration times . Complete radiation survival curves of cells treated with various hypotonic NaCl or KCl solutions are shown in figure 2 . The survival-curve parameters are listed in table 1 . In each case the radiation survival data were corrected for the salt toxicity (i .e . control No . 1) . The toxicity effect of the treatment with 0 . 01 M NaCl or KCl solutions reduced the cell survival to 30 . 9 and 8 . 3 per cent, respectively . The cell toxicity survival after treatment with 0 . 04 M, or the more concentrated solutions of NaCl or KCl, was greater than 80 per cent .
G . P . Raaphorst and j . Kruuv
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a U Q
Figure 2 . Survival curves of cells treated with various NaCl (closed symbols) or KCI (open symbols) solutions for 20 . 0 min at room temperature and irradiated in the last minutes of exposure to salt . The survival curve of control cells, irradiated in medium, is represented by the closed circles ; cells exposed to 0 . 01 and 0 . 04 M salt solutions are signified by diamonds and squares, respectively . The open and closed triangles represent results with cells exposed to 0 . 15 M KCl and 0 . 116 M NaCl, respectively .
From table 1 it can be observed that the changes in cell survival are characterized mainly by changes in Do of the survival curve, while n does not change significantly. In all cases the treatment with hypotonic solutions caused an increase in cellular radiosensitivity . The cells treated with 0 . 01 or 0 . 04 M KCl solutions have a lower survival level than the cells treated with the corresponding concentrations of NaCl . Description
Control 0 . 010 M 0 . 040 M 0 . 116 M 0 . 010 M 0 . 040 M 0 . 150 M Table 1 .
NaCl NaCl NaCl KCl KCl KCl
Do (rad)
Da (rad)
n
124 48 72 112 45 58 113
540 210 280 440 180 280 450
60 60 50 50 60 70 60
D,, salt/ Do control
0 . 39 0 . 58 0 . 90 0 . 36 0 . 46 0 . 90
Parameters from the NaCl and KCI survival curves .
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-3 510
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Figure 3 . Survival of cells exposed for 20 . 0 min to 0. 10 M NaCl solutions and irradiated with a dose of 1380 rad before this exposure time (negative time on the abscissa), during this exposure (0-20 . 0 min on the abscissa) or after the salt exposure (20 . 0-50 . 0 min on the abscissa) . All salt, radiation and medium exposures were carried out at room temperature . The cross represents the control irradiated in whole medium . The horizontal bars represent the time-span of radiation exposure (3 . 0 min) . The experiment depicted in figure 3 (termed a ` window' experiment) shows the radiation survival of cells irradiated either before, during, or after, a 20 min exposure to a 0 . 10 M NaCl solution . The horizontal bars on the data points represent the time required (3 min) to administer the 1380 rad dose of radiation . Large changes in cell survival occur if irradiation is carried out immediately before or after the addition of the NaCl solution . The survival ratio (SR), i .e . the ratio of the experimental point to control No . 2 (cross), is 0 . 208 for cells irradiated immediately before the addition of the salt and 0 . 014 if irradiation is carried out immediately after the salt addition . Likewise, there is an abrupt increase in the SR if cells are irradiated immediately after the salt solution is removed (0 . 317) compared with immediately before the salt is removed (0 . 137) . Also as the cells are irradiated at later times in the period of exposure to salt (between 0 and 20 min on figure 3), the cell survival increases from a SR of 0 . 014 All points have a significantly lower survival at 0-3 min to 0 . 137 at 17-20 min . level than control No . 2 . Figures 4 to 6 display the survival of cells treated with hypotonic salt or sucrose solutions for various times before administering a dose of 1380 rad in the last three minutes of the exposure time . In the case of KCl the radiation dose was 1012 rad and was given in the last 2. 2 min of exposure to solution. The curves of the irradiated cells (closed symbols ; left axis) have been normalized to the toxicity control (open symbols ; right axis) . R .B .
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Figure 4 . Isodose survival curves (closed symbols, left axis) of cells irradiated with a 1380 rad dose in the last 3 . 0 min of various exposure times to 0 . 10 M sucrose (circles) or 0 . 13 M LiCl (squares) solutions as indicated on the abscissa . All pre-irradiation solution exposure times from 0 to 80 . 0 min were carried out at room temperature . The open symbols (right axis) represent the toxicity effect on cells equilibrated in 0 . 10 M sucrose (circles) or 0 . 13 M LiCl (squares) solutions for the various times indicated on the abscissa . The cross (left axis) represents the survival of cells irradiated in whole medium .
