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UBC Theses and Dissertations

Effect of separated chemical carcinogen treatments in vitro Warren, Peggy Margaret 1974

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THE EFFECT OF SEPARATED CHEMICAL CARCINOGEN TREATMENTS J_N VITRO by Peggy Margaret Warren B . S c , U n i v e r s i t y o f B r i t i s h Columbia, 1972  A T h e s i s Submitted i n P a r t i a l F u l f i l m e n t o f the Requirements f o r the Degree o f MASTER OF SCIENCE i n Zoology We accept t h i s t h e s i s as conforming t o the required standard  THE UNIVERSITY OF BRITISH COLUMBIA J4jly,  1974-  In  presenting  this  an a d v a n c e d  degree  the  shall  I  Library  f u r t h e r agree  for  scholarly  by h i s of  this  written  thesis at  it  purposes  for  freely  permission may  representatives. thesis  partial  the U n i v e r s i t y  make that  in  is  financial  of  Z o o l  Columbia,  British  by  for  gain  Columbia  shall  the  that  not  requirements I  agree  r e f e r e n c e and copying  t h e Head o f  understood  °gy  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada  of  for extensive  permission.  Department  of  available  be g r a n t e d  It  fulfilment  of  or  that  study.  this  thesis  my D e p a r t m e n t  copying  for  or  publication  be a l l o w e d w i t h o u t  my  ABSTRACT P r e v i o u s l y , i n v i t r o s t u d i e s of chemical carcinogens have been focussed on determining the e f f e c t s o f s i n g l e , high doses; however, c e l l s i n vivo are exposed to v a r y i n g low doses of numerous chemicals a t v a r y i n g i n t e r v a l s . Consequently,  t h i s study was i n i t i a t e d to i n v e s -  t i g a t e the e f f e c t s of separated, low doses of a chemical in v i t r o .  carcinogen  Monolayer c u l t u r e s o f human s k i n f i b r o b l a s t s were exposed  to 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0) and were challenged a t v a r y i n g i n t e r v a l s (1 1/2, 2, 3, 5, 9 and 13 hours) with a second 4NQ0 treatment. To evaluate the e f f e c t s , three end p o i n t s were employed:  DNA r e p a i r  c a p a c i t y , c e l l s u r v i v a l , and chromosome a b e r r a t i o n s . Following exposure to an i n i t i a l , s i n g l e dose o f 4NQ0, the time course o f DNA r e p a i r s y n t h e s i s (as measured by was determined.  HTdR i n c o r p o r a t i o n )  The peak of r e p a i r s y n t h e s i s was e v i d e n t i n the second  and t h i r d hour a f t e r a d d i t i o n of the carcinogen. was v i r t u a l l y complete a t 12 hours  DNA r e p a i r s y n t h e s i s  post-treatment.  When c e l l s r e c e i v e d a second 4NQ0 treatment w i t h i n 3 hours o f the f i r s t , the l e v e l o f r e p a i r s y n t h e s i s induced by t h i s second dose was f a r below an expected value.  With 9 hours i n c u b a t i o n between t r e a t -  ments, r e p a i r s y n t h e s i s a f t e r the second dose was a t the expected Replacement o f the f i r s t 4NQ0 treatment  with a UV  level.  treatment  produced analogous r e s u l t s . The clone forming c a p a c i t y of c e l l s exposed to s p l i t 4NQ0 t r e a t ments was i n v e s t i g a t e d . A p o t e n t i a t i o n of e f f e c t s was e v i d e n t when  ii  the two treatments were spaced l e s s than 2 hours a p a r t .  With a 9 hour  i n t e r v a l between treatments the c l o n i n g c a p a c i t y was again a t the expected  value. A d i r e c t p r o p o r t i o n a l i t y between an i n c r e a s e i n the  frequency  of chromosome a b e r r a t i o n s and a r e d u c t i o n i n the i n t e r v a l between t r e a t ments was observed.  As the i n t e r v a l between treatments increased (up  to 9 hours) the frequency o f chromosome a b e r r a t i o n s  decreased.  The data i n d i c a t e t h a t when a second 4NQ0 treatment i s a p p l i e d c l o s e to the f i r s t , complete r e p a i r o f the r e s u l t a n t damage does not occur.  T h i s absence o f DNA r e p a i r may i n c r e a s e the c a r c i n o g e n i c poten-  t i a l of the chemical  carcinogen.  ACKNOWLEDGEMENTS I would l i k e t o express my s i n c e r e a p p r e c i a t i o n t o Dr. H. F. S t i c h f o r h i s i n t e r e s t and advice throughout t h i s r e s e a r c h , and t o Mrs. S t i c h f o r h e r many words o f encouragement. I a l s o wish t o thank Lydia Chen, Dorothee K i e s e r and B r i a n Laishes f o r t h e i r s t i m u l a t i n g d i s c u s s i o n s , C h a r l i e Yoshizawa f o r h e r undying patience and e x c e l l e n t t e c h n i c a l a s s i s t a n c e , and e s p e c i a l l y Richard San f o r h i s numerous, h e l p f u l s u g g e s t i o n s . I am a l s o g r a t e f u l t o Dr. R. L. Noble f o r p r o v i d i n g the opport u n i t y and t h e f a c i l i t i e s t o c a r r y o u t t h i s research p r o j e c t .  iv  TABLE OF CONTENTS Page ABSTRACT  .  .........  i i  ACKNOWLEDGEMENTS  iv  LIST OF TABLES LIST OF FIGURES  . .....  ..............  viii  .........  1  ............  5  INTRODUCTION MATERIALS AND METHODS  vii  I. C e l l C u l t u r e s  5  I I . Chemical Treatment  ......  I I I . UV Treatment  6 6  IV. C e l l S u r v i v a l S t u d i e s V. Chromosome S t u d i e s  ........  7  ............. .  7  ..............  8  ............  10  I. The Repair o f 4NQ0-induced DNA Damage . . . . . . . . . .  10  I I . The E f f e c t o f S p l i t 4NQ0 Treatments on DNA Repair S y n t h e s i s  15  VI. Autoradiography RESULTS  ........  I I I . The Repair o f 4NQ0-induced DNA Damage f o l l o w i n g UV irradiation  .  IV. Time Course o f DNA Repair a f t e r a Second 4NQ0 Treatment. . V. C e l l S u r v i v a l and Chromosome S t u d i e s DISCUSSION I. The DNA Repair Process I I . I n t e r p r e t a t i o n o f HTdR I n c o r p o r a t i o n S t u d i e s v  26 36 41 52 52 54  vi Page I I I . S p l i t Doses and C e l l S u r v i v a l  63  IV. Outlook SUMMARY REFERENCES  65 •  67 ......  69  LIST OF TABLES Table  Page  1  E f f e c t o f 2 1/2% MEM on c e l l s u r v i v a l  2  E f f e c t o f a 1 hour 4NQ0 treatment on c e l l s u r v i v a l . .  44  3  The e f f e c t on c e l l s u r v i v a l o f i n c u b a t i o n i n 2 1/2% MEM, p r i o r t o a s i n g l e 4NQ0 treatment . . . . . . . . The e f f e c t on c e l l s u r v i v a l o f i n c u b a t i o n i n 2 1/2%  46  MEM, f o l l o w i n g treatment with 4NQ0  46  5  E f f e c t o f s p l i t 4NQ0 treatments on c e l l s u r v i v a l . . .  48  6  E f f e c t o f s p l i t 4NQ0 treatments on chromosome a b e r r a t i o n s 51  4  vii  ........  44  LIST OF FIGURES Fi gure 1 2  Page Experimental d e s i g n . Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g 4NQ0-induced DNA damage Repair o f 4NQ0-induced DNA damage. E f f e c t o f concent r a t i o n and d u r a t i o n o f exposure  12 13  3  Experimental d e s i g n . V a r i a t i o n o f t h e i n t e r v a l s between s p l i t 4NQ0 t r e a t m e n t s , and e f f e c t on DNA r e p a i r s y n t h e s i s 17  4  Histogram i l l u s t r a t i n g t h e l e v e l o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a second 4NQ0 treatment. Experiment c a r r i e d out i n 5% ADM  18  Histogram i l l u s t r a t i n g t h e l e v e l o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a second 4NQ0 treatment. Experiment c a r r i e d out i n 5% MEM  22  5 6 7  Time c o u r s e o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a s i n g l e 4NQ0 treatment i n e i t h e r 5% ADM o r 5% MEM . . . . . . . 24 Time course o f r e p a i r s y n t h e s i s f o l l o w i n g UV-induced DNA damage . 2 8  8 9 10  Experimental d e s i g n . V a r i a t i o n o f t h e i n t e r v a l s between UV i r r a d i a t i o n and a subsequent 4NQ0 treatment; e f f e c t on DNA r e p a i r s y n t h e s i s  30  Histogram i l l u s t r a t i n g t h e l e v e l o f DNA r e p a i r s y n t h e s i s when a UV dose was followed ( a t varying i n t e r v a l s ) by a 4NQ0 treatment. Experiment c a r r i e d o u t i n 5% ADM ...  31  Histogram i l l u s t r a t i n g t h e l e v e l o f DNA r e p a i r s y n t h e s i s when a UV dose was followed ( a t varying i n t e r v a l s ) by a 4NQ0 treatment. Experiment c a r r i e d out i n 5% MEM. ...  33  11  Experimental d e s i g n . Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a second 4NQ0 treatment 38  12  Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a second 4NQ0 treatment vi i i  39  ix Figure 13  Page Experimental d e s i g n . V a r i a t i o n o f t h e i n t e r v a l s between s p l i t 4NQ0 treatments; e f f e c t on c e l l survival  42  Experimental design. V a r i a t i o n o f t h e i n t e r v a l s between s p l i t 4NQ0 treatments; e f f e c t on chromosome aberrations  50  15  A model f o r DNA r e p a i r  53  16  4-Nitroquinoline  14  1-Oxide (4NQ0)  ....  54  INTRODUCTION Both a u t o r a d i o g r a p h i c and biochemical data i n d i c a t e t h a t most carcinogens i n t e r a c t with n u c l e a r DNA i n such a manner t h a t the DNA 48 i s a l t e r e d . T h i s a l t e r e d DNA then becomes a s u b s t r a t e upon which co  r e p a i r enzymes can a c t  c-j  ' ' In s p i t e o f the abundance o f r e s e a r c h  focussed on the problem, the question o f how these i n t e r a c t i o n s are r e l a t e d to c a r c i n o g e n e s i s s t i l l remains unsolved.  An  understanding  o f these i n t e r a c t i o n s may w e l l provide an i n s i g h t as t o the mechanisms o f chemical c a r c i n o g e n e s i s . Repair of DNA damage c e r t a i n l y plays a major r o l e i n r e s t o r i n g OO  OO  OC  CO  a c e l l to i t s normal f u n c t i o n a l s t a t e ' ' ' . However, when one attempts to account f o r c a r c i n o g e n e s i s , the amounts o f r e s i d u a l , un12 r e p a i r e d DNA damage may be o f even g r e a t e r importance . Treatment o f human f i b r o b l a s t s i n c u l t u r e w i t h a h i g h l y c a r c i n o 74  genie compound w i l l induce high l e v e l s o f DNA r e p a i r s y n t h e s i s  , yet  t h i s does not n e c e s s a r i l y prove t h a t the normal a c t i v i t y o f the c e l l w i l l be r e s t o r e d . When l a r g e amounts o f DNA damage are present, there i s an i n c r e a s e d p o s s i b i l i t y t h a t unrepaired segments w i l l remain or t h a t imperfect r e p a i r w i l l occur.  I f a c e l l enters i n t o DNA r e p l i c a t i o n  with damaged and/or unrepaired DNA molecules and s u r v i v e s , i t c o u l d become g e n e t i c a l l y a l t e r e d and i n turn a c q u i r e the p o t e n t i a l to undergo 12 33 1 neoplastic transformation ' .  2  Direct proof of this particular hypothesis has not yet been obtained, however an almost complete lack of DNA repair capacity has 8 9  been correlated with tumorigenesis ' .  Patients with Xeroderma P i g -  mentosum, have an increased s e n s i t i v i t y to sunlight, numerous skin tumors in the exposed areas.  resulting in  The c e l l s of these patients  show a decreased capacity for repairing certain types of DNA damage '  '  '  , and i t has been shown that the lack of an endonuclease Q 68  a c t i v i t y i s responsible for this effect '  .  Returning to normal human c e l l s , i f high levels of DNA damage (and concomitant DNA repair synthesis), imply increased carcinogenic potential, then one should be able to demonstrate a correlation between 33  these two factors.  This has proven to be the case  .  Highly carcino-  genic chemicals (as indicated by in vivo studies), e l i c i t high levels of DNA repair synthesis (as shown by the unscheduled uptake of  HTdR),  DNA breaks (as measured by alkaline sucrose gradients), decreased c e l l s u r v i v a l , and increased chromosome damage.  