Br. J. Cancer (1978) 37, Suppl. III, 42
RADIATION DAMAGE TO DNA IN AQUEOUS SOLUTION: EFFECTS OF RADIOSENSITIZERS J. F. WARD From the Laboratory of NVuclear Medicine and Radiation Biology, University of California, Los Angeles, California 90024, and Department of Pathology, School of MHedicine, University of California, Los Angeles, California 90024
Summary.-Systems are described for examining the molecular mode of action of anoxic radiosensitizers with DNA-OH- radicals. Pulse radiolysis showed that NPPN reacts with these radicals even in the presence of 0 5 mm oxygen. Analysis of low molecular weight products liberated from DNA by irradiation showed that NPPN does not act in an oxygen-like way but that misonidazole does. Thus a molecular rationale exists for the use of combinations of radiosensitizers.
THE effectiveness of anoxic radiosensitizers has been related to their ability to act in an oxygen-like way, either by adding to radical sites or by chemically reducing them. Although the exact significant mechanisms by which oxygen itself acts at a molecular level have not been defined, several suggestions have been made. We are attempting to define mechanisms of oxygen at a molecular level using a biomolecular model system DNA in vitro. Previous studies (Ward and Kuo, 1976) showed that low-molecular-weight products released from macromolecular DNA are direct indicators of damage to base and to deoxyribose moieties. Release of intact base occurs as a result of OH- radical attack on the sugar moieties, while damaged base release is caused by OH- attack on the bases. In this work the OH- radical adducts reacted with oxygen. More recently (Ward and Kuo, submitted to Radiat. Res.) we have shown that the yields of the low-molecular-weight products are markedly reduced when radiation is carried out in the absence of oxygen. Intact base release remains the same but the damaged base release yield is reduced to almost zero. Since an oxygen effect is evident in this system, it seems well fitted for examining anoxic radiosensitizer effects at the molecular level. We have studied
effects of representative radiosensitizers of the N-oxyl type, nor-pseudo-pelletierene N-oxide (NPPN), and the electron affinic imidazole type, misonidazole. EXPERIMENTAL
All reagents (DNA, etc.) were as described in a previous communication (Ward and Kuo, 1976). NPPN was a gift from Roche Products, England. TAN was a gift from W. B. G. Jones and misonidazole was obtained from NCI. Pulse radiolysis was carried out at the Center for Fast Kinetic Research, Austin, Texas, with the kind cooperation of Dr E. L. Powers and Dr M. A. J. Rodgers. RESULTS AND DISCUSSION
It has been suggested that anoxic radiosensitizers react in an oxygen-like way. One of the mechanisms for oxygen reactions is its attachment to radical site, fixing the damage. However, the reaction of oxygen with the DNA-OH- transient has not been observed by pulse radiolysis (reviewed by Ward, 1975) but peroxide radiation products of DNA have been detected. On the other hand Roberts and Fielden (1971) have shown that NPPN does react with -,50%o of the DNA-OHtransient in the absence of oxygen. Thus we attempted to detect any unobserved
43
RADIATION DAMAGE TO DNA
NPPN
_..
NPPN+02
102
30
26
x ci
I-
0
2
4
6 0
2
4
0
2
4
6
TIME AFTER PULSE (x 104s) Fi.
1.-
Decay of DNA-OH- transient at 320 nm as a function of time in the and NPPN + oxygen.
