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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 of 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 to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J 4 j l y , 1974-In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g r e e t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Depar tment o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f Z o o l ° g y  The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada 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 o f 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 v i v o are exposed to v a r y i n g low doses o f 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 o f s e p a r a t e d , low doses of a chemical carcinogen i n v i t r o . 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 c h a l l e n g e d 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 e v a l u a t e the e f f e c t s , t h r e e 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 . F o l l o w i n g 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 HTdR i n c o r p o r a t i o n ) was determined. The peak o f 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 o f the c a r c i n o g e n . DNA r e p a i r s y n t h e s i s was v i r t u a l l y complete a t 12 hours 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 v a l u e . 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 l e v e l . Replacement o f the f i r s t 4NQ0 treatment with a UV treatment produced analogous r e s u l t s . The c l o n e forming c a p a c i t y o f 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 o f e f f e c t s was e v i d e n t when i i 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 v a l u e . 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 o f 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 i n c r e a s e d (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 o f the chemical c a r c i n o g e n . 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 to Dr. H. F. S t i c h f o r h i s i n t e r e s t and ad v i c e throughout t h i s r e s e a r c h , and to Mrs. S t i c h f o r her many words o f encouragement. I a l s o wish to thank L y d i a 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 her undying p a t i e n c e 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 oppor-t u n i t y and the f a c i l i t i e s t o c a r r y out t h i s r e s e a r c h p r o j e c t . i v TABLE OF CONTENTS Page ABSTRACT . . . . . . . . . . i i ACKNOWLEDGEMENTS i v LIST OF TABLES . v i i LIST OF FIGURES . .... . . . . . . . . . . . . . . v i i i INTRODUCTION . . . . . . . . . 1 MATERIALS AND METHODS . . . . . . . . . . . . 5 I. C e l l C u l t u r e s 5 I I . Chemical Treatment . . . . . . 6 I I I . UV Treatment 6 IV. C e l l S u r v i v a l S t u d i e s . . . . . . . . 7 V. Chromosome S t u d i e s . . . . . . . . . . . . . . 7 VI. Autoradiography . . . . . . . . . . . . . . 8 RESULTS . . . . . . . . . . . . . . . . . . . . 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 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 . 26 IV. Time Course o f DNA Repair a f t e r a Second 4NQ0 Treatment. . 36 V. C e l l S u r v i v a l and Chromosome St u d i e s 41 DISCUSSION 52 I. The DNA Repair Process 52 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 54 v v i 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 65 SUMMARY • 67 REFERENCES . . . . . . 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 . . . . . . . . 44 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 . . . . . . . . 46 4 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, 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 v i i LIST OF FIGURES Fi gure Page 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 12 2 Repair o f 4NQ0-induced DNA damage. E f f e c t o f concen-t r a t i o n and d u r a t i o n o f exposure 13 3 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, 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 the 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 5 Histogram i l l u s t r a t i n g the 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 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 . . . . . . . 24 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 . 2 8 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 s y n t h e s i s 30 9 Histogram i l l u s t r a t i n g the 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 f o l l o w e d ( a t v a r y i n g 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% ADM ... 31 10 Histogram i l l u s t r a t i n g the 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 f o l l o w e d ( a t v a r y i n g 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 3 9 v i i i i x F i g u r e Page 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 42 14 Experimental design. 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 chromosome a b e r r a t i o n s 50 15 A model f o r DNA r e p a i r 53 16 4 - N i t r o q u i n o l i n e 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 c o c-j r e p a i r enzymes can a c t ' ' 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 q u e s t i o n 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 p r o v i d e 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 t o 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 , un-12 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 , y e t 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 p r e s e n t , 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 u n r e p a i r e d 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 w i t h damaged and/or u n r e p a i r e d 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 t u r n a c q u i r e the p o t e n t i a l to undergo 12 33 n e o p l a s t i c t r a n s f o r m a t i o n ' . 