On the other hand, chemicals  with low carcinogenicity e l i c i t l i t t l e or no DNA repair synthesis, few DNA breaks, normal levels of c e l l survival (as compared to that of untreated controls), and few to none chromosome aberrations. When screening for the carcinogenic potential of a chemical compound in v i t r o , these correlations are usually exploited. However, the major drawback of a l l such studies is that they have been employed to investigate only the effects of exposure to a single dose of one carcinogen.  Furthermore, the doses used were so high that c e l l survival  was negligible.  Yet c e l l s in vivo are usually exposed to varying low  3 doses o f numerous chemicals a t d i f f e r e n t i n t e r v a l s . The e f f e c t o f such exposures on the c e l l and on t h e DNA r e p a i r mechanisms has n o t been investigated.  Perhaps many o f these chemicals act s y n e r g i s t i c a l l y  with the net r e s u l t o f i n c r e a s i n g t h e i r c a r c i n o g e n i c p o t e n t i a l .  Since  i t has a l r e a d y been demonstrated i n v i v o t h a t c e r t a i n chemicals (named c o - c a r c i n o g e n s ) , do possess the c a p a c i t y t o enhance the 79 p o t e n t i a l o f a carcinogen  tumorigenic  , and s i n c e the u l t i m a t e goal i n s c r e e n i n g  carcinogens i n v i t r o i s the determination o f the t o t a l c a r c i n o g e n i c p o t e n t i a l o f a chemical compound; i t i s obvious t h a t an i n v i t r o assay system must be developed such t h a t the e f f e c t s o f more than one c a r c i n o gen treatment can be c a r e f u l l y examined. The main o b j e c t i v e o f t h i s study then, was t o make an attempt a t developing such a system. Rather than examining the e f f e c t s o f various combinations o f chemicals i t was decided t o i n v e s t i g a t e m u l t i p l e dosages o f one chemical. The system was designed t o answer one major q u e s t i o n :  i n what manner  does a c e l l t h a t i s already i n t h e process o f r e p a i r i n g DNA damage respond t o f u r t h e r damage, i . e . as t h e i n t e r v a l between treatments i n c r e a s e s , what happens t o the r e p a i r c a p a c i t y : i s there a p e r i o d i n which i t i s reduced and/or enhanced? The chemical chosen f o r t h i s study was 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0). T h i s c h o i c e was made f o r two reasons.  Firstly, its biological  e f f e c t s have been e x t e n s i v e l y s t u d i e d ; previous i n v e s t i g a t i o n s have 35 51 22 50 shown 4NQ0 t o be h i g h l y oncogenic i n v i v o ' , mutagenic i n v i t r o ' , and c e l l - t r a n s f o r m i n g i n v i t r o . 4NQ0 a l s o binds t o D N A ' > > > , e l i c i t s DNA r e p a i r s y n t h e s i s 74 , and produces chromosome a b e r r a t i o n s 72. 6 4  39  41  44  47  77  4  Secondly, i t i s h i g h l y s o l u b l e i n water, does not p r e c i p i t a t e nor degrade 45  r e a d i l y when placed i n medium . To achieve the d e s i r e d end p o i n t , c e l l c u l t u r e s were exposed to a s i n g l e  4NQ0  treatment, and were then c h a l l e n g e d a t varying i n t e r v a l s  to a second dose o f  4NQ0.  Levels o f r e p a i r s y n t h e s i s were determined  as well as the e f f e c t on c e l l s u r v i v a l and chromosome a b e r r a t i o n s .  MATERIALS AND METHODS I.  Cell Cultures a) Media.  For these s t u d i e s the c e l l s were maintained i n two types  of media; Eagles Minimal E s s e n t i a l Medium (MEM)  (Grand I s l a n d B i o l o g i c a l  Co.) and MEM d e f i c i e n t i n a r g i n i n e ( a r g i n i n e d e f i c i e n t medium, ADM). Both ADM and MEM were r o u t i n e l y supplemented with the f o l l o w i n g : 1) A n t i b i o t i c s :  streptomycin s u l f a t e , 29.6 yg/ml (General Biochemicals) p e n i c i l l i n G, 204 units/ml (General Biochemicals) kanamycin, 100 yg/ml ("GIBCO") fungizone, 2.5 yg/ml ("GIBCO")  2) 1.8% sodium b i c a r b o n a t e : 16 mis per 800 ml media 3) f e t a l c a l f serum ("GIBCO"): f o r stock c u l t u r e s 15% f e t a l c a l f serum was added to MEM (15% b) C e l l s .  MEM).  A s k i n punch biopsy was taken from a normal  Caucasian  female (23). Monolayer f i b r o b l a s t c u l t u r e s were d e r i v e d from t h i s biopsy and f i r s t to f i f t h t r a n s f e r passages were used throughout these studies.  Stock c u l t u r e s were maintained i n 15% MEM i n 100 mm p e t r i  dishes ( F a l c o n P l a s t i c s ) , and kept i n a water s a t u r a t e d Co^ i n c u b a t o r at 37°C. 5  6 II.  Chemical Treatment 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0) was obtained from D a i i c h i Pure  Chemical Co., Tokyo.  Immediately p r i o r t o use 1.9 mg o f 4NQ0 was d i s -  solved i n 0.4 ml e t h a n o l , t h i s was warmed s l i g h t l y t o ensure t h a t t h e _3 chemical was completely d i s s o l v e d . To g i v e a 10 M s o l u t i o n , 9.6 mis of ADM o r MEM was added; s e r i a l d i l u t i o n s were then made t o o b t a i n t h e desired concentrations.  For the s u r v i v a l s t u d i e s , c e l l s were t r e a t e d  with 3 mis o f t h e a p p r o p r i a t e 4NQ0 s o l u t i o n i n 2 1/2% MEM.  F o r chromo-  some and DNA r e p a i r s t u d i e s , the c e l l s were t r e a t e d with 1 ml o f t h e chemical s o l u t i o n i n 5% MEM o r ADM. A l l treatments were f o r 1 hour unless otherwise s t a t e d . The chemical was removed by a s t e r i l e p i p e t t e attached t o a s u c t i o n d e v i c e .  A f t e r removal o f t h e chemical t h e c e l l s  were washed twice with 2 mis o f MEM o r ADM (without f e t a l c a l f serum). Medium supplemented with f e t a l c a l f serum was added and t h e p e t r i dishes returned t o the CO2 i n c u b a t o r . I I I . UV Treatment One aspect o f t h e DNA r e p a i r s t u d i e s i n v o l v e d t r e a t i n g t h e c e l l s with UV i r r a d i a t i o n .  In t h i s case a S y l v a n i a g e r m i c i d a l lamp  (G15T8) was used as t h e UV l i g h t source.  A t 20" i t emitted a dose  o f 8 ergs/mm /sec as measured by a UV l i g h t meter ( U l t r a v i o l e t Products, Inc.). Cover s l i p s c o n t a i n i n g c e l l s f o r i r r a d i a t i o n were dipped twice i n s t e r i l e phosphate b u f f e r s a l i n e (PBS) c o n t a i n i n g no phenol red, t o  7 ensure complete removal o f any UV absorbing m a t e r i a l . For i r r a d i a t i o n , cover s l i p s were placed i n an empty p e t r i d i s h , and upon completion o f i r r a d i a t i o n were r e t u r n e d to a p e t r i d i s h c o n t a i n i n g medium.  IV.  Cell Survival Studies 1,600-2,000 c e l l s were seeded i n t o 60 mm p e t r i d i s h e s , and  covered w i t h 4 mis o f 2 1/2% MEM and allowed t o s e t t l e down as s i n g l e c e l l s f o r 16-20 hours.  T h i s low c o n c e n t r a t i o n o f f e t a l c a l f serum  was chosen i n o r d e r t o slow down the m e t a b o l i c r a t e o f the c e l l s such t h a t c e l l d i v i s i o n s would not occur d u r i n g the course o f the experiments. (The e f f e c t o f 2 1/2% MEM on c e l l s u r v i v a l w i l l be d i s c u s s e d more f u l l y i n the r e s u l t s . ) The chemical treatments were i n 2 1/2% MEM and t h e recovery p e r i o d was a l s o i n 2 1/2% MEM.  Once the chemical treatments  were complete 15% MEM was added and the c e l l s were allowed t o d i v i d e and form c o l o n i e s . When the clones had reached the 50-60 c e l l stage (approximately 7 days post-treatment) the p r e p a r a t i o n s were f i x e d w i t h Carnoy's s o l u t i o n (3:1 a l c o h o l a c e t i c a c i d ) ; washed i n 70% e t h a n o l , and d i s t i l l e d water; a i r d r i e d , and s t a i n e d with a 2% aqueous s o l u t i o n o f T o i u i d i n e Blue ( F i s c h e r S c i e n t i f i c Co.). The c o l o n i e s were counted under a r e g u l a r d i s s e c t i n g scope.  V.  Chromosome S t u d i e s C e l l s were seeded onto 20 mm sq c o v e r s l i p s (Corning) i n 35 mm  p e t r i dishes and covered with 2 mis o f 15% MEM.  In o r d e r t o o b t a i n  8  well spread metaphase plates, the c e l l s were used before they reached 80% confluency.  The c e l l s were treated twice with 4NQ0 in 5% MEM and  allowed to recover between doses in 15% MEM. Once c e l l divisions were detected (by observation under an inverted microscope) 0 . 2 mis of a 0.01% solution of colchicine (BDH Chemicals, England) was added for 5 hours.  The covers l i p s were then transferred to petri dishes containing  1% sodium citrate solution for 20 minutes.  This hypotonic treatment  causes the c e l l s to s w e l l , producing chromosomes that are well spread out and separated. dried.  The c e l l s were then fixed with Carnoy's and a i r -  Once dry, they were stained for 5 minutes with 2% aceto-orcein,  dehydrated through alcohol, butanol, butanol-xylol, x y l o l , and mounted on glass slides with Permount (Fischer S c i e n t i f i c Co.).  VI.  Autoradiography In order to distinguish between DNA repair replication and  semiconservative DNA r e p l i c a t i o n , only nuclei undergoing repair synthesis should become l a b e l l e d . entering S-phase.  To achieve t h i s , c e l l s must be prevented from  This was accomplished by placing the cultures in  ADM for 2 1/2 days, at which time approximately 90% of the c e l l s are arrested at G^. Cells were seeded onto 20 mm sq coverslips in 35 mm petri dishes, and covered with 15% MEM.  Upon reaching 80% confluency, the c e l l s were  put into 5% ADM. This was done by dipping the coverslips into two beakers of ADM (no serum), with subsequent transferral to new petri  9 dishes c o n t a i n i n g 2 mis o f 5% ADM.  The experiment was conducted 2 1/2-  3 days l a t e r . T r i t i a t e d thymidine ( HTdR) was obtained from New England Nuclear (Chicago) and was d i l u t e d to a c o n c e n t r a t i o n o f 10 yCi/ml i n e i t h e r 5% MEM o r 5% ADM.  The c e l l s were pulsed w i t h 1 ml of t h i s s o l u t i o n f o r  2 hours, a t which time the c o v e r s ! i p s were moved from the p e t r i d i s h and dipped i n 3 changes o f Hanks balanced s a l t s o l u t i o n to remove any excess  HTdR. They were then immersed i n 1% sodium c i t r a t e f o r 15  minutes, f i x e d i n Carnoy's, r i n s e d i n 100% ethanol and a i r d r i e d . To f a c i l i t a t e h a n d l i n g , t h e c o v e r s l i p s ( c e l l s i d e up) were mounted on g l a s s s l i d e s with melted p a r a f f i n .  Excess Carnoy's was removed by  passing the s l i d e s through a graded a l c o h o l s e r i e s , 95% EtOH, 70% EtOH, 20% EtOH (10 minutes each), 2 changes o f d i s t i l l e d water, one change o f PBS, two more changes d i s t i l l e d water (10 minutes each) and were then l e f t to a i r dry. The s l i d e s were coated w i t h NTB3 emulsion (Kodak) ( a t 4 3 ° C ) , allowed to dry f o r 1 hour and then s t o r e d a t 4°C i n l i g h t - t i g h t boxes f o r 2 weeks. The autoradiograms were processed i n Kodak Dl9 developer (3 minutes), Kodak f i x e r (10 minutes) and r i n s e d i n running water f o r 1 hour.  The c e l l s were then s t a i n e d with 2% o r c e i n f o r 5 minutes, dehy-  drated through s u c c e s s i v e immersion i n e t h a n o l , b u t a n o l , b u t a n o l / x y l o l , x y l o l (2 minutes each) and mounted i n Permount (by p l a c i n g another c o v e r s l i p over the exposed c e l l s ) .  