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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,  U n i v e r s i t y o f 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 i n the D i v i s i o n o f M e d i c a l Genetics  We accept t h i s t h e s i s as conforming t o the r e q u i r e d  standard.  THE UNIVERSITY OF BRITISH COLUMBIA MAY 1969  In presenting  this thesis in p a r t i a l fulfilment of the requirements for  an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t f r e e l y available for reference  and  Study.  I further agree that permission for extensive copying of this thesis for scholarly purposes may by his representatives.  be granted by the Head of my  Department or  It is understood that copying or publication  of this thes.is for f i n a n c i a l gain shall not be allowed without written  permission.  Department of The University of B r i t i s h Columbia Vancouver 8, Canada  my  - i I The  ABSTRACT  frequency o f chromosome breaks was  increased i n r e p l i -  cate c u l t u r e s from each o f ten i n d i v i d u a l s when l y s e r g i c  acid  d i e t h y l a m i d e a t a c o n c e n t r a t i o n o f 1 ug/ml o f c u l t u r e was 24 hours p r i o r to the h a r v e s t o f the c e l l s .  The  between the c o n t r o l and t r e a t e d c u l t u r e s ranged  added  differences from +3.00 to  +7.93 w i t h a mean o f +4.63, i n d i c a t i n g 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 o f chromosomes o f the complement. No  s i g n i f i c a n t d i f f e r e n c e i n e i t h e r the number o f c e l l s  w i t h a b e r r a t i o n s , or the number of.breakage events was  observed  between c e l l s c u l t u r e d from a p a t i e n t w i t h Fanconi's anaemia b e f o r e and 24 hours a f t e r treatment w i t h 250 ug. o f growth h o r mone.  Both were s i g n i f i c a n t l y i n c r e a s e d over the c o n t r o l .  After  treatment w i t h growth hormone, the number o f breaks per aberrant c e l l was  decreased, and the d i s t r i b u t i o n o f f r e q u e n c i e s o f s p e c i -  f i c types o f a b e r r a t i o n s was  changed.  Non-homologous exchange  f i g u r e s were the o n l y two break events observed i n c u l t u r e s the p a t i e n t .  None were observed i n c o n t r o l c e l l s .  The  t i o n o f breaks among the seven groups o f chromosomes was The  frequency o f chromosome a b e r r a t i o n s was  from  distriburandom.  increased i n  c u l t u r e s from a s i n g l e i n d i v i d u a l when t r e a t e d w i t h 1 ug/ml o f mitomycin-C  f o r one hour a t the b e g i n n i n g o f the c u l t u r e p e r i o d .  In the t r e a t e d c u l t u r e s , 181 breaks were observed i n 64 o f the 100 c e l l s examined, whereas o n l y 5 breaks were observed i n three o f the 100 c e l l s s c o r e d i n the c o n t r o l samples.  Forty-seven  exchange c o n f i g u r a t i o n s were observed i n the t r e a t e d c u l t u r e s , 42.56% o f these b e i n g 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 o f chromosomes o f 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 p r o v i d e d by h i s committee chairman, sion o f Medical Genetics.  Dr. M. J . Corey o f the D i v i -  Dr. Corey a l s o deserves s p e c i a l thanks  f o r h e r c o n t i n u e d i n t e r e s t and enthusiasm r e s e a r c h , and a s s i s t a n c e  i n d i r e c t i n g the  i n the p r e p a r a t i o n o f the t h e s i s .  S p e c i a l thanks are a l s o g i v e n t o Dr. J . R. M i l l e r , Head o f the D i v i s i o n o f M e d i c a l G e n e t i c s , f o r h i s d i r e c t i o n and encouragement i n a l l a s p e c t s o f the p r o j e c t . Appreciation  i s expressed t o Dr. S. I s r a e l s , Head o f the  Department o f P a e d i a t r i c s , Dr. T. B i s a l p u t r a , Department o f Botany,  and Dr. C. Finnegan, Department o f Zoology f o r t h e i r  suggestions and comments. Technical  assistance  was r e c e i v e d  the D i v i s i o n o f M e d i c a l G e n e t i c s .  from s e v e r a l people i n  I am e s p e c i a l l y g r a t e f u l t o  Mrs. L. McCarthy and Miss J . McLeod both o f whom gave f r e e l y o f t h e i r time and understanding o f c u l t u r e technique procedures. My w i f e Karen a s s i s t e d i n s e v e r a l phases o f the t h e s i s .  i n the p r e p a r a t i o n  Without her continued support and encouragement  t h i s work would n o t have been p o s s i b l e . S p e c i a l thanks a l s o go t o Mrs. J u l i a Andrews and Mrs. Deanna Dong f o r t y p i n g o f the manuscript. T h i s work was supported by a grant from the M e d i c a l Research C o u n c i l o f Canada t o Dr. M. J . Corey.  - iv TABLE OF CONTENTS I II III IV V  ABSTRACT  i  ACKNOWLEDGEMENTS  i i i  LIST OF TABLES  vii  LIST OF FIGURES  viii  INTRODUCTION A.  1  E f f e c t s of Lysergic Diethylamide  (LSD)  Acid In V i t r o  B.  E f f e c t s o f LSD  In Vivo  C.  E f f e c t s o f LSD In Utero  D.  Lysergic  Chromosomes  E.  Fanconi's Anaemia  F.  E f f e c t s of. M i t o m y c i n C In_ V i v o 8  METHODS  11  Experimental Procedures 1. 2.  C.  6  r  MATERIALS AND  B.  5  7  and ln_ V i t r o  A.  3  A c i d Diethylamide - E f f e c t s  on M e i o t i c  VI  2  Experiments Using L y s e r g i c A c i d Diethylamide (LSD) Treatment o f Leukocytes from a P a t i e n t w i t h Fanconi's Anaemia w i t h Growth Hormone  3.  Experiments  4.  Blinding  5.  Microscopy  11 11 12  U s i n g Mitomycin-C  12  and Coding o f S l i d e s  13  C r i t e r i a Used to D e f i n e Chromosome Aberrations Determination o f the D i s t r i b u t i o n o f Breakage Among the Groups o f Chromosomes  13 14 14  -  VII  V  -  RESULTS A.  15 L y s e r g i c A c i d Diethylamide In V i t r o Study  15  1.  S i n g l e Break Events  18  2.  Two  22  3.  Gaps  Break Events  22  4. B.  D i s t r i b u t i o n o f Breakage Among Chromosome Groups E f f e c t s o f Growth Hormone on a P a t i e n t  24  w i t h Fanconi's Anaemia  25  1.  S i n g l e Break Events  31  2.  Two  31  3.  Gaps  4.  D i s t r i b u t i o n o f Breaks Among  Break Events  31  Chromosome Groups C.  VIII  33  Mitomycin-C  35  1.  S i n g l e Break Events  35  2.  Two  40  3.  Gaps  40  4.  D i s t r i b u t i o n o f Break Events Among Chromosome Groups  42  Break Events  DISCUSSION  44  A.  Lysergic Acid Diethylamide  44  B.  Fanconi's Anaemia  46  C.  Mitomycin-C  49  - vi IX  X  SUMMARY  53  A.  L y s e r g i c A c i d Diethylamide  53  B.  Fanconi's Anaemia  53  C.  Mitomycin-C  54  REFERENCES  56  APPENDIX  A. -  Leukocyte C u l t u r e  APPENDIX  B. -  Detailed Results  APPENDIX  C. -  D e t a i l e d R e s u l t s o f Before and A f t e r Treatment w i t h Growth Hormone on a P a t i e n t w i t h Fanconi's Anaemia  74  D e t a i l e d A n a l y s i s o f Treatment w i t h Mitomycin-C  76  APPENDIX  D. -  Technique f o r LSD 1-10  60 63  - vii III  LIST OF TABLES  LYSERGIC ACID DIETHYLAMIDE (LSD) I II III IV  FANCONI'S V  VI  VII VIII  Summary o f the R e s u l t s o f Chromosome A n a l y s i s i n Treated and C o n t r o l C u l t u r e s  16  D e v i a t i o n s Between Treated and C o n t r o l Cultures  17  Frequency o f S i n g l e and Two Break Events Observed i n Treated and C o n t r o l C u l t u r e s  19  D i s t r i b u t i o n o f I d e n t i f i a b l e Breaks Among the Chromosome Groups  23  ANAEMIA Summary o f R e s u l t s o f Chromosome A n a l y s i s Before and A f t e r Treatment with Growth Hormone, and i n a 'Standard'  26  Frequency o f S i n g l e and Two Break Events Observed i n C u l t u r e s Before and A f t e r Treatment with Growth Hormone, and i n a 'Standard'  28  I d e n t i f i c a t i o n o f Chromosomes Involved i n Exchange F i g u r e s ;  32  D i s t r i b u t i o n o f I d e n t i f i a b l e Breaks  Before  and A f t e r Treatment w i t h Growth Hormone  34  MITOMYCIN-C IX X XI XII  Summary o f R e s u l t s o f Chromosome A n a l y s i s i n Treated and C o n t r o l C u l t u r e s Frequency o f S i n g l e and Two Break Events i n Treated and C o n t r o l C u l t u r e s I d e n t i f i c a t i o n o f Chromosomes Involved i n Exchange F i g u r e s i n Treated C u l t u r e s  41  D i s t r i b u t i o n o f I d e n t i f i a b l e Breaks Among Chromosome Groups  43  36 39  - viii  IV  -  LIST OF FIGURES  Figure 1.  2.  3.  R e p r e s e n t a t i v e Types o f Chromosomal A b e r r a t i o n s Observed i n C o n t r o l C u l t u r e s and i n C u l t u r e s Exposed to L y s e r g i c A c i d Diethylamide  20-21  R e p r e s e n t a t i v e Types o f Chromosomal A b e r r a t i o n s Observed Before and A f t e r Treatment w i t h Growth Hormone i n a P a t i e n t w i t h Fanconi's Anaemia  29-30  R e p r e s e n t a t i v e Types o f Chromosomal A b e r r a t i o n s Observed i n C u l t u r e s Exposed to Mitomycin-C  37-38  - 1 V The  INTRODUCTION  a s s o c i a t i o n o f chromosomal a b e r r a t i o n s w i t h  malformations and malignancy has  congenital  s t i m u l a t e d an i n c r e a s e d i n t e r e s t  i n the study o f the e f f e c t s o f a wide v a r i e t y o f agents on chromosomes.  the  Such s t u d i e s have a p o t e n t i a l p r a c t i c a l , as w e l l  as academic, value  as they p r o v i d e  a means o f i d e n t i f y i n g  t o x i c and p o s s i b l y t e r a t o g e n i c or c a r c i n o g e n i c  cyto-  agents.  Some o f the agents which induce chromosomal a b e r r a t i o n s were f i r s t i d e n t i f i e d by p l a n t c y t o g e n e t i c i s t s .  Extensive  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 o f a v a r i e t y o f chemical cal  agents on the chromosomes have been p u b l i s h e d  Roller,  1947;  Oehlkers,  1952;  physi-  (Darlington  access  and  to m a t e r i a l i n which a  l a r g e number o f c e l l d i v i s i o n s were n a t u r a l l y o c c u r r i n g . the mammalian c y t o g e n e t i c i s t was  hindered  Consequently, mammalian c y t o g e n e t i c s remained a r e s t r i c t e d  However,  by the t e c h n i c a l d i f f i -  c u l t y o f o b t a i n i n g adequate numbers o f d i v i d i n g c e l l s  middle n i n e t e e n  and  R e v e l l , 1952).  P l a n t c y t o g e n e t i c i s t s had  cytogenetics,  review  for a n a l y s i s .  and p a r t i c u l a r l y human f i e l d o f study u n t i l  the  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  d i s c o v e r y t h a t phytohemagglutinin  d i v i s i o n o f c i r c u l a t i n g human white b l o o d  (PHA)  stimulated  c e l l s , made i t p o s s i b l e  to o b t a i n adequate numbers o f r a p i d l y d i v i d i n g c e l l s (Nowell,  1960).  in culture  Refinements were soon made to the i n v i t r o  c u l t u r e technique p r o v i d i n g a convenient method o f chromosome preparation  s u i t a b l e for d e t a i l e d analysis  (Moorhead et a l . 1960).  - 2  -  With the advent o f a technique t h a t enabled r e s e a r c h e r s to study the morphology o f the human chromosome complement, a system  f o r the c l a s s i f i c a t i o n o f the chromosomes t h a t would  become u n i v e r s a l l y r e c o g n i z e d was i n 1960 by a "Proposed Chromosomes" (Denver  T h i s was  effected  System of Nomenclature o f Human M i t o t i c  Report,  i n London and Chicago  established.  1960).  T h i s was  (Chicago Conference,  subsequently  revised  1966).  