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UBC Theses and Dissertations

DNA repair synthesis in cultured human fibroblasts as a bioassay for chemical carcinogens San, Richard Hing-Cheung 1976

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DNA REPAIR SYNTHESIS IN CULTURED HUMAN FIBROBLASTS AS A BIOASSAY FOR CHEMICAL CARCINOGENS by Richard Hing-Cheung San B. Sc., M c G i l l U n i v e r s i t y , 1968 M. Sc., U n i v e r s i t y of B r i t i s h Columbia, 1972 A Thesis 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 of DOCTOR OF PHILOSOPHY i n GENETICS We accept t h i s t h e s i s as conforming to the re q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1976 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 fo r reference and study. I f u r t h e r agree tha t permiss ion for e x t e n s i v e copying 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 granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . R i c h a r d Hing-Cheung San Department of M e d i c a l G e n e t i c s The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 i ABSTRACT The suggestion from e p i d e m i o l o g i c a l s t u d i e s t h a t 80 to 90 per cent of a l l human cancers may have an e n v i r o n -mental f a c t o r i n i t s e t i o l o g y , coupled w i t h the wide use of chemicals i n a modern s o c i e t y c a l l s f o r a simple, r a p i d and economic prescreening programme to i d e n t i f y chemical carcinogens i n the environment. Measures can then be taken to prevent or e f f e c t i v e l y reduce the exposure of human beings to these agents. The standard "rodent p a i n t i n g and f e e d i n g " t e s t f o r c a r c i n o g e n i c i t y of a chemical compound (endpoint being tumour production) i s u n s u i t a b l e f o r a l a r g e screening programme. The cos t and l o g i s t i c s of handling thousands of r a t s or mice (200 - 500 rodents per chemical) i s sta g g e r i n g . Besides, the completion of t h i s t e s t r e q u i r e s a r e l a t i v e l y l ong time (up to 2 y e a r s ) . Most, i f not a l l , chemical carcinogens bind t o DNA, Furthermore, almost a l l DNA-damaging agents, whether p h y s i c a l or chemical, t h a t have been i n v e s t i g a t e d i n the proper t e s t system show evidence of a r e p a i r e f f e c t . This o b s e r v a t i o n r a i s e s the p o s s i b i l i t y of monitoring carcinogen-induced DNA damage and r e p a i r as a screening procedure f o r i d e n t i f y i n g chemical carcinogens. P r e v i o u s l y , the extent of DNA r e p a i r (autoradiographic d e t e c t i o n of unscheduled -^ HTdR i n c o r p o r a t i o n ) i n hamster and human c e l l s f o l l o w i n g exposure to s t r o n g l y , weakly and non-oncogenic isomers and d e r i v a t i v e s of 4 - n i t r o q u i n o l i n e 1-oxide (4NQ0) was examined. A good c o r r e l a t i o n was observed between the o n c o g e n i c i t y of a compound and the l e v e l of DNA r e p a i r s y n t h e s i s . I n the present study, 64 compounds r e p r e s e n t i n g key groups of carcinogens of d i f f e r e n t molecular s t r u c t u r e s were examined f o r the c a p a c i t y to evoke an unscheduled DNA s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . This i n c l u d e s 29 d i r e c t l y a c t i v e proximate or u l t i m a t e carcinogens, 15 precarcinogens, t h a t r e q u i r e metabolic a c t i v a t i o n , 16 non-oncogenic compounds and 4 chemicals of unknown c a r c i n o g e n i c i t y . A l l d i r e c t l y a c t i n g carcinogens t r i g g e r e d a DNA r e p a i r s y n t h e s i s , whereas no unscheduled ^HTdR i n c o r p o r a t i o n was observed f o l l o w i n g the a p p l i c a t i o n of the 16 non-oncogenic compounds. As a r u l e , the precarcinogens (without metabolic a c t i v a t i o n ) d i d not e l i c i t DNA r e p a i r s y n t h e s i s . However, lo n g e r exposures and higher c o n c e n t r a t i o n s of the precarcinogens 2-acetyiaminofluorene, a f l a t o x i n and s t e r i g m a t o c y s t i n evoked an unscheduled - HTdR uptake. The r e s u l t s suggest the s u i t a b i l i t y of u s i n g DNA r e p a i r s y n t h e s i s as endpoint, and c u l t u r e d human c e l l s as s u b j e c t s i n a prescreening programme f o r chemical carcinogens. As a probe i n t o p o s s i b l e v a r i a t i o n s i n s e n s i t i v i t y w i t h i n the human p o p u l a t i o n towards chemical c a r c i n o g e n s , c e l l s from Xeroderma pigmentosum p a t i e n t s (known t o be d e f i c i e n t i n c o r r e c t i n g UV-induced DNA damage) and normal persons were examined f o r t h e i r DNA r e p a i r c a p a c i t y , frequency of chromosome a b e r r a t i o n s and clone forming e f f i c i e n c y f o l l o w i n g exposure to chemical carcinogens. The XP c e l l s show a c o n s i d e r a b l y reduced DNA r e p a i r s y n t h e s i s when exposed to some but not a l l chemical carcinogens. With chemicals f o r which the XP c e l l s e x h i b i t e d a d e f i c i e n c y i n DNA r e p a i r they a l s o e l i c i t e d a higher frequency of chromosome a b e r r a t i o n s and lower clone forming c a p a c i t y than i n normal persons. The advantages, l i m i t a t i o n s and p o s s i b l e m o d i f i c a t i o n s of the DNA r e p a i r bioassay f o r chemical carcinogens are discussed. ACKNOWLEDGEMENTS I would l i k e to thank Dr. H. F. S t i c h f o r h i s guidance and i n s p i r a t i o n throughout the course of t h i s work. To Mrs. W. S t i c h , I would l i k e to express my si n c e r e g r a t i t u d e f o r her expert t e c h n i c a l a s s i s t a n c e . To Charlee Yoshizawa, I am most g r a t e f u l f o r her superb t i s s u e c u l t u r e work and the frequent t r e a t s of e x c e l l e n t homemade p a s t r i e s . I am g r a t e f u l to past and present members of Dr. S t i c h * s crew, i n p a r t i c u l a r Dorothee K i e s e r , Jim Koropatnick, B r i a n L a i s h e s , P a u l Lam, Peggy Warren and Lan Wei, f o r the coun t l e s s sessions of s t i m u l a t i n g d i s c u s s i o n . To my w i f e , Mimi, I wish to extend my deep a p p r e c i a t i o n of her undying patience a l l these years and i n v a l u a b l e help i n the p r e p a r a t i o n of t h i s manuscript. I a l s o wish to thank Dr. R. L. Noble f o r p r o v i d i n g the f a c i l i t i e s to c a r r y out t h i s research p r o j e c t . Support from the N a t i o n a l Cancer I n s t i t u t e of Canada and the N a t i o n a l Research C o u n c i l of Canada (grants to Dr. S t i c h ) i s g r a t e f u l l y acknowledged. V TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENTS i v TABLE OF CONTENTS v LIST OF FIGURES v i i i LIST OF TABLES x i i ABBREVIATIONS x i i i INTRODUCTION . 1 MATERIALS AND METHODS 9 1. Tissue C u l t u r e Techniques 9 1.1. C u l t u r e Media 9 1.2. C u l t u r e d C e l l s 10 2. U l t r a v i o l e t I r r a d i a t i o n 11 3. Chemicals 12 3.1 . Source of Chemicals 12 3 .2 . S o l u t i o n s of Chemicals 14 4. I n V i t r o A c t i v a t i o n of Precarcinogens . . 15 "4~7l. P r e p a r a t i o n of P o s t - M i t o c h o n d r i a l F r a c t i o n (S9 F r a c t i o n ) of Tissue Homogenates. . . . . . . . . 15 4.2. P r e p a r a t i o n of A c t i v a t i o n Mixture and Treatment of C e l l C u l t u r e s . . 16 5. Measurement of DNA Repair Synthesis (Autoradiography) 17 5.1. P r e p a r a t i o n of C e l l C u l t u r e s and Exposure to Test Compound . . . . 17 5 .2 . R a d i o a c t i v e - L a b e l I n c o r p o r a t i o n . . 18 5.3. Coating w i t h Photographic Emulsion . 19 5.4. P r o c e s s i n g and S t a i n i n g of Autoradiograms 20 5.5. A n a l y s i s of Autoradiograms. . . . 20 6. Chromosome St u d i e s 21 6.1. P r e p a r a t i o n of C e l l C u l t u r e s . . . 21 6.2. Exposure to Test Compound . . . . 21 6 .3 . C y t o l o g i c P r e p a r a t i o n s 22 6.4. A n a l y s i s of Metaphase P l a t e s f o r Ohromosome A b e r r a t i o n s 23 v i Page 7. S u r v i v a l S t u d i e s , „ 23 RESULTS . . . . . 25 1. Unscheduled I n c o r p o r a t i o n of T r i t i a t e d Thymidine pHTdR) i n Mammalian C e l l s as a Measure of Repair of DNA Damage F o l l o w i n g Exposure to Chemical Carcinogens 25 1.1. Dose Response 26 1.2. D u r a t i o n of DNA Repair 37 1.3. DNA Repair I n h i b i t i o n 40 2. DNA Repair D e f i c i e n c y i n Xeroderma Pigmentosum C e l l s 4-5 2.1. Response of XP C e l l s to D i f f e r e n t Carcinogens 46 2.2. V a r i a t i o n i n DNA Repair Capacity. . . 50 2.3. XP wit h Normal DNA Repair C a p a c i t y . . 51 2.4. DNA Repair Capacity i n XP Heterozygotes F o l l o w i n g Exposure to Chemical Carcinogens 54 3. DNA Repair, Chromosome A b e r r a t i o n s and Clone Forming A b i l i t y i n Normal and Xeroderma Pigmentosum C e l l s . . . . . . 55 3.1. Chromosome A b e r r a t i o n s i n Normal and XP C e l l s F o l l o w i n g Exposure to Chemical Carcinogens 56 3.2. Clone-Forming Capacity of Normal and XP C e l l s F o l l o w i n g Exposure to Chemical Carcinogens. . , . . . „ 64 3.3. E f f e c t of Chemical Carcinogens on the Frequency of Chromosome A b e r r a t i o n s and Clone-Forming Capacity i n XP C e l l s w i t h D i f f e r e n t DNA Repair" D e f i c i e n c i e s . 67 3.4. Chromosome A b e r r a t i o n s and Clone-Forming Capacity i n XP Heterozygotes F o l l o w i n g Exposure to Chemical Carcinogens. 73 4. Precarcinogens and Ul t i m a t e Carcinogens . . 77 4.1. P o l y c y c l i c Aromatic Hydrocarbons. . . 79 4.2. Aromatic Amines 80 4.2.1. 2-Acetylaminofluorene. . . . 83 4.2.2. 2-Acetylaminophenanthrene . . 85 4.2.3. 4-Aminostilbene 89 4.2.4. 4-Aminobiphenyl 93 4.2.5. Summary on Aromtic Amines . , 94 4.3. N-Oxides 94 4.4. N i t r o s o Compounds ( A l i p h a t i c Carcinogens) 99 V l l 4 . 5 . E f f e c t of Precarcinogens and Ultimate Carcinogens on Chromosome Ab e r r a t i o n s and Clone-Forming Ca p a c i t y i n C u l t u r e d Human F i b r o b l a s t s 100 5 . Carcinogenic Capacity and DNA Repair L e v e l . 106 6 . Design and T r i a l of a Rapid I n V i t r o Bioassay f o r Chemical Carcinogens. . . . 107 6 . 1 . Concentration of Test Compounds . . 108 6 . 2 . T r i a l of Bioassay 109 6 . 3 . Precarcinogens 109 DISCUSSION 114 1. The Use of Unscheduled DNA Synthesis i n the I d e n t i f i c a t i o n of Chemical Carcinogens. 114 2. The Use of DNA Repair i n the I d e n t i f i c a t i o n of S e n s i t i v e C e l l s . . . . . . . . . 124 3 . DNA Damage, Chromosome A b e r r a t i o n s and Carcinogenesis . . • . . . „ . . . 127 4 . P e r s p e c t i v e s . 129 SUMMARY. c . . . 137 REFERENCES. 140 APPENDICES 157 APPENDIX 1 Cost A n a l y s i s of DNA Repair Bioassay f o r a Chemical Carcinogen „ . . . 157 APPENDIX 2 I n i t i a t i o n of Human F i b r o b l a s t C u l t u r e s from S k i n Punch B i o p s i e s . 160 APPENDIX 3 A r g i n i n e D e f i c i e n t Medium . . . . 162 APPENDIX 4 S t a t i s t i c a l A n a l y s i s of Au t o r a d i o -grams . . . . 164 v i i i LIST OF FIGURES Figur e * Page 1. Unscheduled DNA Synth e s i s of Embryonal Syrian-hamster C e l l s (Maintained i n MEM) Fo l l o w i n g Exposure to 4NQ0 28 2. .'The E f f e c t of A r g i n i n e D e p r i v a t i o n on 4NQ0-Induced DNA Repair Synthesis i n Embryonal Syrian-hamster C e l l s 28 3. Frequency D i s t r i b u t i o n of S y r i a n Hamster C e l l s w i t h Various Amounts of Unscheduled DNA Synthesis , 31 4 . Experimental Design: Unscheduled ^HTdR I n c o r p o r a t i o n i n C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g Exposure to a S i n g l e Dose of a Chemical Carcinogen . 33 5. V a r i a t i o n s i n Concentration of Three C a r c i n o -gens th a t T r i g g e r a DNA Repair Synthesis i n Cu l t u r e d Human F i b r o b l a s t s 33 6. V a r i a t i o n s i n Concentration of Three Carcinogens t h a t A f f e c t the Clone-Forming Capacity of C u l t u r e d Human F i b r o b l a s t s . . . . 33 7. D i f f e r e n t L e v e l s of Unscheduled DNA Synth e s i s i n C u l t u r e d Human F i b r o b l a s t s E l i c i t e d by Carcinogens of D i f f e r e n t Molecular S t r u c t u r e s . 35 8. Experimental Design, D u r a t i o n of Unscheduled DNA Synthesis Induced i n Cu l t u r e d Human F i b r o b l a s t s by a S i n g l e Dose of UV R a d i a t i o n or Chemical Carcinogen . . . . . . . . . 39 9.-11. D u r a t i o n of Unscheduled DNA Synthesis i n Cu l t u r e d Human F i b r o b l a s t s F o l l o w i n g Exposure to a S i n g l e Dose of 4NQ0, MNNG, or UV R a d i a t i o n . 39 12. Time Course of DNA Repair Synthesis i n C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g Short Term Exposure to 4NQ0 and Measured as Unscheduled Incorpora-t i o n of ^ HTdR (Autoradiography) or as a S h i f t i n the Sedimentation Rate i n an A l k a l i n e Sucrose Gradient 39 13. Experimental Design. I n h i b i t i o n of DNA Repair Synthesis i n C u l t u r e d Human F i b r o b l a s t s Follow-i n g the Combined A p p l i c a t i o n of UV R a d i a t i o n and a Chemical Carcinogen . . . . . . . . 4-3 i x F i gure Page 14.-16. I n h i b i t i o n of DNA Repair Synthesis i n Cultur e d Human F i b r o b l a s t s F o l l o w i n g the Combined A p p l i c a t i o n of UV R a d i a t i o n and a Chemical Carcinogen 43 17.-18. E f f e c t of a Combined Treatment of UV R a d i a t i o n and a Chemical Carcinogen on the Clone-Forming Capacity of C u l t u r e d Human F i b r o b l a s t s * UV + N-Acetoxy-4-AAS ( F i g . 1 7 ) , UV + 4NQ0 ( F i g . 18) 44 19.-24. Unscheduled ^ HTdR I n c o r p o r a t i o n i n C u l t u r e d F i b r o b l a s t s of Xeroderma Pigmentosum P a t i e n t s and C o n t r o l Persons F o l l o w i n g Short Term Exposures to Carcinogens and Mutagens: 4NQ0 ( F i g . 19), MNNG ( F i g . 20), N-Acetoxy-4-AAS ( F i g . 21), MMS ( F i g . 22), MCA-6,7-Epoxide ( F i g . 23) and NMU ( F i g . 24) . 48 25. Unscheduled I n c o r p o r a t i o n of 3HTdR i n t o N u c l e i of Normal and Four XP F i b r o b l a s t s Exposed f o r 5 Hours to N-Hydroxy-AAF. Autoradiography . . 53 26.-29. Types of Chromosome A b e r r a t i o n s i n XP F i b r o b l a s t s F o l l o w i n g Exposure to 4NQ0 . . . 57 30.-35. Frequency of Metaphase P l a t e s w i t h Chromosome Ab e r r a t i o n s i n XP and Normal C e l l C u l t u r e s F o l l o w i n g a S i n g l e Dose of a Carcinogen . . . 59 36. Frequency of Metaphase P l a t e s w i t h Chromosome Ab e r r a t i o n s i n XP and Normal F i b r o b l a s t s C u l t u r e s a t Various Times F o l l o w i n g a S i n g l e Dose of 4NQ0 62 37. Frequency of Metaphase P l a t e s with Chromosome Ab e r r a t i o n s i n XP and Normal F i b r o b l a s t C u l t u r e s at Various Times F o l l o w i n g a S i n g l e Dose of BA Epoxide 62 38. E f f e c t of V a r i a t i o n i n Exposure Time to a Chemical Carcinogen on the Frequency of Metaphase P l a t e s w i t h Chromosome A b e r r a t i o n s i n XP and Normal F i b r o b l a s t C u l t u r e s . . . . 63 39. Frequency of Metaphase P l a t e s with Chromosome Abe r r a t i o n s i n C u l t u r e d XP and Normal F i b r o b l a s t s Exposed to Various Concentrations of MNNG . . 66 40. Frequency of Metaphase P l a t e s w i t h Chromosome Ab e r r a t i o n s i n C u l t u r e d XP and Normal F i b r o b l a s t s at Various Times F o l l o w i n g a S i n g l e Dose of MNNG 66 Figure Page 41.-46. Clone-Forming Capacity of Normal and XP F i b r o b l a s t s F o l l o w i n g Exposure to a S i n g l e Dose of a Carcinogen 69 47.-52. Clone-Forming Capacity of Normal and XP F i b r o b l a s t s F o l l o w i n g Exposure t o a S i n g l e Dose of a Carcinogen ^ . . . 71 53. S t r u c t u r a l Formula of the Precarcinogens BA and 20-Methylcholanthrene, the H i g h l y Reactive K-region Epoxide (Proximate Carcinogen) and the Corresponding D i h y d r o d i o l ( I n a c t i v e M e t a b o l i t e ) 82 54. Unscheduled DNA Synth e s i s i n Normal Human F i b r o b l a s t s Exposed to BA, BA-5»6-epoxide or BA-cis -5»6-dihydrodiol . . . . . . . 82 55. Unscheduled DNA Synthesis i n Normal Human F i b r o b l a s t s Exposed to 20-Methylcholanthrene, MCA-6,7-epoxide or MCA-6,7-Dihydrodiol . . . 82 56. S t r u c t u r e of the Carcinogenic Aromatic Amines« 2-Acetylaminofluorene (2-AAF) 2-Acetylaminophenanthrene (2-AAP) 4-Acetylaminobiphenyl (4-AABP) 4-Acetylaminostilbene (4-AAS) . . . . . . 84 57. Unscheduled I n c o r p o r a t i o n of 3HTdR i n t o N u c l e i of Normal Human F i b r o b l a s t s Exposed to the Precarcinogen 2-AAF, Proximate Carcinogen - N-Hydroxy-2-AAF or Ultimat e Carcinogen -N-Ace toxy-2-AAF. . 87 58.-60. Unscheduled I n c o r p o r a t i o n of HTdR i n t o N u c l e i of Normal Human F i b r o b l a s t s Exposed to the Pre c a r c i n o g e n i c , Proximate or Ultimate Carcinogenic Forms of the Aromatic Aminest 2-Acetylaminophenanthrene, 4-Acetylaminobiphenyl and 4-Acetylaminostilbene . . . . . . . 91 61. Unscheduled DNA Synthesis i n C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g 1.5-hr. Exposure to 4NQ0, 4HAQ0 or 3-Fluoro-4NQ0 98 62.-65. Frequency of Metaphase P l a t e s with Chromosome Ab e r r a t i o n s i n C u l t u r e d F i b r o b l a s t s from Xeroderma Pigmentosum P a t i e n t s F o l l o w i n g Short-Term Exposure to Precarcinogens, Proximate or Ultimate Carcinogens 102 x i F i gure Page 66.-69. Clone-Forming Capacity of C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g Short-Term Exposure to Precarcinogens, Proximate or Ultimate Carcinogens 105 7 0 . I n h i b i t i o n of DNA Repair i n Cu l t u r e d Human F i b r o b l a s t s E l i c i t e d by High Concentrations of a Chemical Carcinogen . . . . . . . 110 7 1 . - 7 2 . Unscheduled DNA Synthesis i n C u l t u r e d Human F i b r o b l a s t s Evoked by the Precarcinogens, A f l a t o x i n B i ( F i g . 71) and S t e r i g m a t o c y s t i n ( F i g . 72) . . . . . . . . . . . . 113 7 3 . - 7 4 . DNA Repair Synthesis of C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g Exposure to a Photo-s e n s i t i z i n g Chemical and I r r a d i a t i o n by long wavelength (355nm) UV . . . . . . . . 133 X l l LIST OF TABLES Table Page I E f f e c t of Exposure Time to a Chemical Carcinogen on the Le v e l of Unscheduled 3HTdR I n c o r p o r a t i o n i n C u l t u r e d Human F i b r o b l a s t s 36 I I Unscheduled DNA Synthesis of C u l t u r e d Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to Chemical Carcinogens 4-9 I I I Comparative L e v e l s of DNA Repair Synthesis i n C u l t u r e d Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to UV and a S e l e c t e d Group of Chemical Carcinogens 52 3 IV L e v e l of Unscheduled ^HTdR I n c o r p o r a t i o n , Frequency of Chromosome A b e r r a t i o n s and Clone-Forming C a p a c i t y of C u l t u r e d Homozygous and Heterozygous Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to 4NQ0 7^ 3 V L e v e l of Unscheduled ^HTdR I n c o r p o r a t i o n , Frequency of Chromosome Ab e r r a t i o n s and Clone-Forming C a p a c i t y of C u l t u r e d Homozygous and Heterozygous Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to N-Acetoxy-2-AAF . . . 75 VI L e v e l of Unscheduled ^HTdR I n c o r p o r a t i o n , Frequency of Chromosome A b e r r a t i o n s and Clone-Forming Capacity of C u l t u r e d Homozygous and Heterozygous Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to MNNG . . . . . . . 76 V I I Unscheduled DNA Synthesis i n C u l t u r e d Human F i b r o b l a s t s F o l l o w i n g Exposure to A c t i v e and and I n a c t i v e M e t a b o l i t e s of 2-AAF. . . . . 88 V I I I DNA Repair Synthesis (Unscheduled I n c o r p o r a t i o n of 3HTdR) of C u l t u r e d Human F i b r o b l a s t s Exposed to Precarcinogens, Carcinogens, Non-carcinogenic D e r i v a t i v e s and Mutagens , 111 IX The A p p l i c a t i o n of the DNA Repair Assay to Carcinogens, Precarcinogens and Non-carcinogens 122 • « • X l l l ABBREVIATIONS Chemical Carcinogens and Mutagens 4-AABP 4-Acetylaminobiphenyl 2-AAF 2-Acetylaminofluorene 2-AAP 2-Acetylaminophenanthrene 4-AAS 4-Acetylaminostilbene 4AQ0 M" Aminoquinoline 1-oxide BA Benz(a)anthracene DMN Dimethylnitrosamine EMS Ethylmethanesulfonate FANFT N - / _ 4 - ( 5 - N i t r o - 2 - f u r y l ) - 2 - t h i a z o l y l 7 formamide 4HAQ0 4-Hydroxyaminoquinoline 1-oxide HN2 Nitrogen Mustard ICR-191 I n s t i t u t e f o r Cancer Research ( P h i l a d e l p h i a ) , Compound #191 MCA 2 0-Methylcholanthrene 2-Me-4NQ0 2-Methyl-4NQ0 7-Me-4NQ0 7-Methyl-4NQ0 MMS Methylmethanesulfonate MNNG N-Methyl-N * - n i t r o - N - n i t r o s o g u a n i d i n e NMU Nitrosomethylurea 4-NQO 4 - N i t r o q u i n o l i n e 1-oxide 6NQ0 6 - N i t r o q u i n o l i n e 1-oxide Miscellaneous Terms ADM A r g i n i n e D e f i c i e n t Medium ASG A l k a l i n e Sucrose Gradient FCS F e t a l C a l f Serum G6P Glucose-6-phosphate ^HTdR Tr i t i a t e d ^ T h y m i d i n e (Thymidine-methyl-^H) MEM Eagle's Minimal E s s e n t i a l Medium NADP Nicotinamide Adenine D i n u c l e o t i d e Phosphate (Oxidized Form) NADPH Nicotinamide Adenine D i n u c l e o t i d e Phosphate (Reduced Form) PBS Dulbecco's Phosphate Bu f f e r e d S a l i n e PBS/Sucrose 0 . 2 5 M Sucrose i n Phosphate Bu f f e r e d S a l i n e , pH 7 . 5 S9 P o s t - m i t o c h o n d r i a l ( 9 , 0 0 0 x g) Supernatant, "Crude Microsomes" UV U l t r a v i o l e t X P Xeroderma Pigmentosum X P Q Xeroderma Pigmentosum, Calgary P a t i e n t XPg Xeroderma Pigmentosum, Edmonton P a t i e n t X P H Xeroderma Pigmentosum,Hamilton P a t i e n t XPft Xeroderma Pigmentosum, Kamloops P a t i e n t XPy Xeroderma Pigmentosum, Vancouver P a t i e n t INTRODUCTION 1 The i d e a t h a t chemicals i n man's environment can induce cancers i s not a new one. The high frequency of s c r o t a l s k i n cancer among chimney sweeps i n England, as observed by P e r c i v a l P o t t (1775). was b e l i e v e d to be a s s o c i a t e d w i t h the heavy exposure to soot i n t h a t occupation. S t u d i e s by Rehn i n 1895 (Clayson, 1962) on the e l e v a t e d r i s k of workers i n the a n i l i n e dye i n d u s t r y to develop u r i n a r y bladder tumours r e i n f o r c e d the b e l i e f t h a t chemicals c o u l d a c t as carcinogens. As a r e s u l t of these and many subsequent s t u d i e s , many pure compounds and mixtures have been i d e n t i f i e d as carcinogens f o r man ( M i l l e r , 1970). I t i s d i f f i c u l t t o a s c e r t a i n what p r o p o r t i o n of malignant diseases i n human beings develop as a consequence of exposure to chemicals. However, e p i d e m i o l o g i c a l data suggest t h a t 80 to 90 per cent of a l l human cancers may be environmentally induced (Higginson, 1969. 1971). P h y s i c a l , v i r a l or chemical agents (or a combination of two or more of these) are i n v a r i a b l y i n v o l v e d as an e t i o l o g i c f a c t o r i n c a r c i n o g e n e s i s . C o n s i d e r i n g the wide use of chemicals i n a modern s o c i e t y , (e.g. food a d d i t i v e s , drugs, cosmetic products, p e s t i c i d e s ) , i t i s conceivable t h a t an u n c o n t r o l l e d i n t r o d u c t i o n of chemicals i n t o man's environment does pose a s e r i o u s h e a l t h hazard. A major iss u e now i s to i d e n t i f y carcinogens i n the environment, and prevent or e f f e c t i v e l y reduce the exposure of human 2 beings to these agents. The conventional method of t e s t i n g a chemical compound f o r c a r c i n o g e n i c a c t i v i t y i s conducted i n rodents. One advantage of t h i s t e s t system i s t h a t the endpoint - the development of tumour - i s h i g h l y r e l e v a n t . However, since the u s u a l number of t e s t animals employed i n t e s t i n g one compound i s about 200 to 5°0, weak carcinogens or precarcinogens t h a t may induce one tumour i n 1,000 to 10,000 animals -tumour f r e q u e n c i e s which are t o t a l l y unacceptable f o r man -c o u l d be e a s i l y missed. Such a compound would be considered non-carcinogenic, and t h e r e f o r e a " s a f e " dose f o r t h i s compound could be proposed. The i s s u e of c a l c u l a t i n g " s a f e " l e v e l s i n c a r c i n o g e n i c i t y t e s t s have been adequately discussed by Mantel et a l . (196l, 1975a, 1975b). Another problem b e s e t t i n g the standard "rodent p a i n t i n g and f e e d i n g " t e s t s i s the p r o h i b i t i v e c o s t i n c u r r e d - up to $150,000 f o r t e s t i n g a s i n g l e compound on 200 mice and 200 r a t s (Weisburger, personal communication). I t i s estimated t h a t 1,000 new man-made compounds are p l a c e d on the market a n n u a l l y . This does not i n c l u d e the multitude of n a t u r a l l y - o c c u r r i n g chemicals t h a t enter man's immediate environment. The c o s t of t e s t i n g these compounds f o r c a r c i n o g e n i c i t y w i l l be a s t r o n o m i c a l . Furthermore, these t e s t s r e q u i r e a r e l a t i v e l y long time (up to 2 years) to complete. The h a n d l i n g and screening of m i l l i o n s of mice or r a t s would be a herculean task. There i s thus an urgent need f o r the development of simple, r a p i d and 3 economic bioassays f o r chemical carcinogens. Most, i f not a l l , chemical carcinogens b i n d to DNA, but i t i s not proven whether DNA i s the prime t a r g e t f o r c a r c i n o g e n i c a c t i o n . I n those cases which have been adequately s t u d i e d , chemical carcinogens have been found to bind c o v a l e n t l y w i t h DNA, RNA and p r o t e i n i n the t a r g e t t i s s u e s ( M i l l e r and M i l l e r , 1966 j Matsushima and Weisburger, 1 9 6 9 ; Colburn and B o u t w e l l , 1968} Brookes, 1 9 7 1 ; Grover e t a l . , 1 9 7 1 a ; Jungmann and Schweppe; , 1 9 7 2 ) . The r o l e of RNA and p r o t e i n s i n c a r c i n o g e n e s i s cannot be dismissed. Furthermore, s i n c e most, i f not a l l , carcinogens are a l s o mutagens and DNA i s w e l l accepted t o be the t a r g e t f o r mutagenic a c t i o n , i t i s reasonable to assume (though i t i s by no means proven) t h a t DNA i s the t a r g e t molecule f o r c a r c i n o g e n i c a c t i o n ( M a i l i n g and de S e r r e s , 1969? Cookson e t a l . , 1971; M i l l e r and M i l l e r , 1971as Ames et a l . , 1972j B r u s i c k , 1972} Fahmy and Fahmy, 1 9 7 2 a , 1 9 7 2 b ; Ong and de S e r r e s , 1 9 7 2 ; Matter e t a l . , 1 9 7 2 ; Trosko and Chu, 1 9 7 5 ) . With most chemical DNA-daraaging agents, the nature of the primary l e s i o n i s obscure. However, almost a l l DNA-damaging agents, whether p h y s i c a l or chemical, t h a t have been i n v e s t i g a t e d i n the proper t e s t system show evidence of a r e p a i r e f f e c t (Hahn e t a l . , 1 9 6 8 ; B a l l and Roberts, 1 9 7 0 ; Horikawa e t a l . , 1 9 7 2 ; S t i c h and San, 1 9 7 0 , 1 9 7 3 ; Fox and Ayad,' 1 9 7 1 ; E l k i n d and Chang-Liu, 1 9 7 2 ; Goodman and P o t t e r , 1 9 7 2 ; Lieberman and D i p p l e , 1 9 7 2 ; S t i c h e t a l , , 1 9 7 2 b ; Laishes and S t i c h , 1 9 7 3 ; Hennings e t a l , , 1 9 7 4 ; Regan and Setlow, 1 9 7 4 ; S t i c h and Laishes, 1975). This observation r a i s e s the question whether the monitoring of carcinogen-induced DNA damage and r e p a i r could be adapted as a screening procedure f o r carcinogenic compounds (see Appendix 1 f o r cost a n a l y s i s ) . There are several methods by which the r e p a i r of carcinogen-induced DNA damage can be monitored*-1. One approach to estimate DNA repair i s to measure the rate of disappearance of the a l t e r e d nucleotides, e.g. thymine dimers following UV-irradiation (Setlow and C a r r i e r , 1964; Regan et a l , , 1968); alkylated DNA bases following exposure to an a l k y l a t i n g agent (Crathorn and Roberts, 1966; Lieberman and Dipple, 1972). This procedure usually requires the i s o l a t i o n of DNA, followed by hydrolysis and chromatographic separation of the abnormal nucleotides. This technique reveals the type of molecular changes induced by a carcinogen, but i t i s too cumbersome to be used on a large scale. 2. Another s e n s i t i v e method to monitor DNA r e p a i r i s based on the observation that following X - i r r a d i a t i o n new groups (e.g. phosphate) become exposed and are susceptible to enzymatic attack upon breakage of a DNA strand (Chu and Mailing, 1968). The more single-strand breaks that occur i n a given DNA sample, the more exposed ends e x i s t . The rate of disappearance of the exposed DNA end-groups, which can be measured at various times following exposure to a carcinogen, w i l l reveal the rate; of DNA r e p a i r . This sophisticated method remains to be exploited before 5 i t can be a p p l i e d i n a pre-screening programme. 