RESULTS  I.  The Repair o f 4NQ0-induced DNA Damage P r i o r t o attempting any experiments with double 4NQ0 t r e a t -  ments, i t was necessary t o determine t h e time course o f DNA r e p a i r synt h e s i s a f t e r a s i n g l e 4NQ0 treatment and t o choose a p p r o p r i a t e 4NQ0 c o n c e n t r a t i o n s and lengths o f treatment f o r use i n such experiments. A high 4NQ0 c o n c e n t r a t i o n may not be very t o x i c t o c e l l s when given in a s i n g l e treatment but when given twice c o u l d become extremely t o x i c , and would most l i k e l y a f f e c t the l e v e l s o f r e p a i r s y n t h e s i s . T h e r e f o r e i t was important t o s e l e c t a c o n c e n t r a t i o n o f 4NQ0 t h a t would e l i c i t a moderate, o r even low l e v e l o f DNA r e p a i r a f t e r a s i n g l e treatment (thus i m p l y i n g moderate o r low l e v e l s o f DNA damage). Choosing an exposure time was o f equal importance as i t was e s s e n t i a l t o have very l i t t l e r e p a i r s y n t h e s i s t a k i n g p l a c e d u r i n g t h e f i r s t chemical treatment in o r d e r t h a t the second chemical treatment c o u l d be a p p l i e d w h i l e r e p a i r o f t h e i n i t i a l damage was s t i l l proceeding. An experiment was designed t o i n v e s t i g a t e t h e r e p a i r o f 4NQ0induced DNA damage, and the e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s and d i f f e r e n t treatment times. Two c o n c e n t r a t i o n s o f 4NQ0 were chosen; 5 x 10 MM and 1 x 10-7 M. These had a l r e a d y been shown by S t i c h and San72 t o induce moderate and low l e v e l s o f DNA r e p a i r s y n t h e s i s r e s p e c t i v e l y , 3  a f t e r a 90 minute treatment time. The l e v e l o f HTdR i n c o r p o r a t i o n 10  11 (seen as g r a i n s per nucleus) was used as the measure o f DNA r e p a i r synthesis.  The c e l l s were maintained i n ADM p r i o r t o and throughout  the e n t i r e experiment. Figure 1 o u t l i n e s the p r o t o c o l t h a t was employed.  Illustrated  i s a 60 minute 4NQ0 treatment (10~ M o r 5 x 10~ M), the o t h e r treatment 7  7  times were 30 and 90 minutes; a l l underwent subsequent i n c u b a t i o n i n 3 HTdR, 0, 2, 4, 8, 12 and 24 hours a f t e r removal o f the c a r c i n o g e n . A l l o f t h e chemical s o l u t i o n s were a p p l i e d a t the same time, and t h i s was d e s i g n a t e d as z e r o hour ( f o r g r a p h i c a l p u r p o s e s ) . To determine the amount o f r e p a i r s y n t h e s i s t h a t was i n i t i a t e d d u r i n g t h e chemical treatment, HTdR was added s i m u l t a n e o u s l y w i t h 4NQ0 f o r 30, 60 o r 90 3  minutes.  A two hour  HTdR p u l s e was chosen f o r the remaining i n c u b a t i o n  p e r i o d s and was l a t e r adopted f o r a l l subsequent r e p a i r experiments. This c h o i c e was made because the 4NQ0 c o n c e n t r a t i o n s t h a t were employed 3 did not e l i c i t high l e v e l s o f r e p a i r s y n t h e s i s and an HTdR p u l s e o f 1 o r 1 1/2 hours would have produced low g r a i n counts, making the r e s u l t s d i f f i c u l t to interpret. Three general o b s e r v a t i o n s can be made from these r e s u l t s ( F i g u r e 2 ) ; the peak o f r e p a i r r e p l i c a t i o n f o r a l l chemical treatments occurs i n the f i r s t 4 hours f o l l o w i n g a d d i t i o n o f the c h e m i c a l , and then proceeds a t a s l i g h t l y lowered l e v e l f o r t h e next 6 hours; a t 12 3 hours a low but s i g n i f i c a n t uptake o f HTdR can be d e t e c t e d , and i s s t i l l e v i d e n t a t 24 hours. Upon examining the r e s u l t s more c l o s e l y i t i s n o t i c e a b l e t h a t d u r i n g the 30 minute and 60 minute 4NQ0 treatments, the c e l l s e x h i b i t a very low l e v e l o f ongoing r e p a i r s y n t h e s i s , whereas i n the next two  Time ( h r s . ) 0  2  4  6  8  _j  10 i  12 i  14 1  16 1  18 1  20 1  22 1  24  26 1-  1  4NQ0 3 HTdR +  . 4NQ0 .HTdR . 4NQ0 . . . 4NQ0 . , 4NQ0 , • 4NQ0 , • 4NQ0 . . 4NQ0 .  'HTdR 'HTdR HTdR 'HTdR HTdR , 'HTdR  Figure 1: Experimental d e s i g n . Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g 4NQ0-induced DNA damage.  ro  Figure 2: Repair o f 4NQ0-induced DNA damage. E f f e c t o f c o n c e n t r a t i o n and d u r a t i o n o f exposure. Each p o i n t denotes t h e time when the sample was taken and t h e uptake o f 3HTdR over a two hour p e r i o d p r i o r t o sampling (represented by g r a i n s p e r n u c l e u s ) . T 4NQ0 added v" 4NQ0 removed (a) 5 x 10" M 4NQ0 7  (b)  1 x 10" M 4NQ0 7  HOURS  P O S T - T R E AT M E  NT  15 hour  HTdR p u l s e t h a t f o l l o w s , a h i g h l e v e l o f r e p a i r s y n t h e s i s i s  obtained.  In f a c t t h i s i s t h e peak o f r e p a i r s y n t h e s i s . However, i n  the c e l l s exposed t o a 90 minute 4NQ0 dose t h i s i s not t h e case, f o r the peak o f r e p a i r i s reached d u r i n g the chemical treatment, only t o 3  drop s l i g h t l y i n t h e subsequent two hour  HTdR p u l s e . T h i s seems t o  i n d i c a t e t h a t t h e peak o f r e p a i r occurs i n t h e second o r t h i r d hour f o l l o w i n g a d d i t i o n o f 4NQ0. Since both t h e 60 minute and t h e 90 minute 4NQ0 treatments e l i c i t e d t h e same maximum l e v e l o f r e p a i r s y n t h e s i s , and s i n c e t h e peak o f r e p a i r o c c u r r e d a f t e r r a t h e r than d u r i n g t h e 60 minute 4NQ0 t r e a t ment, i t was decided t o u t i l i z e a 60 minute 4NQ0 treatment (1 x l O ^ M and 5 x 10~^M) f o r a l l subsequent experiments. II.  The E f f e c t o f S p l i t 4NQ0 Treatments on DNA Repair S y n t h e s i s Using t h e p r e v i o u s r e s u l t s as a guide, an attempt was made t o  a s c e r t a i n t h e l e v e l o f r e p a i r s y n t h e s i s a t t a i n e d by c e l l s exposed t o a second 4NQ0 treatment w h i l s t s t i l l r e c o v e r i n g from a f i r s t treatment. B a s i c a l l y two problems were o f i n t e r e s t here; f i r s t how does a c e l l a t the peak o f r e p a i r respond t o a second treatment, and secondly how does a c e l l t h a t has v i r t u a l l y completed r e p a i r s y n t h e s i s respond. In d e s i g n i n g t h e experiment, an a p p r o p r i a t e endpoint f o r comp a r i n g t h e data had t o be chosen.  S i n c e i t was known t h a t t h e peak o f  r e p a i r o c c u r r e d d u r i n g t h e two hours immediately f o l l o w i n g removal o f a s i n g l e 60 minute 4NQ0 treatment, i t was decided t o measure the l e v e l o f r e p a i r s y n t h e s i s obtained i n the two hours immediately f o l l o w i n g  16 removal o f the second treatment and then compare these v a l u e s . The experimental p r o t o c o l o u t l i n e d i n F i g u r e 3 was adopted.  C e l l s were  maintained i n ADM throughout the experiment, t r e a t e d i n i t i a l l y w i t h 10~ M o r 5 x 10" M 4NQ0 f o r 60 minutes, and then t r e a t e d again 1 1/2, 7  7  3  2, 3, 5, 9 o r 13 hours l a t e r .  The two hour  followed removal o f t h e second treatment.  HTdR pulse immediately In order t o determine t h e  amount o f r e p a i r s y n t h e s i s induced by f i r s t treatment a l o n e , c o n t r o l s were run f o r each r e c o v e r y p e r i o d . They were exposed t o t h e i n i t i a l 4NQ0 treatment o n l y , allowed t o r e c o v e r w h i l s t t h e remainder r e c e i v e d 3 a second treatment, and then pulsed with  HTdR a t t h e same time as t h e  " d o u b l y - t r e a t e d " sample. The r e s u l t s , which a r e summarized i n F i g u r e 4, d e p i c t t h e a c t u a l l e v e l s o f r e p a i r f o l l o w i n g r e c o v e r y from a s i n g l e treatment and a f t e r exposure t o two treatments; and the t o t a l expected l e v e l o f r e p a i r f o l l o w i n g two treatments. T h i s expected value was determined by making t h e assumpt i o n t h a t a c e l l should t h e o r e t i c a l l y be a b l e t o r e p a i r a second chemical treatment as e f f e c t i v e l y as i f i t were t h e f i r s t , no matter when i t i s applied.  In p r a c t i s e , t h e t o t a l expected l e v e l o f r e p a i r s y n t h e s i s  o b t a i n e d f o l l o w i n g t h e second treatment should be equal t o t h e sum o f the r e p a i r s y n t h e s i s s t i l l proceeding a f t e r r e c o v e r y from t h e f i r s t treatment plus t h e l e v e l o f r e p a i r t h a t i s obtained immediately a f t e r removal o f a s i n g l e 4NQ0 treatment.  In the histograms ( F i g u r e 4) t h e  f i r s t column d e p i c t s t h e l e v e l o f DNA r e p a i r immediately f o l l o w i n g removal of a s i n g l e 4NQ0 treatment; t h i s value i s then added t o t h e c o n t r o l ( c l e a r column i n each s e t ) t o g i v e the expected value ( b l a c k column i n each s e t ) . I t should be c l a r i f i e d a t t h i s p o i n t t h a t t h e c o n t r o l v a l u e  Time (hrs.) 0 ,4NQ0 .4NQ0  1  2  4NQ0  8 i  9  10  11 i  12 i  13 i  14  'HTdR HTdR .4NQ0  ,  ,4NQ0 ,4NQ0  i  HTdR  4NQ0 ,4NQ0  6  5  HTdR 'HTdR  ,4NQ0  .  HTdR 'HTdR  ,4NQ0  .4NQ0  .4NQ0  ,  HTdR 'HTdR  .4NQ0 recovery period  .4NQ0  4NQ0  .  HTdR 'HTdR  ,4NQ0  Figure 3:  Experimental design. Variation of the intervals between s p l i t 4NQ0 treatments; effect on DNA repair synthesis.  15  Figure 4: Histogram i l l u s t r a t i n g t h e a c t u a l g r a i n s p e r n u c l e i immediately f o l l o w i n g t h e f i r s t 4NQ0 treatment only I | , and a f t e r double 4NQ0 t r e a t m e n t s , and t o t a l expected g r a i n s p e r n u c l e i f o l l o w i n g double treatments Q . C e l l s were maintained i n 5% ADM. (a) 5 x 10" M 4NQ0 7  (b) 1 x 10" M 4NQ0 7  19  HOURS  BETWEEN  TREATMENTS  20 does not i n d i c a t e how much r e p a i r s y n t h e s i s was a c t u a l l y t a k i n g p l a c e d u r i n g the second 4NQ0 treatment ( t h i s can be obtained from F i g u r e 2 ) , i t merely r e p r e s e n t s the DNA r e p a i r s y n t h e s i s s t i l l proceeding a f t e r recovery from t h e f i r s t treatment.  The purpose o f t h e c o n t r o l was t o  serve as an a i d i n o b t a i n i n g t h e expected v a l u e . As i l l u s t r a t e d i n t h e r e s u l t s ( F i g u r e 4 ) , i f a second treatment i s a p p l i e d d u r i n g the t h r e e hours immediately f o l l o w i n g a d d i t i o n o f t h e f i r s t , t h e t o t a l l e v e l o f r e p a i r i s s i g n i f i c a n t l y lower than t h e expected. In f a c t there appears t o be very l i t t l e r e p a i r s y n t h e s i s t a k i n g p l a c e on o r above t h a t f o r t h e f i r s t treatment.  A t t h e lower 4NQ0 concen-  t r a t i o n ( F i g u r e 4 b ) , t h i s e f f e c t i s not as marked, and i s o n l y s i g n i f i c a n t l y below the expected f o r two hours post-treatment. A f t e r t h i s p e r i o d , t h e l e v e l o f r e p a i r begins t o approach the expected, and a t t h e lower dosage reaches t h e expected a t 5 hours post-treatment. However, at the h i g h e r c o n c e n t r a t i o n , t h e expected l e v e l i s never a t t a i n e d ; in f a c t a t 13 hours t h e l e v e l i s even f u r t h e r below t h e expected i t was a t 5 hours.  than  Such was t h e case f o r t h e lower c o n c e n t r a t i o n a l s o .  