Br. J. Cancer (1978) 37, Suppl. III, 29
A FAST KINETICS STUDY OF THE MODES OF ACTION OF SOME DIFFERENT RADIOSENSITIZERS IN BACTERIA B. D. MICHAEL, H. A. HARROP, R. L. MAUGHAN AND K. B. PATEL From the Cancer Research Campaign Gray Laboratory, Mount Vernon Hospital, Northwood, M71iddlesex HA6 2RN
Summary.-Using a fast mixing and irradiation technique, the gas explosion method, with Serratia marcescens, the decay of oxygen-dependent damage is found to consist of a fast and a slow stage, each of which is associated with a sub-component of this damage. In the present work, the interactions of these components with radiosensitizers are examined. At low concentrations, 02, TAN (a nitroxyl) and misonidazole all preferentially sensitize the slow-stage damage. At higher concentrations, 02 and TAN sensitize the fast-stage damage by a fixation reaction that competes with its repair; in contrast, misonidazole appears mainly to operate by reaction with an earlier, even shorter lived form of oxygen-dependent damage.
ALTHOUGH the actions of substances that bacterial monolayers. Each filter was placed sensitize hypoxic cells to radiation are be- inside the gas explosion chamber, flushed with lieved to involve fast chemical reactions, the appropriate humidified gas (N2, or, in much of the investigation of their me- some experiments, N2 containing a small of 02) for 2- min and then chanisms has been carried out using proportion exposed to the 02 -shot and electron pulse. essentially slow-response, end-product Experiments were usually carried out at a assay methods. In the present work, using fixed setting of dose/pulse and survival was a sub-millisecond response time fast- scored as a function of the pre-set time delay mixing and irradiation method, the gas between 02 gas shot contact with the bacexplosion technique (Michael et al., 1973), teria and their irradiation. The time resoluwe have compared the kinetics of radio- tion of the technique is estimated to be about sensitization of S. marcescens by 02, 100 ,ts. After irradiation the bacteria were electron-affinic and stable free radical washed off the filters in buffer solution consensitizers. The principal object was to taining 0.3% Tween-80 (Honeywill Ltd., Surrey), diluted and assayed for compare the extents to which sensitiza- Carshalton, colony-forming ability by a pour-plate tion by each of these agents can be attri- technique. buted to the fixation of oxygen-dependent damage in competition with its fast repair. Kinetic theory of competing fixation and repair MATERIALS AND METHODS
Experiments were carried out using the gas explosion technique (Michael et al., 1973) in conjunction with a single 5ns pulse of electrons from a Febetron 706 electron pulse generator (Hewlett-Packard, McMinnville Division, McMinnville, Oregon). Suspensions of S. marcescens (5 x 107 bacteria/ml) containing appropriate concentrations of sensitizers were applied in ca 20u1 aliquots to 13 mm diameter membrane filters (Millipore S.A., France, Type GSWP 01300) to form
Qualitative agreement with the oxygen fixation hypothesis (Alexander and Charlesby, 1954) and the derivation of the associated Alper and Howard-Flanders formula (Alper and Howard-Flanders, 1956) have been obtained using the gas explosion technique with S. marcescens (Michael et al., 1973); more recently we have found quantitative agreement in this system between the observed decay (i.e. repair) kinetics of 02-dependent damage and the kinetics predicted by the Alper and Howard-Flanders model, provided
30
B. D. MICHAEL ET AL.
it is assumed that there are two types of 02dependent damage that are repaired at different rates. In the present work we examine the influence of added sensitizers upon the decay kinetics of the faster and major component of 02-dependent damage. The theoretical treatment has been extended as below, to predict the relationship between enhancement ratio, ER, and the rate of decay, k, of sensitizer-dependent damage, D, in a model system in which the sensitizer, S, is assumed to act only by fixation of damage in competition with its repair by a repairing substance, X D + X-k REPAIR (1) D + S FIXATION
i
(a)X
LETHAL DAMAGE
1- .
a -
c O
ILr 101e c
10 i ,
2°
F
-*
--
i(e
14
(2)
Then the ER (the radiosensitivity in the preof a concentration [S] of an added sensitizer divided by the radiosensitivity in its absence) is given by
*-
*-
00 05 10 Time between
.
