INDUCTION 0F CHROMOSOME DAMAGE BY ETHYLENIMINES AND RELATED COMPOUNDS Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY I THAMBY NINAN ADANGAPURAM 1967 ABSTRACT INDUCTION OF CHROMOSOME DAMAGE BY ETHYLENIMINES AND RELATED COMPOUNDS by Thamby Ninan Adangapuram A number of ethylenimines and related compounds were employed to induce chromosomal aberrations in the primary root meristems of 1;— cia faba and Pisum sativum. The chromosomal damage was then deter- mined quantitatively by scoring anaphase damage in Pisum, and meta— phase damage in yigia over an extended period of time, consisting of several mitotic cycles. An attempt was made to determine the suscep- tible stage of the mitotic cycle at which these agents exert their in— fluence in causing chromosome damage and cycle time delay. These studies were aimed towards gaining a better understanding of the type of disruption of the mitotic cycle by this group of drugs, as well as an understanding of the structural organization of the mitotic chromo— somes. The comparative mutagenic ability of some of the chemicals were tested on Drosophila melanogaster. Treatment of the root meristems of Migia and Ei§3m_was accom- plished by dissolving the chemicals in an aqueous nutrient medium used to culture the pea roots under standard conditions. By microscopical examination of the root meristems, analysis were made of the type of damage induced as Well as the amount of damage. The relative muta— Thamby Ninan Adangapuram genic ability of some of the chemicals were tested by using the Muller 5 system (to detect recessive lethal X chromosome mutants). The two chemicals studied in detail were apholate and metepa. The results ob- tained are concerned mainly with: l. delayed effects 2. persistence of effects. In all of the experiments using metepa and apholate on peas, a maximum effect did not appear until after a period of time equal to one or two complete mitotic cycles. Hence, these are categorized as delayed effects. This delay may be attributed to unusual delay in damaged cells reaching division, or may have to do with the initial unit of breakage. This delay may be explained if we assume that % chromatids become chromatids in the next division. However, there are some strong objections to these assumptions. Persistence of effect may have to do with the number of subunits that make up the chromosomes and the type of assortment of the centromeres, or a combination of both. The time of susceptibility was tested using the 5 amino-uracil system. we are forced to the conclusion that no particular stage is especially sus- ceptible to the chemicals, although the possibibility exists that they may be retained in sufficient concentration to cause maximum dam- age at a critical period in the cycle. In comparing the chemical damage to X-ray damage, it may be seen that these two agents differ in: Thamby Ninan Adangapuram l. the types of damage induced 2. the time of maximum damage 3. the susceptible stage where the damage occurs. These chemicals thus cannot be called radiomimetic in a strict sense. INDUCTION OF CHROMOSOME DAMAGE BY ETHYLENIMINES AND RELATED COMPOUNDS By Thamby Ninan Adangapuram A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1967 To my father and mother ii ACKNOWLEDGMENTS I wish to express my deep sense of gratitude to Professor G. B. Wilson for his inspiring guidance and encouragement. My thanks go to Dr. R. Hoopingarner for his help and ad- vice, and to Dr. J. Van't Hof for doing the X-ray standards in EiéEfl.3t the Brookhaven National Laboratory, and for supplying the Vicia seeds used in this study. I would also like to ex- press my sincere appreciation to Drs. I. Knobloch and'W. B. Drew for their helpful advice. To the cytology group at the Biology Research Center, I offer Special thanks for many late hours, hard work, and co- operation given during this investigation. Thanks also go to Mrs. Joanna Alcorn and Mrs. Doris Brooker for their assistance in typing part of the manuscript. To my wife, Janice, for understanding, encouragement, and willing assistence at all times, and for typing the manuscript and final c0py of this thesis. Final thanks go to the National Institute of Health and to the M. S. U. Agricultural Experiment Station for providing the funds which made this investigation possible. iii TABLE OF CONTENTS _ ,Page DEDICATION............... ........ . ........ .. ............ E. ii ACKNOWLEDGMENTS. .......... . ..... . ............... . ....... . iii LIST OF FIGURES, PLATES, AND CHARTS ........ . ........... vi LIST OF APPENDIX TABLES....... ........................... ix INTRODUCTION. ...... ... ........ . ......... . ........... ..... 1 LITERATURE REVIEW. ........ . .............. ......... u METHODS.... ....... ..... .............. .... ..... .......... 27. A. Cytological Experimental System ................. 27 B. Genetic Experimental System ..................... q 3H C. Retention of the Chemical ....................... 35 A. AZirane ......................................... 37 B. Ethylene Sulfide ................................ 37 C. Tretamine, or TEM ........................... a... NO D. Aphoxide, or TEPA .......................... . ..... HO E. Hexamethylphosphoric triamide .................. . Ml F. Apholate.... .................................... . Ml G. Metepa .......................................... #7 DISCUSSION ............................................... 67 A. Time of Maximum Damage............ .............. 69 iv DISCUSSION, continued B. Persistence of effect ........................... C. Time of Susceptibility .......................... D. Order of Rearrangements ........ . ................ E. Comparison of Effect ............................ 1. Chromosomal aberrations ..................... 2. Mitotic inhibition .......................... 3. Cycle time delay ............................ H. Mutagenic ability ........................... 5. Comparison with X-ray damage ................ SUMMARY .................................................. LITERATURE CITED ......................................... APPENDIX ................................................. Page 72 75 77 81 81 82 82 83 83 85 87 99 LIST OF FIGURES, PLATES, AND CHARTS Figure l. Azirane damage in which the percentage of anaphase damage is plotted against time in hours.........:. 2. Anaphase damage in Pisum sativum, caused by 3 con- centrations of tretamine, plotted against time in hours ............................................. 3. Chromosome damage caused by 3.1 X 10'2 M apholate. Percent anaphase damage vs. time Shows a 3rd cycle maximum and persistent effect ..................... A. Dose effect curve using apholate. Percent anaphase damage vs. dose in ppm ....................... ..... 5. Cycle time delay caused by apholate treatment on Pisum sativum. Percent polyploidy is plotted against time ...................................... 6. Mitotic index change after 8 hours of 150 ppm of 5 A U on Pisum ...................................... 7. Metepa damage in Pisum sativum. Percent anaphase damage vs. time shows 37 hour (3rd cycle) maximum effect as well as 75 hour;(6th cycle) 2nd maximum effect ............................................ 8. Metepa damage in Pisum sativum. Percent anaphase damage plotted against time shows 27 hour (2nd cycle) maximum effect which is nonpersistent ...... 9. Metepa damage in Pisum sativum. Percent anaphase damage vs. time, 27 hour (2nd cycle) maximum effect which is nonpersistent is obtained ........ lO. Dose effect curve using metepa in Pisum sativum. Per— cent anaphase damage is plotted against dose. In this graph, the results oftwo different experi— ments are plotted ....... .... ...................... vi Page 38 39 H2 “3 45 46 #8 SO 51 52 Figure 11. Cycle time delay caused by metepa treatment on Pisum sativum. Percent polyploidy is plotted against time in hours ................................ .... 12. X-ray damage (250 r) at anaphase in percent vs. time in hours in Pisum sativum ........................ 13. Total metaphase damage in Vicia induced by 0.233 X 10‘2 M metepa. Percent damage vs. time .......... 1h. Chromatid and chromosome d mage in Vicia at metaphase induced by 0.233 X 10' M metepa treatment. Per— cent of damage is plotted against time ........... 15. Percent poluploidy vs. time in Vicia treated with 0.233 X 10'2 M metepa, Showing a delay of 5-6 hours for the lst cycle and no delay for the se— cond cycle after % hour treatment ................ 16a. Diagram of the mitotic cycle in pea root meristems... 16. Model showing half-DNA break in two-stranded chromo- some with random distribution of potential chro- matids ........................................... 17. Dose effect curve, maximum apholate damage in per- cent vs. dose on log scale ....................... 18. Metepa damage plotted against dose on log scale ...... l9. Dose effect curve in which the maximum anaphase dame age in percent is plotted against the correspond- ing dose on log scale (metepa treatment) ......... Plates 1. Karyotypes of (l) Vicia faba, and (2) Pisum sativum ..... 2. Different types of aberrations induced by metepa treat— ment on Vicia faba ................................ 3. Aberrations induced by metepa treatment on Vicia faba.. A. Chromosome damage induced by tretamine and apholate on Pisum sativum ..................................... vii Page 53 55 56 62 6A 68 71 78 79 80 99 59 6O 61 CHARTS Page 1. Classification of chromosome and chromatid aberra- tions induced by ethylenimines, and their end results in subsequent cell cycles .......... ... 58 viii LIST OF APPENDIX TABLES Table l. Anaphase damage in percent and mitotic indices at various hours in Pisum induced by azirane ........ 2. Anaphase damage in percent induced by 3.1 X 10"2 M of apholate in Pisum ................................ 3. Anaphase damage in percent and mitotic indices at various hours in Pisum, treated with 3.1 x 10-3 M apholate ......................................... A. Maximum anaphase damage in percent at peak hours in Pisum induced by different concentrations of apho- late ............................................. 5 a. Anaphase damage and M Is at various hours in control Pisum ............................................ b. Anaphase damage in percent and M Is in Pisum treated with 100 ppm of 5 A.U for 8 hours ................ c. Anaphase damage in percent and M Is in Pisum, treated first with 5 A U for 7% hours and imme- diately transferred to 3.1 X 10‘3 M apholate solu tion ............................................. d. Anaphase damage and M Is in Pisum treated with 5 A U for 8 hours and then transferred to 3.1 X 10‘3 M apholate solution at 12% hours.... ....... . ....... e. Anaphase damage in percent and M Is in Pisum,treated with 5 A.U for 8 hours and with 3.1 x 10-2 M apholate at 15 hours ................... . ......... f. Anaphase damage in percent and M IS in Pisum, treated with 5 A.U for 8 hours and with 3.1 X 10-3 M apholate at 10% hours ............................ 6. Anaphase damage in percent and M Is at various hours in Pisum, treated with different concentrations of metepa ........ . ............................... ix Page 100 101 102 103 10H 105 106 107 108 l09 llO Table Page 7. Anaphase damage in percent and M IS at various hours in Pisum, treated with A different concentra- tions of metepa ................................. 112 8. Anaphase damage in percent and M Is at various hours in Pisum after treatment with different concen- trations of metepa... ........................... 11M 9. Anaphase damage in percent induced by 250 r of X-ray treatment in Pisum .............................. 116 10. Percent aberrations at metaphase in Vicia faba, in- duced by 0.233 x 10‘2 M metepa treatment ........ 117 11. Comparison of damage ................................. 118 INTRODUCTION The study of the effects of drugs on cells and their modes of action assumes considerable importance in this "chemical age" since man uses a larger number of drugs for the treatment of disease as well as for the control of pests than ever before. Plants and animals, in- cluding man, are inadvertently exposed to these chemical hazards in their environment. In our search for more effective drugs in combat- ing constantly changing hordes of pathogens and pests, we are unleash- ing chemicals whose long—term effects are only poorly understood. In selecting drugs for these purposes, it is desirable to gain knowledge concerning the potential metabolic target system, the alterations which the drug is capable of inducing in the system, and the concommi- tant metabolic lesions that may manifest themselves as gross or submi- crosc0pic abnormalities which may be deleterious to the survival of affected organisms, including man. The present study is concerned with a group of chemicals known as ethylenimines which are being tested, extensively elsewhere as chemo— sterilants in controlling insect pests. Ever since the discovery of induced chromosome breakage by nitrogen mustards by Auerbach and Robson (1947), a number of investigators have discovered a wide array of chemi- cals that could induce chromosome breakage and cause gene mutations. Ethylenimines are one of this group of chemicals which are capable of inducing the aboveementioned alterations. Most of the investigations 2 with these chemicals, which are also alkylating agents, were performed with a View to simply ascertain their chromosome-breaking ability, and to some extent, their mode of interference with the DNA precursor syn- thetic system which they are assumed to impair. No quantitave data are available with regard to their dose effect relationships over an extended period of time, involving several cell generations. Another important aSpect of the study of these chemicals is to ascertain the susceptible stage or stages in the mitotic cycle when these chemicals exert their in fluence. Studies of this sort are generally beset with problems, since our inferences have to be made on extrapolations from data obtained by looking at visible alterations in the mitotic cycle. Knowledge regarding their mode of action and time of action can be gained by using known inhibitors which preferen- tially hold up stages of the mitotic cycle. Studies of this nature can throw considerable light on not only the effects of these chemi- cals on specific stages of the mitotic cycle, but also on the structur- al organization of the chromosome. 8 To sum up, the investigations described here were carried out in order to: A. Ascertain quantitatively how much chromosome damage is produced by different concentrations of several compounds in which the active group is an ethyleni- mine. B. Obtain a clue as to the susceptible stage or stages of the mitotic cycle with regard to the action of the chemicals. C. F. G. 3 Gain knowledge as to the type of chromosomal aberrations induced by these chemicals. Gain information on the persistence of the effect of these chemicals over several mitotic cycles, as well as to elicit information regarding the persistence of the chemical as such within the system, which may induce further aberrations in the mitotic cycle at later stages. Gain information regarding the unit of breakage, and thereby gain an insight into the structural organization of the mitotic chromosome. Compare the chemical damage by these chemicals to known doses of X-ray damage induced in the same system under controlled conditions. To determine the mutagenic ability of some of these chemicals. LITERATURE REVIEW I. Classification of Chemical Effects ‘Wilson (1960) classified the effects of chemicals on cells into four types: mutagenic, fragmenting, carcinogenic, and antimi- totic activity. Biesle (1958) reviewed the general field of anti- mitosis on the basis of susceptible stages of the mitotic cycle, on observable damage induced, on the physiological changes induced, and on the biochemical reactions affected. According to Levan (1952), lethal and toxic reactions, reversible and physiological reactions, and mutagenic reactions are the three types of chemical effects. Other classifications include: grouping for chromosome breakage, perfect or partial radiomimisis, faulty chromosome separation, and prolongation of metaphase (Biesle, 1958). __\ II. Types of Chemicals Which Induce Breaks Ever since the studies of Auerbach (19u6), and those of Ford (1949) on nitrogen mustards, a large number of chemicals have been re- ported which produce chromosomal aberrations. These can.be classified according to a variety of criteria. These classifications can be based on: the chemical nature of the chromosome-breaking agents; the types of effects produced by the chemicals; the fac- tors, both physical and chemical, that influence the production of aberrations by chemicals; and the chemical, physico-chemical, and 5 biochemical properties of chromosomeébreaking agents likely to be re- sponsible for their cytological effects. The first type of classification, based on the chemical nature of the chromosome-breaking agents, will be followed here. All the other criteria mentioned above will be discussed as they relate to each of the different types of chemicals. A. DNA Precursors and Related Compounds 1. Adenine or 6 Aminopurine ”Ha N N.// c —H H‘C C N PA This is one of the bases of DNA, and produces chro- ~mosomal aberrations in such plants as Allium, Pigym, Migig, (Kihl- man, 1952), and in mammalian cells in tissue culture (Biesle, gt, 31., 1952 b). 2X10'2Medenine is required to break plant chromosomes. The effect is of the delayed type. The predominant aberrations are chromatid exchanges and isochromatid breaks. Absence of oxygen appears to enhance these aberrations. The following mechanisms of action are suggested: (1) as a chelating agent (Frieden and Allen, 1958); (2) as an inhibitor of P incorporation into DNA and RNA in 4 ll" 6 Vicia faba, (Odmark and Kihlman, 1965), and (3) as an inhibitor of purine synthesis in ascites tumor cells (Henderson, 1962). 2. Z’Deoxy adenosine (ADR) H . - fi//' H“... " 1* I $H29Tl i A 2' deoxy adenosine used in late interphase at a concen- tration above 3X10'3M produced strong Eragmentation of chromosomes (Kihlman, et. a1., 1963 and Kihlman, 19 3 b) which was a nondelayed effect. The types of aberrations were inly gaps and chromatid breaks. Isochromatid breaks and interchApges were rare, unlike the effects of adenine in which these were pretent, indicating that nor- mal rejoining is inhibited. Chromatid exchanges localized in the nucleolar constriction were predominant when treatment was at 10'3M concentration for 24 hours, followed by recovery for 24 hours. Chro- mosome-type aberrations were absent. Oxidative phosphorylation inhibitors and anoxia re- duced its effects in 21913, The ADR effect in Eigi§_is inhibited by adenosine and thymidine. Reichard, et:_gl:,(l96l) reported that deoxy adenosine inhibits reduction of ribonucleoside phosphate to deoxy ribose nucleosidediphosphates, and this is assumed to be the cause of its chromosome-breaking ability. 7 3. Cytosine arabinoside - CA 1"" C n —H o=c 9—H 7' ‘ (53on l H K H I II PI CA is inactive in Vicia (Kihlman), but nondelayed breaks of the same types as with ADR are produced in human leukocytes with no rejoinings (Kihlman, 1963). Like the previous chemical, CA in- hibits the formation of deoxyribonucleotides (Chu and Fischer, 1962). 4. 5 Fluorodeoxyuridine - FUDR 931 O , €14on A N f! 17/ I TI H l I H on During the first 10 hours after a 1 hour treatment with 10'6M FUDR, gaps, chromatid breaks, and nonunion isochromatid breaks are produced in yiggg, and its effects, thus, are nondelayed (Taylor, et. al., 1962). These effects are reduced by anoxia and in- hibitors of oxidative phosphorylation (Kihlman, 1962) . 104m thymidine or thymidine analogue BUDR negates the action of FUDR. FUDR inhibits mitosis to a great extent. This is indicated by very low mitotic in- 8 dex after 10 hours following treatment with lO‘éM in Vicia, which re- covers after a day or two. The cells after this period still have a low percentage of damage, composed mainly of chromatid exchanges and sister union isochromatid rejoinings. Hsu et. a1. (1964) used FUDR on hamster cells and mouse LM cells in tissue culture, and found that it causes mitotic inhibition and chromosome shattering. They also found that the sensitivity to mitotic inhibition of mice LMlcells'was higher than that of the hamster cells,and their chromosome shattering susceptibilities to FUDR were just the Opposite. It took 100 times as much thymidine as FUDR to reverse the mitotic inhibition and inhibition of DNA synthesis caused by FUDR. Treatment with FUDR during the second half of interphase showed that the chromosomes most seriously frag- mented were the late replicating ones, which suggested a correlation between damage and DNA synthetic activity. Cohen (et. al., 1958) showed that the reaction inhibited by FUDR is the methylation of Deoxy- uridilic acid to thymidylic acid, which is catalysed by the enzyme thy- midylate synthetase. Thus, FUDR resembles the previous two chemicals, not only in its cytological effect, but also in its biochemical action. It is not incorporated into DNA to any appreciable extent. Ahnstrom and Natarajan (1966) hypothesized that the chromosome-breaking effect of ADR and FUDR is due to the reversal of the DNA polymerase reactions caused by the deficiency of deoxyribonucleoside triphosphate. 5. 5 Bromodeoxyuridine ~ BUDR 11 H BUDR increases the susceptibility to X-ray damage of Vicia cells, but by itself does not cause any damage in Vicia (Kihl— man, 1962, 1963 a). Similar results are obtained with 5 chlorodeoxy— uridine and 5 iododeoxyuridine. The effect of BUDR on mammalian cells is delayed, in contrast to that of FUDR (Somers and Hsu, 1962; Hsu, 1963). BUDR has to be incorporated into the DNA to be effective. BUDR preferentially breaks chromosome ends in Chinese hamster cells in tissue culture (Somers and Hsu, 1962), and the rejoining of broken ends is not infrequent. The biochemical effects of BUDR, IUDR, and CUDR are as different from those of FUDR as the cytological effects. FUDR is a deoxyuridine analogue, whereas the other halogenated deoxy- uridines are thymidine analogues. Due to the Van der Waals radii of the halogen substitutes, C12, Brg, and I2 radii correSpond to the methyl group, whereas that of Fluorine has a radius which corresponds more to that of hydrogen (Szybalski, 1962). 