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Role of dna repair and chromosome aberrations in neoplastic transformation San, Richard Hing-Cheung 1972

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The Role of DNA Repair and Chromosome A b e r r a t i o n s i i n N e o p l a s t i c Transformation by RICHARD KING-CHEUNG SAN B. Sc., M c G i l l U n i v e r s i t y , 1968 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 MASTER OF SCIENCE 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 U n i v e r s i t y of B r i t i s h Columbia October, 1972 In present ing th i s thes is in pa r t i a l f u l f i lmen t o f the requirements for an advanced degree at the Un ivers i t y of B r i t i s h Columbia, I agree that the L ib ra ry sha l l make i t f r ee l y ava i l ab le for reference and study. I fu r ther agree that permission for extensive copying o f th i s thes i s fo r s cho l a r l y purposes may be granted by the Head of my Department or by h is representa t i ves . It is understood that copying or pub l i c a t i on of th i s thes i s fo r f i nanc i a l gain sha l l not be allowed without my wr i t ten permiss ion. . R i c h a r d Hing-Cheung SAW Department "of Z o o l°gy (Genetics Programme) The Un ivers i t y of B r i t i s h Columbia Vancouver 8, Canada October 1 0 , 1 9 7 2 . Date • ABSTRACT An attempt has been made to demonstrate an a s s o c i a t i o n between the c a r c i n o g e n i c a c t i v i t y o f a c h e m i c a l compound and i t s c a p a c i t y t o i n d u c e DNA damage and chromosome a b e r r a t i o n s which may r e s u l t i n m u t a t i o n s and/or n e o p l a s t i c t r a n s f o r m a t i o n . - T w e n t y - f i v e 4 - n i t r o q u i n o l i n e 1-oxide (4NQ0) d e r i v a t i v e s and f i v e r e l a t e d compounds o f 4 - n i t r o p y r i d i n e 1-oxide (4NP0) o f v a r y i n g c a r c i n o g e n i c i t y were examined. 3NQ0 2-methyl-4NQO 6-n-butyl-4NQ0 4NP0 5NQ0 3-methyl-4NQ0 6-t-butyl-4NQO 4HAP0 6NQ0 5-methyl-4NQ0 &-n-hexyl-4NQ0 3-methyl-4NPO 7NQ0 6-methyl-4NQO 3-fluoro-4NQ0 3-methyl-4HAP0 8NQ0 7-methyl-4NQO 6-chloro-4NQO 2,3-dimethyl-4NPO 8-methyl-4NQ0 6-carboxy-4NQO 2,3-d ime thy1-4HAP0 4 - h y d r o x y a m i n o q u i n o l i n e 1-oxide (4HAQ0) 4 - a m i n o q u i n o l i n e 1-oxide (4-NH2-Q0) 4 - h y d r o x y q u i n o l i n e 1-oxide (4-OH2-QO) 4 - p h e n y l s u l f o n y l q u i n o l i n e 1-oxide Q u i n o l i n e 1-oxide (QO) 4 - n i t r o q u i n o l i n e (4NQ) 4 - h y d r o x y a m i n o q u i n o l i n e (4HAQ) 4 - a m i n o q u i n o l i n e (4-NH2-Q) The i n d u c t i o n o f DNA damage, 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 were used as end p o i n t s . Monolayer c u l t u r e s o f embryonal S y r i a n - h a m s t e r c e l l s and an e s t a b l i s h e d l i n e o f baby hamster k i d n e y c e l l s (BHK-21) were employed i n t h i s s t u d y . DNA damage, as measured by the un s c h e d u l e d i n c o r p o r a t i o n of t r i t i a t e d t h y m idine ( H-TdR), was ass a y e d by the a u t o r a d i o -g r a p h i c p r o c e d u r e . To d i s t i n g u i s h DNA r e p a i r s y n t h e s i s from DNA r e p l i c a t i o n s y n t h e s i s a t S-phase, c u l t u r e d embryonal hamster c e l l s were a r r e s t e d a t G-^  by growing them i n an a r g i n i n e d e f i c i e n t medium (ADM) p r i o r t o the a p p l i c a t i o n o f the v a r i o u s c a r c i n o g e n s . The u n s c h e d u l e d uptake o f r a d i o i s o t o p e was e s t i m a t e d by c o u n t i n g the number o f g r a i n s p e r d i p l o i d n u c l e u s o f c a r c i n o g e n t r e a t e d c e l l s . The h i g h l y o n c o g e n i c d e r i v a t i v e s o f 4NQ0 and 4NP0 e l i c i t e d an e l e v a t e d l e v e l o f u n s c h e d u l e d H-TdR i n c o r p o r a t i o n i n t r e a t e d c e l l s , w h i l e the weakly o n c o g e n i c compounds i n d u c e d o n l y a s m a l l e r amount o f DNA r e p a i r s y n t h e s i s . The non-oncogenic N-oxides f a i l e d t o provoke any d e t e c t a b l e H-TdR uptake. Chromosome a b e r r a t i o n s were s t u d i e d i n ADM-arrested c e l l s which were exposed t o the v a r i o u s compounds and then t r i g g e r e d i n t o d i v i s i o n by t r a n s f e r r i n g them i n t o the r e g u l a r growth medium. A d i r e c t p r o p o r t i o n a l i t y was ob s e r v e d between the degree o f c a r c i n o g e n i c i t y o f a compound and the fre q u e n c y o f i n d u c e d chromosome a b e r r a t i o n s . The c l o n e f o r m i n g a b i l i t y o f t r e a t e d c e l l s was employed as a means to compare the c y t o t o x i c i t y o f the 4NQ0 and 4NP0 d e r i v a t i v e s . P o t e n t c a r c i n o g e n s were h i g h l y c y t o t o x i c ; weakly c a r c i n o g e n i c compounds showed o n l y a s l i g h t l e t h a l e f f e c t and non-oncogenic d e r i v a t i v e s d i d not a f f e c t c e l l s u r v i v a l . T h i s study demonstrated the c a p a c i t y o f c a r c i n o g e n s t o induc e a l t e r a t i o n s at the chromosome and DNA l e v e l . The p o s s i b l e r o l e of DNA r e p a i r and chromosome a b e r r a t i o n s i n n e o p l a s t i c transformation was discussed. The use of DNA r e p a i r synthes as an economic and r e l e v e n t t o o l f o r i d e n t i f y i n g mutagens an or carcinogens has been suggested. ACKNOWLEDGEMENTS I would l i k e t o thank Dr. Hans F. S t i c h f o r h i s g u i d a n c e and i n s p