15'' * 25 * 10 02
--l-
20 30 40 50 contact and irradiation-ms
l
sence
1
rT
o1
(b) s 10
mk2[S] + kl[X] k2[S] -+ k1[X]
(3)
where m is the maximum value of ER that be achieved at high [S] in the system. More usually, the above expression is simplified to (m[S] + K)/([S] + K) where K (= (k1[X])/(k2)) is equal to the value of S that produces an ER equal to (1 + (m-1)). If the damage, R, is all produced instantaneously, then the theory predicts that it will disappear by a pseudo first-order process in which the time-dependent term is exp [-(k1[X] + k2[S])t]. In the gas shot experiment we assume that 02 contact instantaneously fixes any 02-dependent damage remaining at time t after the electron pulse and that the resulting survival versus 02 contact time profile reflects the decay kinetics of 02dependent damage. Thus, in the presence of added sensitizer, it is possible to measure the first-order rate of disappearance, k, of 02dependent damage where k = kiEX] + k2[S]. Substitution of k into equation (3) leads to can
ER= ±+(m -1)
ko
(4)
where ko = kl[X], i.e. the experimentally measured first-order rate of disappearance in the absence of added sensitizer. The experiments were designed to test whether the actions of various types of sensitizer conformed to equation (4) above,
Lt
--&-I
-49.
c
I
---*
id'
.c
>S 10 s
0
int u
00-
.jZ025
0
100
05
15
.
2510
20
j-
20
-j-1r1000
flush Time between
0,
contact and irradiation-ms
m
10
I&-
(C)
10t
lC,-
1, a
-
A
-20
" 2 -01
00
Time
0102 between
03
04
0, contact
05
and
06
07
08
irradiation-ms
FiG. 1.-Time courses for the action of the 02 gas shot upon the survival of S. marcescens irradiated with a single 180 Gy pulse of electrons. (a) 0 with no additional sensitizer, * with 2- 5 nim misonidazole, (b) 0 with no additional sensitizer; * with 0 * 4% 02 present in the flushing gas; (c) with 0 1, * 1 *0 and * 4*0 mM TAN.
FAST KINETICS OF RADIOSENSITIZATION
i.e. whether they act by fixing damage in competition with its repair.
31
Damage fixation kinetics
Fig. 2 shows the results of experiments designed to test the applicability of equaRESULTS AND DISCUSSION tion (4) in order to determine the extent to Components of damage which sensitization of the fast, major comFig. 1 shows results of 02 gas-shot ponent of damage can be attributed to experiments, including some in which S. fixation in competition with repair. For marcescens were maintained in contact with each sensitizer, at a number of different various concentrations of added sensitizer. concentrations, values of ER were meaAll irradiations shown in each diagram sured together with corresponding values were carried out at a fixed value of dose/ of k obtained by a first order (i.e. time pulse. Each point represents the surviving exponential) fit to the fast component fraction resulting from a single pulse irra- decay profile. The value of k with no diation with 02 shot contact occurring at sensitizer added, ko, measured 2100 s-l. the time delays shown, negative times and positive times representing 02 contact before and after the electron pulse respectively. In all curves the observed rise of log survival with increasing time delay of 02 shot contact represents the decay of 02dependent damage, provided it is assumed E R. that 02 penetration to and reaction with the damage are relatively fast. This decay corresponds to the kinetics of repair of the damage under hypoxic conditions in a manner that is consistent with the predick-FIRST ORDER RATE OF DECAY OF 0- DEPENDENT DAMAGE-s tions of the oxygen fixation hypothesis. FIG. 2. Sensitizer enhancement ratio, E.R., However, in the curves of Figs. l(a) and versus measured values of k, the first order (b) that were determined without added rate of decay of the fast component of 02sensitizer, there appear to be fast (1-5 ms) stages of the added metronidazole; * with added misorepair. These stages appear to represent nidazole. the repair, at differing rates, of two types of 02-dependent damage. Similar biphasic The experimental points for 02 (0-2decay kinetics