6. N—Methylated Oxypurines 10 3- ethoxycafeezne, 1.3.7.9.Tetramethy1uric acid. Kihlman and Levan (1949) described the chromosome- breaking ability of the naturally occuring methylated oxypurines, caffeine, theophylline, and theobromine is A1__:_1.j._u_h_1_ 993g. Synthetic oxypurines, like 8 ethoxycaffeine (EOC) and tetramethyluric acid (TMU) used at 2 to lOXlO'3 M produced nondelayed effects. Exchanges anisister union isochromatid breaks occur with the same relative fre- quency as produced by X—radiation, indicating noninhibition of re- joining. 'Woodard et. a1. (1961) studied the effect of E00 and com- pared it with that of X-rays. E00 and X-rays are similar in inducing subchromatid, chromatid, and probably chromosome ~type aberrations . 0n the other hand, alkylating agents produce only chromatid-type aberra- tions. However, although G2(post-DNA synthesis) cells Show the highest yield of chromatid-type aberrations with both X-rays and ECG; the cells most sensitive to EOC treatment are cells in late G2, whereas those most sensitive to X-ray treatment are mid—G2. Besides, a considerable pro- portion of EOC aberrations are localized in the nucleolar constriction region, and this has led Kihlman (1952 a and 1961 b) to suggest that EOC may be absorbed on the surface of the nucleolus. This suggestion is supported by the high yield of subchromatid changes which could be associated with the release of EOC following the dissolution of the 11 iaucleolus at prophase. EOC thus does not interfere with DNA synthesis <3r chromosome replication, although its radiomimetic effect may in some way be related to other facets of chromosome synthesis. The aberrations induced by TMU were during prOphase, when sub- chromatid exchanges were induced, and during G2 when chromatid aberra- tions were induced (Kihlman, 1961 b). The effect of TMU, in contrast to EOC, is dependent on mitotic activity during treatment. Kihlman (1951) suggests that this differential ability of EOC and TMU to affect cells may be due to their differences in ability to penetrate into the nucleus, which, in turn, is dependent on their relative lipid solubi- lity. Only EOC is able to penetrate into the cell nucleus during early and mid-interphase, and only EOC is capable of being absorbed onto G1 (pre DNA synthesis) and S (DNA sysnthesis) chromosomes (Kihlman, 1961 b). Methylated oxypurines are not incorporated into DNA (Koch, 1956; Greer, 1958). They are rather inert biochemically, as well. Lieb (1961) reported a temporal DNA synthesis inhibition with caffeine. 0d- mark and Kihlman (1965) reported that both DNA and RNA synthesis in Vicia faba is inhibited by EOC. B. Antibiotics 1. Azaserine ll N27:- CH’C‘O‘CHg" CFH COOH NHz 12 Azaserine is an antibiotic isolated from a strain of Streptomyces (Bartz, et. al., 1954). It produces chromosomal aberra- tions in Tradescantia paludosa root tip cells (Tanaka and Sugimura, 1956). It produces delayed chromatid exchanges predominantly in yigig_ with 10‘” M treatment for l to 2 hours. This effect is completely suppressed by inhibitors of oxadative phosphorylation and anoxia (Khil- man, 1964). Purine biosynthesis, according to Handschwmacher and ‘Welch (1960), is inhibited by azaserine, and it also disturbs amino acid metabolism. Freese (1963) suggested that it may also act as an alkylating agent, as well. 0* II | 2 . Mitomycin Hg,“ CHZAOéNHz ’0' NH Merz (1961), by treating Migiggwith .001% solution, produced delayed effects, predominantly chromatid breaks. Anoxia and inhibi- tion of oxidative phosphorylation does not reduce the effect of mito- mycin (Merz, 1961), and the effects are independent of pH and tempera- ture. Nowell (1964) found that mitomycin C. causes chromosome breaks in human leukocyte cultures mainly of the chromatid type when treated in the mitotically inactive stage (GO) of interphase. Treatments of cells in G2 did not produce any effect, but cells in '8' showed a low amount of damage. Shiba, et.al. (1959) and Reich,et.al. (1961) showed 13 mitomycin produces DNA synthesis inhibition and degradation in bac- teria. Iyer and Szybalski (1963) showed that mitomycin cross links complimentary DNA strands. Schwartz (1963) suggested that mitomycin may act as an alkylating agent (after activation in liyg_by unmasking the fused aziridine ring). 3. Streptonigrin Cohen, et. a1. (1963) observed mitotic inhibition and extensive damage to chromosomes of human leukocytes in culture by .0001% to .1 gram/liter of the antibiotic, and these aberrations are produced as early as two hours after treatment, showing that G2 is the period when the effect is produced. Puck (1964), however, showed that streptonigrin is inactive in mammalian cells in which DNA synthe- sis is completed. In Eigig.f§b§_Kihlman (1964) showed that strepto- nigrin had a nondelayed effect. It produces chromosome breaks in G1’ chromatid breaks in "8", G2, and subchromatid breaks in prophase. This effect was independent of temperature and pH, and is not reduced by anoxia or by oxidative phosphorylation inhibitors. Rao, et. al., 14 (1963) showed that both streptonigrin and mitomycin contain the same O-aminoquinone moity, but the former has a nondelayed effect, and the latter has only a delayed effect, in spite of their chemical similari- ty. Iyer and Szybalski (1964) pointed out that this similarity is only superficial, since the reactivity of streptonigrin with DNA in Mitrg, in contrast to that of mitomycin C, is not influenced by chemi- cal reduction. C. Nitroso compounds 1. N-NitrosoéNamethylurethan /J¢Cb Et3Ch-t{\q:C) CX:2IE5 In Vicia faba, 0.5 - leO'3 M of this compound pro— duced delayed chromatid-type aberrations with normal rejoining fre~ quency, and these were randomly distributed between S and M chromo- somes. This effect is independent of pH and temperature. Respiratory inhibitors and anoxia inhibit its action, but DNP, which uncouples oxidative phosphorylation without affecting respiration, does noNgin- fluence its effect (Kihlman, 1961 d). The nitrosamdnes are known as carcinogenic agents (Druckrey, et. al., 1961, a, b, and c). At least one alkyl group is necessary for this carcinogenic effect, which re- quires the presence of 02 (Brouwers and Emmelot, 1960). The active carcinogenic groups(alkylating agents) are formed in 2212 by an en- zyme-catalyzed oxidative dealkylation. But this mechanism.may not explain its chromosomeebreaking ability, since cupferron, which is 15 devoid of an alkyl group, is effective in producing chromosome breakage. 2. N-Hydroxylphenylnitrosamine -ammonium or cupferron -N—NO ._ 7 - ' Cflblti4, Even 10‘3 M cupferron produced only low effects. De- 1ayed chromatid aberrations (chromatid exchanges and sister union iso- chromatid breaks)were prevalent, showing normal rejoining. The above effect is enhanced at low pH and high temperature, and so is the toxic effect. It is inactive in the absence of 02. The effect of cupferron may well be due to its chelating prOperties, or to a spontaneous decom- position of this nitrosamine to yield a phenylcarbonium ion (Kihlman, 1959 b and 1961 a). 3. léMethy1~3-Nitro-I-Nitrosoguanidine (MNNG) 1R1}! Hsc- n-c—S‘I—H No N02. Gichner, et. a1. (1963), using Vicia faba, obtained delayed chromatid type aberrations with a peak effect between 24 and 48 hours after 1 hour treatment with 5X10"+ M of this chemical. The effect was enhanced by low pH,and temperature from 18 - 24°C. The effect was reduced by anoxia and Sggggm.§gigg, DNP uncoupling was in- effectual. 16 'D. Miscellaneous 1. Maleic Hydrazide R li-tl -§{ H—N c—n Maleic hydrazide produced in Eigig'fgbg_a delayed effect, giving chromatid aberrations with normal rejoining upon 2 hour treatment with 10"LL M at pH 5.8; the effect being localized in the heterochromatic segment, close to the centromere in the nucleolar arm of the M chromosome (Darlington and McLeish, 1951). The highest fre~ quency was obtained between 24 and 36 hours after treatment. Evans and Scott (1964) found that cells in 'S', exposed to maleic hydrazide, were delayed considerably, and contained chromatid aberrations. (The cells in G1 during treatment had chromatid aberrations, while G2 cells were not delayed and did not contain any aberrations. Its effect was reduced by anoxia and uncoupling of oxidative phosphorylation, but was increased with temperature increase, and was 4X higher at pH 4.7 than at pH 7.3 (Kihlman, 1956). Its biological activity may be due to its reaction with SH groups in the cell (Muir and Hansch, 1953), inhibiting enzymes requiring free SH groups (Hughes and Spragg, 1958). 2. Potassium Cyanide (KCN) Lalo" M KCN produces chromatid-type aberrations in Vicia faba of the delayed type with peak frequency between 24 - 36 hours after 1 hour treatment (Lilly and Thoday, 1956). These rejoin 1? normally. Its effect is independent of temperature, and it is in- active in anoxia. .It has only very little effect at 02 concentration below 10%. Above 10%, the effect increases with increasing 02 con- centrations, and is not influenced by inhibitors of oxidative phos- phorylation (Kihlman, 1957). KCN is an effective reSpiratory inhi- bitor, since it reacts with iron or copper in cytochromes, and also with catalase and peroxidase. The damaging effect may be due to H202 accumulation in cells in the presence of KCN. 3. Hydroxylamine (NHZOH) Hydroxylamine produced nonrandom chromatid and chro~ mosome aberrations localized in the centromere region. Exchanges were frequent (Somers and Hsu, 1962). Borenfreund, et. a1. (1964) suggested that chromosomal aberrations induced by this chemical is the result of main chain. scission of DNA rather than from a reaction with cytosine in DNA. Cohn (1964) reported chromosome breakage and shattering l - 2 days after treatments of onion and Eigig_roots for one-half hour with 10'"3 M.NH20H. Exchanges were rare. 4. Ethyl Alcohol, Coca Cola, Coffee, and Antinauseants All of the above agents were capable of inducing chromosomal aberrations (Sax and Sax, 1966). A concentration of 0.5% ethyl alcohol was equivalent to about 20 r/day of chronic gamma radia- tion, or an accumulated dose of 75r. Caffeine has long been known to 18 be radiomimetic for plant chromosomes (Kihlman, 1949). Strong coffee (2 T. per cup) produced more aberrations than did 50 r per day of chro- nic gamma radiation. Coca cola had to be diluted 50% to permit growth of onion roots. It is a fourth as potent as weak coffee in producing chromosomal aberrations. Antinauseant drugs like thalidomide produced a small but significant increase in chromosome aberrations. Dramamine produced a slight but consistent increase in chromosome aberrations. 5. Lysergic Acid Diethylamide (LSD) CHsza fiiIDCZ bl}! CH2 —V 1?