i r a t i o n t h roughout the c o u r s e o f t h i s work. To Mrs. W. S t i c h , M i s s Ruth Sheuing and M i s s C h a r l o t t e Yoshizawa, I would l i k e to ex p r e s s my s i n c e r e g r a t i t u t e f o r t h e i r e x p e r t t e c h n i c a l a s s i s t a n c e . Support from the N a t i o n a l Cancer I n s t i t u t e o f Canada and the N a t i o n a l R e s e a r c h C o u n c i l o f Canada ( g r a n t s t o Dr. S t i c h ) i s d e e p l y a p p r e c i a t e d . V TABLE OF CONTENTS ABSTRACT i ACKNOWLEDGEMENTS i v TABLE OF CONTENTS v LIST OF FIGURES v i LIST OF TABLES v i i i INTRODUCTION 1 MATERIALS AND METHODS 9 I Tissue C u l t u r e Techniques 9 I I Chemical Treatment 11 I I I U l t r a v i o l e t I r r a d i a t i o n 13 IV C y t o l o g i c Preparations 14 V Autoradiographic Technique 16 VI Measurement of DNA Repair Synthesis 17 RESULTS 19 I DNA Repair Synthesis and DNA R e p l i c a t i o n 19 I I DNA Repair Synthesis and C a r c i n o g e n i c i t y of a Compound 26 I I I Chromosome Ab e r r a t i o n s and C a r c i n o g e n i c i t y <. of a Compound 37 IV Clone Forming Capacity of C e l l s and C a r c i n o g e n i c i t y of a Compound ^ 42 DISCUSSION 46 SUMMARY 59 REFERENCES 62 APPENDICES .81 v i LIST OF FIGURES Figure 1. Main Types of DNA Repair Mechanisms. 2 Figure 2. P o s t u l a t e d Steps i n the E x c i s i o n Repair of Damaged DNA. 4 Figure 3. DNA Repair Synthesis Induced i n Syrian-Hamster C e l l s by a S i n g l e Dose of U l t r a v i o l e t I r r a d i a t i o n . 20 Figure 4. The %-TdR I n c o r p o r a t i o n P a t t e r n i n Syr i a n -Hamster C e l l s . 20 Figure 5. 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 Embryonal Syrian-Hamster C e l l s . 22 Figure 6. 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 . 22 Figure 7. Amount of 3H-TdR and DNA Content of C e l l s . 25 Fig u r e 8. Frequency D i s t r i b u t i o n of C e l l s w i t h Various Amounts of DNA Repair S y n t h e s i s . 25 Figure 9. 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0) and Related Compounds. 27 Figure 10. 4 - N i t r o p y r i d i n e 1-Oxide (4NP0) and Related Compounds. 29 V l l Figure 11. DNA Repair Synthesis i n ADM-Arrested Syrian-Hamster C e l l s Exposed to 4NQ0 D e r i v a t i v e s . 33 Figure 12. Chromosome Ab e r r a t i o n s i n Syrian-Hamster C e l l s a f t e r Exposure to 4NQ0. 38 Figure 13. Clone Forming Capacity of Syrian-Hamster C e l l s Exposed to Various 4NQ0 D e r i v a t i v e s . 44 V LIST OF TABLES In c o r p o r a t i o n of -5H.-TdR i n t o N u c l e i of Hamster and Human C e l l s A r r e s t e d by A r g i n i n e D e p r i v a t i o n . DNA Repair Synthesis i n Syrian-Hamster C e l l s Exposed to Various 4NQ0 D e r i v a t i v e s . C o r r e l a t i o n between C a r c i n o g e n i c i t y of Some 4NQ0 D e r i v a t i v e s and Induced DNA Repair Synthesis i n Syrian-Hamster C e l l s (A Summary). DNA Repair Synthesis i n Syrian-Hamster C e l l s Exposed to Various 4 - N i t r o p y r i d i n e l-Oxide (4NP0) D e r i v a t i v e s . DNA Repair Synthesis i n Syrian-Hamster C e l l s Exposed to N-Methyl-N'-Nitro-N-Ni t r o s o g u a n i d i n e (MNNG) and 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0). Frequency of Chromosome Breaks and Exchanges i n Syrian-Hamster C e l l s Exposed to Various 4NQ0 and 4NP0 D e r i v a t i v e s . Clone Forming Capacity of Syrian-Hamster C e l l s a f t e r Exposure to Various 4NQ0 and 4NP0 D e r i v a t i v e s . Table V I I I . Response of Syrian-Hamster C e l l s to S t r o n g l y , Weakly and Non-Oncogenic D e r i v a t i v e s of 4NQ0 and 4NP0. Table IX. E f f e c t of 8NQ0 (A Non-Carcinogenic Compound) on DNA Repair Synthesis INTRODUCTION In s p i t e o f numerous s t u d i e s on s p o n t a n e o u s l y o c c u r r i n g tumors and e x p e r i m e n t a l neoplasms i n d u c e d by v a r i o u s v i r u s e s , c h e m i c a l s , and i o n i z i n g o r n o n - i o n i z i n g r a d i a t i o n , the i n v o l v e -ment o f genes i n n e o p l a s t i c t r a n s f o r m a t i o n i s s t i l l l a r g e l y unknown. R e c e s s i v e o r dominant m u t a t i o n s a f f e c t the d e v e l o p -ment o f few r e l a t i v e l y r a r e human tumors. Abnormal chromosome complements c o n s t i t u t e a common f e a t u r e i n many, i f not a l l , p r i m a r y sarcomas and carcinomas o f man and i n most c h e m i c a l or v i r a l i n d u c e d tumors o f r o d e n t s 1 1 ^ . Moreover, t h e r e i s growing e v i d e n c e o f a p o s i t i v e 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 p r o p e r t y and mutagenic c a p a c i t y o f p h y s i c a l and c h e m i c a l a g e n t s 1 ' 2 ' 3 6 ' 3 7 ' 6 9 ' 7 0 ' 7 1 . The o l d q u e s t i o n must be r a i s e d a g a i n as to whether n e o p l a s t i c t r a n s f o r m a t i o n i s a d i r e c t o r i n d i r e c t r e s u l t o f a damage t o the g e n e t i c m a t e r i a l . The p r e s e n t study i s an attempt to f i n d a l i n k between the l e v e o f o n c o g e n i c i t y o f a compound and i t s c a p a c i t y to i n d u c e a g e n e t i c change at the chromosomal o r the m o l e c u l a r l e v e l . S e v e r a l p h y s i c a l and c h e m i c a l agents a r e known t o i n d u c e d i r e c t breakage o f DNA s t r a n d s ( e . g . X - r a y s , a l k y l a t i n g agents) o r i n f l i c t a DNA damage i n an i n d i r e c t way ( e . g . UV). Once the DNA damage i s p r o d u c e d , the c e l l p