Br. J. Cancer (1978) 37, Suppl. III, 38
RAPID-MIXING STUDIES OF THE MECHANISMS OF CHEMICAL RADIOSENSITIZATION AND PROTECTION IN MAMMALIAN CELLS D. W. WHILLANS AND J. W. HUNT Physics Division, Ontario Cancer Institute, Toronto, Canada M4X 1K9
Summary.-A continuous flow, rapid-mixing system has been constructed to measure the degree of radiosensitization or protection observable after incubation of Chinese hamster ovary cells at room temperature for periods from about 5 to 1000 ms with a number of radiation modifiers. In agreement with Adams et al. (1975) full sensitization by oxygen is obtained in only a few tens of milliseconds. Misonidazole (10 mM) sensitizes at very nearly the steady-state level (E.R. ~23) in about 300 ms. Similarly near maximum sensitization by 10 mM metronidazole is seen within the same period. For large effects to be observed in such short times makes unlikely the involvement of any biochemical modification of either the drug or cells. Radioprotection by lM dimethyl sulphoxide reaches about 80% of the equilibrium control value within 850 ms. However if its mode of action involves scavenging of hydroxyl radicals in competition with cellular components, the necessity for very high concentrations in the immediate region of the cellular target would be expected to require longer diffusion times. Cysteamine (10 mM) shows no evidence of its substantial protective ability even after 850 ms incubation. This molecule, which alone in this group exists as an ion in neutral solution, may encounter considerable difficulty in entering the cells.
SUBSTANTIAL, indirect evidence has accumulated that radiosensitization and protection of mammalian cells by externally-added chemical modifiers results in part from direct interactions with free radical damage produced in critical targets of the cell. This hypothesis can be tested by measuring the incubation times, on a millisecond scale, in which these effects can be observed. Direct interactions will occur as soon as the agent reaches the site of damage, whereas effects resulting from interactions of biochemically-modified drugs or damage require from several seconds to many hours. Shenoy et at. (1975) exploited this approach using a rapid-mixing system to demonstrate the direct nature of oxygen sensitization of mammalian cells. While this group (Adams et al. (1975)) also examined several other sensitizers, in all cases full effects were found to lie outside the resolution (,.'40 ms) of that system. We have continued this approach using a similar mixing system which allows incubation periods
from about 5 to 1000 ms. In this report we provide evidence for modification of cellular radiosensitivity after short incubations with the hypoxic cell sensitizers, misonidazole and metronidazole, and the radioprotector, dimethyl sulphoxide. MATERIALS AND METHODS
The continuous flow mixing system which has been constructed uses a hydraulic unit containing a precision flow control to drive the contents of two 50 ml glass syringes into the mixing tubes. Such a unit allows convenient and reproducible control of the flow rate. The mixing system consists of two 2-4 mm ID pyrex capillaries coupled to the syringes which join at a shaped mixing junction together with a 3*4 mm ID tube 500 mm in length. Solutions driven from the syringes meet at the mixing junction and pass up the larger tube for collection in a third glass syringe at the top. Irradiations can be carried out through a well-shielded 10 mm aperture at any point above the junction, or just before the junction in one tube. The radiation source is a pulsed beam of 25 MeV electrons
RAPID-MIXlNG STUDIES OF MECHANISMS
from a Varian Clinac 35. Dosimetry results from a beam-stop, charge collection device which is calibrated against Fricke solution flowing through the mixing system under the same conditions as for cell irradiations. This secondary dosimetry allows convenient monitoring of the individual pulses delivered to different segments of the flowing solution. The minimum time resolution, 5 ms, is set by the maximum available pulse rate for a stable beam, 200 Hz, and the maximum incubation time, 1,000 ms, is constrained by flow parameters which ensure uniform motion through the tubes. Chinese hamster ovary cells were suspended in complete cy medium lacking serum at concentrations of 2-5 x 105 cells/ml. Drugs were prepared in the same medium and both samples were equilibrated with the appropriate gases before delivery to the mixing system. Survival ability of cells collected at the top was measured using standard colony-forming techniques. In all cases results were consistent with a dose-modification response. Control irradiations were performed with a 60Co source at a dose rate of 2 Gy/min. (a)
RESULTS
Initial studies of the protection of oxygenated cells demonstrated no difficulty in obtaining control survival curves virtually identical to those seen following 60Co irradiations. Fig. 1 shows the results of mixing 1M (in the mixed solution) dimethyl sulphoxide (DMSO) with cells at various times before irradiation. A progressive increase in protection with incubation time occurs, such that at 850 ms 80% of the control level is obtained. Based largely on the high concentrations necessary to produce protection and the reactivity of DMSO with hydroxyl radicals (-OH), Chapman et al. (1973) attributed this protection to scavenging of -OH in competition with cellular targets, a direct mechanism. Our data is entirely consistent with such a mechanism. On the other hand, although 10 mM cysteamine produces an even larger degree of protection under equilibrium conditions, no modi(b)
S ~~~~~~~20
_
100
5X
400
0
800
lncubaotin time (ms)
\ o
RAPID-MIXING STUDIES OF THE MECHANISMS OF CHEMICAL RADIOSENSITIZATION AND PROTECTION IN MAMMALIAN CELLS D. W. WHILLANS AND J. W. HUNT Physics Division, Ontario Cancer Institute, Toronto, Canada M4X 1K9
Summary.-A continuous flow, rapid-mixing system has been constructed to measure the degree of radiosensitization or protection observable after incubation of Chinese hamster ovary cells at room temperature for periods from about 5 to 1000 ms with a number of radiation modifiers. In agreement with Adams et al. (1975) full sensitization by oxygen is obtained in only a few tens of milliseconds. Misonidazole (10 mM) sensitizes at very nearly the steady-state level (E.R. ~23) in about 300 ms. Similarly near maximum sensitization by 10 mM metronidazole is seen within the same period. For large effects to be observed in such short times makes unlikely the involvement of any biochemical modification of either the drug or cells. Radioprotection by lM dimethyl sulphoxide reaches about 80% of the equilibrium control value within 850 ms. However if its mode of action involves scavenging of hydroxyl radicals in competition with cellular components, the necessity for very high concentrations in the immediate region of the cellular target would be expected to require longer diffusion times. Cysteamine (10 mM) shows no evidence of its substantial protective ability even after 850 ms incubation. This molecule, which alone in this group exists as an ion in neutral solution, may encounter considerable difficulty in entering the cells.