‘ CH3 The effect of LSD 25 was studied in cultures of human leukocytes at concentrations ranging from 10 - 0.001.,ng/m1 for 4, 24, and 48 hours. Cytogenetic investigations of a patient extensively treated with this drug over a 4 year period showed similar increase in chromosomal aberrations. It was found to/induce different kinds of chro- mosomal aberrations and showed an affinity for certain regions of chro- mosomes, like the centromeres and secondary constrictions (Cohen and Marinello, 1967). Chromosomal abnormalities in leukocytes in LSD 25 users was studied by Irwin and Egozcue (1967), who found a significant increase of chromosomal abnormalities as compared to nonuser controls. l9 6. Irradiated Carbohydrates Swaminathan, et. a1. (1962) and Holsten, et. a1. (1965), found chromosome aberrations such as stickiness, lagging ana- phase fragments, nuclear disintegration, etc. induced in plant cells in culture by irradiated carbohydrates in the medium. Shaw and Hayes (1966), studied the effects of irradiated sucrose on the chromosomes of human lymphocytes in Iitgg, These showed chromosome breaks of the isochromatid type; gaps, exchanges, stickiness, endoreduplication, etc. Breaks were nonrandom along the chromosomes. 7. Alkylating Agents Since the ethylenimines belong to the class of com- pounds known as alkylating agents, these will be discussed in detail. There are many agents that carry one, two, or more alkyl groups in a reactive form. These are called mono, bi, or polyfunctional alkylat- ing agents. The various groups on a polyfunctional agent may either act separately or they may cause the crosslinking of molecules. The chemical structure of the most commonly used classes of alkylating agents are given below: PU-C1 .. s< CI-Al-N’Al C‘ Al -Cl \Al -Cl Sulfer mustard Nitrogen mustard 20 S? AI—O— é$5» o-AL A\—o—S -AI Dialkyl sulfates Alkylalkane sulfonates Ii 11 R- c-‘é—R HC-ICH \ / \ / Epoxides R‘ R Ethylenimines IIII‘- IT Ib3::PJ—¥K. [TKF'C) D—' :0 Other —Pr0piolactone Diazo compounds reactive oxygens Stacey, et. a1. (1958) reported that all alkylating agents that prevent cell duplication have in common their reactivity with nucleophylic, i.e., slightly negatively-charged groups. Price (1958) described the various chemical reactions by which alkylation can occur. Ross (1958) described the various chemical groups. Most reactive are sulfhydryl and thioester groups. When isolated DNA is treated in solution, the reactivity of alkylating agents depend on their charge; negatively-charged molecules being least reactive. Alexander and Stacey (1958) prOposed that polyfunctional mustards exert their lethal effect by crosslinking DNA molecules, rather than by merely breaking the sugar phOSphate backbone of DNA, but the latter mechanism does not seem negligible, since monofunctional alkylating agents also have lethal effects. 21 Most alkylating agents have a mutagenic effect, whether mono or poly~functional. Whether their effects are due to direct action on DNA or chromosomes, or whether their effects are indirect are not known. In the case of Triethylenemelamine or tretamine, chemical spe- cificity has been correlated with mutagenecity. Tretamine reacts with free pyrimidine bases to form a base analog; the reaction product being mutagenic with thymine and not with cytosine (Lorkiewicz and Szybalski (1961). Fahmy and Fahmy (1958) reported that tretamine induces domi- nant lethals in Drosophila by causing chromosome aberrations. DNA can be altered in five different ways by ethylating and methylating agents: (1) The alkylation of the phosphate groups of nucleic acids has been measured for many alkylating agents (Alexander, 1952; Reiner and Zamenhof, 1951; Stacey, et. al., 1958). The phosphate triester thus formed is unstable, and hydrolyzes mostly to return the free alkyl groups. DNA duplication might be inhibited if enough alkyl groups remain attached up to the time at which DNA attempts to duplicate. The attached alkyl group might interfere with the DNA du- plication in such a way that some noncomplimentary base would be incor- porated into the new strand. (2) The DNA "backbone" is broken if the phosphate triester hydrolyzes between the sugar and the phosphate. The relative frequen- cies with which the alkyl group becomes removed, or if the chain is broken, are not known. This kind of chain breakage might induce larger 22 alterations to be lethal. (3) Some of the DNA bases are alkylated. The preferential formation of 7-alkylguanine has been found for nitrogen mustard (Brookes and Lawley, 1960 a). Dimethyl sulfate produced mainly seven methyl guanine (Reiner and Zamenhof, 1957). Lett, et. a1. (1962) agreed that alkylation of the base does not occur directly, but transalkylation, the alkyl group changing from the phosphate to the base, takes place. Thus, the alkylated base might inhibit DNA duplication or cause base pairing mistakes during DNA duplication. (4) Depurination: Unstable quaternary nitrogen are pro- duced by the alkylation of the purines in the 7~position, in which case the alkyl group itself hydrolyzes away from the purines, or else the alkylated purine separates from the deoxyribose, leaving it de- purinated (Freese, 1963). Bautz and Freese (1960) observed the libera- tion of ethylated and methylated purines from DNA. The gap might in~ terfere with DNA duplication, or cause the incorporation of a wrong base. (5) At high pH, the depurinated DNA is labile, and may occasionally break even at neutral pH, thus inducing larger altera- tions or be lethal (Freese, 1963). The majority of the known chro~ mosomeebreaking agents are believed to act by one or the other of these mechanisms. The cytological effects produced by these agents are mainly exchanges of the chromatid type, and isolocus breaks of 23 the delayed type. Base analog and alkylating agents may, according to Lorkiewicz and Szybalski (1961) produce aberrations in chromosomes by rather similar mechanisms, namely, incorporation into DNA during "S" period. It was pointed out earlier that tretamine causes chromosome aberrations by this mechanism. Thus, breakage by alkylating agents occur during "8" period (Scott and Evans, 1964). But this mechanism of aberrations production by alkylation of DNA precursors rather than of DNA is not widely accepted. It is generally agreed that DNA is, the most sensitive material to alkylation'within the cell, and is probably the primary site of alkylation (Wheeler, 1962). It is then difficult to understand why alkylating agents are effective only dur- ing "8", if it is true that DNA rather than DNA precursors is the primary site of alkylation. Kihlman (1966) suggests several reasons why this could be so: (1) It may be that alkylation of DNA is possible only during "8" because the chromosomes are protected at other stages by other sub- stances (2) It is also possible that although DNA may be alkylated at any stage, the alkylation results in chromosome aberrations only during "S" (3) It is possible that the direct cause of aberrations is not the alkylation of DNA, but the inhibition of DNA synthesis, which the alkylation has produced. This inhibition could be due to the reduced primer activity of alkylated DNA, or due to competition for sites in the DNA polymerase enzyme between normal and alkylated 24 deoxyribonucleotide triphosphates. The above possibilities are shown schematically in the following (Kihlman, 1966): / Alkylation of DNAr—>Inhibition of DNA synthesis.l::::::> Chromosomal I/fl ~Aberrations Alkylation of DNA—precursors-—>Incorporation into DNA However, for alkylating agents, there appears to be no corre- lation between the ability to inhibit DNA synthesis and production of chromosomal aberrations (Wheeler, 1962). The alkylating agents are very reactive, and combine readily with nucleophylic centers in other molecules, such as SH groups, io- nized acid groups, and nonionized amino groups, and their affinity to- ‘wards nucleophylic groups is a result of their ability to form positive carbonium ions in polar solvents; e.g., RZNCHZCHZCIQifiRZNCHZEHZ + Cl” (Kihlman, 1966) Some of the alkylating agents are discussed below with regard to their chromosomeebreaking ability. a. Di (Z-chlorethyl) methylamine or Nitrogen mustard /CH2CH2,C| H3C—N \CHZCHQG Darlington and Koller (l9fl7) studied the chromosome Abreaking effects of nitrogen mustards on mitotic cells in wheat, and Ford (1940) studied their effects on roots of Vicia faba. With 10-5 M HNZ produced delayed nonrandom chromatid-type aberrations that first appeared between 8 - 10 hours, and these rejointed normally. The 25 effect was independent of 02 tension during treatment (Kihlman, 1955). b . Di ( —2 -3 -Epoxypropyl) ether (DEPE) O /CHzC<1é1-la ‘ (Racy/CH2 Loveless and Revell 0(1949), and Reve11(l9 53), studied the chromosomeebreaking effect of epoxides. ReveIl(l953), and Kihlman (19 56) treated vigig root tips with 2x104F M DEPE for one hour and obtained chromatid aberrations ten hours after treatment with a maximum effect between 24 and 36 hours. Rejoining frequency was nor- mal. The heterchromatic segment in the middle of the long arm of "S" chromosomes appeared to be more often affected (Revell, 1953). This effect is temperature dependent and is independent of 02 tension (Kihlman, 1956). c. P-Propiolactone Cle-‘gfiz 0—co Smith and Srb (19 51), and Swanson and Merz (1959) studied its effect. Delayed chromatid-type aberrations with high re— joining frequency with a peak effect at 1+8 hours after treatment, were obtained. The breakage was nonrandom within the chromosomes. Higher temperature increases the effect, but it is independent of pH and 02 tension. d. Ethylenimines and other Pesticides The effect of apholate on Aggie-g aegypti was studied by Rai (19614). It was found to induce aberrations such as stickiness, 26 deletions, ring chromosomes, dicentrics, and anaphase bridges in so- matic cells. wu and Grant (1966), carried out a study on the root tips of Cl and C2 generations of Hordeum vulagre, after treating seeds (CO generation) with one of 15 pesticides, including metepa, sevin, and cytrol. In the C1 generation, the percentage of affected cells ranged from 2.9 to 17.7%. Treatments with metepa induced 17.7% aberrations, which exceeded the frequency of chromosome aberrations (9.1%) found in root tip cells from X—rayed seeds (5500 R). In the C2 generation, chro- mosome aberrations were found in over half of the different treatments. A number of 'mutant' C2 seedlings were found, which included albino and yellow seedlings, dwarfs, and striped and narrow thick-leaved seedlings. Comparative mutagenic ability of TEPA and HEMPA was tested in the Sperm of Bracon hebetor by Palmquist and LaChance (1966). They found that TEPA is 100 times more effective than its nonalkylating analog, HEMPA, in inducing recessive lethal mutations. METHODS I. Cytological Experimental System A standardized experimental system was employed to determine the cytological effects of ethylenimines. The root apical meristem of the pea, Pisum sativum Varietv Alaska, (obtained from the Farm Bureau, Lansing, Michigan), and the broad bean Yigia fgbg, (obtained from Sutton and Company of England) were used in the study. The dried peas were rinsed (three times) and soaked in de— ionized double distilled water for five hours at 22.50 C. The soaked peas were then spread evenly on absorbant paper toweling, moistened with deionized double distilled water (DDW), rolled, and then wrapped with wax paper. The rolls were placed in an upright position in 600 m1 beakers containing about one inch of DDW. The beakers were then placed in an incubator at 22.5°C., and the peas allowed to germinate for 