have been observed in fully 2.2%) present in the flushing gas show hydrated Bacillus megaterium spores, al- that sensitization is accompanied by a though on a much slower timescale (Strat- pronounced increase in k. The curve drawn ford et al., 1977; Tallentire et al., 1977). In through the points is derived from equathe present work the slower stage of decay tion (4) by inserting m - 2-8. The ER is preferentially removed by added mison- values shown for 02 were determined for idazole (2.5 mM), 02 (0.4%) or TAN (1*0 the fast component only, for which a mM). This supports the view that there are maximum ER of 2-8 was found. Thus, two types of 02-dependent damage, each sensitization of the fast component by 02 associated with a stage of decay, and that concentrations in the range 0.2-2.2% the slow-stage damage is more effectively can be accounted for by fixation in comsensitized by these agents. Other observa- petition with repair. tions of multiple actions associated with The behaviour of TAN (0 1-4'0 mM), the 02 effect have been reported (Powers, Figure 2, appears similar to that of 02, i.e. Webb and Ehret, 1960; Alper, 1963; its sensitization is achieved by fixation of Tallentire, Jones and Jacobs, 1972; Shenoy 02-dependent damage. The ER values et al., 1975; Ewing and Powers, 1976). shown are for overall (fast + slow) sen-
32
B. D. MICHAEL ET AL.
sitization and the observed maximum value of 1P9 has been used to generate the curve. Other evidence for competition between 02 and nitroxyl radiosensitizers has been reported (Johansen, 1974). With metronidazole (2-47 mM), within the moderate range of enhancement that could be achieved, there is little to distinguish its behaviour from that of 02, although the results suggest that its sensitization is accompanied by less increase of k than that found in 02. Misonidazole (1-25 mM) behaves quite differently from oxygen and TAN. This is evident because a high degree of sensitization is obtained with only about a 1 5-fold increase in k; therefore, little of the sensitization by misonidazole is due to fixation of post-effect 02-dependent damage. However, although kinetically 02 and misonidazole would appear to act differently, most of their overall effect (Asquith et al., 1974) is clearly within the same pool of damage. This suggests the reaction scheme below in which the formation of post-effect 02-dependent damage, D, occurs through an earlier, shorter-lived stage D'. The reactivity of D' with misonidazole is much greater than that of D. A similar reaction scheme has been proposed at this conference as a possible explanation for certain additivity effects of electron-affinic sensitizers and oxygen in Chinese hamster cells (McNally and de Ronde, 1977). Fixation -
misonidazole /
D'
Lethal Damage
TAN
>D
\~X
Repair
In the above scheme misonidazole does not compete directly with the repair reaction. This is in accord with the finding that misonidazole has relatively little effect on the first order rate of disappearance of D. Other evidence has been obtained, using repair deficient mutants of Escherichia coli, that electron-affinic and organic nitroxyl sensitizers do not compete directly for the same lesion (Sapora et al., 1977; Johansen et al., 1977).
The above scheme is simplified and intended to represent only the major actions of the agents on the fast component of 02-dependent damage over the ranges of concentration tested. The approximately 1*5-fold increase in k found with misonidazole, as shown in Fig. 2, indicates that there is also some reaction between this compound and D, but this contributes only a minor part of the sensitizing effect and this reaction has therefore not been included in the scheme. Also omitted are any reactions between D' and either TAN or 02, although their relatively low redox potentials (Greenstock et al., 1974) would be expected to favour, at sufficiently high concentrations, appreciable reaction with the short-lived D'. The authors wish to acknowledge the support of the Cancer Research Campaign.
