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Repair processes in germinating seeds: Caffeine enhancement of damage induced by gamma-radiation and alkylating chemicals
Mulalio~2 Research, 26 (I974) 99-1o3 i© Elsevier Scientific Publishing .Company, Amsterdam - Printed in The Netherlands
99
R E P A I R PROCESSES IN GERMINATING SEEDS: CAFFEINE ENHANCEMENT OF DAMAGE INDUCED BY GAMMA-RADIATION AND ALKYLATING CHEMICALS
GUNNAR AHNSTR(')M
U1ziversily of Slockholm, lVallenberg LaboratoJ,y, Lilla Frescali, S-zo 4 05 Stockholm 50 (Swcden) (Received November ist, 1973)
I~#roductio¢¢ Caffeine enhances the biological effect of UV and mutagenic chemicals in many organisms; tile frequency of mutations in bacteria after UV ~, the lethal effect of UV and alkylating agents in mammalian cells", ~a, the frequency of chromosomal aberrations in plant cells after treatment with mutagenic compoundsT, 8. Conflicting results have been reported about damage produced by ionizing radiations: no effect of caffeine was found by WOLFF AND SCOTT~5in their split-dose experiments On Vicia (irradiated in G1) ; on the other hand, a clear effect was observed when resting barley seeds were irradiated and allowed to germinate in the presence of caffeine~. A clear effect was also observed by YAMAMOTOAND YAMAGUCHI~", who studied X-ray-induced chromosomal aberrations in barley seeds (G2).
I~¢hibitio~, of seedling growth and chromosomal aberration, Inhibition of seedling growth after treatment of seeds is a convenient and reproducible technique for studying the effects of ionizing radiations and radiomimetic chemicals in plants. The inhibition of seedling growth seems also to be well correlated with tile amount of chromosomal damage. This was shown by CoNG~I~ANDSTEVENSEN (ref. 4), who investigated the variation in seedling height within one dose of ~-rays and found an almost perfect correlation between inhibition of the first leaf and frequency of chromosomal aberrations in a root-tip from the same seedling.
Repair processes in germinating seeds The resistance of resting seeds to ~ r a y s indicates that efficient repair processes take place early during germination. To elucidate this problem we have tried to increase the radiation-induced damage by treating the gen~nating seeds with compounds believed to interfere with the repair of damage to the genetic material. One such compound found to be effective was caffeine, as demonstrated in Fig. I. Caffeine increased the y-ray-induced damage by a factor of two, if present during the first 5 h of germination. The treatment also doubled the frequency of chromosomal aberrations. A more detailed study showed that the critical stage occurred between 2 and 5 h from the beginning of germination, indicating that the first 2 h could involve water uptake Abbreviations: EM8, ethyl methanesulfonatc; MMS, methyl methanesul~onate; iso-PMS, isopropyl mcthmlesulfonate.
GUNNAR AHNSTROM
ZOO 100
8 8
5O
.E
~"
caffeineO-Sh%
o
"~
I 20 40I Gammadose(krad)
60I
Fig. x. Seedling g r o w t h inhibition after y - i r r a d i a t i o n of resting seeds, Effect of a caffeine postt r e a t m e n t for 5 h (z m g I m l ) a t different stages of germination,
1.0~./'~X~x,,
F ;
J_ --
:~ o.1
~
/ /
o
/
neincorporatlor ]/ 8a'rnrnarays // -E3H]t.~Lyrnl -in barle.diyeF~bryos / (SJJC~/ml2h} 103~
p
/
~0,05 o.o2,
;
lb
....