37 hours. At the end of this period, the paper toweling was unrolled and the seedlings were selected for the modal length of the population. This length was usually between 2 to 2.5 cms. These seedlings were suspended by paraffin—coated grids and the roots immersed in nutrient solutions contained in 350 m1 crystall- izing dishes, 100 mm X 50mm. The nutrient solution was modified Hoag- lands, made according to the following schedule. Each stock solution was made in 500 m1 of DDW. 27 A. B. C. D. E. 28 3.80 grams of Calcium Nitrate Ca (N03)2 Lmzo 5.16 gms. of Ammonium Nitrate NHuNO3 7.20 gms. of Magnesium Sulfate MgSOn 7H20 5. gms. of Potassium monobasic Phosphate KH2P04 0.28 gms. of Potassium dibasic Phosphate K2HP04 12.5 ml of each of the above stock solutions were used, and 970 m1 of DDW was added to make 1,000 m1 of nutrient. The pH of this solution was found to be between 5.6 and 5.7. In order to keep the nutrient solution well-oxygenated and stirred, filtered air was con- stantly bubbled through the solution. The dishes containing the peas 'were kept in a constant temperature water bath at 22.5°C., located in an air-conditioned room kept at 22.500. The temperature of the water bath was monitored by means of a recording thermograph. The peas were allowed to equilibrate in the culture conditions for at least 4 hours. After this period, the wire grids carrying the peas were transferred to the treatment solutions contained in similar dishes. The chemical under investigation was dissolved in the above nutrient solution according to the desired concentrations and placed in the treatment dishes. All of the ethylenimines were readily solu- ble in water to the extent required. The time that the treatments were begun was considered to be the zero hour. The chemical treatments were given for only one half hour, since prolonged treatment was likely to produce complex results. .After the one half hour period, the wire grids containing the pea seed- lings were thoroughly rinsed in DDW and returned to the nutrient solu- 29 tions. Samples were taken at predetermined hours that coincided with the mitotic cycle times, determined by experimentation; the cycle time delay being known (from experiments described on page 32) for each chemical studied in detail. Samples taken at critical hours in peas were scattered. Scattering was accomplished by transferring the seed- lings to 120 ppm of rulene solution mixed in water, using a wetting agent. They were allowed to stay in that solution for 45 minutes be— fore they were fixed and squash preparations made as described below. The scattered metaphases and anaphases were studied for chromatid and chromosome damage, and were photographed (Plates I and IV ). The first three cm. of the pea root was cut off by means of tweezers, placed in a small vial containing 5 m1 of Pienaar's fixative (Pienaar, 1955) (6 partsof methanol, 3 parts chloroform, and 2 parts of propionic acid). Five pea root tips were placed in vacuum for ten minutes, after which time they were refrigerated for 12 hours before slides were made. The Feulgen technique (Lillie, 1951) was employed to stain the fixed material. This technique consisted of pouring off the fixa- tive and refilling the vials with one normal HCL, kept in an oven at 60° 0., and hydrolyzing the tips for 16 to 17 minutes. After this time, the acid was poured off, and leucobasic fuchsin was added. The meristematic region developed a deep purplish color in 15 to 20 min- utes. Squash preparations were made from the darkly-stained region. The slides were then transferred to 90 parts tertiary butyl alcohol and 10 parts 100% ethyl alcohol solution for dehydration. After 10 to 12 hours in the TBA solution, the slides were made permanent, using 30 diaphane as the mounting medium. These slides were then scored for the type of data desired. The dired Eigi§.f§b§_seeds were soaked 15 to 20 hrs. in DDW. The soaked seeds were peeled in order to facilitate germination. These were then placed in large dishes containing moistened absorbant paper towel- ing, and stacked one over the other to prevent drying. Occasionally, water'was added to the dishes. After 48 hrs., the primary roots ac- quired a length of 1.to 2". During this period, the seeds were thor- oughly washed several times and returned to fresh absorbant paper in clean dishes. These washings helped to remove secretions from cotyle— dons that inhibited growth of the roots. They were then transferred to wire grids, placed over 600 ml beakers containing modified Hoagland so- lution, and equilibrated for at least 4 hrs. After equilibration, the seedlings were transferred to other 600 ml beakers containing the che— micals dissolved in the nutrient solution. The treatments were given for % hr., using the desired concentrations of chemicals. After this time, the seedlings were transferred to the nutrient solution. Samples were taken at predetermined hours from these dishes. Only 2 root tips were taken from each group and fixed in Pienaar's fixative. 1 hr. pri- or to the predetermined sampling times, the seedlings were transferred to a saturated paradichloro benzene solution (PDB), and allowed to re- main there for 1 hr. before the tips were excised and fixed. The Feulgen technique was employed to stain the slides. Each root apex was split into 3 equal parts longitudinally, and transferred to 3 slides, macerated, and squash preparations were made from them. These were made permanent according to the procedure used for Pisum. 31 The chemicals under investigation belong to the general group 162., ethylenimines . dinyl ring. They are: cnz CH _ 2 “Q SQ CH - CH 1. Azirane 2. Ethylene sulfide Ti “2. ' | N—P___ 0 H9, I N lkfl%{::CI{z 4. Tris (1-aziridiny1) phosphine oxide or TERA Metepa 7. The active group in these chemicals is the aziri- 32°sz N 1% Pl N H2 2H2: 3. Triethylenemela- mine or Treta- mine or TEM. 3 “3 N H, 1t3C: ) I¢———4FE==C> 113C. ' Ti ligCV/\\Cnfl3 5. Hexamethyl phosphoric triamide or HEMRA Apholate 32 Detailed studies were made using apholate and metepa. Most of the quantitative data using peas as test organisms were obtained by scor- ing anaphase bridges and fragments. A total of 200 anaphases were scored whenever possible from each of the five slides, and these were then averaged and converted to percent damage. In beans, the quanti- tative data were obtained by analyzing metaphases that were scattered, using PDB treatment for one hour prior to the predetermined sampling times. Chromosome and chromatid fragments and exchanges were scored, and good preparations were photographed. Mitotic index, which is the total number of dividing cells for a total of 1,000 cells, was also determined for each slide whenever those data were deemed necessary. Random fields were chosen, and in each field the total number of di- viding and interphase cells were counted. These counts were continued until 1,000 cells were obtained. Mitotic indices were usually obtained from at least four slides, and the average computed. For determining the cycle time delay caused by treatment with the chemicals under investigation, a continuous colchicine method Was employed. The peas were first treated with the chemical with the de- sired concentration for one half hour, and immediately rinsed and transferred to a colchicine solution (75 ppm), and allowed to remain there for the rest of the experiment. Controls with and without col- chicine treatments were also run simultaneously. Samples were taken at one, two, and four hours to check the colchicine effect. These root tips were fixed, and Squash preparations were made and scored for clumps, scatters, and normal post-prophase stages. A colchicine 33 index (Greenberg, 1966) was then calculated, using the formula 2X # of clumps + 1 X the # of scatters + 0 X normal postprophase cells postprophase cells The index value has a peak at about two hours. At 1.8 or above the colchicine is assumed to be good. If the effect was low, a new batch of colchicine was obtained for experimentation. Samples were taken at predetermined hours. Slides were made from these, and scored for per- cent polyploidy. As time increases, the percent polyploidy also in- creases. On a plot of time versus ploidy, at least four points were ob- tained, and these were then joined to meet the X-axis. Where they meet is assumed to be the minimum cycle time (Bekken, 1966). For determining the duration of the second cycle after treat- ment, the roots were treated with the chemical for one half hour, rinsed, and transferred to the nutrient solution until the end of the first cycle, already determined by experimentation. These were then transferred to a continuous colchicine solution (75 ppm), and were allowed to stay there for the rest of the experiment. Samples were taken from these dishes at predetermined time intervals. The results were plotted as before, and cycle time delay was thus determined. The 5 amino-uracil (5 AU) system was employed in order to ascertain the susceptible stage of the mitotic cycle at which the ethylenimines exert their influence in causing chromosome breakage. The pea system was exclusively employed in these experiments. After 34 four hours of equilibration, the peas were transferred to dishes con- taining 100 - 150 ppm of 5 AU suspended in the nutrient solution. Since 5 AU was not readily soluble in the nutrient solution, the tem- perature was raised to about 40°C with constant stirring. This im- proved solubility. The peas were left in 5 amino-uracil for 8 hours, at which time they were taken out and thoroughly rinsed in DDW, and returned to the nutrient solution. Treatment with apholate 3.1 X 10‘3 M was made at various times after the 5 AU treatment. These treatments were made at 7%, 10%, 12, l5, l6, and 17 hours after treat- ment with 5 AU. Samples were taken up to 63 hours after treatment, and slides were scored for anaphase damages as well as for mitotic indices. X-ray standards, using peas, were also run. This was done at Brookhaven National Laboratories by Dr. Van't Hoff. A dose (250 r) was given, and samples were taken at 3, 6, 9, 12, 15, and 27 hours. Slides were made and scored for damage. 2. Genetic Experimental System The Muller 5 system was used to study the mutagenic ability of certain of the chemicals. Cultures of Oregon R Droso- Phil? melanogaster were started in fresh culture media. A cylindri- cal piece of white bond paper, 3"X 4", was placed in contact with the medium in the bottles. The larvae climbed this paper before pupation. When the larvae reached the third instar stage, the paper with the larvae attached to it was lifted from the medium and dipped into a 1% 35 solution of the chemical for one minute. It was then dried with ab- sorbant paper toweling and transferred to fresh media. When the.flies emerged, they were etherized, and the males were mated to virgin Muller 5 females. The F1 progeny were then pair—mated in smaller vials to get the F2. These were screened for eye color. If in any F2 progeny the full red-eyed males were missing, this was considered to be induced by a lethal mutation. Such cultures were retested by mating the red-eyed females, which are the carriers of the lethal, to wild type red-eyed males. The progeny of these retested pair matings were then screened for eye color. If no full red—eyed males occured in this progeny, then the existence of a lethal mutation was considered confirmed. 