Aside from b e i n g a u s e f u l d i a g n o s t i c a i d i n determining chromosome a b n o r m a l i t i e s r e s p o n s i b l e f o r some c o n g e n i t a l malf o r m a t i o n s , the three day PHA  s t i m u l a t e d leukocyte c u l t u r e o f  a few drops o f human b l o o d p r o v i d e s a r a p i d and convenient  system  f o r s t u d y i n g the e f f e c t s o f a g r e a t v a r i e t y o f agents on the chromosomes. The  study presented here was  c a r r i e d out to i n v e s t i g a t e the  frequency and type o f chromosomal a b e r r a t i o n s observed i n human p e r i p h e r a l leukocytes exposed in_ v i t r o to (a) l y s e r g i c d i e t h y l a m i d e , and  (b) mitomycin-C.  A comparison  acid  o f the  frequency  and types o f chromosomal a b e r r a t i o n s i n samples o b t a i n e d from a p a t i e n t w i t h Fanconi's Anaemia b e f o r e and a f t e r treatment w i t h growth hormone i s a l s o i n c l u d e d . A.  E f f e c t s o f L y s e r g i c A c i d Diethylamide  (LSD)  In V i t r o  L i t t l e work has been r e p o r t e d on the e f f e c t s o f LSD i n v i t r o i n comparison  t o the number o f r e p o r t s p u b l i s h e d on i n  v i v o s t u d i e s ; o n l y two r e p o r t s , both by Cohen and h i s a s s o c i a t e s , have appeared  i n the literature„(Cohen, M a r i n e l l o , and Black,  -  1967;  Cohen, H i r s c h h o r n ,  3  -  and Frosch,  ing a marked d e p r e s s i o n o f mitoses  1967).  They r e p o r t e d  find-  in dividing c e l l polutions,  and an i n c r e a s e i n chromosomal a b e r r a t i o n s when leukocyte c u l t u r e s were t r e a t e d with LSD.  Most the chromosome a b e r r a t i o n s  i n these s t u d i e s were chromatid apparent  and  observed  i s o c h r o m a t i d breaks,  although  exchange f i g u r e s and d i c e n t r i c chromosomes were a l s o  observed. The exchange c o n f i g u r a t i o n s observed by Cohen ejt a_l. (l967_)t i n the LSD  t r e a t e d c u l t u r e s resembled those i n c u l t u r e s exposed  to mitomycin-C found  i n two  (Nowell,  1964;  Cohen and  Shaw, 1964)  and  autosomal r e c e s s i v e d i s e a s e s , Fanconi's  (Bloom e_t aJL. 1966;  German and C r i p p a , 1966)  (German, A r c h i b a l d , and Bloom, 1965). i s , however, much lower  i n LSD  The  those  anaemia  and Bloom's syndrome frequency o f a b e r r a t i o n s  treated cultures.  N e i t h e r the mechanism nor the phase o f the m i t o t i c c y c l e a t which LSD B.  i s e f f e c t i v e has been i n v e s t i g a t e d .  E f f e c t s o f LSD The  years had  In V i v o  c o n s i d e r a b l e use o f LSD by young people  i n the l a s t  l e d r e s e a r c h e r s to i n v e s t i g a t e not o n l y i t s e f f e c t s  few on  chromosomes but a l s o i t s p o s s i b l e mutagenic and t e r a t o g e n i c effects. At the p r e s e n t time,  s e v e r a l authors have r e p o r t e d  finding  an i n c r e a s e d l e v e l o f chromosome damage i n 'users ' o f LSD  (Cohen,  Hirschhorn,  Irwin  and Frosch, 1967;  and Egozcue, 1967;  H i r s c h h o r n and Cohen, 1967;  Egozcue, Irwin and Maruffo,  1968).  Cohen e t a l . (l967) observed t h a t the frequency o f chromosomal a b e r r a t i o n s ranged from 5.3% to 25.1% i n the l e u k o c y t e s from 18 LSD  'users'.  i n a s e r i e s o f drug f r e e c o n t r o l s , the frequency o f  chromosomal a b e r r a t i o n s ranged from 2.3% to 5.5%,  indicating a  t h r e e to four times i n c r e a s e i n damage i n t r e a t e d  cultures.  Irwin and Egozcue chromosome breakage  (1967) observed an i n c r e a s e d l e v e l o f  i n 6 out o f 8 LSD  'users . 1  C o n t r o l breakage  f r e q u e n c i e s ranged from 7 to 25% whereas p a t i e n t s i n g e s t i n g demonstrated a 12 t o 38% breakage  LSD  rate.  Egozcue e t a l . (1968) i n a l a t e r s e r i e s o f drug f r e e cont r o l s and LSD from 6.0  'users', observed c o n t r o l breakage r a t e s to v a r y  to 16.5% w h i l e i n d i v i d u a l s who  had i n g e s t e d LSD demon-  s t r a t e d breakage v a l u e s r a n g i n g from 8 to 45%. i n c r e a s e i n chromosome damage was  A two  observed i n LSD  fold  'users . 1  N e i t h e r Cohen e t 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 o f chromosome breakage and the number o f doses taken, the amount o f the dose, or the p e r i o d o f time which had e l a p s e d between the l a s t dose and the time a t which the study was Hungerford et. a_l.  undertaken.  (1968) r e p o r t e d f i n d i n g a t r a n s i e n t  i n c r e a s e o f chromosomal a b n o r m a l i t i e s when LSD was  injected  i n t r a v e n e o u s l y , w i t h a r e t u r n to c o n t r o l f r e q u e n c i e s one month a f t e r a d m i n i s t r a t i o n o f the f i n a l  dose.  A number o f r e s e a r c h e r s have r e p o r t e d f i n d i n g no i n chromosome damage i n i n d i v i d u a l s i n g e s t i n g LSD 1967; 1968).  Bender  and Sankar,  1968;  increase  (Loughman e t a l .  Sparkes, Melnyk, and B o z z e t t i ,  - 5 Loughman e t aJL. (1967) r e p o r t e d t h a t leukocytes p a t i e n t s r e c e i v i n g l a r g e doses o f LSD  (4,000 ug.)  from 8  f a i l e d to show  a s i g n i f i c a n t l y higher breakage r a t e than i n the c o n t r o l c u l t u r e s . Bender and  Sankar  (1968) r e p o r t e d t h a t seven c h i l d r e n t r e a t e d f o r  p e r i o d s o f 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 i n c r e a s e i n chromosome breakage.  Chromosome s t u d i e s were not  performed u n t i l 20 to 48 months a f t e r the l a s t dose.  Sparkes  e t a l . (1968) a l s o found no s i g n i f i c a n t i n c r e a s e i n chromosome damage i n a group o f  'users' and m e d i c a l l y t r e a t e d i n d i v i d u a l s  over the c o n t r o l s . The  reported  t i o n o f LSD, whether LSD  f i n d i n g s o f chromosome damage caused by  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 does or does not  i n c r e a s e the frequency  damage i s not p o s s i b l e at t h i s C.  E f f e c t s o f LSD The  time.  on the chromosomes o f c h i l d r e n whose  taken the drug d u r i n g pregnancy have been r e p o r t e d  (Cohen, H i r s c h h o r n , Zellweger  and Frosch,  e t a l . 1967;  1967;  Egozcue ejt a l .  Cohen e t a l . 1968).  observed a high frequency  o f four c h i l d r e n exposed to LSD  and  f o u r t h months o f pregnancy.  (range 22-28%)  Cohen e t a l . (1968)  i n utero d u r i n g the  showed a h i g h  19%)  in  third  Egozcue e_t a l . (1968) a l s o  s t u d i e d four c h i l d r e n whose mothers took LSD Three o f these  1968;  o f chromosome damage (13 and  two  frequency  o f chromosome  In Utero  e f f e c t s o f LSD  mothers had  inges-  frequency  d u r i n g pregnancy.  o f chromosome  breaks,  Cohen et_ a l . (1968) r e p o r t e d f i n d i n g an  o f breakage among c h i l d r e n exposed to LSD  increased  i n utero when  - 6 compared to matched c o n t r o l s . Although there i s a great d e a l o f c o n t r o v e r s y over  the  p o s s i b l e t e r a t o g e n i c e f f e c t s o f i n - u t e r o exposure to LSD, one  only  r e p o r t o f c o n g e n i t a l malformations i n a c h i l d whose mother  i n g e s t e d LSD colleagues  d u r i n g pregnancy has  Zellweger  and h i s  (1967) d e s c r i b e d a case o f a c h i l d born w i t h  d e f o r m i t i e s , and  i t was  e a r l y i n her pregnancy. sonable  appeared.  known t h a t the mother had The  authors  lower l e g  ingested  f e l t t h a t i t was  LSD  not unrea-  to assume a c a s u a l r e l a t i o n s h i p between the LSD  and  the  limb d e f o r m i t i e s . Sato and Pergament  (1968) found no  i n a c h i l d born to a mother who  had  foetal  i n g e s t e d LSD  abnormalities e a r l y i n the  pregnancy. Teratogenic reported  e f f e c t s o f LSD  (Alexander  Rugowski, 1967).  e t a l . 1967;  The  i n r a t s and hamsters have been Geber, 1967;  Auerbach  and  most common a b n o r m a l i t i e s have been  of the b r a i n , s p i n a l cord and  l i v e r , w i t h a b o r t i o n and  those  under-  developed o f f s p r i n g a l s o p r e v a l e n t . Warkany and Takacs  (1968) r e p o r t e d f i n d i n g no  i n c r e a s e i n a b n o r m a l i t i e s when LSD was  significant  i n j e c t e d i n t o 55 pregnant  rats. The  mutagenic e f f e c t s of LSD  r e p o r t e d by Browning found no evidence D.  (1968) .  i n D r o s o p h i l a have been  However, Grace et_ a_l.  (1968)  o f chromosome breakage or mutations.  L y s e r g i c A c i d Diethylamide  - E f f e c t s on M e i o t i c Chromosomes  Damage to m e i o t i c chromosomes o f mice i n j e c t e d w i t h  LSD V  - 7  has been r e p o r t e d .  -  Skakkebaek e t aJL. (1968) r e p o r t e d f i n d i n g  s e v e r a l breaks, gaps, and u n i d e n t i f i a b l e  fragments  i n treated  animals, but w i t h few e x c e p t i o n s , not i n the c o n t r o l s . and Mukherjee  Cohen  (1968) observed a ten f o l d i n c r e a s e i n chromosome  damage i n spermatogonial c e l l s and bone marrow c e l l s o f mice t r e a t e d w i t h LSD E.  i n comparison  to the c o n t r o l s .  Fanconi's Anaemia Fanconi's anaemia i s a syndrome o f m u l t i p l e c o n g e n i t a l mal-  formations and p r o g r e s s i v e pancytopenia which i s thought to be i n h e r i t e d as an autosomal  recessive.  C y t o g e n e t i c a l l y the c o n d i -  t i o n i s c h a r a c t e r i z e d by a v a r i e t y o f chromosomal anomalies s i s t i n g o f chromatid and i s o c h r o m a t i d gaps and breaks, and exchange c o n f i g u r a t i o n s .  (Schmid e_t a l . 1965;  con-  fragments,  Bloom e t a l .  1966);. Schmid e t a l . (1964) were the f i r s t to r e c o g n i z e the chromosomal damage i n p a t i e n t s w i t h Fanconi's anaemia.  Bloom e_t al_. (1966) s t u d i e d the p e r i p h e r a l leukocytes o f 12 p a t i e n t s w i t h t h i s d i s o r d e r .  A v a r i e t y o f s t r u c t u r a l exchanges,  e n d o r e d u p l i c a t i o n s and other chromosomal a b e r r a t i o n s were observed i n ten o f the twelve p a t i e n t s . cells,  (19.43%) o f the 1,621  o f one type or another.  In the leukocyte c u l t u r e s ,  315  c e l l s s c o r e d had a breakage event  In bone marrow p r e p a r a t i o n s from  one  o f the p a t i e n t s , 18% o f the c e l l s examined possessed a breakage event.  Seventy-eight chromatid exchanges were observed i n the  leukocyte c u l t u r e s , and two were observed  i n the c e l l s o f the  - 8 -  bone marrow. A l a r g e number o f c o n t r o l c e l l s were s t u d i e d t o determine  (approximately  15,000)  the frequency o f a b e r r a t i o n s i n  p a t i e n t s w i t h o u t t h i s form o f c o n s t i t u t i o n a l a p l a s t i c anaemia. E i g h t chromatid exchanges and o n l y a few e n d o r e d u p l i c a t i o n s were  observed. S w i f t and H i r s c h h o r n (1966) s t u d i e d two p a t i e n t s w i t h  disorder.  this  In both cases the p e r i p h e r a l l e u k o c y t e s and f i b r o b l a s t s  were s t u d i e d , and i n one case the bone marrow.  In the leukocyte  c u l t u r e s o f both cases, 4 9 % o f the c e l l s were abnormal. a l s o observed  I t was  i n the f i b r o b l a s t c u l t u r e s o f case I t h a t 4 1 % o f  the c e l l s were abnormal w h i l e i n case I I , 3 6 % o f the c e l l s were aberrant.  