1 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 Pig -mentosum, have an increased sensi t iv i ty to sunlight, resulting in numerous skin tumors in the exposed areas. The ce 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 act iv i ty 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 ce l l surv ival , 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 ce 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 ro , 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 ce l l survival was negligible. Yet cel ls 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 the DNA r e p a i r mechanisms has not been i n v e s t i g a t e d . Perhaps many o f these chemicals a c t 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 . S i n c e 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 to enhance the tumorigenic 79 p o t e n t i a l o f a ca r c i n o g e n , 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 d e t e r m i n a t i o n 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 to make an attempt a t developing such a system. Rather than examining the e f f e c t s o f v a r i o u s combinations o f chemicals i t was decided to i n v e s t i g a t e m u l t i p l e dosages o f one ch e m i c a l . 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 a l r e a d y i n the process o f r e p a i r i n g DNA damage respond to f u r t h e r damage, i . e . as the 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 the r e 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. F i r s t l y , i t s b i o l o g i c a l 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 to 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 6 4 . 4NQ0 a l s o binds to D N A 3 9 ' 4 1 > 4 4 > 4 7 > 7 7 , 74 72 e l i c i t s DNA r e p a i r s y n t h e s i s , and produces chromosome a b e r r a t i o n s . 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 v a r y i n g 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 w e l l 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. C e l l C u l t u r e s a) Media. For these s t u d i e s the c e l l s were maintained i n two types o f 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 : s treptomycin s u l f a t e , 29.6 yg/ml (General B i o c h e m i c a l s ) p e n i c i l l i n G, 204 units/ml (General B i o c h e m i c a l s ) kanamycin, 100 yg/ml ("GIBCO") fung i z o n e , 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% MEM). b) C e l l s . 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 s t u d i e s . Stock c u l t u r e s were maintained i n 15% MEM i n 100 mm p e t r i d i shes ( 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 a t 37°C. 5 6 I I . 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 to use 1.9 mg o f 4NQ0 was d i s -s o l v e d 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 to ensure t h a t the _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 or MEM was added; s e r i a l d i l u t i o n s were then made to o b t a i n the d e s i r e d c o n c e n t r a t i o n s . 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 the 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. For 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 w i t h 1 ml o f the 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 to a s u c t i o n d e v i c e . A f t e r removal o f the chemical the c e l l s were washed twice with 2 mis o f MEM or 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 the p e t r i d i s h e s r e t u r n e d t o the CO2 i n c u b a t o r . I I I . UV Treatment One a s p e c t o f the 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 the 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 the UV l i g h t s o u r c e . 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, I n c . ) . 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 r e d , t o 7 ensure complete removal of 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 re 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. C e l l S u r v i v a l S t u d i e s 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 to 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 to 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 the 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 to 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 d ishes and covered with 2 mis o f 15% MEM. In or d e r to o b t a i n 8 well spread metaphase plates, the ce l ls were used before they reached 80% confluency. The ce l ls were treated twice with 4NQ0 in 5% MEM and allowed to recover between doses in 15% MEM. Once ce 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 ips were then transferred to petri dishes containing 1% sodium citrate solution for 20 minutes. This hypotonic treatment causes the ce l ls to swel l , producing chromosomes that are well spread out and separated. The ce l ls were then fixed with Carnoy's and a i r -dried. 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 Sc ient i f i c Co.). VI. Autoradiography In order to distinguish between DNA repair replication and semiconservative DNA repl icat ion, only nuclei undergoing repair synthesis should become label led. To achieve th i s , ce l ls must be prevented from entering S-phase. This was accomplished by placing the cultures in ADM for 2 1/2 days, at which time approximately 90% of the ce l ls 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 ce l ls 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 or 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 p a s s i n g 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 to attempting any experiments with double 4NQ0 t r e a t -ments, i t was necessary t o determine the time course o f DNA r e p a i r syn-t h e s i s a f t e r a s i n g l e 4NQ0 treatment and to 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 to c e l l s when given i n 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 to 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 or 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 to 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 the f i r s t chemical treatment i n 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 the i n i t i a l damage was s t i l l p roceeding. An experiment was designed to i n v e s t i g a t e the r e p a i r o f 4NQ0-induced 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 -7 72 and 1 x 10 M. These had a l r e a d y been shown by S t i c h and San to 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 s y n t h e s i s . The c e l l s were maintained i n ADM p r i o r to and throughout the e n t i r e experiment. F i g u r e 1 o u t l i n e s the p r o t o c o l t h a t was employed. I l l u s t r a t e d i s a 60 minute 4NQ0 treatment (10~ 7M o r 5 x 10~ 7M), the o t h e r treatment 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 the 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 designated as zero hour ( f o r g r a p h i c a l purposes). 