For both the 0 . 13 M LiCl and 0 . 10 M sucrose curves in figure 4, the cell survival is the lowest when irradiation is carried out immediately after the addition of the solution (i .e . SRs of 0 . 021 and 0 . 122 for the sucrose and LiC1 curve, respectively) . However, if the length of exposure to solution before irradiation is increased, survival increases . Initially the increase is very rapid, but with longer pre-exposure to solution, the curves appear to approach asymptotically a constant value with SR of 1 . 040 and 0 . 556 at 80 min for sucrose and LiCl, respectively . The curve of cells treated with 0 . 10 M NaCl solutions for various times (figure 5) displays the same characteristics as the LiCl or sucrose curves of figure 4 . The SRs for irradiation immediately after the addition of salt and at 80 min after are 0 . 041 and 0 . 413, respectively . However, the curve of cells treated with ouabain and 0 . 10 M NaCl solutions shows a slight decrease in cell survival as the length of exposure to solution before irradiation is increased, the SRs being 0 . 037 and 0 . 023 for irradiation carried out at 0 and 80 min exposure times, respectively . The open triangle represents the survival of cells treated with ouabain in an identical manner to the experimental cells, except that treatment
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Figure 5 . Isodose survival curves (closed symbols, left axis) of cells irradiated with a 1380 rad dose in the last 3 . 0 min of various exposure times to 0 . 10 M NaCl (circles) or 0 . 10 M NaCl-ouabain (squares) solutions as indicated on the abscissa . All pre-irradiation solution exposure times from 0 to 80 . 0 min were carried out at room temperature . The open symbols (right axis) represent the toxicity effect on cells equilibrated in 0 . 10 M NaCl or 0 . 10 M NaCl-ouabain solutions for the various times indicated on the abscissa . The closed and open triangles (left axis) represent the survival of cells irradiated in untreated whole medium and survival of cells pretreated for 12 hours in medium-plus-ouabain solutions at 37°C before irradiation, respectively .
with a salt solution containing ouabain was omitted ; thus, the cells were irradiated in whole medium containing ouabain . It should be noted that ouabain treatment by itself does radiosensitize cells ; the SR is 0 . 12 at the 1380 rad dose level . Figure 6 shows that the radiation survival of cells decreases as the 0 . 12 M KCl exposure to solution before radiation is increased ; the SRs are 0 . 016 and 0 . 00035 for the 0 and 80 min exposure times, respectively. Even after 80 min, the curve appears to be still decreasing . This effect is opposite to the results obtained for cells treated with hypotonic LiCl, NaCl or sucrose, but similar to those with cells treated with 0 . 10 M NaCl plus ouabain solutions . Control No . 2 for the KC1 experiment, which is not shown on figure 6, had a fraction survival level of 6 . 66 x 10-2 . The radiation survival curve of cells treated with 0 . 12 M NH 4C1 does not show as large an increase with time as cells treated with hypotonic NaCl, LiCl or sucrose solutions . Also the initial drop in survival is not very large compared with the LiC1, NaCl, KCl or sucrose results . The SRs at the 0 and 40 min are
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16,
0-1
1
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a
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1
1 1
10 0
20
40 TIME
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Figure 6 . The survival of cells irradiated with a dose of 1012 rad in the last 2 . 2 min of various exposure times to 0 . 12 M KCl solutions (closed circles, left axis) or with a dose of 1380 rad in the last 3 . 0 min of various exposure times to 0 . 12 M NH 4C1 solutions (closed squares, left axis) . All solution exposures were carried out at room temperature . The open symbols (right axis) represent the toxicity effect on cells exposed for various times to solutions of 0 . 12 M KCl (circles) or 0 . 12 M NH 4 C1 (squares) . The fraction survival of the radiation control (not shown) of cells irradiated in whole medium with a 1012 rad dose was 6 . 66 x 10 -2 . The cross (left ordinate) represents the survival of cells irradiated in whole medium with a dose of 1380 rad .