r o c e e d s to r e p a i r i t a l m o s t i m m e d i a t e l y ^ . There a r e s e v e r a l ways by which DNA r e p a i r can take p l a c e 1 1 6 ( F i g u r e 1). In t h i s s t u d y , we a r e c o n c e r n e d w i t h e x c i s i o n r e p a i r ("dark r e p a i r " ) . T h i s e x c i s i o n DECAY OF DAMAGE (Hydration of Pyrimidines) REVERSION TO ORIGINAL FORM in situ DIRECT (Pyrimidine PHOTOREVERSAL Dimers) DNA REPAIR REJOINING OF SINGLE STRAND BREAKS ENZYME ("Light MEDIATED Repair") PHOTOREACTI VATI ON DAMAGED SEGMENTS REMOVED AND REPLACED EXCISION REPAIR RECOMBINATION REPAIR ("Dark Repair") Figure 1. Main types of DNA re p a i r mechanisms. (Courtesy of Stic h and Laishes, 1972) 3 r e p a i r process, which can occur i n a l l stages of the c e l l c y c l e 9 0 , has a l s o been coined "unscheduled DNA s y n t h e s i s " ^ to d i s t i n g u i s h i t from the r e g u l a r DNA r e p l i c a t i o n o c c u r r i n g d u r i n g S-phase. The type of DNA l e s i o n s induced by U V - i r r a d i a t i o n and the subsequent r e p a i r of t h i s damage has been stud i e d i n great d e t a i l i n m a m m a l i a n 1 1 ' 9 4 ' 1 0 3 ' 1 3 0 and b a c t e r i a l 9 6 ' 1 0 0 systems. There i s sound evidence to s u b s t a n t i a t e the f o l l o w i n g o p e r a t i o n a l steps i n the " e x c i s i o n r e p a i r " of a l t e r e d DNA bases (Figure 2 ) : (1) D i m e r i z a t i o n of adjacent p y r i m i d i n e molecules occurs, OA followed by r e c o g n i t i o n of t h i s a l t e r a t i o n i n the DNA (2) Nicks are made i n the p r o x i m i t y of the dimers, probably by the a c t i o n of an endonuclease. (3) The "nicked" p o r t i o n i s removed from the DNA, probably by the a c t i o n of an e x o n u c l e a s e 1 1 ' 1 0 0 ' 1 0 9 . (4) The e x c i s e d DNA i s degraded. (5) I n s e r t i o n of short sequences of new purine and p y r i m i d i n e bases then takes p l a c e , probably by the a c t i o n of a DNA p o l y m e r a s e 9 0 ' 9 6 ' 1 0 3 ' 1 1 1 . (6) The i n s e r t e d segment i s j o i n e d up w i t h the r e s t of the DNA molecule by a l i g a s e - t y p e enzyme. . To study the e x c i s i o n r e p a i r of UV-type induced DNA l e s i o n s i n mammalian c e l l s , s e v e r a l methods may be used. 3 The DNA can be p r e l a b e l l e d w i t h K-TdR p r i o r to UV i r r a d i a t i o n or chemical treatment. Then, broken DNA strands and i n t a c t DNA may be separated by c e n t r i f u g i n g through an a l k a l i n e sucrose g r a d i e n t 6 ^ ' 1 1 0 ' 1 1 1 ] This technique i s e s s e n t i a l l y F U N C T I O N P R O B A B L E M U T A N T S E N Z Y M E S I . R E C O G N I T I O N S . I N C I S I O N E N D D N U C L E A S E E X C 3 . E X C I S I O N E X O N U C L E A S E 4 . D E G R A D A T I O N S . R E P A I R R E P L I C A T I O N P O L Y M E R A S E R E S 6 . R E J O I N I N G • A W V W F i g u r e 2. P o s t u l a t e d steps i n the e x c i s i o n r e p a i r of damaged DNA. (Courtesy of S t i c h and L a i s h e s , 1972) 5 a measurement of the f i r s t step i n e x c i s i o n r e p a i r and r e f l e c t s the number of strand s c i s s i o n s i n the DNA. 65 99 The s e l e c t i v e p h o t o l y s i s of 5-bromodeoxyuridine (BrdU) ' 95 and bromouracil (BrUra) has been used as a r a p i d method f o r d e t e c t i n g r e p a i r r e p l i c a t i o n i n mammalian and m i c r o b i a l systems. When these thymidine analogs are i n c o r p o r a t e d i n t o DNA, they Q become s e n s i t i v e to f l u o r e s c e n t l i g h t or long-wave u l t r a v i o l e t i r r a d i a t i o n6" * and are rendered a l k a l i n e - l a b i l e . Exposure of DNA w i t h BrUra- or BrdU-containing r e p a i r e d regions to f l u o r e s c e n t or 313 nm l i g h t reduces the sedimentation r a t e c f the DNA i n a l k a l i n e sucrose. From the degree of s h i f t i n the DNA peak f o l l o w i n g 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 g r a d i e n t , the extent of DNA breakage and r e p a i r can be deduced. DNA r e p a i r s y n t h e s i s can a l s o be measured by the combined 19 96 use of a den s i t y and r a d i o a c t i v e l a b e l ' . DNA allowed to r e p l i c a t e i n the presence of 5-bromodeoxyuridine (BrdU), a base analog of thymidine, w i l l be denser than the n o n - r e p l i c a t e d p a r e n t a l DNA. I f the DNA i s then exposed to a p h y s i c a l or 3 chemical agent followed by H-TdR, any unscheduled i n c o r p o r a t i o n 3 of H-TdR w i l l appear i n the l i g h t , h y b r i d , and heavy DNA, separable through c e n t r i f u g a t i o n on a cesium c h l o r i d e g r a d i e n t . Autoradiography i s another means whereby DNA r e p a i r s y n t h e s i s may be assayed. The unscheduled i n c o r p o r a t i o n of H-TdR i n the nucleus of c e l l s a f t e r i r r a d i a t i o n or treatment wit h a chemical agent r e f l e c t s the amount of DNA r e p a i r s y n t h e s i s that has taken p l a c e . In essence, the autoradio-graphic technique measures the l a t e r stages of the r e p a i r process: namely, the i n s e r t i o n of n u c l e o t i d e bases i n t o places vacated by e x c i s e d p y r i m i d i n e dimers. Detection of r a d i o a c t i v i t y by l i q u i d s c i n t i l l a t i o n counting of DNA f r a c t i o n s c o l l e c t e d from an a l k a l i n e sucrose gradient (ASG) i s more r a p i d than autoradiography. However, the use of a r e l a t i v e l y l a r g