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Induced and spontaneous chromosomal aberrations in cultured human leukocytes Andrews, John Charles 1969

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INDUCED AND SPONTANEOUS CHROMOSOMAL ABERRATIONS IN CULTURED HUMAN LEUKOCYTES by JOHN CHARLES ANDREWS B.Sc, University of B r i t i s h Columbia, 1965 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN UKNKTKS in the D i v i s i o n of Medical Genetics We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA MAY 1969 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and Study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thes.is for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada - i I ABSTRACT The frequency of chromosome breaks was increased i n r e p l i -cate cultures from each of ten individuals when l y s e r g i c acid diethylamide at a concentration of 1 ug/ml of culture was added 24 hours p r i o r to the harvest of the c e l l s . The differences between the control and treated cultures ranged from +3.00 to +7.93 with a mean of +4.63, indicating no v a r i a t i o n i n response between i n d i v i d u a l s . The breaks were randomly d i s t r i b u t e d among the seven groups of chromosomes of the complement. No s i g n i f i c a n t difference i n either the number of c e l l s with aberrations, or the number of.breakage events was observed between c e l l s cultured from a patient with Fanconi's anaemia before and 24 hours after treatment with 250 ug. of growth hor-mone. Both were s i g n i f i c a n t l y increased over the contr o l . After treatment with growth hormone, the number of breaks per aberrant c e l l was decreased, and the d i s t r i b u t i o n of frequencies of s p e c i -f i c types of aberrations was changed. Non-homologous exchange figures were the only two break events observed i n cultures from the patient. None were observed i n control c e l l s . The d i s t r i b u -t i o n of breaks among the seven groups of chromosomes was random. The frequency of chromosome aberrations was increased in cultures from a single i n d i v i d u a l when treated with 1 ug/ml of mitomycin-C for one hour at the beginning of the culture period. In the treated cultures, 181 breaks were observed in 64 of the 100 c e l l s examined, whereas only 5 breaks were observed in three of the 100 c e l l s scored i n the control samples. Forty-seven exchange configurations were observed in the treated cultures, 42.56% of these being non-homologous exchanges. No marker or d i c e n t r i c chromosomes were observed. Breaks were randomly d i s t r i b u t e d among the seven groups of chromosomes of the complement. II ACKNOWLEDGEMENTS The author g r a t e f u l l y acknowledges the support and d i r e c t i o n provided by his committee chairman, Dr. M. J . Corey of the D i v i -sion of Medical Genetics. Dr. Corey also deserves s p e c i a l thanks for her continued inte r e s t and enthusiasm in d i r e c t i n g the research, and assistance in the preparation of the thes i s . Special thanks are also given to Dr. J . R. M i l l e r , Head of the D i v i s i o n of Medical Genetics, for his d i r e c t i o n and encourage-ment in a l l aspects of the project. Appreciation i s expressed to Dr. S. Israels, Head of the Department of Paediatrics, Dr. T. Bisalputra, Department of Botany, and Dr. C. Finnegan, Department of Zoology for their suggestions and comments. Technical assistance was received from several people in the D i v i s i o n of Medical Genetics. I am e s p e c i a l l y g r a t e f u l to Mrs. L. McCarthy and Miss J . McLeod both of whom gave f r e e l y of the i r time and understanding of culture technique procedures. My wife Karen assisted in several phases in the preparation of the thes i s . Without her continued support and encouragement t h i s work would not have been possible. Special thanks also go to Mrs. J u l i a Andrews and Mrs. Deanna Dong for typing of the manuscript. This work was supported by a grant from the Medical Research Council of Canada to Dr. M. J. Corey. - iv -TABLE OF CONTENTS I ABSTRACT i II ACKNOWLEDGEMENTS i i i III LIST OF TABLES v i i IV LIST OF FIGURES v i i i V INTRODUCTION 1 A. E f f e c t s of Lysergic Acid Diethylamide (LSD) In V i t r o 2 B. E f f e c t s of LSD In Vivo 3 C. E f f e c t s of LSD In Utero 5 D. Lysergic Acid Diethylamide - E f f e c t s on Meiotic Chromosomes 6 E. Fanconi's Anaemia 7 F. E f f e c t s of. Mitomycin rC In_ Vivo and ln_ V i t r o 8 VI MATERIALS AND METHODS 11 A. Experimental Procedures 11 1. Experiments Using Lysergic Acid Diethylamide (LSD) 11 2. Treatment of Leukocytes from a Patient with Fanconi's Anaemia with Growth Hormone 12 3. Experiments Using Mitomycin-C 12 4. Blinding and Coding of Slides 13 5. Microscopy 13 B. C r i t e r i a Used to Define Chromosome Aberrations 14 C. Determination of the D i s t r i b u t i o n of Breakage Among the Groups of Chromosomes 14 - V -VII RESULTS 15 A. Lysergic Acid Diethylamide -In V i t r o Study 15 1. Single Break Events 18 2. Two Break Events 22 3. Gaps 22 4. D i s t r i b u t i o n of Breakage Among Chromosome Groups 24 B. E f f e c t s of Growth Hormone on a Patient with Fanconi's Anaemia 25 1. Single Break Events 31 2. Two Break Events 31 3. Gaps 31 4. D i s t r i b u t i o n of Breaks Among Chromosome Groups 33 C. Mitomycin-C 35 1. Single Break Events 35 2. Two Break Events 40 3. Gaps 40 4. D i s t r i b u t i o n of Break Events Among Chromosome Groups 42 VIII DISCUSSION 44 A. Lysergic Acid Diethylamide 44 B. Fanconi's Anaemia 46 C. Mitomycin-C 49 - v i -IX SUMMARY 53 A. Lysergic Acid Diethylamide 53 B. Fanconi's Anaemia 53 C. Mitomycin-C 54 X REFERENCES 56 APPENDIX A. - Leukocyte Culture Technique 60 APPENDIX B. - Detailed Results for LSD 1-10 63 APPENDIX C. - Detailed Results of Before and After Treatment with Growth Hormone on a Patient with Fanconi's Anaemia 74 APPENDIX D. - Detailed Analysis of Treatment with Mitomycin-C 76 - v i i -III LIST OF TABLES LYSERGIC ACID DIETHYLAMIDE (LSD) I Summary of the Results of Chromosome Analysis i n Treated and Control Cultures 16 II Deviations Between Treated and Control Cultures 17 III Frequency of Single and Two Break Events Observed i n Treated and Control Cultures 19 IV D i s t r i b u t i o n of I d e n t i f i a b l e Breaks Among the Chromosome Groups 23 FANCONI'S ANAEMIA V Summary of Results of Chromosome Analysis Before and After Treatment with Growth Hormone, and i n a 'Standard' 2 6 VI Frequency of Single and Two Break Events Observed i n Cultures Before and After Treatment with Growth Hormone, and i n a 'Standard' 28 VII I d e n t i f i c a t i o n of Chromosomes Involved i n Exchange Figures ; 32 VIII D i s t r i b u t i o n of I d e n t i f i a b l e Breaks Before and After Treatment with Growth Hormone 34 MITOMYCIN-C IX Summary of Results of Chromosome Analysis in Treated and Control Cultures 36 X Frequency of Single and Two Break Events in Treated and Control Cultures 39 XI I d e n t i f i c a t i o n of Chromosomes Involved in Exchange Figures i n Treated Cultures 41 XII D i s t r i b u t i o n of I d e n t i f i a b l e Breaks Among Chromosome Groups 43 - v i i i -IV LIST OF FIGURES Figure 1. Representative Types of Chromosomal Aberrations Observed in Control Cultures and i n Cultures Exposed to Lysergic Acid Diethylamide 20-21 2. Representative Types of Chromosomal Aberrations Observed Before and After Treatment with Growth Hormone in a Patient with Fanconi's Anaemia 29-30 3. Representative Types of Chromosomal Aberrations Observed i n Cultures Exposed to Mitomycin-C 37-38 - 1 -V INTRODUCTION The association of chromosomal aberrations with congenital malformations and malignancy has stimulated an increased interest in the study of the e f f e c t s of a wide v a r i e t y of agents on the chromosomes. Such studies have a p o t e n t i a l p r a c t i c a l , as well as academic, value as they provide a means of i d e n t i f y i n g cyto-toxic and possibly teratogenic or carcinogenic agents. Some of the agents which induce chromosomal aberrations were f i r s t i d e n t i f i e d by plant cytogeneticists. Extensive review a r t i c l e s o u t l i n i n g the e f f e c t s of a v a r i e t y of chemical and physi-c a l agents on the chromosomes have been published (Darlington and Roller, 1947; Oehlkers, 1952; Revell, 1952). Plant cytogeneticists had access to material in which a large number of c e l l d i v i s i o n s were natu r a l l y occurring. However, the mammalian cytogeneticist was hindered by the technical d i f f i -c u l t y of obtaining adequate numbers of d i v i d i n g c e l l s for analysis. Consequently, mammalian cytogenetics and p a r t i c u l a r l y human cytogenetics, remained a r e s t r i c t e d f i e l d of study u n t i l the middle nineteen f i f t i e s when a s a t i s f a c t o r y i n v i t r o system was devised. The discovery that phytohemagglutinin (PHA) stimulated d i v i s i o n of c i r c u l a t i n g human white blood c e l l s , made i t possible to obtain adequate numbers of r a p i d l y d i v i d i n g c e l l s in culture (Nowell, 1960). Refinements were soon made to the i n v i t r o culture technique providing a convenient method of chromosome preparation suitable for detailed analysis (Moorhead et a l . 