0116 M
0. 0775 M
Salt Volume KCl NaCl CaCl2 LiCI Na 2 SO 4
2290 1550 1110 1600 1400
SR
µ3 1 .8 9.1 8.0 6 .0 7.0
x x x x x
10-2 10 -2 10 -1 10 -2 10 -'
I Table 2
Volume p 3 2660 1580 1190 1680 1580
SR 1.0 3.8 3.0 6.4 1 .4
x x x x x
10-3 10-3 10-1 10 -3 10 -1
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0 . 43 and 0-78, respectively ; no further increase is observed at times between 40 and 80 min . The cross represents control No . 2 for the NH 4 C1 curve only . Results for cells treated with hypotonic (0 . 10 M) NaNO 3 solutions (not shown) are similar to the results observed for NH 4 C1 treatment in that the radiosensitivity does not decrease rapidly after the initial salt solution treatment and that the total decrease is not very large (i .e . SRs range from 0 . 072 to 0 . 152 at 0 and 80 min, respectively) . The data presented in table 2 are from the cell-volume curves of Raaphorst et al. (1975) and from the radiation survival data of Raaphorst and Kruuv (1976, 1977 a) . The numbers were obtained by either direct measurement or interpolation from the curves for cell-volume or radiation survival in the various solutions . Generally, the SR of cells increases as the cell volume decreases in the various salt solutions . However, if SR is plotted versus cell-volume (semilog), the slope of this plot is different for each salt solution . That is, even though radiosensitivity is generally directly related to cell-volume, there are ion-specific effects . 4.
Discussion The data show that the treatment of cells with hypotonic NaCl solutions for 20 min has no effect on the long-term growth of the cells . Roti Roti and Rothstein (1973 b) have shown that mammalian cells could grow indefinitely in hypotonic media with osmolalities as low as 185 mosm (equivalent to 0 . 101 M NaCl solution) and that when the hypotonic medium was removed, normal growth was resumed . Furthermore, it was shown that cells, exposed for 15 min to 60 mosm medium (equivalent to 0 . 031 M NaCl solution), undergo an initial decrease in cell survival but resume normal growth when returned to isotonic medium . Our results with cells treated for 20 min with hypotonic NaCl solutions at 0 . 05 M are similar . The treatment of cells with hypotonic solutions causes substantial increases in cellular radiosensitivity, as can be observed from the large changes in D o of the survival-curve data . These changes in radiosensitivity may be associated with changes in the amount and structure of cellular water . Raaphorst and Kruuv (1977 b) have shown that when cells are subjected to hypotonic solutions the water-content of the cell increases and the total water structure of the cell decreases, as measured by N .M .R . The data in table 2 generally support the hypothesis that, in hypotonic solutions, the radiation survival is related to the cell-volume . That is, cells with a smaller volume, such as those treated with CaCl2 , are less radiosensitive than cells treated with hypotonic KCl solutions, which results in a much larger volume . However, as pointed out previously, it is also possible that the various ions may have differing effects on cellular radiosensitivity over and above their effects on cell-volume . It has been shown for several different cell-types that in hypotonic solutions single cells rapidly swell, then equilibrate in size with time, eventually shrinking to a volume smaller than expected from the Boyle-van't Hoff relationship by losing cellular potassium (Roti Roti and Rothstein 1973 a, Doljanski et al. 1974) . This final shrinking phase was reported to be complete in 10 to 30 min (Doljanski et al . 1974, Hendil and Hoffman 1974, Shank et al. 1973, Roti Roti and Rothstein 1973 a, Rosenberg, Shank and Gregg 1972) . It should be pointed out that some
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of the results that are referenced above were derived from experiments carried out at 25°C, while others were done at 37 . 5°C ; however, the cell swelling and shrinking dynamics during hypotonic treatment were not greatly different at these different temperatures . Thus the cell may lose about 47 per cent of its K` equilibrating in 189 mosmolar K+-free medium for 16 min (Doljanski et al . 1974) . No substantial protein loss occurs during this process . It should be mentioned that the dynamics of the cell-volume adaption are such that the swelling phase of the cell requires 2-3 min at 25 ° C and that no K+ is lost from the cell during this phase . It can be seen from figures 4 and 5 that the radiosensitivity of the hypotonic sucrose, LiCI or NaCl treated cells has almost recovered to the control level after 30 min in the salt solution . This time correlates well with the time required for the cells to shrink to a new equilibrium phase due to a loss of potassium and water . When investigators exposed cells to hypotonic phosphate-buffered potassium solutions, the cells continued to swell beyond the largest size observed for the initial swelling phase when cells had been subjected to hypotonic phosphatebuffered saline solutions . This swelling phase was completed in 40 min and no subsequent shrinking phase was observed (Doljanski et al . 