e number of c e l l s and h i g h dosage of the p h y s i c a l or chemical agents are necessary to provide s u f f i c i e n t 6 ^  DNA fragments to be d e t e c t a b l e on an a l k a l i n e sucrose gradient The ASG technique f u r t h e r s u f f e r s from the l i m i t a t i o n that only a small number ( u s u a l l y s i x ) of samples can be handled at one time. There i s evidence that the ASG technique can introduce s i n g l e strand breaks by v i r t u e of the a l k a l i n e l a b i l i t y of m o d i f i e d regions of DNA . Autoradiography permits the c y t o l o g i c a l 3 l o c a l i z a t i o n of the i n c o r p o r a t e d H-TdR. Even a low l e v e l of DNA r e p a i r i n a few c e l l s can be e a s i l y detected. For massive study of dosage e f f e c t and the time-course of DNA r e p a i r , 3H-TdR i n c o r p o r a t i o n combined w i t h autoradiography i s the method of choice. The b i n d i n g of carcinogens to DNA spurs an i n t e r e s t i n 12 the study of damage to the genetic m a t e r i a l . Brookes and Lawley found a p o s i t i v e c o n - e l a t i o n between the c a r c i n o g e n i c a c t i v i t y of pol y c y . c l i c aromatic hydrocarbons and t h e i r a b i l i t y to bind to DNA. Subsequently, these f i n d i n g s were extended to i n c l u d e 68 the c a r c i n o g e n i c compounds: dimethylnitrosamine , aminoazo 26 2^ 53 dyes ,/3-propiolactone 7 ,12-dimethylbenz(a)anthracene , 53 59 74 N-hydroxy-N-fluorenylacetamide > > and epoxides o f p o l y c y c l i c h y d r o c a r b o n s4 1. This r a i s e s the question whether the b i n d i n g of carcinogens to DNA leads to a l t e r a t i o n i n the genome. Strand s c i s s i o n s i n DNA f o l l o w e d by r e j o i n i n g of the DNA were observed i n mammalian c e l l s a f t e r exposure to sulphur and n i t r o g e n mu stards ' ' , ^ 3-propiolactone , N-acetoxy-2-acetylamino-f l u o r e n e6 6 and 3'-methy1-4-dimethyl aminoazobenzene^0. In view of the f o r e g o i n g biochemical evidence, the p o s s i b i l i t y t hat damage of DNA and i t s subsequent r e p a i r i s i n f a c t i n v o l v e d i n c a r c i n o g e n e s i s cannot be r u l e d out. To continue the search f o r a p o s s i b l e l i n k between DNA r e p a i r 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 , the carcinogen and mutagen 4 - n i t r o q u i n o l i n e 1-oxide and i t s d e r i v a t i v e s were chosen f o r the present study. The choice of these N-oxides i s based mainly on two f a c t s : F i r s t , these compounds are more water s o l u b l e than most other carcinogens. Second, because of the r e l a t i v e l y simple s t r u c t u r e and chemical r e a c t i v i t y of n i t r o q u i n o l i n e 1-oxides, r e l a t e d compounds w i t h a v a r y i n g degree of c a r c i n o g e n i c a c t i v i t y can be e a s i l y s y n t h e s i z e d . F i r s t synthesized by O c h i a i and h i s a s s o c i a t e s i n 1940, 4NQ0 was subjected to t r i a l s as an a n t i c a n c e r d r u g "0 4. Apart from i t s t u m o r i c i d a l a c t i v i t y on E r h l i c h a s c i t e s t u m o r1 0 4, i t was found to have mutagenic a c t i o n on m i c r o o r g a n i s m s3" * ' ^1. 52 I t a l s o i n a c t i v a t e s phages and destroys the transforming a c t i o n of i s o l a t e d DNA . I t s a d m i n i s t r a t i o n to rodents in v i v o has r e s u l t e d i n tumors i n the l u n g , ovary, u t e r u s , tongue, stomach, 8 31 91 92 skin, gingiva and other organs » ' . In vit r o , i t has been found to cause neoplastic transformation of hamster 60,105 85 and rat c e l l s . At the cytologic level, 4NQ0 produces chromosome 1171 ' } ? 7Q aberrations^'»J-J*• and causes unequal nuclear division . At the molecular level, i t binds to DNA^> 5 1» 5 7' 6^' 7 3> 8 2, induces strand scission in the DNA3'^9 and e l i c i t s DNA repair s y n t h e s i s 3 , From a comparison of the effects of 4NQ0 and i t s related derivatives, i t is hoped to establish a possible relationship between the carcinogenicity, the induction of DNA repair synthesis, frequency of chromosome aberrations, and the rate of c e l l survival. MATERIALS AND METHODS I . Tissue C u l t u r e Techniques (a) C u l t u r e Media Eagle's Minimum E s s e n t i a l Medium (MEM). Eagle's minimum 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 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 minimum e s s e n t i a l medium ( M E M )2 8 , 7 6. 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 exception 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 2 ) . The n o n - e s s e n t i a l amino aci d s ( i n the form of 100X concentrated m i x t u r e ) , as w e l l as v i t a m i n s , were obtained from Flow L a b o r a t o r i e s I n c . (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 : p e n i c i l l i n G (General B i o -chemicals, 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 Biochemicals; 29.6 pg/ml), Kanamycin (Grand I s l a n d B i o l o g i c a l Co., B e r k e l e y , C a l i f o r n i a ; 100 /ig/ml), and Fungizone ("Gibco", 2.5 ^ug/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 1 s t o c k . For c e l l c u l t u r e s maintained i n Leighton tubes, the c u l t u r e medium was supplemented 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). In the case o f 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 stored at -20°C. I t was i n a c t i v a t e d by hea t i n g at 56°C f o r 30 minutes before use. MEM was