3 . Sedimentation of DNA through a l k a l i n e sucrose g r a d i e n t s provides a d i r e c t measure of s i n g l e - s t r a n d e d DNA breakage. O r i g i n a l l y designed by McGrath and W i l l i a m s ( 1 9 6 6 ) , t h i s technique i n v o l v e s l y s i n g i n t a c t c e l l s d i r e c t l y on top of an a l k a l i n e sucrose g r a d i e n t , whereby mechanical shearing of DNA i s kept to a minimum. The i d e a i s based on the assumption t h a t DNA w i t h carcinogen-induced s i n g l e - s t r a n d breaks w i l l not sediment as f a s t as i n t a c t DNA strands and th a t the r e t u r n of the broken DNA strands t o the o r i g i n a l molecular weight f o l l o w i n g a carcinogen a p p l i c a t i o n i s a measure of DNA r e p a i r . This technique has been s u c c e s s f u l l y employed i n measuring DNA damage and r e p a i r i n v i v o and i n v i t r o (Cox et, a l . , 1973? Horikawa et a l . , 1972} Laishes and S t i c h , 1 9 7 3 ; L a i s h e s , 1 9 7 * 0 . k. The most wid e l y used methods of e s t i m a t i n g DNA r e p a i r s y n t h e s i s i n v o l v e the ' r e s y n t h e s i s ' of short s e c t i o n s of the DNA molecule which were e l i m i n a t e d by endo- and exo-nuclease enzymes f o l l o w i n g exposure to exogenous DNA damaging agents (Rasmussen and P a i n t e r , 1 9 6 6 ) . The i n -c o r p o r a t i o n of 5-hromo-deoxyuridine (BrdU) i n t o n u c l e a r DNA of ca r c i n o g e n - t r e a t e d c e l l s f o l l o w e d by buoyant d e n s i t y c e n t r i f u g a t i o n through a cesium c h l o r i d e g r a d i e n t provides an elegant means to d i s t i n g u i s h DNA r e p l i c a t i o n from DNA r e p a i r (Cleaver, 1 9 7 0 , 1973 ). However, t h i s procedure i s not a p p l i c a b l e as a r a p i d a n a l y t i c a l method to screen l a r g e numbers of c a r c i n o g e n i c agents. Incorporation of BrdU into DNA regions undergoing r e p a i r coupled with long-wavelength u l t r a v i o l e t r a d i a t i o n produces a photosensitizing e f f e c t which can be used to estimate the extent of DNA r e p a i r (Regan et a l . , 1971? Kimble et a l . , 1974). Cultured mammalian c e l l s are treated with a chemical carcinogen, allowed to undergo re p a i r r e p l i c a t i o n i n the presence of BrdU and thereafter exposed to a pulse of 313 nm l i g h t . Repair-replicated regions containing BrdU are rendered a l k a l i n e - l a b i l e by the i r r a d i a t i o n . The extent of DNA breakage and r e p a i r i s deduced by the , degree of s h i f t i n the DNA peak following c e n t r i f u g a t i o n i n an a l k a l i n e sucrose gradient. Only recently introduced, t h i s approach requires further evaluation before i t can be considered i n a large-scale prescreening programme. The technique of choice appears to be the autoradio-graphic detection of radioactive precursors applied during the period of DNA r e p a i r synthesis. By exposing non-p r o l i f e r a t i n g c e l l s to exogenous phy s i c a l (Cleaver, 1970) or chemical agents (Cleaver, 1973 l S t i c h and San, 1970, 1973; S t i c h et a l . , 1971, 1972a, 1972b) and then allowing them to engage i n DNA r e p a i r synthesis i n the presence of ^HTdR, a measure of unscheduled r e p a i r synthesis can be r e a d i l y obtained. Since hundreds of autoradiographic preparations can be handled simultaneously, and the evaluation of the preparations can be semi-me-chanized, t h i s procedure can be applied i n large-scale screening tests (Bootsma et a l . , 1970). 7 The i d e a of u s i n g DNA r e p a i r s y n t h e s i s of mammalian c e l l s as an i n d i c a t o r f o r a c a r c i n o g e n i c a c t i v i t y o f a compound i s based on the assumption t h a t c h e m i c a l c a r c i n o g e n s i n t e r a c t w i t h DNA and t h a t the ensuing DNA changes w i l l r e s u l t i n a DNA r e p a i r s y n t h e s i s . The working h y p o t h e s i s was p r e v i o u s l y examined by comparing the ex t e n t of DNA r e p a i r i n hamster and human c e l l s f o l l o w i n g exposure t o s t r o n g l y , weakly and non-oncogenic isomers and d e r i v a t i v e s of 4 - n i t r o q u i n o l i n e 1-oxide (4NQ0) ( S t i c h e t a l . , 1971). A good c o r r e l a t i o n was ob t a i n e d between the o n c o g e n i c i t y o f a compound and the l e v e l o f DNA r e p a i r s y n t h e s i s . C e l l s respond t o h i g h l y and weakly oncogenic d e r i v a t i v e s o f 4-NQO wi t h a h i g h and low l e v e l o f unscheduled DNA s y n t h e s i s r e s p e c t i v e l y . The non-oncogenic 4NQ0 d e r i v a t i v e s l a c k the c a p a c i t y t o evoke a d e t e c t a b l e DNA r e p a i r s y n t h e s i s . One o f the o b j e c t i v e s o f t h i s t h e s i s was t o ev a l u a t e the f e a s i b i l i t y o f u s i n g DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s as a b i o a s s a y f o r c h e m i c a l c a r c i n o g e n s . An attempt i s made t o i n v e s t i g a t e the f o l l o w i n g p r o b l e m s i -1. Whether the c o r r e l a t i o n between the c a r c i n o g e n i c i t y o f a ch e m i c a l compound and the i n d u c t i o n o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s can be e s t a b l i s h e d f o r c h e m i c a l s o f d i f f e r e n t m o l e c u l a r s t r u c t u r e s . 2 . Whether the p r e c a r c i n o g e n i c , proximate and/or u l t i m a t e c a r c i n o g e n i c forms o f a ch e m i c a l c a r c i n o g e n v a r y i n t h e i r c a p a c i t y t o e l i c i t unscheduled DNA s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . 8 A second objective i n the present study concerned the possible v a r i a t i o n i n s e n s i t i v i t y within the human population towards chemical carcinogens. Such differences must be considered when deciding on "permissible" or "safe" l e v e l s of a chemical compound. To i l l u s t r a t e whether differences i n s u s c e p t i b i l i t y to chemical carcinogens do ex i s t i n man, c e l l s from Xeroderma pigmentosum patients (who are d e f i c i e n t i n r e p a i r i n g UV-induced DNA damage) and normal persons are compared with respect to t h e i r DNA r e p a i r capacity, frequency of chromosome aberrations and clone forming e f f i c i e n c y following exposure to a chemical carcinogen. MATERIALS AND METHODS 9 1. Tissue C u l t u r e Techniques 1.1. C u l t u r e Media -Eagle's Minimal E s s e n t i a l Medium (MEM). Eagle's minimal e s s e n t i a l medium was purchased i n powder form from Grand I s l a n d B i o l o g i c a l Company, Berkeley, C a l i f o r n i a . The powder was r e c o n s t i t u t e d w i t h d i s t i l l e d water and s t e r i l i s e d by passage through a m i l l i p o r e f i l t e r (pore s i z e : 0.22 microns; M i l l i p o r e F i l t e r C o r p o r a t i o n , Mass., U.S.A.). A r g i n i n e D e f i c i e n t Medium (ADM). A r g i n i n e d e f i c i e n t medium was prepared according to the standard formula f o r Eagle's minimal e s s e n t i a l medium (Eagle, 1959} Merchant et a l . , 1964). The v a r i o u s e s s e n t i a l amino a c i d s , w i t h the e x c e p t i o n of a r g i n i n e , were weighed out and d i s s o l v e d i n the manner as d i r e c t e d (see Appendix 3). The n o n - e s s e n t i a l amino a c i d s ( i n the form of lOOx concentrated m i x t u r e ) , as w e l l as the v i t a m i n s , were obtained from Flow L a b o r a t o r i e s , Inc. (Inglewood, C a l i f o r n i a ) and added to the c u l t u r e medium. A n t i b i o t i c s . C u l t u r e media were r o u t i n e l y supplemented w i t h the f o l l o w i n g a n t i b i o t i c s J P e n i c i l l i n G (General Biochemicals, Chagrin F a l l s , Ohio} 204 u n i t s / m l . , f i n a l c o n c e n t r a t i o n ) , Streptomycin s u l f a t e (General B i o c h e m i c a l s j 29.6 jag/ml.), Kanamycin (Grand I s l a n d B i o l o g i c a l Co.; 100 ug/ml.), and Fungizone (Grand I s l a n d B i o l o g i c a l Co.; 2.5 ^g/ml.). Sodium Bicarbonate. A 7>5% s o l u t i o n , s t e r i l i s e d by f i l t r a t i o n , was prepared as a standard stock. For c u l t u r e s maintained i n Leighton tubes, the c u l t u r e medium was supple-mented w i t h 4 ml. of the 7-5% stock s o l u t i o n ( f o r 800 ml. of c u l t u r e medium). I n the case of p e t r i p l a t e c u l t u r e s , 16 ml. of sodium bicarbonate was added to 800 ml. of medium. F e t a l C a l f Serum (FCS). F e t a l c a l f serum was purchased from Grand I s l a n d B i o l o g i c a l Company and s t o r e d a t -20°C. I t was i n a c t i v a t e d by h e a t i n g at 56°C f o r 3 0 minutes before use. MEM was supplemented w i t h 10% FCS whereas ADM was supplemented w i t h only 5% FCS. 1.2. C u l t u r e d C e l l s Embryonal Hamster C e l l s . Primary c u l t u r e s of 12-day-o l d Syrian-hamster embryos were i n i t i a t e d as des c r i b e d by Cooper (1968). D i p l o i d embryonal c e l l s from the f i r s t t o f o u r t h passage were used i n the present i n v e s t i g a t i o n . Human S k i n F i b r o b l a s t s . Human s k i n f i b r o b l a s t s were grown from e x p l a n t s of s k i n punch b i o p s i e s from Xeroderma pigmentosum p a t i e n t s (age: 11-22) and normal Caucasian donors (age: 18-24):-X P C 1 (male, 12 years o l d , J.T., Calgary) X P C 2 (female, 11 years o l d , M.T., Calgary) XP E (female, 18 years o l d , N/A, Edmonton) XP^^ (female, 20 years o l d , N/A, Hamilton) X PH2 ( m a l e» 2 1 years o l d , N/A, Hamilton) XP K (male, 22 years o l d , C.F., Kamloops) XP V (female, 21 years o l d , D.S., Vancouver) F i b r o b l a s t c u l t u r e s of X P H 1 and X P H 2 were kindly-provided by Drs. L. Skarsgard ( U n i v e r s i t y of B r i t i s h Columbia, Canada) and S. Mak (McMaster U n i v e r s i t y , Canada). C u l t u r e s o f f i b r o b l a s t s from the other XP p a t i e n t s were i n i t i a t e d i n t h i s l a b o r a t o r y . p A 3 to k mm. skin-punch biopsy was taken from the forearm of the donor, teased i n t o t i n y fragments, sandwiched between two cover g l a s s e s and incubated i n MEM (15% f e t a l c a l f serum) f o r 2 - 3 weeks at 37°C i n a C0 2 i n c u b a t o r . Growth medium was changed a t weekly i n t e r v a l s . When f i b r o b l a s t s migrated from the t i s s u e fragments, the "sandwich was opened and the t i s s u e fragments were removed, l e a v i n g a p a r t i a l monolayer of f i b r o b l a s t s on the cover g l a s s e s . Upon f u r t h e r i n c u b a t i o n , the f i b r o b l a s t s on i n d i v i d u a l cover g l a s s were allowed to grow up i n t o a monolayer which c o u l d then be s u b c u l t u r e d by standard techniques. A d e t a i l e d d e s c r i p t i o n of the biopsy and explant c u l t u r e techniques are g i v e n i n Appendix 2. Stock C u l t u r e s . Stock c u l t u r e s were grown i n mono-l a y e r s i n 10 cm. diameter p e t r i p l a t e s (Falcon p l a s t i c ) and maintained i n Eagle's Minimal E s s e n t i a l Medium (MEM) supplemented w i t h a n t i b i o t i c s and 10$ f e t a l c a l f serum. The c u l t u r e s were kept i n a h u m i d i f i e d incubator (37°C) s t a f f e d w i t h 5% C0o and 95% a i r . 2. U l t r a v i o l e t I r r a d i a t i o n A S y l v a n i a g e r m i c i d a l lamp (G15 T8) was employed as a source f o r UV l i g h t . At a distance of seven inches, i t gave a dose of 30 ergs/ram /sec. as measured by a UV l i g h t meter ( U l t r a v i o l e t Products, Inc., San G a b r i e l , C a l i f o r n i a ) . The UV meter was c a l i b r a t e d a g a i n s t E. c o l i , s u r v i v a l r a t e s f o l l o w i n g UV i r r a d i a t i o n by Dr. J . Kemp (Simon F r a s e r U n i v e r s i t y , Burnaby, B. C ) . C e l l c u l t u r e s on c o v e r - s l i p s to be i r r a d i a t e d were taken out of the L e i g h t o n tube, dipped three times i n s t e r i l e phosphate b u f f e r s a l i n e (see Appendix 3) t o t remove serum i n the c u l t u r e medium adhering to the c e l l s . Serum and phenol red (the pH i n d i c a t o r used i n c u l t u r e medium) are known to absorb UV i r r a d i a t i o n . H a l f of each c o v e r - s l i p was s h i e l d e d by a t i n p l a t e , so t h a t these c e l l s p r o t e c t e d from UV l i g h t c o u l d serve as c o n t r o l s . A f t e r i r r a d i a t i o n , ^ HTdR l a b e l l i n g and the autoradiographic procedures were c a r r i e d out as f o r c e l l c u l t u r e s exposed to chemical carcinogens. 3. Chemicals 3.1. Source of Chemicals 4 - N i t r o q u i n o l i n e 1-oxide, i t s isomers and d e r i v a t i v e s as w e l l as n i t r o s o m e t h y l u r e a (NMU), were the generous g i f t of Dr. Y. Kawazoe ( N a t i o n a l Cancer Centre Research I n s t i t u t e , Tokyo, Japan). Benz(a)anthracene, 20-methylcholanthrene and d e r i v a t i v e s were k i n d l y provided by Dr. C. Heidelberger (McArdle Laboratory f o r Cancer Research, U n i v e r s i t y of Wisconsin, U.S.A The aromatic amines (2-acetylaminofluorene, 4 - a c e t y l -aminostilbene, 4-acetylaminobiphenyl, 2-acetylamino-phenanthrene and the corresponding N-hydroxy- and N-acetoxy- d e r i v a t i v e s , N-myristoyloxy-2-AAF, N-acetoxy-2-m y r i s t o y l - A F , and N-myristoyloxy-2-myristoyl-AF), d i p h e n y l -c a r b i n o l s and s a f r o l e d e r i v a t i v e s were k i n d l y provided by Drs. J.A. M i l l e r , E.C. M i l l e r (McArdle Laboratory f o r Cancer Research, U n i v e r s i t y of Wisconsin, U.S.A.) and H. Bar t s c h (IARC, Lyons, France). N-Methyl-N'-nitro-N-nitrosoguanidine ( A l d r i c h Chemical Co. Inc., Milwaukee, Wisconsin) was k i n d l y provided by Dr. C T . Beer ( U n i v e r s i t y of B r i t i s h Columbia, Canada). Ethylmethanesulfonate and methylmethanesulfonate (Eastman Organic Chemicals, Rochester, New York) were k i n d l y provided by Dr. D.T. Suzuki ( U n i v e r s i t y of B r i t i s h Columbia, Canada). ICR-191 was k i n d l y provided by Dr. H.J. Creech ( I n s t i t u t e f o r Cancer Research, P h i l a d e l p h i a , U.S.A.). S t r e p t o n y g r i n was k i n d l y provided by Dr. J.W. Lown ( U n i v e r s i t y of A l b e r t a , Canada). L u t e o s k y r i n and r u g u l o s i n were k i n d l y provided by Dr. S. Sh i b a t a ( U n i v e r s i t y of Tokyo, Japan). The n i t r o f u r a n , N - / ^ - ( 5 - n i t r o - 2 - f u r y l ) - 2 - t h i a z o l y l 7 formamide (FANFT) was k i n d l y provided by Dr. D.R. McCalla (McMaster U n i v e r s i t y , Canada). Nitrogen mustard was obtained as Mustargen (mechlore-thamine h y d r o c h l o r i d e ) from Merck,' Sharpe and Dohme Canada 14 L t d . ( K i r k l a n d , Que.). A n a t o x i n s B^, B 2, G^, G 2 and s t e r i g m a t o c y s t i n were purchased from Makor Chemicals, Jerusalem, I s r a e l . Dimethylnitrosamine (DMN) was purchased from K and K L a b o r a t o r i e s , P l a i n v i e w , N.Y. Anhydrous methylguanidine hy d r o c h l o r i d e and ethidium bromide were purchased from Sigma Chemical Co., S t . L o u i s , Mo. A c r i f l a v i n e N e u t r a l was purchased from Mann Research L a b o r a t o r i e s , New York N.Y. <?<-Terthienyl and cow p a r s n i p were k i n d l y provided by Drs. G.F.Q. Chan, G.H.N. Towers and E. Camm ( U n i v e r s i t y of B r i t i s h Columbia, Canada). Daunomycin was obtained as Cerubidine from Specia, P a r i s . 3.2. S o l u t i o n s of Chemicals Several of the chemicals t e s t e d are r e a d i l y s o l u b l e i n water. These i n c l u d e MNNG, NMU, ICR - 1 9 1 , HN 2, a c r i f l a v i n e n e u t r a l , daunomycin, ethidium bromide and methylguanidine. An appropriate amount of the t e s t compound was d i s s o l v e d i n t i s s u e c u l t u r e medium (MEM or ADM as r e q u i r e d ) . EMS, MMS and DMN are l i q u i d s at room temperature. They are r e a d i l y m i s c i b l e w i t h water and d i l u t i o n s were made d i r e c t l y w i t h c u l t u r e medium. 4NQ0 and i t s d e r i v a t i v e s are not r e a d i l y s o l u b l e i n water. To prepare a 10 -^M s o l u t i o n (the standard stock used throughout t h i s s t u d y ) , 0.4 ml. of 100$ ethanol was added to an appropriate amount of s o l i d i n a disposable p l a s t i c tube, f o l l o w e d by a d d i t i o n of 9 . 6 ml. of t i s s u e c u l t u r e medium. The f i n a l c o n c e n t r a t i o n of e t h a n o l d i d not exceed 5%» The p o l y c y c l i c hydrocarbons (3A, MCA and d e r i v a t i v e s ) , the aromatic amines (2-AAF, 4-AAS, 4-AABP, 2-AAP and d e r i v a t i v e s ) , s a f r o l e (and d e r i v a t i v e s ) , d i p h e n y l c a r b i n o l s , a f l a t o x i n s , s t e r i m a t o c y s t i n . l u t e o s k y r i n and r u g u l o s i n were d i s s o l v e d i n dim e t h y l s u l f o x i d e (DMSO) p r i o r t o d i l u t i o n w i t h c u l t u r e medium to the d e s i r e d c o n c e n t r a t i o n s . The f i n a l c o n c e n t r a t i o n o f DMSO d i d not exceed 1$. -3 Unless h i g h e r c o n c e n t r a t i o n s were r e q u i r e d , a 10 ^M stock s o l u t i o n was u s u a l l y prepared immediately before use. 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 f i n a l c o n c e n t r a t i o n s . I f ADM was used throughout the e n t i r e experiment, then the s o l u t i o n was prepared i n ADM, otherwise i n MEM. 4. I n V i t r o A c t i v a t i o n of Pr e c a r c i n o g e n s 4.1. P r e p a r a t i o n of P o s t - M i t o c h o n d r i a l F r a c t i o n (S9 F r a c t i o n ) o f T i s s u e Homogenates Young a d u l t Swiss mice (males w i t h average body weight of 20 gm.) ob t a i n e d from the Animal U n i t , F a c u l t y of Medicine, U n i v e r s i t y of B r i t i s h Columbia, were k i l l e d by c e r v i c a l d i s l o c a t i o n and were immediately d i s s e c t e d . L i v e r s were removed, trimmed of ad h e r i n g c o n n e c t i v e t i s s u e and p l a c e d i n t o a beaker c o n t a i n i n g PBS/sucrose b u f f e r a t 4°C (PBS, with c a l c i u m and magnesium i o n s , c o n t a i n i n g 0.25M sucrose, pH 7 . 5 ) . A l l subsequent s t e p s were conducted w i t h s o l u t i o n s and c o n t a i n e r s maintained a t 4°C. L i v e r from s e v e r a l mice were pooled, washed w i t h PBS/sucrose b u f f e r , dabbed dry on absorbent t i s s u e , weighed, and q u i c k l y t r a n s f e r r e d to separate beakers of f r e s h PBS/ sucrose b u f f e r such th a t 6 gm. of l i v e r were contained i n 10 ml. b u f f e r . The l i v e r s were minced w i t h s c i s s o r s and the contents of each beaker were t r a n s f e r r e d to homogenization v e s s e l s . The t i s s u e s were then homogenized by 10 up-and-down s t r o k e s of a l o o s e - f i t t i n g Potter-Elvejhem Homogenizer op e r a t i n g at 1,000 rev./min. The homogenate was c e n t r i f u g e d a t 9,000 x g f o r 10 min. (at 4°C) i n a pre-cooled r o t o r (Beckman, Type 40) and n i t r o c e l l u l o s e c e n t r i f u g e tubes. The r e s u l t i n g p o s t - m i t o c h o n d r i a l supernatant f r a c t i o n s (sometimes r e f e r r e d t o as S9 f r a c t i o n s ) were pooled, mixed thoroughly to ensure homogeneity of each batch, and then d i s t r i b u t e d i n measured a l i q u o t s t o polypropylene tubes, capped, immediately f r o z e n i n l i q u i d n i t r o g e n and s t o r e d i n a Revco f r e e z e r at -70°C ( L a i s h e s , 1974; S t i c h and L a i s h e s , 1975). 4.2. P r e p a r a t i o n of A c t i v a t i o n Mixture and Treatment  of C e l l C u l t u r e s The a c t i v a t i o n system was prepared by d i s s o l v i n g , f o r each c e l l c u l t u r e to be t r e a t e d , 4.0 umoles NADPH or NADP, 25 umoles MgCl 2, and 20 pmoles G6P i n 0.4 ml. of S9 f r a c t i o n (thawed immediately before use) and a d j u s t i n g the pH to 7.2 w i t h 0.1 N NaOH. Equal volumes of precarcinogen s o l u t i o n (at twice the d e s i r e d f i n a l c o n c e ntration) and a c t i v a t i o n system ( u s u a l l y 0 . 5 ml. each) were mixed, vortexed q u i c k l y , and the r e s u l t i n g a c t i v a t i o n mixture was added to c e l l c u l t u r e s w i t h i n 5 seconds of mixing. The c u l t u r e s were incubated at 3 7 ° . At the end of the treatment, the c e l l s were washed three times w i t h c u l t u r e medium and normal c u l t u r e medium (MEM or ADM as required) was r e p l a c e d . 5 . Measurement of DNA Repair S y n t h e s i s (Autoradiography) 5 . 1 . P r e p a r a t i o n of C e l l C u l t u r e s and Exposure to Test  Compound C e l l s were seeded onto 1 0 x 3 5 mm. cover g l a s s e s (Clay Adams) kept i n 1 6 x 8 5 mm. L e i g h t o n t i s s u e c u l t u r e tubes ( B e l l c o G l a s s , I n c . , V i n e l a n d , New Jersey) a t approximately k 5 x 1 0 c e l l s per tube and covered w i t h MEM (supplemented w i t h 1 5 % f e t a l c a l f serum). The c e l l c u l t u r e s were always used f o r experimentation before the monolayer has reached confluency. This permits b e t t e r c y t o l o g i c p r e p a r a t i o n , b e t t e r c e l l exposure to carcinogens and, i n the case of c u l t u r e s processed f o r autoradiography, a lower background count. To d i s t i n g u i s h between DNA r e p l i c a t i o n a t S-phase and DNA r e p a i r s y n t h e s i s , m o d i f i c a t i o n of the procedure developed by Freed and Schatz (1969) was employed. By d e p r i v i n g the c e l l c u l t u r e of the e s s e n t i a l amino a c i d a r g i n i n e , i t was observed t h a t DNA s y n t h e s i s a s s o c i a t e d w i t h chromosome r e p l i c a t i o n was d r a s t i c a l l y reduced ( S t i c h and San, 1970). Upon reaching 80% confluency ( 2 - 3 days a f t e r seeding), the c e l l s were placed i n t o an a r g i n i n e - d e f i c i e n t medium (5% FCS). This was done by d i p p i n g the c o v e r s l i p s about f i v e times i n t o each of two beakers of ADM (no serum), and then t r a n s f e r r i n g them to new Leighton tubes c o n t a i n i n g 1 ml. of ADM (5% FCS). A f t e r 2 | to 3 days i n ADM, more than 90% of the c e l l s would be a r r e s t e d at G^. Chemical treatment was c a r r i e d out by r e p l a c i n g the medium i n the L e i g h t o n tube c u l t u r e w i t h a s o l u t i o n of the t e s t compound a t the d e s i r e d c o n c e n t r a t i o n . I f r a d i o a c t i v e -l a b e l l i n g was done immediately a f t e r exposure to the t e s t compound, the chemical s o l u t i o n was decanted, and ^HTdR ( i n ADM, 5% FCS) was added a f t e r the c e l l c u l t u r e had been r i n s e d three times w i t h ADM (no serum). Otherwise, the c e l l c u l t u r e was put back i n t o ADM f o l l o w i n g chemical treatment. 5.2. R a d i o a c t i v e - L a b e l I n c o r p o r a t i o n T r i t i a t e d thymidine ( s p e c i f i c a c t i v i t y 20Ci/mmol), obtained as thymidine (methyl-^H) i n aqueous s o l u t i o n from New England Nuclear (Dorval, Que.), was used i n a l l p u l s e - l a b e l experiments. D i l u t i o n down to a f i n a l concentra-t i o n of 10 u c i / m l . was made by mixing with ADM (5$ FCS). Unless otherwise s p e c i f i e d , c h e m i c a l l y - t r e a t e d or U V - i r r a d i a t e d c e l l s c u l t u r e s were p u l s e - l a b e l l e d w i t h ^HTdR 3 f o r 1 .5 hours. The -^HTdR-containing medium was then poured o f f and any unincorporated r a d i o i s o t o p e removed by r i n s i n g two or three times w i t h Hank's balanced s a l t s o l u t i o n (see Appendix 3). The c o v e r s l i p s were then taken out of the Leighton tubes, immersed i n 1% sodium c i t r a t e f o r 12-15 minutes, f o l l o w e d by f i x a t i o n i n a c e t i c a c i d / ethanol (1«3» V/V), and allowed to a i r dry. 5.3. Coating w i t h Photographic Emulsion C o v e r s l i p s are very f r a g i l e and l i a b l e to breakage. To f a c i l i t a t e t h e i r m a n i p u l a t i o n , they were mounted on g l a s s s l i d e s . A s m a l l q u a n t i t y of p a r a f f i n wax was placed on a g l a s s s l i d e and warmed over a flame u n t i l i t began to melt. An a i r - d r i e d c o v e r s l i p was placed on top of the wax w i t h the c e l l monolayer f a c i n g upwards. Once the wax r e s o l i d i f i e d , the c o v e r s l i p became anchored to the g l a s s s l i d e . A c e t i c A c i d has been r e p o r t e d to r e a c t w i t h photographic emulsions, r e s u l t i n g i n an i n c r e a s e i n background g r a i n count (Stocker and M u l l e r , 1967). As a p r e c a u t i o n , excess a c e t i c a c i d was removed by passing the s l i d e s through a graded a l c o h o l s e r i e s , 95$ EtOH, 70$ EtOH, 20$ EtOH (10 minutes each), two changes of d i s t i l l e d water, one change of PBS, two more changes of d i s t i l l e d water (10 minutes each) and were then l e f t t o a i r dry. The s l i d e s were coated w i t h photographic emulsion i n the dark-room. A Kodak Wratten S e r i e s 2 red f i l t e r was used. Kodak NTB-3 n u c l e a r - t r a c k emulsion was thawed at 43 °C i n a water-bath i n the dark, and d i l u t e d w i t h an equal volume of d i s t i l l e d water. Glass s l i d e s were then i n d i v i d u a l l y dipped i n the emulsion, a i r - d r i e d i n a v e r t i c a l p o s i t i o n , 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 14 days. 5.4. P r o c e s s i n g and S t a i n i n g of Autoradiograms Autoradiograms were brought back to room-temperature a f t e r 14 days of exposure. P r o c e s s i n g was done a t 18°C i n Kodak D-19 developer (3 minutes), stop bath (30 seconds), Kodak f i x e r (10 minutes) and h y p o c l e a r i n g agent (1 minute). A f t e r a 30-minute r i n s e i n running water (18°C), the s l i d e s were s t a i n e d w i t h 2% a c e t o - o r c e i n f o r 10 minutes, dehydrated through successive immersion (1 . 5 minutes each) i n 95$ 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 , two changes of x y l o l and mounted i n Permount ( F i s h e r S c i e n t i f i c Co.) by super-imposing another c o v e r s l i p over t h a t bearing the c e l l monolayer. 5.5 . A n a l y s i s of Autoradiograms The amount of DNA r e p a i r s y n t h e s i s was estimated by counting the number of g r a i n s over each nucleus. Care was taken t h a t n u c l e i of comparable s i z e were s e l e c t e d so t h a t only c e l l s of the same p l o i d y were used. R o u t i n e l y , g r a i n counts were made on small interphase n u c l e i . Back-ground count was taken i n t o c o n s i d e r a t i o n by reckoning the number of g r a i n s over an area equal i n s i z e to t h a t of the nucleus. At l e a s t 30 n u c l e i , at random l o c a t i o n s throughout the e n t i r e c o v e r s l i p c u l t u r e , were analysed f o r g r a i n number. When the g r a i n number appeared below 10 g r a i n s / n u c l e u s , at l e a s t 100 n u c l e i were scored. Based on s t a t i s t i c a l c a l c u l a t i o n s , Rogers and England (1973) have demonstrated t h a t the accuracy of e s t i m a t i n g the r a d i o a c t i v i t y per nucleus w i l l depend, not on the number of n u c l e i counted, nor on the t o t a l area of emulsion scanned, but on the t o t a l number of s i l v e r g r a i n s counted i n one sampling of the p o p u l a t i o n . A more d e t a i l e d c o n s i d e r a t i o n of the s t a t i s t i c a l a n a l y s i s of autoradiograms i s i n c l u d e d as an appendix i n t h i s t h e s i s (see Appendix 4 ) . 6. Chromosome S t u d i e s 6.1. P r e p a r a t i o n of C e l l C u l t u r e s 2 C u l t u r e d human f i b r o b l a s t s were seeded onto 20 mm . c o v e r s l i p s (Corning) i n 35 mm. p e t r i dishes a t approximately 5 x 10** c e l l s per d i s h and covered w i t h MEM (15$ FCS). I n order to o b t a i n w e l l spread metaphase p l a t e s , the c e l l s were used before they reached 80$ confluency. 