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 the chemical treatment, 3HTdR 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 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 d i d 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 pu 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 t o i n t e r p r e t . 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 the 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 10 12 14 16 18 20 22 24 26 _j i i 1 1 1 1 1 1 1-4NQ0 3 + HTdR . 4NQ0 .HTdR  . 4NQ0 . .  . 4NQ0 .  , 4NQ0 ,  • 4NQ0 ,  • 4NQ0 .  . 4NQ0 . 'HTdR 'HTdR HTdR 'HTdR HTdR , 'HTdR Fig u r e 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 the time when the sample was taken and the uptake o f 3HTdR over a two hour p e r i o d p r i o r to sampling ( r e p r e s e n t e d by g r a i n s per n u c l e u s ) . T 4NQ0 added v" 4NQ0 removed (a) 5 x 10" 7M 4NQ0 (b) 1 x 10" 7M 4NQ0 H O U R S P O S T - T R E A T M E N T 15 hour HTdR pu l s e t h a t f o l l o w s , a high l e v e l o f r e p a i r s y n t h e s i s i s obta i n e d . In f a c t t h i s i s the 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 to a 90 minute 4NQ0 dose t h i s i s not the case, f o r the peak o f r e p a i r i s reached during the chemical treatment, only to 3 drop s l i g h t l y i n the subsequent two hour HTdR p u l s e . T h i s seems to i n d i c a t e t h a t the peak o f r e p a i r occurs i n the second or 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 the 60 minute and the 90 minute 4NQ0 treatments e l i c i t e d the 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 the 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 the 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. 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 Using the previous r e s u l t s as a guide, an attempt was made to a s c e r t a i n the 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 the experiment, an a p p r o p r i a t e endpoint f o r com-p a r i n g the data had to be chosen. S i n c e i t was known t h a t the peak o f r e p a i r o c curred during the 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 to 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 Fi 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~ 7M or 5 x 10" 7M 4NQ0 f o r 60 minutes, and then t r e a t e d again 1 1/2, 3 2, 3, 5, 9 or 13 hours l a t e r . The two hour HTdR pulse immediately f o l l o w e d removal o f the second treatment. In order to determine the 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 recovery p e r i o d . They were exposed t o the i n i t i a l 4NQ0 treatment o n l y , allowed to re c o v e r w h i l s t the remainder r e c e i v e d 3 a second treatment, and then pulsed with HTdR a t the same time as the "d o u b l y - t r e a t e d " sample. The r e s u l t s , which are summarized i n F i g u r e 4, d e p i c t the 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 recovery from a s i n g l e treatment and a f t e r exposure to 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 the assump-t 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 to 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 the f i r s t , no matter when i t i s a p p l i e d . In p r a c t i s e , the 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 the second treatment should be equal t o the 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 covery from the f i r s t treatment plus the 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) the f i r s t column d e p i c t s the 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 o f a s i n g l e 4NQ0 treatment; t h i s value i s then added to the c o n t r o l ( c l e a r column i n each s e t ) to 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 the c o n t r o l v a l u e Time (hrs.) 0 1 2 ,4NQ0 HTdR 5 6 i 8 i 9 10 11 i 12 i 13 i 14 15 .4NQ0  4NQ0 4NQ0 'HTdR HTdR ,4NQ0 .4NQ0 , HTdR ,4NQ0 'HTdR ,4NQ0 ,4NQ0 . HTdR ,4NQ0 'HTdR .4NQ0 .4NQ0 , HTdR .4NQ0 'HTdR .4NQ0 recovery period 4NQ0 . HTdR ,4NQ0 'HTdR Figure 3: Experimental design. Variation of the intervals between s p l i t 4NQ0 treatments; effect on DNA repair synthesis. F i g u r e 4: Histogram i l l u s t r a t i n g the a c t u a l g r a i n s per n u c l e i immediately f o l l o w i n g the f i r s t 4NQ0 treatment o n l y 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 per 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" 7M 4NQ0 (b) 1 x 10" 7M 4NQ0 19 H O U R S B E T W E E N T R E A T M E N T S 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 the f i r s t treatment. The purpose o f the c o n t r o l was to serve as an a i d i n o b t a i n i n g the expected v a l u e . As i l l u s t r a t e d i n the 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 th r e e hours immediately 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 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 the expected. In f a c t t here 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 or above t h a t f o r the f i r s t treatment. At the 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 , the l e v e l o f r e p a i r begins to approach the expected, and a t the lower dosage reaches the expected a t 5 hours post-treatment. However, at the hig h e r c o n c e n t r a t i o n , the expected l e v e l i s never a t t a i n e d ; i n f a c t a t 13 hours the l e v e l i s even f u r t h e r below the expected than i t was a t 5 hours. Such was the case f o r the 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 to assume t h a t once the c e l l s had regained the p o t e n t i a l to r e p a i r the DNA damage i n f l i c t e d by the second treatment, they would c o n t i n u e to do so. 