Chromatid  and i s o c h r o m a t i d gaps and breaks,  fragments,  exchange f i g u r e s and d i c e n t r i c chromosomes were observed i n l e u k o c y t e and f i b r o b l a s t c u l t u r e s .  Ten percent o f the bone  marrow c e l l s c o n t a i n e d a b n o r m a l i t i e s , i n c l u d i n g chromatid and i s o chromatid breaks and gaps, fragments r i n g or d i c e n t r i c  and exchange f i g u r e s but no  chromosomes.  . To date, there has been no apparent success i n determining 'the u n d e r l y i n g causes r e s p o n s i b l e f o r the degenerative a p l a s i a or the a s s o c i a t e d c o n g e n i t a l malformations F.  observed i n t h i s d e f e c t .  E f f e c t s o f Mitomycin-C In Vivo and In V i t r o Wakaki e_t a l . (1958) f i r s t  f r a c t i o n o f the mitomycin subsequently used  i s o l a t e d mitomycin-C as a d i s t i n c t  group o f a n t i b i o t i c s .  The drug was  f o r the treatment o f n e o p l a s t i c d i s e a s e s , but  because o f i t s t o x i c e f f e c t on the bone marrow, and the subse-  - 9 quent  thrombocytopenia  and l e u k o p e n i a , i t s u s e was d i s c o n t i n u e d  (Watne, Moore, a n d B e d r e t t i a , Mertz  (1961) was t h e f i r s t  compound o n t h e chromosomes. Vicia  f a b a , he o b s e r v e d  exposed  1967; J o n e s ,  to a solution  1959) .  to observe  the e f f e c t  U s i n g r o o t t i p chromosomes o f  c h r o m a t i d a b e r r a t i o n s when c e l l s  o f 0.001% m i t o m y c i n - C  f i g u r e s were e v i d e n t 24 h o u r s  greatest. still  after  division  t h e f r e q u e n c y o f c h r o m a t i d a b e r r a t i o n s was  This high  f r e q u e n c y 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  e v i d e n t 96 h o u r s  after  exposure.  The d a t a i n d i c a t e d  that  to the a c t i o n o f  mitomycin. Mitomycin-C  h a s b e e n r e p o r t e d t o be an e f f e c t i v e b r e a k i n g  a g e n t o f human chromosomes Shaw to  Few  He  treatment w i t h the drug,  most o f t h e i n t e r p h a s e s t a g e s were s e n s i t i v e the  were  f o r one h o u r .  a l s o o b s e r v e d a marked i n h i b i t i o n o f m i t o s i s .  b u t a t 72 h o u r s  of this  (1964),  and Shaw and Cohen  l e u k o c y t e s i_n v i t r o  f i r s t hour  figurations.  Large  observed  that  that  markedly.  t o the drug  Treatment  o r more c o m p l e t e l y i n h i b i t e d  induced  o f exchange c o n -  fragments  52nd, 62nd o r 6 8 t h t o 72nd h o u r ,  C f o r 24 h o u r s  mitomycin-C  t h e drug  chromosomes were uncommon.  i f c e l l s were e x p o s e d  decreased quite  activity.  By a d d i n g  as a l a r g e number  and s m a l l a c e n t r i c  and d i c e n t r i c  (1964) , Cohen and  a t a c o n c e n t r a t i o n o f 1 ug/ml f o r t h e  as w e l l  but r i n g  tions  (1965).  i n c u l t u r e , Nowell observed  chromatid breaks  the 24th,  in_ v i t r o b y N o w e l l  were a l s o I t was  found, further  f o r one h o u r a t  t h e number  of aberra-  o f c e l l s with subsequent  mitomycin-  mitotic  - 10 Cohen and Shaw (1964) and Shaw and Cohen treating cells  (1965) r e p o r t e d  i n c u l t u r e w i t h 0.1, and 1.0 ug/ml f o r the l a s t  24 hours o f c u l t u r e .  Again, l a r g e numbers o f breaks and exchange  f i g u r e s were produced, n o t a b l y  a t the secondary  construction  r e g i o n o f chromosomes #1, #9, and #16. The most unusual e f f e c t o f mitomycin-C of exchange f i g u r e s t h a t were produced. Shaw (1964), and Shaw and Cohen rearrangements  was the l a r g e number  Nowell  (1964), Cohen and  (1965), a l l noted the s t r u c t u r a l  f r e q u e n t l y i n v o l v e d members o f apparent homologous  p a i r s o f chromosomes. Experiments  c a r r i e d out on E_. c o l i i n d i c a t e t h a t  causes s e l e c t i v e i n h i b i t i o n o f DNA and p r o t e i n s y n t h e s i s Takagi,  appear  mitomycin-C  (Shiba et_ a l . 1959) w h i l e RNA  to be u n a f f e c t e d .  (Sekiguchi and  1960).  Iyer and S z b a l s k i l i n k i n g o f DNA which  (1964) a t t r i b u t e d the i n h i b i t i o n to cross  i n t e r f e r e d with r e p l i c a t i o n .  - 11 VI A.  Experimental  MATERIALS AND  METHODS  Procedures  A l l experiments were c a r r i e d out on c u l t u r e d human leukocytes u s i n g a m o d i f i c a t i o n o f the technique used by Moorhead et_ a l . (1960), adapted  f o r micro-amounts o f whole b l o o d .  t e c h n i q u e see Appendix In  complete  A.)  each o f the experiments  b l o o d was  (For  performed,  0.25  added to 5 ml. o f c u l t u r e medium.  cc o f whole  Replicate cultures  were s e t up f o r each o f the t r e a t e d and c o n t r o l experiments,  and  these c u l t u r e s were then incubated f o r approximately 72 hours a t 37°C.  One  and a h a l f to two hours b e f o r e the c e l l s were h a r v e s t e d ,  Colcemid was  added a t a c o n c e n t r a t i o n o f 0.02  arrest c e l l division.  ug/ml o f c u l t u r e , to  At the end o f the i n c u b a t i o n p e r i o d , c e l l s  were t r e a t e d w i t h a hypotonic s o l u t i o n and f i x e d i n 3:1 a b s o l u t e a l c o h o l and g l a c i a l a c e t i c a c i d .  Flame d r i e d s l i d e s were then  prepared from each o f the c u l t u r e s . 1.  Experiments  Using L y s e r g i c A c i d Diethylamide  (LSD)  Ten v o l u n t e e r s , f i v e males and f i v e females were used f o r i n v i t r o experiments w i t h LSD. students, and one was  Nine o f the ten v o l u n t e e r s were  a professor.  These i n d i v i d u a l s were  i n t e r v i e w e d and asked whether they had r e c e i v e d x-rays o f any k i n d f o r d i a g n o s i s or f o r treatment, viral  infections.  and whether they had r e c e n t  The two p a t i e n t s who  are i d e n t i f i e d i n Table I.  had r e c e i v e d r e c e n t x-rays  - 12 Three cc's o f whole b l o o d were o b t a i n e d from each  individual,  and 12 r e p l i c a t e c u l t u r e s were e s t a b l i s h e d . Lysergic a c i d diethylamide, was  ( D e l y s i d , LSD-25, Sandoz Ltd.)  o b t a i n e d i n s e a l e d g l a s s v i a l s c o n t a i n i n g 0.1 mg LSD i n 1.0  ml o f aqueous s o l u t i o n .  The s o l u t i o n was  diluted with s t e r i l e  d i s t i l l e d water to a c o n c e n t r a t i o n o f 20 ug/ml, and 24 hours p r i o r to the t e r m i n a t i o n o f the c u l t u r e s , 0.25  cc (5 ug)  added to s i x o f the r e p l i c a t e c u l t u r e s y i e l d i n g a f i n a l t r a t i o n o f 1 ug/ml. (0.25 cc) was 2.  was concen-  An e q u a l volume o f s t e r i l e d i s t i l l e d water  added to each o f the remaining s i x c u l t u r e s .  Treatment o f Leukocytes from a P a t i e n t w i t h Fanconi's Anaemia w i t h Growth Hormone Blood was  o b t a i n e d from a p a t i e n t immediately b e f o r e t r e a t -  ment w i t h growth hormone, and e i g h t r e p l i c a t e c u l t u r e s  (0.25 cc  whole blood/5 cc o f medium) were e s t a b l i s h e d . Twenty four hours a f t e r treatment w i t h 250 ug. o f growth hormone, another sample o f b l o o d was to the procedure o u t l i n e d A sample  taken and s e t up a c c o r d i n g  above.  from a normal boy,  incubated i n the same media  at the same time, was used as a c o n t r o l p r i m a r i l y to ensure t h a t environmental f a c t o r s d i d not c o n t r i b u t e to the breakage frequency i n c u l t u r e s from the p a t i e n t . 3.  Experiments U s i n g Mitomycin-C A 3 cc sample o f whole b l o o d was  i n d i v i d u a l , and 0.25 replicate  cultures.  o b t a i n e d from a s i n g l e  cc o f whole b l o o d was  added t o each o f twelve  - 13 Mitomycin-C o b t a i n e d from N u t r i t i o n a l B i o c h e m i c a l s Corporat i o n , C l e v e l a n d , Ohio was  d i s s o l v e d i n s t e r i l e d i s t i l l e d water to  a c o n c e n t r a t i o n o f 20 ug/ml. p e r i o d , 0.25  At the b e g i n n i n g o f the i n c u b a t i o n  cc (5 ug.) o f the s o l u t i o n was  added t o s i x o f the  r e p l i c a t e c u l t u r e s to y i e l d a f i n a l c o n c e n t r a t i o n o f 1 ug/ml. equal volume o f s t e r i l e d i s t i l l e d water o f the remaining s i x r e p l i c a t e  (0.25 cc) was  An  added to each  cultures.  A f t e r one hour o f i n c u b a t i o n c e l l s i n a l l c u l t u r e s were washed twice i n c u l t u r e medium, resuspended i n f r e s h media,  and  r e p l a c e d i n the i n c u b a t o r f o r the remainder o f the 72 hour  cul-  ture period. 4.  B l i n d i n g and Coding o f S l i d e s In each study (LSD, Mitomycin-C and Fanconi's anaemia,) the  ten b e s t s l i d e s from each o f the c o n t r o l and t r e a t e d samples were s e l e c t e d and coded to ensure the examiner c o u l d 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 Z e i s s  automatic photomicroscope equipped w i t h phase c o n t r a s t condenser and o b j e c t i v e l e n s e s .  Good, w e l l spread metaphase f i g u r e s were  s e l e c t e d under low power ( x l 6 o b j e c t i v e ) and once a c e l l s e l e c t e d i t was  i n c l u d e d i n the study.  was  Wherever p o s s i b l e ,  10  c e l l s on each o f 10 s l i d e s were s e l e c t e d to y i e l d a t o t a l o f 100 c e l l s  f o r each c o n t r o l or treatment.  Once a c e l l was  s e l e c t e d i t was  (xlOO) f o r s t r u c t u r a l a b e r r a t i o n s .  analyzed under o i l immersion  The c e l l s were then photographed  - 14 on h i g h c o n t r a s t , f i n e g r a i n f i l m B.  (Adox 135, KB-14), and r e a n a l y z e d .  C r i t e r i a Used to Define Chromosome A b e r r a t i o n s A l l c e l l s were analyzed f o r chromosome number and s t r u c t u r a l  aberrations,  i n c l u d i n g gaps, breaks, a c e n t r i c fragments,  deletions,  d i c e n t r i c s , marker chromosomes and exchange c o n f i g u r a t i o n s . A break was.defined  as a d i s c o n t i n u i t y i n an arm i n which  the a c e n t r i c fragment was d i s p l a c e d from i t s alignment w i t h the proximal o r c e n t r i c p a r t o f the arm. the d i s t a l fragment  remained  Achromatic  segments i n which  i n alignment w i t h the proximal arm  were s c o r e d as gaps, and were not c o n s i d e r e d to be breakage events. C o n f i g u r a t i o n s t h a t i n v o l v e d more than one chromosome were presumed to r e s u l t  from chromatid i n t e r c h a n g e s .  Throughout t h i s  study, these c o n f i g u r a t i o n s w i l l be r e f e r r e d to as 'exchanges'. Breaks,  a c e n t r i c fragments  and d e l e t i o n s were scored as  s i n g l e break events, w h i l e d i c e n t r i c chromosomes and exchanges were scored as two break C.  events.  Determination o f the D i s t r i b u t i o n o f Breakage Events Among the Groups o f Chromosomes A measure o f whether or not the breakage events were randomly  d i s t r i b u t e d throughout  the chromosome complement was o b t a i n e d by  comparing the d i s t r i b u t i o n o f observed a b e r r a t i o n s w i t h the number expected on the b a s i s o f the r e l a t i v e l e n g t h o f the chromosomes i n each group.  