supplemented w i t h 10% FCS whereas ADM was supplemented w i t h only 5% FCS. (b) Cultured C e l l s Embryonal Hamster C e l l s . Primary c u l t u r e s of 12-day-old Syrian-hamster embryos were i n i t i a t e d as described by Cooper D i p l o i d embryonal c e l l s from the f i r s t to fo u r t h passage were used i n the present i n v e s t i g a t i o n . Baby Hamster Kidney C e l l s (BHK-21). An e s t a b l i s h e d l i n e of Syrian-hamster c e l l s obtained from Flow L a b o r a t o r i e s , I n c . , Berk e l e y , C a l i f o r n i a , BHK-21 was used f o r comparative purposes. Fortner Tumor C e l l s . This e s t a b l i s h e d l i n e of Syrian-hamster c e l l s was obtained from Flow L a b o r a t o r i e s , I n c . , R o c k v i l l e , Maryland. Human Skin 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 explants of s k i n punch b i o p s i e s from v o l u n t a r y donors. Human Embryos. F i b r o b l a s t c u l t u r e s were obtained from spontaneously aborted embryos, k i n d l y s u p p l i e d by Dr. B.J. Poland Grzybowski, Department of O b s t e t r i c s and Gynaecology, F a c u l t y of M e d i c i n e , U n i v e r s i t y of B r i t i s h Columbia. (c) C ulture Methods Stock C u l t u r e s . Stock c u l t u r e s were maintained i n "Sani-g l a s s " screw-cap p r e s c r i p t i o n b o t t l e s (Brown's B o t t l e s and S u p p l i e s , I n c . , M o n t r e a l , Quebec) ranging i n s i z e s from 6 to 32 o z . Leighton Tube C u l t u r e s . For DNA r e p a i r and chromosome s t u d i e s , c e l l s were grown on 10 x 35 mm c o v e r - s l i p s (Clay Adams) i n 16 x 85 mm Leighton 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) and maintained i n MEM. 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 s and, i n the case of s l i d e s used f o r autoradiographic p r e p a r a t i o n s , a lower background count. P e t r i P l a t e C u l t u r e s . For c l o n i n g experiments i n s u r v i v a l s t u d i e s , 300-600 BHK-21 or embryonal hamster c e l l s were seeded i n 5 cm s t e r i l e disposable p e t r i p l a t e s (Falcon P l a s t i c s Co., Los Angeles, C a l i f o r n i a ) . R o u t i n e l y , 4 ml o f MEM were added. The c e l l s were allowed to s e t t l e down overnight (16-24 hours) before chemical treatment. The c u l t u r e s were then harvested ! a f t e r 10-14 days when clones comprising 60 or more c e l l s were v i s i b l e . I I . Chemical Treatment 4NQ0 was obtained from D a i i c h i Pure Chemical Co., Tokyo. D e r i v a t i v e s of the compound were k i n d l y s u p p l i e d by Dr. Y. Kawazoe of the N a t i o n a l Cancer Center Research I n s t i t u t e , Tokyo, Japan (see Figures 9 and 10). 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"3M 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% EtOH was added to an appropriate amount of s o l i d i n disposable p l a s t i c tube, followed 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 (MEM or ADM as r e q u i r e d ) . D i l u t i o n s were made w i t h c u l t u r e medium to give the d e s i r e d 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. A few of the d e r i v a t i v e s , notably the n i t r o - , a l k y l - and halogeno- s u b s t i t u t e d compounds, p r e c i p i t a t e d out upon d i l u t i o n w i t h c u l t u r e medium. Dimethyl s u l f o x i d e was t r i e d as a solvent i n these cases though again without much success. However, the suspension i n many instances remained i n the form of micro-p a r t i c l e s d ispersed throughout the medium f o r a cons i d e r a b l e p e r i o d of time and has been found to induce DNA r e p a i r s y n t h e s i s as i n the case of n - b u t y l , t - b u t y l and n-hexyl 4NQ0. I t i s thus reasonable to assume that enough of these compounds may have been d i s s o l v e d to t r i g g e r DNA r e p a i r s y n t h e s i s . *• i. N - M e t h y l - N ' - n i t r o - n i t r o s o g j i a n i d i n e (MNNG) was purchased from Ald r i c h , C h e m i c a l Company, I n c . (Milwaukee, W i s c o n s i n ) . S o l u t i o n s were prepared by d i s s o l v i n g the s o l i d i n water and d i l u t i n g w i t h t i s s u e c u l t u r e medium to give the appropriate f c o n c e n t r a t i o n s . ' • / 13 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 Leighton tube c u l t u r e w i t h a s o l u t i o n of 4NQ0 or i t s d e r i v a t i v e s . 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 che m i c a l , the s o l u t i o n was decanted and JH-TdR-ADM added. 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 treatment. Exposure of p e t r i p l a t e c u l t u r e s to the 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 d e v i c e . Four m i l l i l i t r e s of the chemical s o l u t i o n was added f o r 1% hours 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, the c e l l s were covered w i t h 4 ml of medium and returned to the CO2 i n c u b a t o r . I I I . 