,&
Soakingtime (h)
2'0
2'5
o
Fig. 2. (a) Sensitivity of barley seeds (seedling g r o w t h inhibition) to ElVIS a n d y - r a y s a t different stages of presoaking at 25 °. 50°,/0 inhibition dose a t o h: y-rays, 5 ° kraal; EMS, t r e a t m e n t for I h in 0.4 M solutions. (b} R a t e of D N A - s y n t h e s i s in b a r l e y seeds as a f u n c t i o n of p r e s o a k i n g t i m e at 25 ° .
and hydration of various structures in the cell, and the main repair took place and was completed during the following 3 h. The effect of caffeine was also investigated on seeds irradiated in presoaked conditions. Seeds soaked for 4 h showed the same caffeine enhancement factor as seeds irradiated in the resting state. This factor, however, was reduced at prolonged presoaking times, and after I5 h of germination only a small caffeine effect remained ~ (enhancement factor = 1.2). In contrast to the findings with 7-radiation, no enhancement was obtained when resting seeds were treated for I h in solutions of EMS, MMS or iso-PMS and then soaked for the next 5 h in the presence of caffeine. On the other hand, caffeine was found to have an effect if applied later during germination, i.e. after I5-2o h of soaldng when the cells have entered the S-phase (Fig. 2). The alkylating agents tested differed somewhat with respect to the caffeine enhancement. In experiments with EMS the effect was hardly significant but the effects of ~IMS and iso-PMS were significantly increased. With MMS the potentiating factor was L2 and with iso-PMS it was L6.
EFFECT OF CAFFEINE WITH ),-RADIATION AND ALKYLATING CHEMICALS
IOI
Discussion In an earlier report ~ we tried to explain why caffeine has a potentiating effect with y-radiation only during the early stages of germination. This stage is characterized by a steep increase in radiosensitivity (Fig. ~). When the seeds have germinated :for 8-12 h a constant level of radiosensitivity is reached, and during this period caffeine has less effect. The increase in sensitivity is dependent on metabolic processes. If the seeds are soaked under conditions where little or no metabolism takes place~in the absence of oxygen and at low tenlperature---the seeds keep their radioresistance for a long time 5. We therefore assumed that the main factors that control radiosensitivity during germination are the organization and mobility of the chromosomal material. At the beginning of germination, interactions between different chromosomes and chromosomal regions may be limited but will continuously increase during germination. This will lead to an increasing probability of interactions between distant lesions causing more misrepair and explessed as exchange aberrations of various types. The concentration of caffeine applied in our experiments (I mg/ml) did not reduce normal growth of the seedlings. Since the caffeine increased the radiationinduced danlage, it was obvious that the caffeine treatment interfered with repair processes. This interference could take place at the DNA level (it is, for example, known that caffeine increases the so-called helix-coil transitions in DNA ~a) and it is probable that this kind of denaturation effect could lead to an enhancement of the frequency of misrepair. If this is true, however, one would also expect caffeine to be effective during the later stages of germination. It was therefore assmned that caffeine could slow down repair processes without markedly affecting the ratio misrepair to correct repair. As mentioned above, we believe that the frequency of misrepair continuously increases during germination up to a level that is reached after about 12 h soaking in water. Lesions induced in seeds presoaked for 5 h seem to have I0 times more "weigl~t" than lesions produced during the first hour of soaking (Fig. 2). A slowing down of the repair processes at this stage, without affecting the metabolic processes controlling the increase in sensitivity, will leave more unrepaired lesions in the following more radiosensitive stage. Hence, even a moderate reduction in the rate of repair could lead to a substantial increase in the number of misrepaired lesions. On the other hand, if a cell had reached a constant level of radiosensitivity, a retardation of the repair processes would not be expected to increase the damage drastically. That caffeine has a small effect even in seeds tl~at have reached a constant level of sensitivity could mean that caffeine is able to interact with radiation-induced lesions in more than one way. The effect of caffeine on seeds treated with alkylating agents was not unexpected in view of earlier findings. SWIETLIt~'SKAn, for example, reported a strong caffeine potentiation of diepoxybutane-induced aberrations in Vicia. The caffeine effect was only obtained when the cells were exposed to caffeine during the S-phase.