The r e l a t i v e lengths o f the chromosomes used are  those e s t a b l i s h e d a t the Denver and London Conferences Conference,  1966).  (Chicago  - 15  VII A.  RESULTS  L y s e r g i c A c i d Diethylamide - In V i t r o Study The  cultures B.  -  r e s u l t s o f chromosome a n a l y s i s from each o f the ten s u b j e c t s  for t r e a t e d and  are presented i n Appendix  These r e s u l t s are summarized i n Table I . A t o t a l o f 1010  were a n a l y z e d .  An  c u l t u r e s contained  c o n t r o l c e l l s and  average o f 3.91% aberrations  1094  c e l l s exposed to  o f the c e l l s i n the  whereas 7.89%  t r e a t e d c u l t u r e s were s i m i l a r l y a f f e c t e d .  control  This indicates a  i n the t o t a l number o f c e l l s  a breakage event.  2  (X  demonstrating  t e s t . p<0.001)  A comparison o f the t o t a l number o f breaks i n the c o n t r o l cultures indicated a s i g n i f i c a n t increase  treated cultures.  The  number o f breaks per  100  i n the c o n t r o l c u l t u r e s ranged from 0 to 15.12 4.72. per  In the c u l t u r e s t r e a t e d w i t h LSD, 100  LSD  o f the c e l l s i n the  s i g n i f i c a n t increase  and  control  c e l l s ranged from 4.0  to 18.70  treated  i n the  c e l l s observed w i t h a mean o f  the number o f breaks  w i t h a mean o f  A n e u p l o i d l e v e l s i n both the t r e a t e d and  9.37.  control cultures  were s i m i l a r , w i t h 90.03% o f the t r e a t e d c e l l s and  89.23% o f  the  c o n t r o l c e l l s h a v i n g a modal number o f f o r t y - s i x chromosomes. As  c o n t r o l 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 a c c u r a t e measure o f damage a s s o c i a t e d LSD was and  o b t a i n e d by a n a l y s i s o f d e v i a t i o n s between the  c o n t r o l sample for each i n d i v i d u a l .  The  deviations  p a i r e d t - t e s t v a l u e s f o r s i n g l e break events, two t o t a l break events and  with  treated and  break events,  gaps are presented i n Table I I .  TABLE I and C o n t r o l C u l t u r e s  Summary o f the R e s u l t s o f Chromosome A n a l y s i s i n Treated o  CD to  03 rrj H •P • H 0 0 CD  ^ a u c  u  0  a  & CD  rH X  «  H  a  CO •P O l O rH O rO O CD EH O  H  co  ret 10 H 01 U C rO rH CD 0 O CD "H & •H EH U ^ < •P  CO H  (tf  i  U  M  o  o  rH  CO  rd H CD U rH U CD CD  T  c  T  c  T  ffl a u c  T  J.A.  m  86  123  12.79  19.51  11.63  15.45  15.12  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  1010  1094  184.44  254.99  39.05  79.60  47.21  93.72  TOTAL *  10  Received chest or d e n t a l x-rays w i t h i n s i x months o f t e s t i n g .  18.70 *  TABLE I I D e v i a t i o n s Between Treated and C o n t r o l C u l t u r e s  Gaps  S i n g l e 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 d i f f e r e n c e  +  7.00  + 3.96  + 0.697  + 4.64  3.49  9.86  T value Prob.  ^0.01- >0.001  <0.001  1.44 < 0.2- >0.1  9.51 <0.001  - 18 In each o f the ten c u l t u r e s t r e a t e d w i t h LSD, t h e r e was an increase  i n the number o f s i n g l e break events and the t o t a l  number  o f break events, w i t h the amount o f d e v i a t i o n r a n g i n g from 2.06 to 6.00 breaks per 100 c e l l s f o r s i n g l e break events, and from 3.00 to 7.93 breaks per 100 c e l l s f o r t o t a l break events. number o f two break events was i n c r e a s e d  The  i n four i n d i v i d u a l s ,  decreased i n one, and no d i f f e r e n c e was observed i n f i v e .  (Table  II) A paired t-test analysis indicated a significant  increase  i n s i n g l e break events and t o t a l break events, but not i n two break events. An a n a l y s i s o f s p e c i f i c  types o f a b e r r a t i o n s  i n Table I I I , and r e p r e s e n t a t i v e 1.  i s presented  f i g u r e s are presented i n F i g . 1.  S i n g l e Break Events S i n g l e break events comprised the g r e a t e s t number o f aber-  r a t i o n s i n both the t r e a t e d and c o n t r o l c u l t u r e s , 88.46% o f the t o t a l number o f break events i n the t r e a t e d and 91.30% o f the t o t a l number o f breaks i n the c o n t r o l b e i n g o f t h i s Chromatid breaks  ( F i g . 1-D,  1-E, and 1-F) were the most  common o f the s i n g l e break events s c o r e d . tures,  type.  In the t r e a t e d  cul-  64.33% o f the t o t a l number o f break events were o f t h i s  type, whereas 54.35% o f the t o t a l number o f break events i n the c o n t r o l s were chromatid b r e a k s .  However, 6.12 chromatid breaks  per 100 c e l l s were observed i n the t r e a t e d c u l t u r e s , to 2.47 per 100 c e l l s i n the c o n t r o l .  1094  1010  T o t a l No. of C e l l s  67  6.12 /100  25  2.47 /100  M  24  2.19 /100  16  1.58 /100 0.099 /100  \  T o t a l chromatid breaks  Q  Acentrics td >  O  H  M •  O O O ^£>  Deletions  H  3 "-3 CO  tO  to  O  (-3  0 Hi  cn  PJ 0) 3 Cu Cb  Rings  O  CD < CD 3 Oi n H - ^<  ro H fu 3 ti- iQ roH & CD  No. o f B. E .  &  t-i to ro ro  3  No. o f Aberrations  to  o  O  s 0 rt t-i dd O H h-1 CD  0 O  Dicentrics  rO  NJ  O  Exchanges  K>  No. o f Aberrations  (Ti  O  I'd  to  No. o f B. E.  00  TOTAL NO. OF ABERRATIONS  1— 1  I—  1  cn  o  TOTAL NO. OF B. E .  - 61 -  s  * P  H r t <! CD H 3 CD r t CO Cfl  i-3  > LJ  H H H  - 20  -  FIGURE 1.  R e p r e s e n t a t i v e Types o f Chromosomal A b e r r a t i o n s Observed i n C o n t r o l C u l t u r e s and i n C u l t u r e s Exposed to L y s e r g i c A c i d Diethylamide  A and B - chromatid gaps C - isochromatid gap D, E, and F - chromatid breaks G, H, and I - a c e n t r i c  fragments  J K  -  exchange f i g u r e from t r e a t e d c u l t u r e exchange f i g u r e from c o n t r o l c u l t u r e  L  -  d e l e t e d G group chromosome  ti  - 22 Acentric  fragments  ( F i g . 1-G,  both t r e a t e d and c o n t r o l c u l t u r e s . o f a b e r r a t i o n was i n c r e a s e d to 2.19 a c e n t r i c fragments  1-H,  1-1) were observed i n  The frequency o f t h i s type  from 1.58 per 100 c e l l s  i n the c o n t r o l  per 100 c e l l s i n the t r e a t e d  cultures.  Only two d e l e t i o n s t h a t c o u l d be d e f i n i t e l y i d e n t i f i e d were observed. control.  One was found i n the t r e a t e d c u l t u r e s and one i n the These d e l e t i o n s were observed as c e n t r i c fragments, and  were s c o r e d as s i n g l e break events, 2.  ( F i g . 1-L).  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 i n c r e a s e between the t r e a t e d c u l t u r e s and c o n t r o l s . S i x were observed i n the t r e a t e d c u l t u r e s and 2 i n the c o n t r o l s . The two observed i n the c o n t r o l c u l t u r e s , and four o f the s i x observed i n the t r e a t e d c u l t u r e s were exchange f i g u r e s ,  (Fig.  1-J and 1-K). Two d i c e n t r i c chromosomes were a l s o observed i n the t r e a t e d  cultures.  In the t e n r e p l i c a t e c u l t u r e s two break events were observed i n both t r e a t e d and c o n t r o l c u l t u r e s  from one i n d i v i d u a l , and i n  three i n d i v i d u a l s they were observed o n l y 3.  i n the t r e a t e d r e p l i c a t e s .  Gaps The d i f f e r e n c e i n the frequency o f gaps between the t r e a t e d  and c o n t r o l c u l t u r e s  (Table  II) v a r i e d from -1.89 to +18.00, w i t h  a mean d i f f e r e n c e o f 7.05 and a standard d e v i a t i o n o f 6.39. represents a s i g n i f i c a n t difference  This  ( t - t e s t . p < 0^.01- >0.001)  i n the gaps i n the t r e a t e d and c o n t r o l c u l t u r e s .  TABLE IV Distribution LSD  EXPECTED  A  B  C  D  E  F  G  13  3  8  8  3  1  2  3.86  3. 34  1.77  1. 64  8.99  4.64  B  OBSERVED  8  7  EXPECTED  8.19  4.19  X CONTROL MALES 2  OBSERVED  P <o .25C  > 0.10  Not  s i g . a t 0.05  E  F  G  14  4  1  0  1  13. 99  3.49  3. 02  1.60  1. 16  p <0 .75-  Not  >0.50  s i g . a t 0.05  A  B  C  D  E  F  G  4  5  4  1  1  0  0  1.83 3.55 X2 t o t a l = 7.51 CONTROL FEMALES  level  D  t o t a l = 5.16  EXPECTED LSD  13. 74  X2 t o t a l = 9.60 TREATED FEMALES A  LSD  Breaks Among the Chromosome Groups  TREATED MALES  OBSERVED  LSD  of Identifiable  5. 43  1.52 P < 0 .5-> 0.25  1. 32 Not  level  0.70 0.65 s i g . a t 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 . a t 0.05 l e v e l  - 24 4.  D i s t r i b u t i o n o f Breakage  Among Chromosome  Groups  The d i s t r i b u t i o n o f observed break events among the seven groups o f chromosomes f o r t r e a t e d and c o n t r o l males and females are p r e s e n t e d i n Table IV. significant deviation  Chi-square a n a l y s i s  from random.  i n d i c a t e d no  - 25 B.  E f f e c t s o f Growth Hormone on a P a t i e n t w i t h Fanconi's Anaemia T h i s study was c a r r i e d out on a p a t i e n t w i t h Fanconi's  anaemia who had been assessed a n n u a l l y a t the H e a l t h Centre f o r C h i l d r e n , and who had not r e q u i r e d any treatment other than the a d m i n i s t r a t i o n o f growth hormone. indicated  Previous chromosome  studies  a h i g h frequency o f chromosome a b e r r a t i o n s (Corey and  Andrews,  1968).  In t h i s study, b l o o d samples were o b t a i n e d b e f o r e , and twenty  immediately  four hours a f t e r the a d m i n i s t r a t i o n o f growth  hormone. A normal boy o f approximately the same age was used as a •standard . 1  The d e t a i l e d C.  r e s u l t s o f t h i s study are presented i n Appendix  These r e s u l t s are summarized i n Table V. When the number o f a b e r r a n t c e l l s were compared: i n the  c u l t u r e s b e f o r e and a f t e r treatment w i t h growth hormone, there was a s i g n i f i c a n t i n c r e a s e both b e f o r e (25.49%) and a f t e r ment  treat-  (28.03%) over the standard (10.98%), but no s i g n i f i c a n t  d i f f e r e n c e was observed between the two samples taken from the p a t i e n t w i t h Fanconi's anaemia. Although the t o t a l number o f breakage  events per 100 c e l l s  was i n c r e a s e d i n both samples from the p a t i e n t , t h e r e was a decrease a f t e r the treatment w i t h growth hormone.  In the c e l l s  observed p r i o r to the treatment w i t h the hormone, 46.07 breaks per 100 c e l l s were observed, whereas i n the sample a f t e r ment, 35.51 breaks per 100 c e l l s were observed.  treat-  TABLE V Summary o f R e s u l t s o f Chromosome A n a l y s i s Before and A f t e r Treatment w i t h Growth Hormone/and i n a 'Standard'  Total No. Cells  Total No. Gaps  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  91  21 23.07/100  11 12.08/100  10 10.98/100  1.10  •Standard'  Total No. Breaks  T o t a l No. C e l l s with Aberrations  Br. f r e q . per aberr, Cell  0>  - 27 This i s r e f l e c t e d aberrant c e l l .  i n the number o f break events per  (The r a t i o o f breakage events t o the number o f  aberrant c e l l s . )  In the c u l t u r e s examined p r i o r t o growth h o r -  mone treatment, 1.80 breaks per a b e r r a n t c e l l were observed, whereas a breakage  frequency o f 1.26 breaks per aberrant c e l l  was observed a f t e r  treatment, s u g g e s t i n g t h a t the growth hormone  may have an e f f e c t on the number o f a b e r r a t i o n s found i n each o f the a b e r r a n t c e l l s . I t was f u r t h e r observed b e f o r e treatment w i t h growth hormone t h a t i n d i v i d u a l  c e l l s demonstrated up to and  four breaks per c e l l , but a f t e r  treatment w i t h growth hormone,  no more than two breaks per c e l l were observed. of  including  0, 1, 2, 3, and 4 breaks per c e l l  The d i s t r i b u t i o n  i n the c u l t u r e s b e f o r e  treatment d i d not f i t a p o i s s o n d i s t r i b u t i o n o f r a r e events (X  2  t e s t , p < 0.005).  