6.2. Exposure to Test Compound Exposure of p e t r i p l a t e c u l t u r e s to the t e s t chemicals was performed i n the f o l l o w i n g manner. The medium was removed w i t h a s t e r i l e pasteur p i p e t t e connected to a s u c t i o n device. One ml. of the chemical s o l u t i o n was added f o r the time p e r i o d d e s i r e d ( maintained at 37°C i n a C0 2 incubator) and then removed by s u c t i o n . A f t e r r i n s i n g twice w i t h MEM (no serum), the c e l l s were covered w i t h 2 ml. of MEM (15% FCS) and returned to the C0 2 i n c u b a t o r . 6.3 . C y t o l o g i c P r e p a r a t i o n s 6 . 3 . 1 . Reagents C o l c h i c i n e (BDH Chemicals L t d . , Poole, England). A 1% stock s o l u t i o n i n d i s t i l l e d water was prepared from which ap p r o p r i a t e d i l u t i o n to a 0.01% s o l u t i o n c ould be made. Human c e l l s were t r e a t e d wrth 0.1 ml. of the 0.01% s o l u t i o n per ml. of medium ( i . e . a f i n a l c o l c h i c i n e concen-t r a t i o n of 10 ug/ml.). Sodium C i t r a t e . Sodium c i t r a t e ( F i s h e r S c i e n t i f i c Co.) was used as a 1% s o l u t i o n (W/v) i n d i s t i l l e d water. F i x a t i v e . A mixture of three p a r t s of 100% ethanol w i t h one p a r t of g l a c i a l a c e t i c a c i d (V/V) was used as a f i x a t i v e . I t was u s u a l l y prepared 1 t o 2 hours: before use. Aceto-Orcein. A c e t o - o r c e i n , used as a 2% s o l u t i o n , was prepared by r e f l u x i n g f o r 6 hours the appropriate amount of o r c e i n (BDH Chemicals L t d . , Poole, England) i n 45% a c e t i c a c i d . The s o l u t i o n so obtained was always f i l t e r e d (with Whatman No. 1 paper) before use. 6 . 3 . 2 . Chromosome P r e p a r a t i o n s Once c e l l d i v i s i o n s were detected (by ob s e r v a t i o n under an i n v e r t e d microscope) f o l l o w i n g exposure to a t e s t compound, 23 0.2 ml. of a 0.01$ s o l u t i o n of c o l c h i c i n e was added to c o v e r s l i p c u l t u r e s ( c o n t a i n i n g approximately 2 ml. c u l t u r e medium) f o r 4-5 hours ( f i n a l c o n c e n t r a t i o n of c o l c h i c i n e i 10 jug/ml.). The c o v e r s l i p s were then t r a n s f e r r e d to p e t r i dishes c o n t a i n i n g a 1$ sodium c i t r a t e s o l u t i o n . This hypotonic treatment (20 minutes) enables the c e l l s t o increase i n volume and permits a n i c e spread of the metaphase chromosomes. F i x a t i o n of the c e l l s was c a r r i e d out i n a 3*1 mixture (V/V) of 100$ ethanol and g l a c i a l a c e t i c a c i d (10 minutes). Once a i r d r i e d , the c e l l s were s t a i n e d w i t h 2$ a c e t o - o r c e i n , dehydrated through immersion i n 100$ EtOH, but a n o l , b u t a n o l / x y l o l , two changes of x y l o l and mounted i n Permount ( F i s h e r S c i e n t i f i c Co.). 6.4. A n a l y s i s of Metaphase P l a t e s f o r Chromosome A b e r r a t i o n s For each sample about 30 to 50 w e l l - s p r e a d metaphase p l a t e s were analyzed f o r chromatid breaks, exchanges and fragmentation. When the frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s appeared to be below $%, a t l e a s t 150 metaphases were scored. 7. S u r v i v a l S t u d i e s About 2,000 human d i p l o i d f i b r o b l a s t s were seeded i n 5 cm. s t e r i l e d i sposable p e t r i p l a t e s (Falcon P l a s t i c ) , covered w i t h 4 ml. of MEM (10$ FCS) and allowed to s e t t l e down as s i n g l e c e l l s overnight (16-20 hours) before chemical treatment. I t was observed t h a t c e l l d i v i s i o n d i d not 24 u s u a l l y occur w i t h i n t h i s p e r i o d of time. Exposure to the t e s t chemical was performed by s u c t i o n i n g o f f the c u l t u r e medium and r e p l a c i n g i t w i t h 3 ml. of the chemical s o l u t i o n i n MEM (5% FCS). F o l l o w i n g chemical treatment, the c u l t u r e s were r i n s e d twice w i t h MEM (no serum) and incubated i n f r e s h MEM (15% FCS) which was changed a f t e r 7 days. When the clones from s u r v i v i n g c e l l s had reached the 50 - 60 c e l l stage (approximately 10 - 14 days post-treatment) the c u l t u r e s were f i x e d w i t h e t h a n o l - a c e t i c a c i d (3*1, V/V) f o r 10 minutes, washed i n 70% e t h a n o l , a i r d r i e d and s t a i n e d w i t h a 2% aqueous s o l u t i o n of T o l u i d i n e Blue ( F i s h e r S c i e n t i f i c Co.). Excess dye was removed by r i n s i n g i n d i s t i l l e d water. The clones c o n t a i n i n g 50 or more c e l l s were counted under a r e g u l a r d i s s e c t i n g microscope. U s u a l l y , counts from three to s i x c u l t u r e s were averaged f o r each c o n c e n t r a t i o n of the t e s t chemical. The data were expressed as a percentage of the clone count i n the untreated c o n t r o l . RESULTS 1. Unscheduled Incorporation of T r i t i a t e d Thymidine  (•^ HTdR) i n Mammalian C e l l s as a Measure of Repair  of DNA Damage Following Exposure to Chemical Carcinogens One of the objectives of t h i s thesis was to evaluate the f e a s i b i l i t y of using DNA r e p a i r synthesis i n cultured human f i b r o b l a s t s as a bioassay f o r chemical carcinogens. An important question that arises i s whether the unscheduled incorporation of t r i t i a t e d thymidine i n carcinogen-treated f i b r o b l a s t s can be taken as a measure of the ensuing' DNA r e p a i r . Furthermore, i n order to design a protocol to monitor the r e p a i r of carcinogen-induced DNA damage, the following problems must be consideredi - whether a d i s t i n c t i o n can be made between DNA r e p a i r synthesis and DNA synthesis associated with chromosome r e p l i c a t i o n . - whether a l l or only a f r a c t i o n of the carcinogen-treated c e l l s e x h i b i t a DNA r e p a i r synthesis. - whether d i f f e r e n t carcinogen doses ( i n terms of exposure time or concentration) have any e f f e c t on the l e v e l and duration of the ensuing DNA r e p a i r synthesis. - whether the capacity of cultured c e l l s to r e p a i r DNA damage can be temporarily reduced or blocked by chemicals. These questions w i l l be examined i n the following three chaptersi 1.1. Dose Response 1.2. Duration of DNA Repair 1.3. DNA Repair I n h i b i t i o n 1.1. Dose Response To separate DNA-repair synthesis (unscheduled incorporation 3 of -\HTdR) from DNA-replication synthesis associated with chromosome r e p l i c a t i o n at S-phase, cultures of mammalian c e l l s were kept i n a r g i n i n e - d e f i c i e n t medium (ADM) f o r 3 days p r i o r to t h e i r exposure to carcinogens. The ADM suppresses the flow of c e l l s from G^ into S-phase, Thus the DNA-repair synthesis can be examined i n non-dividing c e l l s without any interference by a DNA synthesis associated with chromosome r e p l i c a t i o n (Fig, 1 and 2 ) . At f i r s t we examined the proportion of cultured c e l l s that showed a DNA r e p a i r synthesis following exposure to carcinogens. About 2x10** Syrian-hamster c e l l s from primary to t e r t i a r y cultures, 10^ c e l l s from a d i p l o i d human embryo and 5x10^ c e l l s from a t r i p l o i d t h e r apeutically aborted embryo and 10^ cultured c e l l s from a 22 year old female were screened f o r c e l l s l a c k i n g the capacity of DNA r e p a i r . Less than 1 i n 100,000 n u c l e i showed no incorporation of ^HTdR following exposure to> x l 0 ~ »M 4NQ0 f o r 1.5 h, or U V - i r r a d i a t i o n (900 ergs/mm ). Nuclei that show no unscheduled incorporation of ^HTdR were small and stained heavily with orcein. They probably represent pycnotic n u c l e i of dying or dead c e l l s . I f viable mammalian c e l l s lacking DNA-repair synthesis e x i s t at a l l , t h e i r frequency i n a population of Figure 1 Embryonal Syrian-hamster c e l l s cultured i n MEM and exposed to kNQ0 (4xlO" 6M) and 3HTdR f o r 1.5 hours. Autoradiograph. The S-phase nu c l e i are heavily l a b e l l e d , whereas a l l other n u c l e i show a r e s t r i c t e d number of grains. Figure 2 Embryonal Syrian-hamster c e l l s kept f o r 3 days i n a r g i n i n e - d e f i c i e n t medium (ADM) and exposed to 4NQ0 (4xlO" 6M) and 3HTdR f o r 1.5 hours. Autoradio-graph. Note the absence of heavily l a b e l l e d S-phase nu c l e i and the -'HTdR incorporation i n a l l n u c l e i of the non-p r o l i f e r a t i n g c e l l s . 29 of normal human c e l l s must be extremely low. The amount of ^ HTdR i n c o r p o r a t i o n i n t o n u c l e a r DNA of c e l l s exposed to a c t i v e carcinogens depends among many other f a c t o r s on the p l o i d y of c e l l s or more p r e c i s e l y expressed on the DNA content of a nucleus ( F i g . 3 a ) . The histogram of F i g . 3D shows the d i s t r i b u t i o n of g r a i n s per nucleus of c e l l s w i t h a 2C, 4C or 8C amount of DNA. This r e l a t i o n s h i p between DNA content per nucleus and amount of ^HTdR i n c o r p o r a t i o n must be kept i n mind when autoradiographs of c e l l stages w i t h v a r i o u s DNA l e v e l s (e.g. Gl versus G2 n u c l e i ; metaphase p l a t e s versus telophase n u c l e i ) or c e l l s w i t h d i f f e r e n t chromosome numbers are compared w i t h each other. The e f f e c t of v a r i o u s carcinogens on DNA r e p a i r s y n t h e s i s was examined by f o l l o w i n g an i n i t i a l carcinogen treatment 3 w i t h 1.5 hours of HTdR ( F i g , 4). The average number of g r a i n s per nucleus i n c e l l s exposed to a s i n g l e dose of v a r i o u s proximate or u l t i m a t e carcinogens are shown i n F i g s . 5 and 7. The r e s u l t s shown examplify the most common types of dose response curves. Three d i s t i n c t i v e f e a t u r e s can be observed:-(1) The range of co n c e n t r a t i o n s of v a r i o u s carcinogens t h a t t r i g g e r d e t e c table l e v e l s of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s v a r i e s g r e a t l y ( F i g . 5)« (2) The l e t h a l dose of v a r i o u s carcinogens a l s o d i f f e r ranging from about 10~8M to 10"2M ( F i g . 6), (3) P r e v i o u s l y , we pointed out a good c o r r e l a t i o n between the l e v e l of DNA r e p a i r evoked by a carcinogen and the Figure 3(a) Unscheduled "'HTdR i n c o r p o r a t i o n i n c u l t u r e d Syrian-hamster c e l l s f o l l o w i n g exposure to a carcinogen. Autoradiograph. The small nucleus i s probably i n the G^ phase (one arrow) and the l a r g e r one i n G 2 phase (two arrows). Figure 3(b) Frequency d i s t r i b u t i o n of c e l l s w i t h v a r i o u s numbers of g r a i n s above t h e i r n u c l e i . C u l t u r e d S y r i a n -hamster c e l l s were p r e t r e a t e d f o r 3 days w i t h ADM t o block DNA r e p l i c a t i o n and m i t o s i s , thereupon exposed to kNQO (4xlcT 6M) and ^ HTdR f o r 1.5 hours. Autoradiograph, g r a i n counts. 2C, 4C and 8C r e f e r to d i p l o i d , t e t r a p l o i d and o c t o p l o i d n u c l e i . Figure 4 Experimental design: Unscheduled HTdR i n c o r p o r a t i o n i n c u l t u r e d human f i b r o b l a s t s exposed to one dose of a chemical carcinogen f o l l o w e d by 3HTdR (10 uCi/ml., 1.5 hours). Figure 5 V a r i a t i o n s i n c o n c e n t r a t i o n of three carcinogens t h a t t r i g g e r a DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . Exposure to carcinogen was f o l l o w e d by ^HTdR (1.5 hours). Figure 6 V a r i a t i o n s i n c o n c e n t r a t i o n of three carcinogens t h a t a f f e c t the clone forming c a p a c i t y of c u l t u r e d human f i b r o b l a s t s . Experimental Designs I -Carcinogen HTdR (1.5 nr.) 'sampling t ISO-ISO. (0 D Ul -I u 3 Z cc Ul a. to z < a Q <ao< ? o N a a i i - S H R ) i ICT IO" —rr IO C O N C E N T R A T I O N C M ) IOO--I 4 > > a • a • H . O l -4 N Q O C I - 5 H R ) \ „ • ^ o - o - o — o _ 0 _ 0 N - D H - A A R 5 H W O M M S C I - 5 H F O ° IO i-7 C O N C E N T R A T I O N C M ) degree of i t s c a r c i n o g e n i c p o t e n t i a l when the a c t i o n of stron g and weak c a r c i n o g e n i c 4-NQO isomers and d e r i v a t i v e s was compared ( S t i c h e t a l . , 1971). However, no such c o r r e l a t i o n became obvious when carcinogens of d i f f e r e n t molecular s t r u c t u r e s were i n c l u d e d i n the comparative study. For example, 4NQ0 e l i c i t e d a hig h l e v e l of unscheduled 3 -^ HTdR i n c o r p o r a t i o n a t r e l a t i v e l y low doses, w h i l e the 6,7-epoxide of MCA r e q u i r e d about 1,000 times h i g h e r c o n c e n t r a t i o n s to t r i g g e r a r e l a t i v e l y low l e v e l of DNA r e p a i r s y n t h e s i s ( F i g . 7) . Obviously 4NQ0 i s not 1,000 times more c a r c i n o g e n i c than MCA or i t s epoxide. A d i f f e r e n t type of dose-response study concerns the e f f e c t of d i f f e r e n t exposure times t o a carcinogen on DNA-repair s y n t h e s i s (Table I ) . With s e v e r a l carcinogens (e.g. N-OH-AAF), l i t t l e or no DNA r e p a i r s y n t h e s i s was observed a f t e r a s h o r t exposure time. One e x p l a n a t i o n i s t h a t these chemicals may r e q u i r e more time f o r i n t e r a c t i o n w i t h DNA t o come about. With some carcinogens (e.g. 4NQ0, N-Ac-AAF) a longer exposure time r e v e a l s l i t t l e or no incr e a s e i n the l e v e l of ^ HTdR i n c o r p o r a t i o n . One e x p l a n a t i o n i s t h a t the carcinogen remains a c t i v e only f o r a sh o r t time and no f u r t h e r DNA damage i s i n f l i c t e d upon prolonged exposure. Another p o s s i b i l i t y i s t h a t once the r e p a i r of the i n i t i a l DNA damage i s underway, f u r t h e r DNA damage w i l l not be cat e r e d to because the r e p a i r system i s overtaxed. This aspect w i l l be considered i n some d e t a i l i n connection w i t h the d u r a t i o n of DNA r e p a i r and the e f f e c t of carcinogens 35 o j 4 N Q O ( 1 . 3 H R ) ' f •Jo"7 io"6 1 0 5 -io 4 io1 C O N C E N T R A T I O N C M ) Figure 7 D i f f e r e n t l e v e l s of unscheduled DNA synthesis i n cultured human f i b r o b l a s t s e l i c i t e d by carcinogens of d i f f e r e n t molecular structures. TABLE I E f f e c t of Exposure Time to a Chemical Carcinogen on the Level of Unscheduled ^HTdR Incorporation i n Cultured Human Fi b r o b l a s t s * N-Hydroxy- N-Acetoxy- 4NQ0 2-AAF 2-AAF 5 x 1 0 " % 2 . 5 x l 0~ 5M 5xlO" 6M Exposure Time - Grains Per Nucleus 1 .5 Hours 3 33 90 3 Hours 12 28 72 5 Hours 20 25 58 * Exposure to carcinogen was followed by 1 .5 hour 3HTdR (10 /iCi/ml.). on chromosome a b e r r a t i o n s and c e l l s u r v i v a l . 37 1.2. D u r a t i o n of DNA Repair C u l t u r e d human f i b r o b l a s t s were kept i n ADM p r i o r to and throughout the e n t i r e experiment. I n t h i s way i t was p o s s i b l e to estimate the d u r a t i o n of DNA r e p a i r s y n t h e s i s i n n o n - d i v i d i n g c e l l s . The d u r a t i o n of DNA r e p a i r s y n t h e s i s was examined by exposing n o n - d i v i d i n g c e l l s to 3 1.5 h pu l s e s of -^ HTdR a t v a r i o u s time i n t e r v a l s a f t e r the a p p l i c a t i o n of carcinogen ( F i g . 8). Samples f o r the auto-radiographs were taken a t the end of each ^HTdR p u l s e . For the f i r s t sample carcinogen and ^ HTdR were a p p l i e d simultaneously f o r 1.5 h. The time course of r e p a i r s y n t h e s i s f o l l o w i n g exposure t o d i f f e r e n t chemical carcinogens i s shown i n F i g . 9. The major p a r t of r e p a i r s y n t h e s i s appears to be completed by about 8 t o 10 h post-treatment. However, a low but s i g n i f i c a n t uptake of ^ HTdR i n t o n u c l e a r DNA can be detected f o r a prolonged p e r i o d . Repair s y n t h e s i s f o l l o w s a s i m i l a r time course i n c e l l s whether they are exposed to higher or lower concentrations of a carcinogen ( F i g . 10). For comparative purposes the course of 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 dose of UV (900 ergs/mm ) was added i n F i g . 11. The design of experiments was s i m i l a r to the previous ones i n which a chemical carcinogen was used t o induce r e p a i r s y n t h e s i s . The d u r a t i o n of the main phase of r e p a i r s y n t h e s i s i s s i m i l a r i n c e l l s i r r a d i a t e d w i t h UV (100-900 ergs/mm ) or exposed to v a r i o u s chemical Figure 8 Experimental Design. Duration of unscheduled DNA s y n t h e s i s induced i n c u l t u r e d human f i b r o b l a s t s by a s i n g l e dose of UV r a d i a t i o n or chemical carcinogen. Figure 9 - 1 1 D u r a t i o n of unscheduled DNA s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s f o l l o w i n g exposure to a s i n g l e dose of 4NQ0, MNNG or UV r a d i a t i o n . Each p o i n t denotes the time when the sample was taken 3 and the uptake of ^ HTdR over a 1 .5-hour p e r i o d p r i o r t o sampling (represented by g r a i n s per n u c l e u s ) , (Figure 9) 4NQ0 (8xlO" 7M, 1 . 5 hours), • , or MNNG ( 5 x 1 0 " % , 1 .5 h o u r s ) , * . I 1 = Exposure to carcinogen, (Figure 10) D i f f e r e n t c o n c e n t r a t i o n s of 4NQ0, 5 x l 0" 6M ( O ) , 8 x l 0~ 7M ( • ) . (Figure 11) UV r a d i a t i o n (260 nm, 900 ergs/mm 2). Figure 12 Time course of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s f o l l o w i n g short term exposure to 4NQ0 (5xl0~^M, 1 .5 hours) and measured 3 as unscheduled i n c o r p o r a t i o n of -^ HTdR (autoradiography, v ) or as a s h i f t i n the sedimentation r a t e i n an a l k a l i n e sucrose g r a d i e n t ( • ). By about 12 hours, post-treatment DNA r e p a i r s y n t h e s i s ceases and the sedimentation p r o f i l e approaches the o r i g i n a l p a t t e r n . (Sedimentation* ordinate denotes f r a c t i o n number corresponding to peak). Protocol! Time (hours) 0 r 1.5 — C 4 T 6 T 8 T 10 " T 12 14 16 Carcinogen + ^ HTriR I « 3 Carcinogen -'HTdR | ^ 1 C a r c inogen I Ci~T C a r c i n o g e n 18 T r T C a r c i n o g e n I ^ I Carcinogen^ C a r c i n o g e n C a r c i n o g e n C a r c i n o g e n 'HTdR HTdR 1 'HTdR I 'HTdR 1 HTdR 1 HTdR , 3HT<^ \ © © T 1 S IE .T H O J f l a A F T E R T R E A T M E N T 1 O 3 Ul O I « N a z 4 ® H O U R S A F T E R T R E A T M E N T ^ 6 z carcinogens. One c r i t i c i s m of the autoradiographic technique i s 3 whether the unscheduled ^HTdR uptake could be equated to DNA r e p a i r s y n t h e s i s . The unscheduled i n c o r p o r a t i o n of 3 ^HTdR i n d i c a t e d DNA s y n t h e t i c a c t i v i t y but does not provide any evidence t h a t the newly synt h e s i s e d DNA segments are j o i n e d a t a l l . To answer t h i s q u e s t i o n , one can r e s o r t t o the a l k a l i n e sucrose g r a d i e n t (ASG) technique. I n t h i s procedure, a s h i f t i n the sedimentation r a t e i n d i c a t e s t h a t the DNA has been fragmented whereas a r e t u r n t o the normal sedimentation r a t e i m p l i e s t h a t the DNA molecule has been r e s t o r e d . For comparison, an ASG a n a l y s i s of DNA from kNQO-treated human f i b r o b l a s t s has been i n c l u d e d ( F i g . 12). I t i s of i n t e r e s t t o note t h a t the time course of DNA r e p a i r (measured as unscheduled i n c o r p o r a t i o n of ^HTdR) resembles the p e r i o d of r e j o i n i n g of the fragmented DNA t o i t s o r i g i n a l s i z e (measured as s h i f t i n sedimentation r a t e i n an a l k a l i n e sucrose g r a d i e n t ) . 1.3. DNA Repair I n h i b i t i o n There i s a p l e t h o r a of evidence showing t h a t a g e n e t i c a l l y impaired DNA r e p a i r mechanism i n c r e a s e s the s e n s i t i v i t y of b a c t e r i a l and mammalian c e l l s towards the l e t h a l and mutagenic e f f e c t of p h y s i c a l and chemical agents. The q u e s t i o n must be r a i s e d whether the c a p a c i t y of c u l t u r e d c e l l s t o r e p a i r DNA can be t e m p o r a r i l y reduced or blocked by chemicals and whether exposure to one carcinogen would s e n s i t i z e c e l l s 41 towards the e f f e c t of other chemical carcinogens. I n t h i s chapter the dual a c t i o n of carcinogens i s examined: namely t h e i r c a p a c i t y to induce DNA l e s i o n s and t h e i r c a p a c i t y to a f f e c t a normal DNA r e p a i r s y n t h e s i s . The f o l l o w i n g experimental p r o t o c o l was used* non-d i v i d i n g human c e l l s were e i t h e r U V - i r r a d i a t e d and t h e r e a f t e r exposed t o chemical carcinogens, or t r e a t e d w i t h the chemical carcinogen p r i o r t o , as w e l l as a f t e r , U V - i r r a d i a t i o n ( F i g . 13). F o l l o w i n g t h i s combined a p p l i c a t i o n of U V - i r r a d i a t i o n and a chemical carcinogen, the c e l l s were placed i n t o c u l t u r e medium w i t h ^HTdR to measure DNA r e p a i r s y n t h e s i s ( F i g . 14, 15 and 16) or maintained i n MEM (15% FCS) to estimate the clone-forming c a p a c i t y ( F i g . 17 and 18). The r e s u l t s show th a t a l l the a c t i v e chemical carcinogens examined i n h i b i t DNA r e p a i r s y n t h e s i s and t h a t v a r i o u s c a r c i n o g e n i c compounds g r e a t l y d i f f e r i n t h e i r c a p a c i t y to induce DNA l e s i o n s and to i n h i b i t DNA r e p a i r . Un-f o r t u n a t e l y , i t i s somewhat d i f f i c u l t to assess the r e s u l t s of the combination experiments, because the chemical carcinogens add new DNA l e s i o n s to those induced by U V - i r r a d i a t i o n . Thus the ensuing unscheduled DNA s y n t h e s i s i s a product of the DNA l e s i o n s induced by UV, DNA l e s i o n s induced by the chemical, and the extent to which DNA r e p a i r s y n t h e s i s i s a d v e r s e l y a f f e c t e d by UV and the chemical carcinogen. I n s p i t e of these t e c h n i c a l r e s t r i c t i o n s , one can grade carcinogens as s t r o n g or weak inducers of DNA l e s i o n s and strong or weak i n h i b i t o r s of DNA r e p a i r . Figure 13 Experimental Design. I n h i b i t i o n of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s f o l l o w i n g the combined a p p l i c a t i o n of UV r a d i a t i o n and a chemical carcinogen. F i g u r e s 14-16 I n h i b i t i o n of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s f o l l o w i n g the combined a p p l i c a t i o n of UV r a d i a t i o n and a chemical carcinogen 3 • = unscheduled ^HTdR i n c o r p o r a t i o n f o l l o w i n g 1.5 hours of N-acetoxy-4-AAS (Figure 14), n i t r o g e n mustard (Figure 15), or FANFT (Figure 16). 3 • = unscheduled ^HTdR i n c o r p o r a t i o n f o l l o w i n g UV i r r a d i a t i o n (900 ergs/mm ) and one of the three compounds. • = t h e o r e t i c a l curves showing a s t r i c t a d d i t i v e e f f e c t of UV and one of the three compounds (an a d d i t i v e e f f e c t occurs at the lowest c o n c e n t r a t i o n s employed). ® Protocol: Carcinogen \ UV + ^HTdR Sampling iooi s o N - A C - A A S IO 5 . I O - 5 ro D UJ -I u 3 z a Ul a CD 2 < a a sa HN. 5 Q 2 - & I O - 5 F A I M F T A -IO" 4 a - s - i o - 4 25 I O O © M © uglml 43 ® IO • Ol 1-6 ® N - A C - A A S IO C O N C E N T R A T I O N ( M J IO-5M I O " 8 I O " 7 M C O N C E N T R A T I O N ( M J Figures 17-18 E f f e c t of a combined treatment of UV r a d i a t i o n and a chemical carcinogen on the clone-forming capacity of cultured human f i b r o b l a s t s . (Figure 17) UV r a d i a t i o n (16 ergs/mm ) followed by N-acetoxy-4-AAS (5 hours) (Figure 18) UV r a d i a t i o n (16 ergs/mm ) followed by 4NQ0 (1.5 hours) The absence of a 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 a d m i n i s t r a t i o n of a compound co u l d be due to the l a c k of i t s i n t e r a c t i o n w i t h DNA (e.g. non-oncogenic compounds) or due t o a blockage of r e p a i r processes by a chemical w i t h a stro n g i n h i b i t o r y p roperty (e.g. a c r i f l a v i n e , daunomycin, mitomycin C). An unanswered q u e s t i o n remains as t o whether a chemical could have a ca r c i n o g e n i c e f f e c t by merely i n h i b i t i n g r e p a i r of 'spontaneously' o c c u r r i n g DNA l e s i o n s and i n t h i s manner c o n t r i b u t e to g e n e t i c anomalies and n e o p l a s t i c t r a n s f o r m a t i o n . Extensive s t u d i e s by Gaudin e t a l . (1972a, 1972b) have r e v e a l e d t h a t a l l co-carcinogens examined i n h i b i t DNA r e p a i r s y n t h e s i s . 2. DNA Repair D e f i c i e n c y i n Xeroderma Pigmentosum C e l l s -Numerous s t u d i e s on m i c r o b i a l systems have r e v e a l e d t h a t an e l e v a t e d s e n s i t i v i t y to U V - i r r a d i a t i o n i s due t o a d e f e c t i n one of the steps i n the DNA e x c i s i o n - r e p a i r or 'cut and patch' procedure ( W i t k i n , 1969; Kondo et a l . , 1970? Ganesan and Smith, 1971). The e x c i s i o n - d e f i c i e n t b a c t e r i a l mutants and UV - s e n s i t i v e yeast c e l l s a l s o show an i n c r e a s e d s e n s i t i v i t y t o the l e t h a l a c t i o n of exogenous chemicals and an e l e v a t e d mutation r a t e when exposed to U V - r a d i a t i o n or c h a l l e n g e d w i t h s e v e r a l chemical mutagens (Haynes et a l . . 1968} Kondo and Kato, 1968). These m i c r o b i a l systems have proven t o be good models f o r normal and r e p a i r - d e f i c i e n t human c e l l s i n which the r o l e of DNA r e p a i r and i t s g e n e t i c c o n t r o l are only now being s l o w l y u n r a v e l l e d . For example, 46 homozygous r e c e s s i v e Xeroderma pigmentosum (XP) c e l l s are known to be d e f i c i e n t i n c o r r e c t i n g UV-induced thymine dimers and p o s s i b l y c y t o s i n e dimers i n the DNA molecules (Cleaver, 1 9 6 8 : Setlow et a l . , 1 9 6 9 ) . I n t h i s chapter, an attempt i s made to answer three questions concerning the s e n s i t i v i t y of Xeroderma pigmentosum c e l l s to chemical carcinogens. The f i r s t q u e s t i o n i s whether XP c e l l s respond t o chemical carcinogens w i t h a reduced DNA r e p a i r c a p a c i t y . A second p o i n t of i n t e r e s t i s the wide spectrum of DNA r e p a i r d e f i c i e n c i e s found i n c e l l s of u n r e l a t e d p a t i e n t s . The q u e s t i o n i s t h e r e f o r e whether XP c e l l s , which g r e a t l y d i f f e r i n t h e i r c a p a c i t y to r e p a i r UV-induced DNA changes, a l s o d i f f e r i n t h e i r response to c h e m i c a l l y induced DNA a l t e r a t i o n s . T h i r d l y , the q u e s t i o n must be r a i s e d as to the DNA r e p a i r c a p a c i t y of XP-heterozygous persons. The u l t i m a t e g o a l i s to explore the p o s s i b l e use of the DNA r e p a i r bioassay i n the i d e n t i f i c a t i o n of persons h y p e r s e n s i t i v e towards chemical carcinogens. Unless otherwise s t a t e d , XP]? c e l l s were used i n comparative s t u d i e s w i t h c e l l s from c o n t r o l persons, 2 , 1 . Response of XP C e l l s t o D i f f e r e n t Carcinogens The response of XP c e l l s towards chemical or v i r a l carcinogens has remained unknown u n t i l r e c e n t l y (Cleaver, 1971aj Setlow and Regan, 1972} S t i c h and San, 1971} S t i c h e t a l . , 1 9 7 2 a , 1 9 7 2 b ) . To g a i n i n f o r m a t i o n on t h i s important q u e s t i o n , key carcinogens of d i v e r s e molecular s t r u c t u r e s were s e l e c t e d and added to c u l t u r e d XP f i b r o b l a s t s and normal human c e l l s f o r short time p e r i o d s (1.5 - 5 hours). The unscheduled i n c o r p o r a t i o n of ^ HTdR i n t o n u c l e a r DNA of no n - d i v i d i n g c e l l s was then estimated "by the p r e v i o u s l y described autoradiographic technique. The r e s u l t s are summarized i n F i g s . 19 - 2 k. The XP c e l l s show a c o n s i d e r a b l y reduced DNA r e p a i r s y n t h e s i s when exposed to some but not a l l chemical carcinogens (e.g. kNQO, N-Ac-AASf MCA-Epoxide) ( F i g . 19, 21, 2 3 ) . Thus, i t i s m i s l e a d i n g to r e f e r t o XP c e l l s as d e f i c i e n t i n DNA r e p a i r without mentioning the 'agent provocateur'. As can be seen from F i g . 