Molecular basis for a &~ere~tial repair of 7-i~dueed lesions and damage produced by alkylati,~¢g age~l,ts Io~¢izi~g radiatio1~s. Ionizations in the DNA, as well as the attack of radiolytic products from water, lead to the breakage of covalent bonds. Some of the destructive events which take place in the backbone area of the DNA--the sugar-phosphate moiety--will result in strand breaks. This will happen either if tile sugar-phosphate
I02
GUNNAR
AHNSTROM
ester bond is hydrolyzed or if one of the carbon-carbon bonds in the deoxyribose ring is broken. Most of the breaks are so-called single-strand breaks which, although they weaken the DNA molecule, do not immediately affect the linear integrity of the molecule. However, parallel to the single-strand breaks, a substantial number of double-strand breaks also occm'. Free DNA molecules in solution are disrupted by this latter event, but in the cell this may not happen because DNA occurs in complexes with other macromolecules. The attack on the bases in the DNA results in a variety of chemical changes: deaminations, breakage of the cyclic structure, loss of whole bases. In the presence of oxygen, some unstable pyrimidine peroxides are formed. Alkylatilig agents. Alkylating agents are more specific in their action than ionizing radiations. The primary event consists of the addition of an alkyl group to the DNA, this addition occurring most frequently on the purines 9. Indirect evidence also points to phosphate alkylation ~. Strand breaks do not occur as a primary event but can arise as a result of secondary processes. The alkylation of the purines at certain positions weakens the base-sugar bond and results in depurination. The sugar-phosphate bond at the depurinated site is also unstable. After hydrolysis, a single-strand break is produced. (For references, see ref. 9). Strand interruptions can also arise as a result of an enzymatic attack on the damaged part of the DNA. In living cells the alkylated DNA is subjected to repair processes similar to those existing after UV treatment. Excision repair enzymes introduce a cut adjacent to the defect, the defect is removed and the gap is filled by repair synthesis. Another process that creates interruptions in the DNA has been discussed by RUPP AND HOWARD-FLANDERS ~°, who found that some excision-deficient bacterial strains were able to replicate their DNA after UV irradiation without prior excision of the pyrimidine dimers. In these bacteria the newly synthesized DNA contained interruptions that occurred at the same frequency as the existing pyrimidine dimers. Apparently, the interruptions occurred opposite the dimers, and were subsequently filled in by a rather slow post-replication repair process. CLEAVEI~AND T~IO~IAS~ have demonstrated the existence of similar processes in mammalian cells, and they also showed the gap-filling reaction was inhibited by caffeine. This was in contrast to excision repair which is not affected by caffeine 2. It is suggested that the damage induced by radiation in plant seeds is to a large extent caused by strand breaks in DNA. These breaks are detected and repaired at an early stage during germination. The extent of lmsrepair, expressed as chromosomal aberrations, increases during the course of germination, and this is believed to depend on the increasing probability of interaction between lesions in different chromosomes and chromosomal regions. It is not known whether both single and double strand breaks contribute to the biological effects. It is possible that alkylating agents induce defects in DNA which are subject to the same post-replication repair as the defects caused by UV. Alkylating agents produce more subtle changes in the DNA than ionizing radiation. The effect of caffeine on seed treated with iso-PMS--no potentiation in GI, potentiation in S--indicates that the lesions are detected and repaired only during the S.phase.
EFFECT OF CAFFEINE WITH ~-RADIATION AND ALKYLATING CHEMICALS
I0~