However, a f t e r  treatment, the d i s t r i b u t i o n  o f c e l l s w i t h up to two breaks per c e l l d i d not d e v i a t e from random ( X  2  t e s t , p <0.25- >0.10).  A comparison o f the d i f f e r e n t types o f a b e r r a t i o n s observed b e f o r e and a f t e r  treatment w i t h growth hormone i s presented i n  Table VI and r e p r e s e n t a t i v e f i g u r e s  are presented i n F i g . 2.  There was a s i g n i f i c a n t d i f f e r e n c e i n the d i s t r i b u t i o n o f s i n g l e break events patient.  (X  2  t e s t . p < 0.005) i n the two samples  from the  The number o f s i n g l e break events and two break events  was e l e v a t e d i n both the samples to the 'standard'.  from the p a t i e n t i n comparison  > a  CO  H O  VD  16  T o t a l No. Chromatid Breaks  w CD  cn  H SI Q  M \  CO  CO  H• O to O IX)  \ M h- • O O O VD  1—  1  1  \ to h-" • O 00 O O  co  \ M H• O 00 O CTi  to  M  CO  1—  1  CO  1—  1  17  o o  T o t a l No. Cells  16.66 /100  o  •  15.68 /100  •a A  H• O CTi O (XI  29  to  27.10 /100  X 10 CQ H3 vQ H CD  H O to  \ H H• O VO o m  to  Acentrics  a  M  12  < Deletions  CO  Hi 0 H H CD CD £2 s3 CD  0» 3  CD  rt  cn  No. o f Aberrations  co cn  No. o f B.E.  O  Rings  O  > Hi 0 rt Hi CD H CO Hi-3 3 H vQ CD H PJ CD rt 3 (U 3 3  CO  3  &  H- 0 rt 3 rjd H O CD H fu Q *T J  el- t d s' < CD  3 ffi  O  O  1-3  o O  O  O  Dicentrics  X to A  O O to  tf I-S (D  O  H- to t- • O 00 O (Ti 1  1  \ cn rji H• O 00 O 00  V  CD  to  O  <T>  rt o 01  o H O  H to  O O H.  1—  1  CO cn  4^1  I—  No. o f Aberrations No. o f B.E. TOTAL NO. OF ABERR. TOTAL NO. OF B.E.  CO 00  - 82 -  3 0 3  CD  O  &  CO CD H  to is w  »  H  H-H-  CO  <  CD 0» & 3  3  •3  U i CD  I  Exchanges  rt H CO  0  3  fu O cn  C  1—  1  rt rt P J 3 O- CD  fu CO H O,  _ 29. -  FIGURE 2. R e p r e s e n t a t i v e Types o f Chromosomal A b e r r a t i o n s Observed Before and A f t e r Treatment w i t h Growth Hormone i n a P a t i e n t w i t h Fanconi's Anaemia  A, B, and C - chromatid gaps D, E, and F - chromatid G and H  - acentric  breaks fragments  I, J , and K - non-homologous exchange figures d e l e t e d G group chromosome  - 31 1.  S i n g l e Break Events Although the t o t a l number o f s i n g l e break events was  s i m i l a r i n both c u l t u r e s from the p a t i e n t , the d i s t r i b u t i o n o f types o f a b e r r a t i o n s was s i g n i f i c a n t l y d i f f e r e n t due t o the change i n the r e l a t i v e , f r e q u e n c i e s o f chromatid breaks and a c e n t r i c fragments. fragments  Before treatment, breaks and a c e n t r i c  o c c u r r e d w i t h approximately equal f r e q u e n c i e s ,  whereas a f t e r treatment, chromatid breaks o c c u r r e d w i t h a much h i g h e r frequency than a c e n t r i c  fragments.  D e l e t i o n s were r a r e and o c c u r r e d w i t h equal frequency i n both 2.  cultures. Two Break Events Exchange c o n f i g u r a t i o n s were the o n l y two break events  observed i n the c e l l s c u l t u r e d b e f o r e or a f t e r treatment w i t h growth hormone.  The chromosomes i n v o l v e d i n each o f the exchange  c o n f i g u r a t i o n s are presented i n Table V I I . No two break events were observed i n the 'standard'. S i x exchange f i g u r e s were observed i n the c e l l s  cultured  b e f o r e hormone treatment w h i l e o n l y two exchanges were observed i n the c u l t u r e s a f t e r a d m i n i s t r a t i o n o f the hormone. The number o f break e q u i v a l e n t s r e s u l t i n g from two break events was decreased a f t e r treatment w i t h the growth hormone. 3.  Gaps P r i o r t o growth hormone treatment, 56.86 gaps per 100 c e l l s  TABLE V I I  I d e n t i f i c a t i o n o f Chromosomes Involved  i n Exchange F i g u r e s  BGH  AGH  'Standard  lxC 3xB  CxG lxC  None  2xB  lxB  not  identifiable  -  33  were observed, w h i l e 45.10 gaps per 100 c e l l s were observed treatment.  after  The frequency o f gaps was decreased a f t e r the t r e a t -  ment w i t h the hormone but both the c u l t u r e s from the p a t i e n t w i t h Fanconi's anaemia demonstrated  a h i g h e r frequency o f gaps i n com-  p a r i s o n to the 21 gaps observed i n the c e l l s 4.  from the 'standard'.  D i s t r i b u t i o n o f Breaks Among Chromosome Groups In the c e l l s observed p r i o r t o , and a f t e r treatment w i t h  growth hormone, the expected d i s t r i b u t i o n o f 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 o f the chromosome groups d i d not d e v i a t e from random.  (Table VIII)  TABLE V I I I D i s t r i b u t i o n o f I d e n t i f i a b l e Breaks Before and A f t e r Treatment w i t h Growth Hormone  B .G .H.  A  B  C  11  5  7  2.93  8.69  5.68 X  2  t o t a l = 11.87  D  E  F  G  1  0  0  0  2.44  2.11  1.12  1.04  p <0.10- >0.05  Not s i g . a t 0.05 l e v e l  A  B  C  D  E  F  G  11  4  12  4  1  0  1  4.03  11.94  2.90  1.54  1.43  A.G.H. 7.81 X  2  t o t a l = 4.33  . 3.35  p<0.75->0.50  Not s i g . a t 0.05 l e v e l  - 35 C.  Mitomycin-C An i n c r e a s e i n the number and types o f chromosomal a b e r r a -  t i o n s was observed a f t e r exposure o f human leukocytes _in v i t r o f o r one hour t o a low c o n c e n t r a t i o n o f mitomycin-C  (1 ug/ml).  The d e t a i l e d r e s u l t s o f t h i s study are found i n Appendix D and are summarized i n Table IX.  R e p r e s e n t a t i v e f i g u r e s are presented  i n F i g . 2. In 64 o f the 100 c e l l s exposed  to t h i s drug, 181 breakage  events were observed, whereas o n l y 5 breakage  events were observed  i n 3 o f the 100 c o n t r o l c e l l s examined. A d e t a i l e d a n a l y s i s o f the types o f a b e r r a t i o n i n the t r e a t e d and c o n t r o l c u l t u r e s i s presented i n Table X. the 181 breakage  Of  events observed a f t e r treatment, 85 were s i n g l e  break events and 96 were two break events.  The 5 breakage  events  observed i n the c o n t r o l were a l l s i n g l e break events. Aneuploid l e v e l s d i f f e r e d markedly w i t h 94% o f the c o n t r o l c e l l s and 61% o f the t r e a t e d c e l l s h a v i n g 46 chromosomes. 1.  S i n g l e Break  Events  Chromatid breaks were the most common s i n g l e breakage  events  found i n the t r e a t e d c u l t u r e s ; 65 a b e r r a t i o n s or 35.91% o f the t o t a l number o f break events b e i n g o f t h i s type. In the t r e a t e d sample, 18 a c e n t r i c fragments were observed. F i v e were found i n the c o n t r o l , and t h i s r e p r e s e n t e d the t o t a l number o f breakage  events.  TABLE IX  Summary o f the R e s u l t s o f Chromosome A n a l y s i s i n Treated and C o n t r o l C u l t u r e s  T o t a l No. Cells  TREATED  100  T o t a l No. C e l l s with Breaks 64  T o t a l No. Gaps  41  Total Breaks  181 U)  CONTROL  100  3  17  FIGURE 3 .  R e p r e s e n t a t i v e Types o f Chromosomal A b e r r a t i o n s Observe^ i n C u l t u r e s Exposed to Mitomycin-C.  A, B, and C - chromatid breaks  D, E, F. G, H, and I - homologous figures  exchange  I, J , K, L, M, and N - non-homologous exchange f i g u r e s  TABLE X Frequency o f S i n g l e and Two Break Events i n Treated and C o n t r o l C u l t u r e s SINGLE BREAK EVENTS  rH H (0 -P • 0 o EH S3  H rd -p O  -p rd co E M o fd SH CD ^ S-i  EH O  •  • •  •H  H  CD U  TWO BREAK EVENTS  ffl  O  •  •  m . o u  u  <!  Q  • CD O A S3 <  u  H  CD  m O  •  w  •  0 • S5 m  •  CO  C •H  •  u •H P  • O X w  S3  0 u  m 0  • CD 0 A S3 <  • w 0 • S3 m  MH  .  SH  •  Ct  « W  S3  • w  -3 •  <! eo  EH  O En  EH  O  O  EH  O  EH  fn O  TREATED  100  65  18  2  85  85  0  0  48  48  96  133  181  CONTROL  100  0  5  0  5  5  0  0  0  0  0  5  5  CO  - 40 Two d e l e t i o n s t h a t c o u l d 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 t r e a t e d c u l t u r e s ; none b e i n g observed i n the c o n t r o l s . 2.  Two Break Events The most s t r i k i n g o b s e r v a t i o n i n c u l t u r e s t r e a t e d w i t h  mitomycin-C was the h i g h frequency o f exchange f i g u r e s .  Forty-  seven exchange c o n f i g u r a t i o n s were observed i n the t r e a t e d c u l t u r e s ; none were observed i n the c o n t r o l s . The 47 exchange f i g u r e s r e p r e s e n t e d 96 break events or 53.04% o f the t o t a l number o f breakage events observed. exchange f i g u r e s are l i s t e d  i n Table XI.  The  Twenty-seven or 57.44%  o f the exchange f i g u r e s were between apparent homologous chromosomes  ( F i g . 3., 3D-H.) w h i l e 20 or 42.56% were between chromosomes  o f d i f f e r e n t groups  ( F i g . 3., 3-H to 3-N).  One unusual exchange  f i g u r e appeared to i n v o l v e 4 chromosomes and was scored as a 4 break event.  The chromosomes i n v o l v e d i n t h i s  particular  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 o f the C group were most f r e q u e n t l y  involved  i n the exchange f i g u r e s , 59.57% o f the exchanges i n v o l v i n g one or more chromosomes from t h i s group.  Chromosomes  from the G  group were r a r e l y i n v o l v e d i n exchanges, o n l y 6.38% i n v o l v i n g chromosomes from t h i s group. 3.  Gaps Gaps were more frequent than breaks i n the c o n t r o l  c u l t u r e s and l e s s frequent than breaks i n the t r e a t e d  cultures.  - 41 -  TABLE XI  I d e n t i f i c a t i o n , o f Chromosomes Involved i n Exchange F i g u r e s i n Treated C u l t u r e s  A.  Exchanges I n v o l v i n g Apparent Homologous Chromosomes  3 (lxl)  1 (BxB)  18 (CxC)  2 (ExE)  1 (FxF) SUBTOTAL  B.  2 (DxD)  =27  Exchanges I n v o l v i n g Members o f D i f f e r e n t Pairs  Chromosome  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 i n c r e a s e from 17 gaps i n the c o n t r o l to 41 i n the t r e a t e d c u l t u r e s i n d i c a t e d t h a t the drug s i g n i f i c a n t l y i n c r e a s e d the frequency o f t h i s type o f event. 4.  Distribution  o f Break Events Among Chromosome Groups  The d i s t r i b u t i o n o f i d e n t i f i a b l e breaks among the seven groups o f chromosomes d i d not d e v i a t e from random (Table XII) indicating  t h a t the drug d i d not p r e f e r e n t i a l l y  o f breaks i n any p a r t i c u l a r  cause an excess  group o f chromosomes.  TABLE X I I  Distribution of Identifiable  A  B  C  OBSERVED  32  16  66  EXPECTED  38.58  19.88  1.12  0.75  X  2 s u b  x2  Breaks Among Chromosome Groups  E  F  G  13  19  11  6  58.96  16.54  14.32  7.60  7.04  0.84  0.75  1.52  1.53  0.15  TOTAL = ' 6  I)  6 6  P .05  d  f  0  p <0.50- > 0.25 Not  s i g n i f i c a n t a t 0.05 l e v e l  =  6  =  1 2  '  6  - 44 VIII A.  -  DISCUSSION  L y s e r g i c A c i d Diethylamide Cohen, M a r i n e l l Q , and Black (1967), and Cohen, H i r s c h h o r n  and F r o s c h (1967), observed t h a t LSD a t c o n c e n t r a t i o n s from to 10 ug/ml o f c u l t u r e , w i t h exposure p r i o r to the h a r v e s t o f the c e l l s , chromosome a b e r r a t i o n s .  