The effect of cupferron on recovery from radiation damage in Tradescantia microspores

Radiation Botany, 1970, Vol. 10, pp. 191 to 198. Pergamon Press. Printed in Great Britain.

THE EFFECT OF CUPFERRON ON RECOVERY F R O M R A D I A T I O N DAMAGE IN TRADESCANTIA MICROSPORES* H E I ~ E R T L. DAVIS, JR. Emory University, Department of Biology, Atlanta, Georgia 30322, U.S.A.

(Received t5 October 1969) A b s t r a e t - - C u p f e r r o n was used to determine its effect on the frequency of X-ray induced chromosomal aberrations in microspores of Tradescantia paludosa. Inflorescences were given pre- and post-radiation treatment in various concentrations of cupferron in conjunction with a total dose of 400 rads of X-radiation delivered in a helium atmosphere. Concentrations of cupferron of 10-~ molar or greater proved to be toxic but did not produce any radiomimetic effects in the form of chromosomal aberrations. Pre-treatment or post-treatment with cupferron resulted in a reduction of the number of chromosomal aberrations. Treatment with cupferron in a helium atmosphere demonstrated that its effectiveness in reducing chromosomal aberrations decreased as the period of anoxia increased. The effects of cupferron on cellular respiration are discussed along with the recovery system in Tradescantia mierospores. R6sLma~---On d~termine dam les microspores de Tradescantiapaludosa, les effets du cupferron sur la frdquence des 16sions chromosomiques induites par les rayons X. Les inflorescences sont trait~es par diverses concentrations de cupferron avant ou apr~s irradiation. La dose totale de rayons X est de 400 rads; elle est d~livr6e sons atmosphere d'helium. Des concentrations de cupferron sup6rieures ou dgales ~t 10-~ molaires sont toxiques mais ne produisent pas d'effets radiomim~tiques, tels que des 1Csions de chromosomes. On note une diminution du nombre de l~sions apr~s prdtraitement par le cupferron. U n traitement par le cupferron sous atmosphere d'helium d~montre que son efficacitd marqude par la diminution du hombre de Idsions ddcrolt en m~me temps que la dur~e de l'anoxie augmente. Les effets du cupferron sur la respiration cellulaire sont discutOs de m~me que le syst~me de restauration des microspores de Tradeseantia.

Zusammenfassung--Die Wirkung

von Cupferron auf die durch R6ntgenstrahlen induzierten chromosomalen Aberrationen wurde bei Mikrosporen yon Tradescantia paludosa untersucht. Infloreszenzen wurden mit verschiedenen Konzentrationen Cupferron vor und nach Bestrahlung in Heliumatmosph~ire mit einer Gesamt-Dosis yon 400 tad R6ntgenstrahlen behandelt. I0 -~ molare mad h6here Konzentrationen an Cupferron erwiesen sich als toxisch, hatten aber keine radiomimetischen Wirkungen in Form yon Chromosomenaberrationen. Vor- und Nachbehandlung mit Cupferron bewirkten eine Reduktion chromosomaler Aberrationen. Die Behandlung mit Cupferron in Heliumatmosph~ire zeigte, dass seine Wirksamkeit beztiglich der Reduktion chromosomaler Aberrationen mit zunehmend l~ingerer Anoxie abnimmt. Die Wirkungen von Cupferron aufdie zellul~ire Atmung werden im Zusammenhang. mit dem System der Erholung der Mikrosporen yon Tradescantia diskutiert. *Acknowledgement is given to the National Science Foundation for support as an NSF Science Faculty Fellow and to the Atomic Energy Commission for the use of equipment purchased under contract No. AT-(40-1)-2669. 191

HERBERT L. DAVIS, JR.