REFERENCES I A~NS'rRdM, G., AND A. T, NATARAJAN, Repair of gamma-ray and neutron-induced lesions in germinating barley seeds, Intern. J. Radiation Biol., I9 (1971) 433-443. 2 CLEAV~IZ,J. E., Repair replication of mammalian cell DNA: effects of compounds that inhibit DNA synthesis or dark repair, Radiation Res., 37 (1969) 334-348. 3 CLEAVER, J. E., AND G. H. THOMAS, Single-strand interruptions in DNA and the effects of caffeine in Chinese hamster cells irradiated with ultraviolet light, Biochem. Biophys. Res. Commun., 36 (I969) 2o3-2o8. 4 CONe;OR, A. D., AND H. Q. ST~VENSgN, A correlation of seedling height and chromosomal damage in irradiated barley seeds, Radiation Pot., 9 (1969) I-I4. 5 DAVIES, D. R., AND E. T. WALL, The influence of oxygen and temperature on the radiosensitivity of soaked barley seeds, Intern. J. Radiation Biol., 2 (196o) 26~-267. 6 DOMON, M., AND A. M. RAUTH, Effects of caffeine on ultraviolet-irradiated mouse L cells, Radiation Res., 39 (~969) 2o7-221. 7 KIHLMAN, B. A., S. STURELID, B, HARTLEY-AsPAND I':. NILSBON, Caffeine potentiation of the chromosome damage produced in bean root tips and in Chinese hamster cells by various cheniical and physical agents, Mutation Res., 17 (1973) 271-275. 8 I{IHLMAN, B. A., S. STURIBLID, B. HARTLEY-ASPAND I~, NILSSON, The enhancement by caffeine of the frequencies of chromosomal aberra~:ions iuduced in plant and animal cells by chemical and physical agents, Mutation Res., 26 (t974) IO5-I22. 9 Ross, W. C. J., Biological Alkylating Agents, Butterworth, London, 1962. IO I~UPP, ~V'. D., AND P. HOWARD-FLANDERS, Discontinuitics in the DNA synthesized in an exeision~defective strain of E, coli following ultraviolet irradiation, J, Mol, Biol., 31 (I968) 29I-3O 4. II SWlETLINSKA, Z., High frequency of chrollXOsome aberrations induced by DEB with caffeine posttreatment in Viciafaba var. minor, Mol. Gen. Goner., 112 (197I) 87-9o. 12 Ts6, P. O. P., G. 1,2. HIgLMKAMPAND C. SANDER, Interaction of nucleosides and related compounds with nucleic acids as indicated by the change of helix-coil transition temperature, P~,oc. Natl. Acad. Sci. (U.S.), 48 (1962) 686-698. 13 "WALXEILI. G., AND B. D. REID, Caffeine potentiation of the lethal action of alkylating agents on L-cells, Mutation Res., 12 (197 I) i o i - i o , t. 14 WITKIN, E. M., Post-lrradiatlon metabolism and the timing o[ ultraviolet-induced mutations in bacteria, Proc. zoth Intern. Congr, Genet., I (1958) 28o-e99. 15 WOLFF, S., AND D. SCOTT, Repair of radiatlon-induced damage to chromosomes, Exptl. Cell Res., 55 (I969) 9-I6. 16 ¥AMAMOT0, I':., AND H. YAMAalJCHI, Inhibition by caffeine of the repair of v-ray-induced chromosome breaks in barley, Mutation Res., 8 (I969) 428-43o.
99
R E P A I R PROCESSES IN GERMINATING SEEDS: CAFFEINE ENHANCEMENT OF DAMAGE INDUCED BY GAMMA-RADIATION AND ALKYLATING CHEMICALS
GUNNAR AHNSTR(')M
U1ziversily of Slockholm, lVallenberg LaboratoJ,y, Lilla Frescali, S-zo 4 05 Stockholm 50 (Swcden) (Received November ist, 1973)
I~#roductio¢¢ Caffeine enhances the biological effect of UV and mutagenic chemicals in many organisms; tile frequency of mutations in bacteria after UV ~, the lethal effect of UV and alkylating agents in mammalian cells", ~a, the frequency of chromosomal aberrations in plant cells after treatment with mutagenic compoundsT, 8. Conflicting results have been reported about damage produced by ionizing radiations: no effect of caffeine was found by WOLFF AND SCOTT~5in their split-dose experiments On Vicia (irradiated in G1) ; on the other hand, a clear effect was observed when resting barley seeds were irradiated and allowed to germinate in the presence of caffeine~. A clear effect was also observed by YAMAMOTOAND YAMAGUCHI~", who studied X-ray-induced chromosomal aberrations in barley seeds (G2).
I~¢hibitio~, of seedling growth and chromosomal aberration, Inhibition of seedling growth after treatment of seeds is a convenient and reproducible technique for studying the effects of ionizing radiations and radiomimetic chemicals in plants. The inhibition of seedling growth seems also to be well correlated with tile amount of chromosomal damage. This was shown by CoNG~I~ANDSTEVENSEN (ref. 4), who investigated the variation in seedling height within one dose of ~-rays and found an almost perfect correlation between inhibition of the first leaf and frequency of chromosomal aberrations in a root-tip from the same seedling.