20, 22, 2 k and Table I I , XP c e l l s can cope normally w i t h a DNA damage induced by t y p i c a l a l k y l a t i n g mutagens and carcinogens, i n c l u d i n g MNNG, MMS and NMU. This c e l l - c o n t r o l l e d degree of response t o v a r i o u s chemical agents i s d i f f i c u l t t o i n t e r p r e t a t present. Although the r e a c t i o n between DNA and the v a r i o u s agents used are not f u l l y known the ensuing molecular changes w i l l c e r t a i n l y d i f f e r * thymidine dimers and, t o a l e s s e r degree, cytosine-thymidine dimers, are formed by u l t r a v i o l e t i r r a d i a t i o n (Smith and Hanawalt, 19^9)t a r y l a m i d a t i o n of guanine and, to a l e s s e r extent, of adenine, w i l l f o l l o w exposure t o N-hydroxy and N-acetoxy-AAF ( M i l l e r e t a l . , 1966a; K r i e k et a l . , 1967), and the b i n d i n g between kNQ0 or kHAQ0 and DNA may l e a d to charge t r a n s f e r complexes i n v o l v i n g p urines (Okano et a l . , 1969l P a u l et a l . , 1971? Okano e t a l . , 1972), or to an e l e c t r o p h i l i c a t t a c k of n u c l e i c a c i d bases (Enomoto et a l . , 1968). By c o n t r a s t , X-ray and MNNG seem Figures 19-24 Unscheduled -*HTdR incorporation i n cultured f i b r o b l a s t s of Xeroderma pigmentosum patients ( • ) and c o n t r o l persons ( O ) following short term exposures to carcinogens and mutagenst (Figure 19) 4NQ0 (1 . 5 hours) (Figure 20) MNNG (3 hours) (Figure 21) N-Acetoxy-4-AAS (5 hours) (Figure 22) MMS (1 . 5 hours) (Figure 23) MCA Epoxide (3 hours) (Figure 24) NMU (1 hour) 4-9 TABLE I I Unscheduled DNA Synthesis i n Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to Chemical Carcinogens Normal Repair C a p a c i t y * Reduced Repair C a p a c i t y MNNG 4NQ0 & c a r c i n o g e n i c d e r i v a t i v e s MMS 3-Me-4NP0 EMS MCA-6,7-Epoxide (K-region) NMU BA-5,6-Epoxide (K-region) HN 2 N-Acetoxy-2-AAF ICR-191 N-Hydroxy-2-AAF S t r e p t o n i g r i n N-Acetoxy-4-AAS Methylguanidine N-Hydroxy-4-AAS ( N i t r o s a t i o n ) N-Acetoxy- k-AABP N-Hydroxy-4-AABP N-Acetoxy-2-AAP N-Hydroxy-2-AAP N-Myristoyloxy-2-AAF N-Acetoxy-3-AAF 1'-Hydroxy-Safrole 3'-Hydroxy-Safrole 3'-Acetoxy-Safrole 1,l-Diphenyl-2-propynyl-N-c y c l o h e x y l c a r b i n o l 1 - P h e n y l - l - ( 3 , 4 - x y l y l ) - 2 - p r o p y n y l cyclohexylcarbamate DMN ( a c t i v a t i o n ) A f l a t o x i n ( a c t i v a t i o n ) X-ray UV * Compared to l e v e l of unscheduled JHTdR i n c o r p o r a t i o n i n f i b r o b l a s t s from c o n t r o l persons. to e l i c i t a normal degree of DNA r e p a i r s y n t h e s i s i n XP c e l l s (Cleaver, 1 9 7 1 a ; S t i c h e t a l . , 1 9 7 2 b ) . I t seems l i k e l y t h a t the two groups of agents d i f f e r i n the way i n which they cause s i n g l e - s t r a n d breaks (Regan and Setlow, 1 9 7 4 ) . An i n c i s i o n - p r o d u c i n g enzyme seems to be r e q u i r e d t o induce s t r a n d breakage and i n i t i a t i o n of r e p a i r processes f o l l o w i n g u l t r a v i o l e t , n i t r o q u i n o l i n e oxides and the aromatic amide d e r i v a t i v e s , whereas i t s presence seems unnecessary to produce DNA str a n d breaks and 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 agents of the second group, 2 . 2 . V a r i a t i o n i n DNA Repair C a p a c i t y With respect to UV-induced DNA a l t e r a t i o n s , v a r i o u s l e v e l s of DNA r e p a i r d e f i c i e n c i e s have been found i n c u l t u r e d c e l l s obtained from s e v e r a l u n r e l a t e d XP p a t i e n t s (Bootsma et a l . , 1 9 7 0 ; Cleaver, 1970 , 1972 ; S t i c h e t a l . , 1 9 7 2 b ; Robbins et a l . , 1 9 7 2 , 1 9 7 4 ) . Despite the f a c t t h a t s l i g h t l y d i f f e r e n t techniques were used i n d i f f e r e n t l a b o r a t o r i e s to estimate the degree of d e f i c i e n c y and tha t the r e s u l t s may not be s t r i c t l y comparable, the data c l e a r l y demonstrate t h a t among the d i f f e r e n t XP p a t i e n t s examined the DNA r e p a i r s y n t h e s i s v a r i e s from an undetectable l e v e l t o 100% of t h a t found i n c e l l s of n o n - a f f l i c t e d c o n t r o l persons f o l l o w i n g U V - i r r a d i a t i o n . Complementation s t u d i e s (somatic c e l l h y b r i d i s a t i o n between c e l l s of two u n r e l a t e d XP p a t i e n t s ) suggest the presence of d i f f e r e n t gene mutations among XP p a t i e n t s (De Weerd-Kastelein et a l . , 1 9 7 2 , 1 9 7 3 , 1 9 7 4 ) . The q u e s t i o n then a r i s e s as to whether the XP c e l l s , which g r e a t l y d i f f e r i n t h e i r c a p a c i t y to r e p a i r UV-induced DNA changes, a l s o d i f f e r i n t h e i r response to c h e m i c a l l y induced DNA a l t e r a t i o n s . C e l l c u l t u r e s of s i x XP p a t i e n t s were compared f o r the degree of 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 exposure t o UV and a s e l e c t e d group of chemical carcinogens. Each compound was used a t a c o n c e n t r a t i o n which r e s u l t s i n a very high degree, i f not the maximum, of unscheduled DNA s y n t h e s i s . The r e s u l t s are expressed i n percentages of DNA r e p a i r s y n t h e s i s found i n c u l t u r e d c e l l s of twelve u n a f f e c t e d c o n t r o l persons (Table I I I ) . The l e v e l s of DNA r e p a i r s y n t h e s i s among the u n r e l a t e d XP p a t i e n t s d i f f e r e d s i g n i f i c a n t l y and were b a r e l y a f f e c t e d by the type of i n i t i a t i n g agent used. S i s t e r ( X P ^ ) and brother ( X P ^ ) * however, showed a comparable degree of d e f i c i e n c y ( F i g . 2 5 ) . The s i m i l a r response of s i b members (XP^^, and X P ^ t x p c i a n d X PC2^ f o l l o w i n g such di v e r s e obnoxious agents as N-hydroxy-AAF, N-acetoxy-AAF, k - n i t r o q u i n o l i n e 1-oxide and u l t r a v i o l e t i n d i c a t e s g e n e t i c c o n t r o l of the degree of impairment. 2.3. XP w i t h Normal DNA Repair C a p a c i t y I t has been suggested th a t the c l i n i c a l m a n i f e s t a t i o n s of XP ( h y p e r s e n s i t i v i t y to UV and m u l t i p l e a c t i n i c s k i n l e s i o n s ) could be a t t r i b u t e d to the d e f e c t i v e or d e f i c i e n t DNA r e p a i r mechanism i n these p a t i e n t s . Whether the degree of DNA r e p a i r d e f i c i e n c y v a r i e s w i t h the s e v e r i t y of the TABLE I I I Comparative L e v e l s of DNA Repair Synthesis i n Xeroderma Pigmentosum F i b r o b l a s t s F o l l o w i n g Exposure to UV, 4NQ0, N-Acetoxy-2-AAF and MNNG U l t r a v i o l e t 4NQ0 N-Acetoxy-2-AAF MNNG . P a t i e n t Sex 1000 ergs 2xl0" 6M 5 x 1 0 " ^ 2xlO"%I 2 per mm 1 . 5 Hr 5 Hr 1 . 5 Hr X P H 1 F 9.8 11 . 5 10 . 4 103 X P H 2 M 12.1 13.1 16.0 103 -X P E F 21 . 5 26 . 9 22 . 9 102 X P C 1 M 3 3 . 3 3 2 . 6 31.1 110 X P C 2 F 29.4 3 1 . 9 2 7 . 4 108 XP V F 36.0 3 3 . 6 29.1 105 X P K M 5 6 . 6 53.8 6 0 . 7 107 Heterozygotes X P E Father 99.8 102 . 9 101.1 104 XP £ Mother 102 111 . 5 106 101 X P C Father 104 120.2 98 . 6 112 XP Q Mother 102 122.1 1Q2 106 XP y Mother 101 108 . 5 100 102 XP K Father 1 2 3 . 5 94.2 104 106 X P K Mother 116.1 95.2 106 109 C o n t r o l * M+F 100% 100% 100% 100% * F i b r o b l a s t s of twelve.unaffected s u b j e c t s ( s i x males and s i x females) were used to o b t a i n a c o n t r o l l e v e l of DNA r e p a i r s y n t h e s i s . The unscheduled i n c o r p o r a t i o n of %TdR v a r i e d w i t h i n 11% among the 12 i n d i v i d u a l s examined. The average number of g r a i n s above n u c l e i of c e l l s which were i r r a d i a t e d or exposed to v a r i o u s chemical agents was put at 100%. 53 C O N C E N T R A T I O N C M ) Figure 25 Unscheduled incorporation of ^HTdR into n u c l e i of normal and four XP f i b r o b l a s t s exposed f o r 5 hours to N-hydroxy-AAF. Autoradiography. O Normal person • XP^ (Kamloops) • XPy (Vancouver) A X P H 1 (Hamilton) T X P H 9 (Hamilton) c l i n i c a l p i c t u r e i s d i f f i c u l t t o assess at the moment. The f a c t t h a t a few cases of p a t i e n t s w i t h d e f i c i e n t XP symptoms have a normal DNA r e p a i r c a p a c i t y argues a g a i n s t a c a u s a l r e l a t i o n s h i p between the c l i n i c a l m a n i f e s t a t i o n s and an i n c i s i o n d e fect a t the molecular l e v e l (Robbins et a l . , 1972? C l e a v e r , 1972 ; Robbins and Burk, 1973). 2.4. DNA Repair C a p a c i t y i n XP Heterozygotes F o l l o w i n g  Exposure to Chemical Carcinogens C u l t u r e d c e l l s from XP p a t i e n t s have been shown t o e x h i b i t DNA r e p a i r d e f i c i e n c y f o l l o w i n g exposure to some but not a l l chemical carcinogens ( S e c t i o n 2 . 1 . ) . Furthermore, w i t h r e s p e c t t o UV or chemical-induced DNA a l t e r a t i o n s , v a r i o u s l e v e l s of DNA r e p a i r d e f i c i e n c y have been found i n c u l t u r e d c e l l s obtained from s e v e r a l u n r e l a t e d XP p a t i e n t s . The question' f o l l o w s whether the parents of XP p a t i e n t s , being o b l i g a t e heterozygotes, would manifest a v a r i a t i o n i n DNA r e p a i r c a p a c i t y . The l e v e l of unscheduled ^HTdR i n c o r p o r a t i o n i n c u l t u r e d c e l l s from d i f f e r e n t XP heterozygotes f o l l o w i n g exposure to s e v e r a l chemical carcinogens are not s i g n i f i c a n t l y d i f f e r e n t from t h a t i n the c o n t r o l c e l l s ( S e c t i o n 2 . 2 . , Table I I I ) . Other heterozygote s t u d i e s have r e v e a l e d v a r i a b l e r e s u l t s w i t h r e p a i r of UV-induced DNA damage ranging from 50 to 100 percent of normal (Cleaver, 1969, 1971b, 1972 > Bootsma et a l . , 1970; K l e i j e r e t a l . , 1973). However, si n c e some of the techniques employed d i d not measure r e p a i r a c t i v i t y d i r e c t l y , the f i n d i n g s are only 55 suggestive of d i f f e r e n c e s among the v a r i o u s heterozygotes. 3. DNA Repair. Chromosome A b e r r a t i o n s and Clone Forming  A b i l i t y i n Normal and Xeroderma Pigmentosum C e l l s V a r i a t i o n s i n s e n s i t i v i t y w i t h i n the human p o p u l a t i o n should be considered when d e c i d i n g on " p e r m i s s i b l e " or " s a f e " l e v e l s of c a r c i n o g e n i c or mutagenic agents. At present t h i s aspect of environmental c a r c i n o g e n e s i s and mutagenesis i s somewhat neglected because of the s c a r c i t y of r e l i a b l e i n f o r m a t i o n about the range of s e n s i t i v i t y w i t h i n the human p o p u l a t i o n and the response of s e n s i t i v e c e l l s towards p a r t i c u l a r chemical compounds. I n the preceding s e c t i o n the r e p a i r of DNA damage i n XP c e l l s exposed t o v a r i o u s chemical carcinogens was examined. This chapter reviews the frequency of chromosome a b e r r a t i o n s i n c u l t u r e d c e l l s of p a t i e n t s w i t h Xeroderma pigmentosum and of c o n t r o l persons f o l l o w i n g exposure to v a r i o u s chemical carcinogens. Chromatid breaks, isochromatid breaks and s i n g l e and m u l t i p l e exchanges which can be r e a d i l y q u a n t i f i e d were employed as a s e n s i t i v e i n d i c a t o r of an induced damage to the genome of normal and XP c e l l s . The second q u e s t i o n r a i s e d i s whether any d i f f e r e n c e s between normal and XP c e l l s i n response to chemical carcinogens can a l s o be detected a t the c e l l u l a r l e v e l . Clone forming c a p a c i t y of normal and XP c e l l s f o l l o w i n g exposure to v a r i o u s chemical carcinogens was used as one endpoint. As previously described, XP c e l l s with varying degree of DNA r e p a i r deficiency respond to c e r t a i n chemical carcinogens d i f f e r e n t l y (Section 2.1.). The question then a r i s e s as to whether such differences i n DNA re p a i r capacity are r e f l e c t e d i n the frequency of chromosome aberrations and clone forming e f f i c i e n c y a f t e r exposure to chemical carcinogens, 3.1. Chromosome Aberrations i n Normal and XP C e l l s Following Exposure to Chemical Carcinogens The frequency of metaphase plates with chromosome aberrations i n populations of cultured normal and XP c e l l s following exposure to various chemical carcinogens i s shown i n F i g . 30 - 35. The types of chromosome aberrations observed include single or multiple chromatid breaks, single or multiple chromatid exchanges and chromatid fragmentation (Fig. 26-29). Two types of chemicals were examined* (1) those that induce DNA changes that can be normally repaired by XP c e l l s and (2) compounds that evoke DNA a l t e r a t i o n that cannot be corrected at a normal rate by the r e p a i r d e f i c i e n t XP c e l l s . Among the compounds that lead to r e l a t i v e l y high l e v e l s of DNA r e p a i r synthesis i n control c e l l s but evoke only low l e v e l s of unscheduled -'HTdR uptake i n XP c e l l s , the following are included; 4-NQ0, BA-epoxide, N-hydroxy-2-AAF, N-acetoxy-2-AAF, N-hydroxy-4-AAS, and N-acetoxy-4-AAS. In the XP c e l l s , a s t r i k i n g increase i n chromatid aberrations occurred at doses of carcinogens which did not s i g n i f i c a n t l y 57 and sampled 20 hours post treatment. (Figure 26) Two t r a n s l o c a t i o n f i g u r e s . (Figures 27-28) Chromatid breaks, m u l t i p l e chromatid exchanges and chromatid fragments. (Figure 29) Severe fragmentation of the e n t i r e chromosome complement. Figures 30-3 5 Frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n XP ( • ) and normal ( O ) c e l l c u l t u r e s f o l l o w i n g a s i n g l e dose of a carcinogens-(Figure 30) (Figure 3 D (Figure 32) (Figure 33) (Figure 3 k ) (Figure 35) 4-NQO (1.5 hours) BA Epoxide (3 hours) N-Hydroxy-2-AAF (5 hours) N-Acetoxy-2-AAF (5 hours) N-Hydroxy-4-AAS (5 hours) N-Acetoxy-4-AAS (5 hours) UJ U l Ul 5 B A - S . S - E P O X I O E C O N C E N T R A T I O N I M I N - H Y D R O X Y - 3 - A A F S K I O I O 1 . 6 » 1 Q C O N C E N T R A T I O N IM) N - A C E T O X Y - 2 - A A F 2.5xio"° i o - 5 < j K i ' a " s 3 x i a " ' i o _ " 1 0 C O N C E N T R A T I O N - C M I C O N C E N T R A T I O N I M I I8 s g (I) < m or < 5 < 5 ui • IOOI N - H Y O R O X Y - O - A A S N - A C E T O X Y - 4 - A A S e « i o " 7 3«icr* G . I C T ' O X K T * C O N C E N T R A T I O N C M ) C O N C E N T R A T I O N C M ) e l e v a t e the frequency of chromosome a b e r r a t i o n s i n the . c o n t r o l f i b r o b l a s t s ( F i g . 3 0 - 3 5 ) . About 100 to 1 , 0 0 0 times higher c o n c e n t r a t i o n of these carcinogens must be a p p l i e d to normal c e l l s i n order to o b t a i n frequencies of a b e r r a t i o n s comparable t o those induced i n the s e n s i t i v e XP c e l l s . Since an e x c l u s i v e r e l i a n c e on one sampling p e r i o d could e a s i l y l e a d to erroneous c o n c l u s i o n s , a time study has been performed w i t h 4NQ0 ( F i g . 36) and BA - 6 , 7-epoxide ( F i g . 3 7 ) . For a two-day p e r i o d the frequency of XP metaphase p l a t e s w i t h chromosome a b e r r a t i o n s was above that seen i n e q u a l l y t r e a t e d normal c e l l s . T h e r e a f t e r the l e v e l of chromosome a b e r r a t i o n s of the 4-NQO or BA - 6 , 7-epoxide exposed normal and XP c e l l s was i n the range of untreated c e l l s . Thus the 4NQ0 or BA - 6 , 7-epoxide c o n c e n t r a t i o n used induced a wave of chromosome a b e r r a t i o n s i n the XP c e l l s . To answer the q u e s t i o n whether the l e n g t h of exposure to a chemical carcinogen has any e f f e c t on the frequency of chromosome a b e r r a t i o n s i n the t r e a t e d c e l l s , the data on two examples are presented here. C u l t u r e d normal XP c e l l s exposed to N-acetoxy-2-AAF (5xl0~^M) or N-acetoxy -4-AAS (5xl0~^M) showed an increase i n the frequency of chromosome a b e r r a t i o n s w i t h exposure time ( F i g . 3 8 ) . A p a r t i c u l a r exposure time ( 5 h. i n t h i s i n s t a n c e ) was a r b i t r a r i l y chosen f o r the comparative study between XP and c o n t r o l c e l l s . MNNG was chosen as a r e p r e s e n t a t i v e of the group of chemical carcinogens t h a t e l i c i t comparable l e v e l s of DNA Figure 36 Frequency of metaphase p l a t e s with chromosome a b e r r a t i o n s i n XP ( • ) and normal ( O ) c e l l c u l t u r e s at v a r i o u s times f o l l o w i n g a s i n g l e dose of kNQO (lxlO~'M, 1.5 hours). C= the frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n untreated c u l t u r e s . Figure 37 Frequency of metaphase p l a t e s with chromosome a b e r r a t i o n s i n XP ( • ) and normal ( O) c e l l c u l t u r e s at v a r i o u s times f o l l o w i n g a s i n g l e dose of BA Epoxide ( kxlO~^M, 3 hours). C = the frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n untreated c u l t u r e s . E X P O S U R E T I M E C H O U R S ) Figure 38 E f f e c t of v a r i a t i o n i n exposure time to a chemical carcinogen on the frequency of metaphase plates with chromosome aberrations i n XP ( • ) and normal (O) c e l l c u l t u r e s t -(a) N-Acetoxy-2-AAF (5 x 10"6M) (b) N-Acetoxy-4-AAS (5 x 10~6M) r e p a i r synthesis i n XP c e l l s and controls. The frequency of metaphase plates with chromatid breaks and chromatid exchanges i n the cultured XP c e l l s resembles that found i n control f i b r o b l a s t s treated with equimolar concentrations of MNNG (Figs. 39 and 40). 3.2. Clone-Forming Capacity of Normal and XP C e l l s  Following Exposure to Chemical Carcinogens The response of c e l l s to damaging agents i s to a large extent under genetic c o n t r o l . Bacteria d e f i c i e n t i n the e x c i s i o n mechanism of pyrimidine dimers or i n the recombination process show an increased s u s c e p t i b i l i t y to physical and chemical mutagens (Haynes et a l . , 1968; Kondo and Kato, 1968). The elevated s e n s i t i v i t y was demonstrated by comparing the clone-forming capacity of r e p a i r - d e f i c i e n t mutants with that of normal bacteria. This procedure was s u c c e s s f u l l y applied to cultured c e l l s of patients with XP. The r e p a i r - d e f i c i e n t c e l l s proved to be more sensi t i v e to the l e t h a l e f f e c t of UV-radiation than f i b r o b l a s t s from n o n - a f f l i c t e d persons (Stich et a l . . 1972a; Maher et a l . , 1975b). Somatic c e l l s of XP patients are also defective i n the r e p a i r of DNA a l t e r a t i o n s induced by various chemical carcinogens (Section 2.1.). The question must be r a i s e d whether the reduced r e p a i r l e v e l of chemically induced DNA lesions i n the XP c e l l s i s r e f l e c t e d i n an increased s e n s i t i v i t y to the inducing agents. Figure 39 Frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d XP c e l l s (n ) and normal c e l l s ( O , v ) exposed to v a r i o u s c o n c e n t r a t i o n s of MNNG (3 hours). Figure 40 Frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d XP c e l l s ( • ) and normal c e l l s ( O ) at v a r i o u s times f o l l o w i n g a s i n g l e dose of MNNG (1.2xlO"" 5M, 3 hours). r— 1 . i . 10 4 i o - 10 C O N C E N T R A T I O N C M ) C 12 34 36 4S 60 H O U R S A F T E R T R E A T M E N T I n t h i s chapter the clone-forming c a p a c i t y and l e v e l of DNA r e p a i r of XP and normal c e l l s exposed to v a r i o u s chemical carcinogens i s re p o r t e d . As r e p r e s e n t a t i v e examples of the group of chemical carcinogens which e l i c i t e d a reduced l e v e l of DNA r e p a i r s y n t h e s i s i n XP c e l l s , the f o l l o w i n g have been i n c l u d e d * 4NQ0, 4HAQ0, 2-Me-4NQ0, 7-Me-4NQ0, BA-Epoxide, N-hydroxy-2-AAF, N-acetoxy-2-AAF, N-hydroxy -4-AAS and N-acetoxy -4-AAS. The e f f e c t of these compounds on the c a p a c i t y of normal and XP c e l l s t o form clones i s shown i n F i g s 41 - 4-9. The XP c e l l s are h i g h l y s e n s i t i v e t o the l e t h a l e f f e c t of these carcinogens and t h e i r response i s comparable to t h a t f o l l o w i n g U V - i r r a d i a t i o n ( F i g . 50). A s i m i l a r k i l l i n g e f f e c t i n c o n t r o l c e l l s was apparent only a t much higher c o n c e n t r a t i o n s of the carcinogens used. With r e s p e c t to MNNG and MMS, which induced a comparable l e v e l of unscheduled -^ HTdR uptake i n both XP and c o n t r o l c e l l s , there was only a small d i f f e r e n c e i n clone-forming c a p a c i t y between the XP c e l l s and normal ones ( F i g . 51, 52). 3 . 3 . E f f e c t of Chemical Carcinogens on the Frequency of  Chromosome A b e r r a t i o n s and Clone-Forming Capacity  i n XP C e l l s w i t h D i f f e r e n t DNA Repair D e f i c i e n c i e s I t has been found t h a t f i b r o b l a s t s of u n r e l a t e d XP p a t i e n t s do e x h i b i t d i f f e r e n t DNA r e p a i r c a p a c i t i e s ( S e c t i o n 2 . 2 . ) . For example, w i t h respect to the r e p a i r of UV-induced Figures 4-1-46 Clone-forming capacity of normal human f i b r o b l a s t s ( O ) and XP c e l l s ( • ) following exposure to a single dose of a carcinogen (expressed as percentage of s u r v i v a l i n each c e l l l i n e ' s untreated c o n t r o l s ) . (Figure 41) 4NQ0 (1.5 hours) (Figure 42) 4HAQ0 (1.5 hours) (Figure 43) 2-Methyl-4NQ0 (1.5 hours) (Figure 44) 7-Methyl-4NQ0 (1.5 hours) (Figure 45) N-Hydroxy-2-AAF (5 hours) (Figure 46) N-Acetoxy-2-AAF (5 hours) F i g u r e s 47-62 Clone-forming c a p a c i t y of normal human f i b r o b l a s t s ( O) and XP c e l l s ( • ) f o l l o w i n g exposure to a s i n g l e dose of a carcinogen. (Figure 4-7) (Figure 48 ) (Figure 49 ) (Figure 50) (Figure 5 D (Figure 52) N-Hydroxy -4-AAS (5 hours) N-Acetoxy -4-AAS (5 hours) BA - 5 .6-Epoxide (5 hours) UV r a d i a t i o n (260nm) MNNG (3 hours) MMS (1.5 hours) 71 DNA damage, the l e v e l of unscheduled ^HTdR uptake i n various XPs (as compared to u n a f f l i c t e d controls) were found to be as follows* XP-Hj 9 . 8 $ , XP-H£ 1 2 . 1 $ , XP-E 2 1 $ , XP-C 3 k $ , XP-V 36$ and XP-K 56$ (Table III and Section 2 . 2 . ) . Furthermore, the d i f f e r e n t l e v e l s of DNA r e p a i r synthesis among the unrelated XP patients were not affected by the type of i n i t i a t i n g agent used (Table I I I and Section 2 . 2 . ) . The question which a r i s e s from t h i s observation i s whether such differences i n DNA r e p a i r capacity i s r e f l e c t e d at the chromosome or c e l l u l a r l e v e l . A comparative study was then performed with c e l l s from four unrelated XP patients. Again, a representative compound was selected from the group of chemical carcinogens which e l i c i t e d a reduced l e v e l of unscheduled ^HTdR uptake i n XP c e l l s (e.g. kNQO, N-acetoxy-2-AAF) and those which provoked a comparable l e v e l of DNA r e p a i r synthesis i n both XP and control c e l l s (e.g. MNNG). In each instance, the l e v e l of unscheduled ^HTdR incorporation, the frequency of metaphase plates with chromosome aberrations and the clone-forming capacity were examined. The r e s u l t s are shown i n Tables IV to VI. I t must be pointed out that, f o r te c h n i c a l reasons, the same concentration of a carcinogen cannot be used i f observable r e s u l t s are to be obtained i n the three assay systems. In general, the concentrations required to produce a detectable l e v e l of unscheduled -^ HTdR uptake are higher than those used f o r chromosome aberrations and cloning studies. With reference to the d i f f e r e n t XPs tested, i t appears t h a t the l e v e l o f DNA r e p a i r d e f i c i e n c y c o r r e l a t e s w i t h the f r e q u e n c y o f chromosome a b e r r a t i o n s and c l o n e f o r m i n g c a p a c i t y . As an example, one can compare the r e s p o n s e o f XP-K and XP-E t o 4NQ0 ( T a b l e I V ) . W i t h r e s p e c t t o DNA r e p a i r c a p a c i t y , XP-K i s a p p r o x i m a t e l y 50$ o f normal whereas XP-E i s 25$. The p e r c e n t a g e o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i s 12.8$ i n XP-K but i t i s more t h a n f o u r f o l d h i g h e r (57.1$) i n XP-E (wh i c h i s more DNA-r e p a i r d e f i c i e n t ) . The c l o n e - f o r m i n g c a p a c i t y a f t e r exposure t o 4NQ0 i n XP-K i s 38$, about two f o l d h i g h e r t h a n t h e 16$ i n the more r e p a i r d e f i c i e n t XP-E. The response o f c e l l s f rom d i f f e r e n t XP p a t i e n t s f o l l o w i n g exposure t o N-a c e t o x y -2-AAF r e s e m b l e s t h a t a f t e r kNQO ( T a b l e V ) . There i s no s i g n i f i c a n t d i f f e r e n c e f o l l o w i n g exposure t o MNNG i n t h e f r e q u e n c y o f chromosome a b e r r a t i o n s and c l o n e - f o r m i n g c a p a c i t y among c e l l s from XP p a t i e n t s w i t h d i f f e r e n t DNA r e p a i r c a p a c i t i e s ( T a b l e V I ) . Moreover, the pe r c e n t a g e o f metaphase p l a t e s w i t h chromosome a b e r r a t i o n s and the c l o n e - f o r m i n g c a p a c i t y i n XP c e l l s w i t h d i f f e r e n t DNA r e p a i r d e f i c i e n c y r e s e m b l e s t h a t f o u n d i n c o n t r o l f i b r o b l a s t s t r e a t e d w i t h e q u i m o l a r c o n c e n t r a t i o n s o f MNNG. 3.k. Chromosome A b e r r a t i o n s and C l o n e - F o r m i n g C a p a c i t y  i n XP H e t e r o z y g o t e s F o l l o w i n g Exposure t o C h e m i c a l  C a r c i n o g e n s I n the p r e c e d i n g s e c t i o n (3.3.), i t has been r e p o r t e d t h a t c e l l s from XP p a t i e n t s w i t h d i f f e r e n t l e v e l s o f DNA TABLE IV L e v e l of Unscheduled -'HTdR I n c o r p o r a t i o n , Frequency of Chromosome A b e r r a t i o n s and Clone Forming C a p a c i t y of C u l t u r e d F i b r o b l a s t s From Xeroderma Pigmentosum P a t i e n t s and C o n t r o l Persons F o l l o w i n g Exposure to 4 - N i t r o q u i n o l i n e 1-Oxide. 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0) DNA Repair Grains/Nucleus Metaphase P l a t e s With Chromosome p A b e r r a t i o n s (%) Clone-Forming Capacity-^ X PH1 12 6 8 . 5 __ 28 57.1 16 X P01 34 20 . 3 33 X P K 56 12.8 38 Heterozygotes X P E Father 10? — — XP £ Mother 116 3.8 71 XP C Father 125 — XP C Mother 12? — 86 X P K Father 98 1 . 5 72 X P K Mother 99 1 . 9 80 C o n t r o l 104 0 . 7 79 C o n t r o l 102 0.0 72 1. Unscheduled i n c o r p o r a t i o n of -'HTdR was measured f o l l o w i n g 1 . 5-hr. exposure to 2xlO~Sl 4NQ0. Autoradiography. 2. Chromosome a b e r r a t i o n s (breaks and exchanges) were counted on f i b r o b l a s t c u l t u r e s exposed to 5xl0~^M 4NQ0 ( 1 . 5 h r . ) . _Q 3 . Clone-forming c a p a c i t y a f t e r 1 . 5-hr. exposure to 10" M 4NQ0 was expressed as % of each c e l l l i n e ' s untreated c o n t r o l . TABLE V L e v e l of Unscheduled ^HTdR I n c o r p o r a t i o n , Frequency of Chromosome A b e r r a t i o n s and Clone Forming Capacity of C u l t u r e d F i b r o b l a s t s From Xeroderma Pigmentosum P a t i e n t s and C o n t r o l Persons F o l l o w i n g Exposure to N-Acetoxy - 2-Acetylaminofluorene. N-Acetoxy - 2-Acetylaminofluorene DNA Repair Metaphase P l a t e s Clone-Forming 1 3 Grains/Nucleus With Chromosome Capacity-^ A b e r r a t i o n s (%) X P H 1 3 7 5 . 9 XP E 6 4 7 . 9 29 X P C 1 9 2 6 . 2 17 X P K 17 1 7 . 6 Heterozygotes X P E Father 28 XP £ Mother 33 2 . 0 76 XP C Father 28 — XP C Mother 29 2 . 8 80 X P K Father 29 3 . 5 X P K Mother 30 — C o n t r o l 27 4 . 7 86 C o n t r o l 29 3 . 2 81 1. Unscheduled i n c o r p o r a t i o n of ''HTdR was measured f o l l o w i n g 5-hr. exposure to 5 X 1 0 ~ 5M N-acetoxy-2-AAF. Autoradiography. 2 . Chromosome a b e r r a t i o n s (breaks and exchanges) were scored on f i b r o b l a s t c u l t u r e s exposed to 5 x l 0~^M N-acetoxy-2-AAF (5 h r . ) . 3 . Clone-forming c a p a c i t y a f t e r 5-hr. exposure to 1 0 " % N-acetoxy-2-AAF was expressed as % of each c e l l l i n e ' s untreated c o n t r o l . 76 TABLE VI L e v e l of Unscheduled ^HTdR I n c o r p o r a t i o n , Frequency of Chromosome A b e r r a t i o n s and Clone Forming Capacity of C u l t u r e d F i b r o b l a s t s From Xeroderma Pigmentosum P a t i e n t s and C o n t r o l Persons F o l l o w i n g Exposure to N-Methyl-N'-Nitro-N-Nitrosoguanidine N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG) DNA Repair Metaphase P l a t e s Clone-Forming 1 3 Grains/Nucleus With Chromosome Capacity-' o A b e r r a t i o n s {%) X PH1 4 1 MM X P E 4 1 56 .2 69 X P01 44 46.6 73 X P K 43 4 8 . 2 — Heterozygotes XP £ Father 4 2 -- MM XP £ Mother 4 0 4 6 . 2 70 XP C Father 45 — — XP C Mother 4 2 4 2 . 0 58 X P K Father 4 2 — _ _ X P K Mother 44 — — C o n t r o l 39 50.0 63 C o n t r o l 4 1 47 .2 68 1. Unscheduled i n c o r p o r a t i o n of JHTdR was measured f o l l o w i n g - 4 3-hr. exposure to 2x10 M MNNG. Autoradiography. 