times from 4 to 48 hours  i n c r e a s e d the frequency o f  The h i g h e s t c o n c e n t r a t i o n o f 10 ug/ml  brought about g r e a t e r damage i n a s h o r t e r exposure same e f f e c t was exposure  0.001  time.  The  observed a t a c o n c e n t r a t i o n o f 1 ug/ml w i t h an  time o f 24 hours.  However, a t a c o n c e n t r a t i o n o f  ug/ml, more chromosome damage was  observed a t longer  0.001  exposure  times, w h i l e an exposure o f four hours a t the same c o n c e n t r a t i o n produced  few b r e a k s .  breakage  frequency  3.9%,  Among the t r e a t e d c u l t u r e s , the lowest  (7.7%) was  and the breakage  almost twice the c o n t r o l v a l u e o f  frequency i n t r e a t e d c u l t u r e s ranged to  over four times the c o n t r o l values  (17.5%).  The manner i n which the r e s u l t s are presented by Cohen et_ al.  (1967), y i e l d s no i n f o r m a t i o n concerning p o s s i b l e v a r i a t i o n  i n response.  Since the same authors found t h a t not a l l i n d i v u a l s  had an i n c r e a s e i n breakage  a f t e r i n g e s t i n g LSD,  i n f o r m a t i o n con-  c e r n i n g p o s s i b l e v a r i a b i l i t y i n i n d i v i d u a l response i n v i t r o would appear  to be v a l u a b l e . ,  In the p r e s e n t study, one o f the _in v i t r o  experiments  r e p o r t e d by Cohen, M a r i n e l l o , and Black (1967) was w i t h an e x p e r i m e n t a l design which p e r m i t t e d d i r e c t  repeated but comparison  - 45 o f samples from the same i n d i v i d u a l , c u l t u r e d w i t h and without LSD.  The e f f e c t s o f the drug were measured by the d i f f e r e n c e  between the samples r a t h e r than u s i n g the breakage  frequency,  and the v a r i a t i o n i n d i f f e r e n c e was used as a measure o f v a r i a t i o n in  response. The c o n c e n t r a t i o n o f 1 ug/ml f o r 24 hours was s e l e c t e d  because  p r e v i o u s experiments  (Cohen, M a r i n e l l o , and Black, 1967)  i n d i c a t e d i n c r e a s e d breakage  v a l u e s , and y e t 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  c u l t u r e technique and s c o r i n g system used were s i m i l a r t o those used by Cohen, M a r i n e l l o and Black The average  (1967).  frequency o f breakage  and the range o f breakage  i n the c o n t r o l c u l t u r e s ,  i n the t r e a t e d c u l t u r e s r e p o r t e d here  are v e r y s i m i l a r to those r e p o r t e d by Cohen, M a r i n e l l o , and Black (1967), and Cohen, H i r s c h h o r n , and Frosch (1967). study i t was observed t h a t the breakage c u l t u r e s ranged  In the present  frequency i n t r e a t e d  from 4.0 t o 18.70 w i t h a mean o f 9.57.  I t was  f u r t h e r observed t h a t although t h e r e was a wide range o f values a f t e r treatment, t h e r e was an e q u a l l y wide range b e f o r e treatment, but the d i f f e r e n c e s ranged o n l y from 3.00 t o 7.93 w i t h a mean o f 4.64, to  i n d i c a t i n g v e r y l i t t l e v a r i a t i o n i n response from  individual  individual. While  i t i s t r u e t h a t the breakage  frequency i n t r e a t e d  c u l t u r e s i n c r e a s e d 1.24 t o 4 times, t h i s i s r e l a t i v e to the breakage  frequency i n the c o n t r o l c u l t u r e s and i s not a r e f l e c -  t i o n o f v a r i a t i o n i n the amount o f damage.  In f a c t , the 1.24  times i n c r e a s e r e p r e s e n t e d a change from 15.12 to 18.70, and  - 46 the 4 times i n c r e a s e r e p r e s e n t e d an i n c r e a s e o f from 0 to 4 breaks.  Thus these two extremes r e p r e s e n t i n c r e a s e s o f 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 s i m i l a r to those i n other p u b l i s h e d r e s u l t s .  However, some  minor d i f f e r e n c e s i n the types o f a b n o r m a l i t i e s were observed. Cohen, H i r s c h h o r n , and Frosh  (1967) observed a 'high frequency  o f s m a l l a c e n t r i c fragments * whereas i n t h i s study, the number o f a c e n t r i c fragments was not s i g n i f i c a n t l y i n c r e a s e d i n the t r e a t e d c u l t u r e s when compared to the c o n t r o l s .  T h i s may be  due to the f a c t t h a t Cohen and h i s a s s o c i a t e s i n c l u d e d t e r m i n a l breaks w i t h a c e n t r i c  fragments.  Cohen and h i s coworkers  r e p o r t e d f i n d i n g two break  o n l y i n c u l t u r e s t r e a t e d w i t h LSD.  However,  events  i n the present study,  two break events were observed i n both t r e a t e d and c o n t r o l  cul-  t u r e s and were not s i g n i f i c a n t l y i n c r e a s e d i n the t r e a t e d replicates.  Although two break events are v e r y r a r e , they have  been observed i n normal  individuals  (Bloom et_ a l .  1966) .  As they were observed i n low frequency accompanied by an i n c r e a s e i n s i n g l e break events by Cohen e t a l . and i n the p r e s e n t study, i t seems u n l i k e l y t h a t LSD s p e c i f i c a l l y i n c r e a s e s t h i s type o f a b e r r a t i o n . B.  Fanconi's Anaemia Reports o f h i g h f r e q u e n c i e s o f chromosomal a b e r r a t i o n s are  s u f f i c i e n t l y c o n s i s t e n t t o be c o n s i d e r e d p a r t o f the syndrome  - 47  -  known as Fanconi's anaemia (Swift e t a l . 1966; Varela  and Sternberg, 1967).  Bloom e t a l . 1966;  C e l l death due to chromosome  breakage has been suggested as a p o s s i b l e cause o f the p r o g r e s s i v e pancytopenia  (Bloom e_t ajL. 1966) .  At the p r e s e n t time, p a t i e n t s w i t h t h i s d i s e a s e are t r e a t e d w i t h s t e r o i d hormones such as t e s t o s t e r o n e and c o r t i s o n e , as t h i s form o f treatment appears to r e t a r d the p r o g r e s s i v e bone marrow aplasia  (Shahadi and Diamond, 1959).  However, the  literature  c o n t a i n s no i n f o r m a t i o n o f the e f f e c t s o f treatment on the  fre-  quency o f chromosomal breakage. The a v a i l a b i l i t y o f a p a t i e n t i n whom the a n t i c i p a t e d bone marrow a p l a s i a had not progressed s u f f i c i e n t l y to warrant  treat-  ment, p r o v i d e d an o p p o r t u n i t y to observe the frequency and type o f a b e r r a t i o n s both b e f o r e onset o f the bone marrow a p l a s i a  and  b e f o r e treatment, and to f u r t h e r study the p o s s i b l e e f f e c t s o f treatment on the frequency and type o f a b e r r a t i o n s . A p r e v i o u s study had i n d i c a t e d t h a t a h i g h frequency o f breakage was p r e s e n t study  e v i d e n t i n t h i s p a t i e n t two years p r i o r to the (Corey and Andrews, 1968).  I t was  t h a t time t h a t 34% o f the c e l l s demonstrated  observed at  a breakage  event;  i n the p r e s e n t study, 25.5% o f the c e l l s b e f o r e , and 28.04% a f t e r treatment w i t h the hormone were s i m i l a r l y  affected.  Between the study conducted on t h i s p a t i e n t two years ago, the p r e s e n t one,  and  the p a t i e n t had been given a course o f growth  hormone treatment.  - 48 In  the i n i t i a l  study, and i n the present one, the frequen-  c i e s and types o f a b e r r a t i o n s were s i m i l a r to those r e p o r t e d elsewhere.  Bloom e t a l . (1966) r e p o r t e d t h a t i n 1,621 leukocytes  from p a t i e n t s w i t h Fanconi's anaemia, 16.8% o f the c e l l s demons t r a t e d a breakage  event.  The most frequent a b e r r a t i o n s were  chromatid and i s o c h r o m a t i d breaks, a c e n t r i c fragments and exchange c o n f i g u r a t i o n s . of  In the p r e s e n t study, the number  chromatid breaks observed was i n c r e a s e d from 16 i n the  u n t r e a t e d c u l t u r e s t o 29 a f t e r treatment w i t h the growth hormone; the number o f a c e n t r i c fragments was decreased 17 i n u n t r e a t e d c u l t u r e s to 3 a f t e r exposure  from  t o growth hormone.  Bloom e t a l . (1966) a l s o observed 78 exchange f i g u r e s i n the 1,621 c e l l s o f event.  s c o r e d y i e l d i n g a frequency o f 0.048 f o r t h i s type The frequency o f exchanges i n the p a t i e n t  studied  here p r i o r to growth hormone treatment i s c o n s i s t e n t w i t h the r e s u l t s o f Bloom et_ a l . , the frequency o f these two break events b e i n g 0.059; a f t e r treatment, the frequency o f exchanges was 0.019. Although the number o f c e l l s w i t h a b e r r a t i o n s was unchanged i n samples o b t a i n e d 24 hours a f t e r growth hormone treatment i n comparison  to the samples taken b e f o r e , changes i n the frequency  and types o f a b e r r a t i o n s were observed i n t h a t there was a r e d u c t i o n o f (1) the number o f breaks per a b e r r a n t c e l l , the number o f a c e n t r i c fragments, figures.  (2)  and (3) the number o f exchange  - 49 Due  to the l i m i t e d nature o f the study, i t i s impossible  to assess the s i g n i f i c a n c e o f the r e s u l t s , but the suggested r e d u c t i o n i n the amount o f damage per a b e r r a n t c e l l and the r e d u c t i o n i n two break events encourages o f growth hormone as a treatment.  further  investigation  This i s further  emphasized  by the f a c t t h a t treatment w i t h t e s t o s t e r o n e and c o r t i s o n e has u n d e s i r a b l e s i d e e f f e c t s such as m a s c u l i n i z a t i o n o f the p a t i e n t and an i n h i b i t i o n o f the defence mechanisms o f the body. hormone, however, may  Growth  have the d e s i r e d e f f e c t o f s t i m u l a t i n g the  growth o f these dwarfed p a t i e n t s , and i f i t d i d decrease the chromosome damage and r e t a r d somewhat the p r o g r e s s i o n o f the d i s e a s e , i t would be a more d e s i r a b l e C.  treatment.  Mi tomyc in-C„ The experiment w i t h mitomycin-C was  undertaken  p r o j e c t to develop an induced chromosome breakage  as a p i l o t  system which  c o u l d , i n f u t u r e , be used to assess the e f f e c t s o f s t e r o i d s  and  other hormone used to t r e a t p a t i e n t s w i t h Fanconi's anaemia. Mitomycin-C  was  used because  i t not o n l y has been r e p o r t e d to  cause a tremendous i n c r e a s e o f breakage 1964;  Cohen and Shaw, 1964;  events JLn v i t r o  (Nowell,  Shaw and Cohen, 1965), but the  a b e r r a t i o n s such as chromatid breaks, a c e n t r i c fragments  and  exchange f i g u r e s are s i m i l a r to those found i n Fanconi's anaemia. Although mitomycin-C has been r e p o r t e d to be e f f e c t i v e at a v a r i e t y o f times and c o n c e n t r a t i o n s (Cohen and Shaw, 1964), the one hour treatment o f 1 ug/ml o f mitomycin-C a t the b e g i n n i n g o f the c u l t u r e p e r i o d r e p o r t e d by Nowell  (1964)  - 50 r e s u l t e d i n the g r e a t e s t number o f chromosomal a b e r r a t i o n s w i t h the h i g h e s t m i t o t i c  index.  