REFERENCES ALEXANDER, P. & CHARLESBY, A. (1954) PhysicoChemical Methods of Protection against Ionizing Radiations. In Radiobiology Symposium, Eds. Z. M. Bacq and P. Alexander, London: Butterworth's. p. 49. ALPER, T. (1963) Lethal Mutations and Cell Death. Physic3 Med. Biol. 8, 365. ALPER, T. & HoWARD-FLANDERS, P. (1956) The Role of Oxygen in Modifying the Radiosensitivity of E. coli B. Nature, Lond., 178, 978. ASQUITH, J. C., WATTS, M. E., PATEL, K. B., SMITHEN, C. E. & ADAMS, G. E. (1974) Electronaffinic Sensitization. V: Radiosensitization of Hypoxic Bacteria and Mammalian Cells in vitro by some Nitroimidazoles and Nitropyrazoles. Radiat. Res. 60, 108. EWING, D. & POWERS, E. L. (1976) Irradiation of Bacterial Spores in Water: Three Classes of
Oxygen-Dependent Damage. Science, N.Y., 194,
1049. GREENSTOCK, C. L., CHAPMAN, J. D., RALEIGH, J. A., SHERMAN, E. & REUVERS, A. P. ( 1974) Competitive Radioprotection and Radiosensitization in Chemical Systems. Radiat. Res., 59, 556. JOHANSEN, I. (1974) Competition between Tetramethylpiperidinol N-oxyl and Oxygen in Effects on Single-strand Breaks in Episomal DNA and in Killing after X-irradiation in E. coli. Radiat. Res., 58, 398. JOHANSEN, I., GULBRANDSEN, R., FIELDEN, E. M. & SAPORA, 0. (1977) Additive Effects shown by Combinations of Nitroxyl and Electron-affinic Hypoxic Cell Sensitizers. Radiat. Res., 70, 597. McNALLY, N. J. & DE RONrIE, J. Interactions between Electron-affinic Sensitizers. (This Conference.) Br. J. Cancer, 37, Suppl. III, 90. MICHAEL, B. D., ADAMS, G. E., HEWITT, H. B., JONES, W. B. G. & WATTS, M. E. (1973) A Post-
FAST KINETICS OF RADIOSENSITIZATION effect of Oxygen in Irradiated Bacteria: A Submillisecond Fast Mixing Study. Radiat. Re8., 54, 239. POWERS, E. L., WEBB, R. B. & EHRET, C. F. (1960) Storage Transfer and Utilization of Energy from X-rays in Dry Bacterial Spores. Radiat. Re8. Suppl. 2, 94. SAPORA, O., FIELDEN, E. M. & LOVEROCK, P. S. (1977) A Comparative Study of the Effects of Two Classes of Radiosensitizer on the Survival of E. coli B and K12 Mutants. Radiat. Res., 69, 293. SHENOY, M. A., AsQUITH, J. C., ADAMS, G. E., MICHAEL, B. D. & WATTS, M. E. (1975) Timeresolved Oxygen Effects in Irradiated Bacteria and Mammalian Cells: A Rapid-mix Study. Uadiat. Res., 62, 498.
4
33
STRATFORD, I. J., MAUGHAN, R. L., MICHAEL, B. D. & TALLENTIRE, A. (1977) The Decay of Potentially Lethal Oxygen-dependent Damage in Fully Hydrated B. megaterium Spores Exposed to Pulsed Electron Irradiation. Int. J. Radiat. Biol., 32, 447. TALLENTIRE, A., STRATFORD, I. J., MAUGHAN, R. L. & MICHAEL, B. D. (1977) The Effects of some Hypoxic Cell Radiosensitizers on the Decay of Potentially Lethal Oxygen-dependent Damage in Fully Hydrated Spores. (This Conference.) Br. J. Cancer, 37, Suppl. III, 34. TALLENTIRE, A., JoNEs, A. B. & JACOBS, G. P. (1972) The Radiosensitizing Actions of Ketonic Agents and Oxygen in Bacterial Spores Suspended in Aqueous and Nonaqueous Milieu. I8rael J. Chem., 10, 1185.