192 INTRODUCTION

THE SUGGESTION by WOLFF and LUIPPOLD(13) and BEATTY et al.¢4) that energy derived from oxidative metabolism will reduce the number of X-ray induced chromosomal aberrations has led to numerous investigations of the effects of various agents which affect cell metabolism. This suggestion was substantiated by WOLFF and LUIPPOLD(14) and BEATT'¢ and BEATTY(2) using exogenous A T P to reduce the number of chromosome aberrations. Cupferron (the a m m o n i u m salt of N-nitrosophenylhydroxylamine) is a chelating agent and has been demonstrated to affect cellular respiration.~L8,10,12) LERNER02) showed that if injected directly into leaf tissue, cupferron would non-specifically inhibit respiration. However, if it were translocated through the petioles of these leaves it would stimulate respiration. KIHLMAN(7) working with Vicia seedling roottips, demonstrated that cupferron would inhibit respiration when applied in solution directly to the roottip and, if oxygen were present, would enhance the effectiveness of X-rays in inducing aberrations. The fact that cupferron will either stimulate or inhibit respiration and therefore could effect the number of chromosome aberrations induced by X-radiation suggested the present problem. The question is raised of how cupferron might affect initial damage or recovery when radiation is used to induce chromosomal aberrations in the Tradescantia microspore system. The experiments reported here were conducted in an effort to distinguish between stimulation and inhibition of respiration by studying the effect of cupferron on the production of chromosomal aberrations. Secondly, it was hoped to gain a better understanding of the recovery system operating in Tradescantia microspores by affecting the system rather than by supplying exogenous metabolites.

MATERIALS A N D M E T H O D S

Inflorescences of Tradescantia paludosa (Sax clone 5) were treated by immersing the stems in solutions of cupferron to determine its effects on frequencies of radiation-induced chromosomal aberrations observed at the first microspore division. Various concentrations of eupferron,

variation of treatment duration, and pre- and post-irradiation treatments were employed to distinguish the most effective treatment and to differentiate between modification of initial damage and the recovery process. X-radiation was delivered in the dark in a helium atmosphere at an intensity of 50 rads per rain for a total dose of 400 rads. All inflorescences were allowed to remain in the helium atmosphere for a period of 15 min following the irradiation unless post-treatment was involved, in which case the period in helium corresponded to the post-treatment time. Control experiments using each of the treatment components or modifications separately (i.e. cupferron alone or radiation alone), were conducted to establish that cupferron was responsible for the observed change in aberration frequencies. Following treatment and irradiation the inflorescences were maintained in moist chambers in a greenhouse for 96 hr, after which acetocarmine squashes were made of microspores in division stages of the first microspore division. The slides were sealed with dental wax and, after the stain had intensified sufficiently, metaphase figures were analysed and scored for two-hit chromosome aberrations of the centric ring, dicentric and tricentric types. These exchanges are expressed in terms of average number per cell with standard errors calculated after the method of CATCHESIDE, LEA and THODAY.(5) Unless otherwise noted, five hundred metaphase figures were examined for each of the experiments. Possible radiomimetic effects of cupferron were tested using solutions of 10 -1 , 10 -2 , 10 -3 and 10 -4 molar for 90 min treatment time with no irradiation. These same experiments were used to observe any toxic effects on the inflorescences or microspores. Deletions as well as two-hit aberrations were scored. The effects of various concentrations of cupferron in combination with radiation were determined by treating inflorescences for 90 rain at 30°C in solutions of 10 -1, 10 -3, 10 -3, 10 -4, 10 -s and 10 -n molar prior to irradiation in helium. A 10 -z molar solution was chosen as the optimum concentration to be used in further experiments with the exception of the post-

THE EFFECT OF CUPFERRON ON RECOVERY FROM RADIATION DAMAGE treatment experiments. This concentration produced no observable toxic effects and resulted in the maximum reduction of the number of chromosomal aberrations. This concentration, 10-3 molar, was used for pre-treatment periods of 15, 30, 45, 90, 120 and 240 min to determine the length of time required to produce the maximum effect and to determine if there were any decrease in the maximum effect with prolonged treatment. The inflorescences from these treatments were subsequently irradiated in helium. This same concentration was also used in the series of experiments to determine the residual effects of cupferron treatment. The inflorescences were treated in 10-3 molar cupferron for a period of 90 min and then placed in distilled water for periods of 15, 30, 60 and 90 min prior to irradiation in helium. Post-treatment with cupferron following irradiation was used to differentiate between postirradiation and post-irradiation effects. This series consisted of post-treatment in 10-2 , 10-3 and 10-4 molar solutions for 90 rain and one 45 min post-treatment in 10-3 molar cupferron to determine the post-irradiation effects of both concentration and time. All post-treatments were in helium as well as a control experiment which was allowed to remain for 90 min in helium following irradiation. Information from the preceding experiments indicated that the presence of oxygen or perhaps the duration of anoxia could be involved in the effect of cupferron. To test this further, experiments were conducted using 10 -3 molar solutions for 15, 30, 60 and 90 min periods in a helium atmosphere. The inflorescences were treated and irradiated in darkness. As a control one group ofinflorescences was placed in helium without cupferron for a period of 90 min prior to irradiation in helium. All experiments of this series remained in helium and cupferron during the 8 min of irradiation and a 15 rain post-irradiation period. This makes a total of 23 additional min of anoxia to be added to the treatment time. OBSERVATIONS

AND RESULTS

The experiments to test for possible radiomimetic effects and toxicity of cupferron indicated that there was no radiomimetic effect at

193

the concentrations tested. These results (experiments 1---4, Table 1) show that cupferron does Table 1. Results of investigations of a possible radiomimetic effect of treatment for 90 min in air at 30°C using cupferron in various concentrations

Expt. no.