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 the 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 be f o r e i n 5% ADM f o r 2 1/2 days, but 3 hours p r i o r to the f i r s t 4NQ0 treatment, 21 * the c e l l s were r e t u r n e d to 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 , to p r o v i d e the c e l l s with a chance to e q u i l i b r a t e . The r e s t o f the experiment was then c a r r i e d out 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 the ADM blocked c e l l s are pla c e d i n MEM b r i e f l y (3 hours) p r i o r to 4NQ0 treatment, the 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 the 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 the case when the c e l l s were maintained i n ADM throughout the experiment. Furthermore, the second drop i n r e p a i r c a p a c i t y i s not apparent i n the MEM-maintained c e l l s ; a t 12 hours the l e v e l o f DNA r e p a i r f o l l o w i n g the second 4NQ0 treatment i s i d e n t i c a l to the expected. There was no reason to 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 the 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 s y n t h e s i s a s s o c i a t e d w i t h c e l l d i v i s i o n . The corresponding 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). F u r t h e r -25 more Freed and Schatz have shown t h a t a f t e r removal o f a blo c k such as ADM the c e l l s do not en t e r S-phase u n t i l 16 to 20 hours l a t e r . T h i s i s without a 4NQ0 treatment which tends to 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 the 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 to as MEM-maintained c e l l s . F i g u r e 5: Histogram i l l u s t r a t i n g the a c t u a l g r a i n s per 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 g r a i n s 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" 7M 4NQ0 (b) 1 x 10" 7M 4NQ0 23 (a) m o . life 3 3 S 9 13 H O U R S B E T W E E N T R E A T M E N T S IB 3 UI -J (J O 2 d ui a w z < i 0 7 w / i / 1 / 1 • / 1 / 1 a (b) H O U R S B E T W E E N T R E A T M E N T S 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 the time when the sample was taken; and the uptake o f 3nTdR over a two hour p e r i o d p r i o r to sampling ( r e p -resented by g r a i n s per n u c l e u s ) . i n 5% ADM i n 5% MEM (a) 5 x 10" 7M 4NQ0 • o (b) 1 x 10~'M 4NQ0 • • 26 was used w i t h the e x c e p t i o n t h a t only 1 hour treatments were employed, and t h a t the experiment was run i n 5% MEM as we 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 or 5% ADM. The r e s u l t s are 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 to r e p a i r a second s e t o f DNA damage to the expected l e v e l , whereas c e l l s i n MEM can, even though both are capable o f r e -p a i r i n g the 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 are 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 to 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 the same r e s u l t s . For t h i s purpose i t was decided to 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 to 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 to UV (desig n a t e d as zero hour) and the experiment was run i n both 5% ADM and 5% MEM. 27 2 2 Two dosages were used: 40 ergs/mm and 20 ergs/mm f o l l o w e d 3 by two hour HTdR pulses a t 0, 2, 4, 8, 12 and 22 hours post-treatment. The peak o f r e p a i r was found to occur d u r i n g the two hours imme-d 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 the 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 , the 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 the 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 the 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 f o r 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 the 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 the c e l l s were main-t a i n e d i n 5% ADM f o r the e n t i r e experiment; the 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 before and were exposed t o the UV treatment only o r the 4NQ0 treatment o n l y . The r e s u l t s are summarized i n the histograms o f Figures 9 and 10. Note t h a t the f i r s t column i n each graph r e p r e s e n t s the l e v e l o f r e p a i r o btained immediately f o l l o w i n g a s i n g l e one hour 4NQ0 treatment (5 x 10 - 7M or 10~ 7M). T h i s value was used t o c a l c u l a t e the expected l e v e l o f r e p a i r ( 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 the damage induced by a subsequent 4NQ0 dose (no matter when i t i s a p p l i e d ) to a l e v e l s i m i l a r to t h a t a f t e r a s i n g l e 4NQ0 dose. 28 Fig u r e 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 the time when the 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 to sampling ( r e p r e s e n t e d 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 i i i i • • « • • • i i i i • • 4NQ0 . 3HTdR , 3HTdR 4NQ0 . 3HTdR , . 3HTdR , , 4NQ0 . 3HTdR , . 3HTdR , .4NQ0 . 3HTdR , i • 3HTdR , , ; ; ,4NQ0 . 3HTdR , • 3HTdR , . 4NQ0 . 3HTdR 3HTdR Fi 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 s y n t h e s i s . 31 F i g u r e 9: 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 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 g r a i n s per nucleus a f t e r treatment S 9 . Experiment run i n 5% ADM. (a) 5 x 10' 7M 4NQ0 and 40 ergs/mm 2 (b) 1 x 10" 7M 4NQ0 and 20 ergs/mm 2 H O U R S B E T W E E N T R E A T M E N T S 33 F i g u r e 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" 7M 4NQ0 and 40 ergs/mm 2 (b) 1 x 10" 7M 4NQ0 and 20 ergs/mm 2 H O U R S B E T W E E N T R E A T M E N T S 35 I t i s i n t e r e s t i n g t h a t the r e s u l t s are very s i m i l a r to those o f the previous experiments. When the c e l l s are 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 the 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 , the ADM-maintained c e l l s never t o t a l l y r e g a i n the a b i l i t y t o r e p a i r the second 4NQ0 treatment ( a t both c o n c e n t r a t i o n s ) . 