A FAST KINETICS STUDY OF THE MODES OF ACTION OF SOME DIFFERENT RADIOSENSITIZERS IN BACTERIA B. D. MICHAEL, H. A. HARROP, R. L. MAUGHAN AND K. B. PATEL From the Cancer Research Campaign Gray Laboratory, Mount Vernon Hospital, Northwood, M71iddlesex HA6 2RN
Summary.-Using a fast mixing and irradiation technique, the gas explosion method, with Serratia marcescens, the decay of oxygen-dependent damage is found to consist of a fast and a slow stage, each of which is associated with a sub-component of this damage. In the present work, the interactions of these components with radiosensitizers are examined. At low concentrations, 02, TAN (a nitroxyl) and misonidazole all preferentially sensitize the slow-stage damage. At higher concentrations, 02 and TAN sensitize the fast-stage damage by a fixation reaction that competes with its repair; in contrast, misonidazole appears mainly to operate by reaction with an earlier, even shorter lived form of oxygen-dependent damage.
ALTHOUGH the actions of substances that bacterial monolayers. Each filter was placed sensitize hypoxic cells to radiation are be- inside the gas explosion chamber, flushed with lieved to involve fast chemical reactions, the appropriate humidified gas (N2, or, in much of the investigation of their me- some experiments, N2 containing a small of 02) for 2- min and then chanisms has been carried out using proportion exposed to the 02 -shot and electron pulse. essentially slow-response, end-product Experiments were usually carried out at a assay methods. In the present work, using fixed setting of dose/pulse and survival was a sub-millisecond response time fast- scored as a function of the pre-set time delay mixing and irradiation method, the gas between 02 gas shot contact with the bacexplosion technique (Michael et al., 1973), teria and their irradiation. The time resoluwe have compared the kinetics of radio- tion of the technique is estimated to be about sensitization of S. marcescens by 02, 100 ,ts. After irradiation the bacteria were electron-affinic and stable free radical washed off the filters in buffer solution consensitizers. The principal object was to taining 0.3% Tween-80 (Honeywill Ltd., Surrey), diluted and assayed for compare the extents to which sensitiza- Carshalton, colony-forming ability by a pour-plate tion by each of these agents can be attri- technique. buted to the fixation of oxygen-dependent damage in competition with its fast repair. Kinetic theory of competing fixation and repair MATERIALS AND METHODS
Experiments were carried out using the gas explosion technique (Michael et al., 1973) in conjunction with a single 5ns pulse of electrons from a Febetron 706 electron pulse generator (Hewlett-Packard, McMinnville Division, McMinnville, Oregon). Suspensions of S. marcescens (5 x 107 bacteria/ml) containing appropriate concentrations of sensitizers were applied in ca 20u1 aliquots to 13 mm diameter membrane filters (Millipore S.A., France, Type GSWP 01300) to form
Qualitative agreement with the oxygen fixation hypothesis (Alexander and Charlesby, 1954) and the derivation of the associated Alper and Howard-Flanders formula (Alper and Howard-Flanders, 1956) have been obtained using the gas explosion technique with S. marcescens (Michael et al., 1973); more recently we have found quantitative agreement in this system between the observed decay (i.e. repair) kinetics of 02-dependent damage and the kinetics predicted by the Alper and Howard-Flanders model, provided
30
B. D. MICHAEL ET AL.
it is assumed that there are two types of 02dependent damage that are repaired at different rates. In the present work we examine the influence of added sensitizers upon the decay kinetics of the faster and major component of 02-dependent damage. The theoretical treatment has been extended as below, to predict the relationship between enhancement ratio, ER, and the rate of decay, k, of sensitizer-dependent damage, D, in a model system in which the sensitizer, S, is assumed to act only by fixation of damage in competition with its repair by a repairing substance, X D + X-k REPAIR (1) D + S FIXATION
i
(a)X
LETHAL DAMAGE
1- .
a -
c O
ILr 101e c
10 i ,
2°
F
-*
--
i(e
14
(2)
Then the ER (the radiosensitivity in the preof a concentration [S] of an added sensitizer divided by the radiosensitivity in its absence) is given by
*-
*-
00 05 10 Time between
.