1974) . This agrees well with the data of table 2, where the volume of cells subjected to KCl solutions for 15 min is much larger than that of cells subjected to NaCl solutions for 15 min . Similarly, the large increase in the radiosensitivity of mammalian cells, as the times of exposure to hypotonic KCl solution are increased, correlates well with the continuing increase in cell-volume and the prevention of the loss of potassium and water by the presence of potassium ions in the external bathing solution (Doljanski et al. 1974) . Thus, it appears that the dynamics of the radiosensitivity of cells subjected to hypotonic solutions is closely related to the changes in cell-volume and potassium content ; that is, the initial rapid cell-volume increase due to an increase in cell water-content after hypotonic treatment is accompanied by an increase in radiosensitivity in all experiments (NaCl, KCl, NaCl plus ouabain, NH 4 C1, LiCl and sucrose). Following this, the cells either continue to swell and increase in radiosensitivity if K+ is plentiful in the bathing solution or shrink by losing K+ and water with a concomitant decrease in radiosensitivity if no K+ is present in the external solution . However, the above experiments do not unequivocally prove whether the change in the cell radiosensitivity during this last phase is caused by changes in the amount of intracellular water or K± or both . It has been reported (Lehninger 1975) that NH 4 + can be substituted for K+ in maintaining the N+- K+ ATPase activity in erythrocytes . However, the changes in radiosensitivity of cells exposed to NH 4 Cl solutions are not the same as those observed during KCI treatment . Results of cells treated with hypotonic (0 . 10 M) NaNO 3 indicate that the anion NO3 can produce effects similar to the cation NH 4 +, since the results obtained with hypotonic NaCl solutions (figure 5) are quite different from those with either NaNO 3 or NH 4 CI solutions (figure 6 and § 3) . It has been shown that the cross-linking in DNA irradiated in vitro can be increased by increasing the water-content of the DNA (Alexander 1962) and that bacterial spores become more radiosensitive when their water-content is increased (Iwasaki, Tallentire, Kimler and Powers 1974) . Likewise, in
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hypotonically-treated cells, the amount of water present may be instrumental in the increase in the radiosensitivity . In vivo experiments, injecting distilled water into tumours, have shown that the cells swell and that tumour regression after irradiation is enhanced (Sugiura 1940) . Similarly, in our experiments during the initial rapid swelling phase in hypotonic solutions (i .e . when there is no loss of K+ taking place), whether the external bathing solution consists of hypotonic sucrose, NaCl, LiC1 or NaNO3 there was a dramatic increase in radiosensitivity with respect to the control in the first 3 . 0 min of exposure to salt, and the same applied to KCI solutions . It is only after this initial event that the dynamics of radiosensitivity show ionic specificity . Thus, it seems likely that the rapid influx of water is responsible for the initial increase in radiosensitivity of hypotonically treated cells . The data of the window experiment (figure 3) of cells subjected to 0 . 1 M NaCl solutions support the results of the hypotonic solution experiments discussed above . There is a large initial drop in survival when the cells are subjected to the hypotonic solution and irradiated immediately, but as the time between initial exposure to salt and irradiation is increased, the radiosensitivity of the cells decreases, i .e . the latter temporally corresponds to the cell shrinking phase . The radiosensitivity of cells decreases when the irradiation is carried out immediately after the removal of the salt solution and the addition of the complete medium . It has been shown that when hypotonically-adapted cells are returned to normal medium, an initial shrinking is followed by a re-swelling to normal size, associated with a gain in K+ content (Roti Roti and Rothstein 1973 a) . Thus, the initial shrinking phase is accompanied by a decrease in radiosensitivity and then, as the cells swell and gain K+, the radiosensitivity increases again . Thus, it appears that a gain in K+ and water in the cell corresponds to an increase in radiosensitivity, which agrees with the shrinking phase of the experiment depicted in figure 5, where a loss of cell water and K+ was accompanied by a decrease in radiosensitivity . When cells are restored to whole medium, they may equilibrate back to their normal size after 30 min (Roti Roti and Rothstein 1973 a) ; however, a large increase in radiosensitivity persists (figure 3), but the reasons are not clear . The decrease in survival as the time of irradiation before salt