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 the f i r s t hour. Again the c e l l s i n MEM are capable o f r e p a i r i n g the second treatment to the 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 the 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 the 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 to 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. T h i s 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 ight toxic i ty in the ce l ls 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 re-f lect a lag in repair synthesis. To c la 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, cel ls 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 ce l ls in ADM never seemed to regain the capacity for repairing DNA damage in f l i c ted by the second treatment, and as a result the lat ter 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 Figure 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 given the double dose. The r e s u l t s are summarized i n Figure 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 are 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 o f 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 i n c u b a t i o n p e r i o d ) . 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 the i n t e r v a l between treatments i n c r e a s e s , the peak of r e p a i r i n c r e a s e s and the o v e r a l l p a t t e r n of 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 given 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 a t 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 given 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. I t seems reasonable to conclude then, t h a t i n the p e r i o d imme-d 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, the a p p l i c a t i o n o f a second 4NQ0 dose does not produce the expected response. E i t h e r t h i s second dose does not induce f u r t h e r damage or i f i t does, f o r some reason DNA damage does not occur. Time (hr s . ) 0 2 4 6 8 10 12 14 16 18 20 .4NQ0 , 4NQ0 3,HTdR • .4NQ0 . 3.HTdR L .4NQ0 . 4NQ0 3HTdR -4NQ0 , 3HTdR i .4NQ0 . .4NQ0 • 3HTdR •4NQ0 . 3HTdR 3HTdR 3HTdR .4N0O . .4N00 .4N00 . 3HTdR .4NQ0 . .4NQ0 .4NQ0 . 3HTdR Fi 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 the f i r s t . Other treatments were a p p l i e d 2, 5 or 9 hours a f t e r the f i r s t and were f o l l o w e d by the same sequence o f ^ HTdR p u l s e s . CO 0 0 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 a p a r t (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 Stu d i e s Iii o r der to thoroughly c h a r a c t e r i z e the 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 to r e p a i r experiments ' . T h e r e f o r e i t seemed l o g i c a l t h a t to complete t h i s p a r t i c u l a r study the 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 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 , or chromo-some a b e r r a t i o n s . On the other hand, i f the 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 to 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 to a s c e r t a i n the 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, be 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 to 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, the c e l l s are seeded i n t o 15% MEM, allowed to s e t t l e down f o r 12-16 hours, exposed to a s i n g l e chemical treatment, and then l e f t to d i v i d e and form c o l o n i e s . The c e l l s do not have an o p p o r t u n i t y to d i v i d e p r i o r to treatment and as a r e s u l t o nly s i n g l e c e l l s a re exposed t o the 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 to leav e the c e l l s f o r p e r i o d s o f up to 12 hours a f t e r exposure to the f i r s t dose Time (hrs.) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 • 4NQ0 j i i i • i 1 « 1 1 1 1 i n t o 15% MEM 4NQ0 . 4NQ0 f .4NQ0 4NQ0 . .4NQ0 4NQ0 . .4NQ0 4NQ0 . .4NQ0 4NQ0 . 4NQ0 .4NQ0 .4N0O , 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 to 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 ccur, 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 only 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 efore completion of 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 ex-periment i n 5% ADM, and then r e t u r n the c e l l s to 15% MEM; or secondly, 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 during 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 par-t i c u l a r s u r v i v a l s t u d i e s . C e l l s l e f t i n 2 1/2% MEM f o r periods 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 others 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 0 2 4 8 12 Average number o f c o l o n i e s 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 c e l l s u r v i v a l 4NQ0 c o n c e n t r a t i o n 5 x 10 - 8M 10 _ 8M 5 x 1 0 " % 10"" h 5 x 1 0 _ 1 % % s u r v i v i n g c o l o n i e s 0 9 68 81 90 Treatment o f the c e l l s f o r 1 hour with a range o f 4NQ0 concen-t r a t i o n s gave the 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 to 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 trea t m e n t ) . 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 to 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 or 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 to 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 or 12 hours, a f t e r which time they were r e t u r n e d to 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 Tables 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 . I t i s i n t e r e s t i n g 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 to 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 e x t r a 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 or 13 hours and then r e t u r n e d to 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 previous DNA r e p a i r experiments i t was necessary to 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 ce 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"9M 5 x 10" 1 0M 0 71 86 2 65 79 4 70 88 8 60 76 12 55 68 Table 4. The effect 2 1/2% MEM, on cel l survival of incubation following treatment with 4NQ0 in % surviving colonies at concentrations Hours in 2 1/2% MEM after treatment 5 x 10"9M 5 x 10" 1 0M 0 76 87 2 67 78 4 70 83 8 58 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 per-centage o f c e l l s to 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 to these s u r v i v i n g c e l l s , the same percentage o f these c e l l s would be expected to s u r v i v e . In other words i f 68% o f the c e l l s s u r -v i v e the f i r s t dose, then 68% o f these 68% should be expected to s u r v i v e the second dose ( i . e . 