Molar cone.

No. cells counted

No. interchanges

1

10-1 10-2 10-a 10-4

200 250 500 500

none none none none

2 3 4

not produce chromosomal aberrations when applied in concentrations ranging from 10 -1 to 10 -4 molar. Observations of toxicity, however, show a definite effect of the higher concentrations. Additional experiments which were not scored for chromosomal aberrations show that at concentrations ranging from 10-1 to 2 molar cupferron was toxic as evidenced by extreme loss of turgor, bleaching of leaves at the base and along the midrib, and a high degree of sterility of the microspores. The sterility was particularly pronounced in post-metaphase stage of the first microspore divisions in inflorescences treated with 10-1 and 10-2 molar, and complete sterility was seen of all stages except pre-meiotic in concentrations above 10 -x molar. The use ofcupferron in pre- or post-irradiation treatments resulted in fewer chromosomal aberrations than were observed in control experiments receiving only irradiation in helium. The results of experiments using various concentrations of cupferron during a 90 min pretreatment time (experiments 5-11) prior to irradiation are presented in Table 2, along with results from experiments involving various pre-treatment times in 10-3 molar cupferron solutions (experiments 12-17). Experiment 5 with an aberration frequency of 0-24-[-0"01 served as a control for these experiments. The most effective concentration in reducing chromosome exchanges was 10-3 molar which resulted in 0.15-t-0.02 aberrations per cell compared to the control value of 0"24±0-01 for those inflorescences which had been irradiated but received no cupferron treatment. Treatment

HERBERT L. DAVIS, JR.

194

Table 2. The effects of various concentrations of cupferron and various treatment times on the frequency of two-hit chromosomal aberrations produced by 400 fads of X-rays given in helium Expt. no.

Molar conc.

5 (control)

none

none

6

I0 - I

90 mln

10.3

90 mm 90 mm 90 mm 90 mm 90 mm 15 mm 30 mm 45 mm 90 mm 120 mm 240 mm

7 8 9 I0 11 12 13 14 15 16 17

10 - s 10-4 10 -5 10 -6

10-s 10-s I0 -s 10 -3 10 -s 10 -3

Pre-treatment time

with 10 .3 m o l a r solutions p r i o r to i r r a d i a t i o n i n d i c a t e d t h a t cupferron was effective when a p p l i e d for as short a p e r i o d of time as 15 rain, a n d no d i m i n i s h i n g of this effect was a p p a r e n t after p r e - t r e a t m e n t times o f 240 rain. T h e residual effects of p r e - t r e a t m e n t in 10 -s m o l a r cupferron for 90 m i n followed b y p l a c i n g the inflorescences in w a t e r for various periods of t i m e p r i o r to i r r a d i a t i o n are presented in T a b l e 3 (experiments 27-31). These results show t h a t the cupferron effect is r a p i d l y lost in the inflorescences as e v i d e n c e d b y the fact t h a t there are no significant differences in e x c h a n g e frequencies from inflorescences p l a c e d in w a t e r for periods of 15-90 m i n following cupferron t r e a t m e n t a n d the control v a l u e o f 0"22-[-0"02 from inflorescences p l a c e d in w a t e r for 90 rain w i t h no cupferron p r i o r to i r r a d i a t i o n .