15'' * 25 * 10 02
--l-
20 30 40 50 contact and irradiation-ms
l
sence
1
rT
o1
(b) s 10
mk2[S] + kl[X] k2[S] -+ k1[X]
(3)
where m is the maximum value of ER that be achieved at high [S] in the system. More usually, the above expression is simplified to (m[S] + K)/([S] + K) where K (= (k1[X])/(k2)) is equal to the value of S that produces an ER equal to (1 + (m-1)). If the damage, R, is all produced instantaneously, then the theory predicts that it will disappear by a pseudo first-order process in which the time-dependent term is exp [-(k1[X] + k2[S])t]. In the gas shot experiment we assume that 02 contact instantaneously fixes any 02-dependent damage remaining at time t after the electron pulse and that the resulting survival versus 02 contact time profile reflects the decay kinetics of 02dependent damage. Thus, in the presence of added sensitizer, it is possible to measure the first-order rate of disappearance, k, of 02dependent damage where k = kiEX] + k2[S]. Substitution of k into equation (3) leads to can
ER= ±+(m -1)
ko
(4)
where ko = kl[X], i.e. the experimentally measured first-order rate of disappearance in the absence of added sensitizer. The experiments were designed to test whether the actions of various types of sensitizer conformed to equation (4) above,
Lt
--&-I
-49.
c
I
---*
id'
.c
>S 10 s
0
int u
00-
.jZ025
0
100
05
15
.
2510
20
j-
20
-j-1r1000
flush Time between
0,
contact and irradiation-ms
m
10
I&-
(C)
10t
lC,-
1, a
-
A
-20
" 2 -01
00
Time
0102 between
03
04
0, contact
05
and
06
07
08
irradiation-ms
FiG. 1.-Time courses for the action of the 02 gas shot upon the survival of S. marcescens irradiated with a single 180 Gy pulse of electrons. (a) 0 with no additional sensitizer, * with 2- 5 nim misonidazole, (b) 0 with no additional sensitizer; * with 0 * 4% 02 present in the flushing gas; (c) with 0 1, * 1 *0 and * 4*0 mM TAN.
FAST KINETICS OF RADIOSENSITIZATION
i.e. whether they act by fixing damage in competition with its repair.
31
Damage fixation kinetics
Fig. 2 shows the results of experiments designed to test the applicability of equaRESULTS AND DISCUSSION tion (4) in order to determine the extent to Components of damage which sensitization of the fast, major comFig. 1 shows results of 02 gas-shot ponent of damage can be attributed to experiments, including some in which S. fixation in competition with repair. For marcescens were maintained in contact with each sensitizer, at a number of different various concentrations of added sensitizer. concentrations, values of ER were meaAll irradiations shown in each diagram sured together with corresponding values were carried out at a fixed value of dose/ of k obtained by a first order (i.e. time pulse. Each point represents the surviving exponential) fit to the fast component fraction resulting from a single pulse irra- decay profile. The value of k with no diation with 02 shot contact occurring at sensitizer added, ko, measured 2100 s-l. the time delays shown, negative times and positive times representing 02 contact before and after the electron pulse respectively. In all curves the observed rise of log survival with increasing time delay of 02 shot contact represents the decay of 02dependent damage, provided it is assumed E R. that 02 penetration to and reaction with the damage are relatively fast. This decay corresponds to the kinetics of repair of the damage under hypoxic conditions in a manner that is consistent with the predick-FIRST ORDER RATE OF DECAY OF 0- DEPENDENT DAMAGE-s tions of the oxygen fixation hypothesis. FIG. 2. Sensitizer enhancement ratio, E.R., However, in the curves of Figs. l(a) and versus measured values of k, the first order (b) that were determined without added rate of decay of the fast component of 02sensitizer, there appear to be fast (1-5 ms) stages of the added metronidazole; * with added misorepair. These stages appear to represent nidazole. the repair, at differing rates, of two types of 02-dependent damage. Similar biphasic The experimental points for 02 (0-2decay kinetics