46%). In c a l c u l a t i n g the expected values a t 9 and 13 hours, the c o n t r o l s f o r those time periods were employed i n or d e r to account f o r any l o s s o f c e l l s due to 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 Table 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 the 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 the 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 the r e s u l t s o f the DNA r e p a i r e x p e r i -ments, i n which the below expected r e p a i r l e v e l s are o b t a i n e d i n the 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 , the 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 , the value a t t a i n e d i s above the expected, though not the same; c e l l s exposed to two doses o f the lower concen-t r a t i o n r i s e t o a hig h e r p o i n t above the 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 the c e l l s t h a t s u r v i v e the 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 the f i r s t treatment nears completion, the 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 are given c l o s e t o g e t h e r , DNA damage i s induced by the 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 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 [4NQ0] Time a f t e r f i r s t dose when second dose a p p l i e d (hours) % s u r v i v i n g c o l o n i e s a f t e r second dose Expected valu e (%) D i f f e r e n c e {%) 5 x 10"9M 1 1/2 h r s . 8 50 -42 2 6 50 -44 3 34 50 -16 5 30 50 -20 9 40 30 +10 13 37 32 +5 5 x 10" 1 0M 1 1/2 h r s . 55 79 -24 2 51 79 -28 3 80 79 +1 5 92 79 +13 9 58 44 +14 13 61 38 +23 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 o c c u r 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 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 r ecovered 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 imme-d 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 g i v e n 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 a t 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 . A f t e r f i v e hours, the values approach normal l e v e l s . 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 -9 -8 -7 -6 -5 -4 -3 -2 -1 0 i i • ; i i i i i 1 i 4NQ0 4NQ0 . 4NQ0 4NQ0 .4NQ0 4NQ0 .4NQ0 4NQ0 .4NQ0 .4NQ0 .4NQ0 Fi g u r e 14: Experimental design. 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 chromo-some a b e r r a t i o n s . O 51 T a b l e 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 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 concen-t r a t i o n s 5 x 10" 7M 10" 7M 1 1/2 h r s . 24 15 2 48 21 3 23 11 5 17 5 9 9 3 S i n g l e dose o n l y 11 3 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 o f the r e s u l t s o b t a i n e d i n t h i s study, i t i s e s s e n t i a l t h a t the u n d e r l y i n g mechanisms o f 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 , un-scheduled r e p a i r s y n t h e s i s ) have been we l l 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 O C C 7 systems ' * 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 o f 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 F i g u r e 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 a t l e a s t 4 enzymes and 5 d e f i n e d a c t i v i t i e s : a) UV i r r a d i a t i o n r e s u l t s i n the formation o f p y r i m i d i n e 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 n i c k 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 a t the n i c k e d s i t e and begins 30 to s y n t h e s i z e new DNA using the o p p o s i t e s i d e o f the DNA as a template . d) An exonuclease c l e a v e s 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 o f 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 3 4 42 may have such an exonuclease a c t i v i t y ' ' . 52 2 5 6 I II I I I I I ! II • • • • • • • • I •••. UV ENOONUCLEASE 111111111 11 n 11111 I I I I I I I I I O ONA POLYMERASE IIIIIIIII rffo*i 11111111 • I I M I I I I I i i i i i i i i i i rn i l i i i i i i in • 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 H I I nil POLYNUCLEOTIDE LIGA5E + DPH 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 integ-r i t y of the DNA. The repair of both UV and 4NQ0-induced DNA damage is postulated to be basically s imi lar ; this fact was further substantiated by the data obtained in the present study. However, the induction of damage is not ident ica l . The structure of 4NQ0 is i l lustrated 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. This distortion is the recognition s i te for the endonuclease. Removal of this distortion proceeds in the same manner as removal of pyrimidine dimers. 0 Figure 16. 4-Nitroquinoline 1-Oxide (4NQ0). II. 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 radiat ion. Interpretation of this data was somewhat simplif ied by employing cel l survival and chromosome studies. The results of these were: 1) a drop in cel l survival and an increase in chromosome aberrations when the interval between doses is less than 3 hours; 2) a r ise in ce 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 . This could have originated from any one of three conditions: 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, conse-quently 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; t h e r e f o r e , 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 t h a t 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 t u r n 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 . T h i s 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 o b t a i n e d 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 f i r s t . 