T h i s p e c u l i a r decrease i n r e p a i r c a p a c i t y was not p r e d i c t e d , s i n c e i t seemed l o g i c a l t o assume t h a t once the c e l l s had r e g a i n e d t h e p o t e n t i a l t o r e p a i r t h e DNA damage i n f l i c t e d by t h e second treatment, they would c o n t i n u e t o do s o . To determine whether t h i s drop i n r e p a i r c a p a c i t y may have been due t o t h e a r g i n i n e - d e f i c i e n t c u l t u r e medium, an i d e n t i c a l experiment, with a few m o d i f i c a t i o n s was designed. The c e l l s were blocked as b e f o r e in 5% ADM f o r 2 1/2 days, but 3 hours p r i o r t o the f i r s t 4NQ0 treatment,  21  *  the c e l l s were r e t u r n e d t o 5% MEM.  T h i s 3 hour time p e r i o d was chosen  a r b i t r a r i l y , t o p r o v i d e t h e c e l l s with a chance t o e q u i l i b r a t e . The r e s t o f t h e experiment was then c a r r i e d o u t i n 5% MEM. Comparison o f the histograms f o r c e l l s i n ADM, with those i n MEM r e v e a l some d i f f e r e n c e s ( F i g u r e 5 ) . When t h e ADM blocked c e l l s a r e placed i n MEM b r i e f l y (3 hours) p r i o r t o 4NQ0 treatment, t h e two hour p e r i o d i s again n o t i c e a b l e d u r i n g which t h e DNA r e p a i r l e v e l f a l l s below expected v a l u e s . However, t h i s r e d u c t i o n i n DNA r e p a i r s y n t h e s i s i s not as pronounced as i n t h e case when t h e c e l l s were maintained i n ADM throughout t h e experiment.  Furthermore, t h e second drop i n r e p a i r c a p a c i t y  i s not apparent i n t h e MEM-maintained c e l l s ; a t 12 hours t h e l e v e l o f DNA r e p a i r f o l l o w i n g t h e second 4NQ0 treatment i s i d e n t i c a l t o t h e expected. There was no reason t o suspect t h a t t h i s i n c r e a s e i n r e p a i r c a p a c i t y was t h e r e s u l t o f a concommitant r i s e i n semi c o n s e r v a t i v e DNA synthesis associated with c e l l d i v i s i o n .  The c o r r e s p o n d i n g c o n t r o l s  ( s i n g l e treatment) were c a r e f u l l y examined and d i d not c o n t a i n any n u c l e i with high g r a i n counts ( i n d i c a t i v e o f DNA s y n t h e s i s a t S-phase). 25 more Freed and Schatz  Further-  have shown t h a t a f t e r removal o f a b l o c k such  as ADM t h e c e l l s do not e n t e r S-phase u n t i l 16 t o 20 hours l a t e r .  This  i s without a 4NQ0 treatment which tends t o f u r t h e r slow down e n t r y o f 44  c e l l s i n t o S-phase . For comparative purposes, an experiment was run s i m u l t a n e o u s l y to determine t h e course o f r e p a i r a f t e r a s i n g l e 4NQ0 treatment f o r c e l l s i n ADM and MEM. The p r o t o c o l p r e v i o u s l y o u t l i n e d i n F i g u r e 1 *such c e l l s w i l l be r e f e r r e d t o as MEM-maintained c e l l s .  Figure 5: Histogram i l l u s t r a t i n g t h e actual grains p e r nucleus f o l l o w i n g a s i n g l e 4NQ0 treatment 1 | , and double 4NQ0 treatments 1^1 ; and expected grains per n u c l e i f o l l o w i n g double treatments gffj . The e n t i r e e x p e r i ment was c a r r i e d out i n 5% MEM, a f t e r p r e v i o u s l y being blocked i n 5% ADM. (a) 5 x 10" M 4NQ0 7  (b) 1 x 10" M 4NQ0 7  23  (a) mo.  life  3  HOURS  3  B E T W E E N  S  9  13  TREATMENTS  IB 3  UI -J (J  (b)  O  2  d ui  7w  w  1  a z  < i  0  i 1 / 1 / 1  / / /  •  a  HOURS  B E T W E E N  TREATMENTS  24  Figure 6: Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a s i n g l e 4NQ0 treatment i n e i t h e r 5% ADM o r 5% MEM. Each p o i n t denotes t h e time when t h e sample was taken; and t h e uptake o f 3nTdR over a two hour p e r i o d p r i o r t o sampling (represented by grains p e r n u c l e u s ) . i n 5% ADM i n 5% MEM (a) 5 x 10" M 4NQ0 • o 7  (b) 1 x 10~'M 4NQ0 • •  26 was used w i t h t h e e x c e p t i o n t h a t only 1 hour treatments were employed, and t h a t t h e experiment was run i n 5% MEM as w e l l as 5% ADM.  The same  chemical s o l u t i o n was used f o r both kinds o f medium, with the f i n a l d i l u t i o n being made i n e i t h e r 5% MEM o r 5% ADM. The r e s u l t s a r e summarized i n F i g u r e 6, and from t h i s graph i t can be concluded t h a t r e p a i r o f a s i n g l e 4NQ0 treatment i s not a f f e c t e d by the type o f medium employed. Returning t o F i g u r e s 4 through 6 i t i s apparent then t h a t c e l l s i n ADM a r e not a b l e t o r e p a i r a second s e t o f DNA damage t o t h e expected l e v e l , whereas c e l l s i n MEM can, even though both a r e capable o f r e p a i r i n g t h e f i r s t treatment.  Furthermore c e l l s maintained i n e i t h e r  medium possess a " s e n s i t i v e " two hour p e r i o d f o l l o w i n g c h e m i c a l l y i n duced DNA damage, the s e n s i t i v i t y being more pronounced when c e l l s a r e maintained i n ADM.  I I I . The Repair o f 4NQ0-induced DNA Damage F o l l o w i n g UV I r r a d i a t i o n The q u e s t i o n now a r i s e s as t o whether DNA r e p a i r s y n t h e s i s i n duced by o t h e r agents can produce t h e same r e s u l t s .  For t h i s purpose  i t was decided t o r e p l a c e the f i r s t 4NQ0 treatment with a UV treatment. P r i o r t o running such an experiment, i t was necessary t o c h a r a c t e r i z e the time-course o f r e p a i r f o l l o w i n g a s i n g l e UV exposure.  The p r o t o c o l  used was b a s i c a l l y i d e n t i c a l t o t h a t used f o r a s i n g l e 4NQ0 treatment ( F i g u r e 1) except t h a t the c e l l s were exposed t o UV ( d e s i g n a t e d as zero hour) and t h e experiment was run i n both 5% ADM and 5% MEM.  27 Two dosages were used:  40 ergs/mm 2 and 20 ergs/mm 2 f o l l o w e d  3  by two hour  HTdR p u l s e s a t 0, 2, 4, 8, 12 and 22 hours post-treatment.  The peak o f r e p a i r was found t o occur d u r i n g the two hours immed i a t e l y f o l l o w i n g UV treatment ( F i g u r e 7 ) ; t h i s concurs w i t h t h e time course o f DNA r e p a i r s y n t h e s i s a f t e r a s i n g l e 4NQ0 treatment ( F i g u r e 2 ) . A f t e r t h i s i n i t i a l peak o f DNA r e p a i r s y n t h e s i s , t h e l e v e l decreases markedly such t h a t a t 8 hours o n l y a low but s i g n i f i c a n t amount o f r e p a i r s y n t h e s i s i s d e t e c t a b l e , again i n concordance with t h e time course o f r e p a i r o f 4NQ0-induced DNA damage. Both UV doses produced l e v e l s o f r e p a i r s y n t h e s i s t h a t would serve as s u i t a b l e replacements f o r t h e f i r s t 4NQ0 treatments (40 e r g s / 2 - 7 2 - 7 mm  f o r 5 x 10 M and 20 ergs/mm  f o r 10 M), and were t h e r e f o r e used  for t h i s s e t o f experiments. The experiment was run a c c o r d i n g t o t h e p r o t o c o l o u t l i n e d i n Figure 8, and was d i v i d e d i n t o two p a r t s ; h a l f o f t h e c e l l s were maint a i n e d i n 5% ADM f o r t h e e n t i r e experiment; t h e o t h e r h a l f were p l a c e d i n 5% MEM 3 hours p r i o r t o UV treatment, and remained i n 5% MEM throughout the experiment.  Corresponding c o n t r o l s were run as b e f o r e and were  exposed t o t h e UV treatment o n l y o r t h e 4NQ0 treatment o n l y . The r e s u l t s a r e summarized i n t h e histograms o f F i g u r e s 9 and 10. Note t h a t t h e f i r s t column i n each graph r e p r e s e n t s t h e l e v e l o f r e p a i r obtained immediately f o l l o w i n g a s i n g l e one hour 4NQ0 treatment (5 x 1 0 M -7  or 10~ M). T h i s value was used t o c a l c u l a t e t h e expected l e v e l o f r e p a i r 7  ( l a s t column i n each s e t ) . T h e o r e t i c a l l y a l l UV-treated c e l l s should be capable o f r e p a i r i n g t h e damage induced by a subsequent 4NQ0 dose (no matter when i t i s a p p l i e d ) t o a l e v e l s i m i l a r t o t h a t a f t e r a s i n g l e 4NQ0 dose.  28  Figure 7: Time course o f r e p a i r s y n t h e s i s f o l l o w i n g UV-induced DNA damage. Each p o i n t denotes t h e time when t h e sample i s taken; and the uptake o f ^HTdR over a two hour p e r i o d p r i o r t o sampling (represented by g r a i n s per n u c l e u s ) . i n 5% ADM i n 5% MEM (a) o • 40 ergs/mm p (b) • • 20 ergs/mm  G R A I N S  P E R  N U C L E U S  U.V. i r r a d i a t i o n T  0  i  1  2  i  4NQ0  3  i  .  ,  3  4NQ0  8  •  •  9  10  •  i  11 i  12 i  13  14  i  •  ,  3  .  3  HTdR  .  3  HTdR  ,  .4NQ0  i  ,  7  «  3  ,  ,  6  •  . HTdR . HTdR  ,  5  HTdR  ,  ,  •  HTdR 3  4NQ0  4  i  . HTdR  ,  • HTdR  ,  3  3  ;  ;  ,4NQ0  . HTdR 3  • HTdR 3  . 4NQ0 .  F i g u r e 8: Experimental d e s i g n . V a r i a t i o n o f the i n t e r v a l s between UV i r r a d i a t i o n and a subsequent 4NQ0 treatment; e f f e c t on DNA r e p a i r synthesis.  3  HTdR  3  HTdR  15 •  31  Figure 9: Histogram d e p i c t i n g a c t u a l grains p e r nucleus immediately f o l l o w i n g 4NQ0 treatment YsZ\ , a t v a r y i n g periods o f time a f t e r UV treatment) | , and a f t e r UV treatment plus 4NQ0 treatment i expected grains p e r nucleus a f t e r treatment S9 . Experiment run i n 5% ADM. (a) 5 x 10' M 4NQ0 and 40 ergs/mm  2  (b) 1 x 10" M 4NQ0 and 20 ergs/mm  2  7  7  HOURS  BETWEEN  TREATMENTS  33  Figure 10: Histogram d e p i c t i n g a c t u a l g r a i n s per nucleus immediately f o l l o w i n g 4NQ0 treatment f|jp , a t v a r y i n g p e r i o d s o f time a f t e r UV treatment | | , and a f t e r UV treatment plus 4NQ0 treatment ; expected g r a i n s per nucleus a f t e r UV plus 4NQ0 treatment g§U . Experiment was c a r r i e d out i n 5% MEM a f t e r p r e v i o u s l y being blocked i n 5% ADM. (a) 5 x 10" M 4NQ0 and 40 ergs/mm  2  (b) 1 x 10" M 4NQ0 and 20 ergs/mm  2  7  7  HOURS  BETWEEN  TREATMENTS  35  I t i s i n t e r e s t i n g t h a t t h e r e s u l t s a r e very s i m i l a r to those o f the previous experiments.  When t h e c e l l s a r e maintained i n ADM, DNA  r e p a i r s y n t h e s i s f o l l o w i n g the second dose ( a t e i t h e r c o n c e n t r a t i o n ) again f e l l  below expected, and i n t h i s case (UV-4NQ0 combination)  the decrease i s even more pronounced than i n s p l i t - d o s e 4NQ0 experiments. In f a c t a t h a l f an hour post-treatment r e p a i r o f t h e UV damage appears to be s l i g h t l y i n h i b i t e d .  L i k e w i s e , t h e ADM-maintained c e l l s never  t o t a l l y r e g a i n t h e a b i l i t y t o r e p a i r t h e second 4NQ0 treatment ( a t both concentrations). The p a t t e r n o f r e p a i r f o r MEM-maintained c e l l s does not change a p p r e c i a b l y from before except t h a t a t both c o n c e n t r a t i o n s a s l i g h t i n h i b i t i o n occurs d u r i n g t h e f i r s t hour.  Again t h e c e l l s i n MEM a r e  capable o f r e p a i r i n g t h e second treatment t o t h e expected l e v e l a f t e r an 8 hour recovery p e r i o d . In f a c t t h e r e p a i r s y n t h e s i s a t t h i s p o i n t i s h i g h e r than expected. These r e s u l t s imply then t h a t t h e two hour " s e n s i t i v e " p e r i o d p r e v i o u s l y c h a r a c t e r i z e d i s not p e c u l i a r t o 4NQ0 t r e a t e d c e l l s and t h a t the r e p a i r c y c l e induced by UV a l s o has a s i m i l a r temporal  sequence.  This o b s e r v a t i o n indicates t h a t r e p a i r o f UV-induced damage may be a f f e c t e d to a g r e a t e r extent by a subsequent 4NQ0 treatment than i s r e p a i r o f 4NQ0-induced damage.  36  IV.  Time Course of DNA Repair after a Second 4NQ0 Treatment Interpretation of the aforementioned results is complicated  by the fact that the choice of end point may not have been entirely appropriate ( i . e . comparison of repair levels obtained in the two hour period immediately following removal of the second treatment).  