No. cells counted 1000 500 500 500 500 500 500 500 500 500 500 500 500

No. interchanges 0.244-0.01 0.164-0.02 0.16 4- 0.02 0.15 4-0.02 0.19 4- 0.02 0.194-0-02 0" 19 4- 0.02 0" 17 4-0.02 0" 15 4-0.02 0" 15 4-0.02 0.15 4-0.02 0" 15 4- 0"02 0-18 4- 0-02

T h e s e p a r a t i o n of pre- a n d pos~-irradiation effects o f cupferron as tested b y p o s t - t r e a t m e n t in various concentrations i n d i c a t e d initially t h a t cupferron was not effective w h e n a p p l i e d postr a d i a t i v e l y ( T a b l e 4, e x p e r i m e n t s 18-21), b u t a n a d d i t i o n a l e x p e r i m e n t ( e x p e r i m e n t 46) in w h i c h 10 -z m o l a r cupferron was a p p l i e d in h e l i u m for a p e r i o d o f 45 rain p o s t - t r e a t m e n t shows a definite p o s t - t r e a t m e n t effect. T h i s suggested t h a t the d u r a t i o n o f a n o x i a c o m b i n e d w i t h cupferron t r e a t m e n t was responsible for the lack o f a p o s t - t r e a t m e n t effect w i t h l o n g e r t r e a t m e n t times. T h i s was clearly shown in the results o f the experiments in w h i c h p r e - t r e a t m e n t w i t h 10 -3 m o l a r cupferron took p l a c e in h e l i u m for various periods of time followed b y i r r a d i a t i o n plus a n a d d i t i o n a l 15 rain postt r e a t m e n t in h e l i u m with the cupferron b e i n g

Table 3. Results of investigations of pre-treatmentfor 90 rain in 10-3 molar cupferronfollowed by placing the inflorescences in water for various periods of time prior to irradiation in helium Expt. no.

Time in water

No. cells counted

27 (control) 28 29 30 31

90 rain (water only) 15 min 30 min 60 rain 90 min

500 500 500 500 500

No. interchanges 0.22 4-0"02 0"22 4-0"02 0-20 4-0.02 0-21 4-0-02 0"22 4-0.02

THE EFFECT OF CUPFERRON ON RECOVERY FROM RADIATION DAMAGE

195

Table 4. Post-treatmenteffects of various concentrations of atpferron administered in helium on the frequency of two-hit chromosomalaberrationsprodueed by 400 fads of X-rays in helium

Expt. no.

Molar cone.

Time of treatment

No. cells counted

18 (control)

none

19

I 0-3

20 21 46

10-3 10- 4 10-3

90 min 90 min 90 min 90 rain 45 min

400 500 500 500 500

No. interchanges 0.25 ±0"02 0"26 q- 0"02 0.25 4-0"02 0.24 4-0.02 0"18-4-0"02

Table5. The effectsofpre-treatmentfor variousperiods of time in l O-3 molar solutions of cupferron in a helium atmosphereprior to irradiation with 400 rads of X-rays in helium

Expt. no.

Time of treatment

No. cells counted

22 (control) 23 24 25 26

90 min with no Cupferron 15 min 30 min 60 min 90 min

500 500 500 500 500

present for the duration of the experiment. These results are presented in Table 5 (experiments 22-26) as exchanges per cell with pretreatment time in anoxia. All experiments had an additional 23 min of anoxia beyond the pre-treatment time. This includes the 8 min during which radiation is administered and a 15 min post-irradiation period. T h e effects of the pre-treatment in cupferron with anoxia gradually decrease as the duration of anoxia increases. Pre-treatment times of 15 and 30 min produce a definite effect, 0.154-0.02 and 0"194-0-02 respectively, compared to the control frequency of 0.244-0.02 from inflorescences which were placed in helium 90 min prior to irradiation. With pre-treatment times of 60 and 90 min, the effectiveness of 10 -8 molar cupferron in reducing the n u m b e r of chromosome aberrations was no longer apparent. DISCUSSION

T r e a t m e n t with cupferron concentrations which are non-toxic reduces the frequency of X-rays induced chromosomal aberrations. This effect of cupferron is rapidly lost from the

No. interchanges 0"24t0.02 0.15±0.02 0"194-0-02 0.25 -4-0-02 0-23 +0.02

microspore and is dependent upon the duration of anoxia when cupferron is used in a helium atmosphere. The effectiveness ofcupferron in reducing the number of chromosomal aberrations produced by X-rays is very likely due to a stimulating effect on respiration. The requirement for energy in the rejoining of broken chromosomes has long been known. LERNER(12) has reported that cupferron will stimulate respiration when translocated through the petioles of leaves, but when injected directly into the leaf tissue, respiration is non-specifically inhibited. Similar inhibition has been reported in root tips of Vida seedlings by KII-ILMAN(L8)in conjunction with an analysis of chromatid aberrations produced by X-rays. Results of the present experiments can be taken to support the hypothesis of a stimulatory effect of cupferron on respiration when it is applied through the stems of inflorescences. It m a y be that some alteration or degradation of cupferron is occurring and that the effective agent in reducing chromosomal aberrations is not the intact cupferron molecule but some product.