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 di s t a n c e of seven i n c h e s , i t gave a dose of 30 ergs/mm /sec as measured by a UV l i g h t meter ( S c i e n t i f i c Apparatus, Thomas Co., P h i l a d e l p h i a ) . The UV meter was c a l i b r a t e d agciinst 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 (Sitnan 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 Leighton 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) to removed serum i n the c u l t u r e medium adhering to the c e l l s . Serum and phenol r e d ( t h e pH i n d i c a t o r used i n c u l t u r e medium) a r e known t o abso r b UV i r r a d i a t i o n . H a l f o f 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 s e r v e 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 , JH-TdR l a b e l l i n g and the a u t o r a d i o g r a p h i c p r o c e d u r e s were c a r r i e d out as f o r c h e m i c a l l y t r e a t e d c e l l c u l t u r e s . IV „ C y t o l o g i c P r e p a r a t i o n s (a) Reagents C o l c h i c i n e (BDH Ch e m i c a l s L t d . , P o o l e , . E n g l a n d ) . A 1 % s t o c k s o l u t i o n i n d i s t i l l e d water was p r e p a r e d from which a p p r o p r i a t e d i l u t i o n t o . 0 1 % and . 0 0 1 7 * s o l u t i o n s c o u l d be made. F o r hamster c u l t u r e s , 0 . 1 ml o f the . 0 1 % c o l c h i c i n e s o l u t i o n was added p e r ml o f c u l t u r e medium, g i v i n g a f i n a l c o n c e n t r a t i o n o f 1 0 ug/ml. Human c e l l s were t r e a t e d w i t h 0 . 1 ml o f the . 0 0 1 % , s o l u t i o n p e r ml o f medium ( i . e . a f i n a l c o l c h i c i n e c o n c e n t r a t i o n o f 1 ug/ml). Sodium C i t r a t e . Sodium c i t r a t e was used as a 17o s o l u t i o n (W/V) i n d i s t i l l e d w a t er. F i x a t i v e . Carnoy's s o l u t i o n was p r e p a r e d by m i x i n g t h r e e p a r t s o f 1 0 0 % e t h a n o l w i t h one p a r t o f g l a c i a l a c e t i c a c i d . A c e t o - o r c e i n . A c e t o - o r c e i n , used as a 2%, s o l u t i o n , was pr e p a r e d by r e f l u x i n g f o r 6 hours the a p p r o p r i a t e amount o f o r c e i n (BDH Ch e m i c a l s L t d . , P o o l e , England) i n 4 5 % a c e t i c a c i d . The s u s p e n s i o n so o b t a i n e d was always f i l t e r e d ( w i t h Whatman No. 1 paper) befor'e u s e . T o l u i d i n e Blue 0 ( F i s h e r S c i e n t i f i c Co.). A 2% s o l u t i o n was prepared by adding the appropriate amount of s o l i d to d i s t i l l e water w i t h constant s t i r r i n g f o r two hours. (b) Techniques Chromosome P r e p a r a t i o n s . To ob t a i n metaphase chromosomes, - 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 f o r 4-5 hours. The c o v e r - s l i p was then removed from the Leighton tube and placed i n 1% sodium c i t r a t e s o l u t i o n . This hypotonic treatment (10-15 minutes) enables the c e l l s to incre a s e 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 of 100% Ethanol and g l a c i a l a c e t i c a c i d (10 m i n u t e s ) . 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 27» a c e t o - o r c e i n , dehydrated through immersion i n 100% EtOH, b u t a n o l , b u t a n o l / x y l o l , 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.). Pr e p a r a t i o n of c e l l c u l t u r e s f o r the study of DNA-repair s y n t h e s i s w i l l be described i n a l a t e r s e c t i o n . S u r v i v a l Experiments. For s u r v i v a l experiments, p e t r i p l a t e c u l t u r e s were harvested by decanting the medium and f i x i n g w i t h Carnoy's f o r 10 minutes. This was fo l l o w e d by r e h y d r a t i o n through 70% ethanol (10 minutes) and two r i n s e s i n d i s t i l l e d water. 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 a 2% t o l u i d i n e blue s o l u t i o n (10 minutes). Excess dye was removed by r i n s i n g i n d i s t i l l e d water. 16 V. Autoradiographic Technique (a) 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 3 T r i t i a t e d thymidine ( H-TdR), obtained i n aqueous s o l u t i o n from Schwarz Bio-Research (Orangeburg, New Y o r k ) , 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 concen-t r a t i o n of 10 juCi/ml was made by mixing w i t h ADM. Che 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 c u l t u r e s were 3 3 p u l s e - l a b e l l e d w i t h H-TdR f o r \\ hours. The H-TdR 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 1 ) . The c o v e r - s l i p s were then taken out of the Leighton tube, immersed i n W sodium c i t r a t e f o r 12-15 minutes, followed by f i x a t i o n i n three changes of Carnoy's (10 minutes each) and allowed to a i r d r y . (b) S t a i n i n g 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 small q u a n t i t y of p a r a f f i n wax was placed oh 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 me l t . 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 gl a s s s l i d e . The c e l l c u l t u r e s were s t a i n e d i n 2% ac e t o - o r c e i n (20 m i n u t e s ) . Excess s t a i n was removed by successive immersion i n two changes of 20% ethanol (10 minutes' each) , -two changes of 10% ethanol (10 minutes each) and four changes of d i s t i l l e d water (10 minutes each). (c) Coating Stained c e l l c u l t u r e s were coated wi 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 stored at 4°C i n l i g h t - t i g h t boxes f o r 14 days. (d) Processing 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 at 18°C i n Kodak D-19 developer (3 minutes) and Kodak f i x e r (15 m i n u t e s ) . A f t e r 1% hours r i n s e i n running water (18°C), the s l i d e s were dehydrated through successive immersion (1\ minutes) i n 95% EtOH, b u t a n o l , b u t a n o l / x y l o l , 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 superimposing another c o v e r - s l i p over that bearing the c e l l monolayer. V I . Measurement of DNA Repair