2. Chromosome a b e r r a t i o n s (breaks and exchanges) were scored on f i b r o b l a s t c u l t u r e s exposed to 2.5x10"% MNNG (3 h r . ) . 3. Clone-forming c a p a c i t y a f t e r 3-hr. exposure to 5x10"% MNNG was expressed as $ of each c e l l l i n e ' s untreated c o n t r o l . r e p a i r capacity show d i f f e r e n t s e n s i t i v i t i e s towards 4NQ0 and N-acetoxy-2-AAF with respect to chromosome aberrations and clone-forming capacity. The question follows whether the parents of XP patients, being obligate heterozygotes, would manifest an increased s e n s i t i v i t y towards chemical carcinogens. The l e v e l of unscheduled ^HTdR uptake i n d i f f e r e n t XP heterozygotes following exposure to several chemical carcinogens resembles:that i n control c e l l s (Section 2.2., Table I I I ) . The response of several XP heterozygotes towards 4NQ0, N-acetoxy-2-AAF and MNNG are shown i n Table IV - VI, Section 3 . 3 . . The frequency of chromosome aberrations and clone-forming capacity i n the XP heterozygotes are not s i g n i f i c a n t l y d i f f e r e n t from that i n the control c e l l s . 4. Precarcinogens and Ultimate Carcinogens Many chemical carcinogens are not active at the s i t e of administration but they p r e f e r e n t i a l l y cause tumours i n s p e c i f i c target organs. For instance, 2-acetylamino-fluorene (2-AAF), whether introduced t o p i c a l l y or by subcutaneous i n j e c t i o n , causes predominantly l i v e r tumours ( M i l l e r , 1970). This organotropic phenomenon suggests that the carcinogen requires metabolic a c t i v a t i o n by enzymatic systems i n the target organs. Further support f o r the presence of such a c t i v a t i n g metabolism i s derived from the f a c t that metabolites i s o l a t e d from animals exposed to a chemical carcinogen are more potent than the parent compound ( M i l l e r and M i l l e r , 1969). These me t a b o l i t e s act on l o c a l t i s s u e t a r g e t s as w e l l as the usual remote t a r g e t organs. Chemical carcinogens which r e q u i r e metabolic a c t i v a t i o n are described as precarcinogens while the m e t a b o l i t e s come to be known as proximate ( i f f u r t h e r metabolism i s required) or u l t i m a t e carcinogens. I t now seems c l e a r t h a t the c a r c i n o g e n i c a l k y l a t i n g agents are u l t i m a t e carcinogens i n the form administered ( M i l l e r and M i l l e r , 1971b). They are strong e l e c t r o p h i l e s and r e a c t r e a d i l y and d i r e c t l y w i t h c e l l c o n s t i t u e n t s . Most other chemical carcinogens apparently r e q u i r e metabolic a c t i v a t i o n , u s u a l l y enzymatic, to become u l t i m a t e carcinogens ( M i l l e r and M i l l e r , 1971b). From the f o r e g o i n g d i s c u s s i o n , i t appears l i k e l y t h a t many of the chemical carcinogens the human p o p u l a t i o n comes i n t o contact w i t h are i n the p r e c a r c i n o g e n i c form. I n order to co n s i d e r the use of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s as a pre-screening bioassay f o r environmental chemical carcinogens, i t i s t h e r e f o r e of paramount importance to determine whether t h i s system responds to precarcinogens as w e l l as to proximate and ul t i m a t e carcinogens. I n t h i s chapter, the p r e - c a r c i n o g e n i c , proximate and/or u l t i m a t e c a r c i n o g e n i c forms of key compounds from a few r e p r e s e n t a t i v e groups of chemical carcinogens are examined w i t h respect to t h e i r c a p a c i t y to e l i c i t unscheduled DNA s y n t h e s i s , chromosome a b e r r a t i o n s and r e d u c t i o n i n colony formation i n c u l t u r e d human s k i n f i b r o b l a s t s . These compounds i n c l u d e p o l y c y c l i c aromatic hydrocarbons, aromatic amines, N-oxides and n i t r o s o compounds. 4.1. P o l y c y c l i c Aromatic Hydrocarbons The i d e a t h a t epoxides might be metabolic intermediates i n the o x i d a t i o n of p o l y c y c l i c hydrocarbons was suggested about 25 years ago by Boyland (1950). Based on t h e o r e t i c a l c a l c u l a t i o n s i t i s concluded t h a t the K-region ( f o r m e r l y o f t e n c a l l e d the meso-phenanthrenic region) i s p a r t i c u l a r l y apt t o undergo a d d i t i o n r e a c t i o n s and the chemical r e a c t i v i t y of t h i s double bond has been demonstrated e x p e r i m e n t a l l y (Arcos and Argus, 19?4). Probably because of t h e i r i n s t a b i l i t y , d i r e c t evidence f o r the f o r m a t i o n of the epoxides of c a r c i n o -genic p o l y c y c l i c hydrocarbons i s not a v a i l a b l e . However, the f i n d i n g by Grover e_t a l . (1971t>) t h a t the K-region epoxides of benz(a)anthracene and dibenz(a,h)anthracene transform hamster f i b r o b l a s t s and mouse p r o s t a t e c e l l l i n e s w i t h g r e a t e r e f f i c i e n c y than e i t h e r the parent hydrocarbons, the d i h y d r o d i o l s , or the phenols (which could be d e r i v e d from these epoxides) i s c o n s i s t e n t w i t h the epoxide i n t e r -mediate hypothesis. I n t h i s chapter, the c a p a c i t y of benz(a)anthracene (BA), 20-methylcholanthrene (MCA), t h e i r K-region epoxide and d i h y d r o d i o l to e l i c i t a DNA r e p a i r s y n t h e s i s i s examined. The s t r u c t u r a l formulae of benz(a)anthracene and 20-methylcholanthrene, t h e i r K-region epoxides and the corresponding d i h y d r o d i o l are depicted i n F i g . 5 3 . An unscheduled i n c o r p o r a t i o n of "^ HTdR i n t o n u c l e a r DNA was observed i n n o n - d i v i d i n g c e l l s exposed f o r 5 h. to BA - 5 , 6-epoxide at c o n c e n t r a t i o n s ranging from 10~-* to 5 x 1 0 ' ^ ( F i g . 5 4 ) . At these doses the precarcinogen BA and the metabolite B A - c i s - 5 , 6 - d i h y d r o d i o l d i d not t r i g g e r a d e t e c t a b l e amount of DNA r e p a i r s y n t h e s i s . A s i m i l a r p a t t e r n was observed w i t h 2 0-methylcholanthrene (MCA), i t s a c t i v e K-region epoxide and i n a c t i v e d i h y d r o d i o l ( F i g . 5 5 ) . 4 . 2 . Aromatic Amines Wi t h i n the c l a s s of f u l l y aromatic n o n - s u b s t i t u t e d r i n g systems, i t i s among the penta- and h e x a c y c l i c hydrocarbons t h a t the most potent carcinogens are found. Except f o r tv/o compounds no u n s u b s t i t u t e d p o l y c y c l i c hydrocarbon below f o u r condensed r i n g s possess d e t e c t a b l e c a r c i n o g e n i c a c t i v i t y (Arcos and Argus, 1 9 7 4 ) . However, i f an amino group i s introduced at s p e c i f i c p o s i t i o n s , these r i n g systems o f t e n acquire a very high l e v e l and m u l t i -t a r g e t c a r c i n o g e n i c a c t i v i t y . Furthermore, metabolic i n t e r c o n v e r s i o n of the amino group w i t h the hydroxylamino and n i t r o s o s u b s t i t u e n t s produces d e r i v a t i v e s which are s u b s t a n t i a l l y more a c t i v e and u b i q u i t o u s l y c a r c i n o g e n i c than the amines themselves. The N-hydroxy m e t a b o l i t e s of s e v e r a l aromatic amines have been demonstrated to be proximate carcinogens. N-hydroxylation i s a c r i t i c a l Figure 53 S t r u c t u r a l formula of the precarcinogens BA and 2 0-methylcholanthrene, the h i g h l y r e a c t i v e K-region expoxide (proximate carcinogen) and the corresponding d i h y d r o d i o l ( i n a c t i v e m e t a b o l i t e ) . Figure 5 k Unscheduled DNA s y n t h e s i s i n normal human c e l l s exposed f o r 3 hours to BA ( • ) , BA - 5 , 6-epoxide ( or B A - c i s - 5 , 6 - d i h y d r o d i o l ( • ) . Figure 55 Unscheduled DNA s y n t h e s i s i n normal human c e l l s exposed f o r 3 hours to 2 0-methylcholanthrene (• MCA - 6 , 7-epoxide ( v ) or MC A - 6 , 7 - d i h y d r o d i o l ( • ) . metabolic a c t i v a t i o n step of aromatic amine carcinogens and has been found to take place i n v i r t u a l l y every t i s s u e and species i n which these agents e x e r t a c a r c i n o g e n i c e f f e c t (Arcos and Argus, 1974). A second a c t i v a t i o n by e s t e r i f i c a t i o n y i e l d s d e r i v a t i v e s (e.g. N-acetoxy and N- s u l f a t e ) which are more a c t i v e than the N-hydroxy compounds ( M i l l e r and M i l l e r , 1969). I n t h i s chapter, the f o l l o w i n g aromatic amines (pre-carcinogens) and t h e i r corresponding N-hydroxy and N-acetoxy d e r i v a t i v e s (proximate and u l t i m a t e carcinogens r e s p e c t i v e l y ) are examined f o r t h e i r c a p a c i t y to e l i c i t DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s : 2-acetylamino-f l u o r e n e (2-AAF), 2-acetylaminophenanthrene, 4-acetylaminobi-phenyl and 4 - a c e t y l a m i n o s t i l b e n e ( F i g . 56). 4 . 2 . 1 . 2-Acetylaminofluorene 2-Acetylaminofluorene (or 2-fluorenylacetamide) was used as a h i g h l y e f f e c t i v e i n s e c t i c i d e u n t i l Wilson e t a l . (1941) d i s c o v e r e d t h a t r a t s administered 2-AAF developed tumours i n the l i v e r and i n v a r i o u s t i s s u e s and i n t e r n a l organs. Furthermore, i t was found t o be metabolized i n p a r t to N-hydroxy -2-AAF (Cramer et a l . , i960). This N-hydroxy met a b o l i t e i s a more a c t i v e and v e r s a t i l e carcinogen than the parent compound. Subsequent s t u d i e s s t r o n g l y i n d i c a t e t h a t the i n v i v o r e a c t i v i t y of the N-hydroxy compounds i s dependent, a t l e a s t i n p a r t , on the formation . of e s t e r s of these compounds ( M i l l e r and M i l l e r , 1969). 84 2-Acetylaminofluorene (2-AAF) 2-Acetylaminophenanthrene (2-AAP) 5 6" • 6 5 *(/ \ W / V f l-OOH 3 4 H g 3' 1 2 3 4-Acetylaminobiphenyl (4-AABP) 6 5 0 4-Acetylaminostilbene (4-AAS) 2 3 F i g u r e 56 S t r u c t u r e of the c a r c i n o g e n i c aromatic amines i 2-Acetylaminofluorene (2-AAF) 2-Acetylaminophenanthrene (2-AAP) 4-Acetylaminobiphenyl (4-AA3P) 4-Acetylaminostilbene (4-AAS) The data a l s o suggest t h a t the e s t e r s are more proximate carcinogens than the parent N-hydroxy d e r i v a t i v e s . This l e d to the p o s t u l a t i o n the N-acetoxy and N-hydroxy-2-AAF are the u l t i m a t e and proximate c a r c i n o g e n i c forms of 2-AAF r e s p e c t i v e l y ( F i g . 57). I n t h i s chapter, DNA r e p a i r s y n t h e s i s was examined on normal human f i b r o b l a s t s exposed to the precarcinogen 2-AAF and i t s proximate and u l t i m a t e c a r c i n o g e n i c forms, N-hydroxy-2-AAF and N-acetoxy-2-AAF r e s p e c t i v e l y . The r e s u l t s are shown i n F i g . 57. At e q u i m o l a r i t y , the c h e m i c a l l y r e a c t i v e N-acetoxy-2-AAF i s more a c t i v e than N-hydroxy-2-AAF i n producing DNA l e s i o n s r e s u l t i n g i n a r e p a i r s y n t h e s i s . At h i g h e r c o n c e n t r a t i o n s , N-hydroxy-2-AAF may be a c t i v a t e d by enzymatic e s t e r i f i c a t i o n . This response of c u l t u r e d f i b r o b l a s t s i s s i m i l a r to t h a t observed i n human p e r i p h e r a l lymphocytes (Lieberman et a l . , 1971a). 2-AAF shows a very weak c a p a c i t y f o r e l i c i t i n g a DNA r e p a i r s y n t h e s i s and t h i s only a t very high c o n c e n t r a t i o n s . I n a c t i v e m e t a b o l i t e s of N-hydroxy-2-AAF which were found i n the u r i n e of t r e a t e d r a t s hav^e been i d e n t i f i e d as 1-, 3-, 5- and 7-hydroxy-2-AAF (Arcos and Argus, 197 k). These non-carcinogenic m e t a b o l i t e s were i n c l u d e d i n the present i n v e s t i g a t i o n . No unscheduled DNA s y n t h e s i s was e l i c i t e d i n c u l t u r e d human f i b r o b l a s t s by these compounds (Table V I I ) . 4.2.2. 2-Acetylaminophenanthrene 2-Aminophenanthrene i s a very potent carcinogen i n the Figure 57 Unscheduled i n c o r p o r a t i o n of "'HTdR i n t o n u c l e i of normal human f i b r o b l a s t s exposed f o r 5 hours to the precarcinogen 2-AAF ( • ), proximate carcinogen - N-hydroxy-2-AAF ( v ) or u l t i m a t e carcinogen -N-acetoxy-2-AAF ( O ) . Autoradiography. \ /rA // Hydroxylation HO 0 2-Acetylaminofluorene (2-AAF) (Precarcinogen) N-Hydroxy-2-AAF (Proximate Carcinogen) Ester i f i c a t i o n 0 II N-C-CH3 O-C-CH^ II J 0 N-Acetoxy-2-AAF (Ultimate Carcinogen) 3 0 1 0 1 0 * 1 0 C O N C E N T R A T I O N CM) I O TABLE VII Unscheduled DNA synthesis i n Cultured Human Fibr o b l a s t s Following 5-Hour Exposure to Active and Inactive Metabolites of 2-Acetylaminofluorene (2-AAF) Grains Per Nucleus* Concentration (M) IO' 3 3.3x10 * -4 10 * 5xl0~5 2.5X10"-5 1.2X10*"-5 6xlo" 6 i o " 6 2-AAF 0.1 0.6 0.3 0.4 0.2 — — — N-Hydroxy-2-AAF - - 18.0 13.0 10.0 • 5 3 3 N-Acetoxy-2-AAF - - 27.0 21.0 17.0 8 4 2 l-Hydroxy-2-AAF 1.8 0.5 0.2 0.2 0.1 - -3-Hydroxy-2-AAF 0.6 0.2 0.4 0 0.6 -5-Hydroxy-2-AAF 0 0 0.1 0.1 0.1 - -7-Hydroxy-2-AAF 0.5 0.1 0.3 0.1 0 — - -* '*-" denotes concentration not tested. r a t when administered o r a l l y (Huggins and Yang, 1Q62« Dannenberg and Huggins, 1 9 6 9 ) . The N-acetylated form, 2-acetylaminophenanthrene, appears s l i g h t l y l e s s a c t i v e than the f r e e amine (Hartman e t a l . , 1959s Dannenberg and Huggins, 1 9 6 9 ) . Tumours induced by these compounds i n c l u d e t h a t of the mammary gland ( i n females) and of the ear duct and g a s t r o i n t e s t i n a l t r a c t , and leukemia. N-hydroxy - 2-acetylaminophenanthrene has been demonstrated to be the proximate carcinogen of the parent amide. Rats fe d 2-acetylaminophenanthrene excrete s u b s t a n t i a l amounts of the N-hydroxy m e t a b o l i t e , mostly as glucuronides ( M i l l e r et a l . , 1 9 6 6 b ) . N-hydroxy - 2-acetylaminophenanthrene i s a more potent carcinogen than the parent amide. The N-acetoxy - 2-acetylaminophenanthrene, an a c t i v a t i o n product by e s t e r i f i c a t i o n , was shown to be more a c t i v e than the N-hydroxy d e r i v a t i v e ( M i l l e r and M i l l e r , 1 9 6 9 ) . In t h i s chapter, the c a p a c i t y of 2-acetylaminophenan-threne (2-AAP) and i t s proximate (N-OH) and ultimate(N-Ac) c a r c i n o g e n i c forms to e l i c i t a DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s i s examined. The r e s u l t s are shown i n F i g . 5 8 . At equimolar c o n c e n t r a t i o n s , N-acetoxy-2-AAP i s more a c t i v e than N-hydroxy-2-AAP i n i n f l i c t i n g DNA damage r e s u l t i n g i n a r e p a i r s y n t h e s i s . The parent compound 2-AAP f a i l e d t o e l i c i t any unscheduled DNA s y n t h e s i s even at much higher c o n c e n t r a t i o n s . k . 2 . 3 . 4 -Aminostilbene .90 F i g u r e s 58-60 Unscheduled i n c o r p o r a t i o n of ^ HTdR i n t o n u c l e i of normal human f i b r o b l a s t s exposed f o r 5 hours to the p r e c a r c i n o g e n i c , proximate or u l t i m a t e c a r c i n o -genic forms of aromatic amines. Autoradiography. (Figure 58) 2-Acetylaminophenanthrene ( • ) - precarcinogen. N-Hydroxy-2-AAP ( v ) - proximate carcinogen. N-Acetoxy-2-AAP ( O ) - u l t i m a t e carcinogen. (Figure 59) 4-Acetylaminostilbene ( • ) - precarcinogen. N-Hydroxy-4-AAS ( v ) - proximate carcinogen. N-Acetoxy-4-AAS ( O) - u l t i m a t e carcinogen. (Figure 60) 4-Acetylaminobiphenyl ( • ) - precarcinogen. N-Hydroxy-4-AABP ( v ) - proximate carcinogen. N-Acetoxy-4—AABP ( O ) - u l t i m a t e carcinogen. I n the search f o r tumour i n h i b i t o r y substances, Haddow e t a l . , (1948) discovered 4-aminostilbene. The compound as w e l l as the N-acetylated d e r i v a t i v e , 4-AAS was l a t e r found t o be an a c t i v e carcinogen. Administered subcutaneously or o r a l l y , these two compounds induced a whole spectrum of neoplasms i n the r a t (mammary gland, ear duct, l u n g , kidney, i n t e s t i n e s ) . The nature and d i s t r i b u t i o n of these tumours are very s i m i l a r to those produced by 2-AAF (Haddow, 1953). N-hydroxy-4-AAS i s ex c r e t e d i n the u r i n e , mainly conjugated w i t h g l u c u r o n i c a c i d , i n r a t s f e d 4-AAS (Baldwin and Romerii, 1965). I n the M i l l e r ' s group, the N-hydroxy d e r i v a t i v e was found t o be a d e f i n i t e l y stronger carcinogen than e i t h e r 4-amino or 4-acetylaminostilbene toward the mammary gland, forestomach, subcutaneous t i s s u e and s m a l l i n t e s t i n e i n the r a t , but e q u a l l y c a r c i n o g e n i c toward the ear duct glands (Andersen e t a l . , 1964). I n t h i s chapter, the parent amide, 4-aminostilbene, i t s N -acetylated d e r i v a t i v e , 4 - a c e t y l a m i n o s t i l b e n e , and the presumed proximate and u l t i m a t e c a r c i n o g e n i c forms (N-hydroxy-4-AAS and N-acetoxy-4-AAS) are examined f o r t h e i r c a p a c i t y t o i n f l i c t DNA damage i n c u l t u r e d human f i b r o b l a s t s . The r e s u l t s are shown i n F i g . 59. The presumed proximate and u l t i m a t e c a r c i n o g e n i c -.'orms (N-0H-4-AAS and N-Ac-4-AAS) e l i c i t e d an unscheduled DNA sy n t h e s i s w i t h i n the c o n c e n t r a t i o n range s t u d i e d . Exposure to the parent compound d i d not r e s u l t i n any de t e c t a b l e l e v e l of unscheduled DNA synthesis i n cultured human f i b r o b l a s t s . 4.2.4. 4-Aminobiphenyl Pure biphenyl i s not a carcinogen, but 4-aminobiphenyl (4-ABP), a substantial contaminant i n the dye manufacturing industry, has been shown to be a powerful bladder carcinogen i n man (Arcos and Argus, 1974). Given by the subcutaneous or o r a l route to r a t , 4-aminobiphenyl induces tumours i n the mammary gland, acoustic sebaceous gland, l i v e r and small i n t e s t i n e (Walpole and Williams, 1958). The carcino-genic potencies of 4ABP and 4AABP are roughly comparable (Walpole and Williams, 1958). N-hydroxy-4-acetylamino-biphenyl and N-acetoxy-4-acetylarainobiphenyl e l i c i t a considerable increase i n carcinogenic a c t i v i t y and spectrum of tissue targets ( M i l l e r and M i l l e r , 1969). This f i n d i n g i s consistent with the assumption that N-hydroxy- and N-acetoxy- derivatives are the proximate and ultimate carcinogenic forms re s p e c t i v e l y of the parent compound, 4-acetylaminobiphenyl. In t h i s section, 4-aminobiphenyl, 4-acetylaminobiphenyl and the assumed proximate and ultimate carcinogenic forms (N-hydroxy-4-AABP and N-acetoxy-4-AABP) are examined f o r t h e i r DNA damaging capacity i n cultured human f i b r o b l a s t s . An unscheduled incorporation of ^ HTdR i s observed following exposure to the N-hydroxy- and N-acetoxy- derivatives ( Fig. 60) No unscheduled DNA synthesis was detected i n the cases of 4ABP and 4AABP. 94 4.2.5. Summary on Aromatic Amines Among a l l f o u r groups of aromatic amines t e s t e d , the N-hydroxy- and N-acetoxy- d e r i v a t i v e s (the presumed proximate and u l t i m a t e c a r c i n o g e n i c forms of the parent compounds) possess the c a p a c i t y to induce DNA a l t e r a t i o n s r e s u l t i n g i n an unscheduled DNA s y n t h e s i s . At equiraolar c o n c e n t r a t i o n s the parent compounds (precarcinogens) f a i l e d to e l i c i t any unscheduled i n c o r p o r a t i o n of -'HTdR. Fo l l o w i n g exposure to the parent amines, DNA r e p a i r s y n t h e s i s was observed, i f a t a l l , only when very high doses of the compounds were used. The s i m p l e s t i n t e r -p r e t a t i o n i s to assume t h a t c u l t u r e d human f i b r o b l a s t s c o u l d a c t i v a t e t o a l i m i t e d degree p r e c a r c i n o g e n i c aromatic amines. The precarcinogens would r e q u i r e metabolic a c t i v a t i o n before d i r e c t i n t e r a c t i o n w i t h DNA and DNA r e p a i r s y n t h e s i s c o u l d occur. The r e s u l t s show t h a t the assay system i s s u i t a b l e f o r the d e t e c t i o n of proximate and u l t i m a t e carcinogens o n l y , as i t may y i e l d " f a l s e " n egatives w i t h precarcinogens. 4.3. N-Oxides The N-oxides are i n c l u d e d i n t h i s d i s c u s s i o n because there i s mounting evidence t h a t some of them may r e q u i r e metabolic conversion i n t o proximate carcinogens (Arcos and Argus, 1974). An N-oxide grouping i s formed when the n i t r o g e n i n a chemical compound i s l i n k e d to oxygen by coordinate bonds ( u s u a l l y represented as N-**0 to d i s t i n g u i s h i t from an N = 0 double bond). I n t h i s p a r t i c u l a r s i t u a t i o n , the e l e c t r o n d e n s i t y i s d i s p l a c e d towards oxygen. The N-K) bond i s h i g h l y p o l a r i s e d and presumably very a c t i v e . A notable example i s k - n i t r o q u i n o l i n e N-oxide, a powerful a n t i b a c t e r i a l , f u n g i c i d a l , mutagenic and ca r c i n o g e n i c agent ( M i t a , 1971). I t i s h i g h l y t o x i c toward cancer c e l l s i n v i v o and i n v i t r o (Fukuoka, 1971). The ca r c i n o g e n i c a c t i v i t y of kNQO has been demonstrated i n v a r i o u s s p e cies (mice, r a t , hamster, guinea p i g , r a b b i t and f o w l ) . Depending on the route of a d m i n i s t r a t i o n , kNQO induces tumours i n a wide spectrum of t a r g e t t i s s u e s (Arcos and Argus, 1 9 7 k ) . Although the a c t i v e molecular form remains u n s e t t l e d , kNQO i s r a p i d l y reduced to k-hydroxyaminoquinoline N-oxide, which does not d i f f e r a p p r e c i a b l y from kNQO i n ca r c i n o g e n i c a c t i v i t y (Kawazoe and A r a k i , 1970). On the b a s i s of s i m i l a r c a r c i n o g e n i c potency and t a r g e t t i s s u e spectrum i n 4NQ0 and kHAQ0, i t i s d i f f i c u l t to envisage 4HAQ0 as the proximate carcinogen. The assignment of kHAQ0 as the proximate c a r c i n o g e n i c form i s grounded mainly on metabolic and t i s s u e d i s t r i b u t i o n aspects. A c i r c u m s t a n t i a l piece of evidence i n support of t h i s d e s i g n a t i o n may be der i v e d from a c o n s i d e r a t i o n of the 3-halogen s u b s t i t u t e d d e r i v a t i v e s of kNQ0. In 3-chloro- kNQ0 and 3-fluoro- kNQ0, the halogen i s more r e a c t i v e than the n i t r o group towards n u c l e o p h i l i c reagents. This r e a c t i o n i s expected to compete w i t h metabolic r e d u c t i o n of the n i t r o group t o a hydroxylamino group w i t h concomitant r e d u c t i v e dehalogenation t o the N-hydroxy proximate carcinogen. The halogen i n 3-brorao-4NQO i s l e s s r e a c t i v e towards n u c l e o p h i l e s and i s a c t u a l l y found t o undergo t r a n s f o r m a t i o n t o the proximate carcinogen 4HAQ0 more r e a d i l y than the c h l o r o and f l u o r o d e r i v a t i v e s . 3-Bromo-4NQ0 a l s o proved t o be the most potent carcinogen of the three halogen d e r i v a t i v e s , thus i n d i r e c t l y l e n d i n g support f o r the assignment of 4HAQO as a proximate carcinogen (Kawazoe and A r a k i , 1 9 7 0 ) . I n t h i s chapter, an attempt i s made to i n v e s t i g a t e whether the l e v e l of DNA r e p a i r s y n t h e s i s e l i c i t e d i n c u l t u r e d f i b r o b l a s t s would p a r a l l e l the ease w i t h which the proximate carcinogen i s formed. A comparison i s made among the 3-halogen s u b s t i t u t e d 4NQ0 d e r i v a t i v e s w i t h r e s p e c t t o t h e i r c a p a c i t y t o provoke an unscheduled DNA s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . F o l l o w i n g the same r a t i o n a l i s a t i o n s as i n the preceding paragraph, i f 3-bromo-4-NQO produces a b e t t e r y i e l d of the proximate carcinogen than the corresponding c h l o r o and f l u o r o -d e r i v a t i v e s , i t w i l l a l s o ©licit the h i g h e s t " l e v e l ^ o f DNA r e p a i r synthesis/: The l e v e l s of unscheduled DNA s y n t h e s i s e l i c i t e d by 4NQ0 and 4HAQ0 have been i n c l u d e d f o r comparison. From F i g . 6 1 i t i s evident t h a t both 4NQ0 and 4HAQ0 provoked a comparable l e v e l of unscheduled DNA s y n t h e s i s . The very low l e v e l of unscheduled ^HTdR uptake observed w i t h 3-fluoro- kNQ0 may be exp l a i n e d by the p o s s i b i l i t y t h a t l i t t l e of t h i s compound was transformed i n t o the proximate c a r c i n o g e n i c form. D i f f i c u l t y was experienced w i t h the s o l u b i l i t y of 3-bromo-kNQ.O and 3-chloro-4NQO i n c u l t u r e medium. As a r e s u l t , no unscheduled DNA s y n t h e s i s was observed i n c u l t u r e d human f i b r o b l a s t s exposed t o a suspension of these two compounds. Another piece of i n d i r e c t evidence supporting the n o t i o n t h a t kHAQO i s the proximate c a r c i n o g e n i c form of 4NQ0 may be provided from 3-niethyl- kNQ0. The presence of a methyl s u b s t i t u e n t a t the 3 - p o s i t i o n has rendered t h i s kNQO d e r i v a t i v e a t most m a r g i n a l l y a c t i v e as a carcinogen (Arcos and Argus, 197 k ) . I n chemical terms, the l o s s of a c t i v i t y has been a t t r i b u t e d t o the 3-niethyl group p r e s e n t i n g s t e r i c encumbrance of the access t o the n i t r o group, thereby c r e a t i n g a hindrance t o r e d u c t i o n t o the proximate c a r c i n o g e n i c hydroxylamine compounds. 3-Methyl- kNQ0 a l s o e x h i b i t s a sm a l l c a p a c i t y to induce DNA r e p a i r s y n t h e s i s i n mammalian c e l l s ( S t i c h e t a l . , 1971). Again, the o b s e r v a t i o n may be ex p l a i n e d i n terms of the d i f f i c u l t y f o r 3-methyl- kNQ0 to undergo t r a n s f o r m a t i o n i n t o kHAQO. A p o s s i b l e c o n c l u s i o n from t h i s chapter i s t h a t the l e v e l of unscheduled DNA s y n t h e s i s e l i c i t e d by v a r i o u s p r e c a r c i n o g e n i c kNQO d e r i v a t i v e s i s p a r a l l e l l e d by the ease w i t h which the proximate carcinogen (kHAQO) i s formed. C O N C E N T R A T I O N C M ) F i g u r e 61 Unscheduled DNA s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s f o l l o w i n g 1.5-hour exposure to 4NQ0 ( • ) , 4HAQ0 ( v ) or 3-fluoro-4NQ0 ( • ). Autoradiography. 99 4 . 4 . N i t r o s o Compounds ( A l i p h a t i c Carcinogens) The three preceding chapters have been concerned w i t h the metabolic a c t i v a t i o n of aromatic carcinogens and the c a p a c i t y of the u l t i m a t e c a r c i n o g e n i c forms to provoke DNA damage f o l l o w e d by an unscheduled DNA sy n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . I n t h i s chapter, an example of an a l i p h a t i c carcinogen r e q u i r i n g metabolic a c t i v a t i o n i s presented. D i m e t h y l n i -trosamine i s a potent carcinogen. Upon i n j e c t i o n i n t o mice, i t induces l i v e r and lun g neoplasma and a few kidney tumours (Toth e t a l . , 1964; T e r r a c i n i e t a l . , 1966). DMN i s converted by the mixed f u n c t i o n oxidases of the endoplasmic r e t i c u l u m t o intermediates which decompose r e a d i l y t o y i e l d a l k y l a t i n g s p e c ies (Magee, 1972). I t has been demonstrated by La i s h e s and S t i c h (1973) t h a t DMN alone (precarcinogen) d i d not i n f l i c t any DNA damage (as measured by the a l k a l i n e sucrose g r a d i e n t technique) nor e l i c i t any DNA r e p a i r s y n t h e s i s (as evidenced by the ' unscheduled uptake of.HTdR) i n c u l t u r e d human f i b r o b l a s t s . However, when DMN was mixed w i t h the p o s t - m i t o c h o n d r i a l f r a c t i o n of mouse l i v e r homogenate and then added to c u l t u r e d human f i b r o b l a s t s , DNA damage and an ensuing unscheduled DNA s y n t h e s i s was observed. A p l a u s i b l e e x p l a n a t i o n i s t h a t l i t t l e or no a c t i v a t i o n of DMN took place w i t h the enzymes present i n the c u l t u r e d f i b r o b l a s t s . 