reaction of oxygen with DNA-OH- by competition with NPPN. We found the reaction of NPPN (1.5 x 10-4M) with DNA-OH- to be almost independent of the presence of oxygen (up to 5 x 10-4M). Fig. 1 shows the reaction of NPPN with DNA-0H- in the absence of oxygen; the rate constant for the reaction is 1-75 X 107 m-1 s-1 in agreement with that obtained by Roberts and Fielden. Fig. 1 also shows a slight reaction of DNA-OH- with 02 and reaction of NPPN with DNA-OH- in the presence of oxygen. A slight difference is evident in the reaction of NPPN depending on whether 02 is present, but the extent and rate of the overall reaction are similar. These data indicate that oxygen does not react rapidly with the DNA-OHtransient which reacts with NPPN. (The rate constant for the reaction DNA-OH- -- 02 is lessthan 5 x 105M-1S-1). In the absence of sensitizer the DNA-OHradical would react with oxygen since radical-radical reactions between polymer radicals are unlikely. The DNA-OH- radicals observed by pulse radiolysis are formed on the base moieties: the sugar radicals have a low extinction coefficient. In order to learn more about sugar radical reactions it is necessary to study product yields. The latter are the most significan measure of radiation damage in vivo since they are
presence
of NPPN, oxygen
what the irradiated cell must handle to survive.
The measure of damage we choose to is the release of low-molecular-weight fragments from the macromolecule (Ward and Kuo, 1976). The macromolecule is precipitated with ethanol, the supernate concentrated and column-chromatographed on G1o Sephadex. The column eluent is assayed for UV absorption and 3H counts when 3H thymine DNA is used. Thus yields of intact bases (UV absorption) and damaged thymine products released (3H counts) can be monitored. Fig. 2 shows the elution pattern of 3H thymine counts released from DNA when irradiated in the presence of misonidazole and in the absence of oxygen. The major peak at Fraction 31 is intactthymine. Four minor peaks are evident; Fraction 12 is oligonucleotides, Fraction 18, Fraction 23 and Fraction 27 correspond to damaged thymine products. Fig. 2 also shows the pattern when oxygen is present during irradiation. These data are summarized in Table I, which collates results from DNA irradiated alone, with NPPN and with misonidazole. The yields show G values (molecules changed per 100 eV absorbed) which were obtained from yield dose plots. When DNA is irradiated alone in the absence of oxygen the majority of the lowmolecular-weight product released is thyuse
J. F. WARD
44
MISONIDAZOLE
MISONIDAZOLE + 02
0 x
E CL -J
z
CD 0
8-0
FRACTION NUMBER FIG. 2.-Elution profile of low-molecular-weight 3H-labelled products released from y irradiated (2 krads) DNA (1 -1 M w.r.t. nucleotides) DNA labelled with 3H thymidine. Column Sephadex G 10 40 x 0 -9 cm. Eluent 0 -1 M ammonium formate. Fraction volume 1 -1 ml.
TABLE.- Yields (G Values) ofLow-molcularweight Products from Irradiated DNA (O.D.7.6)
more effective in the presence of oxygen, markedly affecting the yield of free base and reducing the yields of Products A and C to zero and B by a factor of 6. Peak Peak Peak Misonidazole, on the other hand, has C A B (Frac- (Frac- (Fraclittle if any effect on the free base and tion tion tion Thydamaged base yields in the presence of Conditions 18) 22-23)25,26) mine oxygen. It has a major effect only on the DNA 0 0-02 0 03 0-26 DNA + 02 0 04 0-13 0 05 0-39 yields of damaged base products in the DNA + NPPN (10-5M) 0-03 0-03 0-01 0-22 absence of oxygen: increasing the yields. 0-02 0-14 + 02 Thus these two sensitizers act in differDNA + Misonidazole 0-02 0*07 0-02 0-28 (10-5M) ent ways with the DNA-OH- radicals. 0-05 0-14 0-07 0-41 +02 Misonidazole acts in the absence of oxygen, causing increases in damaged base yields, more closely approaching the yields of this mine with a G value of release of 0-26. This yield is correlated to damage produced on damage in the presence of oxygen. It acts the sugar moiety. Damage to the base as a true anoxic radiosensitizer in this moieties is evidenced by release of Pro- systeni. On the other hand NPPN has its major ducts A, B and C which are as yet uncharacterized. Although these yields ap- effect in the presence of oxygen, causing pear small, the significance of the damage only slight changes in its absence. It could cannot be neglected and the distribution be argued that NPPN does not act at of the products is markedly affected by DNA sites in cellular systems. In any the presence of oxygen. Increases in all event, since there appears to be a different damage base product yields occur if the mode of action for these two sensitizers, radiation is carried out in the presence of there is justification at a molecular level oxygen. The thymine base yield increases for using combinations of sensitizers in the manner of Millar, Fielden and Smithen 50% under oxygenated conditions. NPPN shows only slight effects in the (1977). absence of oxygen: reducing the free base The above work was supported by ERDA Conyield slightly and changing the distribu- tract EY-76-C-03-0012, Laboratory of Nuclear tion of the damaged base products. It is Medicine and Radiation Biology, University of
RADIATION DAMAGE TO DNA California, Los Angeles, and the Department of Pathology, School of Medicine, University of California, Los Angeles, California. Part of this work was performed at the Center for Fast Kinetics Research at the University of Texas at Austin. The CFKR is supported by NIH grant RR-00886 from the Biotechnology Branch of the Division of Research Resources and by The University of Texas at Austin.