It is  conceivable that the addition of a second 4NQ0 treatment close to the f i r s t , may i n i t i a l l y produce a s l i g h t toxicity in the c e l l s and thus delay the onset of repair synthesis.  Consequently the peak level of 3  repair would not be detected during the two hour  HTdR pulse.  If this  is the case, then the below expected DNA repair levels would merely ref l e c t a lag in repair synthesis. To c l a r i f y this point, periods of repair incubation following removal of the second dose were employed.  In this manner, i t would be  possible to determine when the peak of repair was occurring. Furthermore, since i t has been shown that at 9 hours post-treatment, cells in MEM respond to a second treatment with a peak level of repair synthesis similar to that following a single treatment, i t seemed logical to determine i f the time course of repair after a second dose at 9 hours, also resembled that of a single dose. In order to achieve both objectives i t was necessary to run the experiment in 5% MEM. This decision was made because c e l l s in ADM never seemed to regain the capacity for repairing DNA damage i n f l i c t e d by the second treatment, and as a result the l a t t e r objective could never be attained.  37 For t h i s experiment, c e l l s were exposed to a second 4NQ0 t r e a t ment, 2, 3, 5 o r 9 hours a f t e r a d d i t i o n o f the f i r s t , and pulsed w i t h 3 HTdR as o u t l i n e d i n F i g u r e 11. One s e t o f c e l l s served as c o n t r o l s r e c e i v i n g the f i r s t treatment o n l y , and were always pulsed s i m u l t a n e o u s l y with samples g i v e n the double dose. The r e s u l t s a r e summarized i n F i g u r e 12. In the graphs f o r " d o u b l y - t r e a t e d " c e l I s the values a r e expressed as g r a i n s per nucleus r e s u l t i n g from the second treatment o n l y . In these cases the l e v e l of r e p a i r f o r the " s i n g l y - t r e a t e d " c o n t r o l was s u b t r a c t e d from the t o t a l l e v e l o f DNA r e p a i r d e t e c t e d a f t e r the second treatment ( f o r each r e p a i r incubation period). A general o b s e r v a t i o n can be made from these r e s u l t s ; as t h e i n t e r v a l between treatments i n c r e a s e s , the peak o f r e p a i r i n c r e a s e s and t h e o v e r a l l p a t t e r n o f r e p a i r more c l o s e l y resembles t h a t o f the i n i t i a l dose.  I t i s i n t e r e s t i n g t h a t i f the second treatment i s g i v e n  2 hours a f t e r the f i r s t , a peak i n DNA r e p a i r s y n t h e s i s does not occur at a l a t e r time. T h i s would then r u l e out the e x i s t e n c e o f a l a g p e r i o d . Furthermore the r e s u l t s show t h a t when a second treatment i s g i v e n 9 hours a f t e r the f i r s t , t h e p a t t e r n o f r e p a i r i s b a s i c a l l y the same as t h a t e l i c i t e d by a s i n g l e treatment. It seems reasonable t o conclude then, t h a t i n the p e r i o d immed i a t e l y f o l l o w i n g i n d u c t i o n o f DNA damage by4NQ0, t h e a p p l i c a t i o n o f a second 4NQ0 dose does not produce the expected response.  Either this  second dose does not induce f u r t h e r damage o r i f i t does, f o r some reason DNA damage does not occur.  Time ( h r s . ) 0 .4NQ0 ,  2  4 4NQ0  .4NQ0 . .4NQ0 .  ,HTdR  •  .HTdR  L  4NQ0  .4NQ0  .4N00  .4N00 . .4NQ0 . .4NQ0 .  10  12  14  16  18  20  3  •4NQ0 . .4N0O .  8  3  -4NQ0 , .4NQ0 .  6  .4NQ0  3  HTdR  3  HTdR  i  3  HTdR  3  HTdR  •  3  HTdR  3  HTdR 3  HTdR  3  HTdR  F i g u r e 11: Experimental d e s i g n . Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a second 4NQ0 treatment. I l l u s t r a t e d i s the sequence o f 3HTdR pulses a f t e r removal o f the second treatment given 3 hours a f t e r a d d i t i o n o f t h e f i r s t . Other treatments were a p p l i e d 2, 5 o r 9 hours a f t e r t h e f i r s t and were f o l l o w e d by t h e same sequence o f ^HTdR p u l s e s . CO  00  39  Figure 12:  Time course o f DNA r e p a i r s y n t h e s i s f o l l o w i n g a second 4NQ0 treatment (5 x 10"7M). (a) i n i t i a l treatment only (b) 2 treatments 2 hours apart (c) 2 treatments 3 hours a p a r t (d) 2 treatments 5 hours a p a r t (e) 2 treatments 9 hours a p a r t  G R A I N S  P E R  N  U  C  L  E  U  S  41 V.  C e l l S u r v i v a l and Chromosome S t u d i e s Iii order t o thoroughly c h a r a c t e r i z e t h e c a r c i n o g e n i c i t y o f a  chemical compound, previous s t u d i e s have employed c e l l s u r v i v a l and 74 75 chromosome s t u d i e s i n a d d i t i o n t o r e p a i r experiments  ' . Therefore  i t seemed l o g i c a l t h a t t o complete t h i s p a r t i c u l a r study t h e e f f e c t of s p l i t 4NQ0 treatments on c e l l s u r v i v a l and chromosome a b e r r a t i o n s should be determined.  I f a p e r i o d does e x i s t i n which a second  4NQ0 treatment does not induce f u r t h e r damage, then one would expect t h a t i n t h i s p e r i o d there would be no change i n c e l l s u r v i v a l , o r chromosome a b e r r a t i o n s . On the o t h e r hand, i f t h e damage does o c c u r , y e t i s not being r e p a i r e d , one would expect t h i s t o be r e f l e c t e d by changes i n c e l l s u r v i v a l and i n chromosome a b e r r a t i o n s . a) C e l l S u r v i v a l S t u d i e s An experiment was designed t o a s c e r t a i n t h e e f f e c t o f s p l i t 4NQ0 treatments on c e l l s u r v i v a l ( F i g u r e 13). However, b e f o r e t h i s experiment c o u l d be c a r r i e d out, i t was necessary t o s o l v e one t e c h n i c a l problem.  In most s u r v i v a l experiments, t h e c e l l s a r e seeded i n t o 15%  MEM, allowed t o s e t t l e down f o r 12-16 hours, exposed t o a s i n g l e chemical treatment, and then l e f t t o d i v i d e and form c o l o n i e s . The c e l l s do n o t have an o p p o r t u n i t y t o d i v i d e p r i o r t o treatment and as a r e s u l t only s i n g l e c e l l s a r e exposed t o t h e chemical. Each i n d i v i d u a l c e l l t h a t s u r v i v e s w i l l then d i v i d e t o produce one colony. But as o u t l i n e d i n the experimental p r o t o c o l ( F i g u r e 13), i t was necessary t o l e a v e t h e c e l l s f o r p e r i o d s o f up t o 12 hours a f t e r exposure t o the f i r s t dose  Time (hrs.) 0 •  1 j  2  3  4  5  6  i  i  i  •  i  .4NQ0 .4NQ0 .4NQ0 .4NQ0  1  8 «  9  10  4NQ0  .  1  1  11 1  12 1  13  14  .4N0O  ,  i n t o 15% MEM  4NQ0 4NQ0  7  .  4NQ0  f  4NQ0  . 4NQ0  . 4NQ0  .  4NQ0 .4NQ0 4NQ0  •  F i g u r e 13. Experimental d e s i g n . V a r i a t i o n o f the i n t e r v a l s between s p l i t 4NQ0 treatments; e f f e c t on c e l l s u r v i v a l .  43  p r i o r t o a p p l i c a t i o n o f the second treatment.  During such a "time p e r i o d ,  c e l l d i v i s i o n s most l i k e l y w i l l o c c u r , and as a r e s u l t two c e l l s i n s t e a d o f one would r e c e i v e the second treatment, i n v a r i a b l y producing f a l s e r e s u l t s as o n l y one c e l l need s u r v i v e to form a c o l o n y . I t i s apparent then t h a t a method was r e q u i r e d t h a t would prevent the c e l l s from d i v i d i n g b e f o r e completion o f the experiment.  Two a l t e r -  n a t i v e s seemed l i k e l y ; f i r s t l y , seed the c e l l s i n t o 5% ADM, run the experiment i n 5% ADM, and then r e t u r n the c e l l s to 15% MEM; o r s e c o n d l y , seed the c e l l s i n t o 2 1/2% MEM, run the experiment i n 2 1/2% MEM, and then r e t u r n the c e l l s t o 15% MEM.  I t was hoped t h a t ADM would t o t a l l y  a r r e s t the c e l l s , and t h a t 2 1/2% MEM would slow down c e l l u l a r processes to a p o i n t , such t h a t c e l l d i v i s i o n s would not occur d u r i n g the course o f the experiment.  The f i r s t a l t e r n a t i v e proved not f e a s i b l e ; when the  c e l l s were seeded i n t o 5% ADM they would not s e t t l e down and adhere to the p e t r i d i s h .  However, c e l l s seeded i n t o 2 1/2% MEM d i d a t t a c h to  the p e t r i d i s h . F u r t h e r experiments were then c a r r i e d out to i n v e s t i g a t e the f e a s i b i l i t y o f u t i l i z i n g c e l l s seeded i n t o 2 1/2% MEM f o r these particular survival studies.  C e l l s l e f t i n 2 1/2% MEM f o r p e r i o d s o f time  a f t e r they had adhered to the p e t r i d i s h were not s i g n i f i c a n t l y a f f e c t e d i n t h e i r a b i l i t y to form c o l o n i e s (Table 1 ) . Close examination o f the c e l l s under the i n v e r t e d microscope a f t e r 12 hours i n 2 1/2% MEM r e v e a l e d no c e l l d i v i s i o n s .  C e l l s undergoing d i v i s i o n can e a s i l y be d e t e c t e d  as they become rounded whereas the o t h e r s remain f l a t .  44 Table 1. E f f e c t o f 2 1/2% MEM on c e l l s u r v i v a l Hours i n 2 1/2% MEM Average number o f c o l o n i e s  0  2  4  8  12  75  70  71  72  73  T a b l e 2. E f f e c t o f a 1 hour 4NQ0 treatment on cell survival 4NQ0 c o n c e n t r a t i o n 5 x 10 M -8  % surviving colonies  0  10 M _8  9  5 x 1 0 " % 10""h 68  81  5 x 10  _ 1  %  90  Treatment o f t h e c e l l s f o r 1 hour with a range o f 4NQ0 concent r a t i o n s gave t h e r e s u l t s o u t l i n e d i n Table 2. C e l l s were seeded i n t o 2 1/2% MEM, allowed t o s e t t l e down, t r e a t e d with chemical i n 2 1/2% MEM and then immediately r e t u r n e d t o 15% MEM. The number o f s u r v i v i n g c o l o n i e s was expressed as a percentage o f the c o n t r o l (no 4NQ0 treatment). From these r e s u l t s , two c o n c e n t r a t i o n s were chosen f o r use i n f u r t h e r -9 -10 experiments (5 x 10 M and 5 x 10  M). These c o n c e n t r a t i o n s were chosen  because one dose allowed enough c e l l s t o s u r v i v e so t h a t a f t e r a p p l i c a t i o n o f a second dose, a reasonable number o f c e l l s would s t i l l s u r v i v e .  45 F i n a l l y c e l l s seeded i n t o 2 1/2% MEM and allowed to adhere t o the p e t r i d i s h , were assessed f o r t h e i r a b i l i t y to r e p a i r 4NQ0-induced DNA damage f o l l o w i n g 12 hours i n 2 1/2% MEM; and t h e i r a b i l i t y t o r e p a i r 4NQ0-induced DNA damage w h i l s t maintained i n 2 1/2% MEM f o r 12 hours. In the f i r s t case the c e l l s were l e f t i n 2 1/2% MEM ( a f t e r the i n i t i a l s e t t l i n g down p e r i o d ) f o r 0, 2, 4, 8 o r 12 hours p r i o r t o 4NQ0 treatment, t r e a t e d and then p l a c e d i n 15% MEM (Table 3 ) . In the second case the c e l l s were exposed t o 4NQ0 ( a f t e r s e t t l i n g down) and then allowed to recover i n 2 1/2% MEM f o r 0, 2, 4, 8 o r 12 hours, a f t e r which time they were r e t u r n e d t o 15% MEM (Table 4 ) . I t i s apparent from the r e s u l t s l i s t e d i n T a b l e s 3 and 4 t h a t r e p a i r o f 4NQ0-induced DNA damage i s not s i g n i f i c a n t l y a f f e c t e d by 2 1/2% MEM u n t i l 8 hours, a t which time t h e r e i s a small but s i g n i f i c a n t drop i n c e l l s u r v i v a l .  It i s interesting  t h a t the e f f e c t i s the same whether o r not the treatment was given before or a f t e r i n c u b a t i o n i n 2 1/2% MEM.  T h i s seems t o imply t h a t a f t e r pro-  longed p e r i o d s i n 2 1/2% MEM, a general t o x i c i t y occurs t h a t i s s l i g h t l y enhanced by exposure to 4NQ0. The f e a s i b i l i t y o f employing t h i s technique was then apparent, and o n l y r e q u i r e d i n c l u s i o n o f e x t r a " s i n g l y - t r e a t e d " c o n t r o l s .  