1 0 0 4.5- E f f e c t of Precarcinogens and Ultimate Carcinogens on Chromosome A b e r r a t i o n s and Clone-Forming Ca p a c i t y  i n C u l t u r e d Human F i b r o b l a s t s One t e n t a t i v e c o n c l u s i o n from the present s e c t i o n i s that an unscheduled DNA sy n t h e s i s i n c u l t u r e d human f i b r o b l a s t s c o u l d be demonstrated only w i t h proximate and u l t i m a t e c a r -cinogens. Precarcinogens as a r u l e f a i l e d to e l i c i t any unscheduled i n c o r p o r a t i o n of ^ HTdR unless they are r e a d i l y a c t i v a t e d i n the f i b r o b l a s t s . The qu e s t i o n a r i s e s as to whether both precarcinogens and u l t i m a t e carcinogens c o u l d induce chromosome a b e r r a t i o n s and reduce the clone-forming c a p a c i t y of c u l t u r e d human f i b r o b l a s t s . The e f f e c t of the precarcinogens benz(a)anthracene, 2-acetylaminofluorene, k - a c e t y l a m i n o s t i l b e n e and k - n i t r o q u i n o l i n e 1-oxide and t h e i r corresponding proximate and u l t i m a t e c a r c i n o g e n i c forms on the frequency of chromosome a b e r r a t i o n s i n c u l t u r e d human f i b r o b l a s t s are shown i n F i g s . 62 - 65. The p r e c a r c i n o -gen benz(a)anthracene and i t s i n a c t i v e degradation product benz(a)anthracene-cis - 5 , 6-dihydrodial d i d not induce any chromosome a b e r r a t i o n s i n c u l t u r e d human f i b r o b l a s t s . Chromosome a b e r r a t i o n s were produced only w i t h the u l t i m a t e carcinogen. With 2-acetylaminofluorene and k - a c e t y l a m i n o -s t i l b e n e , chromosome a b e r r a t i o n s were observed i n c u l t u r e d human f i b r o b l a s t s f o l l o w i n g exposure to the proximate (N-hydroxy d e r i v a t i v e ) and u l t i m a t e (N-acetoxy d e r i v a t i v e ) c a r c i n o g e n i c forms but the precarcinogen i t s e l f was i n a c t i v e . I n the case of the precarcinogen K N Q 0 , because 101 Figures 62-65 Frequency of metaphase p l a t e s w i t h chromosome a b e r r a t i o n s i n c u l t u r e d f i b r o b l a s t s from Xeroderma pigmentosum p a t i e n t s f o l l o w i n g short-terra exposure to precarcinogens, proximate or u l t i m a t e carcinogens. (Figure 62) BA (3 hr.) (•) - precarcinogen. BA -5 ,6-Epoxide ( v ) - proximate carcinogen. B A - c i s - 5 , 6 - d i h y d r o d i o l (•) - i n a c t i v e m e t a b o l i t e . (Figure 63) 2-AAF (5 hr.) (•) - precarcinogen. N-Hydroxy-2-AAF ( v ) - proximate carcinogen. N-Acetoxy-2-AAF ( O ) - u l t i m a t e carcinogen. (Figure 64) 4-AAS (5 hr.) (•) - precarcinogen. N-Hydroxy-4-AAS ( v ) - proximate carcinogen. N-Acetoxy-4-AAS ( O ) - u l t i m a t e carcinogen. . (Figure 65) 4NQ0 (1.5 hr.) (•) - precarcinogen. 4HAQ0 (1.5 hr.) ( v ) - proximate carcinogen. of the ease w i t h which i t i s converted i n t o the proximate carcinogen 4HAQ0, both d e r i v a t i v e s are e q u a l l y e f f e c t i v e i n the p r o d u c t i o n of chromosome a b e r r a t i o n s . The e f f e c t of v a r i o u s precarcinogens and t h e i r corresponding proximate and u l t i m a t e c a r c i n o g e n i c form on the clone-forming c a p a c i t y of c u l t u r e d human f i b r o b l a s t s are d e p i c t e d i n F i g s 66 - 69. F o l l o w i n g short term exposure t o benz(a)anthracene (precarcinogen) and the i n a c t i v e m e t a b o l i t e ( B A - c i s - 5 , 6 - d i h y d r o d i o l ) , the c l o n e -forming c a p a c i t y of c u l t u r e d human f i b r o b l a s t s d i d not d i f f e r from t h a t i n the untreated c o n t r o l . A r e d u c t i o n i n the number of clones formed was observed only a f t e r the c e l l s were exposed to the u l t i m a t e carcinogen, BA -5»6-epoxide. A s i m i l a r phenomenon was observed w i t h 2-AAF and 4-AAS, w i t h the precarcinogen being i n e f f e c t i v e i n reducing clone formation. With regard to 4NQ0, both the precarcinogen and u l t i m a t e carcinogen (4HAQ0) a f f e c t e d the clone-forming c a p a c i t y of c u l t u r e d human f i b r o b l a s t s t o a s i m i l a r extent. The almost i d e n t i c a l response t o %NQO and i t s u l t i m a t e carcinogen (4HAQ0) c o u l d probably be ex p l a i n e d by the f a c t t h a t 4NQ0 i s r e a d i l y transformed i n t o 4HAQ0. From t h i s s e c t i o n , i t may be concluded t h a t , as a r u l e , precarcinogens do not evoke any chromosome a b e r r a t i o n s nor a f f e c t the clone-forming c a p a c i t y of c u l t u r e d human f i b r o b l a s t s unless they are transformed i n t o u l t i m a t e c a r c i n o g e n i c d e r i v a t i v e s . 104 Figures 66-69 Clone-forming capacity of cultured human f i b r o b l a s t s following short-term exposure to precarcinogens, proximate or ultimate carcinogens (expressed as percentage of s u r v i v a l i n untreated c o n t r o l s ) . (Figure 66) BA (3 hr.) (•) - precarcinogen, BA-5»6-e:poxide ( v ) - proximate carcinogen. BA-Cis-5,6-dihydrodiol (•) - non-carcinogenic metabolite. (Figure 67) 2-AAF (5 hr.) (•) - precarcinogen. N-Hydroxy-2-AAF ( v ) - proximate carcinogen. N-Acetoxy-2-AAF ( O) - ultimate carcinogen. (Figure 68) 4-AAS (5 hr.) (•) - precarcinogen. N-Hydroxy-4-AAS ( v ) - proximate carcinogen, N-Acetoxy-k-AAS ( O ) - ultimate carcinogen. (Figure 69) kNQ0 (1.5 hr.) (•) - precarcinogen. 4HAQ0 ( v ) - proximate carcinogen. 106 5 . Carcinogenic C a p a c i t y and DNA Repair L e v e l I n Chapters 3 and 4, data have been presented to support the n o t i o n t h a t the c a r c i n o g e n i c i t y of a chemical i s c o r r e l a t e d w i t h i t s c a p a c i t y to damage DNA as evidenced by the ensuing unscheduled DNA s y n t h e s i s . An important but as y e t unanswered q u e s t i o n concerns the r e l a t i o n s h i p between the l e v e l of DNA r e p a i r t r i g g e r e d by a carcinogen and the degree of i t s c a r c i n o g e n i c p o t e n t i a l . P r e v i o u s l y a good c o r r e l a t i o n between these two f a c t o r s has been demonstrated when the a c t i o n of str o n g and weak c a r c i n o g e n i c 4NQ0 isomers and d e r i v a t i v e s were compared ( S t i c h e t a l . , 1971 i 1974). H i g h l y oncogenic d e r i v a t i v e s i n i t i a t e r e p a i r s y n t h e s i s a t c o n s i d e r a b l y lower doses than weakly oncogenic ones. The maximum l e v e l of ^ HTdR i n c o r p o r a t i o n i n t o c e l l s exposed to weakly oncogenic d e r i v a t i v e s i s always lower than i n those t r e a t e d w i t h h i g h l y oncogenic compounds. The qu e s t i o n then a r i s e s as to whether such a c o r r e l a t i o n between c a r c i n o g e n i c p o t e n t i a l and l e v e l of DNA r e p a i r s y n t h e s i s c o u l d be extended to carcinogens of d i f f e r e n t molecular s t r u c t u r e s . A few carcinogens have been i n c l u d e d i n a comparative study. From F i g . 7, i t i s evident t h a t 4-NQO e l i c i t e d a high l e v e l of unscheduled %TdR i n c o r p o r a t i o n a t r e l a t i v e l y low doses, while the 6,7-epoxide of MCA r e q u i r e d about 1,000 times- higher c o n c e n t r a t i o n t o t r i g g e r a r e l a t i v e l y low l e v e l of DNA r e p a i r s y n t h e s i s . Obviously 4NQ0 i s not 1,000 times more ca r c i n o g e n i c than MCA or i t s epoxide. N-Acetoxy-2-AAF, a l s o a potent carcinogen, i n i t i a t e s a comparatively lower unscheduled DNA s y n t h e s i s than i n the case of 4NQ0 even though much higher c o n c e n t r a t i o n s were used. Thus, the l e v e l of unscheduled DNA s y n t h e s i s e l i c i t e d by carcinogens of d i f f e r e n t molecular s t r u c t u r e s does not r e f l e c t t h e i r r e s p e c t i v e c a r c i n o g e n i c a c t i v i t y . 6 . Design and T r i a l of a Rapid I n V i t r o Bioassay f o r  Chemical Carcinogens To cope w i t h the c a r c i n o g e n i c i t y t e s t i n g of the r e l a t i v e l y l a r g e number of man-made compounds t h a t are p l a c e d on the market a n n u a l l y , and the multitude of n a t u r a l l y - o c c u r r i n g chemicals t h a t enter man's immediate environment, the i n t r o d u c t i o n of f a s t and economic pre-screening procedures has become a n e c e s s i t y . Most of the newly-developed methods th a t appear s u i t a b l e f o r a l a r g e - s c a l e prescreening programme depend on the c a p a c i t y of carcinogens to induce mutations or to a f f e c t the DNA of i n d i c a t o r o r g a n i s m s ( S t o l t z e t a l . , 1 9 7 * 0 . At present, a great emphasis i s being p l a c e d on the use of v a r i o u s b a c t e r i a ( S l a t e r e t a l . , 1 9 7 1 ; Kada e t a l . , 1 9 7 2 ; Ames et a l . , 1 9 7 3 a , 1 9 7 3 b ; McCalla et a l . , 1 9 7 5 ) , yeast (Koske and S t i c h , 1 9 7 3 ; F a h r i g , 1 9 7 * 0 , Neurospora (Ong and de S e r r e s , 1 9 7 2 ; De S e r r e s , 197 * 0 or D r o s o p h i l a (Sobel, 197*+) as s e n s i t i v e , economical i n d i c a t o r organisms. However, i n c o n s i s t e n c y among the v a r i o u s types of t e s t organisms i n t h e i r response to a p a r t i c u l a r carcinogen i s not uncommon. I n a d d i t i o n , the f e a s i b i l i t y of e x t r a p o l a t i n g observations from m i c r o b i a l systems t o the human s i t u a t i o n poses another problem. I n view of the l i m i t a t i o n s of these m i c r o b i a l bioassays, i t may be worthwhile to explore the use of c u l t u r e d human c e l l s as t e s t s u b j e c t s and the a p p l i c a t i o n of DNA r e p a i r s y n t h e s i s estimated by unscheduled ^HTdR i n c o r p o r a t i o n as an endpoint (Rasmussen and P a i n t e r , 1966; S t i c h et a l . , 1971) , to measure the DNA damaging a c t i o n of chemical carcinogens. To t e s t the r e l i a b i l i t y of DNA r e p a i r as a bioassay f o r chemical carcinogens, 64 d i f f e r e n t compounds were examined. These i n c l u d e precarcinogens t h a t r e q u i r e metabolic a c t i v a t i o n , proximate and u l t i m a t e carcinogens, man-made and n a t u r a l l y - o c c u r r i n g compounds, non-carcinogenic but mutagenic chemicals (e.g. daunomycin, ethidium bromide) and non-carcinogenic as w e l l as non-mutagenic chemicals. The responses of c u l t u r e d human f i b r o b l a s t s to these compounds are summarised i n the f o l l o w i n g three s e c t i o n s . 6 . 1 . C o n c e n t r a t i o n of Test Compounds The range of co n c e n t r a t i o n s of v a r i o u s carcinogens t h a t t r i g g e r d e t e ctable l e v e l s of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s v a r i e s g r e a t l y ( F i g . $ ) , S i m i l a r l y , the l e t h a l e f f e c t of v a r i o u s carcinogens O p ranges from about 10 M to 10 M ( F i g . 6). Therefore one cannot suggest the use of a p a r t i c u l a r s et of con-c e n t r a t i o n s t h a t would be a p p l i c a b l e f o r a l l chemical carcinogens. Even the use of the highest t o l e r a b l e dose co u l d produce m i s l e a d i n g r e s u l t s , s i n c e i n h i b i t i o n of the DNA r e p a i r system by high c o n c e n t r a t i o n s of a carcinogen would e l i c i t undetectable l e v e l s of an unscheduled %TdR i n c o r p o r a t i o n ( F i g . 70). The range of con c e n t r a t i o n s i s best s e l e c t e d by f i r s t e s t a b l i s h i n g the dose of a carcinogen t h a t would give an obvious t o x i c e f f e c t w i t h i n the time of the experiment (exposure to t e s t compounds f o r 1.5 to 5 h. f o l l o w e d by 1.5 h. of ^HTdR), and then s t a r t i n g a d i l u t i o n s e r i e s w i t h h a l f t h i s t o x i c dose, 6 . 2 . T r i a l of Bioassay The response of c u l t u r e d human f i b r o b l a s t s to the 64 d i f f e r e n t t e s t compounds (summarised i n Table V I I I ) r e v e a l e d a c o n s i s t e n t p a t t e r n * 1. C e l l s exposed to proximate and u l t i m a t e carcinogens responded w i t h a DNA r e p a i r s y n t h e s i s ; 2. Precarcinogens e i t h e r e l i c i t e d no detectable l e v e l s of unscheduled ^HTdR i n c o r p o r a t i o n , or t r i g g e r e d a DNA r e p a i r a t hig h c o n c e n t r a t i o n s or a t longer exposure times (e.g. 2-AAF, A f l a t o x i n B^); 3 . Non-carcinogenic compounds f a i l e d to i n i t i a t e DNA r e p a i r s y n t h e s i s . I n t h i s connection, i t i s of i n t e r e s t to note the negative response to a c r i f l a v i n e , daunomycin and ethidium bromide which are very potent mutagens but seem to l a c k a c a r c i n o g e n i c c a p a c i t y . 6 . 3 . Precarcinogens In v i t r o t e s t systems using m i c r o b i a l c e l l s respond C O N C E N T R A T I O N C M ) F i g u r e 70 I n h i b i t i o n of DNA r e p a i r i n c u l t u r e d human f i b r o b l a s t s e l i c i t e d by h i g h c o n c e n t r a t i o n s of a chemical carcinogen. TABLE VIII ONA REPAIR SYNTHES I S (UNSCHEOULED INCORPORATION OF ^HTdR) OF CULTURED HUMAN FIBROBLASTS EXPOSED TO PRECARCINOGENS, CARCINOGENS, NON-CARCINOGENIC DERIVATIVES ANO MUTAGENS CARCINO- COMPOUND EXPOSURE CONCENTRATION RANGE WITHIN WHICH MAXIMUM GENICITY TIME UNSCHEDULED ^fTdR UPTAKE OETECTEO (Ml GRAINS IO - 7 IO"* IO - 5 - IO - 4 I O - 3 IO" 2 NUCLEUS-PC 4NQ0 1.3 H n ' ISO UC 4HAOO 1.5 H r « 78 PC 2-METHYL-4NOO I.J H n — — — — — |22 NC 6NQO 1.5 Hrs 0 NC 4AQ0 1.5 H n 0 PC 8A 3 Hrt 0 UC 8A-5.6-EP0XI0E 3 H n _ _ _ _ _ _ _ _ 2 6 NC 8A-cl5-5,6-OIHYOROOIOL 3 Hrs 0 PC MCA 3 Hrs 0 UC MCA-6,7-EPOXIOE 3 Mrs — — — — — 4a NC MCA-6,7-01HYDROOIOL 3 H n 0 PC 2-AAF 3 Hrs • 10 UC N-HYDR0XY-2-AAF 3 H n . 42 UC N-ACET0XY-2-AAF 5 H n • J4 NC I-HYDR0XY-2-AAF 5 H n 0 NC 3-HYDR0XY-2-AAF 3 H n 0 NC 5-HYDROXY-2-AAF 5 H n 0 NC 7-HYDROXY-2-AAF 5 H n 0 PC 4-AAS 3 Hrs 0 UC N-HYDR0XY-4-AAS 5 H n — — — — 14 UC N-ACET0XY-4-AAS 3 H n • 33 PC 4-AABP - 3 H n 0 UC N-HY0R0XY-4-AA.BP 5 Hrs — — — |0 UC N-ACET0XY-4-AABP 5 Hrs — — — — 20 PC 2-AAP . 5 H n 0 UC N-HY0R0XY-2-AAP 5 H n _ _ _ _ _ J Q UC N-ACET0XY-2-AAP 3 H n 4 0 UC N-MYRIST0YL0XY-2-AAF 3 H n . ' 13 UC N-ACLrOXY-2-MYKISTOYl-AF 5 Hrs ' |2 UC N-MYIIISTOYLOXY-2-MYRISTOYL-AF 5 Hrs — — — 14 UC MNNG UC MMS UC EMS UC M l X ICR-I9I UC HN2 PC SAFnoie UC I'-HYDROXY-SAFROLE X 3'-HY0R0XY-SAFR0LE X 3'-ACET0XY-SAFR0LE NC I'-KETO-SAFROLE UC 1.l-DIPHENYL-2-PROPYNYL-N-CYCLOHEXYLCARBINOL UC l-PHENYL-l-(3,4-XYLYL)-2-PR0PYNYL CYCLOHEXYLCARBAMATE NC I,I-DIPHENYL-2-BUTYNYL-N-CYCLOHEXYLCARBAMATE NC DIPHENYLCARBINOL X STREPT0N1GRIN 1.5 Hrs • 30 PC AFLATOXIN B. 0.3 Hr 0 PC AFLATOX1N B| 2 Hrs • UC AFLATOXIN B. (Activation) 0.5 Hr — 16 PC AFLATOXIN G l 0.5 Hr 0 UC AFLATOXIN G, (Activation) 0.5 NC AFLATOXIN G 2 0.5 Hr 0 NC AFLATOXIN Gj (Activation) 0.5 Hr 0 PC STERIGMATOCYST1N 0.5 Hr 0 PC STERIGMATOCYST1N 2 Hrs 15 UC STERIGMATOCYST1N (Activation) 0.5 — 40 PC 0IMETHYLNITRO5AM1 NE 1 Hr 0 UC DIMETHYLN1TROSAM1NE (Activation) 1 Hr — — — — — — — — 23 NC METHYLGUANIDINE 2-5 Hrs 0 UC METHYLGUANIDINE ( N l t r o j » t l o n ) 4 Hrs _ « NC ACRIFLAVINE NEUTRAL 3-6 H n 0 NC DAUNOMYCIN 3-6 H n . 0 NC ETHIDIUM BROMIDE 3-6 H n 0 PC LUTEOSKYRIN 1.3-5 H n UC LUTEOSKYRIN (Activation) 1.5-5 Hrs PC RUGULOSIN 1.5-3 Hrs UC RUGULOSIN (Act ivat ion) 1.5-5 Hrs 3 1.5 3 I 3 3 5 5 5 5 5 Mrs Mrs Hrs Hr Hrs Hrs Hrs Hrs H n H n H n 1.3 1.5 H n Hrs I.5-5 Hrs 1.5-5 Hrs 41 19 20 23 16 13 0 17 14 26 0 38 41 I PC • precarcinogen ; UC • ultimata carclnoqan Including proximate carcinogen ; NC • non-carcinogen J X • unknown carcinogenic i ty to r e a c t i v e forms of c a r c i n o g e n s , but u s u a l l y f a i l t o d e t e c t p r e c a r c i n o g e n s t h a t r e q u i r e m e t a b o l i c a c t i v a t i o n . C u l t u r e d human f i b r o b l a s t s behave s i m i l a r l y . F o r example, short-terra exposures t o MCA, 4-AAS, 4-AABP and 2-AAP and s a f r o l e d i d not r e s u l t i n d e t e c t a b l e unscheduled i n c o r p o r a t i o n of ^HTdR, whereas the hydroxy and acetoxy forms ( r e p r e s e n t i n g the proximate o r u l t i m a t e c a r c i n o g e n s ) d i d t r i g g e r DNA r e p a i r s y n t h e s i s . There were e x c e p t i o n s t o t h i s p a t t e r n . R e l a t i v e l y h i g h doses of 2-AAF ( F i g . 57) or l o n g e r exposures to a f l a t o x i n and s t e r i g m a t o c y s t i n induced a DNA r e p a i r s y n t h e s i s ( F i g . 71 and 72), although these three compounds r e q u i r e d a c t i v a t i o n (Cramer e t a l . , I960; S t i c h and L a i s h e s , 1975). The s i m p l e s t e x p l a n a t i o n i s t h a t c u l t u r e d f i b r o b l a s t s c a r r i e d low l e v e l s o f a c t i v a t i o n enzymes, even though t h i s a c t i v a t i o n c a p a c i t y o f f i b r o b l a s t s was r e s t r i c t e d . I n the case o f DMN, even c o n c e n t r a t i o n s exceeding 10 M d i d not e l i c i t a DNA r e p a i r s y n t h e s i s (San, u n p u b l i s h e d d a t a ) . S i n c e the a c t i v a t i o n p o t e n t i a l o f human f i b r o b l a s t s cannot be p r e d i c t e d from the m o l e c u l a r s t r u c t u r e o f a p r e c a r c i n o g e n , i n c l u s i o n o f an a c t i v a t i o n procedure i n the b i o a s s a y i n mandatory. N e v e r t h e l e s s , t h e r e remains a c e r t a i n drawback t o t h i s approach, because the a c t i v a t i o n o f a compound may e a s i l y be missed by a p p l y i n g the wrong a c t i v a t i n g system or source o f a c t i v a t i o n enzymes. The ca r c i n o g e n s l u t e o s k y r i n and r u g u l o s i n /"which gave a n e g a t i v e response when a p p l i e d d i r e c t l y or added i n combination w i t h the S9 a c t i v a t i o n system (Garner and Hanson, 1971l M a i l i n g , 1971l Ames e t a l . , 1973b)_7 may belong i n t h i s c a t e g o r y . 113 C O N C E N T R A T I O N C M ) F i g u r e s 71-72 Unscheduled DNA s y n t h e s i s i n c u l t u r e d , human f i b r o b l a s t s evoked by the precarcinogens a f l a t o x i n B^.(Figure 71) and s t e r i g r a a t o c y s t i n ( Figure 72). Exposure to carcinogen was f o l l o w e d by ^HTdR ( 3 h o u r s ) . e 2-hour exposure t o ca r c i n o g e n O 0 . 5-hour exposure t o carcin o g e n 114 DISCUSSION One of the o b j e c t i v e s of t h i s t h e s i s was t o evaluate the use of DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s as a bioassay f o r chemical carcinogens. A second o b j e c t i v e concerned the p o s s i b l e v a r i a t i o n i n s e n s i t i v i t y w i t h i n the human p o p u l a t i o n towards chemical carcinogens. I n p a r t i c u l a r , c u l t u r e d c e l l s from Xeroderma pigmentosum p a t i e n t s (known to be d e f i c i e n t i n r e p a i r i n g UV-induced DNA damage) and normal persons were compared w i t h respect to t h e i r DNA r e p a i r c a p a c i t y , frequency of chromosome a b e r r a t i o n s and clone forming e f f i c i e n c y f o l l o w i n g exposure to a chemical carcinogen. Since the d i f f e r e n t questions asked are q u i t e independent of one another, i t seems appropriate to d i s c u s s each i s s u e i n a separate s e c t i o n . The d i s c u s s i o n has t h e r e f o r e been subdivided i n t o the f o l l o w i n g c h a p t e r s t -1. The use of unscheduled DNA s y n t h e s i s i n the i d e n t i f i c a t i o n of chemical carcinogens. 2. The use of DNA r e p a i r i n the i d e n t i f i c a t i o n of s e n s i t i v e c e l l s . 3 . DNA damage, chromosome a b e r r a t i o n s and c a r c i n o g e n e s i s . 4. P e r s p e c t i v e s . 1. The Use of Unscheduled DNA Synthesis i n the I d e n t i f i c a t i o n  of Chemical Carcinogens The r e s u l t s of the present study show the f e a s i b i l i t y of i n t r o d u c i n g DNA r e p a i r s y n t h e s i s of c u l t u r e d human f i b r o b l a s t s as a s e n s i t i v e system f o r the d e t e c t i o n of chemic a l c a r c i n o g e n s . Of 64 c h e m i c a l examined, 29 were d i r e c t l y a c t i v e proximate o r u l t i m a t e c a r c i n o g e n s , 15 would be c l a s s i f i e d as p r e c a r c i n o g e n s and 16 were non-oncogenic compounds. The c a r c i n o g e n i c c a p a c i t y of f o u r chemicals - s t r e p t o n i g r i n , ICR-191, 3 ' - h y d r o x y - s a f r o l e and 3 ' - a c e t o x y - s a f r o l e - i s unknown a t p r e s e n t . A l l the proximate and u l t i m a t e c a r c i n o g e n s t r i g g e r e d DNA r e p a i r syn-3 t h e s i s , w h ile no d e t e c t a b l e l e v e l o f unscheduled -'HTdR i n c o r p o r a t i o n was observed f o l l o w i n g treatment w i t h the 16 non-oneogenic compounds. Of the 15 s o - c a l l e d p r e c a r c i n o g e n s , 5 responded p o s i t i v e l y , w h i l e the remainder d i d not e l i c i t DNA r e p a i r s y n t h e s i s a t the c o n c e n t r a t i o n s and exposure times a p p l i e d i n the p r e s e n t e x p e r i m e n t a l s e r i e s . I t i s v e r y l i k e l y t h a t f i b r o b l a s t s c an a c t i v a t e the f i v e p r e c a r c i n o g e n s , 4NQ0, 2-methyl-4NQ0, 2-AAF, a f l a t o x i n Bj, and s t e r i g m a t o c y s t i n t o a l i m i t e d degree. I n a d d i t i o n t o the 64 compounds examined d u r i n g the p r e s e n t study, we p r e v i o u s l y t e s t e d 12 c a r c i n o g e n i c and 8 n o n - c a r c i n o g e n i c isomers and d e r i v a t i v e s o f 4NQ0 u s i n g S y r i a n hamster c e l l s as s u b j e c t s and unscheduled -'HTdR i n c o r p o r a t i o n as endpoint ( S t i c h e t a l . , 1971). Only the 12 c a r c i n o g e n s t r i g g e r e d d e t e c t a b l e l e v e l s ' d f DNA~repair s y n t h e s i s . R e s u l t s from the t r i a l o f the DNA r e p a i r b i o a s s a y show t h a t t h i s t e s t system i s s u i t a b l e f o r the d e t e c t i o n of proximate and u l t i m a t e c a r c i n o g e n s o n l y , as i t may y i e l d " f a l s e " negatives with precarcinogens. This d i f f i c u l t y can be overcome by mixing the precarcinogens with a post-mitochondrial f r a c t i o n from a l i v e r homogenate (S9 mix). Using t h i s procedure, i t has been possible to activate several precarcinogens i n v i t r o , e.g. dimethylnitrosaraine (Mailing, 1971? Laishes and Stic h , 1 9 7 3 ) , a f l a t o x i n (Garner and Hanson, 1 9 7 1 » S t i c h and Laishes, 1975) and sterigmatocystin (Stich and Laishes, 1 9 7 5 ) . Considering that the aim of the DNA repa i r assay i s to i d e n t i f y environmental carcinogens and mutagens and prevent human populations from being exposed to these agents, the relevance of using human c e l l s as tes t subjects i s an advantage that cannot be ignored. In addition, since the unscheduled DNA synthesis triggered by a carcinogen occurs i n more than 99% of the treated c e l l s , only a small number of c e l l s are required. Furthermore, human f i b r o b l a s t s can be r e a d i l y obtained from skin biopsies of "normal" and cancer predisposed persons. The technique can also be adapted f o r peripheral lymphocytes. Minute quantities of human blood are adequate to provide s u f f i c i e n t numbers of lymphocytes f o r short term cultures (48 - 72 hours) during which time a DNA r e p a i r assay can be performed ( B u r k e t a l . , 1 9 7 1 J Lieberman et a l . , 1 9 7 1 a , 1971b; Clarkson and Evans, 1 9 7 2 ; Jacobs et a l . , 1 9 7 2 ) . DNA repair synthesis can be detected i n human f i b r o b l a s t s following exposure to carcinogens of various chemical structures. The DNA repa i r assay has the advantage that the e n d - p o i n t (unscheduled -'HTdR i n c o r p o r a t i o n ) i s a g e n e r a l phenomenon not l i m i t e d t o one s p e c i f i c type o f DNA damage but encompasses a v a r i e t y o f a l t e r a t i o n s o r l e s i o n s i n the DNA m o l e c u l e s . I n s p i t e of i t s many advantages and i t s h i g h r e l e v a n c e t o man, the DNA r e p a i r b i o a s s a y i s not w i t h o u t i t s l i m i t a t i o n s . The technique s u f f e r s from the f a c t t h a t i t does not throw any l i g h t on the p r e c i s e DNA-carc inogen i n t e r a c t i o n that t r i g g e r s DNA r e p a i r s y n t h e s i s . F o r example, a l k y l a t i o n of DNA may occur a t N I , N3 or N? of a d e n i n e , N3, N7, 06 o f % g u a n i n e , N3 o f c y t o s i n e and N3 and Qk o f thymine (Lawley and B r o o k e s , 19&3; Lawley and T h a t c h e r , 1970; Lawley e t a l . , 1971/1972, 1973; O'Connor e t a l . , 1972; Sarma e t a l . , 197^). Which o f these events i n i t i a t e s r e p a i r and whether a l l a l k y l a t i o n p r o d u c t s are removed by e x c i s i o n r e p a i r and a t what r a t e remains t o be e l u c i d a t e d . There are s e v e r a l u n s o l v e d q u e s t i o n s worthy o f a t t e n t i o n . The l e v e l o f DNA r e p a i r s y n t h e s i s depends on the c o n c e n t r a t i o n of c a r c i n o g e n , d u r a t i o n o f a p p l i c a t i o n and type o f c a r c i n o g e n . F o r s u c c e s s f u l a p p l i c a t i o n o f the DNA r e p a i r technique i n a l a r g e s c a l e p r e s c r e e n i n g programme, s t a n d a r d i z e d t reatment and measuring procedures s h o u l d be i n t r o d u c e d . However, a t o o r i g i d regime c o u l d produce m i s l e a d i n g r e s u l t s . The t o x i c i t y o f the t e s t compound and i t s c a p a c i t y t o i n h i b i t a DNA r e p a i r system must be t a k e n i n t o account when the range of c o n c e n t r a t i o n s and the time o f exposure are determined ( S t i c h e t a l . , 197*0. Therefore a t o x i c i t y 118 t e s t must precede the a c t u a l DNA r e p a i r study. The l e v e l o f DNA r e p a i r s y n t h e s i s may be i n f l u e n c e d by the l e v e l s o f ^iHTdR uptake i n t o c e l l s and the a v a i l a b i l i t y of p r e c u r s o r s . Furthermore, a DNA r e p a i r s y n t h e s i s may r e q u i r e deoxyadenosine o r deoxyguanosine r a t h e r t han thymidine or c y t o s i n e as p r e c u r s o r s ( C l e a v e r , 1973? Lieberman and P o i r i e r , 1973). F o r i n s t a n c e , c e l l s exposed to the c a r c i n o g e n p - p r o p i o l a c t o n e which i n t e r a c t w i t h p u r i n e s i n s e r t o n l y p u r i n e p r e c u r s o r s d u r i n g DNA r e p a i r (Hennings e t a l . , 1 9 7 k ) . The range o f c o n c e n t r a t i o n s o f v a r i o u s c a r c i n o g e n s t h a t t r i g g e r d e t e c t a b l e l e v e l s o f DNA r e p a i r s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s v a r i e s g r e a t l y . There i s a l s o a l a c k o f u n i f o r m i t y i n the exposure times (0.5 t o 5 hours) r e q u i r e d f o r d i f f e r e n t c h e m i c a l c a r c i n o g e n s t o e l i c i t a d e t e c t a b l e l e v e l o f unscheduled ^HTdR i n c o r p o r a t i o n . I n the case of p r e c a r c i n o g e n s , t h i s may be l i n k e d t o the time p e r i o d w i t h i n which a c t i v e i n t e r m e d i a t e s or m e t a b o l i t e s are produced, or ( i n the case of both p r e c a r c i n o g e n s and u l t i m a t e c a r c i n o g e n s ) t o the time r e q u i r e d f o r the f o r m a t i o n of DNA-carcinogen complexes t o b r i n g about an a l t e r a t i o n i n the DNA s t r u c t u r e . The type of exposure poses another problem i n comparing the l e v e l o f unscheduled DNA s y n t h e s i s t r i g g e r e d by d i f f e r e n t c h e m i c a l c a r c i n o g e n s . Warren and S t i c h (1975) showed t h a t i f a second dose o f kNQ0 i s g i v e n w i t h i n 3 hours of the f i r s t one, the DNA r e p a i r c a p a c i t y of the human f i b r o b l a s t s i s s e v e r e l y c u r t a i l e d . However, i f the second dose i s administered more than 5 hours a f t e r the f i r s t one, the unscheduled i n c o r p o r a t i o n of ^ HTdR appears normal. I n t h i s 3-hour p e r i o d the c u l t u r e d c e l l s show an inc r e a s e d s e n s i t i v i t y to the l e t h a l e f f e c t and chromosome-damaging a c t i o n of the second kNQO dose. The p e r i o d of reduced DNA r e p a i r c a p a c i t y seems t o inc r e a s e the mutagenic e f f e c t of the chemical carcinogen. Whether a 5-hour continuous exposure t o a chemical carcinogen i s eq u i v a l e n t t o a double-dose exposure w i t h i n the " r e f r a c t i v e p e r i o d " as de s c r i b e d by Warren and S t i c h remains t o be assessed. The s t a b i l i t y of a chemical carcinogen i n aqueous s o l u t i o n a l s o deserves some c o n s i d e r a t i o n . The r o u t i n e procedure i n the DNA r e p a i r assay i s t o l a b e l w i t h ^HTdR f o l l o w i n g exposure to a carcinogen. Concomitant exposure of c u l t u r e d human f i b r o b l a s t s to both carcinogen and %TdR may p i c k up some unscheduled DNA s y n t h e t i c a c t i v i t y which would have been missed by the post-carcinogen l a b e l l i n g procedure. This i s one aspect t h a t warrants f u r t h e r i n v e s t i g a t i o n . Another as yet unanswered q u e s t i o n concerns the r e l a t i o n s h i p between the l e v e l of DNA