Repair processes in germinating seeds The resistance of resting seeds to ~ r a y s indicates that efficient repair processes take place early during germination. To elucidate this problem we have tried to increase the radiation-induced damage by treating the gen~nating seeds with compounds believed to interfere with the repair of damage to the genetic material. One such compound found to be effective was caffeine, as demonstrated in Fig. I. Caffeine increased the y-ray-induced damage by a factor of two, if present during the first 5 h of germination. The treatment also doubled the frequency of chromosomal aberrations. A more detailed study showed that the critical stage occurred between 2 and 5 h from the beginning of germination, indicating that the first 2 h could involve water uptake Abbreviations: EM8, ethyl methanesulfonatc; MMS, methyl methanesul~onate; iso-PMS, isopropyl mcthmlesulfonate.
GUNNAR AHNSTROM
ZOO 100
8 8
5O
.E
~"
caffeineO-Sh%
o
"~
I 20 40I Gammadose(krad)
60I
Fig. x. Seedling g r o w t h inhibition after y - i r r a d i a t i o n of resting seeds, Effect of a caffeine postt r e a t m e n t for 5 h (z m g I m l ) a t different stages of germination,
1.0~./'~X~x,,
F ;
J_ --
:~ o.1
~
/ /
o
/
neincorporatlor ]/ 8a'rnrnarays // -E3H]t.~Lyrnl -in barle.diyeF~bryos / (SJJC~/ml2h} 103~
p
/
~0,05 o.o2,
;
lb
....
,&
Soakingtime (h)
2'0
2'5
o
Fig. 2. (a) Sensitivity of barley seeds (seedling g r o w t h inhibition) to ElVIS a n d y - r a y s a t different stages of presoaking at 25 °. 50°,/0 inhibition dose a t o h: y-rays, 5 ° kraal; EMS, t r e a t m e n t for I h in 0.4 M solutions. (b} R a t e of D N A - s y n t h e s i s in b a r l e y seeds as a f u n c t i o n of p r e s o a k i n g t i m e at 25 ° .
and hydration of various structures in the cell, and the main repair took place and was completed during the following 3 h. The effect of caffeine was also investigated on seeds irradiated in presoaked conditions. Seeds soaked for 4 h showed the same caffeine enhancement factor as seeds irradiated in the resting state. This factor, however, was reduced at prolonged presoaking times, and after I5 h of germination only a small caffeine effect remained ~ (enhancement factor = 1.2). In contrast to the findings with 7-radiation, no enhancement was obtained when resting seeds were treated for I h in solutions of EMS, MMS or iso-PMS and then soaked for the next 5 h in the presence of caffeine. On the other hand, caffeine was found to have an effect if applied later during germination, i.e. after I5-2o h of soaldng when the cells have entered the S-phase (Fig. 2). The alkylating agents tested differed somewhat with respect to the caffeine enhancement. In experiments with EMS the effect was hardly significant but the effects of ~IMS and iso-PMS were significantly increased. With MMS the potentiating factor was L2 and with iso-PMS it was L6.
EFFECT OF CAFFEINE WITH ),-RADIATION AND ALKYLATING CHEMICALS
IOI
Discussion In an earlier report ~ we tried to explain why caffeine has a potentiating effect with y-radiation only during the early stages of germination. This stage is characterized by a steep increase in radiosensitivity (Fig. ~). When the seeds have germinated :for 8-12 h a constant level of radiosensitivity is reached, and during this period caffeine has less effect. The increase in sensitivity is dependent on metabolic processes. If the seeds are soaked under conditions where little or no metabolism takes place~in the absence of oxygen and at low tenlperature---the seeds keep their radioresistance for a long time 5. We therefore assumed that the main factors that control radiosensitivity during germination are the organization and mobility of the chromosomal material. At the beginning of germination, interactions between different chromosomes and chromosomal regions may be limited but will continuously increase during germination. This will lead to an increasing probability of interactions between distant lesions causing more misrepair and explessed as exchange aberrations of various types. The concentration of caffeine applied in our experiments (I mg/ml) did not reduce normal growth of the seedlings. Since the caffeine increased the radiationinduced danlage, it was obvious that the caffeine treatment interfered with repair processes. This interference could take place at the DNA level (it is, for example, known that caffeine increases the so-called helix-coil transitions in DNA ~a) and it is probable that this kind of denaturation effect could lead to an enhancement of the frequency of misrepair. If this is true, however, one would also expect caffeine to be effective during the later stages of germination. It was therefore assmned that caffeine could slow down repair processes without markedly affecting the ratio misrepair to correct repair. As mentioned above, we believe that the frequency of misrepair continuously increases during germination up to a level that is reached after about 12 h soaking in water. Lesions induced in seeds presoaked for 5 h seem