These  extra c o n t r o l s would r e c e i v e the f i r s t treatment o n l y , were subsequently incubated i n 2 1/2% MEM f o r 9 o r 13 hours and then r e t u r n e d t o 15% MEM along with the " d o u b l y - t r e a t e d " c u l t u r e s .  Other c o n t r o l s r e c e i v e d the  f i r s t treatment and were then p l a c e d immediately i n t o 15% MEM. As i n the p r e v i o u s DNA r e p a i r experiments i t was necessary t o s e l e c t a s u i t a b l e end p o i n t f o r e v a l u a t i n g the r e s u l t s .  Again an expected  46 Table 3.  The effect on c e l l survival of incubation in 2 1/2% MEM, prior to a single 4NQ0 treatment % surviving colonies at concentrations  Hours in 2 1/2% MEM prior to treatment  5 x 10" M 9  5 x 10" M 10  0 2  71 65  86 79  4 8  70 60  88  12  55  68  Table 4.  76  The effect on c e l l survival of incubation in 2 1/2% MEM, following treatment with 4NQ0 % surviving colonies at concentrations  Hours in 2 1/2% MEM after treatment  5 x 10" M 9  5 x 10" M 10  0 2  76 67  87 78  4 8  70 58  83 72  12  53  64  47 value was d e f i n e d : i f a s i n g l e 4NQ0 treatment enables a c e r t a i n percentage o f c e l l s t o s u r v i v e , then t h e o r e t i c a l l y when a second treatment i s a p p l i e d t o these s u r v i v i n g c e l l s , t h e same percentage o f these c e l l s would be expected t o s u r v i v e . In o t h e r words i f 68% o f the c e l l s s u r vive t h e f i r s t dose, then 68% o f these 68% should be expected t o s u r v i v e the second dose ( i . e . 4 6 % ) . In c a l c u l a t i n g t h e expected values a t 9 and 13 hours, the c o n t r o l s f o r those time p e r i o d s were employed i n o r d e r to account f o r any l o s s o f c e l l s due t o t o x i c i t y i n 2 1/2% MEM. The r e s u l t s o u t l i n e d i n T a b l e 5 i n d i c a t e a p e r i o d i n which there i s a p o t e n t i a t i o n o f the e f f e c t s o f t h e second dose.  T h i s occurs  i n the three hours immediately f o l l o w i n g a d d i t i o n o f t h e f i r s t treatment, and c o r r e l a t e s very c l o s e l y with t h e r e s u l t s o f t h e DNA r e p a i r e x p e r i ments, i n which t h e below expected r e p a i r l e v e l s a r e o b t a i n e d i n t h e same 3 hours. C l o s e r examination o f the r e s u l t s r e v e a l t h a t a f t e r t h i s p e r i o d o f decreased c e l l s u r v i v a l , t h e l e v e l s r i s e and reach a p l a t e a u a t 9 hours.  For both c o n c e n t r a t i o n s , t h e value a t t a i n e d i s above t h e expected,  though n o t t h e same; c e l l s exposed t o two doses o f the lower concent r a t i o n r i s e t o a h i g h e r p o i n t above t h e expected v a l u e . The f a c t t h a t c e l l s u r v i v a l does r i s e above the expected may simply r e f l e c t an i n c o r r e c t c h o i c e o f end p o i n t , o r a c a p a c i t y f o r i n c r e a s e d s u r v i v a l i n t h e c e l l s t h a t s u r v i v e t h e f i r s t dose. N e v e r t h e l e s s , i t i s obvious t h a t as the r e p a i r o f t h e f i r s t treatment nears completion, t h e c e l l s r e g a i n t h e i r c a p a c i t y f o r s u r v i v a l . I t i s a l s o apparent t h a t i f two 4NQ0 treatments a r e given c l o s e t o g e t h e r , DNA damage i s induced by t h e second treatment, and furthermore t h a t some o f i t i s not being r e p a i r e d .  48  Table  Effect of split Time a f t e r f i r s t dose when second dose a p p l i e d (hours)  [4NQ0] 5  5.  x 10" M 9  1 1/2  hrs.  2  10  treatments on c e l l s u r v i v a l  % surviving colonies a f t e r second dose  Expected value (%)  Difference {%)  8  50  -42  6  50  -44  3  34  50  -16  5  30  50  -20  9  40  30  +10  37  32  +5  55  79  -24  2  51  79  -28  3  80  79  +1  5  92  79  +13  9  58  44  +14  61  38  +23  13  5 x 10" M  4NQ0  1 1/2  13  hrs.  49 b) Chromosome S t u d i e s Since i n the previous experiments, a drop i n c e l l s u r v i v a l was o b t a i n e d when the two treatments were given c l o s e t o g e t h e r , i t would be i n t e r e s t i n g to a s c e r t a i n i f a simultaneous i n c r e a s e i n chromosome a b e r r a t i o n s a l s o occurs.  The experimental p r o t o c o l o u t l i n e d i n F i g u r e  14 was designed to i n v e s t i g a t e t h i s .  A 13 hour s p l i t treatment was  not done f o r 2 reasons; f i r s t l y , the c e l l s have more o r l e s s t o t a l l y recovered i n t h e i r a b i l i t y to r e p a i r the second dose at 9 hours, and secondly the main i n t e r e s t o f the experiment i n v o l v e d the 3 hours immed i a t e l y f o l l o w i n g the f i r s t dose. The c e l l s were t r e a t e d i n 5% MEM, and allowed to r e c o v e r i n 15% MEM.  C o l c h i c i n e was added a t 20 and 25 hours post-treatment  (second  dose). The r e s u l t s are o u t l i n e d i n Table 6.  A marked i n c r e a s e i n chromo-  some a b e r r a t i o n s i s e v i d e n t i f the second treatment i s given i n the two hours f o l l o w i n g a d d i t i o n o f the f i r s t .  The e f f e c t i s not as dramatic  at the lower c o n c e n t r a t i o n s but i s s t i l l e v i d e n t . the values approach normal l e v e l s .  A f t e r f i v e hours,  Again a c o r r e l a t i o n with the DNA  r e p a i r experiments i s o b t a i n e d , and provides f u r t h e r evidence t h a t DNA damage i s not being r e p a i r e d .  Time (hrs.) -10 i  -9  -8  i  -7 •  ;  -6 i  -5 i  -4 i  -3  -2  i  -1  i  1  0  i  4NQ0 4NQ0  .  4NQ0 4NQ0 4NQ0 .4NQ0  4NQ0 .4NQ0 .4NQ0 .4NQ0 .4NQ0  F i g u r e 14: Experimental design. V a r i a t i o n o f t h e i n t e r v a l s between s p l i t 4NQ0 treatments; e f f e c t on chromosome a b e r r a t i o n s .  O  51 Table 6.  E f f e c t o f s p l i t 4NQ0 treatments on chromosome aberrations  Time a f t e r f i r s t dose when second dose a p p l i e d (hours)  Frequency o f metaphase p l a t e s with chromosome a b e r r a t i o n s a t concentrations 5 x 10" M  10" M  1 1/2 h r s . 2 3 5 9  24 48 23 17 9  15 21 11 5 3  S i n g l e dose only  11  3  7  7  DISCUSSION I.  The DNA Repair  Process  Before embarking on any d i s c u s s i o n or i n t e r p r e t a t i o n of the r e s u l t s obtained i n t h i s study, i t i s e s s e n t i a l that the underlying mechanisms of the DNA r e p a i r process be d e f i n e d .  The enzymes i n v o l v e d  i n e x c i s i o n - r e p a i r ( a l s o c a l l e d dark r e p a i r , r e p l i c a t i o n r e p a i r , unscheduled r e p a i r s y n t h e s i s ) have been well c h a r a c t e r i z e d i n b a c t e r i a l fi  OO  systems '  C7  OC  *  and i t i s b e l i e v e d t h a t a s i m i l a r enzymatic process 13 56 58  e x i s t s i n mammalian c e l l s  '  ' . The e s s e n t i a l f e a t u r e s of e x c i s i o n -  r e p a i r i n b a c t e r i a are o u t l i n e d i n Figure 15.  B a s i c a l l y , the mechanism  can be d e s c r i b e d as a c y c l e i n v o l v i n g at l e a s t 4 enzymes and 5 d e f i n e d activities: a) UV i r r a d i a t i o n r e s u l t s i n the formation of pyrimidine dimers; each producing a l o c a l i z e d s t r u c t u r a l d i s t o r t i o n i n the DNA h e l i x . b) These d i s t o r t i o n s serve as r e c o g n i t i o n s i t e s f o r a t t a c k by an endonuclease; and a nick i s e s t a b l i s h e d i n the DNA c l o s e to the dimer on the 5' s i d e . c) DNA polymerase binds to the DNA at the nicked s i t e and  begins 30  to s y n t h e s i z e new DNA using the opposite s i d e o f the DNA as a template d) An exonuclease cleaves the 3' s i d e of the dimer r e l e a s i n g the damaged p o r t i o n of the DNA; i t i s b e l i e v e d t h a t the polymerase i t s e l f 52 3 4 42 may have such an exonuclease a c t i v i t y ' ' .  .  I II I I I I I ! II • • • • • • • • I • • • .  UV ENOONUCLEASE  2  11 n 11111  111111111  I I I I I I I I I  O ONA POLYMERASE  IIIIIIIII •  rffo*i 11111111 I I M I I I I I  iiiiiiiiiirniliiiiiiin •  5  1  1  1  1  1  1  1  *****  I k 1111111 M M1111 i 111111 I l l l l l M i l l l l l l l HI I  nil  POLYNUCLEOTIDE LIGA5E + DPH  6  111111 M i n 1111111111111 I I I I I I I H I I I H I I I I I I I I I I  Figure 15. A model for DNA repair. (From Molecular and Cellular Repair Processes. Baltimore: John Hopkins, 1972, p. 15).  54 e) Simultaneous synthesis and degradation continues' in the 5' to 3' direction until the ligase displaces the polymerase and seals the last bond. The net result of this process is restoration of the functional integr i t y of the DNA. The repair of both UV and 4NQ0-induced DNA damage is postulated to be basically s i m i l a r ; this fact was further substantiated by the data obtained in the present study. is not i d e n t i c a l .  However, the induction of damage  The structure of 4NQ0 is i l l u s t r a t e d in Figure 16, 67 77  and probably binds covalently to a purine base in the DNA  '  .  As a  result depurination of the DNA occurs followed by distortion of the sugar phosphate backbone. for the endonuclease.  This distortion is the recognition s i t e  Removal of this distortion proceeds in the same  manner as removal of pyrimidine dimers.  Figure 16.  II.  0 4-Nitroquinoline 1-Oxide (4NQ0).  Interpretation of  HTdR Incorporation Studies  The results of the DNA repair experiments can be summarized as follows:  55  1) DNA repair synthesis is below the expected levels when a second treatment is given within 3 hours of the f i r s t , 2) DNA repair synthesis reached the expected levels i f the second treatment is applied as repair of the f i r s t approaches completion, and 3) analogous results when the f i r s t 4NQ0 treatment is replaced with UV i r r a d i a t i o n . Interpretation of this data was somewhat simplified by employing cell survival and chromosome studies.  The results of these were:  1) a drop in cell survival and an increase in chromosome aberrations when the interval between doses is less than 3 hours; 2) a rise in c e l l survival and a drop in chromosome aberrations approaching the expected when the interval between doses is more than 3 hours. Five interpretations of this data warrant consideration and w i l l be discussed individually in the subsequent paragraphs. The most logical explanation for the below-expected repair levels is that further damage did not ensue when the second dose was applied close to the f i r s t . of three conditions:  This could have originated from any one  the enzyme pools necessary for activation of the  chemical were depleted, the DNA was already overloaded with damage, or the 4NQ0 was somehow prevented from entering the c e l l .  Yet the  data does not support any of these suggestions. The results of the chromosome studies indicate that damage is not being repaired, consequently i f the second dose does not induce further damage, one would expect the level of repair synthesis following the second treatment  56  to be below the c o n t r o l l e v e l ( s i n g l e dose).  But i t i s not;  therefore,  f u r t h e r damage must occur, though a l l of i t i s not n e c e s s a r i l y r e p a i r e d . The next p o s s i b i l i t y i s that the enzymes necessary f o r the r e p a i r process are being i n h i b i t e d by the 4NQ0, which i n turn would be r e f l e c t e d by low r e p a i r l e v e l s . This does not seem very p l a u s i b l e because one would expect such an i n h i b i t i o n to occur a f t e r each 4NQ0 treatment.  