The treatment method o f exposing l e u k o c y t e s i i i v i t r o to 1 ug/ml o f mitomycin-C d u r i n g the f i r s t hour of c u l t u r e has advantages breakage  f o r an i n v i t r o model system i n t h a t  several  (1) i t i s a  system where the b r e a k i n g agent can be removed from  the c u l t u r e system b e f o r e treatments such as growth hormone i n the experiment w i t h Fanconi's anaemia are added, system i n which -the c e l l s exposed  (2) i t i s a  to the drug go through more  than one c e l l d i v i s i o n , so t h a t an attempt can be made to measure the s u r v i v a l o f c e l l s ,  and  (3) the drug i n c r e a s e s the  v a r i e t y as w e l l as the frequency o f chromosome and  chromatid  aberrations. Many o f the chromosome a b e r r a t i o n s observed w i t h w i t h mitomycin-C were u n u s u a l .  treatment  Apart from chromatid and i s o -  chromatid breaks, and a c e n t r i c fragments, exchange c o n f i g u r a t i o n s were observed.  a l a r g e number o f  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 o f exchanges per c e l l o f 0.67.  In the present study, the mean  number o f exchanges per c e l l  i n t r e a t e d c e l l s was  be 0.47.  None were observed i n the c o n t r o l  observed to  cultures.  D i f f e r e n t types o f exchange c o n f i g u r a t i o n s may d i f f e r e n t consequences  i n subsequent  cell divisions.  have Therefore,  examination o f chromosome a b e r r a t i o n s a f t e r treatment w i t h mitomycin-C can p r o v i d e a measure o f c e l l s u r v i v a l by of  comparison  the f r e q u e n c i e s and types o f exchange c o n f i g u r a t i o n s w i t h the  - 51 types and frequencies  of aberration  a f t e r the c e l l s have proceeded  through s e v e r a l d i v i s i o n s . Exchanges between chromosomes o f d i f f e r e n t groups, or s i t u a t i o n s i n which chromosomes o f the same groups have undergone unequal exchange to y i e l d  asymmetrical f i g u r e s were c o n s i d e r e d as non-  homologous exchange c o n f i g u r a t i o n s .  In c o n t r a s t t o Fanconi's  anaemia where a l l o f the exchanges observed were non-homologous, mitomycin-C treatment r e s u l t e d i n both homologous and nonhomologous exchanges.  Shaw and Cohen (1965) observed 46.36%  o f the exchanges i n mitomycin-C t r e a t e d c u l t u r e s t o be homologous exchanges; i n the present study, 57.44% were o f t h i s type. Homologous exchanges would n o t produce d e t e c t a b l e changes i n subsequent c e l l d i v i s i o n s .  karyotype  Non-homologous exchanges,  l i k e those found i n Fanconi's anaemia and i n c e l l s  treated with  mitomycin-C should have r e s u l t e d i n balanced t r a n s l o c a t i o n s , d u p l i c a t i o n s and d e f i c i e n c i e s , and marker chromosomes, as w e l l as a c e n t r i c 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 t r e a t e d c u l t u r e s , b u t a c e n t r i c fragments and d e l e t i o n s were found.  Acentric  fragments and d e l e t i o n s c o u l d a l s o have been  produced by s i n g l e break events r a t h e r  than b e i n g the end  r e s u l t s o f non-homologous exchange c o n f i g u r a t i o n s . (1964) r e p o r t e d extremely r a r e , 1  that  Nowell  ' d i c e n t r i c and r i n g chromosomes were  and Cohen and Shaw (1964) and Shaw and Cohen  (1965) make no mention o f f i n d i n g d i c e n t r i c chromosomes. However as they t r e a t e d c e l l s  l a t e i n the c u l t u r e  period,  - 52 presumably  i n 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 o f marker and d i c e n t r i c  chromosomes suggests  t h a t c e l l s w i t h 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 i n the daughter c e l l s may be r e s p o n s i b l e f o r the death o f the c e l l s .  T h i s s u g g e s t i o n i s t e n a t i v e i n t h a t the  c u l t u r e s were terminated a t 72 hours and t h e r e i s no whether or not these a b e r r a t i o n s occur i n the f i r s t after  treatment.  indication division  IX  SUMMARY  A. L y s e r g i c A c i d Diethylamide The  frequency and types o f chromosomal a b e r r a t i o n s were  observed i n r e p l i c a t e c u l t u r e s from each o f t e n i n d i v i d u a l s c u l t u r e d w i t h and w i t h o u t the a d d i t i o n o f 1 ug/ml o f l y s e r g i c a c i d d i e t h y l a m i d e d u r i n g the l a s t 24 hours o f the c u l t u r e p e r i o d . Approximately  100 c e l l s  from each o f the r e p l i c a t e s were analyzed  and the d i f f e r e n c e between u n t r e a t e d and t r e a t e d r e p l i c a t e s was used as a measure o f the e f f e c t s o f the treatment. The  frequency o f breaks i n u n t r e a t e d r e p l i c a t e s  ranged  from 0 to 15.12 breaks per 100 c e l l s , w i t h a mean o f 4.72. In the t r e a t e d r e p l i c a t e s , the frequency o f breaks per : 100 c e l l s ranged  from 4.00 t o 18.70 w i t h a mean o f 9.37. The d i f f e r e n c e between u n t r e a t e d and t r e a t e d  ranged  cultures  from +3.00 t o +7.93 w i t h a mean o f +4.65. A paired t - t e s t analysis indicated a s i g n i f i c a n t increase  i n both t o t a l break events and s i n g l e break events, b u t n o t i n a b e r r a t i o n s due t o two break events. The chromosome breaks were randomly d i s t r i b u t e d among the seven groups o f chromosomes o f the complement. B.  Fanconi's Anaemia The  frequency and types o f chromosome a b e r r a t i o n s were  observed i n a p a t i e n t w i t h Fanconi's anaemia immediately b e f o r e and 24 hours a f t e r a d m i n i s t r a t i o n o f 250 ug. o f growth hormone,  - 54 and i n a c o n t r o l .  Approximately  100 c e l l s  from each sample  were examined. F o r t y - s e v e n breaks i n 102 c e l l s were observed i n u n t r e a t e d cultures, to  38 breaks i n 107 c e l l s b e i n g observed a f t e r  the hormone.  exposure  E l e v e n breaks i n 91 c e l l s were observed i n  c e l l s o f the 'standard'. There was no s i g n i f i c a n t d i f f e r e n c e i n the number o f aberrant c e l l s  i n the c u l t u r e s from the p a t i e n t , but the number  o f breaks per a b e r r a n t c e l l were decreased from 1.80 i n the c u l t u r e s b e f o r e treatment t o 1.26 i n the c e l l s a f t e r  exposure  to the hormone. S i n g l e break events were the most common a b e r r a t i o n s i n both samples from the p a t i e n t , but 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 o f s i n g l e break events between the two c u l t u r e s was  observed.  Exchange c o n f i g u r a t i o n s were the o n l y two break  events  observed i n both samples w i t h the number o f break events b e i n g decreased from 12 to 4 a f t e r treatment. events were observed i n the c e l l s  No two break  from the 'standard'.  Chromosome breaks were randomly d i s t r i b u t e d among the seven groups o f chromosomes i n a l l samples. C.  Mitomycin-C An i n c r e a s e i n the frequency and types o f chromosomal  a b e r r a t i o n s was observed a f t e r exposure o f human leukocytes to a low c o n c e n t r a t i o n o f mitomycin-C f o r one hour a t the  - 55 b e g i n n i n g o f the c u l t u r e p e r i o d when compared t o the u n t r e a t e d cultures. One hundred exposed  and e i g h t y one breaks i n 64 o f the 100 c e l l s  to the drug were observed.  Only 5 breaks were observed  i n 3 o f the 100 c o n t r o l c e l l s examined. S i n g l e break events r e p r e s e n t e d 46.96% o f the t o t a l number o f break events observed i n the t r e a t e d c u l t u r e s .  The  f i v e break events observed i n the c o n t r o l c u l t u r e s were a l l s i n g l e break events. Exchange f i g u r e s c o n s t i t u t e d a l l o f the two break events i n the t r e a t e d  c u l t u r e s ; 27 homologous and 20 non-homologous  exchanges b e i n g observed. i n the c o n t r o l  No two break events were observed  cultures.  The chromosome breaks were randomly d i s t r i b u t e d among the  seven groups o f chromosomes o f the complement.  - 56 -  X  REFERENCES  A l e x a n d e r , G.J., M i l e s , B.E., G o l d , G.M., and A l e x a n d e r , R.B. 1967. 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Science 148:506-507. German, J . , and C r i p p a , 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., C a r l s o n , E.F., and Goodman, P. 1968. P r o s o p h i l a melanogaster t r e a t e d w i t h LSD: absence o f mutation and chromosomal breakage. Science 161:694-696. H i r s c h h o r n , K., and Cohen, M.M. 1967. Non-psychic e f f e c t s o f LSD: (Chromosome damage human). Ann. i n t e r n . Med. 6^(5): 1109-1111. Hungerford, D.A., T a y l o r , K.M., Shagass, C , LaBadie, G.U., Balaban, G.B., and Paton, G.R. 1968. C y t o g e n e t i c e f f e c t s of LSD therapy i n man. J . Am. Med. Assn. 206(10):2287^2291. Irwin, S., and Egozcue, J . 1967. Chromosome a b n o r m a l i t i e s i n l e u k o c y t e s from LSD u s e r s . Science 157:313-316. Iyer, V.N., and S z y b a l s k i , W.A. 1964. A molecular mechanism o f mitomycin a c t i o n : l i n k i n g o f complementary DNA s t r a n d s . Proc. natn. Acad. S c i . U.S.A. 50:355. Jones, R. 1959. A p r e l i m i n a r y r e p o r t o f human pharmacology and i n i t i a l t h e r a p e u t i c t r i a l w i t h mitomycin-C. Cane. Chem. Rep.. 2z 3. Loughman, W., Sargent, T.W., I s r a e l s t a m , D.M. 1967. of humans exposed t o l y s e r g i c , a c i d d i e t h y l a m i d e . chromosome damage. Science 158:508-510.  Leukocytes Lack o f  Mertz, T. 1961. E f f e c t s o f mitomycin-C on l a t e r a l r o o t t i p chromosomes o f V i c i a faba. Science 133:329. Moorhead, P.C., Nowell, P.C., Mellman, W.J., B a t t i p s , D.M., and Hungerford, D.A. 1960. Chromosome p r e p a r a t i o n s o f l e u k o c y t e s c u l t u r e d from human p e r i p h e r a l b l o o d . E x p l C e l l Res. 20:613-616.  - 58 Nowell, P.C. 1960. Phytohemagglutinin: An i n i t i a t o r o f m i t o s i s i n c u l t u r e s o f normal human l e u k o c y t e s . Cancer Res. 20: 462-466. Nowell, P.C. 1964. M i t o t i c i n h i b i t i o n and chromosome damage by mitomycin-C i n human leukocyte c u l t u r e s . E x p l C e l l Res. 33: 445_449. Oehlkers, F. 1952. Chromosome breaks H e r e d i t y 6(Supp):95-105.  i n f l u e n c e d by chemicals.  R e v e l l , S.H. 1952. Chromosome breakage by x-rays and r a d i o mimetic substances i n V i c i a . H e r e d i t y 6_ (supp) : 107-124. Sato, H., and Pergament, E . 1968. Lancet II:639-640.  Is l y s e r g i d e a teratogen?  Schmid, W., Scharer, K., Baumann, T., Fanconi, G. 1965. Chromosomenbruchigkeit b i e der f a m i l a r e n Panmyelopathie (Typus F a n c o n i ) . Schweiz..Med. Wochen. 45:1461-1464. S e k i g u c h i , M., and Takagi, Y. 1960. N o n - i n f e c t i o u s bacteriophage produced by the a c t i o n o f mitomycin-C. V i r o l o g y 10:160-161. S h a h i d i , N.T., and Diamond, L.K. 1959. Testosterone-induced r e m i s s i o n i n a p l a s t i c anemia. A.M.A.J. D i s . C h i l d . 98: 293-302. Shaw, M.W., and Cohen, M.M. 1965. Chromosome exchanges i n human l e u k o c y t e s induced by mitomycin-C. Genetics 51: 181-190. Shiba, S., T i r a w a k i , A., Taguchi, T., and Kawamata, S. 1959. S e l e c t i v e i n h i b i t i o n o f formation o f DNA i n E. c o l i by mitomycin-C. Nature 183:1056-1057. ~~ Skakkebaek, W.E., P h i l i p s , J . , and R a f a e l s e n , O.J. 1968. LSD i n mice: a b n o r m a l i t i e s i n m e i o t i c chromosomes. Science.160: 1246-1248. Sparkes, R.S., Melnyk, J . , and B o z z e t t i , L.P. 1968. Chromosomal e f f e c t in. v i v o o f exposure to l y s e r g i c a c i d d i e t h y l a m i d e . Science.160:1343-1344. S w i f t , M.R., and H i r s c h h o r n , K. 1966. Fanconi's anaemia: i n h e r i t e d s u s c e p t i b i l i t y o f chromosome breakage i n v a r i o u s t i s s u e s . Ann. i n t e r n . Med. 65:496-503. V a r e l a , M.A., and Sternberg, W.H. 1967. Preanaemic s t a t e i n 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. I s o l a t i o n o f new f r a c t i o n s o f antitumour mitomycins. Antibiotics Chemother. £: 228-240. Warkany, J . , and Takacs, E . 