1960). - 2 -With the advent of a technique that enabled researchers to study the morphology of the human chromosome complement, a system for the c l a s s i f i c a t i o n of the chromosomes that would become u n i v e r s a l l y recognized was established. This was effected in 1960 by a "Proposed System of Nomenclature of Human Mi t o t i c Chromosomes" (Denver Report, 1960). This was subsequently revised in London and Chicago (Chicago Conference, 1966). Aside from being a useful diagnostic aid in determining chromosome abnormalities responsible for some congenital mal-formations, the three day PHA stimulated leukocyte culture of a few drops of human blood provides a rapid and convenient system for studying the e f f e c t s of a great va r i e t y of agents on the chromosomes. The study presented here was car r i e d out to investigate the frequency and type of chromosomal aberrations observed in human peripheral leukocytes exposed in_ v i t r o to (a) l y s e r g i c acid diethylamide, and (b) mitomycin-C. A comparison of the frequency and types of chromosomal aberrations in samples obtained from a patient with Fanconi's Anaemia before and after treatment with growth hormone i s also included. A. E f f e c t s of Lysergic Acid Diethylamide (LSD) In V i t r o L i t t l e work has been reported on the effects of LSD i n  v i t r o i n comparison to the number of reports published on i n  vivo studies; only two reports, both by Cohen and his associates, have appeared i n the literature„(Cohen, Marinello, and Black, - 3 -1967; Cohen, Hirschhorn, and Frosch, 1967). They reported f i n d -ing a marked depression of mitoses in d i v i d i n g c e l l polutions, and an increase in chromosomal aberrations when leukocyte cultures were treated with LSD. Most the chromosome aberrations observed in these studies were chromatid and isochromatid breaks, although apparent exchange figures and d i c e n t r i c chromosomes were also observed. The exchange configurations observed by Cohen ejt a_l. (l967_)t in the LSD treated cultures resembled those i n cultures exposed to mitomycin-C (Nowell, 1964; Cohen and Shaw, 1964) and those found in two autosomal recessive diseases, Fanconi's anaemia (Bloom e_t aJL. 1966; German and Crippa, 1966) and Bloom's syndrome (German, Archibald, and Bloom, 1965). The frequency of aberrations i s , however, much lower i n LSD treated cultures. Neither the mechanism nor the phase of the mitotic cycle at which LSD i s e f f e c t i v e has been investigated. B. E f f e c t s of LSD In Vivo The considerable use of LSD by young people in the l a s t few years had led researchers to investigate not only i t s effects on chromosomes but also i t s possible mutagenic and teratogenic e f f e c t s . At the present time, several authors have reported finding an increased l e v e l of chromosome damage in 'users ' of LSD (Cohen, Hirschhorn, and Frosch, 1967; Hirschhorn and Cohen, 1967; Irwin and Egozcue, 1967; Egozcue, Irwin and Maruffo, 1968). Cohen et a l . (l967) observed that the frequency of chromosomal aberrations ranged from 5.3% to 25.1% i n the leukocytes from 18 LSD 'users'. i n a series of drug free controls, the frequency of chromosomal aberrations ranged from 2.3% to 5.5%, indicating a three to four times increase i n damage i n treated cultures. Irwin and Egozcue (1967) observed an increased l e v e l of chromosome breakage i n 6 out of 8 LSD 'users 1 . Control breakage frequencies ranged from 7 to 25% whereas patients ingesting LSD demonstrated a 12 to 38% breakage rate. Egozcue et a l . (1968) in a later series of drug free con-t r o l s and LSD 'users', observed control breakage rates to vary from 6.0 to 16.5% while individuals who had ingested LSD demon-strated breakage values ranging from 8 to 45%. A two f o l d increase i n chromosome damage was observed in LSD 'users 1. Neither Cohen et a l . (1967) nor Irwin and Egozcue (1967) observed a r e l a t i o n s h i p between the frequency of chromosome break-age and the number of doses taken, the amount of the dose, or the period of time which had elapsed between the l a s t dose and the time at which the study was undertaken. Hungerford et. a_l. (1968) reported finding a transient increase of chromosomal abnormalities when LSD was injected intraveneously, with a return to control frequencies one month after administration of the f i n a l dose. A number of researchers have reported finding no increase i n chromosome damage i n individuals ingesting LSD (Loughman et a l . 1967; Bender and Sankar, 1968; Sparkes, Melnyk, and Bozzetti, 1968). - 5 -Loughman et aJL. (1967) reported that leukocytes from 8 patients receiving large doses of LSD (4,000 ug.) f a i l e d to show a s i g n i f i c a n t l y higher breakage rate than in the control cultures. Bender and Sankar (1968) reported that seven children treated for periods of from 5^ to 35 months, f a i l e d to show a s i g n i f i c a n t increase in chromosome breakage. Chromosome studies were not performed u n t i l 20 to 48 months after the l a s t dose. Sparkes et a l . (1968) also found no s i g n i f i c a n t increase i n chromosome damage in a group of 'users' and medically treated individuals over the controls. The reported findings of chromosome damage caused by inges-t i o n of LSD, are c o n f l i c t i n g , and a d e f i n i t i v e statement as to whether LSD does or does not increase the frequency of chromosome damage i s not possible at t h i s time. C. E f f e c t s of LSD In Utero The e f f e c t s of LSD on the chromosomes of children whose mothers had taken the drug during pregnancy have been reported (Cohen, Hirschhorn, and Frosch, 1967; Egozcue ejt a l . 1968; Zellweger et a l . 1967; Cohen et a l . 1968). Cohen et a l . (1968) observed a high frequency of chromosome damage (13 and 19%) in two of four children exposed to LSD i n utero during the t h i r d and fourth months of pregnancy. Egozcue e_t a l . (1968) also studied four children whose mothers took LSD during pregnancy. Three of these showed a high frequency of chromosome breaks, (range 22-28%) Cohen et_ a l . (1968) reported finding an increased frequency of breakage among children exposed to LSD i n utero when - 6 -compared to matched controls. Although there i s a great deal of controversy over the possible teratogenic e f f e c t s of in-utero exposure to LSD, only one report of congenital malformations in a c h i l d whose mother ingested LSD during pregnancy has appeared. Zellweger and h i s colleagues (1967) described a case of a c h i l d born with lower leg deformities, and i t was known that the mother had ingested LSD early i n her pregnancy. The authors f e l t that i t was not unrea-sonable to assume a casual r e l a t i o n s h i p between the LSD and the limb deformities. Sato and Pergament (1968) found no f o e t a l abnormalities in a c h i l d born to a mother who had ingested LSD early i n the pregnancy. Teratogenic e f f e c t s of LSD i n rats and hamsters have been reported (Alexander et a l . 1967; Geber, 1967; Auerbach and Rugowski, 1967). The most common abnormalities have been those of the brain, s p i n a l cord and l i v e r , with abortion and under-developed o f f s p r i n g also prevalent. Warkany and Takacs (1968) reported finding no s i g n i f i c a n t increase in abnormalities when LSD was injected into 55 pregnant r a t s . The mutagenic e f f e c t s of LSD i n Drosophila have been reported by Browning (1968) . However, Grace et_ a_l. (1968) found no evidence of chromosome breakage or mutations. D. Lysergic Acid Diethylamide - E f f e c t s on Meiotic Chromosomes Damage to meiotic chromosomes of mice injected with LSD V - 7 -has been reported. Skakkebaek et aJL. (1968) reported finding several breaks, gaps, and u n i d e n t i f i a b l e fragments i n treated animals, but with few exceptions, not i n the controls. Cohen and Mukherjee (1968) observed a ten f o l d increase in chromosome damage in spermatogonial c e l l s and bone marrow c e l l s of mice treated with LSD i n comparison to the controls. E. Fanconi's Anaemia Fanconi's anaemia i s a syndrome of multiple congenital mal-formations and progressive pancytopenia which i s thought to be inherited as an autosomal recessive. Cytogenetically the condi-t i o n i s characterized by a va r i e t y of chromosomal anomalies con-s i s t i n g of chromatid and isochromatid gaps and breaks, fragments, and exchange configurations. (Schmid e_t a l . 1965; Bloom et a l . 1966);. Schmid et a l . (1964) were the f i r s t to recognize the chromo-somal damage i n patients with Fanconi's anaemia. Bloom e_t al_. (1966) studied the peripheral leukocytes of 12 patients with t h i s disorder. A va r i e t y of s t r u c t u r a l exchanges, endoreduplications and other chromosomal aberrations were observed in ten of the twelve patients. In the leukocyte cultures, 315 c e l l s , (19.43%) of the 1,621 c e l l s scored had a breakage event of one type or another. In bone marrow preparations from one of the patients, 18% of the c e l l s examined possessed a breakage event. Seventy-eight chromatid exchanges were observed i n the leukocyte cultures, and two were observed i n the c e l l s of the - 8 -bone marrow. A large number of control c e l l s (approximately 15,000) were studied to determine the frequency of aberrations in patients without t h i s form of c o n s t i t u t i o n a l a p l a s t i c anaemia. Eight chromatid exchanges and only a few endoreduplications were observed. Swift and Hirschhorn (1966) studied two patients with t h i s disorder. In both cases the peripheral leukocytes and fibr o b l a s t s were studied, and i n one case the bone marrow. In the leukocyte cultures of both cases, 49% of the c e l l s were abnormal. I t was also observed i n the f i b r o b l a s t cultures of case I that 41% of the c e l l s were abnormal while i n case II, 36% of the c e l l s were aberrant. Chromatid and isochromatid gaps and breaks, fragments, exchange figures and d i c e n t r i c chromosomes were observed in leukocyte and f i b r o b l a s t cultures. Ten percent of the bone marrow c e l l s contained abnormalities, including chromatid and i s o -chromatid breaks and gaps, fragments and exchange figures but no r i n g or d i c e n t r i c chromosomes. . To date, there has been no apparent success i n determining 'the underlying causes responsible for the degenerative aplasia or the associated congenital malformations observed i n t h i s defect. F. E f f e c t s of Mitomycin-C In Vivo and In Vit r o Wakaki e_t a l . (1958) f i r s t i s o l a t e d mitomycin-C as a d i s t i n c t f r a c t i o n of the mitomycin group of a n t i b i o t i c s . The drug was subsequently used for the treatment of neoplastic diseases, but because of i t s toxic e f f e c t on the bone marrow, and the subse-- 9 -quent thrombocytopenia and leukopenia, i t s use was d i s c o n t i n u e d (Watne, Moore, and B e d r e t t i a , 1967; Jones, 1959) . Mertz (1961) was the f i r s t to observe the e f f e c t o f t h i s compound on the chromosomes. Using r o o t t i p chromosomes o f V i c i a faba, he observed chromatid a b e r r a t i o n s when c e l l s were exposed to a s o l u t i o n o f 0.001% mitomycin-C f o r one hour. He a l s o observed a marked i n h i b i t i o n o f m i t o s i s . Few d i v i s i o n f i g u r e s were e v i d e n t 24 hours a f t e r treatment w i t h the drug, b u t a t 72 hours the frequency o f chromatid a b e r r a t i o n s was g r e a t e s t . T h i s h i g h frequency o f i s o c h r o m a t i d a b e r r a t i o n s was s t i l l e v i d e n t 96 hours a f t e r exposure. The data i n d i c a t e d t h a t most o f the i n t e r p h a s e stages were s e n s i t i v e to the a c t i o n o f the mitomycin. Mitomycin-C has been r e p o r t e d to be an e f f e c t i v e b r e a k i n g agent o f human chromosomes in_ v i t r o by Nowell (1964) , Cohen and Shaw (1964), and Shaw and Cohen (1965). By adding mitomycin-C to l e u k o c y t e s i_n v i t r o a t a c o n c e n t r a t i o n o f 1 ug/ml f o r the f i r s t hour i n c u l t u r e , Nowell observed t h a t the drug induced chromatid breaks as w e l l as a l a r g e number o f exchange con-f i g u r a t i o n s . Large and s m a l l a c e n t r i c fragments were a l s o found, but r i n g and d i c e n t r i c chromosomes were uncommon. I t was f u r t h e r observed t h a t i f c e l l s were exposed to the drug f o r one hour a t the 24th, 52nd, 62nd or 68th to 72nd hour, the number o f a b e r r a -t i o n s decreased q u i t e markedly. Treatment o f c e l l s w i t h mitomycin-C f o r 24 hours or more completely i n h i b i t e d subsequent m i t o t i c a c t i v i t y . - 10 -Cohen and Shaw (1964) and Shaw and Cohen (1965) reported treating c e l l s in culture with 0.1, and 1.0 ug/ml for the l a s t 24 hours of culture. Again, large numbers of breaks and exchange figures were produced, notably at the secondary construction region of chromosomes #1, #9, and #16. The most unusual e f f e c t of mitomycin-C was the large number of exchange figures that were produced. Nowell (1964), Cohen and Shaw (1964), and Shaw and Cohen (1965), a l l noted the s t r u c t u r a l rearrangements frequently involved members of apparent homologous pairs of chromosomes. Experiments c a r r i e d out on E_. c o l i indicate that mitomycin-C causes s e l e c t i v e i n h i b i t i o n of DNA (Shiba et_ a l . 1959) while RNA and protein synthesis appear to be unaffected. (Sekiguchi and Takagi, 1960). Iyer and Szbalski (1964) attributed the i n h i b i t i o n to cross l i n k i n g of DNA which interfered with r e p l i c a t i o n . - 11 -VI MATERIALS AND METHODS A. Experimental Procedures A l l experiments were carr i e d out on cultured human leukocytes using a modification of the technique used by Moorhead et_ a l . (1960), adapted for micro-amounts of whole blood. (For complete technique see Appendix A.) In each of the experiments performed, 0.25 cc of whole blood was added to 5 ml. of culture medium. Replicate cultures were set up for each of the treated and control experiments, and these cultures were then incubated for approximately 72 hours at 37°C. One and a h a l f to two hours before the c e l l s were harvested, Colcemid was added at a concentration of 0.02 ug/ml of culture, to arrest c e l l d i v i s i o n . At the end of the incubation period, c e l l s were treated with a hypotonic solution and fixed i n 3:1 absolute alcohol and g l a c i a l acetic acid. Flame dried s l i d e s were then prepared from each of the cultures. 1. Experiments Using Lysergic Acid Diethylamide (LSD) Ten volunteers, fiv e males and fiv e females were used for in v i t r o experiments with LSD. Nine of the ten volunteers were students, and one was a professor. These individuals were interviewed and asked whether they had received x-rays of any kind for diagnosis or for treatment, and whether they had recent v i r a l i n f e c t i o n s . The two patients who had received recent x-rays are i d e n t i f i e d in Table I. - 12 -Three cc's of whole blood were obtained from each ind i v i d u a l , and 12 r e p l i c a t e cultures were established. Lysergic acid diethylamide, (Delysid, LSD-25, Sandoz Ltd.) was obtained i n sealed glass v i a l s containing 0.1 mg LSD i n 1.0 ml of aqueous solu t i o n . The solution was d i l u t e d with s t e r i l e d i s t i l l e d water to a concentration of 20 ug/ml, and 24 hours p r i o r to the termination of the cultures, 0.25 cc (5 ug) was added to s i x of the r e p l i c a t e cultures y i e l d i n g a f i n a l concen-t r a t i o n of 1 ug/ml. An equal volume of s t e r i l e d i s t i l l e d water (0.25 cc) was added to each of the remaining s i x cultures. 2. Treatment of Leukocytes from a Patient with Fanconi's  Anaemia with Growth Hormone Blood was obtained from a patient immediately before t r e a t -ment with growth hormone, and eight r e p l i c a t e cultures (0.25 cc whole blood/5 cc of medium) were established. Twenty four hours after treatment with 250 ug. of growth hormone, another sample of blood was taken and set up according to the procedure outlined above. A sample from a normal boy, incubated i n the same media at the same time, was used as a control primarily to ensure that environmental factors did not contribute to the breakage frequency in cultures from the patient. 3. Experiments Using Mitomycin-C A 3 cc sample of whole blood was obtained from a single i n d i v i d u a l , and 0.25 cc of whole blood was added to each of twelve r e p l i c a t e cultures. - 13 -Mitomycin-C obtained from N u t r i t i o n a l Biochemicals Corpora-tion , Cleveland, Ohio was dissolved i n s t e r i l e d i s t i l l e d water to a concentration of 20 ug/ml. At the beginning of the incubation period, 0.25 cc (5 ug.) of the solution was added to s i x of the r e p l i c a t e cultures to y i e l d a f i n a l concentration of 1 ug/ml. An equal volume of s t e r i l e d i s t i l l e d water (0.25 cc) was added to each of the remaining s i x r e p l i c a t e cultures. After one hour of incubation c e l l s in a l l cultures were washed twice i n culture medium, resuspended i n fresh media, and replaced i n the incubator for the remainder of the 72 hour c u l -ture period. 4. Blinding and Coding of Slides In each study (LSD, Mitomycin-C and Fanconi's anaemia,) the ten best s l i d e s from each of the control and treated samples were selected and coded to ensure the examiner could not i d e n t i f y the source. 5. Microscopy The s l i d e s were examined and photographed on a Zeiss automatic photomicroscope equipped with phase contrast condenser and objective lenses. Good, we l l spread metaphase figures were selected under low power (xl6 objective) and once a c e l l was selected i t was included i n the study. Wherever possible, 10 c e l l s on each of 10 s l i d e s were selected to y i e l d a t o t a l of 100 c e l l s for each control or treatment. Once a c e l l was selected i t was analyzed under o i l immersion (xlOO) for s t r u c t u r a l aberrations. The c e l l s were then photographed - 14 -on high contrast, fine grain f i l m (Adox 135, KB-14), and reanalyzed. B. C r i t e r i a Used to Define Chromosome Aberrations A l l c e l l s were analyzed for chromosome number and s t r u c t u r a l aberrations, including gaps, breaks, acentric fragments, deletions, d i c e n t r i c s , marker chromosomes and exchange configurations. A break was.defined as a discontinuity in an arm in which the acentric fragment was displaced from i t s alignment with the proximal or cen t r i c part of the arm. Achromatic segments i n which the d i s t a l fragment remained i n alignment with the proximal arm were scored as gaps, and were not considered to be breakage events. Configurations that involved more than one chromosome were presumed to r e s u l t from chromatid interchanges. Throughout this study, these configurations w i l l be referred to as 'exchanges'. Breaks, acentric fragments and deletions were scored as single break events, while d i c e n t r i c chromosomes and exchanges were scored as two break events. C. Determination of the D i s t r i b u t i o n of Breakage Events  Among the Groups of Chromosomes A measure of whether or not the breakage events were randomly d i s t r i b u t e d throughout the chromosome complement was obtained by comparing the d i s t r i b u t i o n of observed aberrations with the number expected on the basis of the r e l a t i v e length of the chromosomes in each group. The r e l a t i v e lengths of the chromosomes used are those established at the Denver and London Conferences (Chicago Conference, 1966). - 15 -VII RESULTS A. Lysergic Acid Diethylamide - In V i t r o Study The r e s u l t s of chromosome analysis for treated and control cultures from each of the ten subjects are presented i n Appendix B. These r e s u l t s are summarized i n Table I. A t o t a l of 1010 control c e l l s and 1094 c e l l s exposed to LSD were analyzed. An average of 3.91% of the c e l l s in the control cultures contained aberrations whereas 7.89% of the c e l l s i n the treated cultures were s i m i l a r l y affected. This indicates a s i g n i f i c a n t increase i n the t o t a l number of c e l l s demonstrating a breakage event. (X 2 t e s t . p<0.001) A comparison of the t o t a l number of breaks i n the treated and control cultures indicated a s i g n i f i c a n t increase in the treated cultures. The number of breaks per 100 c e l l s observed in the control cultures ranged from 0 to 15.12 with a mean of 4.72. In the cultures treated with LSD, the number of breaks per 100 c e l l s ranged from 4.0 to 18.70 with a mean of 9.37. Aneuploid levels i n both the treated and control cultures were si m i l a r , with 90.03% of the treated c e l l s and 89.23% of the control c e l l s having a modal number of f o r t y - s i x chromosomes. As control values vary considerably from i n d i v i d u a l to i n d i v i d u a l , a more accurate measure of damage associated with LSD was obtained by analysis of deviations between the treated and control sample for each i n d i v i d u a l . The deviations and paired t - t e s t values for single break events, two break events, t o t a l break events and gaps are presented i n Table I I . TABLE I Summary of the Results of Chromosome Analysis in Treated and Control Cultures X CD to rH 03 rrj H •P • H 0 0 CD ^ a u o u & CD a H CO (tf CO H •P O l O rH O rO O CD EH O H U « 0 a i ret H 01 U rO rH CD O CD "H & EH U ^ < 10 C 0 •H •P o co o M rH CO rd H CD U rH U CD CD ffl a u c T c T c T c T J.A. m 86 123 12.79 19.51 11.63 15.45 15.12 18.70 * K.A. f 120 123 22.50 39.02 2.50 7.32 4.17 8.94 P.H. f 104 136 21.15 21.32 1.92 5.15 1.92 5.15 E.M. f 100 122 12.00 18.03 6.00 9.02 6.00 13.93 G.R. m 100 100 9.00 27.00 4.00 10.00 4.00 10.00 * S.S. m 100 100 29.00 32.00 00.00 4.00 00.00 4.00 G.T. f 100 100 13.00 23.00 3.00 7.66 5.00 9.00 J.C. f 100 90 23.00 21.11 3.00 7.00 4.00 10.00 W.G. m 100 100 25.00 32.00 3.00 6.00 3.00 6.00 T.B. m 100 100 17.00 22.00 4.00 8.00 4.00 8.00 TOTAL 10 1010 1094 184.44 254.99 39.05 79.60 47.21 93.72 * Received chest or dental x-rays within s i x months of testing. TABLE II Deviations Between Treated and Control Cultures Gaps Single B. E. Two Break Events Total Breaks 1. + 6.72 + 2.06 + 1.62 + 3.58 2. + 16.52 + 3.15 + 1.62 + 4.67 3. + 0.17 + 3.23 00.00 + 3.23 4. + 6.08 + 4.65 + 3.29 + 7.93 5. + 18.00 + 6.00 00.00 + 6.00 6. + 3.00 + 4.00 00.00 + 4.00 7. + 10.00 + 6.00 - 2.00 + 4.00 8. - 1.89 + 3.55 + 2.44 + 6.00 9. + 7.00 + 3.00 00.00 + 3.00 10. + 5.00 + 4.00 00.00 .