REFERENCES MIILLAR, B. C., FIELDEN, E. M. & SMITHEN, C. M. (1977) Polyfunctional Radiosensitizers III, Effect
45
of the Biradical (Ro-03-6061) in Combination with Other Radiosensitizers on the Survival of Hypoxic V-79 Cells. Radiat. Res., 69, 489. ROBERTS, P. B. & FIELDEN, E. M. (1971) Pulse Radiolysis Studies of the radiosensitizer Norpseudo pelletierene N-oxyl (NPPN) II Reactions involving Biological Radicals. Int. J. Radiat. Biol., 20, 363. WARD, J. F. (1975) Molecular Mechanisms of Radiation Induced Damage to Nucleic Acids. Adv. in Radiat. Biol. 5, 181. WARD, J. F. & Kuo, I. (1976) Strand Breaks, Base release, and Post-irradiation Changes in DNA y-Irradiated in Dilute 02-Saturated Aqueous Solution. Radiat. Res., 66, 485.
RADIATION DAMAGE TO DNA IN AQUEOUS SOLUTION: EFFECTS OF RADIOSENSITIZERS J. F. WARD From the Laboratory of NVuclear Medicine and Radiation Biology, University of California, Los Angeles, California 90024, and Department of Pathology, School of MHedicine, University of California, Los Angeles, California 90024
Summary.-Systems are described for examining the molecular mode of action of anoxic radiosensitizers with DNA-OH- radicals. Pulse radiolysis showed that NPPN reacts with these radicals even in the presence of 0 5 mm oxygen. Analysis of low molecular weight products liberated from DNA by irradiation showed that NPPN does not act in an oxygen-like way but that misonidazole does. Thus a molecular rationale exists for the use of combinations of radiosensitizers.
THE effectiveness of anoxic radiosensitizers has been related to their ability to act in an oxygen-like way, either by adding to radical sites or by chemically reducing them. Although the exact significant mechanisms by which oxygen itself acts at a molecular level have not been defined, several suggestions have been made. We are attempting to define mechanisms of oxygen at a molecular level using a biomolecular model system DNA in vitro. Previous studies (Ward and Kuo, 1976) showed that low-molecular-weight products released from macromolecular DNA are direct indicators of damage to base and to deoxyribose moieties. Release of intact base occurs as a result of OH- radical attack on the sugar moieties, while damaged base release is caused by OH- attack on the bases. In this work the OH- radical adducts reacted with oxygen. More recently (Ward and Kuo, submitted to Radiat. Res.) we have shown that the yields of the low-molecular-weight products are markedly reduced when radiation is carried out in the absence of oxygen. Intact base release remains the same but the damaged base release yield is reduced to almost zero. Since an oxygen effect is evident in this system, it seems well fitted for examining anoxic radiosensitizer effects at the molecular level. We have studied
effects of representative radiosensitizers of the N-oxyl type, nor-pseudo-pelletierene N-oxide (NPPN), and the electron affinic imidazole type, misonidazole. EXPERIMENTAL
All reagents (DNA, etc.) were as described in a previous communication (Ward and Kuo, 1976). NPPN was a gift from Roche Products, England. TAN was a gift from W. B. G. Jones and misonidazole was obtained from NCI. Pulse radiolysis was carried out at the Center for Fast Kinetic Research, Austin, Texas, with the kind cooperation of Dr E. L. Powers and Dr M. A. J. Rodgers. RESULTS AND DISCUSSION
It has been suggested that anoxic radiosensitizers react in an oxygen-like way. One of the mechanisms for oxygen reactions is its attachment to radical site, fixing the damage. However, the reaction of oxygen with the DNA-OH- transient has not been observed by pulse radiolysis (reviewed by Ward, 1975) but peroxide radiation products of DNA have been detected. On the other hand Roberts and Fielden (1971) have shown that NPPN does react with -,50%o of the DNA-OHtransient in the absence of oxygen. Thus we attempted to detect any unobserved
43
RADIATION DAMAGE TO DNA
NPPN
_..