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 o f 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, wi t h an almost t o t a l i n h i b i t i o n o f r e p a i r s y n t h e s i s 1 0 . T h i s was not e v i d e n t i n the present study. Consequently, i n h i b i t i o n o f the r e p a i r enzymes by 4NQ0 can be d i s c o u n t e d . 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 o f 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 ob s e r v a t i o n s d i s a g r e e with such an i n t e r p r e t a t i o n ; i n the f i r s t p l a c e i t i m p l i e s t h a t 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 a t a l a t e r time. The r e s u l t s o f 57 the time course of repair after a second dose do not verify th 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 -d u c e d 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 ce l l in quantities capable of repairing most of DNA damage. Treatment of the ce l ls with cycloheximide for up to 8 hours before exposure to UV did not al ter the repair leve ls ; even though protein synthesis had been almost total ly blocked . Cells l e f t in cycloheximide for 20 hours prior to UV irradiat ion 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 •j o cc of X-ray induced damage ' . 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 art i facts of the technique employed. During the i n i t i a l peak of repair synthesis, there is a l imi 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 d u r i n g the e a r l y stages of r e c o v e r y , 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 s u b j e c t e d to f u r t h e r damage du r i n g t h i s time, an a l k y l a t e d base c o u l d 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 100 n u c l e o t i d e s 11,17,59,61^ T n t h i s m a n n e r j a i o w e r than expected g r a i n count c o u l d ensue. I f a second dose i s given when most o f 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 o b t a i n e d . 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 o f the chromosome and c e l l s u r v i v a l s t u d i e s . Moreover, c u r r e n t r e s e a r c h 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 r e g i o n ' . 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 o f the enzymes themselves has a l r e a d y been di s c o u n t e d , one o f the few remaining s o l u t i o n s i s t h a t 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 . T h i s 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 , d u r i n g 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 o b t a i n e d 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 t h a t i n d u c t i o n o f damage and i n h i b i t i o n o f r e p a i r occur s i m u l t a n e o u s l y upon a p p l i c a t i o n o f 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 o c c u r , f o r as each r e p a i r c y c l e p r o g r e s s e s , many s u s c e p t i b l e s i t e s would be exposed. B i n d i n g o f molecules a t t h i s time c o u l d e a s i l y take p l a c e 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 a t some o t h e r 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 o f DNA i s never r e p a i r e d ; the b i n d i n g would have o c c u r r e d i n such a manner t h a t movement o f enzymes along the segment i s permanently b l o c k e d . 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 o f r e p a i r o f 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 b i n d i n g 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 o f damage. T h i s combined e f f e c t would account f o r the below expected l e v e l s o f r e p a i r . As r e p a i r o f 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 o f damage and consequently r e p a i r s y n t h e s i s would be o b t a i n e d . 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 o f 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 d u r i n g 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 o f the b i n d i n g 60 w i l l have taken place before repair synthesis reaches i ts peak; there-fore only a s l ight inhibit ion 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 p r e v i o u s l y 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 acr i f lavine and ch lo roqu ine 1 0 ' 2 7 which do not induce DNA repair synthesis but considerably inhib i t the repair of UV damage. Acrif lavine preferential ly k i l l s irradiated bacteria, and enhances UV mutagenesis, i fi fifi most l i ke l y by inhibit ing 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, testos-terone, and diethylst i1bestrol . 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 ign i -?fi ?8 f icant effect on DNA repair induced by UV ' . Most of these compounds have been c lass i f ied as co-carcinogens because they have the ab 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 inhibit ing 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 ce l ls 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 o f 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 , i m p l y i n g t h a t the e f f e c t s was due to the l a c k o f 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 o f semi-5 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 . I t i s b e l i e v e d t h a t a c c u r a t e 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 o f 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 i n c u b a t i o n 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 conse-q u e n t l y the t o x i c i t y o f 4NQ0 i s enhanced. Repair o f the f i r s t treatment 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 t o g e t h e r , the response o f 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 o f 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. T h i s phenomenon warrants f u r t h e r i n v e s t i g a t i o n . 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 o f r e p a i r t h a t o c c u r r e d when UV exposure was c l o s e l y f o l l o w e d by a 4NQ0 treatment. T h i s was not e v i d e n t i f the f i r s t treatment i n v o l v e d ex-posure to 4NQ0. 