196

HERBERT L. DAVIS, JR,

The majority of experiments involved the use of 10 -8 molar concentrations of cupferron. The results of the experiments to determine the toxicity and possible radiomimetic effects of cupferron show that this concentration is not toxic by the standards used and no radiomimetic effects were observed. Radiomimetic effects of cupferron have been reported by KIHLMAN(a,g) in which case chromatid aberrations were produced. This difference in results is understandable since only chromosomal aberrations were studied in the present series of experiments and cupferron did not result in the production of any chromosomal aberrations in these experiments. Most radiomimetic agents so far studied produce chromatid aberrations since the most sensitive portion of the cell cycle is during D N A replication. The very slight increase in aberrations with 240 rain treatment, although not statistically significant, may well be due to some inhibition of respiration by the intact cupferron molecule. The residual effects of cupferron are of very short duration as no reduction of aberration frequency is produced when inflorescences are placed in water for as little as 15 min following 90 rain pre-treatment in 10 -a molar solutions. I f as is suggested, cupferron is being degraded, the effective component is either subject to rapid leaching or is rapidly metabolized. Initial results of post-treatment with various concentrations of cupferron for 90 min were misleading in that they suggested that the effect of cupferron is on initial damage only. Posttreatment for 45 min, however, resulted in a reduction of chromosomal aberrations. This finding suggested the experiments in which pre-treatment with cupferron took place in helium. The results of these experiments show that the duration ofanoxia governs the effectiveness of eupferron. T h e agreement of results from the experiment using 15 min pre-treatment in helium plus the additional 23 min of anoxia (8 min irradiation, 15 min post-radiation period), with the results from the experiment using 45 rain post-treatment in helium, shows that there is no difference in this case between pretreatment and post-treatment. This could be true only if the cupferron is acting on recovery rather than on initial damage.

BEATTY and BEATTY(1) have reported a gradual increase in the number of aberrations when inflorescences are kept in helium for periods of 0-534 min prior to irradiation in helium. They found little variation from 0 to 80 min pre-treatment, but a rather sharp increase after 133 min. This indicates that the endogenous energy supply in the microspore is rather constant or does not drop below a critical level for periods of up to 133 rain. T h e y later confirmed this( s) in their work with labeled ATP. Thus results of the present experiments in which pre-treatment in helium was limited to 90 min should not be affected significantly by endogenous variation and the decrease in aberrations when cupferron is present is an effect of the cupferron. The elimination of a cupferron effect when pre- or post-treatments are applied with prolonged anoxia demonstrated that "cupferron is acting on an aerobic system. Agents which stimulate respiration m a y act to supply additional energy required for the recovery of broken chromosomes. This requirement for additional energy has been demonstrated by BEATTY and BEATTY(3) in a series of experiments in which they showed that in irradiated anthers there was an uptake of exogenous A T P into the anther above that of non-irradiated controls. T h e y suggested that under stress of radiation more A T P was used. In the same work they demonstrated that the A T P content of the periplasmodium was increased by exogenous treatment while that of the microspore was not. A possible explanation for other differences in the effects observed with the use of cupferron in Tradescantia and those observed by K i h l m a n in Vicia can be offered by noting the difference in the two types of tissue used and the method of treatment. T h e Tradescantia system involves treatment of entire inflorescences and cupferron must be translocated up through the stem and through the anther tissue into the locule before reaching the microspores. Thus it m a y be subject to degradation by the tissue through which it must pass. Vicia seedling root tips on the other hand, are immersed directly into the cupferron solutions and due to the intercellular spaces in the young root, virtually every cell is in direct contact with the cupferron solution.