NPPN+02
102
30
26
x ci
I-
0
2
4
6 0
2
4
0
2
4
6
TIME AFTER PULSE (x 104s) Fi.
1.-
Decay of DNA-OH- transient at 320 nm as a function of time in the and NPPN + oxygen.
reaction of oxygen with DNA-OH- by competition with NPPN. We found the reaction of NPPN (1.5 x 10-4M) with DNA-OH- to be almost independent of the presence of oxygen (up to 5 x 10-4M). Fig. 1 shows the reaction of NPPN with DNA-0H- in the absence of oxygen; the rate constant for the reaction is 1-75 X 107 m-1 s-1 in agreement with that obtained by Roberts and Fielden. Fig. 1 also shows a slight reaction of DNA-OH- with 02 and reaction of NPPN with DNA-OH- in the presence of oxygen. A slight difference is evident in the reaction of NPPN depending on whether 02 is present, but the extent and rate of the overall reaction are similar. These data indicate that oxygen does not react rapidly with the DNA-OHtransient which reacts with NPPN. (The rate constant for the reaction DNA-OH- -- 02 is lessthan 5 x 105M-1S-1). In the absence of sensitizer the DNA-OHradical would react with oxygen since radical-radical reactions between polymer radicals are unlikely. The DNA-OH- radicals observed by pulse radiolysis are formed on the base moieties: the sugar radicals have a low extinction coefficient. In order to learn more about sugar radical reactions it is necessary to study product yields. The latter are the most significan measure of radiation damage in vivo since they are
presence
of NPPN, oxygen
what the irradiated cell must handle to survive.
The measure of damage we choose to is the release of low-molecular-weight fragments from the macromolecule (Ward and Kuo, 1976). The macromolecule is precipitated with ethanol, the supernate concentrated and column-chromatographed on G1o Sephadex. The column eluent is assayed for UV absorption and 3H counts when 3H thymine DNA is used. Thus yields of intact bases (UV absorption) and damaged thymine products released (3H counts) can be monitored. Fig. 2 shows the elution pattern of 3H thymine counts released from DNA when irradiated in the presence of misonidazole and in the absence of oxygen. The major peak at Fraction 31 is intactthymine. Four minor peaks are evident; Fraction 12 is oligonucleotides, Fraction 18, Fraction 23 and Fraction 27 correspond to damaged thymine products. Fig. 2 also shows the pattern when oxygen is present during irradiation. These data are summarized in Table I, which collates results from DNA irradiated alone, with NPPN and with misonidazole. The yields show G values (molecules changed per 100 eV absorbed) which were obtained from yield dose plots. When DNA is irradiated alone in the absence of oxygen the majority of the lowmolecular-weight product released is thyuse
J. F. WARD
44
MISONIDAZOLE
MISONIDAZOLE + 02
0 x
E CL -J
z
CD 0
8-0
FRACTION NUMBER FIG. 2.-Elution profile of low-molecular-weight 3H-labelled products released from y irradiated (2 krads) DNA (1 -1 M w.r.t. nucleotides) DNA labelled with 3H thymidine. Column Sephadex G 10 40 x 0 -9 cm. Eluent 0 -1 M ammonium formate. Fraction volume 1 -1 ml.