r e p a i r t r i g g e r e d by a carcinogen, and the degree of i t s c a r c i n o g e n i c p o t e n t i a l . P r e v i o u s l y we poi n t e d out a good c o r r e l a t i o n between these two f a c t o r s when the a c t i o n of strong and weak c a r c i n o g e n i c kNQ0 isomers and d e r i v a t i v e s were compared ( S t i c h e t a l . , 1971, 1974). However, no such c o r r e l a t i o n became obvious when carcinogens of d i f f e r e n t molecular s t r u c t u r e s were i n c l u d e d i n the comparative study. For example, 4NQ0 e l i c i t e d a high l e v e l of unscheduled %TdR i n c o r p o r a t i o n a t r e l a t i v e l y low doses, while MMS,- EMS and the 6,7-epoxide of MCA r e q u i r e d about 1,000 times higher c o n c e n t r a t i o n s to t r i g g e r a r e l a t i v e l y low l e v e l of DNA r e p a i r s y n t h e s i s . Obviously 4NQ0 i s not 1,000 times more ca r c i n o g e n i c than MCA epoxide, MMS or EMS. The absence of a good c o r r e l a t i o n between the l e v e l of DNA r e p a i r t r i g g e r e d by a carcinogen and the degree of i t s c a r c i n o g e n i c p o t e n t i a l c ould be due to the d i f f i c u l t y i n p l a c i n g a q u a n t i t a t i v e value on c a r c i n o g e n i c i t y (from i n v i v o rodent a s s a y s ) . Many chemical carcinogens are known to e x h i b i t s p e c i e s and organ s p e c i f i c i t y . A f l a t o x i n , f o r example, could be considered a potent, weak or non-carcinogenic agent, depending on the species examined (Wogan, 1971). The s o l u b i l i t y of chemical carcinogens i n an aqueous media may i n f l u e n c e t h e i r p e n e t r a t i o n i n t o c e l l s and n u c l e i , which i n t u r n may a f f e c t the extent of DNA a l t e r a t i o n s induced. With a few exceptions (e.g. 4NQ0, MNNG, MMS, EMS, NMN, ICR-191 and HN 2), most carcinogens t e s t e d ( e s p e c i a l l y p o l y c y c l i c aromatic compounds) are hydrophobic. They are u s u a l l y d i s s o l v e d i n DMS0 or 100% EtOH but p r e c i p i t a t i o n occurs upon d i l u t i o n w i t h t i s s u e c u l t u r e medium, r e s u l t i n g i n a micro-suspension. Whether the s o l u b i l i t y problem i s one of the causes l e a d i n g t o a low l e v e l of unscheduled DNA s y n t h e s i s i s not known, although the low l e v e l of unscheduled ^HTdR i n c o r p o r a t i o n obtained w i t h water s o l u b l e carcinogens such as MMS, EMS and HNg tend t o argue a g a i n s t such a simple c o r r e l a t i o n . F e t a l bovine serum, when used a t 10 - 20$ c o n c e n t r a t i o n of the t o t a l volume of c u l t u r e medium, has been demonstrated t o e f f e c t i v l y reduce the DNA damaging c a p a c i t y of a chemical carcinogen ( S t i c h and San, unpublished d a t a ) . B i n d i n g of carcinogen by serum p r o t e i n may prevent the former from i n t e r a c t i n g w i t h DNA. On the other hand, fo r m a t i o n of a c a r c i n o g e n - p r o t e i n complex may a s s i s t the t r a n s p o r t of the carcinogens t o the c e l l nucleus. The i n v i t r o a c t i v a t i o n of pre-carcinogens and carcinogen-conjugates i s another area t h a t s t i l l needs t o be p r o p e r l y explored. R e s u l t s from the t r i a l of the DNA r e p a i r bioassay show t h a t t h i s t e s t system i s s u i t a b l e f o r the d e t e c t i o n of proximate and u l t i m a t e carcinogens o n l y , as i t may y i e l d " f a l s e " n egatives w i t h precarcinogens (Table I X ) . The precarcinogens l u t e o s k y r i n and r u g u l o s i n gave a negative response when a p p l i e d d i r e c t l y or added i n combination w i t h the S9 a c t i v a t i o n system (San and S t i c h , 1975, Table V I I I and I X ) . L i k e w i s e , the a p p l i c a t i o n of the same a c t i v a t i o n procedure was not s u c c e s s f u l w i t h the precarcinogens benz(a)anthracene, 20-methylcholanthrene and 2-acetylaminofluorene ( S t i c h and San, unpublished d a t a ) . I n f a c t , the i n v i t r o a c t i v a t i o n of 2-acetylaminofluorene has not been demonstrated t o date (Heidelberger, 1973). TABLE IX The application of the DNA repair assay to 60 compounds including 29 carcinogens, 1M> precarcinogens and 1? non-carcinogens. CARCINOGENS POSITIVE NEGATIVE PRECARCINOGENS POSITIVE NEGATIVE NON-CARCINOGENIC DERIVATIVES NEGATIVE ^-Kydroxyaminoquinoline 1-oxide EA-5.6-Epoxide y.0A-6.?-Epoxide N-l-y:2roxy-2-AAF N-Ace toxy-2-AAF N-Hydroxy-U-AAS N-Acetoxy-l+-AAS N-Kydroxy-l*-AA3P N-Acetoxy-<*-AAEP N-Hydroxy-2-AAP N-Acstoxy-2-AAP N-Mvristoyloxy-2-AAF N-Ace toxy-2-T.yristoyl-AF N-Vyristoyloxy-C-myristoyl-AF N-Methyl-V-ni tro-N-nitroso-g u a . i i d i n e Ke thylae thane sulfonate Ethylnethanesulfonate Nitrosexethylurea N i t r o g e n musta rd 1' -Ky Jroxy-s : i f rolo 1,l-SIphenyl-2-propynyl-N-cyclohexylcarblnol 1-Phenyl -1- (3. Wly l y l ) -2-propynyl-cyc1ohoxyl-carbamate A f l a t o x i n 3j (activation) A f l a t b x i n Gj (activation) Sterigaatocystin (activation) Diaethylnitrosanine (activation) J'.ethylguanidine (nitrosation) L u t e o s k y r i n ( a c t i v a t i o n ) R u g u l o s i n ( a c t i v a t i o n ) ^-Nitroquinoline 1-oxide 2-Methyl-l*NQ0 A f l a t o x i n Bj^  Sterigmatocystin Benz(a)anthracene 20-Methylcholanthrene 2-Acetylaminofluorene 4-Acetylarainostilbene 4-Acetylaminobiphenyl 2-Acetylarninophenan-threne Safrole Dimethylnitrosamine Luteoskyrin Rugulosin 6NQ0 <t-Aminoquinoline-1-oxide BA-c i 3-5.6-dihydrodiol MCA-6,7-dihydrodiol l-Hydroxy-2-AAP 3-Kydroxy-2-AAF 5-Kydroxy-2-AAF 7-Kydroxy-2-AAF l'-Keto-safrole l,l-Diphenyl-2-butynyl-N-cyclohexylcarbamate Diphenylcarbinol A f l a t o x i n C 2 A f l a t o x i n G 2 (activation) Methylguanidine A c r i f l a v i n e neutral Daunomycin Ethidium bromide 1 T P o s i t i v e i unscheduled incorporation of -tedR into DNA of cultured human f i b r o b l a s t s that were a r r e s t e d a t G,by an a r g i n i n e d e f i c i e n t , low serum (5%) c u l t u r e medium. 1 H The standard method of a p p l y i n g the S 9 l i v e r f r a c t i o n o b v i o u s l y has i t s l i m i t a t i o n s . F o r example, n i t r o f u r a n s need a r e d u c t i v e and not an o x i d a t i v e m e t a b o l i c a c t i v a t i o n ( M c C a l l a and V o u t s i n o s , 1974)j c y c a s i n r e q u i r e s cleavage by |3 - g l u c o s i d a s e (Laqueur and Matsumoto, 1966; Spatz, 1968) and (b - g l u c u r o n i d a s e treatment enhances the mutagenic e f f e c t of p - g l u c u r o n i d e conjugates of some c a r c i n o g e n s (Durston and Ames, 1974). S h o r t term c u l t u r e o f d i f f e r e n t i a t e d c e l l s from v a r i o u s t i s s u e s and organs can now be maintained ( L e f f e r t and P a u l , 1972| S a i j o , 1972, Lewis e t a l . , 1973; Noyes, 1973? S h a p i r o and S c h r i e r , 1973? F u j i t a e t a l . , 1974; S l a v i n s k i e t a l . , 1974). T h i s p e r m i t s the examination of o r g a n - s p e c i f i c a c t i v a t i o n o r i n a c t i v a t i o n of c a r c i n o g e n s . The use o f t i s s u e and organ c u l t u r e s i n a p r e s c r e e n i n g programme i n v a r i a b l y augments the work l o a d , a l t h o u g h i t w i l l prove i n v a l u a b l e i n t e s t i n g c a r c i n o g e n s which f a i l e d t o t r i g g e r any unscheduled DNA s y n t h e s i s i n c u l t u r e d human f i b r o b l a s t s . I t i s e s s e n t i a l , however, to ensure t h a t c e l l s from d i f f e r e n t organs or t i s s u e s do m a i n t a i n t h e i r t i s s u e - s p e c i f i c p r o p e r t i e s under the c o n d i t i o n s p r e v a i l i n g i n c u l t u r e s . Another approach to t a c k l e the problem o f organ s p e c i f i c i t y of c a r c i n o g e n s i s the more complex i n v i v o / i n v i t r o combination system developed by S t i c h and K i e s e r (1974). I n t h i s procedure, a c a r c i n o g e n i s a d m i n i s t e r e d to the t e s t animal by i n j e c t i o n (subcutaneous or i n t r a p e r i t o n e a l ) or f o r c e - f e e d i n g . The t e s t animal i s s a c r i f i c e d 2 hours l a t e r , t i s s u e p i e ces from v a r i o u s organs are removed and maintained i n -'HTdR-containing c u l t u r e medium f o r 3 hours. The t i s s u e p i e c e s are then f i x e d , embedded, sectioned and processed f o r autoradiography. Using t h i s technique, i t has been shown t h a t w i t h 4 - n i t r o q u i n o l i n e 1-oxide, unscheduled DNA s y n t h e s i s was detected only i n the lung, which i s the s i t e of tumour formation. I n c o n t r a s t , dimethylnitrosamine appears to be mainly a c t i v a t e d i n the l i v e r of rodents, and to a much s m a l l e r extent i n other organs i n c l u d i n g kidney and r e s p i r a t o r y t r a c t s . DNA r e p a i r s y n t h e s i s was observed i n those t i s s u e s which give r i s e t o neoplasms i mainly i n c e l l s of the l i v e r and lung and i n c e l l c l u s t e r s of the convoluted kidney t u b u l e s . I t must be mentioned t h a t t h i s i n v i v o / i n v i t r o combination i s not s u i t a b l e i n a l a r g e s c a l e p r e s c r e e n i n g programme of chemical carcinogens, because i t does not s a t i s f y the requirement f o r a simple, r a p i d and inexpensive assay. 2, The Use of DNA Repair i n the I d e n t i f i c a t i o n of S e n s i t i v e  C e l l s . R e s u l t s from the present s e r i e s of s t u d i e s i n d i c a t e d t h a t c u l t u r e d f i b r o b l a s t s from Xeroderma pigmentosum p a t i e n t s e x h i b i t e d a reduced c a p a c i t y t o r e p a i r DNA damage induced by some but not a l l chemical carcinogens. Xeroderma pigmentosum c e l l s a l s o have an e l e v a t e d frequency of chromosome a b e r r a t i o n s and a reduced clone forming c a p a c i t y f o l l o w i n g exposure to chemical carcinogens which e l i c i t e d i n them a reduced l e v e l of unscheduled DNA s y n t h e s i s . I f t h i s type of s e l e c t i v e response a l s o occurs i n c e l l s c a r r y i n g other cancer p r e d i s p o s i n g genes, one can p r e d i c t t h a t the human p o p u l a t i o n must c o n s i s t of numerous groups, subgroups and f a m i l i e s , each being s e n s i t i v e t o a d i f f e r e n t s e t of c a r c i n o g e n i c agents. There i s evidence i n support of such an assumption. For example, c u l t u r e d f i b r o b l a s t s of p a t i e n t s w i t h K l i n e f e l t e r * s syndrome (Mukerjee e t a l . , 1970), Fanconi's anemia (Young, 1 9 7 1 a ) , Down's syndrome (Todaro and M a r t i n , 1 9 6 7 ; Young, 1 9 7 1 b ) , and XY-gonadal dysgenesis (Mukerjee et a l . , 1972) are h i g h l y s u s c e p t i b l e to v i r a l (SV40) induced t r a n s f o r m a t i o n whereas the XP c e l l s . d o not show t h i s phenomenon ( P a r r i n g t o n e t a l . , 1971)* D e f e c t i v e DNA r e p a i r has been demonstrated i n Fanconi's anemia where the d e f i c i e n c y i s b e l i e v e d t o be i n an exonuclease f u n c t i o n which removes the damaged s t r a n d of DNA a f t e r the endonucleocytic s t r a n d s c i s s i o n has been made (Poon e t a l . , 1 9 7 * 0. Lymphocytes from Fanconi's anemia p a t i e n t s are more s u s c e p t i b l e (compared t o c e l l s from u n a f f l i c t e d persons) t o chromosome damage by DNA c r o s s - l i n k i n g agents (S a s a k i and Tonomura, 1 9 7 3 )• A number of cancer-prone and/or s u n - s e n s i t i v e g e n e t i c and nongenetic c o n d i t i o n s have been examined f o r t h e i r c a p a c i t y t o r e p a i r UV-induced DNA damage but no d e f e c t was demonstrated ( E p s t e i n , 1 9 7 4 ) . This v a r i a t i o n i n the type and spectrum of s e n s i t i v i t y among p a t i e n t s w i t h d i f f e r e n t disease c o n d i t i o n s poses a s e r i o u s problem i n c a l c u l a t i n g " s a f e " 126 doses of noxious agents - whether the response of the "average" man or the most s e n s i t i v e one should he used as b a s i s . Another c o m p l i c a t i o n i n s e t t i n g "safe l e v e l s " of a p o t e n t i a l h e a l t h hazard i s the g e n e t i c heterogeneity w i t h i n a p a r t i c u l a r d e f e c t , as i s the case f o r Xeroderma pigmentosum. P a t i e n t s m a n i f e s t i n g t y p i c a l XP c l i n i c a l symptoms may have a DNA r e p a i r l e v e l ranging from over 90% d e f i c i e n c y t o near normal c a p a c i t y . Since they show the c l i n i c a l symptoms, i t i s conceivable t h a t XP p a t i e n t s w i t h normal e x c i s i o n r e p a i r c a p a c i t y s t i l l possess an e l e v a t e d s e n s i t i v i t y towards U V - i r r a d i a t i o n and other DNA-damaging agents. Indeed, recent s t u d i e s have demonstrated t h a t XP c e l l s w i t h normal l e v e l s of DNA e x c i s i o n r e p a i r are d e f i c i e n t i n p o s t - r e p l i c a t i o n r e p a i r of UV-induced damage (Maher e t a l . , 1 9 7 5 a ; Lehmannst a l . . 1975) and have only 70% of the normal c a p a c i t y to r e a c t i v a t e UV-damaged adenovirus-2 (Day, 1974, 1975). Rare tumour p r e d i s p o s i n g genes (a few cases per m i l l i o n ) do not seem to present a major h e a l t h problem. Nevertheless, the number of heterozygous c a r r i e r s of such genes i s by no means n e g l i g i b l e . According t o S w i f t ' s c a l c u l a t i o n on Fanconi's amenia (FA), there should be about 650,000 FA c a r r i e r s among the 200 x 10^ North Americans ( S w i f t , 1 9 7 1 ) o I f these e s t i m a t i o n s are extended to other gene mutants and chromosome anomalies, then the p r o p o r t i o n of persons c a r r y i n g a t l e a s t one cancer p r e d i s p o s i n g g e n e t i c d e f e c t must • • : . I-1 be c o n s i d e r a b l e . I t i s of i n t e r e s t to note t h a t the r e c e s s i v e antosomal gene of Fanconi's anemia i n c r e a s e s (three f o l d ) the r i s k of a heterozygote dying from malignancy. Although XP heterozygotes have not been c o n c l u s i v e l y demonstrated to e x h i b i t any DNA r e p a i r d e f i c i e n c y or s e n s i t i v i t y towards chemical carcinogens, i t remains to be shown whether they are more prone t o tumour forma t i o n than normal persons. The s t u d i e s on Xeroderma pigmentosum have augmented our p e r s p e c t i v e of the whole problem of carcinogen screening. Because of the conceivable v a r i a t i o n i n the type and spectrum of s e n s i t i v i t y towards c a r c i n o g e n i c agents among the human p o p u l a t i o n , an adequate screening programme must encompass t e s t of chemicals i n "normal" persons, g e n e t i c a l l y a f f l i c t e d p a t i e n t s and h i g h cancer r i s k groups. The e s t i m a t i o n of DNA r e p a i r s y n t h e s i s coupled w i t h enumeration of chromosome a b e r r a t i o n s and a n a l y s i s of the clone forming c a p a c i t y should y i e l d i n s i g h t i n t o the spectrum of s e n s i t i v i t y towards c a r c i n o g e n i c agents. 3. DNA Damage. Chromosome A b e r r a t i o n s and C a r c i n o g e n e s i s . A d i s c u s s i o n on the use of DNA r e p a i r s y n t h e s i s i n the i d e n t i f i c a t i o n of chemical carcinogens w i l l be incomplete without mentioning the p o s s i b l e r o l e of DNA r e p a i r i n chemical c a r c i n o g e n e s i s , i'.uriy i f not a l l carcinogens i n t e r a c t w i t h DNA and show mutagenic p r o p e r t i e s . I t i s p o s s i b l e t h a t an unrepaired or m i s r e p a i r e d DNA damage 128 may l e a d to mutations which under c e r t a i n circumstances may r e s u l t i n a n e o p l a s t i c t r a n s f o r m a t i o n . The high s e n s i t i v i t y of XP c e l l s to UV and c e r t a i n chemical carcinogens c o r r e l a t e s w e l l w i t h the d e f i c i e n t DNA r e p a i r c a p a c i t y . I t i s tempting to l i n k the reduced DNA r e p a i r c a p a c i t y (or unrepaired DNA damage) w i t h the common occurrence of s k i n cancers i n XP p a t i e n t s . However, recent r e p o r t s i n d i c a t e t h a t the XP phenotype can occur i n the absence of a d e f i c i e n t DNA r e p a i r c a p a c i t y . Furthermore, DNA r e p a i r d e f i c i e n c y may not be necessary f o r the development of most mammalian cancers, si n c e cancer c e l l s i n general are q u i t e able to r e p a i r UV-as w e l l as chemical-induced DNA damage ( S t i c h and San, 1970; Norman et a l . , 1972; Lieberman and Forbes, 1973? E p s t e i n , 1974; Robbins et a l . , 1975). The DNA r e p a i r d e f i c i e n c y i n XP c e l l s c o r r e l a t e s w i t h the frequency of chromosome a b e r r a t i o n s f o l l o w i n g exposure to c e r t a i n carcinogens. For example, the more d e f i c i e n t the DNA r e p a i r c a p a c i t y of an XP" p a t i e n t , the higher the frequency of chromosome a b e r r a t i o n s f o l l o w i n g exposure to carcinogens such as 4NQ0 and N-acetoxy-AAF (Tables IV & V). An a t t r a c t i v e hypothesis to e x p l a i n the apparent c o r r e l a t i o n between the two phenomena i s t h a t unrepaired DNA damage (or a l t e r a t i o n s ) somehow leads t o a b n o r m a l i t i e s a t the chromosome l e v e l . However, a ca u s a l r e l a t i o n s h i p between unrepaired DNA damage and chromosome a b e r r a t i o n s i s d i f f i c u l t to prove a t the moment. Furthermore, the r o l e of chromosome a b e r r a t i o n s i n c a r c i n o -genesis i s not c l e a r . A preponderant number of human tumours 129 have been found to be accompanied by chromosomal changes ranging from h y p o d i p l o i d y t o hyper p l o i d y (Sandberg, 197 *0. Marker chromosomes are found i n at l e a s t 50°/o of human cancers. However, no c h a r a c t e r i s t i c or s p e c i f i c k a r y o t y p i c p i c t u r e has emerged f o r any tumour and no two tumours wi t h i d e n t i c a l karyotypes have been described to-date ( A t k i n , 197 * 0 . The only e x c e p t i o n may be the c o n s i s t e n t and c h a r a c t e r i s t i c presence of the Ph'-chromosome i n chronic myelocytic leukemia, but even here there i s evidence t h a t does not e n t i r e l y support the view of the abnormal chromosome p l a y i n g a r o l e i n the development of the leukemia. The occurrence of d i p l o i d cancers, though extremely r a r e , suggests t h a t d i p l o i d y and n e o p l a s i a are compatible (Sandberg, 197 *0. Chromosome a b e r r a t i o n s may represent mere epiphenomena to n e o p l a s t i c t r a n s f o r m a t i o n . Thus, the r o l e of chromosomal changes i n the c a u s a t i o n of human tumours remains a problem t h a t i s f a r from being s e t t l e d . 4. P e r s p e c t i v e s • Except f o r i n d i v i d u a l s who are engaged i n a p a r t i c u l a r i n d u s t r i a l concern, the average person i s not l i k e l y to be exposed to excessive doses of a s i n g l e chemical compound. The involvement of s e v e r a l mutagenic and ca r c i n o g e n i c agents seems more probable under n a t u r a l l y o c c u r r i n g c o n d i t i o n s . I n a d d i t i o n , the exposure may not be l i m i t e d to chemical carcinogens alone; v i r a l or p h y s i c a l agents could a l s o come i n t o p l a y . One prom i s i n g f e a t u r e o f the DNA r e p a i r b i o a s s a y i s t h a t i t p e r m i t s i n v i t r o s i m u l a t i o n of n a t u r a l l y o c c u r r i n g c o n d i t i o n s i n man. F o r example, the f o r m a t i o n of potent c a r c i n o g e n i c compounds from n i t r i t e ( n i t r a t e ) and secondary amines, amides, ureas, g u a n i d i n e s , carbamates e t c , a t a p p r o p r i a t e a c i d i c c o n d i t i o n s (e.g. i n the human stomach) i s w e l l s u b s t a n t i a t e d by v a r i o u s s t u d i e s (e.g. M i r v i s h , 1972; IARC S c i e n t i f i c P u b l i c a t i o n No. 3 ) . I n view of t h i s o b s e r v a t i o n , the massive consumption of food p r o d u c t s and n i t r o s a t a b l e drugs by the human p o p u l a t i o n does pose a p o t e n t i a l h e a l t h hazard. The DNA r e p a i r s y n t h e s i s system p r o v i d e s a procedure f o r d e t e c t i n g the f o r m a t i o n o f c a r c i n o g e n i c n i t r o s a t i o n s produced under a v a r i e t y o f c o n d i t i o n s : v a r i o u s c o n c e n t r a t i o n s of n i t r i t e and of n i t r o s a t a b l e compound, v a r i o u s pH l e v e l s , v a r i o u s l e n g t h s of n i t r o s a t i o n e t c . The r o l e of e l e c t r o n scavengers on the n i t r o s a t i o n r e a c t i o n can a l s o be examined. For example, asc o r b a t e t h a t i s consumed d a i l y by man can a t the r i g h t molar r a t i o t o n i t r i t e suppress the f o r m a t i o n of n i t r o s a t i o n p r o d u c t s ( M i r v i s h e t a l . , 1972; G r e e n b l a t t , 1973t Fan and Tannenbaum, 1973; A r c h e r e t a l . , 1975). Furthermore, a s c o r b a t e , upon o x i d a t i o n , l o s e s i t s i n h i b i t o r y e f f e c t on the n i t r o s a t i o n r e a c t i o n . The o x i d a t i o n p r o c e s s i n t u r n ++ i s a c c e l a r a t e d by the presence of metal i o n s such as Cu , F e + + + . The m u l t i t u d e of complex i n t e r a c t i o n s , , which w i l l determine whether or not a c a r c i n o g e n i c compound w i l l be formed, can be s i m u l a t e d i n v i t r o by u s i n g the DNA r e p a i r b i o a s s a y (Lo and S t i c h , 1975). The i n t e r a c t i o n between p h y s i c a l and chemical agents can be r e a d i l y detected by the DNA r e p a i r bioassay. This may be i l l u s t r a t e d by 8-methoxypsoralen, a p h o t o s e n s i t i z i n g chemical commonly found i n the p l a n t kingdom (Baden et a l , , 1972). 8-Methoxypsoralen i s a potent mutagen ( I g a l i e t a l . . 1970; Bridges, 1971) and carcinogen (Grube et a l . , 1975) when combined w i t h long-wave (355 nm) UV r a d i a t i o n . Other p h o t o s e n s i t i z i n g chemicals can be e x t r a c t e d from such common p l a n t s as marigold (Tagetis erectus) ( F i g . 73) and cow par s n i p (Heracleum lanatum) ( F i g . 7*0 which i s a f a v o u r i t e vegetable among west coast Indians (Turner and B e l l , 1973). The p o s s i b i l i t y of employing the DNA r e p a i r assay i n d e t e c t i n g mutagenic agents i n crude p l a n t e x t r a c t s w i l l f a c i l i t a t e the screening of environmental c a r c i n o -gens, or the p o s s i b l e g e n e r a t i o n of carcinogens from complex mixtures found i n s p i c e s , herbs, e x o t i c f r u i t s , teas e t c . I t i s estimated t h a t 80% to 90% of a l l human cancers are environmentally induced (Higginson, 19^9, 1971). The p o s s i b i l i t y of monitoring the environment f o r c a r c i n o g e n i c agents w i l l be an i n v a l u a b l e step towards cancer p r e v e n t i o n . Adaptation of the DNA r e p a i r assay f o r e x t r a c t s from human faeces, u r i n e , g a s t r i c j u i c e s and n a s a l exudates may permit the i d e n t i f i c a t i o n of groups i n the human p o p u l a t i o n t h a t may run a higher r i s k to develop cancer as a r e s u l t of i n d u s t r i a l or other environmental exposure to obnoxious agents. S e v e r a l i n t e r n a t i o n a l programmes, F i g u r e s 7 3 - 7 4 DNA r e p a i r s y n t h e s i s of c u l t u r e d human f i b r o b l a s t s f o l l o w i n g exposure (1 hr.) to a p h o t o s e n s i t i z i n g chemical and i r r a d i a t i o n by long wave (355 nm) UV at 7 inches from an F15T8-BL S y l v a n i a B l a c k l i t e . A u t o r a d i o -graphy of unscheduled i n c o r p o r a t i o n of ^HTclR (10 uCi/ml.. f o r 2 h r . ) . (Figure 73) cK-Terthienyl from marigold (Tagetis e r e c t u s ) . (Figure 74) Cowparsnip (Heracleum lanaturn Michx.) e x t r a c t . 133 UV DOSE t MINUTES) UV- DOSE (MINUTES) including the U.S.-Japan Cooperative Medical Science Programme and workshops on carcinogenesis assays and rapid screening tests sponsored by the Internations Agency f o r Research on Cancer have indicated i n t e r e s t i n some of these areas. The DNA r e p a i r assay may be modified i n a pre-screening programme f o r chemical carcinogens i n the following fashion. In the event of a negative response obtained i n the f i r s t assay procedure, the next step may be undertaken*-Step 1 Assay f o r unscheduled DNA synthesis i n cultured human f i b r o b l a s t s following exposure to t e s t compound or combination of chemical (and/or physical) agents. Step 2 Repetition of Step 1 with i n v i t r o a c t i v a t i o n employing l i v e r microsomal enzymes (S9 mix). Step 3 Repetition of Step 1 coupling exposure to t e s t compound with UV i r r a d i a t i o n ( p r i o r to or a f t e r administration of t e s t compound) to check f o r i n h i b i t o r y e f f e c t of chemical on UV-induced rep a i r . Step k Administration of t e s t compound to rodents i n vivo, followed by monitoring unscheduled DNA synthesis i n various organ pieces i n v i t r o to assay f o r any organotropic e f f e c t of te s t compound. The DNA rep a i r assay on human c e l l s permits simulation of actual human s i t u a t i o n s . The i n c l u s i o n of c e l l s from normal and high cancer r i s k persons i n t h i s t e s t system 135 may throw some l i g h t on the v a r i a t i o n i n s e n s i t i v i t y towards carcinogens and w i l l be of great value i n s e t t i n g "safe" l e v e l s . A d d i t i o n a l information on the test compound may be obtained through some of the following proceduresi-- i n v i t r o transformation of rodent or human c e l l s (a highly relevant endpoint since i t makes use of a r e a c t i o n involved i n carcinogenesis) (Heidelberger, 1 9 7 3 ; Kakunaga, 1973J Sanford, 1 9 7 k ; Evans and.DiPaolo, 1 9 7 5i Pienta, 1 9 7 5 ) . - recessive mutant te s t of Drosophila melanogaster (a g e n e t i c a l l y well-defined system with high s e n s i t i v i t y and the capacity to activate chemical precarcinogens and premutagens may elucidate the type of genetic changes induced i n a m u l t i c e l l u l a r organism; Sobel, 1 9 7 k ) . - Ames' Salmonella t e s t with s t r a i n s susceptible to frameshift mutations and base-pair substitutions combined with the S 9 or other a c t i v a t i o n mixtures f o r precarcinogens and premutagens (a rapid, economic and quantitative t e s t system which at the same time enables one to gain an i n s i g h t into the type of DNA changes induced by the t e s t compound; Ames et a l . , 1 9 7 3 a , 1973b'; McCann et a l . , 1 9 7 5 ) . The average man i n an i n d u s t r i a l society i s exposed to a multitude of chemical, physical and v i r a l oncogens rather than to a single agent. While the i d e n t i f i c a t i o n of i n d i v i d u a l carcinogenic agents must be continued i n the search f o r e f f e c t i v e .measures i n cancer prevention, i t i s not unreasonable to predict that the etiology of neoplastic transformation i s a m u l t i f a c t o r i a l one. The t o t a l carcino-genic or mutagenic load of our environment i s d i f f i c u l t to assess through the use of assay systems f o r single or even combinations of several carcinogenic agents. An epidemio-l o g i c a l approach appears the only one able to cope with such complex n a t u r a l l y occurring s i t u a t i o n s . The e s t a b l i s h -ment of tumour r e g i s t r i e s and improved d a t a - c o l l e c t i n g procedures w i l l provide adequate information on the patients' ethnic o r i g i n , occupation, v i r a l i n f e c t i o n s , prenatal exposure to drugs, etc. and w i l l g reatly help i n i d e n t i f y i n g environments of high cancer r i s k s . The use of " b u i l t i n " i n d i c a t o r or accumulator organisms f o r chemical carcinogens provides an "early warning" system that responds to the integrated "carcinogenic load" of a p a r t i c u l a r environment. For example, the recent i n t e r n a t i o n a l study on the occurrence of skin papillomas i n various f l a t f i s h species with respect to t h e i r d i s t r i b u t i o n i n i n d u s t r i a l l y contaminated as well as non-polluted areas o f f e r s a promising approach i n t h i s d i r e c t i o n (Stich and Acton, 1976; S t i c h et a l . , 1976). SUMMARY 1 . The primary objective of t h i s study was to evaluate the f e a s i b i l i t y of using DNA r e p a i r synthesis i n cultured human f i b r o b l a s t s as a simple, rapid and economic bioassay f o r chemical carcinogens. DNA rep a i r synthesis i n human f i b r o b l a s t s was measured by the autoradiographic detection of unscheduled ^HTdR incorporation following short term exposure to strongly, weakly or non-oncogenic compounds. 