treatment is decreased may be related to repair of potentially-lethal damage occuring at room temperature (Winans, Dewey and Dettor 1972) . The cells were irradiated and then held at room temperature until exposed to the salt solution at room temperature . Thus, the longer the interval between radiation and salt treatment, the greater the time in which repair of potentially-lethal damage could occur . The studies with ouabain-treated cells exposed to hypotonic NaCl-ouabain solutions show a similar behaviour in cell radiosensitivity as a function of the duration of exposure to salt as the KCI-treated cells . Roti Roti and Rothstein (1973 a) and Doljanski et al. (1974) have shown that cells treated with ouabain and exposed to hypotonic solutions containing ouabain undergo the initial swelling phase but not a subsequent shrinking phase, nor do they continue to swell to the same extent as cells treated with hypotonic phosphate-buffered potassium chloride (PBK) solutions . The survival of the ouabain-treated cells in the 0 . 10 M NaCl-ouabain solution undergoes a large initial drop corresponding to the initial cell swelling phase (figure 5), but then the small subsequent decrease in survival corresponds to the phase in which no large subsequent shrinking or
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swelling phase occurs . This seems to support the argument that the cellular radiosensitivity of hypotonic cells is related to the amount of water in the cells . Treatment with ouabain with no subsequent exposure to salt also increases the radiosensitivity of cells (figure 5) . It has been reported that treatment of cells with ouabain in growth-medium may increase the cell-volume by 10 per cent (Rosenberg et al. 1972) ; however, others have reported slight decreases (Roti Roti and Rothstein 1973 a, McGann, Kruuv, Frim and Frey 1974) . Ouabain is also known to alter many other cell characteristics, some of which are the electrolyte balance, growth-rate and rate of macromolecular synthesis (McDonald et al . 1972) . Thus, it is possible that, during ouabain treatment and ouabainwith-0 . 10 M-NaCl treatment, some of the changes in cellular radiosensitivity were due to the above-mentioned factors as well as to changes in the watercontent of the cell . However, if we limit our discussion to the relative importance of the K+ ion versus water-content in influencing radiosensitivity of hypotonic cells, it should be pointed out that cells pre-treated with ouabain in growth medium may lose two-thirds of their K+, while gaining a somewhat smaller amount of Na+ (Roti Roti and Rothstein 1973 a, McDonald et al. 1972) . When these low K+-high Na+ cells are exposed to hypotonic medium containing ouabain, they suffer a further loss in K+ and gain in Na+ . The K+ content of these cells may be only 12 per cent of that of control cells (Roti Roti and Rothstein 1973 a) . Since the loss of the K+ takes place as a function of time concomitant with the gain of the Na+ ion (McDonald et al. 1972), and since the cell-volume and the radiosensitivity (figure 5) are constant over this time, we conclude that the radiosensitivity of hypotonic cells is related to water-content . Thus, in summary, the increase in radiosensitivity of hypotonically-treated cells may be caused by the increase in the cellular water content with a concomitant decrease in cellular water structure and possibly a change in the ionic composition of the cellular protoplasm. More water in the cell would allow an increase in OH° production by radiation ; more ' free water ' would allow faster diffusion rates of these OH° radicals to their target sites. ACKNOWLEDGMENT
This work was supported by grants from the National Research Council of Canada . Les cellules (V79) d'hamsters chinois furent traitees avec des solutions de NaCl hypertoniques, NaCl-ouabain, KCl, LiC1, NH 4 C1, NaNO 3 ou des solutions de sucrose . Elles furent irradiees a differentes reprises pendant 1'exposition a la solution . L'augmentation extreme de la radiosensibilite de ces cellules, marquee surtout de changements de Do , pourrait resulter des augmentations du contenu de Peau cellulaire et peut-titre de la diminution de la structure de Peau cellulaire . Des ions peuvent exercer leurs effets specifiques sur la radiosensibilite en influencant les facteurs ci-dessus relatifs a Peau . Chinesische Hamsterzellen (V79) wurden mit hypotonischen NaCL-, NaCl-Ouabin-, KCl-, LiCI-, NH4 C1-, NaNO 3 -, oder Saccharose-Losungen behandelt and zu verschiedenen Zeiten wahrend der Behandlung bestrahlt . Die extreme Erhohung der Strahlenempfindlichkeit dieser Zellen, besonders charakterisiert durch Anderungen von D o , konnte auf einen Anstieg des Zellwassergehalts and moglicherweise auf eine Abnahme der Zellwasserstruktur zuruckgefuhrt werden . Die verschiedenen Ionen konnten vielleicht ihre spezifischen Wirkungen auf die Strahlenempfindlichkeit durch Einwirkung auf die obengenannten Faktoren ausiiben .
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