3. Retention of the Chemical Pea seedlings were solely employed in this study. A large quantity of peas were equilibrated for four hours in modified Hoaglands nutrient solution, and treated with 1.4X10'2 M of the chemi~ cal (metepa) for a half hour. These were rinsed thoroughly in DDW. Samples of 75 roots were taken at %, l, 2, and 4, 8, 12 and 24 hours after treatment and ground in a homogenizer, using 1.5 m1 of acetone as extracting medium. This extract was then centrifuged at 8,000 rpm. The supernatant was then carefully poured into a small test tube and injected into a gas chromatographic column for detecting the presence of the phosphate group, using a hydrogen flame ionization detector with a sodium thermionic shield (Giuffrida, 1964) in a Packard 800 36 gas chromatograph. Extracts from control pea roots were also run simultaneously. Metepa standardstof known concentrations were also run as comparisons, and quantitative estimations could be made by comparing the peaks made by the recorder on the graph paper. RESULTS Azirane The damage caused by different concentrations of azirane treatment on the pea root meristem was scored at anaphase. It was found that azirane damage, as indicated by the presence of bridges and fragments, was highest at 1.83X10‘2 M. The maximum effect was found at 39 hours. Low doses up to 0.73XlO“2 M failed to show appre- ciable damage above control level. Using 1.22X1O‘2 M, the damage reached a maximum at 27 hours, and a high level was maintained at 39 hours. Doses above 1.83X10-2 M were lethal, and mitotic inhibi- tion as shown by mitotic indices at different sample times was very great. Even 1.22X10'2 and 1.83X10“2 M treatments showed mitotic in— hibition. The M Is (mitotic indices) at 15 hours were low (14). At 27 and 39 hours the M Is were at control level. At later hours (51 and 63) it was of the order of 20 (see Figure 1). The effects of azirane with respect to M I and damage are summarized in Table l in the appendix. Ethylene Sulphide This was tested on the pea system at concentrations ranging from 0.42X10‘”3 M to 8.32X1O'2 M. There was no significant damage in any of the concentrations employed. The solutions of this chemical turned cloudy. It seems that this compound is very unstable and disso- 37 Percent anaphase damage 10 38, H— - 1.22 X 10"2 M azirane —o—o— - 1.83 X 10-2 M azirane - control peas L I I l L l I I l - I I 15 2O 25 3O 35 4O 45 5O 55 60 65 I Time in hours Figure l. Azirane damage in which the percentage of anaphase damage is plotted against time in hours. Percent anaphase damage 50 1+5 1+0 35 3O 25 20 15 10 39 ____. — control peas o—o - u.9 X 10-4 M tretamine —-—— -2.L+5 X 10-1L M " H— 1.2 X 10-1+ M " I I I I L l I 1 l 10 15 20 25 3o 35 ho us 50 Time in hours Figure 2. Anaphase damage in Pisum sativum, caused by 3 concentrations of tretamine, plotted against time in hours° 40 ciates easily at the pH employed, which might account for its insigni- ficant damaging ability. Tretamine or TEM The chromosomal damage induced by tretamine treatment in peas was measured by scoring anaphase damage. Of the concentrations used, the range between 1.23X1O“4M -4.9X1O'4 M was found to induce the maxi- mum damage without causing lethality. The maximum effect was at 27 hours, and dropped off at later hours, as shown in Figure 2. The maximum effect (60% damage) was obtained with 3.68XlOJIPI -4M, the maximum effect obtained at 27 hours at 27 hours. With 4.9X1O was 40%, and at 39 hours, the damage was only 15%. The maximum damage using 2.45X10-4 M was 16.5%, and at 40 hours the damage dropped to 4%. Exposure to 1.23X1O‘4 M produced the lowest percentage of damage; 15, 27, and 40 hours had respectively; 3.6%, 9.6%, and 2.4% damage (Fig. 2) M Is were also determined at various hours at different treatment levels. The M Is were considerably lower at later hours. The M I showed a decline at 39 hours, and at 51 and 63 hours, M Is were of the order of 15 — 20. Aphoxide or TEPA The effect of TEPA was studied using the pea system. The maximum damage was found at 27 hours, and showed lower values at 39 and subsequent hours. 41 Hexamethyl phosphoric triamide or HEMPA Various concentrations of HEMPA, ranging from 5.58X10'3 M — 1.675X10‘2 M were tested on the pea root meristem. None of the con- centrations tested had any significant chromosome—breaking effect. Inhibition of mitosis as shown by M I was present. HEMPA was tested on the Muller 5 system by treating Drosophila larvae. It was found to induce lethal mutations. f of pair matings f of lethal mutations % 153 3 1.95 The percentage of lethal mutations was considerably higher than can be accounted for by spontaneous mutations, since our particular stock of Oregon R flies have a rate of less than .01% X chromosome recessive lethals.) Apholate The chromosome—damaging effect of apholate was studied more extensively on the pea root system. The apholate used had only 40% active ingredient, and doses were based on this. The damaging effect of apholate was independent of pH between the range of 5 — 7. Two types of results were obtained by using apholate: 1. two experiments showed maximum damage at 39 hours, and the damage persisted for a considerable period of time, viz up to 95 hours, and 2. in other experiments the maximum damage was at 27 hours, and did not persist at such high levels as before, although a level of damage above control was still present up to 95 hours. Percent anaphase damage 30 .. 1+2 25 — 2O _ 15 — lO — I— 5 - 2 I l I I n L 1 l l 1 l 10 2O 3O 4O 5O 6O 7O 8O 90 100 Time in hours Figure 3. Chromosome damage caused by 3.1 X 10"2 M apho- late. Percent anaphase damage vs. time, shows the 3rd cycle maximum and persistent effect. 2O 18 H e $7 ox l-J I'D Percent anaphase damage 00 '5 O\ 1+3 I I I I I I I I I 1 2 1+ 6 8 10 12 1h 16 Dose (ppm) in hundreds. Figure 4. Dose effect curve using apholate. Percent anaphase damage vs. dose in parts per million. 42., The concentrations of apholate employed and their corresponding maxi- mum damages were: 0.26x1o-3 M, 2.5%; 0.52X1o-3 M, 4.5%; 1.03x10'3 M, 8.6%; 2.1X10'3 M, 11.6%; 3.1X10-3 M, 16.4%; and 4.1X10'3 M, 18.4%. Another experiment in which 3.1X10"3 M concentration was em- ployed, the damage at 12 hours was 4.5%. At 24 hours, the damage rose to 18%, and at 36 hours reached a peak at 27.6%. This level of damage then persisted up to 61 hours, after which it dropped to 16% and 16.9% at 72 and 84 hours, respectively. A high percent of damage (15.3%) was still present at 95 hours. The average control damage was of the order of 2% or less (See Figure 3 and Table 2). In a different experiment using 3.11X10'3 M of apholate, it was found that the amount of damage at 15 hours was still at control level, i.e., below 2%. However, at 24 hours the maximum damage of 15% was obtained. At 36 hours the amount of damage was lOWer (7.5%). At 48 and even 61 hours, the damage was persistent at the level of 5% (Table 3). Mitotic indices were determined in both control and treated pea roots. It was found that the M I remained rather steady at con— trol level (M I 60) for up to 30 hours. It then showed a rise, and again fluctuated around the mean 60. The dose effect relationship for the peak hours are shown in Figure 4 and Table 4. Mitotic cycle time delay caused by treatment with apholate (3.1}{10'3 M was also determined (see Figure 5). Continuous treat- ment with colchicine was employed in this experiment, and percent polyploidy was plotted against time. In control peas the polyploids Percent polyploidy 16 ._l 4: }_I I'D ’_l O 00 O\ 4:- Figure 5. 45 III 6 7 8 9 10 12 14 16 18 20 Time in hours C%-—-<) - control peas O——O - 3.1 X 10'3 M apholate (% hour treatment). Cycle time delay caused by apholate treatment on Pisum sativum. Percent polyploidy is plotted against time. Polyploids did not appear until 15 hours in treated peas. 46 appeared earlier. Significant amounts of polyploids appeared in the apholate-treated peas only at 15 hours, and the delay was estimated to be about 2.5 to 3 hours. 5 Amino-Uracil System The 5 amino-uracil system was utilized in order to determine the particular stage or stages or interphase or active mitosis where the damage is taking place. 5 A U is known to inhibit DNA synthesis (Perensky and Smith, 1965). It is also known that it stops cells from passing through the mitotic c cle, at the end of G1, and also in S (Van't Hoff, 1966). The roots were treated with 5 A U continuously for 8 hours, after which the roots were removed from the the 5 A U solution, rinsed, and returned to the nutrient solution. The pattern of M I change after 8 hours of 100 ppm of 5 A U is shown in the following figure (Figure 6, and Tables 5a to 5f). 180 — 16o — 140 _ 120 - 54100 - t“; s 80 - -r-I E 60 - g 40 - -.—I 2 2O ’ A l 1 2 3 4 5 6 7 8 9 10 12 14 16 18 2o ', _ _ . Time in he rs [Figure 6. Mitotic 1ndex change after 8 hrp. of 150 ppm of 5 AU on Pisum. The roots were treated with apholate for 7 hour at various times with—— 8 hours in 5 A U. The different times were chosen so that the cell 47 population undergoing treatment was in several different stages of the mitotic cycle. These treatment times and the corresponding stages that the cell population was assumed to be in were: 7% hours, G18 (actually the end of G1); 10—:- hours, midsynthesis, '5' stage; 12% hours, early G2; and at 15 hours, active mitosis. The peak M I (200) at 15 hours (as shown in Figure 6) was obtained because of the syn- chrony induced by the 5 A U treatment. This synchrony, however, does not persist even for the next cycle. The damages induced at these treatment times were then scored, to determine whether there was any significant difference between them. The data for each time of treatment, showing both M I and the percentage of damage, are given in the tables 5a through 5f. Drosophila larvae were treated with apholate, and were tested against the Muller 5 system to detect any lethal mutations induced by treatment. These data are as follows: LATE LARVAE TREATED BY DIPPING IN 1% APHOLATE FOR 30-60 SECONDS Negative: 104 + 132 + 49 + 103 + 97 = 485 Positive: 8 + 2 + 1 + 9 + 0 = 20 3.9% mutations Metepa Extensive studies were made using Metepa. These studies were conducted on both Pisum sativum and Vicia faba. A. Peas The anaphase damage using metepa at different concen— trations was determined for the system. In this case, two types of results were obtained, as well: .oewm may cams 8mm OOOm one 8mm OOmH mo wooepmep Isoonoo_pe mowowep .m>afinoemoem. .zmuAemm ooomv 2 NIOH N :.H mmonAsmm OOmHv S NIOH N Nm0.0 mmmnAamm daemoospv 2 NIOH N mm:.o eo>h50 can soon: moeeodm .pdopmwmnom we pooemo mna .poommo soaflxma pom Aoaozo mpmv soon me me Ham3.me epoommm SSSMxes Aoaoao ohmv moon em webbm oswp momno> oweswo meanness mo pcooeom .Es>flpem ssmflm cw oweswo omopoz .gfi mesmflm meson ca mafia OJH OMH ONH OHH OOH om ow OF ow Om o: Om Om OH AI _ a — fl ~ _ _ _ d 4 _ DI. _ 48 o moon Honpqoo iIIl e oaoeos z m-oa x mma.o e adores z m-oa x emm.o pdospmoap