+ 4.0.0 Mean difference + 7.00 + 3.96 + 0.697 + 4.64 T value 3.49 9.86 1.44 9.51 Prob. ^0.01- >0.001 <0.001 < 0.2- >0.1 <0.001 - 18 -In each of the ten cultures treated with LSD, there was an increase in the number of single break events and the t o t a l number of break events, with the amount of deviation ranging from 2.06 to 6.00 breaks per 100 c e l l s for single break events, and from 3.00 to 7.93 breaks per 100 c e l l s for t o t a l break events. The number of two break events was increased in four individuals, decreased i n one, and no difference was observed in f i v e . (Table II) A paired t - t e s t analysis indicated a s i g n i f i c a n t increase i n single break events and t o t a l break events, but not i n two break events. An analysis of s p e c i f i c types of aberrations i s presented in Table I I I , and representative figures are presented i n F i g . 1. 1. Single Break Events Single break events comprised the greatest number of aber-rations in both the treated and control cultures, 88.46% of the t o t a l number of break events in the treated and 91.30% of the t o t a l number of breaks in the control being of t h i s type. Chromatid breaks (Fig. 1-D, 1-E, and 1-F) were the most common of the single break events scored. In the treated c u l -tures, 64.33% of the t o t a l number of break events were of thi s type, whereas 54.35% of the t o t a l number of break events in the controls were chromatid breaks. However, 6.12 chromatid breaks per 100 c e l l s were observed i n the treated cultures, to 2.47 per 100 c e l l s in the contr o l . 1010 1094 T o tal No. of C e l l s 25 2.47 /100 67 6.12 /100 T o tal chromatid breaks 16 1.58 /100 24 2.19 /100 A centrics 0.099 /100 \ O H M • O O O £^> Deletions tO to No. of Aberrations to & No. of B. E. O O Rings O rO Dicentrics K> Exchanges NJ (Ti No. of Aberrations 1—1 to No. of B. E. 00 TOTAL NO. OF ABERRATIONS cn I—1 o TOTAL NO. OF B. E. M Q td > H 3 "-3 CO O I'd s o t-i to ro ro < CD CD 3 Oi n ^< H-3 0 Hi (-3 cn ro H-fu 3 i-3 ti- iQ > ro H & CD PJ 0) L J 3 H Cu Cb H H O 0 s 0 r t t-i dd O H h-1 CD P O * H r t <! CD H 3 CD r t CO Cfl - 61 -- 20 -FIGURE 1. Representative Types of Chromosomal Aberrations Observed i n Control Cultures and in Cultures Exposed to Lysergic Acid Diethylamide A and B - chromatid gaps C - isochromatid gap D, E, and F - chromatid breaks G, H, and I - acentric fragments J - exchange figure from treated culture K - exchange figure from control culture L - deleted G group chromosome ti - 22 -Acentric fragments (Fig. 1-G, 1-H, 1-1) were observed i n both treated and control cultures. The frequency of this type of aberration was increased from 1.58 per 100 c e l l s in the control to 2.19 acentric fragments per 100 c e l l s in the treated cultures. Only two deletions that could be d e f i n i t e l y i d e n t i f i e d were observed. One was found i n the treated cultures and one i n the control . These deletions were observed as centric fragments, and were scored as single break events, (Fig. 1-L). 2. Two Break Events Two break events were r a r e l y observed, and there was no s i g n i f i c a n t increase between the treated cultures and controls. Six were observed in the treated cultures and 2 in the controls. The two observed i n the control cultures, and four of the six observed i n the treated cultures were exchange figures, (Fig. 1-J and 1-K). Two d i c e n t r i c chromosomes were also observed i n the treated cultures. In the ten r e p l i c a t e cultures two break events were observed in both treated and control cultures from one in d i v i d u a l , and i n three individuals they were observed only i n the treated r e p l i c a t e s . 3. Gaps The difference in the frequency of gaps between the treated and control cultures (Table II) varied from -1.89 to +18.00, with a mean difference of 7.05 and a standard deviation of 6.39. This represents a s i g n i f i c a n t difference ( t - t e s t . p < 0^.01- >0.001) i n the gaps in the treated and control cultures. TABLE IV Dis t r i b u t i o n of Id e n t i f i a b l e Breaks Among the Chromosome Groups LSD TREATED MALES A B C D E F G OBSERVED 13 3 8 8 3 1 2 EXPECTED 8.99 4.64 13. 74 3.86 3. 34 1.77 1. 64 LSD TREATED X2 t o t a l FEMALES = 9.60 P <o .25- > 0.10 Not s i g . at 0. 05 l e v e l A B C D E F G OBSERVED 8 7 14 4 1 0 1 EXPECTED 8.19 4.19 13. 99 3.49 3. 02 1.60 1. 16 LSD CONTROL X 2 t o t a l MALES = 5.16 p <0 .75- >0.50 Not s i g . at 0. 05 l e v e l A B C D E F G OBSERVED 4 5 4 1 1 0 0 EXPECTED 3.55 1.83 5. 43 1.52 1. 32 0.70 0. 65 LSD CONTROL X2 t o t a l FEMALES = 7.51 P < 0 .5-> 0.25 Not s i g . at 0. 05 l e v e l A B C D E F G OBSERVED 6 1 6 1 0 0 0 EXPECTED 3.25 1.68 5. 36 1.40 1. 21 0.64 0. 46 X 2 t o t a l = 5.24 p<0.75->0.50 Not s i g . at 0.05 l e v e l - 24 -4. D i s t r i b u t i o n of Breakage Among Chromosome Groups The d i s t r i b u t i o n of observed break events among the seven groups of chromosomes for treated and control males and females are presented in Table IV. Chi-square analysis indicated no s i g n i f i c a n t deviation from random. - 25 -B. E f f e c t s of Growth Hormone on a Patient with Fanconi's  Anaemia This study was carried out on a patient with Fanconi's anaemia who had been assessed annually at the Health Centre for Children, and who had not required any treatment other than the administration of growth hormone. Previous chromosome studies indicated a high frequency of chromosome aberrations (Corey and Andrews, 1968). In t h i s study, blood samples were obtained immediately before, and twenty four hours after the administration of growth hormone. A normal boy of approximately the same age was used as a •standard 1. The detailed r e s u l t s of th i s study are presented in Appendix C. These r e s u l t s are summarized i n Table V. When the number of aberrant c e l l s were compared: in the cultures before and after treatment with growth hormone, there was a s i g n i f i c a n t increase both before (25.49%) and after t r e a t -ment (28.03%) over the standard (10.98%), but no s i g n i f i c a n t difference was observed between the two samples taken from the patient with Fanconi's anaemia. Although the t o t a l number of breakage events per 100 c e l l s was increased in both samples from the patient, there was a de-crease after the treatment with growth hormone. In the c e l l s observed prior to the treatment with the hormone, 46.07 breaks per 100 c e l l s were observed, whereas i n the sample after t r e a t -ment, 35.51 breaks per 100 c e l l s were observed. TABLE V Summary of Results of Chromosome Analysis Before and After Treatment with Growth Hormone/and in a 'Standard' Total No. Ce l l s Total No. Gaps Total No. Breaks Total No. Cell s with Aberrations Br. freq. per aberr, C e l l B ,G .H. 102 58 56.86/100 47 46.07/100 26 25.49/100 1.80 A.G.H. 107 46 45.10/100 38 35.51/100 30 28.03/100 1.26 0> •Standard' 91 21 11 10 23.07/100 12.08/100 10.98/100 1.10 - 27 -This i s r e f l e c t e d i n the number of break events per aberrant c e l l . (The r a t i o of breakage events to the number of aberrant c e l l s . ) In the cultures examined prior to growth hor-mone treatment, 1.80 breaks per aberrant c e l l were observed, whereas a breakage frequency of 1.26 breaks per aberrant c e l l was observed after treatment, suggesting that the growth hormone may have an e f f e c t on the number of aberrations found in each of the aberrant c e l l s . I t was further observed before treatment with growth hormone that i n d i v i d u a l c e l l s demonstrated up to and including four breaks per c e l l , but after treatment with growth hormone, no more than two breaks per c e l l were observed. The d i s t r i b u t i o n of 0, 1, 2, 3, and 4 breaks per c e l l i n the cultures before treatment did not f i t a poisson d i s t r i b u t i o n of rare events (X 2 test, p< 0.005). However, after treatment, the d i s t r i b u t i o n of c e l l s with up to two breaks per c e l l did not deviate from random (X 2 t e s t , p <0.25- >0.10). A comparison of the d i f f e r e n t types of aberrations observed before and after treatment with growth hormone i s presented in Table VI and representative figures are presented in F i g . 2. There was a s i g n i f i c a n t difference i n the d i s t r i b u t i o n of single break events (X 2 t e s t . p< 0.005) i n the two samples from the patient. The number of single break events and two break events was elevated in both the samples from the patient i n comparison to the 'standard'. CO > a X 10 CQ H-3 vQ H CD to •a A o • o o A O X to tf I-S (D O to U i CD I V o o H O CD rt 01 O VD H O H O to Total No. C e l l s H • O CTi O (XI 29 27.10 /100 16 15.68 /100 T otal No. Chromatid Breaks \ CO CO H • O to O IX) \ to co h-" • O 00 O O 17 16.66 /100 A centrics \ M 1—1 h-1 • O O O VD \ M to H • O 00 O CTi \ H to H • O VO o m Deletions M 1—1 CO CO cn No. of Aberrations 1—1 CO co cn No. of B.E. O O O Rings O O O Dicentrics O H-1 to t-1 • O 00 O (Ti \ cn rji H • O 00 O 00 Exchanges O to <T> No. of Aberrations O H to No. of B.E. H. CO cn 4^ I—1 TOTAL NO. OF ABERR. 