Synthesis 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 s y n t h e s i s at S-phase and DNA r e p a i r s y n t h e s i s i n a p r o l i f e r a t i n g c e l l c u l t u r e , we followed in. p r i n c i p l e the p r e - l a b e l l i n g procedure developed by Cleaver . C o v e r - s l i p c u l t u r e s were placed i n H-TdR-co n t a i n i n g medium f o r 1% hours p r i o r to exposure to 4NQ0 or d e r i v a t i v e s ( a l s o i n H-TdR-containing medium) f o r \\ hours. With t h i s technique, S-phase n u c l e i are extremely h e a v i l y l a b e l l e d whereas n u c l e i w i t h unscheduled DNA sy n t h e s i s have only a l i m i t e d number of gr a i n s above them. A l t e r n a t i v e l y , i n t e r f e r e n c e by DNA sy 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 may be d r a s t i c a l l y reduced by a r g i n i n e 38 d e p r i v a t i o n . This procedure was developed by Freed and Schatz and. was used w i t h m o d i f i c a t i o n s i n the present i n v e s t i g a t i o n . Embryonal Syrian-hamster c e l l s were seeded on c o v e r - s l i p s (Leighton t u b e s ) , kept i n MEM supplemented w i t h 10% f e t a l c a l f serum f o r two days t i l l the c e l l c u l t u r e s were i n t h e i r l o g a r i t h m i c growth phase. They were then r i n s e d thoroughly i n MEM without a r g i n i n e (ADM) and the c o v e r - s l i p s t r a n s f e r r e d to Leighton tubes c o n t a i n i n g a r g i n i n e - f r e e medium. A f t e r two to three days i n ADM, more than 9970 of the c e l l s would be a r r e s t e d at (estimated from the autoradiogram o f a r r e s t e d c u l t u r e s ) . For DNA r e p a i r s t u d i e s , the c e l l c u l t u r e s were kept i n ADM throughout the experiment. 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 gr a i n s over each n u c l e u s . Care was taken that n u c l e i of comparable s i z e were s e l e c t e d so that only c e l l s of the same, p l o i d y we re used. Background count was taken i n t o c o n s i d e r a t i o n by reckoning the number o f gr a i n s over an area equal i n s i z e to that of the. n u c l e u s . RESULTS DNA Repair Synthesis and DNA R e p l i c a t i o n 3 The unscheduled i n c o r p o r a t i o n of H-TdR i n t o the. DNA of n u c l e i was used to estimate DNA r e p a i r i n c e l l s exposed to chemical mutagens or carcinogens. This procedure has been p r e v i o u s l y employed to measure r e p a i r of UV-induced thymine 20,96,100 T * * ..u- * u • u dimers ' . I n the present study t h i s technique has been adopted to examine the response of mammalian c e l l s t r e a t e d w i t h chemical mutagens known to a f f e c t DNA. 3 The question arose as to whether the unscheduled H-TdR i n c o r p o r a t i o n r e f l e c t e d DNA r e p a i r s y n t h e s i s . I t was necessary to prove ( i ) that the r a d i o i s o t o p e was i n the DNA and ( i i ) that n u c l e i w i t h DNA sy 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 at S-phase could be d i s t i n g u i s h e d from n u c l e i undergoing DNA r e p a i r s y n t h e s i s . To prove that the H-TdR i n c o r p o r a t i o n was i n the DNA, 2 c e l l c u l t u r e s were i r r a d i a t e d w i t h UV (900 ergs/mm ) , pulse-l a b e l l e d w i t h 3H-TdR f o r 1% hours and t r e a t e d w i t h deoxyribonuclease f r e e of rib o n u c l e a s e contamination (General B i o c h e m i c a l s , Chagrin F a l l s ) before s u b j e c t i n g to the autoradiographic procedures. The autoradiogram of deoxyribonuclease t r e a t e d p r e p a r a t i o n s showed a g r a i n count over n u c l e i approximating that of the background (Figure 3b), whereas the non-treated p r e p a r a t i o n s had an i n t e n s i t y of g r a i n s which resembled those i n Figure 3a. 3 This observation suggests that the detected H-TdR was incorporated Figure 3. DNA r e p a i r s y n t h e s i s induced i n Syrian-hamster c e l l s by a s i n g l e dose of u l t r a v i o l e t i r r a d i a t i o n . Autoradiographs. (a) UV (900 ergs/mm2) followed by 3H-TdR f o r \\ hours. (b) Treatment as i n (a) followed by exposure to deoxyribonuclease. Figure 4. The H-TdR i n c o r p o r a t i o n p a t t e r n i n Syrian-hamster c e l l s . Autoradiographs. (a) An S-phase nucleus. (b) A nucleus at l a t e stage of S-phase. (c) Two n u c l e i e x h i b i t i n g DNA r e p a i r s y n t h e s i s induced by 4NQ0 (4xl0"6M, 1\ h o u r s ) . 20 i n t o the nuclear DNA. The autoradiograms of c o l c h i c i n e blocked metaphases a l s o showed g r a i n s over the chromosomes . This i n d i c a t e s that %-TdR was incorporated i n t o the chromosomal DNA. S-phase n u c l e i undergoing DNA r e p l i c a t i o n were h e a v i l y l a b e l l e d i n autoradiograms (Figure 4 a ) . E a r l y or l a t e S-phase n u c l e i presented a c h a r a c t e r i s t i c c o n c e n t r a t i o n of g r a i n s i n and around the e a r l y or l a t e r e p l i c a t i n g regions (Figure 4 b ) . N u c l e i w i t h DNA r e p a i r s y n t h e s i s are c h a r a c t e r i s e d by a r e l a t i v e l y uniform d i s t r i b u t i o n of g r a i n s as shown i n Figure 4 c. The l e v e l of %-TdR i n c o r p o r a t i o n and the d i s t r i b u t i o n of 3K-TdR can be used to d i s t i n g u i s h c e l l s i n DNA r e p l i c a t i o n from c e l l s i n DNA r e p a i r s y n t h e s i s . The a n a l y s i s of DNA r e p a i r s y n t h e s i s can be made much e a s i e