62 Th i s o b s e r v a t i o n 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 are i n v o l v e d i n the r e p a i r o f these two types o f damage are p o s t u l a t e d to be the same ' ' ' , i n i t i a t i o n o f r e p a i r 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 the two s e t s o f data may not be t r u l y comparable. Returning to Fi g u r e 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 a l r e a d y be i n progress h a l f an hour a f t e r removal o f the 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 on l y 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, the 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 are some l i m i t a t i o n s 3 to the 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 to 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 o c c u r r e d , nor was i t p o s s i b l e to d e f i n e the peak o f " s e n s i t i v i t y . " N e v e r t h e l e s s , a f o u n d a t i o n 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 . C h a r a c t e r i z a t i o n o f 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 spacin g the two treatments 15, 30, 45, 60 and 90 minutes a p a r t . A l s o 3 a d d i t i o n o f HTdR si m u l t a n e o u s l y with the second 4NQ0 treatment, would perhaps g i v e 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 d u r i n g the second treatment. 63 III . Spl i t Doses and Cell Survival Since treatment of cel ls 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- irradiat ion on cel l survival has been reported and perhaps can be compared to the effects of s p l i t 4NQ0 treatments on ce l l survival that have been reported in this study. 37 38 Humphrey et a l . using a l ine of Chinese hamster cel ls detected an apparent increase in cytotoxicity ( i . e . decrease in ce 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 ce l l l ine employed. The concomitant r ise in survival after 2 hours was thought to be caused by selection of a radiation-resistant portion of the cel l 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 ine of Chinese hamster c e l l s . A str ik ing 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 to the value expected, as d i d c y t o t o x i c i t y a t 5 hours. Both values reached a s t a b l e expected l e v e l a f t e r 9-12 hours. 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 the 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 of 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 ob t a i n e d i n the f i r s t two hours, and remains c o n s t a n t f o r the next 5 hours, t o 1 19 ?n l e v e l o f f a t a hig h e r value a f t e r 10 hours ' ' . No s i g n i f i c a n t decrease i n c e l l s u r v i v a l was observed. These r e s u l t s are 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 the 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 2 3 ' 5 4 ' 5 6 ' 7 0 , 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 -l i m i t i n g cleavage s t e p , only a small amount of 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 are exposed to 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 to 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 to the r e p a i r of 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 the 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 e v i d e n t . 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 to bases, whereas others produce s i n g l e 7 fx1? s t r a n d breaks ' ; and the types o f DNA r e p a i r processes induced, i t i s very probable t h a t the 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 . F u r t h e r 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 , u n r e p a i r e d 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 the 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 the 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 to 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 to be employed i n s c r e e n i n g f o r ca 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 or d e r t h a t 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 cel ls 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 cel l l i ne . 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 ve 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 lust rated any capacity to induce DNA damage , can be assessed using such techniques. Perhaps their carcinogenicity is due to their a b i l i t y to al ter 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 inves-tigating the interactions of various combinations and sequences of different carcinogens, in v i t ro . 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 object ive of t h i s study was to invest igate the e f fec ts 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 repa i r syn-thesis (as measured by HTdR incorporat ion) , c e l l surv iva l and chromo-some aberrat ions . 2. Following a s ing le one hour treatment with 4NQ0, the peak of repai r synthesis was evident in the second and t h i r d hours a f te r addi t ion 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 wi th in 3 hours of the f i r s t , the level of repai r synthesis induced by t h i s second dose was below a defined expected value. Following 9 hours incubation between treatments, repai r synthesis a f te 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. Cel l surv ival dropped s i g n i f i c a n t l y when the second dose was applied wi th in one hour of the f i r s t . An increase in the frequency of chromosome aberrations was detected when the two treatments were given less than two hours apart . Both values had returned to expected leve ls 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 s e t s o f data i n d i c a t e d t h a t 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 a p p l i e d 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 p r o g r e s s . As r e p a i r o f 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 e x p l a n a t i o n s were d i s c u s s e d and i t was con-cluded 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 o f 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 c a r c i n o g e n was a l s o presented. REFERENCES 1 . A r l e t t , C. F . , and P o t t e r , J . Mutat ion to 8 -azaguan ine r e s i s t a n c e induced 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 . 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