1—1 CO 00 TOTAL NO. OF B.E. w CD Hi 0 H cn H CD H CD £2 SI s3 Q 0» CD 3 3 M O a > Hi 0 M rt Hi 12 CD H CO H-i-3 3 < H vQ CD H PJ CD rt CO 3 (U CD 3 3 & rt H-0 rt 3 J rjd H O CD H fu Q *T el- td s ' < CD ffi 3 0 rt H CO 1-3 3 0 O o 3 & CD CO to » CD i s H w < 3 CD 0» & H H-H-3 3 fu O • 3 C CO cn 1—1 rt rt PJ 3 O- CD fu CO H O, - 82 -_ 29. -FIGURE 2. Representative Types of Chromosomal Aberrations Observed Before and After Treatment with Growth Hormone in a Patient with Fanconi's Anaemia A, B, and C - chromatid gaps D, E, and F - chromatid breaks G and H - acentric fragments I, J, and K - non-homologous exchange figures deleted G group chromosome - 31 -1. Single Break Events Although the t o t a l number of single break events was similar i n both cultures from the patient, the d i s t r i b u t i o n of types of aberrations was s i g n i f i c a n t l y d i f f e r e n t due to the change i n the relative, frequencies of chromatid breaks and acentric fragments. Before treatment, breaks and acentric fragments occurred with approximately equal frequencies, whereas after treatment, chromatid breaks occurred with a much higher frequency than acentric fragments. Deletions were rare and occurred with equal frequency i n both cultures. 2. Two Break Events Exchange configurations were the only two break events observed i n the c e l l s cultured before or after treatment with growth hormone. The chromosomes involved in each of the exchange configurations are presented in Table VII. No two break events were observed in the 'standard'. Six exchange figures were observed in the c e l l s cultured before hormone treatment while only two exchanges were observed in the cultures after administration of the hormone. The number of break equivalents r e s u l t i n g from two break events was decreased after treatment with the growth hormone. 3. Gaps Prior to growth hormone treatment, 56.86 gaps per 100 c e l l s TABLE VII I d e n t i f i c a t i o n of Chromosomes Involved in Exchange Figures BGH AGH 'Standard lxC CxG None 3xB lxC 2xB lxB not i d e n t i f i a b l e - 33 were observed, while 45.10 gaps per 100 c e l l s were observed after treatment. The frequency of gaps was decreased after the t r e a t -ment with the hormone but both the cultures from the patient with Fanconi's anaemia demonstrated a higher frequency of gaps i n com-parison to the 21 gaps observed i n the c e l l s from the 'standard'. 4. D i s t r i b u t i o n of Breaks Among Chromosome Groups In the c e l l s observed p r i o r to, and after treatment with growth hormone, the expected d i s t r i b u t i o n of i d e n t i f i a b l e breaks based on the r e l a t i v e lengths of the chromosome groups did not deviate from random. (Table VIII) TABLE VIII Di s t r i b u t i o n of Id e n t i f i a b l e Breaks Before and After Treatment with Growth Hormone A B C D E F G B .G .H. 11 5 7 1 0 0 0 5.68 2.93 8.69 2.44 2.11 1.12 1.04 X 2 t o t a l = 11.87 p <0.10- >0.05 Not s i g . at 0.05 l e v e l A B C D E F G 11 4 12 4 1 0 1 A.G.H. 7.81 4.03 11.94 . 3.35 2.90 1.54 1.43 X 2 t o t a l = 4.33 p<0.75->0.50 Not s i g . at 0.05 l e v e l - 35 -C. Mitomycin-C An increase i n the number and types of chromosomal aberra-tions was observed after exposure of human leukocytes _in v i t r o for one hour to a low concentration of mitomycin-C (1 ug/ml). The detailed r e s u l t s of thi s study are found in Appendix D and are summarized i n Table IX. Representative figures are presented in F i g . 2. In 64 of the 100 c e l l s exposed to this drug, 181 breakage events were observed, whereas only 5 breakage events were observed in 3 of the 100 control c e l l s examined. A de t a i l e d analysis of the types of aberration i n the treated and control cultures i s presented i n Table X. Of the 181 breakage events observed after treatment, 85 were single break events and 96 were two break events. The 5 breakage events observed i n the control were a l l single break events. Aneuploid levels d i f f e r e d markedly with 94% of the control c e l l s and 61% of the treated c e l l s having 46 chromosomes. 1. Single Break Events Chromatid breaks were the most common single breakage events found i n the treated cultures; 65 aberrations or 35.91% of the t o t a l number of break events being of this type. In the treated sample, 18 acentric fragments were observed. Five were found i n the control, and thi s represented the t o t a l number of breakage events. TABLE IX Summary of the Results of Chromosome Analysis in Treated and Control Cultures Total No. Total No. Total No. Total C e l l s Cells with Gaps Breaks Breaks  TREATED 100 64 41 181 CONTROL 100 3 17 U) FIGURE 3. Representative Types of Chromosomal Aberrations Observe^ i n Cultures Exposed to Mitomycin-C. A, B, and C - chromatid breaks D, E, F. G, H, and I - homologous exchange figures I, J , K, L, M, and N - non-homologous exchange figures TABLE X Frequency of Single and Two Break Events in Treated and Control Cultures SINGLE BREAK EVENTS TWO BREAK EVENTS TREATED CONTROL • • • rH • H O Ct O H -p S3 « S3 • CD rd co m . m • MH . m W w H U H E M o u O • CO • 0 u 0 -3 • (0 rd o fd • u w • SH • <! eo -P • -p SH CD • H • CD • C u O • CD • w EH EH 0 o O ^ S-i u CD O A 0 • •H •H X 0 A 0 • O En O fn EH S3 EH O ffl <! Q S3 < S5 m P w S3 < S3 m EH O EH O 1 0 0 65 1 8 2 8 5 8 5 0 0 4 8 4 8 9 6 1 3 3 1 8 1 1 0 0 0 5 0 5 5 0 0 0 0 0 5 5 CO - 40 -Two deletions that could be d e f i n i t e l y i d e n t i f i e d were found i n the treated cultures; none being observed i n the controls. 2. Two Break Events The most s t r i k i n g observation i n cultures treated with mitomycin-C was the high frequency of exchange figures. Forty-seven exchange configurations were observed i n the treated cultures; none were observed i n the controls. The 47 exchange figures represented 96 break events or 53.04% of the t o t a l number of breakage events observed. The exchange figures are l i s t e d in Table XI. Twenty-seven or 57.44% of the exchange figures were between apparent homologous chromo-somes (Fig. 3., 3D-H.) while 20 or 42.56% were between chromosomes of d i f f e r e n t groups (Fig. 3., 3-H to 3-N). One unusual exchange figure appeared to involve 4 chromosomes and was scored as a 4 break event. The chromosomes involved i n t h i s p a r t i c u l a r exchange were t e n t a t i v e l y i d e n t i f i e d as CxCxDxG. Chromosomes of the C group were most frequently involved in the exchange figures, 59.57% of the exchanges involving one or more chromosomes from t h i s group. Chromosomes from the G group were r a r e l y involved i n exchanges, only 6.38% involving chromosomes from th i s group. 3. Gaps Gaps were more frequent than breaks in the control cultures and less frequent than breaks i n the treated cultures. - 41 -TABLE XI Identification, of Chromosomes Involved in  Exchange Figures in Treated Cultures A. Exchanges Involving Apparent Homologous Chromosomes 3 (lxl) 1 (BxB) 18 (CxC) 2 (DxD) 2 (ExE) 1 (FxF) SUBTOTAL =27 B. Exchanges Involving Members of Different Chromosome  Pairs 2 (lxB), 1 (3xB), 1 (FxB), 4 (lxC), 1 (BxC), 2 (ExC), 1 (DxC), 1 (FxC), 1 (lxF), 1 (FxE), 1 (lxG), 1 (GxE), 1 (DxE), 1 (DxG), 1 (CxCxDxG) • SUBTOTAL =20 TOTAL NUMBER OF EXCHANGES =47 - 42 -The increase from 17 gaps i n the control to 41 in the treated cultures indicated that the drug s i g n i f i c a n t l y increased the frequency of this type of event. 4. D i s t r i b u t i o n of Break Events Among Chromosome Groups The d i s t r i b u t i o n of i d e n t i f i a b l e breaks among the seven groups of chromosomes did not deviate from random (Table XII) indicating that the drug did not p r e f e r e n t i a l l y cause an excess of breaks in any p a r t i c u l a r group of chromosomes. TABLE XII Di s t r i b u t i o n of Id e n t i f i a b l e Breaks Among Chromosome Groups A B C I) E F G OBSERVED 32 16 66 13 19 11 6 EXPECTED 38.58 19.88 58.96 16.54 14.32 7.60 7.04 X 2 s u b 1.12 0.75 0.84 0.75 1.52 1.53 0.15 x 2TOTAL = 6 ' 6 6 P0.05 d f = 6 = 1 2 ' 6 p <0.50- > 0.25 Not s i g n i f i c a n t at 0.05 l e v e l - 44 -VIII DISCUSSION A. Lysergic Acid Diethylamide Cohen, MarinellQ, and Black (1967), and Cohen, Hirschhorn and Frosch (1967), observed that LSD at concentrations from 0.001 to 10 ug/ml of culture, with exposure times from 4 to 48 hours p r i o r to the harvest of the c e l l s , increased the frequency of chromosome aberrations. The highest concentration of 10 ug/ml brought about greater damage in a shorter exposure time. The same e f f e c t was observed at a concentration of 1 ug/ml with an exposure time of 24 hours. However, at a concentration of 0.001 ug/ml, more chromosome damage was observed at longer exposure times, while an exposure of four hours at the same concentration produced few breaks. Among the treated cultures, the lowest breakage frequency (7.7%) was almost twice the control value of 3.9%, and the breakage frequency i n treated cultures ranged to over four times the control values (17.5%). The manner i n which the r e s u l t s are presented by Cohen et_ a l . (1967), yields no information concerning possible v a r i a t i o n i n response. Since the same authors found that not a l l indivuals had an increase i n breakage after ingesting LSD, information con-cerning possible v a r i a b i l i t y i n i n d i v i d u a l response in v i t r o would appear to be valuable., In the present study, one of the _in v i t r o experiments reported by Cohen, Marinello, and Black (1967) was repeated but with an experimental design which permitted d i r e c t comparison - 45 -of samples from the same in d i v i d u a l , cultured with and without LSD. The effects of the drug were measured by the difference between the samples rather than using the breakage frequency, and the v a r i a t i o n i n difference was used as a measure of v a r i a t i o n in response. The concentration of 1 ug/ml for 24 hours was selected because previous experiments (Cohen, Marinello, and Black, 1967) indicated increased breakage values, and yet the dosage was con-s i s t e n t with s u r v i v a l of s u f f i c i e n t c e l l s for analysis. The culture technique and scoring system used were similar to those used by Cohen, Marinello and Black (1967). The average frequency of breakage i n the control cultures, and the range of breakage in the treated cultures reported here are very similar to those reported by Cohen, Marinello, and Black (1967), and Cohen, Hirschhorn, and Frosch (1967). In the present study i t was observed that the breakage frequency i n treated cultures ranged from 4.0 to 18.70 with a mean of 9.57. I t was further observed that although there was a wide range of values after treatment, there was an equally wide range before treatment, but the differences ranged only from 3.00 to 7.93 with a mean of 4.64, in d i c a t i n g very l i t t l e v a r i a t i o n i n response from in d i v i d u a l to i n d i v i d u a l . While i t i s true that the breakage frequency i n treated cultures increased 1.24 to 4 times, t h i s i s r e l a t i v e to the breakage frequency i n the control cultures and i s not a r e f l e c -t i o n of v a r i a t i o n i n the amount of damage. In fact, the 1.24 times increase represented a change from 15.12 to 18.70, and - 46 -the 4 times increase represented an increase of from 0 to 4 breaks. Thus these two extremes represent increases of 3.58 and 4 breaks respectively. In the study presented here, the o v e r a l l r e s u l t s were similar to those in other published r e s u l t s . However, some minor differences in the types of abnormalities were observed. Cohen, Hirschhorn, and Frosh (1967) observed a 'high frequency of small acentric fragments * whereas in th i s study, the number of acentric fragments was not s i g n i f i c a n t l y increased i n the treated cultures when compared to the controls. This may be due to the fact that Cohen and h i s associates included terminal breaks with acentric fragments. Cohen and h i s coworkers reported finding two break events only i n cultures treated with LSD. However, i n the present study, two break events were observed i n both treated and control c u l -tures and were not s i g n i f i c a n t l y increased i n the treated r e p l i c a t e s . Although two break events are very rare, they have been observed i n normal individuals (Bloom et_ a l . 1966) . As they were observed i n low frequency accompanied by an increase i n single break events by Cohen et a l . and in the present study, i t seems u n l i k e l y that LSD s p e c i f i c a l l y increases t h i s type of aberration. B. Fanconi's Anaemia Reports of high frequencies of chromosomal aberrations are s u f f i c i e n t l y consistent to be considered part of the syndrome - 47 -known as Fanconi's anaemia (Swift et a l . 1966; Bloom et a l . 1966; Varela and Sternberg, 1967). C e l l death due to chromosome breakage has been suggested as a possible cause of the progressive pancytopenia (Bloom e_t ajL. 1966) . At the present time, patients with t h i s disease are treated with s t e r o i d hormones such as testosterone and cortisone, as t h i s form of treatment appears to retard the progressive bone marrow aplasia (Shahadi and Diamond, 1959). However, the l i t e r a t u r e contains no information of the effects of treatment on the f r e -quency of chromosomal breakage. The a v a i l a b i l i t y of a patient in whom the anticipated bone marrow aplasia had not progressed s u f f i c i e n t l y to warrant t r e a t -ment, provided an opportunity to observe the frequency and type of aberrations both before onset of the bone marrow aplasia and before treatment, and to further study the possible effects of treatment on the frequency and type of aberrations. A previous study had indicated that a high frequency of breakage was evident i n t h i s patient two years prior to the present study (Corey and Andrews, 1968). I t was observed at that time that 34% of the c e l l s demonstrated a breakage event; in the present study, 25.5% of the c e l l s before, and 28.04% after treatment with the hormone were s i m i l a r l y affected. Between the study conducted on t h i s patient two years ago, and the present one, the patient had been given a course of growth hormone treatment. - 48 -In the i n i t i a l study, and in the present one, the frequen-cies and types of aberrations were similar to those reported elsewhere. Bloom et a l . (1966) reported that in 1,621 leukocytes from patients with Fanconi's anaemia, 16.8% of the c e l l s demon-strated a breakage event. The most frequent aberrations were chromatid and isochromatid breaks, acentric fragments and exchange configurations. In the present study, the number of chromatid breaks observed was increased from 16 i n the untreated cultures to 29 after treatment with the growth hormone; the number of acentric fragments was decreased from 17 in untreated cultures to 3 after exposure to growth hormone. Bloom et a l . (1966) also observed 78 exchange figures in the 1,621 c e l l s scored y i e l d i n g a frequency of 0.048 for thi s type o f event. The frequency of exchanges i n the patient studied here prior to growth hormone treatment i s consistent with the res u l t s of Bloom et_ a l . , the frequency of these two break events being 0.059; after treatment, the frequency of exchanges was 0.019. Although the number of c e l l s with aberrations was unchanged i n samples obtained 24 hours after growth hormone treatment i n comparison to the samples taken before, changes in the frequency and types of aberrations were observed i n that there was a reduction of (1) the number of breaks per aberrant c e l l , (2) the number of acentric fragments, and (3) the number of exchange figures. - 49 -Due to the limited nature of the study, i t i s impossible to assess the significance of the r e s u l t s , but the suggested reduction i n the amount of damage per aberrant c e l l and the reduction i n two break events encourages further investigation of growth hormone as a treatment. This i s further emphasized by the fact that treatment with testosterone and cortisone has undesirable side e f f e c t s such as masculinization of the patient and an i n h i b i t i o n of the defence mechanisms of the body. Growth hormone, however, may have the desired e f f e c t of stimulating the growth of these dwarfed patients, and i f i t did decrease the chromosome damage and retard somewhat the progression of the disease, i t would be a more desirable treatment. C. Mi tomyc in-C„ The experiment with mitomycin-C was undertaken as a p i l o t project to develop an induced chromosome breakage system which could, in future, be used to assess the effects of steroids and other hormone used to treat patients with Fanconi's anaemia. Mitomycin-C was used because i t not only has been reported to cause a tremendous increase of breakage events JLn v i t r o (Nowell, 1964; Cohen and Shaw, 1964; Shaw and Cohen, 1965), but the aberrations such as chromatid breaks, acentric fragments and exchange figures are similar to those found i n Fanconi's anaemia. Although mitomycin-C has been reported to be e f f e c t i v e at a v a r i e t y of times and concentrations (Cohen and Shaw, 1964), the one hour treatment of 1 ug/ml of mitomycin-C at the beginning of the culture period reported by Nowell (1964) - 50 -resulted in the greatest number of chromosomal aberrations with the highest mitotic index. The treatment method of exposing leukocytes i i i v i t r o to 1 ug/ml of mitomycin-C during the f i r s t hour of culture has several advantages for an in v i t r o model system i n that (1) i t i s a breakage system where the breaking agent can be removed from the culture system before treatments such as growth hormone in the experiment with Fanconi's anaemia are added, (2) i t i s a system i n which -the c e l l s exposed to the drug go through more than one c e l l d i v i s i o n , so that an attempt can be made to measure the s u r v i v a l of c e l l s , and (3) the drug increases the v a r i e t y as w e l l as the frequency of chromosome and chromatid aberrations. Many of the chromosome aberrations observed with treatment with mitomycin-C were unusual. Apart from chromatid and i s o -chromatid breaks, and acentric fragments, a large number of exchange configurations were observed. Cohen and Shaw (1964) observed 7 6 exchanges i n 114 c e l l s y i e l d i n g a mean number of exchanges per c e l l of 0.67. In the present study, the mean number of exchanges per c e l l in treated c e l l s was observed to be 0.47. None were observed in the control cultures. D i f f e r e n t types of exchange configurations may have d i f f e r e n t consequences in subsequent c e l l d i v i s i o n s . Therefore, examination of chromosome aberrations after treatment with mitomycin-C can provide a measure of c e l l s u r v i v a l by comparison of the frequencies and types of exchange configurations with the - 51 -types and frequencies of aberration after the c e l l s have proceeded through several d i v i s i o n s . Exchanges between chromosomes of d i f f e r e n t groups, or s i t u a -tions i n which chromosomes of the same groups have undergone unequal exchange to y i e l d asymmetrical figures were considered as non-homologous exchange configurations. In contrast to Fanconi's anaemia where a l l of the exchanges observed were non-homologous, mitomycin-C treatment resulted in both homologous and non-homologous exchanges. Shaw and Cohen (1965) observed 46.36% of the exchanges i n mitomycin-C treated cultures to be homologous exchanges; i n the present study, 57.44% were of th i s type. Homologous exchanges would not produce detectable karyotype changes i n subsequent c e l l d i v i s i o n s . Non-homologous exchanges, l i k e those found i n Fanconi's anaemia and in c e l l s treated with mitomycin-C should have resulted i n balanced translocations, duplications and d e f i c i e n c i e s , and marker chromosomes, as well as acentric fragments and d i c e n t r i c chromosomes. No d i c e n t r i c chromosomes or marker chromosomes were observed i n mitomycin treated cultures, but acentric fragments and deletions were found. Acentric fragments and deletions could also have been produced by single break events rather than being the end res u l t s of non-homologous exchange configurations. Nowell (1964) reported that 'dicentric and ring chromosomes were extremely r a r e 1 , and Cohen and Shaw (1964) and Shaw and Cohen (1965) make no mention of finding d i c e n t r i c chromosomes. However as they treated c e l l s late in the culture period, - 52 -presumably in the l a s t c e l l d i v i s i o n , no two break events would have been expected. The absence of marker and d i c e n t r i c