THE EFFECT OF (3UPFERRON ON RECOVERY FROM RADIATION DAMAGE This means that they would be subject to the more direct action of cupferron. The reduction of chromosomal aberrations observed with pre-treatment in cupferron was unexpected in view of the results obtained by KIItLMAN et al.,Oo) who reported that cupferron enhanced the effectiveness of X-rays in producing chromatid aberrations in Vicia seedling root tips. He subsequently foundO) that this enhancement was indirect and was due to an oxygen effect when cupferron was used with low oxygen tension. This he attributed to inhibition of respiration and an oxygen effect on initial damage. The present experiments in which a reduction of chromosome aberrations was observed, involved inflorescences irradiated in helium, thus eliminating an initial oxygen effect. Production of a cupferron effect requires that oxygen be present at a time other than during irradiation. This is demonstrated by the effects of the duration of anoxia in conjunction with cupferron treatment, with prolonged anoxia resulting in a reduction and final elimination of the cupferron effect. T h e pre-treatment experiments with cupferron demonstrate that I0 -s molar solutions produce the m a x i m u m reduction in chromosome aberrations, and that cupferron is effective when applied for as little as 15 min with only a slight loss in effectiveness when pre-treatment is for as long as 240 min. The m a x i m u m reduction is realized with 30 rain pre-treatment. Cupferron apparently is producing no toxic effects as there is no appreciable change in the frequency of chromosomal aberrations when treatment is prolonged to 240 rain. The nutritive function of the periplasmodium demonstrated by the Beattys greatly increases the complexity of the microspore system for analysis and interpretation of radiation recovery, and suggests a possible explanation of the results of the present experiments. Cupferron acting as a stimulant of cellular respiration m a y result in the production of additional energy above that normally required by the cell. This additional energy could be used in recovery from radiation stress. In the case of the microspore and its apparent dependence on the periplasmodium for some nutritive function, the periplasmodium likely acts as an energy reservoir which is

197

reduced under anoxic conditions. Agents such as cupferron could provide additional energy to the microspore system as long as the level of energy in the periplasmodium is greater than that of the microspore and recovery would bc enhanced. On the other hand, after the reduction of the energy reservoir of the periplasmodium, stimulation of respiration would be meeting an energy deficit and the effect on recovery would be reduced and finally eliminated depending upon the duration of the anoxia. REFERENCES

I. BEATTY A. V. and BEATTYJ. W. (1957) Chromosome breakage and rejoining in Tradescantia microspores. Am. 07. Botany 44, 778-783. 2. BEATTY A. g. and BEATTYJ. W. (1960) Potassium gluconatc and A T P effects on chromosome aberration yield. Proc. Natl Acad. Sci. U.S. 46, 1488-1492. 3. BEATTY A. g. and BEATTYJ. W. (1966) Assay for adcnosine triphosphatc in X-irradiated Trades-

¢antia anthers. Radiation Res. 27, 347-354. 4. BEATTV A. V., BEATT~J. W. and COLLXNS(2. (1956) Effects of various intensities of X-radiation on chromosomal aberrations. Am. 07. Botany 43, 328-332. 5. CATCHESIDE C. G., LEA D. E. and THODAYJ. M. (1946b) Thc production of chromosome structural changes in Tradescantia microspores in relation to dosage, intensity and temperature. 07. Genet. 47, I13-136. 6. KIHLMAN B. A. (1957) Experimentally induced chromosome aberrations in plants. I. The production of chromosome aberrations by cyanide and other heavy metal complexing agcnts. 07.

Biophys. Biochem. Cytol. 3, 363-380. 7. KIHLMAN B. A. (1958) The effect of oxygen, nitric oxide and respiratory inhibition on the production of chromosome aberrations by Xrays. Exptl Cell Res. 14, 639-642. 8. KmLMAN B. A. (1958) Respiration and radiosensitivity of broad bean roots. Nature 182, 730-73 I. 9. KXHLMANB. A. (1959) On the radiomimetic effects of cupferron and potassium cyanide. 07. Biophys. Biochem. Cytol. 5, 351-353. 10. KIHLMANB. A. (1959) The effect of respitatory inhibitors and chelating agents on the frequencies of chromosome aberrations produced by X-rays in Vicia. 07. Biophys. Biochem. Cytol. 5, 481--490.

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HERBERT L. DAVIS, JR.

11. KIHLMAN B. A., MERZ T. and SWANSON(~. P. (1957) Experimentally induced chromosome aberrations in plants. II. The effect of cyanide and other heavy metal complexing agents on the production of chromosome aberrations by X-rays. 07. Biophys. Biochem. Cytol. 3~ 381-390. 12. LER~raRN. H. (1954) Polyphenoloxidase and the respiration by ivy leaves, ft. Exptl Botany 59 79-90.

13. WOLFF S. and LUIPFOLD H. E. (1955) Metabolism and chromosome-break rejoining. Science 1229 231-232. 14. WOLFF S. and LmPPOLD H. E. (1956) The biochemical aspects of chromosome rejoining, pp. 217-221. h~ J. S. MITCHELL, B. E. HOLMES and C. L. SMITH (eds.), Progress in Radiobiology. Oliver and Boyd, Edinburgh.