TABLE.- Yields (G Values) ofLow-molcularweight Products from Irradiated DNA (O.D.7.6)
more effective in the presence of oxygen, markedly affecting the yield of free base and reducing the yields of Products A and C to zero and B by a factor of 6. Peak Peak Peak Misonidazole, on the other hand, has C A B (Frac- (Frac- (Fraclittle if any effect on the free base and tion tion tion Thydamaged base yields in the presence of Conditions 18) 22-23)25,26) mine oxygen. It has a major effect only on the DNA 0 0-02 0 03 0-26 DNA + 02 0 04 0-13 0 05 0-39 yields of damaged base products in the DNA + NPPN (10-5M) 0-03 0-03 0-01 0-22 absence of oxygen: increasing the yields. 0-02 0-14 + 02 Thus these two sensitizers act in differDNA + Misonidazole 0-02 0*07 0-02 0-28 (10-5M) ent ways with the DNA-OH- radicals. 0-05 0-14 0-07 0-41 +02 Misonidazole acts in the absence of oxygen, causing increases in damaged base yields, more closely approaching the yields of this mine with a G value of release of 0-26. This yield is correlated to damage produced on damage in the presence of oxygen. It acts the sugar moiety. Damage to the base as a true anoxic radiosensitizer in this moieties is evidenced by release of Pro- systeni. On the other hand NPPN has its major ducts A, B and C which are as yet uncharacterized. Although these yields ap- effect in the presence of oxygen, causing pear small, the significance of the damage only slight changes in its absence. It could cannot be neglected and the distribution be argued that NPPN does not act at of the products is markedly affected by DNA sites in cellular systems. In any the presence of oxygen. Increases in all event, since there appears to be a different damage base product yields occur if the mode of action for these two sensitizers, radiation is carried out in the presence of there is justification at a molecular level oxygen. The thymine base yield increases for using combinations of sensitizers in the manner of Millar, Fielden and Smithen 50% under oxygenated conditions. NPPN shows only slight effects in the (1977). absence of oxygen: reducing the free base The above work was supported by ERDA Conyield slightly and changing the distribu- tract EY-76-C-03-0012, Laboratory of Nuclear tion of the damaged base products. It is Medicine and Radiation Biology, University of
RADIATION DAMAGE TO DNA California, Los Angeles, and the Department of Pathology, School of Medicine, University of California, Los Angeles, California. Part of this work was performed at the Center for Fast Kinetics Research at the University of Texas at Austin. The CFKR is supported by NIH grant RR-00886 from the Biotechnology Branch of the Division of Research Resources and by The University of Texas at Austin.
REFERENCES MIILLAR, B. C., FIELDEN, E. M. & SMITHEN, C. M. (1977) Polyfunctional Radiosensitizers III, Effect
45
of the Biradical (Ro-03-6061) in Combination with Other Radiosensitizers on the Survival of Hypoxic V-79 Cells. Radiat. Res., 69, 489. ROBERTS, P. B. & FIELDEN, E. M. (1971) Pulse Radiolysis Studies of the radiosensitizer Norpseudo pelletierene N-oxyl (NPPN) II Reactions involving Biological Radicals. Int. J. Radiat. Biol., 20, 363. WARD, J. F. (1975) Molecular Mechanisms of Radiation Induced Damage to Nucleic Acids. Adv. in Radiat. Biol. 5, 181. WARD, J. F. & Kuo, I. (1976) Strand Breaks, Base release, and Post-irradiation Changes in DNA y-Irradiated in Dilute 02-Saturated Aqueous Solution. Radiat. Res., 66, 485.