chromosomes suggests that c e l l s with non-homologous exchanges cannot proceed through another c e l l d i v i s i o n , or, i f the d i v i s i o n i s complete chromosome imbalances in the daughter c e l l s may be responsible for the death of the c e l l s . This suggestion i s tenative in that the cultures were terminated at 72 hours and there i s no indication whether or not these aberrations occur i n the f i r s t d i v i s i o n after treatment. IX SUMMARY A. Lysergic Acid Diethylamide The frequency and types of chromosomal aberrations were observed in r e p l i c a t e cultures from each of ten individuals cultured with and without the addition of 1 ug/ml of ly s e r g i c acid diethylamide during the l a s t 24 hours of the culture period. Approximately 100 c e l l s from each of the re p l i c a t e s were analyzed and the difference between untreated and treated r e p l i c a t e s was used as a measure of the effects of the treatment. The frequency of breaks i n untreated replicates ranged from 0 to 15.12 breaks per 100 c e l l s , with a mean of 4.72. In the treated r e p l i c a t e s , the frequency of breaks per : 100 c e l l s ranged from 4.00 to 18.70 with a mean of 9.37. The difference between untreated and treated cultures ranged from +3.00 to +7.93 with a mean of +4.65. A paired t - t e s t analysis indicated a s i g n i f i c a n t increase in both t o t a l break events and single break events, but not in aberrations due to two break events. The chromosome breaks were randomly d i s t r i b u t e d among the seven groups of chromosomes of the complement. B. Fanconi's Anaemia The frequency and types of chromosome aberrations were observed in a patient with Fanconi's anaemia immediately before and 24 hours after administration of 250 ug. of growth hormone, - 54 -and i n a control. Approximately 100 c e l l s from each sample were examined. Forty-seven breaks i n 102 c e l l s were observed i n untreated cultures, 38 breaks i n 107 c e l l s being observed after exposure to the hormone. Eleven breaks in 91 c e l l s were observed in c e l l s of the 'standard'. There was no s i g n i f i c a n t difference in the number of aberrant c e l l s in the cultures from the patient, but the number of breaks per aberrant c e l l were decreased from 1.80 i n the cultures before treatment to 1.26 i n the c e l l s after exposure to the hormone. Single break events were the most common aberrations in both samples from the patient, but a s i g n i f i c a n t difference in the d i s t r i b u t i o n of single break events between the two cultures was observed. Exchange configurations were the only two break events observed in both samples with the number of break events being decreased from 12 to 4 after treatment. No two break events were observed in the c e l l s from the 'standard'. Chromosome breaks were randomly d i s t r i b u t e d among the seven groups of chromosomes in a l l samples. C. Mitomycin-C An increase in the frequency and types of chromosomal aberrations was observed after exposure of human leukocytes to a low concentration of mitomycin-C for one hour at the - 55 -beginning of the culture period when compared to the untreated cultures. One hundred and eighty one breaks in 64 of the 100 c e l l s exposed to the drug were observed. Only 5 breaks were observed in 3 of the 100 control c e l l s examined. Single break events represented 46.96% of the t o t a l number of break events observed in the treated cultures. The five break events observed in the control cultures were a l l single break events. Exchange figures constituted a l l of the two break events in the treated cultures; 27 homologous and 20 non-homologous exchanges being observed. No two break events were observed in the control cultures. The chromosome breaks were randomly d i s t r i b u t e d among the seven groups of chromosomes of the complement. - 56 -X REFERENCES Alexander, G.J., M i l e s , B.E., Gold, G.M., and Alexander, R.B. 1967. LSD i n j e c t i o n e a r l y i n pregnancy produces a b n o r m a l i t i e s i n the o f f s p r i n g o f r a t s . Science 157;459-460. Auerbach, R., and Rugowski, J.A. 1967. L y s e r g i c a c i d d i e t h y l a m i d e : e f f e c t on embryos. Science 157:1325. 1 Bender, L., and Sankar, S.D.V. 1968. Chromosome damage not found i n l e u k o c y t e s o f c h i l d r e n t r e a t e d w i t h LSD-25. Science 159: 749. Bloom, G.E., Warner, S., Ger a l d , P.S., and Diamond, L.K. 1966. Chromosome a b n o r m a l i t i e s i n c o n s t i t u t i o n a l a p l a s t i c anaemia. New E n g l . J . Med. 274(1):8-14. Browning, L.S. 1968. L y s e r g i c a c i d d i e t h y l a m i d e : mutagenic e f f e c t s i n D r o s o p h i l a . Science 161:1022-1023. 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Corey, M.J., and Andrews, J.C. 1968. Chromosome breakage i n mixed leukocyte c u l t u r e s from a p a t i e n t w i t h Fanconi's anaemia. Can. J . Genet. C y t o l . 10_:767. ( A b s t r a c t ) . D a r l i n g t o n , C.D., and K o l l e r , P.C. 1947. The chemical breakage o f chromosomes. H e r e d i t y .]__: 187-221. Denver Report. 1960. A proposed system o f nomenclature o f human m i t o t i c chromosomes. Lancet 1:1063-1065. - 57 -Egozcue, J., Irwin, S., and Maruffo, C.A. 1968. Chromosome damage in LSD users. J. Am. Med. Assn. 204:214-218. Geber, W.F. 1967. Congenital malformations induced by mescaline, l y s e r g i c acid diethylamide and bromolysergic a c i d . Science 158:265-267. German, J., Archibald, R., and Bloom, D. 1965. Chromosomal breakage in a rare and probably gen e t i c a l l y determined syndrome of man. Science 148:506-507. German, J . , and Crippa, L.P. 1966. Chromosome breakage i n d i p l o i d c e l l l i n e s from Bloom's syndrome and Fanconi's anaemia„ Ann. de Genet. 9^:143-154. Grace, D., Carlson, E.F., and Goodman, P. 1968. Prosophila  melanogaster treated with LSD: absence of mutation and chromosomal breakage. Science 161:694-696. Hirschhorn, K., and Cohen, M.M. 1967. Non-psychic e f f e c t s of LSD: (Chromosome damage human). Ann. intern. Med. 6^(5): 1109-1111. Hungerford, D.A., Taylor, K.M., Shagass, C , LaBadie, G.U., Balaban, G.B., and Paton, G.R. 1968. Cytogenetic effects of LSD therapy i n man. J . Am. Med. Assn. 206(10):2287^2291. Irwin, S., and Egozcue, J . 1967. Chromosome abnormalities i n leukocytes from LSD users. Science 157:313-316. Iyer, V.N., and Szybalski, W.A. 1964. A molecular mechanism of mitomycin action: l i n k i n g of complementary DNA strands. Proc. natn. Acad. S c i . U.S.A. 50:355. Jones, R. 1959. A preliminary report of human pharmacology and i n i t i a l therapeutic t r i a l with mitomycin-C. Cane. 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Sato, H., and Pergament, E. 1968. Is lysergide a teratogen? Lancet II:639-640. Schmid, W., Scharer, K., Baumann, T., Fanconi, G. 1965. Chromosomenbruchigkeit bie der familaren Panmyelopathie (Typus Fanconi). Schweiz..Med. Wochen. 45:1461-1464. Sekiguchi, M., and Takagi, Y. 1960. Non-infectious bacteriophage produced by the action of mitomycin-C. Virology 10:160-161. Shahidi, N.T., and Diamond, L.K. 1959. Testosterone-induced remission i n a p l a s t i c anemia. A.M.A.J. Dis. Ch i l d . 98: 293-302. Shaw, M.W., and Cohen, M.M. 1965. Chromosome exchanges i n human leukocytes induced by mitomycin-C. Genetics 51: 181-190. Shiba, S., Tirawaki, A., Taguchi, T., and Kawamata, S. 1959. Selective i n h i b i t i o n of formation of DNA in E. c o l i by mitomycin-C. Nature 183:1056-1057. ~~ Skakkebaek, W.E., P h i l i p s , J., and Rafaelsen, O.J. 1968. LSD in mice: abnormalities in meiotic chromosomes. Science.160: 1246-1248. Sparkes, R.S., Melnyk, J., and Bozzetti, L.P. 1968. Chromosomal e f f e c t in. vivo of exposure to ly s e r g i c acid diethylamide. Science.160:1343-1344. Swift, M.R., and Hirschhorn, K. 1966. Fanconi's anaemia: inherited s u s c e p t i b i l i t y of chromosome breakage i n various tissues. Ann. in t e r n . Med. 65:496-503. Varela, M.A., and Sternberg, W.H. 1967. Preanaemic state in Fanconi's anaemia. Lancet.II:566-567. - 59 -Wakaki, S., Marumo, H. Tomioka, K., Shimizu, G., Kato, E., Kamada, H., Kudo, S., and Fujimoto, Y. 1958. Isolation of new fractions of antitumour mitomycins. A n t i b i o t i c s Chemother. £: 228-240. Warkany, J., and Takacs, E. 1968. Lysergic acid diethylamide (LSD). No teratogenicity i n r a t s . Science 159:731-732. Watne, A.L., Moore, D., and Bedrettia, G. 1967. So l i d tumour chemotherapy with mitomycin-C. Archs. Surg. 95(2): 175-178. Zellweger, H., McDonald, J.S., and Abbo, G. 1967. Is lyse r g i c acid diethylamide a teratogen? Lancet.II:1066. - 60 -APPENDIX A. LEUKOCYTE CULTURE TECHNIQUE - 61 -.APPENDIX A. LEUKOCYTE CULTURE TECHNIQUE 1. Cultures were grown i n s t e r i l e r o l l e r tubes containing 5 cc of GIBCO Chromosome Medium 1A. 0.25 cc of heparinized venous blood was added to each tube, and the cultures were then incubated at 37QC for approximately 72 hours. 2. Colcemid i n a f i n a l concentration of 0.02 ug/ml was added to the cultures approximately two hours p r i o r to the har-vest of the c e l l s . 3. At the end of the incubation period, the c e l l s were centrifuged at 800 rpm for 8 minutes. The supernatant was removed and discarded, and the c e l l s were suspended in 5 cc of hypotonic solution (5:1 d i s t i l l e d water: f e t a l c a l f serum), and incubated at 37°C for 15 minutes. 4. A drop of f i x a t i v e (3:1 absolute ethyl alcohol: g l a c i a l acetic acid) was added, and the cultures were centrifuged at 800 rpm for 8 minutes. 5. The supernatant was discarded leaving a large drop over the c e l l s . 4 to 5 cc of 3:1 f i x a t i v e was slowly added and the c e l l s gently agitated. 6. The tubes were then stoppered and r e f r i g e r a t e d at 4°C for approximately h a l f an hour and then l e f t at room temperature for one hour or longer. 7. The c e l l s were then centrifuged at 800 rpm for 8 minutes and the supernatant removed and discarded. 0.5 to 1.0 cc of fresh f i x a t i v e was then c a r e f u l l y added so as not to disturb the button of c e l l s . After two minutes the excess f i x a t i v e was removed and replaced with fresh 3:1 f i x . This procedure was repeated several times. 