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 (LSD). No t e r a t o g e n i c i t y i n r a t s . Science 159:731-732. Watne, A.L., Moore, D., and B e d r e t t i a , G. 1967. S o l i d tumour chemotherapy w i t h mitomycin-C. Archs. Surg. 95(2): 175-178. Zellweger, H., McDonald, J.S., and Abbo, G. 1967. Is l y s e r g i c a c i d d i e t h y l a m i d e a teratogen? Lancet.II:1066.  - 60 -  APPENDIX A.  LEUKOCYTE CULTURE TECHNIQUE  - 61 .APPENDIX A. LEUKOCYTE  CULTURE TECHNIQUE  1.  C u l t u r e s were grown i n s t e r i l e r o l l e r tubes c o n t a i n i n g 5 cc o f GIBCO Chromosome Medium 1A. 0.25 cc o f h e p a r i n i z e d venous b l o o d was added to each tube, and the c u l t u r e s were then incubated a t 37QC f o r approximately 72 hours.  2.  Colcemid i n a f i n a l c o n c e n t r a t i o n o f 0.02 ug/ml was added to the c u l t u r e s approximately two hours p r i o r to the h a r v e s t o f the c e l l s .  3.  At the end o f the i n c u b a t i o n p e r i o d , the c e l l s were c e n t r i f u g e d a t 800 rpm f o r 8 minutes. The supernatant was removed and d i s c a r d e d , and the c e l l s were suspended i n 5 cc o f h y p o t o n i c s o l u t i o n (5:1 d i s t i l l e d water: f e t a l c a l f serum), and incubated a t 37°C f o r 15 minutes.  4.  A drop o f f i x a t i v e (3:1 a b s o l u t e e t h y l a l c o h o l : g l a c i a l a c e t i c a c i d ) was added, and the c u l t u r e s were c e n t r i f u g e d a t 800 rpm f o r 8 minutes.  5.  The supernatant was d i s c a r d e d l e a v i n g a l a r g e drop over the c e l l s . 4 t o 5 cc o f 3:1 f i x a t i v e was s l o w l y added and the c e l l s g e n t l y a g i t a t e d .  6.  The tubes were then stoppered and r e f r i g e r a t e d a t 4°C f o r approximately h a l f an hour and then l e f t a t room temperature f o r one hour o r l o n g e r .  7.  The c e l l s were then c e n t r i f u g e d a t 800 rpm f o r 8 minutes and the supernatant removed and d i s c a r d e d . 0.5 to 1.0 cc o f f r e s h f i x a t i v e was then c a r e f u l l y added so as not to d i s t u r b the b u t t o n o f c e l l s . A f t e r two minutes the excess f i x a t i v e was removed and r e p l a c e d w i t h f r e s h 3:1 fix. This procedure was repeated s e v e r a l times.  8.  The c e l l s were f i n a l l y suspended i n f r e s h f i x a t i v e and a drop o f the suspension was p l a c e d on p r e c l e a n e d c o l d wet s l i d e . The s l i d e s were then flamed, a i r d r i e d and stored u n t i l required.  Staining of Slides A few drops o f a c e t o - o r c e i n ( 2 % i n 60 cc g l a c i a l a c i d ) were p l a c e d on a s l i d e ,  and a c l e a n c o v e r s l i p was  acetic applied.  _ 62 The s l i d e was l e f t t h i s way s t a i n was removed by p l a c i n g  f o r 1-2 minutes, and then the excess the s l i d e between a few l a y e r s o f  paper t o w e l l i n g and a p p l y i n g p r e s s u r e to the c o v e r s l i p . c o v e r s l i p was then s e a l e d w i t h  paraffin.  The  - 63 -  APPENDIX B.  DETAILED RESULTS FOR LSD 1-10  o  i-3  86  td  Iso g.  1-3  Cd  123 H3  o  o  Hi  H CO 0  NO. OF CELLS 'i:  TYE'ES 01 GAPS AN!) BRE/^KS ?  H3  a  to  to  cn  o  CO  co  o  to  o  o  <]  o  -J  o  O  o  to  o  O  o  1—  K  t  o  co  o  o  to  o  o  M  M  M  o  4^  co  o  O  o  o  o  o  o  O  o  o  o  M  o  O  o  o  o  O  O  O  o  o  o  O  o  o  o  O  o  O  o  o  M  cn  o  cn  4^  M CO  CO  o  M CO  1  cd  L. S. D.  cd  CHROMOSOME  cd  -  to  M  1  1  to  GROUPS  t—  1  O TOTAL ID. GAPS ID. BREAKS  O  O  O  IDENTIFIABLE EXCHANGES  O  O  DELETIONS  cn  4^>  ACENTRICS  o  1—  DICENTRICS  13/11  23/24  NON-IDENTIF. EXCHANGES  O  o  VO  0.1512  0.1870  i—•  1  -V9-  TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  o  i-3  12 0 H  H  0  0  ro  o  td  ro  •  1-3  o  •  o  *s  o  o  o  M  o  I— to  1  M  to  M  -J  O  to  O  to  h-  ^  O  to  O  r-  1  1  > td  H H  O  LO  o  o  o  O  to  O  o  O  o  o  o  M  o  M  o  O  O  to  td  o  o  o  O  o  O  o  O  O  O  *]  o  o  o  O  o  O  o  O  M  M  O  o  LO  o  -J  o  to  CTi  M  to  to -J  LO  CO  r—  CO  1  LO LO  2  o  L. S. D. -  to  CHROMOSOME GROUPS  o  o  TYPES OF GAPS AND BREAKS  CO  to  to  NO. OF CELLS  123  TOTAL ID. GAPS ID. BREAKS  o  O  N ON-1DENTIF. EXCHANGES  o  O  IDENTIFIABLE EXCHANGES  o  O  DELETIONS  to  M  ACENTRICS  o  H-*  DICENTRICS  11/48  5/2 7  CELLS WITH ABERRATIONS  LO  •  o  0. 089^  O  TOTAL BREAKS AND GAPS  -S9-  TOTAL BREAKAGE FREQUENCY  o  ^3  CO  H to  co  O  NO. OF CELLS  136  104  H  i-3  iQ  Q  H3 Cd  CO  to  0 •  OJ  o  O  o  o  CO  o  o  O  o  co  cn  o  O  o  o  o  O  o  o  O  o  o  !-•  O  >  O  <1  to  o  H-*  CO  n  o  o  O  I—  o  o  o  o  to  o  td  CO  o  o  o  O  o  *i  o  o  o  o  o  O  o  CD  cn  cn  o  co  o  CO  o  Cn  to  o  o  o  o  o  o  t->  o  o  o  o  1—'  o  to to  o  •1  to  iCo  CD  1  to  3  o  AND BREAKS  I iQ C  L. S. D. -  M  TYPES OF GAPS  O  CHROMOSOME GROUPS  o  1-3  TOTAL ID. GAPS ID. BREAKS  o  o  NON-IDENTIF.' • EXCHANGES "' '  o  o  IDENTIFIABLE EXCHANGES  o  o  DELETIONS ACENTRICS  o  o  2/22  7/29  to  -j  0.0192  0.0514 -99-  DICENTRICS TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  o  i-3  100  122  H CO  W  i-3  td  0  1-3  O  H 10  W  i-3  W  NO. OF CELLS  f-3  0  TYPES OF GAPS  o  AND BREAKS  iQ  •  •  o  M  o  O  o  O  .£»  >  o  H  to  O  to  o  M  Y->  W  o  O  I-  O  4^  o  o  CO  Q  O  o  o  O  o  O  4^  o  o  H  D  O  o  o  O  to  o  O  o  o  h-  O  o  o  O  o  o  O  o  o  O  O  o  9  O  o  o  O  o  o  O  H  CO  o  tn  to  4^  O  1  M  o  1  O  r-  1  o  O  to to  O IO || M a  td  4  to  L. S. D. -  O  {.CHROMOSOME GROUPS  O  M  TOTAL ID. GAPS ID. BREAKS NON-IDENTIF. 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OF CELLS  O  •-3  O  M  4^  iQ  TYPES OF GAPS AND BREAKS  •  •  >  OJ  OJ  M  o  o  o  o  OJ  O  o  O  o  o  o  OJ  1—  O  o  M  OJ  CO  o  o  o  o  M  OJ  o  O  O  OJ  o  o  o  NO  O  o  o  M  O  o  to  M  o  o  O  O  o  o  O  O  o  O  o  o  o  O  o  o  O  O  o  o  &  o  o  to  OJ  Ol  Ui  to to  TOTi\L  1  to  o to  o  M  4^  .  OJ  4*  to  M  cn  6  o  L. S. D. -  o  CHROMOSOME GROUPS  o  ID. GAPS ID. BREAKS NON-'."I DENT IF.; EXCHANGES' IDENTIFIABLE EXCHANGES  o  o  o  o  o  o  DELETIONS  o  o  ACENTRICS  o  o  DICENTRICS  0/2 9  4/32  o  4^  0. 04  0.00  -69-  TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE •FREQUENCY  o  i-3  100  o  NO. OF CELLS  H  H CO  to  to  100  o  iQ  r-3 td  cn O  to  i-3  cQ  Q  TYPES OF GAPS AND BREAKS  •  o  to  co  o  o  o  o  I—  o  o  o  1  as  >  o  o  to  o  CD  co  o  O  o  O  4^  o  h-  o  o  r—  o  to  o  4^  o  o  o  1  1  1  O  o  o  o  o  O  o  O  O  td  o  O  o  o  o  o  O  o  o  O  *1  o  O  o  o  o  o  O  o  o  o  O  o  co  o  as  o  M  M CO  CD  to co  co  co  O  7  o  L. S. D. -  p-  CHROMOSOME GROUPS  o  TOTAL ID. GAPS ID. BREAKS  o  NON-1DENTIF. EXCHANGES  o  IDENTIFIABLE EXCHANGES  t—  o  DELETIONS  h-  to  ACENTRICS  o  o  DICENTRICS  o  1  1  9/2 3  5/13 CO  0. 09  0. 05  -0L-  TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  o  1-3  100 H td  H  01  td  i-3  0  iQ  vQ  o  01  cd  i-3 td  vQ  o  o  o  H o  O  AND BREAKS  •  cn  >  to  td  1—  -j  o  o  o  o  O  to  o  O  o  o  O  O  cn  O  I—  to  -J  o  o  O  1  .  1  O  o  O  r  o  o  1—  M  o  o  O  o  \->  o  o  o  o  O  o  o  O  o  o  o  o  o  o  O  o  o  O  o  H  CO  o  cn  1—  1  to  to CO  O TOTAL  1  H  cn  8  o  co  TYPES OF GAPS  L. S. D.  o  iQ  CHROMOSOME GROUPS  H  O  i-3  0  •  o  NO. OF CELLS  90  «£>  ID. GAPS ID. BREAKS  o  NON-1 DENT IF.T. EXCHANGES  1—  to  IDENTIFIABLE EXCHANGES  o  h->  DELETIONS  H  to  ACENTRICS  o  o  DICENTRICS  o 1  4/23  9/19  co  ~j  0.10  0.04  -TL-  TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  o  100  100  H CQ 0  H CQ 0  Cd  rjd  i-3  •  i-3  a  in  i-3 Cd  Cd  NO. OF CELLS  ^3 iQ  o  TYPES OF GAPS AND BREAKS  to  co  o  >  o  o  H  Cd  •  O  to  cn  o  o  o  o  to  to  o  o  to  CO  o  t—  o  CO  VO  O  o  M  M  o  o  o  o  H  O  O  O  o  o  o  o  CO  M  M  o  1—  M  rrj  o  I—  o  o  1  O  o  o  o  o  O  O  O  o  o  o  O  M  O  o  o  o  to  H  CO  cn  o  to  -  o  L. S. D.  H  CHROMOSOME  O  9  to  to CO  co to  to Cn  1  GROUPS  (-•  1  o TOTAL 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  CO  cn  0.06  0.03  -ZL-  TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  o  H3  100  100  H CQ  H CQ 0  td  td  1-3  0  O  i-3  w  td  NO. OF CELLS  i-3  O  cQ  TYPES OF GAPS AND BREAKS  •  cQ  •  o  M  4^  cn  >  I—  o  O  to  o  o  O  O  LO  td  o  o  o  H  CTi  o  LO  r-  H  4^  o  M  o  o  LO  O  M  M  O  O  o  o  O  O  O  O  O  o  o  O  O  o  O  o  M  O  O  LO  M  to  1  1  H to  J  o  1—  o  O  o  r-  w  O  O  o  o  M  O  o  o  to  Ln  tLn  1  1  •  o 1  to  to to  h-  1  -0  4^  O  10  o-  -  4^  L. S. D.  O  o  CHROMOSOME GROUPS  o  O  TOTAL 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  4^  03  0.08  0.04  -ZL-  TOTAL BREAKS AND GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  - 74 -  APPENDIX C.  DETAILED RESULTS OF BEFORE AND AFTER TREATMENT WITH GROWTH HORMONE ON A PATIENT WITH FANCONI'S ANAEMIA  td cn  1—  1  o  to  to  CO  to  o  CO  (->  o  .I-  o  o  to  l—  o  o  o  o  M  o  H  o  o  o  o  o  o  to  Cn  o  cn  o  1  CTi  O  O  i-3  te  1  td  0  td  0  O  -j  0  co  1—  >  cn  td  to  0  to  CD  0  cn  0  l—  H  0  to  M to  0  -J  CO  lO  CO  r->  0  CO  0  M  0  O  o  M  O  H  CO  0  O  0  l—  o  o  O  O  O  O  0  O  o  o  H  O  O  1—  0  M  CO  cn  4^  1—  cn  1  to  hO  1  co  CO CO  1  co  1  1  1  1—  1  M  0  1  M  M  0  I-  O  O  0  O  O  to O  co  cn  CO VD  1  cn  to  4^  CO  4^  cn  TYPES OF GAPS & BREAKS  >-3  0  1  to  .('  i-3  ANAEMIA  co  td  NO. OF CELLS  FANCONI'S  to  o  102  O TOTAL  o  o  BGH  o  o  107  Isog.  Isog.  td  td  AHG  91  ID. GAPS ID. BREAKS NONIDENTIFIABLE EXCHANGES  o  O  4^ II to CO  o  to  4i>  IDENTIFIABLE EXCHANGES  I—  to  to  DELETIONS  co  CO  M -J  ACENTRICS  o  O  O  DICENTRICS  1  47/58  38/46  s h-  1  o  0.110  0.461 1.8 0  • to  to  cn  0.355I 1.26  o  co  o  -5L-  TOTAL BREAKS & GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY BREAK FREQ/ ABERRANT CELL  - 76  APPENDIX D.  DETAILED ANALYSIS OF TREATMENT WITH MITOMYCIN-C  o  f o  100 w  a  H CO 0  NO. OF CELLS  100 r3  cQ  o  H CQ  to  tu  o  •  i-3 cQ  O  to  O  t-  00  TYPES OF GAPS AND BREAKS  •  o  o  Ui  UJ  to OY  o  o  o  O  to  u>  w  o  o  o  to  o  o  o  O  o  o  O  o  o  o  o  o  o  o  M  1  cn  o  to  to  CD  O  o  M UI  to  o  o  o  H  o  Ui  (-• to  M «J  o  H  > to  to  o  o  OJ  o  O  (-•  M  K  CD  O  o  to  H  Ui  O  to  00  <Js o  M  4^  .  o  .  00  CTi  4^  M  I  H Oi p-  .£> M  1  o  MITOMYCIN - C  o  CHROMOSOME GROUPS  o  •Q  TOTAL 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  U)  CT> 4^  1.81  0.05  -LL-  TOTAL BREAKS AND 3GAPS CELLS WITH ABERRATIONS TOTAL BREAKAGE FREQUENCY  

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