But i t does not; low r e p a i r l e v e l s are only  obtained  f o l l o w i n g the second treatment and only when i t i s a p p l i e d c l o s e to the first. Furthermore, i f U V - i r r a d i a t e d c e l l s are t r e a t e d with a chemical t h a t i s known to i n h i b i t one or more of the enzymes i n v o l v e d i n DNA r e p a i r ( i . e . i o d o a c e t a t e ) , the e f f e c t i s very pronounced, with an almost total i n h i b i t i o n of repair s y n t h e s i s . 1 0  present study. can be  This was not evident i n the  Consequently, i n h i b i t i o n o f the r e p a i r enzymes by 4NQ0  discounted. The t h i r d i n t e r p r e t a t i o n i s t h a t the r e p a i r enzymes are being  degraded a f t e r a s i n g l e c y c l e of r e p a i r .  As a r e s u l t , when a second  dose of 4NQ0 i s given c l o s e to the f i r s t , the r e p a i r l e v e l s would be below the expected as not enough enzymes would be present.  The r e p a i r  c a p a c i t y would be' r e s t o r e d when s y n t h e s i s of new enzymes i n c r e a s e s , and would account f o r normal r e p a i r l e v e l s when the i n t e r v a l between the two treatments i n c r e a s e s . However, many observations  disagree with such an i n t e r p r e t a t i o n ;  in the f i r s t place i t implies that renewal of the enzyme pool would r e s u l t i n the damage being r e p a i r e d at a l a t e r time. The r e s u l t s of  57 the time course of repair after a second dose do not verify t h i s ; i f two doses are given close together the peak of repair occurs immediately after removal of the second treatment, i t is not delayed.  The results  of the chromosome and survival studies also do not indicate that the damage is repaired at a later time. Secondly, there is very l i t t l e evidence to substantiate the concept that the degradation of the repair enzymes occurs after one cycle of repair.  In fact most of the work indicates that the enzymes  for repair are very stable, possess a long h a l f - l i f e and are not i n duced ' . 2 9  6 0  Experiments with puromycin and cycloheximide demonstrated  that protein synthesis was not a prerequisite for DNA repair; the enzymes were already present in the c e l l in quantities capable of repairing most of DNA damage.  Treatment of the c e l l s with cycloheximide for up  to 8 hours before exposure to UV did not a l t e r the repair l e v e l s ; even though protein synthesis had been almost t o t a l l y blocked .  Cells l e f t  in cycloheximide for 20 hours prior to UV irradiation showed levels of repair replication that were s t i l l 65% of the untreated, irradiated cultures.  In other studies, puromycin also had no effect on repair • jo  of X-ray induced damage  '  cc  .  In view of these arguments, i t seems highly unlikely that the results of this study can be accounted for by degradation of the repair enzymes. Alternatively, i t is possible that the below expected levels of repair synthesis are a r t i f a c t s of the technique employed.  During  the i n i t i a l peak of repair synthesis, there is a l i m i t to the number  58 2 14 of i n c i s i o n s t h a t can occur at one time along the DNA ' . T h i s i m p l i e s t h a t even though many segments o f the DNA are undergoing r e p a i r s y n t h e s i s during the e a r l y stages of recovery, numerous s i t e s w i l l s t i l l c o n t a i n damaged p o r t i o n s .  I f the DNA i s subjected to f u r t h e r damage during  t h i s time, an a l k y l a t e d base could occur c l o s e to a p r e v i o u s l y a l k y l a t e d base (but as y e t u n r e p a i r e d ) .  When such a DNA segment i s f i n a l l y r e p a i r e d ,  t h e o r e t i c a l l y more than one base c o u l d be removed per 100 n u c l e o t i d e s ( e x c i s i o n r e p a i r i n v o l v e s the removal of approximately 11,17,59,61^ ensue.  T n t h i s m a n n e r j  a  i  o w e r  100 n u c l e o t i d e s  than expected g r a i n count c o u l d  I f a second dose i s given when most of the i n i t i a l damage has  been r e p a i r e d , such an o v e r l a p would not occur, and consequently  the  expected number of g r a i n s would be obtained. The major drawback of t h i s e x p l a n a t i o n i s t h a t even though the g r a i n counts are not up to the expected, the damage i s s t i l l r e p a i r e d ; again t h i s c o n t r a d i c t s the r e s u l t s of the chromosome and c e l l s u r v i v a l studies.  Moreover, c u r r e n t research does not i n d i c a t e t h a t more than 21 59  one dimer i s e x c i s e d per region  ' .  The f i n a l e x p l a n a t i o n concerns the p o s s i b i l i t y t h a t the r e p a i r process i t s e l f i s somehow being i n h i b i t e d by the second 4NQ0 treatment. Since d i r e c t i n h i b i t i o n of the enzymes themselves has already been discounted, one of the few remaining s o l u t i o n s i s that the  ongoing  r e p a i r process i t s e l f i s being a f f e c t e d . This would be e s p e c i a l l y apparent when many s i t e s along the DNA are being r e p a i r e d ( f o r i n s t a n c e , during the peak of r e p a i r ) . However, i f d i r e c t i n h i b i t i o n of r e p a i r by the second dose i s o c c u r r i n g , the l e v e l s obtained a f t e r t h i s second  59  dose would be f a r below the c o n t r o l s .  As t h i s i s not the case, i t  seems l o g i c a l to conclude that i n d u c t i o n of damage and i n h i b i t i o n of r e p a i r occur simultaneously  upon a p p l i c a t i o n of the second dose.  I t i s not d i f f i c u l t to envisage how t h i s i n h i b i t i o n would occur, f o r as each r e p a i r c y c l e progresses, many s u s c e p t i b l e s i t e s would be exposed.  Binding of molecules at t h i s time could e a s i l y take place  i n such a manner t h a t c o n t i n u a t i o n o f the c y c l e i s prevented.  The  enzymes themselves would remain u n a f f e c t e d , and would e v e n t u a l l y f a l l o f f i n i t i a t i n g a new c y c l e o f r e p a i r s y n t h e s i s at some other l o c a t i o n . I m p l i c i t i n t h i s e x p l a n a t i o n i s t h a t the p a r t i a l l y r e p a i r e d segment of DNA i s never r e p a i r e d ; the binding would have occurred i n such a manner that movement o f enzymes along the segment i s permanently blocked. The net r e s u l t when the two treatments  are a p p l i e d c l o s e t o -  gether would be i n h i b i t i o n of r e p a i r of the f i r s t s e t o f damage; and i n d u c t i o n and r e p a i r o f a second s e t .  P o s s i b l y the amount o f damage  produced by the second dose would be l e s s than usual because some o f the binding would have r e s u l t e d i n i n h i b i t i o n , not i n d u c t i o n of damage. This combined e f f e c t would account f o r the below expected l e v e l s o f repair.  As r e p a i r of the f i r s t treatment nears completion, i n h i b i t i o n  by the second dose would not be as e v i d e n t , and normal l e v e l s of damage and consequently  r e p a i r s y n t h e s i s would be obtained.  I f t h i s i s the case, then i t suggests t h a t the c a r c i n o g e n i c i t y o f 4NQ0 may be due i n p a r t to i t s a b i l i t y of s l i g h t l y i n h i b i t i n g damage induced by i t s e l f .  Such an e f f e c t may not be as n o t i c e a b l e during a  s i n g l e treatment, because most l i k e l y a major p o r t i o n of the binding  60 w i l l have taken place before repair synthesis reaches i t s peak; therefore only a s l i g h t inhibition would take place.  Accordingly, i f a  second dose is applied right when the peak of repair is in progress the effect may be very marked. Inhibitors of DNA repair have been reported  previously ' ' ' , 1 0  1 6  2 7  4 0  and f a l l into two categories, those that bind to DNA, and those that show a wide spectrum of effects.  The f i r s t group includes such compounds  as acriflavine and c h l o r o q u i n e ' 10  27  which do not induce DNA repair  synthesis but considerably i n h i b i t the repair of UV damage.  Acriflavine  preferentially k i l l s irradiated bacteria, and enhances UV mutagenesis, i fi fifi  most l i k e l y by inhibiting excision of pyrimidine dimers  '  .  In vivo, 79  chloroquine and caffeine have been found to enhance tumorigenesis  .  In the second group are such compounds as progesterone, testosterone, and diethylsti1bestrol.  These compounds do not bind to DNA  and do not i l l i c i t DNA repair synthesis, but again they have a s i g n i ?fi ?8  ficant effect on DNA repair induced by UV ' . Most of these compounds have been c l a s s i f i e d as co-carcinogens because they have the a b i l i t y 35 to enhance the tumorigenie action of other carcinogens  .  It is believed  that their target is some aspect of the repair process. In view of such evidence i t does not 'seem too unlikely that 4NQ0 could be inhibiting some aspect of the DNA repair process. 3 Discussion of the  HTdR incorporation studies would be somewhat  incomplete i f two additional aspects were not mentioned.  The f i r s t  concerns the data obtained with c e l l s in 5% ADM. When a second treatment was given to these c e l l s , the level of repair never reached the expected.  61  In f a c t as the i n t e r v a l s between the two doses i n c r e a s e d , the l e v e l of r e p a i r f o l l o w i n g the second dose decreased.  I d e n t i c a l experiments  employing c e l l s t r a n s f e r r e d to 5% MEM d i d not y i e l d these r e s u l t s , implying t h a t the e f f e c t s was due to the l a c k of a r g i n i n e i n the medium. Amino a c i d d e p r i v a t i o n causes a f a i r l y r a p i d c e s s a t i o n of semi5  c o n s e r v a t i v e DNA s y n t h e s i s . The mechanism behind t h i s i s not c l e a r , f o r even though s y n t h e s i s of new enzymes i s blocked, enough enzymes are present to enable DNA s y n t h e s i s to take p l a c e .  It is believed that  accurate DNA r e p l i c a t i o n r e q u i r e s u n i n t e r r u p t e d , c o - o r d i n a t e d , de novo 25 29 34 s y n t h e s i s of p r o t e i n s  .  . Perhaps the DNA r e p a i r process i s a f f e c t e d  i n the same manner. T h i s seems h i g h l y u n l i k e l y f o r one would expect both treatments to be a f f e c t e d , not j u s t the second. A more l o g i c a l e x p l a n a t i o n i s t h a t a f t e r prolonged  incubation  i n ADM, the c e l l s are not i n an optimal p h y s i o l o g i c a l s t a t e , and quently the t o x i c i t y o f 4NQ0 i s enhanced.  Repair o f the f i r s t  consetreatment  may be normal, but most l i k e l y a f t e r 8 hours i n c u b a t i o n , the c e l l s begin to d i e , and t h e r e f o r e cannot respond to a d d i t i o n a l c h e m i c a l l y induced damage. When the two treatments are c l o s e together, the  response  of c e l l s i n ADM and MEM was almost, but not completely s i m i l a r ; f o r the c e l l s i n ADM showed a l a r g e r d e c l i n e i n l e v e l s of r e p a i r s y n t h e s i s . This c o u l d r e f l e c t e i t h e r c e l l s i n e a r l y stages o f death or an e f f e c t due to a r g i n i n e d e f i c i e n t medium. This phenomenon warrants f u r t h e r investigation. The second r e s u l t t h a t must be mentioned i s the s l i g h t i n h i b i t i o n of r e p a i r t h a t occurred when UV exposure was c l o s e l y f o l l o w e d by a 4NQ0 treatment.  This was not evident i f the f i r s t treatment i n v o l v e d ex-  posure to 4NQ0.  62 This observation may simply r e f l e c t the d i f f e r e n c e s i n the manner whereby damage was induced by 4NQ0 and UV; 4NQ0 treatment was f o r 1 hour, whereas UV treatment was f o r a few seconds.  