emopos z.muoa x :.H \O CO 01 0 e1 e4 eSemep eseqdeue iueoxeg .d' :—l \O H ma 49 l. in which the maximum damage was at 27 hours, corres- ponding to the second cycle after treatment, and 2. in which the maximum damage was at 39 hours, corres- ponding to the 3rd cycle after treatment. The concentration of mete- pa used in these experiments ranged from .465X1O'2 M to 1.4)(10'2 M. In one experiment where three different concentrations were employed, the maximum damage in all three concentrations appeared at 39 hours (see Figure 7). These concentrations and their corresponding peak damages were: 0.4651(10‘2 M, 5.9%; 0.697x10"2 M, 14.4%; 1.4X10‘2 M, 17.2%. In this experiment a second maximum was obtained at the 6th cycle, although with the highest concentration it was not so pro- nounced. With the lower concentrations this second maximum was very well pronounced, however, and nearly equaled the first (see Table 6). The number of squares under the curves were also determined. Concentrations of .465X10“2 M, .697X10'2 M, and 1.4X10"2 M had values of 23, 68, and 64, respectively. These last two values are not signi- ficantly different from each other, which indicates that these two concentrations induced nearly equal amounts of damage (see Figure 7 and Table 6). The ratios of fragments to bridges were determined at .465X10"2 M and l-4X10'2 M, and these, also, were found to be the same. In another experiment in which a series of concentrations were used, ranging from 0.58X1O"2 M to 1.16X10"2 M, the maximum effects were obtained at 27 hours, which corresponds to the second cycle after treat- 5O .psopwHweo@20d mH moHss apoommo SostmS moHoho ocmv noon Pm mzosm oSHp pmeHmmm poppOHm omesmo omesmmde pdooeom .85>HHMm somHm dH omoamo cameo: mason QH oSHB oe mm on mm Om m: 0: mm om mm om .w made& mH OH H _ _ _ _ _ _ _ A _ _ H _ mood Hoapqoo I macros z NIOH x 3.0 - adopts 2 NIOH x ms.o wages 2 mIOH x mad 1 serves 2 NIOH x OH.H OH NH :H OH wH ON eSemep eseudeue iueoieg 51 695.390 mHSEoo. ImHmHomqoo mun nod? Poomwo gauges Echo ENV .30: pm .933 .m> owmseo endowment pdooaom .aoeHoeem dumHm 9H mefimd adopts .m 253% mason :H 259 mm 0.8 mm On 3 0.: mm cm mm om ma . ca m _ a _‘ _ _ _ . _ _ . _ A _ _ _ .... t _/ t \ (/ o \\ $ ... z \\ /{/¥. + x J \ 2 /~ \+ \ /+/ \ II . N \ It. a \ I o ... \+ \\ 3., \.. \ /» K. \ x.» \ \ i/ s \ x/i ... \ moom Hoepcoo I III... //$ xx \\ mgopoa S NIOH x wmd I 0'0. . x/a \ \\ emopoa S NIOH x FOOOI I . //$ \x\\ amoeba 2 NIOH x $30 I 0. 0 xx/ \\\ emopos 2 NIOH x mad I.X|XLX A/ t\-\ m . I III.II r \ m opus z NIOH x OH H /«\\ .OH NH :H OH mH ON eSemep eseqdeue iueoaeg 52 .UoHHOHm who mpdoSHthXo psoHomme 03¢ mo mpHomoa one smeaw mHsp QH .omoo pquemm ooppOHm mH omwswc madameqe pdoosom .85>Hpem abmHm sH emopoa wQHm: o>ado poommo omom .mooepqsn :H mammv omog mm em mm mm Hm cm ma ma ea ma ma _ _ _ _ _ _ _ _ _ a _ .mss am so oooeao room e do: o>0bw one mo spom .m oedeh aoam ommgo omwnmese Mama I 0 .w oeome scam mmeMU omenmeme xcom I nu mH _ .oa essmaa mH wH ON eSemep eseudeue iueoxeg 53 .oHoho,mH£p sH hmHop deoHMHGmHm on qupeOdeH .meom oopeohp one Hopscoo neon.dH mHoho pom one QH oaHp 08mm 839 pSOQN pe.HMomme mbHOHthom one .oHozo pmeHw one QH mason N paced mH heHoo obH .msoos OH HHp Ins homage poo UHU mpHOHthom «mood ompeoep cH .maoon QH oEHp pmcHewe toHPOHm mH thOHthom psooaom .85>Hpem abmHm so pdoSpwmep emopoa an comoeo heHoo oSHp oHoAO .HH meome meson QH oSHB 0: mm Om :m Nm Om. ON -ON :N NN ON .mHI OH 3H NH OH H _ _ _ H _. _ _\ H _ _. ,.j _ \\o I \\ nu.\\\ \ I If \. nu \. .\ nu.\\\ nu \\\. u x I I \\. I. .\ \ .\ \ \ - \\, .oHoho onooom .L 38585 metres 2 TS x mmm. - II..- .oHoho oqooom .HofiEoo I UIIID .oHoho pmHHO £818.83 oaoeos 2 Tea x mmm. I I .oHoho 0.93.“ eHoaedoo I OIIIO om 0: Om 0O Ob ow om Kptotdfitod iueoxea 54 ment. These peaks were 11.6%, 15.6%, 16.4%, and 19.5% for the re- spective concentrations of 0.58X10'2 M, 0.79X10'“2 M, 0.93X10'2 M, and 1.16XlO"3 M (see Figure 8 and Table 7). In another experiment which duplicated the previous one (ex- cept that one more concentration of metepa l:,0.697X10"'2 M] was used), the results obtained were comparable. Second cycle nonpersistent effect was obtained in this case, also (see Figure 9 and Table 8). The peak effects were plotted against the respective doses, as shown in the dose effect curve in Figure 10. Mitotic indices were determined in all of the above experi— ments (see Tables 6, 7, and 8[M IQ). It was observed that the M Is remained rather steady (at about control level) in the experiment in which a third cycle peak was obtained. However, in two experiments which showed a second cycle peak damage, the M Is dropped considerably below control level after 39 hours. Cycle time delay for both the first and second cycle after treatment with .692x10-2 M of metepa were determined (see Figure 11) . In the control peas, polyploids appeared earlier than in the treated ones. In both treated and control peas, the percentage of polyploidy was determined for at least three different hours. The delay in the first cycle, due to 0.692X10'2 M treatment, was between 2.5 and 3.5 hours. ‘When percent polyploidy was determined for the second cycle, it was observed that in both control and treated peas the polyploids appeared at about the same time. Thus, there was no delay during the second cycle after metepa treatment. if. m. I .mwom GH mhdon QH oEHp momeo> psooaom 3H emanated pm As OmNV omeaep heaIx .NH mesmHm mason QH oSHB Om ON ON :N NN ON wH OH 3H NH OH O O z N OI _ _ _ _ _ _ _ _ . _ _ _ _ _ 55 neon Hospdoo I wmesep ommsmmnm 8.2-x a 0mm - OIO III om mm eSemep eseqdeue iueoaeg Percent anaphase damage 72‘ 64‘ 56 48 40 32 24 16 56 O—-O- total damage ----- control peas -- - --_---------- -----—-—------*--—- 5 I 5 I l 4 8 16 24 32 40 48 56 64 72 80 Time in hours Figure 13. Total metaphase damage in Vicia, induced by 0.233 X 10-2 M metepa. % damage vs. time. 57 figtention of Metepa Acetone extracts were made of the treated roots at %, l, 2, 4, 8, 12, and 24 hours. These were then injected into a column in a gas chromotograph to detect the presence of metepa. It was found that within the limits of the detecting mechanism, .004 micrograms of mete- pa was present per root tip at 8 hours. No metepa could be detected beyond 8 hours after treatment. X-Ra Data 250 r of X-ray was used and sampled at 3, 6, 9, 12, 15, and 27 hours. The percent corresponding damages for the above hours were respectively; 25%, 24.2%, 9.9%, 11.3%, 13.3%, and 7% (see Figure 12 and Table 9). has Vicia primary roots were used mainly to determine the type of damage, time of first appearance, and cycle time delay. The concen- tration used in these studies were .233x10-'2 M. The damage was scored at metaphase. The total damage at 10, 25, 28, 45, 61, and 72 hours were respectively; 13, 57, 63, 57, 33, and 15% (see Figure13). The total damage was classified into: 1. chromatid exchanges 2. chromatid breaks 3. chromosome exchanges,and 4. chromosome breaks (see chart on page 58). 58 .moHoho_HHeo pnonoem Ipnm nH mpHnmen one nHenp one nmonHEHneHunnpe an oeononH mnoHpeneobe oHpeSonno one mnemoeonno mo nOHpeonHmweHO mpnosmenm one momoHnn manponapm eEOmoSonno moEOmoSonno op oeeH a oenopHe op oeeH muneswenw oanneo peonm one A A. one moonnp menoamenw oHpea mownenoxe newnenoxo mpnes OpeweoH Ionno N ow.oeoH omne>mnenp HenHoanwnOH Imenw oHymEOHno manHOWeH Meenp - MWWMWflMMM‘ Meomn oHpeEonnoome .o oHpeSonnoomMIhhmll, oHpe onno .9 oneSoano eHmnHm .e emeswo one aowso .m meonnn eanoo memoHnQ no oHNMMM/I\\\mwmenme endponnpm mace memoHMp manponnpm oaomoeonno IoSoeno $mnepHe on.%eeH pmowzpew oehepHe Op oeeH meH pow mpnesmeam nonemosonno mpnosmesm menoSMWHH mpneamenw OHMHnoo phone OHMHneoHo oannooe oannwmllll(\\\\anpneue ownenoxe on monoano .9 neonp enemosonno .e ./.\\\I\. oweseo mnemononno .H H Bm .— 510.. 'd/V/ Q) U) “3.. '8. 2 (6— ..p a- 0) C) $4 Q)— a. II LIIJLLII III 12 13 1h 15 16 18 2o 25 3o Dose (ppm) in hundreds (Log scale). Figure 19. Dose effect curVe, in which the maximum anaphase damage in percent is plotted against the corres- ponding dose on log scale (metepa treatments). 81 some breaks. Similarly, the appearance of a large number of centric fragments at later cycles may also be explained, by the same mechan— ism. Comparison of Effects A. Chromosomal aberrations Table 11 summarizes the comparative anaphase damage induced by all of the chemicals employed. Xigig.§§b§ is far more sensitive to damage than Eiggm_sativum. Tretamine is the most potent in caus~ ing chromosome damage, and the next in order of potency are apholate, metepa, TEPA, azirane, ethylenesulfide, and HEMPA. Ethylenesulfide easily dissociates at the pH employed, and is noneffective. On the basis of comparisons made in terms of molar concentrations related to damage, we found that tretamine is about 6 times as effective as apho- late. Apholate is about 2 to 2.5 times as effective as metepa. Azir- ane is only 1/17 times as effective as apholate in causing chromoso- somal damage. The poor chromosome-breaking ability of azirane is perhaps, due to its inability to cause cross linking of DNA, in addi- tion to alkylation. When the dose versus effect was plotted on Log Log paper (see Figures 17, 18, and 19) it is observed that in the case of both metepa and apholate we get a straight line, indicating the exponen- tial nature of the curve, which is the type of effect we would ex— pect from a chemical reaction of this nature involving a biological system. The straight line aspect of the Log Log curve in both of 82 these cases may also justify the assumption that both apholate and metepa have a similar mechanism of action. This is not surprising, since both are polyfunctional alkylating agents, and their reactive groups consist of the aziridinyl ring structures. B. Mitotic Inhibition Mitotic inhibition caused by the different chemicals was measured by means of mitotic index change after treatment. It was found that mitotic inhibition produced by tretamine and azirane is quite comparable, although the anaphase damage caused by the two were quite different. Apholate and.metepa are next in order of their abi- lity to induce mitotic inhibition. The high potency of azirane and tretamine in causing mitotic inhibition is probably due to their low molecular weight and consequent ease with which they can enter the treated cells. It is probably due to the extreme toxicity of azir- ane, (which is a monofunctional alkylating agent) that its chromosome- breaking ability is not so pronounced as that of the polyfunctional alkylating agents. C. Cycle Time Delay In peas, the first cycle was delayed for 2% to 3 hours, with % hour apholate treatment. In yigia, metepa caused a delay of 6 to 7 hours in the first cycle. The second cycle was not delayed in either Vicia or Pisum to any appreciable extent. It is suggested that this delay in the first cycle is caused in the G1 stage of interphase owing to the actual presence of the chemical within the system. I... 83 D. Mutagenic Ability In comparing the mutagenic ability of HEMPA and apholate, it is observed that 1% solution of both chemicals used for 1 minute on Drosophila larvae showed 3.9% lethal mutations in the X chromosome with apholate and only 1.9% in the case of HEMPA. This is not surpris- ing when we consider the molecular structure of both the chemicals. Apholate is a polyfunctional alkylating agent with a very reactive aziridynl ring structure. In HEMPA this ring structure does not exist, Since the CH2 group in the structure is replaced by CH3 groups which are far less reactive. This would also account for the difference in chromosome-breaking ability of these two chemicals. E. Comparison with X-ray Damage When we compare the damage caused by ethylenimines to X-ray induced damage, it soon becomes apparent that the term "radiomimetic" as applied to this group of chemicals loses its significance. The only similarity between the two types of damage is that both cause chromosomal aberrations, but the time of peak effects, the time of first appearance and the kinds and persistence of damage induced, are very different in the two cases. Probably the conditions affecting the production of damage and the susceptible stages of damage are also quite different. Wilson and Sparrow (1960) showed that irradia— tion of early interphase nuclei produces chromosomal aberrations, late interphase produces chromatid aberrations, and late prophase induces subchromatid aberrations, indicating the four-stranded condition of 8h the chromosome. It has already been pointed out that this type of se- quence is not found when these chemicals were used. So the peak effects due to X-ray treatment is obtained in the immediate mitosis following treatment. In our comparison to X-ray damage, about 250 r of X~ray treatment is comparable to 2500 ppm of apholate, and 3,000 ppm of me- tepa in causing chromosome damage (see Figure 12). 