have been observed in fully 2.2%) present in the flushing gas show hydrated Bacillus megaterium spores, al- that sensitization is accompanied by a though on a much slower timescale (Strat- pronounced increase in k. The curve drawn ford et al., 1977; Tallentire et al., 1977). In through the points is derived from equathe present work the slower stage of decay tion (4) by inserting m - 2-8. The ER is preferentially removed by added mison- values shown for 02 were determined for idazole (2.5 mM), 02 (0.4%) or TAN (1*0 the fast component only, for which a mM). This supports the view that there are maximum ER of 2-8 was found. Thus, two types of 02-dependent damage, each sensitization of the fast component by 02 associated with a stage of decay, and that concentrations in the range 0.2-2.2% the slow-stage damage is more effectively can be accounted for by fixation in comsensitized by these agents. Other observa- petition with repair. tions of multiple actions associated with The behaviour of TAN (0 1-4'0 mM), the 02 effect have been reported (Powers, Figure 2, appears similar to that of 02, i.e. Webb and Ehret, 1960; Alper, 1963; its sensitization is achieved by fixation of Tallentire, Jones and Jacobs, 1972; Shenoy 02-dependent damage. The ER values et al., 1975; Ewing and Powers, 1976). shown are for overall (fast + slow) sen-
32
B. D. MICHAEL ET AL.
sitization and the observed maximum value of 1P9 has been used to generate the curve. Other evidence for competition between 02 and nitroxyl radiosensitizers has been reported (Johansen, 1974). With metronidazole (2-47 mM), within the moderate range of enhancement that could be achieved, there is little to distinguish its behaviour from that of 02, although the results suggest that its sensitization is accompanied by less increase of k than that found in 02. Misonidazole (1-25 mM) behaves quite differently from oxygen and TAN. This is evident because a high degree of sensitization is obtained with only about a 1 5-fold increase in k; therefore, little of the sensitization by misonidazole is due to fixation of post-effect 02-dependent damage. However, although kinetically 02 and misonidazole would appear to act differently, most of their overall effect (Asquith et al., 1974) is clearly within the same pool of damage. This suggests the reaction scheme below in which the formation of post-effect 02-dependent damage, D, occurs through an earlier, shorter-lived stage D'. The reactivity of D' with misonidazole is much greater than that of D. A similar reaction scheme has been proposed at this conference as a possible explanation for certain additivity effects of electron-affinic sensitizers and oxygen in Chinese hamster cells (McNally and de Ronde, 1977). Fixation -
misonidazole /
D'
Lethal Damage
TAN
>D
\~X
Repair
In the above scheme misonidazole does not compete directly with the repair reaction. This is in accord with the finding that misonidazole has relatively little effect on the first order rate of disappearance of D. Other evidence has been obtained, using repair deficient mutants of Escherichia coli, that electron-affinic and organic nitroxyl sensitizers do not compete directly for the same lesion (Sapora et al., 1977; Johansen et al., 1977).
The above scheme is simplified and intended to represent only the major actions of the agents on the fast component of 02-dependent damage over the ranges of concentration tested. The approximately 1*5-fold increase in k found with misonidazole, as shown in Fig. 2, indicates that there is also some reaction between this compound and D, but this contributes only a minor part of the sensitizing effect and this reaction has therefore not been included in the scheme. Also omitted are any reactions between D' and either TAN or 02, although their relatively low redox potentials (Greenstock et al., 1974) would be expected to favour, at sufficiently high concentrations, appreciable reaction with the short-lived D'. The authors wish to acknowledge the support of the Cancer Research Campaign.