to have I0 times more "weigl~t" than lesions produced during the first hour of soaking (Fig. 2). A slowing down of the repair processes at this stage, without affecting the metabolic processes controlling the increase in sensitivity, will leave more unrepaired lesions in the following more radiosensitive stage. Hence, even a moderate reduction in the rate of repair could lead to a substantial increase in the number of misrepaired lesions. On the other hand, if a cell had reached a constant level of radiosensitivity, a retardation of the repair processes would not be expected to increase the damage drastically. That caffeine has a small effect even in seeds tl~at have reached a constant level of sensitivity could mean that caffeine is able to interact with radiation-induced lesions in more than one way. The effect of caffeine on seeds treated with alkylating agents was not unexpected in view of earlier findings. SWIETLIt~'SKAn, for example, reported a strong caffeine potentiation of diepoxybutane-induced aberrations in Vicia. The caffeine effect was only obtained when the cells were exposed to caffeine during the S-phase.
Molecular basis for a &~ere~tial repair of 7-i~dueed lesions and damage produced by alkylati,~¢g age~l,ts Io~¢izi~g radiatio1~s. Ionizations in the DNA, as well as the attack of radiolytic products from water, lead to the breakage of covalent bonds. Some of the destructive events which take place in the backbone area of the DNA--the sugar-phosphate moiety--will result in strand breaks. This will happen either if tile sugar-phosphate
I02
GUNNAR
AHNSTROM
ester bond is hydrolyzed or if one of the carbon-carbon bonds in the deoxyribose ring is broken. Most of the breaks are so-called single-strand breaks which, although they weaken the DNA molecule, do not immediately affect the linear integrity of the molecule. However, parallel to the single-strand breaks, a substantial number of double-strand breaks also occm'. Free DNA molecules in solution are disrupted by this latter event, but in the cell this may not happen because DNA occurs in complexes with other macromolecules. The attack on the bases in the DNA results in a variety of chemical changes: deaminations, breakage of the cyclic structure, loss of whole bases. In the presence of oxygen, some unstable pyrimidine peroxides are formed. Alkylatilig agents. Alkylating agents are more specific in their action than ionizing radiations. The primary event consists of the addition of an alkyl group to the DNA, this addition occurring most frequently on the purines 9. Indirect evidence also points to phosphate alkylation ~. Strand breaks do not occur as a primary event but can arise as a result of secondary processes. The alkylation of the purines at certain positions weakens the base-sugar bond and results in depurination. The sugar-phosphate bond at the depurinated site is also unstable. After hydrolysis, a single-strand break is produced. (For references, see ref. 9). Strand interruptions can also arise as a result of an enzymatic attack on the damaged part of the DNA. In living cells the alkylated DNA is subjected to repair processes similar to those existing after UV treatment. Excision repair enzymes introduce a cut adjacent to the defect, the defect is removed and the gap is filled by repair synthesis. Another process that creates interruptions in the DNA has been discussed by RUPP AND HOWARD-FLANDERS ~°, who found that some excision-deficient bacterial strains were able to replicate their DNA after UV irradiation without prior excision of the pyrimidine dimers. In these bacteria the newly synthesized DNA contained interruptions that occurred at the same frequency as the existing pyrimidine dimers. Apparently, the interruptions occurred opposite the dimers, and were subsequently filled in by a rather slow post-replication repair process. CLEAVEI~AND T~IO~IAS~ have demonstrated the existence of similar processes in mammalian cells, and they also showed the gap-filling reaction was inhibited by caffeine. This was in contrast to excision repair which is not affected by caffeine 2. It is suggested that the damage induced by radiation in plant seeds is to a large extent caused by strand breaks in DNA. These breaks are detected and repaired at an early stage during germination. The extent of lmsrepair, expressed as chromosomal aberrations, increases during the course of germination, and this is believed to depend on the increasing probability of interaction between lesions in different chromosomes and chromosomal regions. It is not known whether both single and double strand breaks contribute to the biological effects. It is possible that alkylating agents induce defects in DNA which are subject to the same post-replication repair as the defects caused by UV. Alkylating agents produce more subtle changes in the DNA than ionizing radiation. The effect of caffeine on seed treated with iso-PMS--no potentiation in GI, potentiation in S--indicates that the lesions are detected and repaired only during the S.phase.