Even though  the processes t h a t a r e i n v o l v e d i n t h e r e p a i r o f these two types o f damage a r e p o s t u l a t e d to be the same  ' ' ' , i n i t i a t i o n of repair  s y n t h e s i s probably occurs a t d i f f e r e n t times.  As a r e s u l t t h e two  s e t s o f data may not be t r u l y comparable. Returning to Figure 2, i t can be noted t h a t the peak o f r e p a i r s y n t h e s i s may already be i n progress h a l f an hour a f t e r removal o f t h e 4NQ0,  But f o l l o w i n g UV treatment the peak o f r e p a i r s y n t h e s i s may only  j u s t be i n i t i a t e d h a l f an hour a f t e r treatment.  Consequently, t h e  e f f e c t o f a 4NQ0 treatment h a l f an hour a f t e r a UV treatment may have a more profound e f f e c t , f o r i t would be present when r e p a i r o f UV^ induced damage i s a t a maximum. In r e t r o s p e c t , i t i s obvious t h a t there a r e some l i m i t a t i o n s 3 to t h e HTdR i n c o r p o r a t i o n experiments; i t was not p o s s i b l e t o determine e x a c t l y when, and f o r how long the peak o f r e p a i r occurred, nor was i t p o s s i b l e t o d e f i n e the peak o f " s e n s i t i v i t y . " f o r f u r t h e r i n v e s t i g a t i o n has been e s t a b l i s h e d .  Nevertheless, a foundation Characterization of  the two hour " s e n s i t i v e " p e r i o d i n more d e t a i l should be attempted by spacing the two treatments 15, 30, 45, 60 and 90 minutes apart. A l s o 3 a d d i t i o n o f HTdR simultaneously with t h e second 4NQ0 treatment,  would  perhaps give a b e t t e r i n d i c a t i o n o f when and to what extent i n h i b i t i o n may be o c c u r r i n g during the second  treatment.  63 III.  S p l i t Doses and Cell Survival Since treatment of c e l l s in vitro with s p l i t doses of chemical  carcinogens has not been reported before, many aspects of this study are unique.  Consequently i t is d i f f i c u l t to make any direct comparisons  between the results of this work and those of others.  Nevertheless,  a few indirect comparisons can be made and are possibly relevant to this discussion. The effects of s p l i t doses of UV and X-irradiation on c e l l survival has been reported and perhaps can be compared to the effects of s p l i t 4NQ0 treatments on c e l l survival that have been reported in this study. 37 38 Humphrey et a l .  using a line of Chinese hamster c e l l s detected  an apparent increase in cytotoxicity  ( i . e . decrease in c e l l survival)  when double doses of UV were separated by 30-120 minutes.  However,  the results were not interpreted in terms of potentiation but as a lack of repair in the c e l l line employed.  The concomitant rise in survival  after 2 hours was thought to be caused by selection of a radiationresistant portion of the cell population.  Such an interpretation was  not warranted as a simultaneous study of repair synthesis was not attempted. 78  A more comprehensive study has recently been published  , in  which the effects of s p l i t UV treatments on both survival and mutation rates was investigated in a different l i n e of Chinese hamster c e l l s . A s t r i k i n g increase in the frequency of cytotoxicity and mutations was obtained when the intervals between doses were 15, 30, and 60 minutes.  64 At an i n t e r v a l o f 3 hours, the mutation r a t e r e t u r n e d t o the value expected, as d i d c y t o t o x i c i t y a t 5 hours. expected l e v e l a f t e r 9-12 hours.  Both values reached a s t a b l e  I t was concluded t h a t the second  dose was somehow i n t e r f e r i n g w i t h an aspect o f t h e c e l l ' s r e p a i r p r o c e s s . Analogous data was not r e p o r t e d when s p l i t doses o f X - i r r a d i a t i o n were employed.  In these s t u d i e s , i f s u r v i v a l i s p l o t t e d as a f u n c t i o n  o f the time between exposures, a sharp r i s e i n c e l l s u r v i v a l i s o b t a i n e d i n the f i r s t two hours, and remains constant f o r t h e next 5 hours, t o 1 19  ?n  l e v e l o f f a t a h i g h e r value a f t e r 10 hours ' ' . decrease i n c e l l s u r v i v a l was observed.  No s i g n i f i c a n t  These r e s u l t s a r e not s u r p r i s i n g  though, because they most l i k e l y r e f l e c t t h e i n h e r e n t d i f f e r e n c e s between the kinds o f damage and r e p a i r processes induced by UV and X - i r r a d i a t i o n . UV damage produces p y r i m i d i n e dimers, induces a r e p a i r c y c l e t h a t i n v o l v e s i n c o r p o r a t i o n o f approximately 100 new n u c l e o t i d e s and which i s v i r t u a l l y complete a t 6 h o u r s ' 1 3  2 4 , 6 7  .  On the other hand, the major product o f 53  X-ray damage i s s i n g l e - s t r a n d breaks , which induces a r e p a i r c y c l e i n v o l v i n g i n c o r p o r a t i o n o f 2-3 n u c l e o t i d e s ' ' ' , and t h a t i s 43 49 65 69 r a p i d l y completed ' ' ' . T h i s r e p a i r does not r e q u i r e a r a t e 2 3  5 4  5 6  7 0  l i m i t i n g cleavage s t e p , only a small amount o f s y n t h e s i s p r i o r to r e 9 15 52 j o i n i n g ' ' . I t i s c o n c e i v a b l e t h a t when c e l l s undergoing t h i s p a r t i c u l a r r e p a i r a r e exposed t o a second treatment, any e f f e c t t h a t does occur i s probably d i f f i c u l t t o d e t e c t . Two o b s e r v a t i o n s emerge; f i r s t l y , s i n c e the r e p a i r induced by UV damage i s b a s i c a l l y s i m i l a r t o the r e p a i r o f damage induced by 4NQ0, a l e g i t i m a t e c o r r e l a t i o n can perhaps be made between t h e r e s u l t s o f the c e l l s u r v i v a l experiments i n t h i s present study and those r e p o r t e d above  65  i n v o l v i n g s p l i t UV doses.  In both cases a p e r i o d o f i n c r e a s e d s e n s i -  t i v i t y i s evident. Secondly, s i n c e chemicals d i f f e r i n the type o f DNA damage they produce, eg. some bind t o bases, whereas others produce s i n g l e 7  fx ? 1  s t r a n d breaks ' ; and t h e types o f DNA r e p a i r processes induced, i t is  very  probable t h a t t h e e f f e c t s o f s p l i t treatments on these  two kinds o f DNA r e p a i r s y n t h e s i s a l s o d i f f e r .  Further i n v e s t i g a t i o n  o f t h i s phenomenon should be the next s t e p . IV.  Out!ook I t was suggested p r e v i o u s l y t h a t r e s i d u a l , unrepaired DNA damage  may be o f more importance than the l e v e l s o f r e p a i r s y n t h e s i s t h a t take p l a c e upon treatment with a chemical c a r c i n o g e n .  Continuing on t h i s  assumption, i f t h e r e p a i r o f DNA damage i s then a l s o p a r t i a l l y i n h i b i t e d , the frequency o f n e o p l a s t i c t r a n s f o r m a t i o n may be f u r t h e r i n c r e a s e d . In t h i s study, exposure o f c e l l s t o s p l i t treatments o f a c a r c i n o gen r e v e a l e d a p o t e n t i a t i o n o f the e f f e c t s when t h e doses were given c l o s e t o g e t h e r , and was i n t e r p r e t e d t o r e f l e c t a l a c k o f r e p a i r s y n t h e s i s . I f t h i s i s the case, then care must be e x e r c i s e d i n e v a l u a t i n g e x p e r i ments t h a t i n v o l v e only s i n g l e doses o f c a r c i n o g e n s , f o r the t r u e c a r c i n o genic p o t e n t i a l o f the chemical may not be e v i d e n t . In d e s i g n i n g assay systems t o be employed i n s c r e e n i n g f o r c a r c i n o g e n s , i t i s necessary to i n c o r p o r a t e many exceptions i n order that a " t r u e " value can be o b t a i n e d .  F i r s t l y , the e x i s t e n c e o f c e l l  /  66  lines that cannot repair certain types of DNA damage cannot be ignored; the addition of chemicals to these c e l l s in some instances can produce a false negative result.  To circumvent this d i f f i c u l t y thorough screening  should employ more than one c e l l l i n e .  Secondly, some chemical compounds 48  require metabolic activation before they can induce DNA damage  .  Con-  sequently, measures must be taken to allow for activation of these compounds; this usually involves the addition of a l i v e r microsomal 46  preparation simultaneously with the chemical It is obvious from the evidence presented in this study, that a more meaningful evaluation of the carcinogenic potential of a compound can be obtained by inclusion of the technique designed in this study, into future screening programs.  Many carcinogens that previously have CO  not i l l u s t r a t e d any capacity to induce DNA damage using such techniques.  , can be assessed  Perhaps their carcinogenicity i s due to their  a b i l i t y to a l t e r the repair of DNA damage.  Furthermore, chemicals  that have been previously tested using only single doses, should be re-evaluated using double doses, for they may in fact have a greater carcinogenicity than previously imagined. Additional research in this area should be directed at investigating the interactions of various combinations and sequences of different carcinogens, in v i t r o .  These studies should increase our  understanding of the processes involved in the repair of DNA damage and i t s role in chemical carcinogenesis.  SUMMARY  1. The primary o b j e c t i v e of t h i s study was to i n v e s t i g a t e the e f f e c t s of repeated exposure to a chemical carcinogen, 4NQ0, in human skin f i b r o b l a s t s .  Three end points were employed:  DNA r e p a i r syn-  t h e s i s (as measured by HTdR i n c o r p o r a t i o n ) , c e l l s u r v i v a l and chromosome a b e r r a t i o n s . 2. Following a s i n g l e one hour treatment with 4NQ0, the peak of r e p a i r synthesis was evident i n the second and t h i r d hours a f t e r a d d i t i o n of the carcinogen.  Repair synthesis was v i r t u a l l y complete at 12 hours  post-treatment. 3. When c e l l s were challenged with a second 4NQ0 treatment w i t h i n 3 hours of the f i r s t , the l e v e l of r e p a i r synthesis induced by t h i s second dose was below a defined expected value.  Following 9 hours  incubation between treatments, r e p a i r synthesis a f t e r the second dose had returned to the expected value. 4. Replacement of the f i r s t 4NQ0 treatment with a UV treatment produced analogous r e s u l t s . 5. Cell s u r v i v a l dropped s i g n i f i c a n t l y when the second dose was applied w i t h i n one hour of the f i r s t .  An increase i n the frequency of  chromosome aberrations was detected when the two treatments were given less than two hours apart.  Both values had returned to expected l e v e l s  when treatments were separated by more than 5 hours.  67  68  6. E v a l u a t i o n of a l l three sets o f data i n d i c a t e d that i n s u f f i c i e n t DNA r e p a i r s y n t h e s i s was o c u r r i n g when the second treatment was w h i l s t r e p a i r o f the f i r s t was s t i l l i n progress.  applied  As r e p a i r of the  f i r s t treatment neared completion, t h i s e f f e c t was no longer e v i d e n t . 7. Several p o s s i b l e explanations were d i s c u s s e d and i t was concluded t h a t the second dose was p o s s i b l y i n h i b i t i n g the ongoing r e p a i r process. 8. I n t e r p r e t a t i o n o f t h i s c o n c l u s i o n i n terms of the i n c r e a s e d c a r c i n o g e n i c p o t e n t i a l of a chemical carcinogen was a l s o  presented.  REFERENCES  1.  A r l e t t , C. F . , and P o t t e r , J . Mutation to 8-azaguanine r e s i s t a n c e i n d u c e d by y - i r r a d i a t i o n i n Chinese hamster c e l l l i n e s . M u t a t i o n R e s . , 1_3:59, 1971.  2.  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