REFERENCES ALEXANDER, P. & CHARLESBY, A. (1954) PhysicoChemical Methods of Protection against Ionizing Radiations. In Radiobiology Symposium, Eds. Z. M. Bacq and P. Alexander, London: Butterworth's. p. 49. ALPER, T. (1963) Lethal Mutations and Cell Death. Physic3 Med. Biol. 8, 365. ALPER, T. & HoWARD-FLANDERS, P. (1956) The Role of Oxygen in Modifying the Radiosensitivity of E. coli B. Nature, Lond., 178, 978. ASQUITH, J. C., WATTS, M. E., PATEL, K. B., SMITHEN, C. E. & ADAMS, G. E. (1974) Electronaffinic Sensitization. V: Radiosensitization of Hypoxic Bacteria and Mammalian Cells in vitro by some Nitroimidazoles and Nitropyrazoles. Radiat. Res. 60, 108. EWING, D. & POWERS, E. L. (1976) Irradiation of Bacterial Spores in Water: Three Classes of
Oxygen-Dependent Damage. Science, N.Y., 194,
1049. GREENSTOCK, C. L., CHAPMAN, J. D., RALEIGH, J. A., SHERMAN, E. & REUVERS, A. P. ( 1974) Competitive Radioprotection and Radiosensitization in Chemical Systems. Radiat. Res., 59, 556. JOHANSEN, I. (1974) Competition between Tetramethylpiperidinol N-oxyl and Oxygen in Effects on Single-strand Breaks in Episomal DNA and in Killing after X-irradiation in E. coli. Radiat. Res., 58, 398. JOHANSEN, I., GULBRANDSEN, R., FIELDEN, E. M. & SAPORA, 0. (1977) Additive Effects shown by Combinations of Nitroxyl and Electron-affinic Hypoxic Cell Sensitizers. Radiat. Res., 70, 597. McNALLY, N. J. & DE RONrIE, J. Interactions between Electron-affinic Sensitizers. (This Conference.) Br. J. Cancer, 37, Suppl. III, 90. MICHAEL, B. D., ADAMS, G. E., HEWITT, H. B., JONES, W. B. G. & WATTS, M. E. (1973) A Post-
FAST KINETICS OF RADIOSENSITIZATION effect of Oxygen in Irradiated Bacteria: A Submillisecond Fast Mixing Study. Radiat. Re8., 54, 239. POWERS, E. L., WEBB, R. B. & EHRET, C. F. (1960) Storage Transfer and Utilization of Energy from X-rays in Dry Bacterial Spores. Radiat. Re8. Suppl. 2, 94. SAPORA, O., FIELDEN, E. M. & LOVEROCK, P. S. (1977) A Comparative Study of the Effects of Two Classes of Radiosensitizer on the Survival of E. coli B and K12 Mutants. Radiat. Res., 69, 293. SHENOY, M. A., AsQUITH, J. C., ADAMS, G. E., MICHAEL, B. D. & WATTS, M. E. (1975) Timeresolved Oxygen Effects in Irradiated Bacteria and Mammalian Cells: A Rapid-mix Study. Uadiat. Res., 62, 498.
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STRATFORD, I. J., MAUGHAN, R. L., MICHAEL, B. D. & TALLENTIRE, A. (1977) The Decay of Potentially Lethal Oxygen-dependent Damage in Fully Hydrated B. megaterium Spores Exposed to Pulsed Electron Irradiation. Int. J. Radiat. Biol., 32, 447. TALLENTIRE, A., STRATFORD, I. J., MAUGHAN, R. L. & MICHAEL, B. D. (1977) The Effects of some Hypoxic Cell Radiosensitizers on the Decay of Potentially Lethal Oxygen-dependent Damage in Fully Hydrated Spores. (This Conference.) Br. J. Cancer, 37, Suppl. III, 34. TALLENTIRE, A., JoNEs, A. B. & JACOBS, G. P. (1972) The Radiosensitizing Actions of Ketonic Agents and Oxygen in Bacterial Spores Suspended in Aqueous and Nonaqueous Milieu. I8rael J. Chem., 10, 1185.