8. The c e l l s were f i n a l l y suspended i n fresh f i x a t i v e and a drop of the suspension was placed on precleaned cold wet s l i d e . The s l i d e s were then flamed, a i r dried and stored u n t i l required. Staining of Slides A few drops of aceto-orcein (2% in 60 cc g l a c i a l acetic acid) were placed on a s l i d e , and a clean cov e r s l i p was applied. _ 62 -The s l i d e was l e f t t h i s way for 1-2 minutes, and then the excess s t a i n was removed by placing the s l i d e between a few layers of paper towelling and applying pressure to the cove r s l i p . The coversl i p was then sealed with p a r a f f i n . - 63 -APPENDIX B. DETAILED RESULTS FOR LSD 1-10 o i-3 86 123 NO. OF CELLS 1-3 td Cd Iso g. H3 o o Hi cd cd H CO 0 t 'i-: H3 a TYE AN! 'ES 01 ) BRE/ ? GAPS K^S o co o to to cn o CO co CHROMOSOME GROUPS L. S. D. - 1 o to o o M o to o o <] 1 cd CHROMOSOME GROUPS L. S. D. - 1 M M o 4^ co M o -J o CHROMOSOME GROUPS L. S. D. - 1 o O o o o o CO O o to o CHROMOSOME GROUPS L. S. D. - 1 o O o o o M O o 1—1 K CHROMOSOME GROUPS L. S. D. - 1 o O o o o O O O o o CHROMOSOME GROUPS L. S. D. - 1 o O o o o O o O o o O CHROMOSOME GROUPS L. S. D. - 1 M cn o cn 4^ M CO CO to o TOTAL L. S. D. - 1 t—1 M to ID. GAPS ID. BREAKS O O NON-IDENTIF. EXCHANGES O O IDENTIFIABLE EXCHANGES O O DELETIONS cn 4^ > ACENTRICS o 1—1 DICENTRICS 13/11 23/24 TOTAL BREAKS AND GAPS i—• o VO CELLS WITH ABERRATIONS 0.1512 0.1870 TOTAL BREAKAGE FREQUENCY -V9-o i-3 12 0 123 NO. OF CELLS to ro H to 0 • o ro td H CO 0 • 1-3 o TYPES OF GAPS AND BREAKS o to o *s M to M I—1 to > CHROMOSOME GROUPS L. S. D. - 2 o o o M -J O to O to td CHROMOSOME GROUPS L. S. D. - 2 o o h-1 ^ O to O r-1 H H O CHROMOSOME GROUPS L. S. D. - 2 o o o O to O o O LO o CHROMOSOME GROUPS L. S. D. - 2 o o o M o M o O O to td CHROMOSOME GROUPS L. S. D. - 2 o o o O o O o O O O *] CHROMOSOME GROUPS L. S. D. - 2 o o o O o O o O M M O CHROMOSOME GROUPS L. S. D. - 2 o LO o -J to o to CTi M r—1 LO LO TOTAL L. S. D. - 2 LO to -J CO CO ID. GAPS ID. BREAKS o O N ON-1DENTIF. EXCHANGES o O IDENTIFIABLE EXCHANGES o O DELETIONS to M ACENTRICS o H-* DICENTRICS 5/2 7 11/48 TOTAL BREAKS AND GAPS LO CELLS WITH ABERRATIONS O • o 0. 089^ TOTAL BREAKAGE FREQUENCY - S 9 -o 3^ 104 136 NO. OF CELLS CO co H to O i-3 iQ Q H3 Cd to H CO 0 • 1-3 I iQ C O TYPES OF GAPS AND BREAKS o M o OJ o o Cn !-• O > CHROMOSOME GROUPS L. S. D. - 3 o O o o CO o to o O <1 to CHROMOSOME GROUPS L. S. D. - 3 o O o c o cn o o o H-* CO n CHROMOSOME GROUPS L. S. D. - 3 o O o o o o o o O I—1 o CHROMOSOME GROUPS L. S. D. - 3 o O o o t-> o o o to o td CHROMOSOME GROUPS L. S. D. - 3 o O o o CO o o o O o * i CHROMOSOME GROUPS L. S. D. - 3 o o o o o o o o O o CD CHROMOSOME GROUPS L. S. D. - 3 o 1—' o cn cn o c o o CO to TOTAL L. S. D. - 3 to to •1 iCo to CD ID. GAPS ID. BREAKS o o NON-IDENTIF.' • EXCHANGES "' ' o o IDENTIFIABLE EXCHANGES o o DELETIONS ACENTRICS o o DICENTRICS 2/22 7/29 TOTAL BREAKS AND GAPS to - j CELLS WITH ABERRATIONS 0.0192 0.0514 TOTAL BREAKAGE FREQUENCY -99-o i-3 100 122 NO. OF CELLS i-3 td W H CO 0 • 1-3 O i-3 W W H 10 0 iQ • f-3 o TYPES OF GAPS AND BREAKS M o o M O o O .£» > {.CHROMOSOME GROUPS L. S. D. - 4 O o H to O to o M Y-> W {.CHROMOSOME GROUPS L. S. D. - 4 O to o O I-1 O 4^  o o CO Q {.CHROMOSOME GROUPS L. S. D. - 4 O o o O o O 4^  o o H D {.CHROMOSOME GROUPS L. S. D. - 4 O o o O to o O o o h-1 td {.CHROMOSOME GROUPS L. S. D. - 4 O o o O o o O o o O {.CHROMOSOME GROUPS L. S. D. - 4 O o 9 O o o O o o O O {.CHROMOSOME GROUPS L. S. D. - 4 H CO o tn o M O o r-1 TOTAL L. S. D. - 4 4^  to O to to ID. GAPS ID. BREAKS O IO || M a NON-IDENTIF. EXCHANGES o ;I DENT IFI ABLE EXCHANGES o o DELETIONS to Ul ACENTRICS O o DICENTRICS 6/12 17/22 TOTAL BREAKS AND GAPS 1—' CELLS WITH ABERRATIONS 0.060C 0.1393 TOTAL BREAKAGE FREQUENCY -L9-n 3^ 100 100 NO. OF CELLS td td H CO 0 • >-3 o t-3 td td H to 0 • i-3 iQ O TYPES OF GAPS AND BREAKS o o o to M I—1 to o -J co > CHROMOSOME GROUPS • L. S. D. - 5 o o o IO O o o co ; i— 1 I—1 Cd CHROMOSOME GROUPS • L. S. D. - 5 o H" o ;. t i-1 to o o o> co o CHROMOSOME GROUPS • L. S. D. - 5 o O o i— 1 O o to o co o a CHROMOSOME GROUPS • L. S. D. - 5 I-1 O o o O o o o o o td CHROMOSOME GROUPS • L. S. D. - 5 O o o o O o o o o o CHROMOSOME GROUPS • L. S. D. - 5 O o o M O o o o o o O CHROMOSOME GROUPS • L. S. D. - 5 h-1 I-1 o to CO 4^  co -J TOT AL • L. S. D. - 5 tO to - J ID. GAPS ID. BREAKS O o NON- IDENTIE i EXCHANGES o o IDENTIFIABLE EXCHANGES o o DELETIONS CO ACENTRICS o o DICENTRICS 4^  \ VD 10/2 7 TOTAL BREAKS AND GAPS 4^  M O CELLS WITH ABERRATIONS 0.04 0.100 TOTAL BREAKAGE FREQUENCY -89-o H3 100 100 NO. OF CELLS W tu H w 0 • iQ o H3 td bd H cn O • •-3 iQ O TYPES OF GAPS AND BREAKS o o o OJ OJ M 4^ M 4^ > CHROMOSOME GROUPS L. S. D. - 6 o o o o O J O o O . M cn CHROMOSOME GROUPS L. S. D. - 6 o o o OJ M 1—1 O o M OJ CO o CHROMOSOME GROUPS L. S. D. - 6 o o o M O J o O O OJ o CHROMOSOME GROUPS L. S. D. - 6 o o NO O o o M O o to M CHROMOSOME GROUPS L. S. D. - 6 o o O O o o O O o O CHROMOSOME GROUPS L. S. D. - 6 o o o O o o O O o o & CHROMOSOME GROUPS L. S. D. - 6 o o to to o O J Ol Ui to to TOTi \L L. S. D. - 6 o to 4* OJ to ID. GAPS ID. BREAKS o o NON-'."I DENT IF.; EXCHANGES' o o IDENTIFIABLE EXCHANGES o o DELETIONS o o ACENTRICS o o DICENTRICS 0/2 9 4/32 TOTAL BREAKS AND GAPS o 4^ CELLS WITH ABERRATIONS 0.00 0. 04 TOTAL BREAKAGE •FREQUENCY -69-o i-3 100 100 NO. OF CELLS to to H CO o • i Q o r-3 td to H cn O i-3 cQ Q TYPES OF GAPS AND BREAKS o p-1 o to co h-1 o as > CHROMOSOME GROUPS L. S. D. - 7 o o o o I—1 o r—1 o o o to CHROMOSOME GROUPS L. S. D. - 7 o o to o 4^ o CD co o CHROMOSOME GROUPS L. S. D. - 7 o o o o O o O 4^ o CHROMOSOME GROUPS L. S. D. - 7 o O o o o o O o O O td CHROMOSOME GROUPS L. S. D. - 7 o O o o o o O o o O *1 CHROMOSOME GROUPS L. S. D. - 7 o O o o o o O o o o O CHROMOSOME GROUPS L. S. D. - 7 o co o CD as o M O M CO TOTAL co co to co ID. GAPS ID. BREAKS o o NON-1DENTIF. EXCHANGES o IDENTIFIABLE EXCHANGES t—1 o DELETIONS h-1 to ACENTRICS o o DICENTRICS 5/13 9/2 3 TOTAL BREAKS AND GAPS CO CELLS WITH ABERRATIONS 0. 05 0. 09 TOTAL BREAKAGE FREQUENCY -0L-o 1-3 100 90 NO. OF CELLS td td H 01 0 vQ • i-3 iQ o i-3 td cd H 01 0 vQ • i-3 iQ O TYPES OF GAPS AND BREAKS o H o O to o O . cn > CHROMOSOME GROUPS L. S. D. 8 o o o O cn O o o O to td CHROMOSOME GROUPS L. S. D. 8 H I—1 to -J o 1—1 - j o CHROMOSOME GROUPS L. S. D. 8 O o o O o O O o o r o CHROMOSOME GROUPS L. S. D. 8 o o o 1—1 M o o O o \-> CHROMOSOME GROUPS L. S. D. 8 o o o o O o o O o o CHROMOSOME GROUPS L. S. D. 8 o o o o O o o O o H O CHROMOSOME GROUPS L. S. D. 8 to CO o cn 1—1 TOTAL co to CO cn H «£> ID. GAPS ID. BREAKS o o NON-1 DENT IF.T. EXCHANGES 1—1 to IDENTIFIABLE EXCHANGES o h-> DELETIONS H to ACENTRICS o o DICENTRICS 4/23 9/19 TOTAL BREAKS AND GAPS co ~ j CELLS WITH ABERRATIONS 0.04 0.10 TOTAL BREAKAGE FREQUENCY -TL-o i-3 100 100 NO. OF CELLS rjd Cd H CQ 0 • i-3 in a i-3 Cd Cd H CQ 0 • ^3 iQ o TYPES OF GAPS AND BREAKS O H O to cn o o to co o > CHROMOSOME GROUPS L. S. D. - 9 o o o to to o o o H Cd CHROMOSOME GROUPS L. S. D. - 9 o o to CO o t—1 o CO VO O CHROMOSOME GROUPS L. S. D. - 9 o O o M M o o o o H O CHROMOSOME GROUPS L. S. D. - 9 o o O O o o o o CO M CHROMOSOME GROUPS L. S. D. - 9 o o O O O o M o 1—1 M rrj CHROMOSOME GROUPS L. S. D. - 9 o o O M O o o o I—1 o CHROMOSOME GROUPS L. S. D. - 9 o to H CO (-• cn o to to CO TOTAL L. S. D. - 9 t o to Cn co to ID. GAPS ID. BREAKS o o NON-IDENTIF. EXCHANGES o o IDENTIFIABLE EXCHANGES o o DELETIONS M to ACENTRICS o o DICENTRICS 3/25 6/32 TOTAL BREAKS AND GAPS CO cn CELLS WITH ABERRATIONS 0.03 0.06 TOTAL BREAKAGE FREQUENCY -ZL-o H3 100 100 NO. OF CELLS td td H CQ 0 cQ • 1-3 O i-3 td w H CQ 0 • i-3 cQ O TYPES OF GAPS AND BREAKS O o o O 4^ o - o M 4^ cn > CHROMOSOME GROUPS L. S. D. - 10 to I—1 o O to o o O O LO td CHROMOSOME GROUPS L. S. D. - 10 o o o H 1 CTi o LO r-J H 4^ o CHROMOSOME GROUPS L. S. D. - 10 M o o LO O M M O O o 1—1 o CHROMOSOME GROUPS L. S. D. - 10 O o o O O O O O o r-1 w CHROMOSOME GROUPS L. S. D. - 10 O o o O O o O O o o • CHROMOSOME GROUPS L. S. D. - 10 O o M O O M O o o o CHROMOSOME GROUPS L. S. D. - 10 LO M H to to to Ln t-1 Ln TOTAL 4^ h-1 -0 to to ID. GAPS ID. BREAKS o o NON-1DENTIF. EXCHANGES o o IDENTIFIABLE EXCHANGES o o DELETIONS o H ACENTRICS o O DICENTRICS 4/17 8/22 TOTAL BREAKS AND GAPS 4^ 03 CELLS WITH ABERRATIONS 0.04 0.08 TOTAL BREAKAGE FREQUENCY -ZL-- 74 -APPENDIX C. DETAILED RESULTS OF BEFORE AND AFTER TREATMENT WITH GROWTH HORMONE ON A PATIENT WITH FANCONI'S ANAEMIA AHG BGH 91 107 102 NO. OF CELLS td td Isog. o o td td .(' Isog. i-3 O O i-3 td td te1 0 >-3 O TYPES OF GAPS & BREAKS o o o to co cn CTi 0 - j 0 co 1—1 > o 1—1 o to to CO 0 to CD 0 cn 0 l— 1 cn td FANCONI'S ANAEMIA to o CO (-> H 0 to M to 0 -J CO l -1 O to 1—1 0 FANCONI'S ANAEMIA o .I-1 o o to l— 1 CO r-> 0 CO 0 M 0 O M 0 FANCONI'S ANAEMIA o o o o M o M O H CO 0 O 0 l— 1 M M FANCONI'S ANAEMIA o H o o o o O O O O 0 O 0 I-1 O FANCONI'S ANAEMIA o o o o o o H O O 1—1 0 O 0 O O O FANCONI'S ANAEMIA to Cn o cn 1—1 cn h-1 O to co M CO co cn 4^ to O co cn CO VD TOTAL FANCONI'S ANAEMIA to CO CO 4^  cn to 4^  cn CO ID. GAPS ID. BREAKS o O 4^  II to CO NON-IDENTIFIABLE EXCHANGES o to 4i> IDENTIFIABLE EXCHANGES I—1 to to DELETIONS co CO M -J ACENTRICS o O O DICENTRICS s 38/46 47/58 TOTAL BREAKS & GAPS h-1 o co o to cn CELLS WITH ABERRATIONS o • to 0.355I 0.461 TOTAL BREAKAGE FREQUENCY 0.110 1.26 1.8 0 BREAK FREQ/ ABERRANT CELL -5L-- 76 APPENDIX D. DETAILED ANALYSIS OF TREATMENT WITH MITOMYCIN-C o f o 100 100 NO. OF CELLS a w H CO 0 • r3 cQ o tu to H CQ o • i-3 cQ O TYPES OF GAPS AND BREAKS o o o o Ui UJ to OY M to M O > CHROMOSOME GROUPS MITOMYCIN - C o o o O to u> w t-1 00 to CHROMOSOME GROUPS MITOMYCIN - C o o o to <Js cn o to to H to o CHROMOSOME GROUPS MITOMYCIN - C o o o o 4^  CD O o OJ o CHROMOSOME GROUPS MITOMYCIN - C O o o O o M UI O (-• M K CHROMOSOME GROUPS MITOMYCIN - C o o o to o CD O o to CHROMOSOME GROUPS MITOMYCIN - C o o o o H H Ui O . o . o • Q CHROMOSOME GROUPS MITOMYCIN - C o o o Ui (-• to to 00 4^  CTi 00 M TOTAL MITOMYCIN - C o M «J H Oi p-1 I .£> M ID. GAPS ID. BREAKS „o O NON- IDENT IF. EXCHANGES o 00 IDENTIFIABLE EXCHANGES o to DELETIONS Ui M 00 ACENTRICS o O DICENTRICS 5/17 181/41 TOTAL BREAKS AND 3GAPS U) CT> 4^  CELLS WITH ABERRATIONS 0.05 1.81 TOTAL BREAKAGE FREQUENCY -LL-

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