85 SUMMARY Ethylenimines and related compounds were employed in the pre— sent study to determine the amount and type of damage induced on Eiggm sativum and—yigig faba chromosomes. It was observed that these chemicals produce two main types of effects. These are primarily concerned with time of maximum damage and persistence of damage. Using apholate and metepa on Pisum, a maxi- mum damage did not appear until a delay equal to at least one or two complete mitotic cycles; the average mitotic cycle time being 12 hours. The delay could be caused by unusual delay in treated cells entering division, or due to the initial unit of breakage. There are, however, some objections to these explanations. The time of susceptibility was tested using the 5 A U system. we are forced to conclude from the re— sults that there is no particular stage which is especially suscep— tible to these chemicals. Cycle time delay caused by treatment was ddtermined for the first and second cycle after treatment in both Pisum and Vicia. It was found that the first cycle in Vicia was delayed approximately 6 to 7 hours, and in Pisum this delay was approximately 3 hours. In both Vicia and Pisum the second and subsequent cycles after treat- ment Were not delayed to any extent. 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APPENDIX 99 100 ma I I we mm m ew.o I I I H m.o mo ON I I I mm mm ea.a I I I I H.H Hm em I I I on om e:.m I I H.m m.o w.s mm we I I 0: mm om ea.m I I w.m w.e s.: em ma I I I H mm eo.a I a I I a ma mm I I I mm we em.m I I I I m.m me we I om oa m mm em.m I I I . m.m am as I I I me me em.m I w.o m.s m.o o.m am am I I ms em as em.m I I m.m m.w m.m em ea I am s m m ea.a H.H I I I I me I I I I I I I I I I I I me I I I I I I I I I I I I am I I I I I I I I I I I I am we I I os so me em.a I I I a m.a em m.mm I mm mm em an I I I I I I ma .H .2 .>< m e m m a a .mooa m e m m a .mem a_ooaam a.ooaam xmozH OHBOBHS mezmzeama .oooaflnm 2 NIOH N mw.H I w .xm ”compass 2 NIOH x NN.H I b .xm mApmoepoomp oqv Hoapooo I @ .xm "worsens an dooocsw «somwm cw mason mSOHam> pm mooflcefi OHpOpHE com pdooaom ea owoeme ommnmde< .H canoe . .. , I... a .. ... » um uI. . .1qu I IIIIIII.IoI Table 2. Anaphase damage in percent)induced by 3.1 X 10'2 M 101 of apholate in Pisum. FD 19 FRAGMENTS Slide # Hrs 1 2 3 4 5 Frag. % 11 3.7 1 1 — 2.1 ' - 12 — — — — - }- 4.5% 13 4.2 — 11.6 - — 23 - 23 1 - — 18.3 24 - - - - 15.3 18.0% 25 26.1 - 7.5 — - 35 23.8 31.4 17.3 - - 36 22 34.5 - - - 27.6% 37 25 39. - - - 47 31.5 22.4 - _ _ 48 34.6 - - - - 23.4% 49 14.8 13.8 - - - 59 31.3 - - 34 - 60 14.5 - - - - 26.2% 61 22.6 28.4 - - _ 71 14.5 14 - _ _ 72 22.2 15.1 - - - 16.6% 73 17.2 - - - - 83 12.8 11 — - - 84 10.2 — - - - 16 9% 85 32.3 14.7 9.5 27.8 - 954 8.2 37.8 11.1 7.2 12.2} 15.3% 102 am mm om I I mm em 6 a I I m an mm 0: mm ON I I go we m I I e we mm mm I I mm mm em.s I I m.m 6 s mm mm I I mm m: om ewe I we we we :a em em I I e: or On ea I I I a a ea .H .2 .sa m e m m a a .wooa m e m m a .oem Ema Eel... xmozH OHBQBHZ mezmseama me me IIIII .opoaoedo z mIoH x H.m eons oooooeo “ESmHm GH mason mSOHam> pm movaoca oepOpHe pom poooaom ow owoeme omeQmQ< .m magma Table 4. Maximum anaphase damage in percent, at peak hours in P_i__sum induced by different concentrations of apholate: _gD 24 - control (no treatment); FD 25 - 4.1 X 10 M apholate treatment; FD 26 - 2.1 X 10 3 M apholate treatment; FD 27 - 1.03 x 10-3 M apholate treatment; FD 28 - 0.52 x 10-3 M apholate treatment; FD 29 - 0.26 X 10'3 M apholate treatment; __ and FD 30 - 0.13 x 10-3 M apholate treatment. FRAGMENTS Slide-# Hrs. 1 2 3 4 Frag. % FD 24 35 620 2 3.6 - 36 ~ 015 — — — 2.1% 37 0.5 - - - FD 25 35 18.5 32 - — 36 29.5 18 12.6 15.4 37 20.3 27.6 15.6 6.1 18.4% 38 17 19.6 9.8 14.2 FD 26 35 8.5 - - - 36 10.5 14.8 10 13.9 11.6% 37 8.3 18.8 15.3 3.4 FD 27 35 11 13 2 10.8 - 36 5.8 - 6.8 - 8.6% 37 6.6 6 9.4 - FD 28 35 6 - - _ 36 5.4 14 3 4 0 - 37 8.7 - - - 7.9% 38 9.2 - - - FD 29 35 5.1 - 3 - 36 5.3 - 4 3 3 3 37 4.5 - - 4.5% 38 4 - 6.5 FD 3O 35 2 1 — 36 1.5 — 1.9% 37 2 - - 103 104 .H .2 .3. m e m m a e .mona m e m m a .22 % 93.8 $2 obwam xHQZH OHHOBHE mezmzweemm Edam .Hoapqoobe mm .Ssmflm 2H mason mdoflao> pm moowcsfl oHQOpHS com owosme ommmmoe< .m m canoe 105 em I or me an o: I I I I I an no - - - an an I I I I I e. I I I I I I I I I I I mm I I I I l l I I I I I 0m mm I I I mm mm I I I I I am so I I mm or am I I I I I mm mm mma mma mm me me I I I I I am m.aw I I am om or I I I I I om am I I I or m: I I I I I em I I I I I I I I I I I mm ow I I I or mm I I I I I mm I I I I I I I I I I I am I I I I I I I I I I I om mm I :6 mm mm mm I I I I I ma me I I I me am I I I I I we mma so am msa oma oma I I I I I ea msa oma can one omm mam em.a I m.m a m.o ma mma :am am as mm Hma I I I I I ma 2.0: H: mm am am mm I I I I I Mme m.:a em om aa a o I I I I I 4» .H .2 .>< m a m N a a .mona m m m a .oem % ooeam a ooeam meZH 6HeoeH2 222c< m H m m H .I16ooa m H 6 m H .oem a ooHHm % ooHHm xHHHH OHHOHH2 6H2H2oHpoc msHHSU opmaonmm HHHs empower I mm mm .mnsoe mH Ho oeoHoedo 2 mIoH x H.m HHHs one meson 6 How D a m 29H: popcoap somHm 2H mooHeoH UHPOHHE new Hsooaom sH 666866 commemoa .6 m pomH 109 6N 6H I 6N sN mN sm.6 I I I m.s 6.0H Hm NH HH Hm 6N mm 6m s6.NH I I I 6H m.NH NH 6.0m I I I 0N 6s mm s6.NH I I m.m H.NH 6.mH mm Hm I I Hm s6 6H sm.NH I H.0H H.6H m.mH I N6 6.mH om 6H mm 6H sm 66.6H N.mH H.HH I I I 06 m.mm I os 66 6s 6N as.m I s.6 H.s H.NH I sN oH I 6m HH 6H mm 26.6 I I I I 6.6 6N m.6H I NN Hm NH I ss.m I m N.6 I I HN m.6m I I I 66 Hm 6N.m I I I m.m m HN mH I 6m Hm 6m sN sm.N I H 6.H 6.N 6.6 6H m.mmH I I omH osH 6HH _H6.N I I m 6.H m.m sH s6H I I I HsH 06H I I I I I I 6H sON H6H osH NmH mHN s6N .66.H m 6.H s.H m.6 H.N mH m.m I I I N m I I I I I I NH H I I I H H I I I I I I oH 6.m 6 m m m m I I I I I I 6 .H .2 .>2 m H m N H s .monH m H m N H .622 H.66HH6 a ooHHm 2 H H 2 H o H H o H H 2 m H 2 H 2 c H H 2 6m HH .ommpm .m. wHHH56 pooEpooHp oHoHoHHoI6m HH .oneoe «0H 66 oHoHoHHo 2 NIOH 2 H.m 26H: oeo meson 6 too D < m Hsz 66pmmap somHm 2H mooH6cH oHHopHE 626 peooamm 2H 666866 mmmsmmo< .H m pomB HH I I mH _s6, ss.H I I H ..m H maN NHH H6 I I 0s 6m 6N.N I I H m.N 6 00H m.06 I I 66 6m 60.6 I 6 N NH 6.N 66 66 I I s6 m6 ss.H I I 6 m.m m 6s m.NH I I mH 0H ss.N I I N 6 6 H6 N.6m I I Nm mm so.m I I 6 m.s m Nm m.66 I I sH 06 ss.m I 6.m H 6 N.s HH 6m I I I 6m s6.m I I 6 m.H sN m.6m I I 66 06 sm.N I I 6 N NH 6H I I Hm HH I I I I I 0 Hs 2H I I I I I I I I I I NHH I I I I I I I I I I 00H I I I I I I I I I I 66 6m I I 6H sm sm.H I I H N 6s I I I I I I I I I I H6 m I I I I I I I I I I Nm 1 I I I I I I I I I I H: 6s I 60 sH ss 60.N I m.H 6 m.H 0.N sN I I I I I I I I I I NH m.6m I I I m.6m s6.H I I H m.H 0 6s 2H .H .2 .ea m 6 N H s .monH m H N H .622 H oeHHm % ooHHm o H H O H H 2 m H z m S 0 < m m NI0H 2 s06.0 I ms 0H .peospooHp mgopms 2 NI0H x H.H I ms Dm mpHoEPdoHp wmopos S NI0H x mmz.o I :2 DH mApsoEHmep osv HOHpHoo I mm DH "mmmpoe mo mHOHpmHHQoocoo pooHoHHH6 HHHS 66pmoHp «Hoofipmoap 690208 2 “somHm 2H mason meHHm> pm m60H6HH oHPOHHE 6cm peooawm 2H 666266 mmosmmqa .6oiaH 111 s6 0m N6 m.6H NH H.s.N N 6 6 NHH m.6 m.H H 00H m.N H m.N 66 m.s m.6 m.6H 6s H 0H s H6 m.6 m m.6 Nm m.0H m.6H m.sH HH N m.HH m.s sN m.0H 6.H s NH I I I 0 I I m.N 6HH I m.m m.6 HNH 6.H m m.6 NHH m.6 s m.HH 00H 0 m.s 0 66 m.sH 6H 6 6s I 6 m.6 H6 HH HH 6 Nm I m.6H m.mH HH m.0H 6 6 sN I I m.N NH 0 eoseHHooo n6 oHoeH ms. DH ms Dm 6H I I I 6H 6H I I I I I I ms sN I I 6H H6 H6 ss.N I I 6.H 6.6 6. 66 m.NN I I I 6H sN s6IN I I 6 m.N 6.H Hm H6 I I I 06 N6 s6.m I m.m m.6 6 m.H m6 m6 I I I H6 06 s6.HH I 6 m.HH HH m.NH sN E I I I 6H 6H 6.6.6 I I .6 m.6 6 H 66 on m.6H I I I 0N 6H I I I I I I ms H.HH I s6 0H 0m HH I I I I I I Hm m6 I I I N6 6H I I I I I I 06 2 06 I I I H6 Ns ss.N I I 6 0 N sN u Hs I I I 0s 6s s6.N I I m.6 H.H N mH s6 62 .H .2 .>< m H 6 N H s .6onH m H 6 N H .622 6 oeHHm H oeHHm 2HH2H 0HH0HH2 6H2H20 Pm moOH62H oHPOPHS 6cm PmooHom 2H 666866 ommSQmH¢ .2 66968 113 H6 m.HH sH m.sm Hm m.m 6H NN m.66 m.mm :H m.HH Hm ms 6H OH Hs e. Hm m6 HH 6H sN 66 mH Hm sH 66 mH Hm mm mm mH mm mm mm 6N6 sm.mH HH.6H 6.6.6 6.1.3 6N.HH 6N.s 66.3 66.6 ,m.HH NH 6 s H.6H 6.6 6622Hpsoo m.m @H HH 6 m.sH s ON m.6H ms 66 Hm m6 sm mH ms 66 Hm m6 sm mH ms 66 Hm m6 sm mH oHQmB HH 02 0: mm mm 62 6. I 06 - - - 6m 66 66.6 - - 6.6 6.6 6.0 66 6.66 - - - 6 6H 66.6 - - :.: 6.6 6.0 66 6.66 - - 66 - 66 66.6 - - 6.6 6.6a 6.6 66 j: - 6: - - 0: 66.06 - 6.06 6.HH 6.6 NH 66 66 - - 6m :6 :6 66.6 - - - 6 6.06 66 N6 66 6: - - - 66 66 66.: - - z 6 6.6 66 6.66 - - - 66 66 60.06 - - 06 6.6 66 66 6.66 - - 66 66 6: 66.6 - - 6 6.HH 6.6 6H 66 66 6.66 - - - 6H 06 - - - - - - 66 66 - - l 6H 66 - - - - - I H6 66 66 - - - 6H 6 - - - - - - 66 1 66 - - - 06 06 60.6 - - - m a 66 z: - - - - x: 60.6 - - - m H 66 06 66 .H .2 .>< 6 z 6 m H 6 .6666 6 z 6 m H .whm % @6666 6.66666 xmnzH OHeoaHz 662620666 5688866666 ammoba E NIOH x 66.6 - 66 cm can mpcmapmmhp 666668 2 6-06 x 66.0 - :6 66 606 MPQmEpamgp ammpms z -06 x 66.0 - 66 66 6626566666 mmmpme 2 6-06 x 666. - 66 cm mpsmSpmmhp magma 2 NIOH x 66.0 I Hm 6.m 26:66:60,66ch 05 6.0.3080 I Om 6.m “566m G6 .696me .60 3060766636800 2986.666 :96: 68080166666 3026.6 6.30: 6506.866 026 6606626 0306.68 665 0,060th ca. 6668.66 666696.36. .6 66966.. 115 66 m.wm 0: 6 .66 6 .66 66 66 6.0m 6 .66 H6 66. 66 6: Om 66 66 66.06 6:.66 66.66 I I ®.® 6.mH m.6m 6.66 I I 6.6 I I m.m I w 6.: I m.m 6.66 I 6H 6.66 I I m.:H I m.m m I ma MA I m.NH H.HH dwdmflpcoo 66 66 .3 Lr\I-i I-—|C\J P (\1 me0 00806061 LAP I—€(\J N31 r—i L\ (\J .6 6366 mm mm :6 am mm om Table 9. 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