EFFECT OF CAFFEINE WITH ~-RADIATION AND ALKYLATING CHEMICALS
I0~
REFERENCES I A~NS'rRdM, G., AND A. T, NATARAJAN, Repair of gamma-ray and neutron-induced lesions in germinating barley seeds, Intern. J. Radiation Biol., I9 (1971) 433-443. 2 CLEAV~IZ,J. E., Repair replication of mammalian cell DNA: effects of compounds that inhibit DNA synthesis or dark repair, Radiation Res., 37 (1969) 334-348. 3 CLEAVER, J. E., AND G. H. THOMAS, Single-strand interruptions in DNA and the effects of caffeine in Chinese hamster cells irradiated with ultraviolet light, Biochem. Biophys. Res. Commun., 36 (I969) 2o3-2o8. 4 CONe;OR, A. D., AND H. Q. ST~VENSgN, A correlation of seedling height and chromosomal damage in irradiated barley seeds, Radiation Pot., 9 (1969) I-I4. 5 DAVIES, D. R., AND E. T. WALL, The influence of oxygen and temperature on the radiosensitivity of soaked barley seeds, Intern. J. Radiation Biol., 2 (196o) 26~-267. 6 DOMON, M., AND A. M. RAUTH, Effects of caffeine on ultraviolet-irradiated mouse L cells, Radiation Res., 39 (~969) 2o7-221. 7 KIHLMAN, B. A., S. STURELID, B, HARTLEY-AsPAND I':. NILSBON, Caffeine potentiation of the chromosome damage produced in bean root tips and in Chinese hamster cells by various cheniical and physical agents, Mutation Res., 17 (1973) 271-275. 8 I{IHLMAN, B. A., S. STURIBLID, B. HARTLEY-ASPAND I~, NILSSON, The enhancement by caffeine of the frequencies of chromosomal aberra~:ions iuduced in plant and animal cells by chemical and physical agents, Mutation Res., 26 (t974) IO5-I22. 9 Ross, W. C. J., Biological Alkylating Agents, Butterworth, London, 1962. IO I~UPP, ~V'. D., AND P. HOWARD-FLANDERS, Discontinuitics in the DNA synthesized in an exeision~defective strain of E, coli following ultraviolet irradiation, J, Mol, Biol., 31 (I968) 29I-3O 4. II SWlETLINSKA, Z., High frequency of chrollXOsome aberrations induced by DEB with caffeine posttreatment in Viciafaba var. minor, Mol. Gen. Goner., 112 (197I) 87-9o. 12 Ts6, P. O. P., G. 1,2. HIgLMKAMPAND C. SANDER, Interaction of nucleosides and related compounds with nucleic acids as indicated by the change of helix-coil transition temperature, P~,oc. Natl. Acad. Sci. (U.S.), 48 (1962) 686-698. 13 "WALXEILI. G., AND B. D. REID, Caffeine potentiation of the lethal action of alkylating agents on L-cells, Mutation Res., 12 (197 I) i o i - i o , t. 14 WITKIN, E. M., Post-lrradiatlon metabolism and the timing o[ ultraviolet-induced mutations in bacteria, Proc. zoth Intern. Congr, Genet., I (1958) 28o-e99. 15 WOLFF, S., AND D. SCOTT, Repair of radiatlon-induced damage to chromosomes, Exptl. Cell Res., 55 (I969) 9-I6. 16 ¥AMAMOT0, I':., AND H. YAMAalJCHI, Inhibition by caffeine of the repair of v-ray-induced chromosome breaks in barley, Mutation Res., 8 (I969) 428-43o.