2 . The e f f e c t of various carcinogen doses on DNA rep a i r synthesis was examined. Five d i s t i n c t i v e features could be observed: 2 . 1 . The range of concentrations of various . carcinogens that triggered detectable l e v e l s of DNA rep a i r synthesis i n cultured human f i b r o b l a s t s varied greatly. 2 . 2 . The l e t h a l dose of various carcinogens _Q p also d i f f e r e d ranging from about 1 0 ~ M to 1 0 M. 2.3. A good c o r r e l a t i o n was observed between the l e v e l of DNA re p a i r evoked by a carcinogen and the degree of i t s carcinogenic p o t e n t i a l when the action of strong and weak carcinogenic 4 N Q 0 isomers and derivatives were compared. However, no such c o r r e l a t i o n became obvious when carcinogens of d i f f e r e n t molecular structures were included i n the comparative study. 2.4. The major part of DNA rep a i r synthesis following exposure to d i f f e r e n t chemical carcinogens appeared to be completed by about 8 - 1 0 hours post treatment. 2.5. A l l the active chemical carcinogens examined i n h i b i t e d DNA r e p a i r synthesis. 2 . 6 . Various carcinogenic compounds d i f f e r g r e a t l y i n t h e i r capacity to induce DNA lesions and to i n h i b i t DNA r e p a i r . 3 . As a t r i a l of the DNA r e p a i r bioassay, 64 compounds representing key groups of carcinogens of d i f f e r e n t molecular structures were examined f o r the capacity to evoke an unscheduled DNA synthesis i n cultured human f i b r o b l a s t s . This includes. 29 d i r e c t l y active proximate or ultimate carcinogens, 15 precarcinogens that require metabolic a c t i v a t i o n , 16 non-oncogenic compounds and 4 chemicals of unknown carcinogenicity. A l l d i r e c t l y acting carcinogens triggered a DNA r e p a i r synthesis, whereas no unscheduled •^ HTdR incorporation was observed following the a p p l i c a t i o n of the 16 non-oncogenic compounds. As a r u l e , the precarcinogens (without metabolic activation) did not e l i c i t DNA r e p a i r synthesis. 4 . The advantages, l i m i t a t i o n s and possible adaptations of the DNA r e p a i r bioassay f o r chemical carcinogens were presented and discussed. 5. The second objective of t h i s study was to investigate the p o s s i b i l i t y of v a r i a t i o n s i n s e n s i t i v i t y within the human population towards chemical carcinogens. C e l l s i from Xeroderma pigmentosum patients (known to be d e f i c i e n t i n c o r recting UV-induced DNA damage) and "normal" persons were examined f o r t h e i r DNA r e p a i r capacity, frequency of 139 chromosome a b e r r a t i o n s and clone forming e f f i c i e n c y f o l l o w i n g exposure t o chemical carcinogens. The XP c e l l s show a c o n s i d e r a b l y reduced DNA r e p a i r s y n t h e s i s when exposed t o some but not a l l chemical carcinogens. With chemicals f o r which the XP c e l l s e x h i b i t e d a d e f i c i e n c y i n DNA r e p a i r they a l s o e l i c i t e d a higher frequency of chromosome a b e r r a t i o n s and lower clone forming c a p a c i t y than i n normal persons. 6 . C e l l s from u n r e l a t e d XP p a t i e n t s have been found to vary i n t h e i r c a p a c i t y t o r e p a i r UV-induced DNA damage. With chemicals f o r which XP c e l l s e x h i b i t e d a d e f i c i e n c y i n DNA r e p a i r , a s i m i l a r v a r i a t i o n i n r e p a i r c a p a c i t y among u n r e l a t e d XP p a t i e n t s was observed. When c e l l s from two XP p a t i e n t s w i t h d i f f e r e n t DNA r e p a i r c a p a c i t i e s were compared, the one w i t h the more severe r e p a i r d e f i c i e n c y e x h i b i t s a h i g h e r frequency of chromosome a b e r r a t i o n s and lower clone forming c a p a c i t y than i n the l e s s r e p a i r - d e f i c i e n t p a t i e n t . 7. C e l l s from s e v e r a l XP heterozygotes i n c l u d e d i n the present study d i d not e x h i b i t any DNA r e p a i r d e f i c i e n c y or s e n s i t i v i t y towards chemical carcinogens. 8. 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T e r r a c i n i , B., Palestro, G., G i g l i a r d i , M.R., and Montesano, R. 1966. Carcinogenicity of dimethylnitrosamine i n Swiss mice. B r i t . J . Cancer 20; 871-876. Todaro, G.J., and Martin, G.M. 1967. Increased s u s c e p t i b i l i t y of Down's syndrome f i b r o b l a s t s to transformation toy SV^. Proc. Soc. Expt. B i o l . Med. 124; 1232-1236. u Toth, B., Magee, P.N., and Shubik, P. 1 9 6 4 . Carcinogenesis study with dimethylnitrosamine administered o r a l l y to adult and subcutaneously to newborn BAL3/C mice. Cancer Res. 24 i 1 7 1 2 - 1 7 2 1 . Trosko. J.E., and Chu, E.H.Y. 1 9 7 5 . The role of DNA rep a i r and somatic mutation i n carcinogenesis. In "Advances i n Cancer Research", G. K l e i n and S. Weinhouse (Eds.), v o l . 2 1 , pp. 3 9 1 - 4 2 5 . Academic Press, New York. Turner, N.C., and B e l l , M.A.M. 1 9 7 3 . The ethnobotany of the Southern Kwakiute Indians of B r i t i s h Columbia. Economic Botany 27» 2 5 7 - 3 1 0 . 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Metcalf (Eds.), Vol. I I , pp. 3 2 1 - 3 5 0 . Wiley Interscience, New York. Young, D, 1 9 7 1 a . SV^ 0 transformation of c e l l s from patients with Fanconi vs anaemia. Lancet l i 2 9 4 - 2 9 5 . - Young, D. 1971b. The s u s c e p t i b i l i t y to SV^ 0 virus transformation of f i b r o b l a s t s obtained from patients with Down's syndrome. European J. Cancer 7» 3 3 7 - 3 3 9 . APPENDIX 1 Cost Anaylsis of DNA Repair Bioassay for a Chemical Carcinogen. The following c a l c u l a t i o n s are based on the assumption that 50 p e t r i plate cultures of human skin f i b r o b l a s t s are required per t e s t compound. (The cost of i n i t i a t i n g an explant f i b r o b l a s t culture from a skin biopsy has not been included). Materials Tissue Culture Cost ($) 50 35 mm. P e t r i plates (seeding) @ $61.74-/500 ) 6 17 50 35 mm. P e t r i plates (ADM transfer) @ $61.74/500 ) * 50 22 mm. Cover glass (seeding) @ $28.6o/lO oz. (1 oz. = 150 cover glasses) .48 40 ml. 0.25% Trypsin s o l u t i o n @ $4.80/25 gm. .02 40 ml. Hank's Balanced S a l t Solution @ $2.44/10 l i t r e s .10 300 ml. MEM (seeding) @ $70.00/100 l i t r e s .21 For 1400 ml. ADM. 200 ml. (blocking c e l l culture) 200 ml. (chemical solutions) 200 ml. (washing, no serum) 800 ml. (cold chase, no serum, no a n t i b i o t i c s ) 14 ml. Vitamins (lOOx concentrate) @ $4/100 ml. .56 14 ml. Non-essential amino acids (lOOx concentrate) @ $6/100 ml. .84 E s s e n t i a l amino acids @ $2.32/10 l i t r e s ADM .32 Hank's Balanced S a l t Solution @ $2.44/10 l i t r e s ADM .34 A n t i b i o t i c s f o r 900 ml. MEM + ADM P e n i c i l l i n G (Final-concentration @ 204 units/ml.) $11/10° units .02 Streptomycin ( F i n a l concentration© 29.6 ug/ml.) $4.80/25 gm. .005 Fungizone (Amphtericin B) ( F i n a l concentration @ 2.5 ug/ml.) $7.60/100 ml. (250 ug/ml.) .68 Kanamycin ( F i n a l concentration @ 100 ug/ml.) $10.00/100 ml. (10,000 ug./ml.) .90 34 ml. 7.5% Sodium bicarbonate s o l u t i o n for 1700 ml. MEM + ADM (@ 16 ml. per 800 ml. medium) $31/5 l b . .03 20 ml. F e t a l c a l f serum f o r 400 ml. ADM (5% FCS) 45 ml. F e t a l c a l f serum f o r 300 ml. MEM (15% FCS) $43.00/500 ml. 5.59 0 . 5 mCi ^HTdR (50 ml. @ 10 ;jCi/ml.) $ 8 0 . 0 0 / 5 mGi 8.00 Sampling of Treated Cultures: 800 ml. 1% Sodium C i t r a t e Solution $26/5 l b . .09 1400 ml. EtOH (100%) $ 5/gal 1,84 200 ml. G l a c i a l Acetic Acid $ 6 0 / 5 0 pt. .#10 50 Gold Seal Microscope s l i d e s $4 . 3 5/gross 1 .51 NTB-3 Emulsion (Coating @ 200 s l i d e s / 2 oz.) $ 5 0 / 4 o z . 6 . 0 0 Processing and Staining of Autoradiograms: 500 ml. Kodak D-19 Developer $ 1 . 9 8 / g a l . 750 ml. Fixer $ 1 1 . 9 5 / 5 g a l . 200 ml. Hypoclearing Agent $ 2 . 3 5 / 2 g a l . 4 gm. Orcein ( f o r 200 ml. _ 200 ml. G l a c i a l a c e t i c acid $ 6 0 / 5 0 pt. 200 ml. 100 fo EtOH $ 5/gal. 300 ml. Butanol $ 5 4 / 5 g a l . 500 ml. Xylene $ 3 0 / 5 g a l . Permount (1000 s l i d e s per 4 — 50 22 mm. Cover Glass $28.60/ 10 cover glasses) aceto-orcein) $ 2 5 / 2 5 gm. (for 200 ml. 2% aceto-orcein) f l . oz.) $4/4fl. oz. oz. (1 oz. = 150 .26 . 4 7 .06 4.00 .10 .26 . 8 5 . 7 9 .20 . 4 8 Total Cost of Materials: $41.38 Labour Man-Hours Preparation of 40 ml. Trypsin (@ 2 h r . / l i t r e ) oTolJ Preparation of 300 ml. MEM (@ 2 hr./20 l i t r e s ) 0 . 0 3 Preparation of 1400 ml. ADM (@ 4 hr./lO l i t r e s ) O .56 Preparation of 200 ml. Aceto-orcein (@ 1 h r . / l . 5 l i t r e s ) 0 . 1 3 Seeding and changing culture medium 2.00 Transfer of cultures into ADM 1.00 Planning experiment 4.00 Running experiment 8.00 Mounting, washing and coating of c e l l cultures 6.00 Developing and st a i n i n g autoradiograms 4.00 Analysis of autoradiograms (@ 3-4 s l i d e s per hour) if, on and tabulation of data (compilation) ' 41.80 Labour cost (based on the service of a technician earning $850 per month, 37 .5-hr. work week) $220.34 Cost of Materials $ 41.38 $261.72 159 A d d i t i o n a l Cost f o r A c t i v a t i o n with Mouse L i v e r Microsomes  (S9 Mix). ~ ~ Microsomal Preparation (12 gm. mouse l i v e r required) & 1.3 gm. liver(approx.)/adult mouse - 9 mice @ $1.50 $13.50 NADP 150 mg. @ $89.75/gm. 13.50 NADPH (For DMN control) 12 mg. @ $120.00/500 mg. 4.80 Clucose-6-Phosphate 225 mg. @ $30.00/5 gm. 1.35 MgCl 2.6H 2) 203 mg. @ $4.35/ l b . (.002) Labour (2 man-hours) 10.50 $43.65 APPENDIX 2 160 Human Skin Fibroblasts The great advantage of d i p l o i d human skin f i b r o b l a s t o i d c e l l cultures i s that they permit co n t r o l l e d studies of i n d i v i d u a l s t r a i n v a r i a t i o n . Except f o r a variable but s i g n i f i c a n t degree of tetraploidy and occasional nondisjunct-i o n a l progeny, the genotype of the c e l l s established i n culture i s that of the donor. Without exception, human skin f i b r o b l a s t s have a l i m i t e d r e p l i c a t i v e l i f e span which places l i m i t s on the types of experiments which can be performed. Biopsy Technique Vigorous cultures of predominantly d i p l o i d f i b r o b l a s t o i d cultures can often be established from a single dermalepidermal punch biopsy about 2-3 mm. i n diameter. The minimum depth of biopsy varies with the thickness of the epidermis. At the usual s i t e of biopsy, the mesial aspect of the upper-forearm, a depth of 1 mm. w i l l usually s u f f i c e . The s i t e was chosen because of cosmetic considerations, the r e l a t i v e ease of access, the minimal k e r a t i n i z a t i o n , and the sparseness of h a i r f o l l i c l e s . However, successful cultures can be established with dermal explants from other regions of skin. For w e l l - c o n t r o l l e d experiments, consistency i n the s i t e of biopsy i s advised. S t e r i l e skin preps are obtained using 70% ethanol and l o c a l anesthetics. Transport and Storage of Tissue The biopsy tissues are best stored and transported i n a standard s t e r i l e tissue culture medium (MEM) with 15-20% f e t a l c a l f serum. The main precaution i n shipment i s the avoidance of freezing temperatures. I n i t i a t i n g Explant Cultures While enzymatic digestion has been successfully employed for both mass cultures and primary cloning, explant cultures are much more convenient and are probably more r e l i a b l e i n the case of small specimens of skin. Given a l i m i t e d amount of material, we prefer a "sandwich" explant technique. I t i s the surest method of obtaining growth since i t minimizes the p o s s i b i l i t y of loss of explants because of detachment, dessication, pH f l u c t u a t i o n or contamination. The specimen i s washed i n 3 changes of WASH MEM (MEM without serum) with a n t i b i o t i c s ( p e n i c i l l i n , streptomycin, fungizone, kanamycin, anti-PPLO agent). The tissue i s then transferred to a 15 cm. s t e r i l e glass p e t r i dish previously loaded with a small amount of medium. The tissue i s then diced, using 2 sharp scapel blades, into approximately p i n -head sized pieces (about 50-60 pieces f o r a 3 nun punch). With a s t e r i l e pasteur pipette, transfer the pieces to 35 ™M p e t r i dishes. Transfer 3-4 pieces per t e t r i dish. Place s t e r i l e 22 mm. square glass c o v e r s l i p s on top of the pieces i n each dish. The tissue fragments are therefore sandwiched between the glass c o v e r s l i p s and the bottom surfaces of the p e t r i dishes. Two mis. of medium (* MEM - supplemented with 15-20% FCS and a n t i b i o t i c s ) are added to each dish. These are then incubated at 37 C i n a CO£ incubator. In the early stages of c e l l migration from the explants one often observes e p i t h e l o i d c e l l s , probably derived from the epidermis. However, these are in v a r i a b l y and r a p i d l y outgrown by the more a c t i v e l y migrating spindle-shaped f i b r o b l a s t o i d c e l l s which eventually cover the surfaces of the p e t r i dishes and co v e r s l i p s . The time f o r confluency varies g r e a t l y depending upon the s t r a i n and the number of viable explants, but i t i s generally of the order of several weeks when the medium i s changed twice weekly a f t e r an i n i t i a l 7-day i n t e r v a l i n the o r i g i n a l explant medium. When the p e t r i dishes reach approximately 4-0-60% confluency, the c e l l s are transferred to 10 cm. p e t r i dishes (Falcon p l a s t i c ) using t r y p s i n . The skin fragments may be used again with the "sandwich" technique. * With bicarbonate-buffered media, our experience has been that open p e t r i dishes ( i n a humidified atmosphere with the appropriate concentration of CO2) are superior to closed systems; t h i s i s probably because of better pH c o n t r o l . 162 APPENDIX 3 Arginine D e f i c i e n t Medium (ADM) To prepare 10 l i t r e s of ADMi-1. Hank's Balanced S a l t Solution (BSS) To prepare 1 l i t r e of lOx stock solutions-1.1. Sodium Chloride (NaCl) .80 gm. Potassium Chloride (KCI) 4 gm. Magnesium Sulphate (MgSOk.7H2O) 1 gm. Sodium Phosphate, dibasic (Na2HP0k) 0.48 gm. Potassium Phosphate, monobasic (KH2PO4) 0.6 gm. Glucose 10.0 gm. (Dissolved i n 800 ml. of d i s t i l l e d water) 1.2. Calcium Chloride (CaCl2) 1.4 gm. (Dissolved i n 100 ml. of d i s t i l l e d water) 1 .3 . Phenol Red 0.1 gm. (Dissolved i n d i s t i l l e d water. Before making up to a f i n a l volume of 100 ml., the pH has to be adjusted to 7.0 with 0,05 N NaOH) 1.4. Mixing of the above three solutions gives 1 l i t r e of lOx stock Hank's BSS. 2. E s s e n t i a l Amino Acids L-Histidine 310 mg. L-Leucine 520 mg. L-Lysine 580 mg. L-Isoleucine 520 mg. L-Methionine 150 mg. L-Phenylalanine 320 mg. L-Threonine 480 mg. L-Tryptophan 100 mg. L-Valine 460 mg. (Dissolved i n 100 ml. 1 x Hank's BSS) L-Tyrosine 360 mg. (Dissolved i n 100 ml. 0.1 N HC1) L-Cystine 240 mg. (Dissolved i n 100 ml. 0.1 N HCl) L-Glutamine 2.92 gm. (Dissolved i n 100 ml. 1 x Hank's BSS) 3. Non-Essential Amino Acids L-Alanine 89 mg. L-Asparagine 150 mg. L-Aspartic Acid 133 mg. L-Glutamic Acid 147 mg. L-Proline 115 mg. L-Serine 105 mg. Glycine 75 mg. (Dissolved i n 100 ml. 1 x Hank's BSS) 4. Vitamins Choline Chloride 100 mg. Nicotinamide 100 mg. i - I n o s i t o l 200 mg. Pyridoxal 100 mg. R i b o f l a v i n 10 mg. D-Ca-Pantothenate 100 mg. Thiamine HCI 100 mg. (Dissolved i n 100 ml. Ix BSS) F o l i c Acid 10 mg. (Dissolved i n 100 ml. Ix BSS) The solutions from ( 2 ) , ( 3 ) , (4) and the amount l e f t from (1) are thoroughly mixed. D i s t i l l e d water i s added to bring the f i n a l volume to 10 l i t r e s . The culture medium can be s t e r i l i s e d by passage through a m i l l i p o r e f i l t e r (pore size* 0.22 microns: M i l l i p o r e F i l t e r Corporation, Mass., U.S.A). A n t i b i o t i c s , f e t a l c a l f serum and sodium bicarbonate are added to the culture just p r i o r to use. In l i e u of weighing out the i n d i v i d u a l items, the vitamins and non-essential amino acids are obtainable i n the form of lOOx concentrated mixture from Flow Laboratories Inc. (Inglewood, C a l i f o r n i a ) . APPENDIX 4 S t a t i s t i c a l Analysis of Autoradiograms In the analysis of autoradiograms, at l e a s t 30 n u c l e i , at random locations throughout the entire c o v e r s l i p culture, were scored f o r grain number. Background count was taken into consideration by reckoning the number of grains over an area equal i n size to that of the nucleus. Routinely, grain counts were made on small interphase n u c l e i . The data from a t y p i c a l experiment are shown i n Table A. As the mean value of grains per nucleus becomes smaller, the c o e f f i c i e n t of v a r i a t i o n increases f o r a given number of nuc l e i analysed (50 i n t h i s example, except f o r the highest concentration). Based on s t a t i s t i c a l c a l c u l a t i o n s , Rogers and England (1973) have demonstrated that the accuracy of estimating the r a d i o a c t i v i t y per nucleus w i l l depend, not on the number of nu c l e i counted, nor on the t o t a l area of emulsion scanned, but on the t o t a l number of s i l v e r grains counted i n one sampling of the population. England and M i l l e r have prepared charts from which the optimal a l l o c a t i o n of e f f o r t i n grain counting can be obtained once a rough estimate of the r a t i o of counts over the l a b e l l e d sources to counts over the background i s known Microscropy, 92; 167 (1970^7. For example, the optimal number of grains to be counted f o r three stated values of the co-e f f i c i e n t of v a r i a t i o n i s given i n Table B. When the background count i s low (e.g. 1 ) , only 7 n u c l e i need to be counted (for a source to background r a t i o of 20) to obtain a c o e f f i c i e n t of v a r i a t i o n not exceeding 10%. R e f e r r i to the f i r s t set of g r a i n counts i n Table A and using England and M i l l e r ' s c a l c u l a t i o n s , only 12 n u c l e i need to be counted to obtain a c o e f f i c i e n t of v a r i a t i o n of 5%. The actual number (35) of n u c l e i scored i s therefore adequate. For the lowest concentration (6xl0~^M), a t o t a l of 1500 grains would have to be counted. From these considerations, i t i s apparent that a r e l a t i v e l y small number of n u c l e i need to be counted when the average grains per ^nucleus and the source to background r a t i o are high. Many nuc l e i have to be scored i f the counts are low. However, since the main objective of an experiment i s to obtain a dose response p r o f i l e rather than the s t a t i s t i c a l s i g n i f i c a n c e of mean value f o r grains per nucleus (e.g. 2 ) , the analysis of 30 to 50 n u c l e i per sample ( i r r e s p e c t i v e of mean grains per nucleus) may be j u s t i f i e d . Another problem a r i s e s when data from d i f f e r e n t experiments are to be compared, e.g. the l e v e l of un-scheduled DNA synthesis induced by two d i f f e r e n t chemical carcinogens. To obtain a meaningful comparison, a control s l i d e has been included i n each experiment to ensure that any differences between two experiments are not due to technical v a r i a t i o n s alone. Table C shows the grain counts from control s l i d e s of 5 experiments. Before a comparison i s made between two sets of data from d i f f e r e n t experiments, the control s l i d e s are f i r s t analysed f o r any s t a t i s t i c a l l y s i g n i f i c a n t difference. Adjustments can then be made when the two sets of data are compared. 167 Table A. Unscheduled vHTdR Incorporation i n Cultured Human Fibroblasts Following Exposure to N-Hydroxy-2-AAF (5 hr.) and JHTdR (1.5 hr.) CONCENTRATION (M) 10" k 5xlO" 5 2 . 5 x l O " 5 1.2xl0~ 5 6x10" GRAIN COUNT* S+B B S+B .B S+3 B S+B B S+B B S+B B S+B B S+B B S+B B S+B B 4-5 2 40 1 34 2 26 2 12 2 16 2 7 0 6 1 6 8 7 5 42 0 31 0 29 0 31 0 12 1 14 4 10 3 9 5 10 8 6 3 44 1 29 0 17 0 17 2 13 1 16 4 6 2 10 4 8 5 9 4 37 2 34 0 18 0 18 1 12 0 17 2 8 2 10 4 9 4 2 2 35 2 40 1 19 1 27 1 11 1 18 3 10 2 7 1 4 3 3 4 47 0 29 0 21 1 30 1 11 2 17 4 10 2 10 4 7 4 4 4 50 1 42 0 28 0 25 0 15 1 19 0 11 1 13 3 7 3 8 5 47 1 43 1 24 1 15 0 8 2 22 3 5 0 10 4 6 4 5 4 37 0 36 0 22 1 21 0 16 1 22 3 14 4 5 2 5 2 5 5 40 1 38 1 14 0 24 4 10 1 10 2 12 5 10 5 7 7 7 4 48 0 12 0 26 3 19 3 15 2 14 5 8 3 4 2 4 3 50 1 34 2 17 1 9 4 11 1 7 1 4 2 5 4 5 3 45 1 32 1 24 1 16 1 12 5 13 4 8 2 5 4 3 3 46 0 22 2 25 4 13 1 23 5 7 4 12 3 6 4 4 2 40 0 15 0 24 3 19 4 10 4 8 3 8 2 7 4 4 4 35 1 24 1 23 1 11 0 11 0 5 0 8 3 7 4 9 5 41 2 17 0 28 1 12 0 10 1 2 4 10 5 5 6 7 4 26 2 19 0 24 2 13 0 12 2 8 3 5 3 9 6 6 3 45 1 18 1 22 1 14 1 17 2 11 3 7 0 5 3 6 4 38 2 27 0 14 2 10 1 11 2 8 2 7 0 8 4 4 2 37 0 22 0 21 1 8 1 7 2 8 2 8 3 4 3 5 2 37 0 18 0 19 2 15 2 11 3 12 4 12 4 6 3 3 3 38 0 15 2 20 1 10 0 . 9 0 8 4 7 1 7 4 4 2 36 1 21 1 24 1 9 0 9 1 7 1 12 4 9 6 5 2 28 1 16 1 18 0 16 3 8 4 11 4 5 1 7 4 5 3 10" •4 5x10" 2.5x10" % 1.2x10" 5M 6xlO"6M S+B B S+B B S+B B S+B B S+B B S x i 1376 26 1101 52 661 94 433 134 293 194 x 3 9 . 3 0.7 22.0 U 0 13.2 1 . 9 8.7 2.7 5.9 3.9 S.D.(S) 6 . 3 5 . 5 4.2 3.1 2 .4 S.E.(S) 1. 06 0.77 0.60 0 . 4 4 0 . 33 C.V.(S) 16% 26% 37% 52% 120% * S+B = grains over nucleus due to source including background. B = background count. S.D.(S) = Standard deviation of grain count due to source alone = Vs.D.( S + B ) 2 + S.D. ( B ) 2 S.E.(S) = standard error of the mean of counts due to source alone. C.V.(S) = c o e f f i c i e n t of v a r i a t i o n of counts due to source alone. Table B. Optimal A l l o c a t i o n of E f f o r t i n Grain Counting Between the Labelled Sources and Background Rough Estimate of Grain Count S+B1 B (S+B)/B Ratio Number of Background p Grains to Count Number of Grains Over Sources to Count Number to of Nuclei Count CV<=.10 cv=.05 CV=.025 CV=.10 cv=.05 CV=.025 CV=.10 CV=.05 CV=.025 2 5 10 20 4o 1 1 1 1 1 2 5 10 20 40 70 18 5 1.5 0.5 300 70 20 6 2 1200 300 80 25 8 370 210 160 135 120 1500 840 650 540 490 6000 3400 2600 2150 1980 185 42 16 7 3 750 168 65 27 12 3000 680 260 107 49 6 15 30 60 120 3 3 3 3 3 2 5 10 20 40 70 18 5 1.5 0.5 300 70 20 6 2 1200 300 80 25 8 370 210 160 135 120 1500 840 650 540 490 6000 3400 2600 2150 1980 62 14 5 2 1 250 53 22 9 4 1000 226 87 36 17 1. S+3 B 2. CV = grain count over source, including background. = background grain count. = three stated values of the c o e f f i c i e n t of v a r i a t i o n . Table C. UV Control Slide from 5 D i f f e r e n t Experiments (100 ergs/mm2 Followed by 1.5 hr. ^ HTdR) 169 .EXPERIMENT 1 2 3 4 5 GRAIN COUNT* S+B B S+B B S+B B S+B B S+B B 70 3 70 2 80 0 70 0 61 0 54 2 68 3 62 0 62 0 55 4 46 2 60 1 53 0 73 1 47 4 52 1 52 0 62 0 54 0 78 6 59 1 62 1 68 1 80 0 65 4 46 3 57 0 77 0 72 0 86 5 70 1 52 0 67 0 48 1 51 3 59 2 26 0 63 0 66 1 54 0 62 3 35 2 50 0 58 0 76 5 63 2 70 1 68 0 46 0 60 3 54 2 57 2 66 1 56 0 50 5 58 2 52 1 56 1 64 0 60 3 57 2 78 1 64 0 80 0 63 5 60 1 61 0 44 0 48 0 76 6 66 2 82 0 68 1 68 0 72 4 56 • 1 82 2 56 0 60 0 58 4 57 2 48 0 58 0 78 1 74 6 45 0 52 2 64 0 60 0 68 4 43 2 60 1 62 1 54 0 57 2 65 1 58 1 58 0 50 0 65 6 63 0 69 1 72 1 80 0 70 0 64 1 61 0 61 0 62 0 83 0 1142 33 1182 20 1574 6 1604 6 1276 79 X 57.1 1.7 59.1 1.0 63.0 1 0.2 64.2 0.2 63.8 4.0 S.D.(S) 8.0 14.1 7.9 11.0 10.7 S.E.(S) 1.8 3. 2 1.6 2. 2 2 .4 C.V.(S) 14.4% 24. 2% 12.5! % 17. 1% 17.8% * S+B = grains over nucleus due to source including background. B = background count. S.D.(S) = standard deviation of grain count due to source alone = V s . D . ( S + B ) 2 + S.D.(B) 2 S.E.(S) = standard error of the mean of counts due to source alone. C.V.(S) = c o e f f i c i e n t of variation.of counts due to source alone. RICHARD HING-CHEUNG SAN Pub l i c a t i o n s i 1. S t i c h , H.F., and San, R.H.C. DNA repa i r synthesis and chromosome aberrations. Abstract. Can. J. Genet. Cytol. 12: 396-397 (1970). 2. St i c h , H.F., and San, R.H.C. DNA repair and chromatid anomalies i n mammalian c e l l s exposed to 4-nitroquinoline . 1-oxide. Mutation Res. 10. 389-404 (1970). 3. S t i c h , H.F., San, R.H.C,, and Kawazoe, Y. DNA repair synthesis i n mammalian c e l l s exposed to a series of oncogenic and non-oncogenic derivatives of 4-nitro-quinoline 1-oxide. Nature (Lond.) 229: 416-419 (1971). 4. S t i c h , H.F., and San, R.H.C. Reduced DNA repair synthesis i n Xeroderma pigmentosum c e l l s exposed to the oncogenic 4-nitroquinoline 1-oxide and 4-hydroxyaminoquinoline 1-oxide. Mutation Res. 13_i 279-282 (1971). 5. S t i c h , H.F., San, R.H.C., M i l l e r , J.A., and M i l l e r , E.C. Various l e v e l s of DNA repa i r synthesis i n Xeroderma pigmentosum c e l l s exposed to the carcinogenic N-hydroxy-and N-acetoxy -2-acetylaminofluorene. Nature New B i o l . (Lond.) 2_3: 9 (1972). 6. St i c h , H.F., San, R.H.C, and Kawazoe, Y. Increased s u s c e p t i b i l i t y of Xeroderma pigmentosum c e l l s to chemical carcinogens and mutagens. Mutation Res. 17* 127-137 (1972). 7. S t i c h , H.F., and San, R.H.C. DNA repa i r synthesis and c e l l s u r v i v a l of re p a i r d e f i c i e n t human c e l l s exposed to the K-region epoxide of benz(a)anthracene. Proc. Soc. Exp. B i o l . Med. 142i 155-158 (1973). 8. S t i c h , H.F. Stich', W., and San, R.H.C. Elevated frequency of chromosome aberrations i n r e p a i r - d e f i c i e n t human c e l l s exposed to the carcinogen and mutagen 4-nitroquinoline 1-oxide. Proc. Soc. Exp. B i o l . Med. 142; 1141-1144 (1973). 9. S t i c h , H.F., Kieser, D., Laishes, B.A., and San, R.H.C. The use of DNA re p a i r i n the i d e n t i f i c a t i o n of carcinogens, precarcinogens, and target t i s s u e . In "Canadian Cancer Conferencet Proceedings of the Tenth Canadian Cancer Conference" (P.G. Sc h o l e f i e l d , ed.), pp. 83-110. University of Toronto Press, Toronto.(1974). 10. S t i c h , H.F., Kieser, D., Laishes, B.A., San, R.H.C, and Warren, P. DNA repair of human c e l l s as a relevant, rapid and economic assay f o r environmental carcinogens. Int Recent Topics i n Chemical Carcinogenesis: Gann Monograph on Cancer Research 12* 3-15 (1975). 11. San, R.H.C, and S t i c h , H.F. DNA r e p a i r s y n t h e s i s o f c u l t u r e d human c e l l s as a r a p i d b i o a s s a y f o r chemical c a r c i n o g e n s . I n t . J . Cancer l6i 284-291 (1975). 12. S t i c h , H.F.,.-San, R.H.C, Lam, P., Koropatnick, D.J., Lo, L.W., and. L a i s h e s , B.A.. DNA fra g m e n t a t i o n and DNA r e p a i r as an i n v i t r o and i n v i v o assay f o r chemical p r e c a r c i n o g e n s , c a r c i n o g e n s and c a r c i n o g e n i c n i t r o s a t i o n p r o d u c t s . IARC/CEC Workshop on "Rapid Screening T e s t s to P r e d i c t Late T o x i c E f f e c t s of Environmental Chemicals" B r u s s e l s , 9-12 June, 1975. IARC S c i e n t i f i c P u b l i c a t i o n s ( i n p r e s s ) . 13. S t i c h , H.F., Lam, P., Lo, L.W., Koropatnick, D.J., and San, R.H.C. The search f o r r e l e v a n t s h o r t term b i o a s s a y s f o r chemical c a r c i n o g e n s : the t r i b u l a t i o n of a modern Sisyphus ( I n v i t a t i o n P a p e r ) . Can. J . Genet. C y t o l . rr_ 1 4-71-4-92 (1975). 14. S t i c h , H.F. , San, R.H.C, Lam, P., and Koropatnick, D.J. The d e t e c t i o n of n a t u r a l l y o c c u r r i n g and man-made. car c i n o g e n s and mutagens by the DNA r e p a i r assay. P r e s e n t e d a t the j o i n t US/USSR meeting on the "Comprehensive A n a l y s i s o f the Environment, US/USSR P r o j e c t VII-2", Honolulu, Oct. 21-25, 1975 (In P r e s s ) . 15. S t i c h , H.F., San, R.H.C, and Lam, P. The p o s s i b l e use of s h o r t term t e s t s f o r c a r c i n o g e n s i n experimental epidemiology. P r e s e n t e d a t a symposium on "Epidemiology and Cancer R e g i s t r i e s i n the P a c i f i c B a s i n " , Maoi, Hawaii, Nov. 10-17, 1975 (In P r e s s ) . 16. San, R.H.C, S t i c h , W., and S t i c h , H.F. D i f f e r e n t i a l s e n s i t i v i t y o f Xeroderma pigmentosum c e l l s of d i f f e r e n t r e p a i r c a p a c i t i e s towards the chromosome b r e a k i n g a c t i o n of c a r c i n o g e n s and mutagens. I n t . J . Cancer ( I n P r e s s ) . 

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