SUBSTANTIAL, indirect evidence has accumulated that radiosensitization and protection of mammalian cells by externally-added chemical modifiers results in part from direct interactions with free radical damage produced in critical targets of the cell. This hypothesis can be tested by measuring the incubation times, on a millisecond scale, in which these effects can be observed. Direct interactions will occur as soon as the agent reaches the site of damage, whereas effects resulting from interactions of biochemically-modified drugs or damage require from several seconds to many hours. Shenoy et at. (1975) exploited this approach using a rapid-mixing system to demonstrate the direct nature of oxygen sensitization of mammalian cells. While this group (Adams et al. (1975)) also examined several other sensitizers, in all cases full effects were found to lie outside the resolution (,.'40 ms) of that system. We have continued this approach using a similar mixing system which allows incubation periods
from about 5 to 1000 ms. In this report we provide evidence for modification of cellular radiosensitivity after short incubations with the hypoxic cell sensitizers, misonidazole and metronidazole, and the radioprotector, dimethyl sulphoxide. MATERIALS AND METHODS
The continuous flow mixing system which has been constructed uses a hydraulic unit containing a precision flow control to drive the contents of two 50 ml glass syringes into the mixing tubes. Such a unit allows convenient and reproducible control of the flow rate. The mixing system consists of two 2-4 mm ID pyrex capillaries coupled to the syringes which join at a shaped mixing junction together with a 3*4 mm ID tube 500 mm in length. Solutions driven from the syringes meet at the mixing junction and pass up the larger tube for collection in a third glass syringe at the top. Irradiations can be carried out through a well-shielded 10 mm aperture at any point above the junction, or just before the junction in one tube. The radiation source is a pulsed beam of 25 MeV electrons
RAPID-MIXlNG STUDIES OF MECHANISMS
from a Varian Clinac 35. Dosimetry results from a beam-stop, charge collection device which is calibrated against Fricke solution flowing through the mixing system under the same conditions as for cell irradiations. This secondary dosimetry allows convenient monitoring of the individual pulses delivered to different segments of the flowing solution. The minimum time resolution, 5 ms, is set by the maximum available pulse rate for a stable beam, 200 Hz, and the maximum incubation time, 1,000 ms, is constrained by flow parameters which ensure uniform motion through the tubes. Chinese hamster ovary cells were suspended in complete cy medium lacking serum at concentrations of 2-5 x 105 cells/ml. Drugs were prepared in the same medium and both samples were equilibrated with the appropriate gases before delivery to the mixing system. Survival ability of cells collected at the top was measured using standard colony-forming techniques. In all cases results were consistent with a dose-modification response. Control irradiations were performed with a 60Co source at a dose rate of 2 Gy/min. (a)
RESULTS
Initial studies of the protection of oxygenated cells demonstrated no difficulty in obtaining control survival curves virtually identical to those seen following 60Co irradiations. Fig. 1 shows the results of mixing 1M (in the mixed solution) dimethyl sulphoxide (DMSO) with cells at various times before irradiation. A progressive increase in protection with incubation time occurs, such that at 850 ms 80% of the control level is obtained. Based largely on the high concentrations necessary to produce protection and the reactivity of DMSO with hydroxyl radicals (-OH), Chapman et al. (1973) attributed this protection to scavenging of -OH in competition with cellular targets, a direct mechanism. Our data is entirely consistent with such a mechanism. On the other hand, although 10 mM cysteamine produces an even larger degree of protection under equilibrium conditions, no modi(b)
S ~~~~~~~20
_
100
5X
400
0
800
lncubaotin time (ms)
\ o