r i n the absence of DNA r e p l i c a t i o n at S-phase. This can be achieved by suppressing DNA r e p l i c a t i o n w i t h chemical i n h i b i t o r s 19 (e.g. Actinomycin D, Cycloheximide) . However, DNA r e p a i r s y n t h e s i s i s o f t e n a f f e c t e d as w e l l . Only agents known to i n h i b i t s e l e c t i v e l y DNA r e p l i c a t i o n at S-phase should be used (hydroxyurea, c y t o s i n e a r a b i n o s i d e , 5-aminouracil and FUdR). A l t e r n a t i v e l y , S-phase n u c l e i can be e l i m i n a t e d by b l o c k i n g the c e l l s at G-^ . This can be done by d e p r i v i n g the c e l l s of an e s s e n t i a l amino a c i d . In the present study, an a r g i n i n e d e f i c i e n t medium was used. The percentage of S-phase n u c l e i dropped g r a d u a l l y as a r e s u l t of a r g i n i n e d e p r i v a t i o n . A f t e r three, days i n ADM, more than 997B of the c e l l s were a r r e s t e d at G^ (Figures 5a and 5b), A r g i n i n e d e p r i v a t i o n by i t s e l f d i d not Figure 5. 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 embryonal Syrian-hamster c e l l s . Autoradiographs. (a) Syrian-hamster c e l l s c u l t u r e d i n minimum e s s e n t i a l medium (MEM) and exposed to 3H-TdR f o r lk hours. The S-phase n u c l e i are h e a v i l y l a b e l l e d . (b) 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 H-TdR f o r 1% hours. Note the absence of h e a v i l y l a b e l l e d n u c l e i . Figure 6, 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 r e p a i r s y n t h e s i s i n embryonal Syrian-hamster c e l l s . Autoradiographs. (a) Embryonal Syrian-hamster c e l l s from a growing c u l t u r e exposed to 4NQ0 (4x10"6M) and 3H-TdR f o r \\ hours. Note h e a v i l y l a b e l l e d S-phase n u c l e i and the r e s t r i c t e d number of g r a i n s i n a l l other n u c l e i , (b) Embryonal Syrian-hamster c e l l s from an ADM-arrested c u l t u r e (3 days i n ADM) exposed to 4NQ0 (4x10"6M) and 3H-TdR, 3 Note the absence of S-phase n u c l e i and the H-TdR i n c o r p o r a t i o n i n a l l n u c l e i of the n o n - p r o l i f e r a t i n g c e l l s . 22 induce any DNA damage and subsequent DNA r e p a i r s y n t h e s i s (Figure 5b). The autoradiogram of an unarrested c u l t u r e and one blocked by ADM are shown i n Figure 6. Both have been t r e a t e d w i t h 4NQ0. In the ADM a r r e s t e d c u l t u r e , the h e a v i l y l a b e l l e d S-phase n u c l e i are absent. A l l the n u c l e i d e p i c t i n g a uniform d i s t r i b u t i o n of g r a i n s can be s a f e l y assumed to e x h i b i t DNA r e p a i r s y n t h e s i s . In the p r e l i m i n a r y t r i a l s , 4NQ0-induced DNA r e p a i r s y n t h e s i s was s t u d i e d i n v a r i o u s c e l l types and c e l l l i n e s . Primary c e l l c u l t u r e s ( f i b r o b l a s t s , myoblasts and heart c e l l s ) as w e l l as e s t a b l i s h e d c e l l l i n e s (Fortner tumor, BHK-21) of Syrian-hamster were employed. For comparative purposes, primary c e l l c u l t u r e s of spontaneously aborted human 'embryos were a l s o i n c l u d e d . The i n c o r p o r a t i o n of H-TdR a f t e r a 90-minute exposure to 4NQ0, as measured by the autoradiographic technique, are summarised i n Table I . I t i s apparent that 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 d i f f e r e n t c e l l types t e s t e d are q u i t e comparable. S i m i l a r 3H-TdR i n c o r p o r a t i o n was observed i n both human and Syrian-hamster c e l l s a f t e r UV i r r a d i a t i o n . In both hamster and human c e l l s t r e a t e d w i t h 4NQ0, pr o p o r t i o n -o a l i t y was observed between the amount of H-TdR i n c o r p o r a t i o n and the p l o i d y of the c e l l ( o r , more p r e c i s e l y , the DNA content per n u c l e u s ) . Thus, t e t r a p l o i d n u c l e i have approximately twice as many g r a i n s as those over d i p l o i d ones (Figure 7 ) . A q u a n t i t a t i v e e v a l u a 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 i s shown i n the histogram of Fi g u r e 8. 24 Table I. I n c o r p o r a t i o n of -'H-TdR i n t o N u c l e i of Hamster and Human C e l l s A r r e s t e d by A r g i n i n e D e p r i v a t i o n . Labeled n u c l e i (%) Grain counts (%) C o n t r o l3 4NQ0b UVC C o n t r o l3'6 4NQ0b'e UVc'e Syrian-hamster F i b r o b l a s t s young 0.9 100 100 0 100 87 F i b r o b l a s t s o l d o.o 96 94 0 112 90 Myoblasts 0.5 100 100 0 101 85 Heart C e l l s o.o 100 100 0 90 83 F o r t n e r tumor 1.6 100 100 0 84 60 BHK-21 1.1 100 100 0 89 64 Human embryos F i b r o b l a s t s d i p l o i d 0.1 100 100 0 115 107 F i b r o b l a s t s o l d o.o 94 91 0 122 120 F i b r o b l a s t s t r i p l o i d ^ 0.2 100 100 0 183 147 F i b r o b l a s t s abnormal^ 0.6 100 100 0 109 80 F i b r o b l a s t s i n f e c t e d ^ 0.6 98 95 0 84 88 a C e l l c u l t u r e s exposed to a H-TdR pulse of 1% hours but not to 4NQ0 or UV. b C e l l c u l t u r e s exposed f o r lk hours to 4x10" 6M 4NQ0 and simultaneously to 3H-TdR. c C e l l c u l t u r e s exposed to a s i n g l e dose of UV (900 ergs/mm2) and placed immediately i n t o 3H-TdR-containing medium. d The average number of gr a i n s per d i p l o i d nucleus of embryonal Syrian-hamster f i b r o b l a s t s exposed to 4NQ0 (4xl0~ M, lk hours) was assumed as 1007„ e The few heavy l a b e l e d n u c l e i at S-phase were excluded from the counts, f F i b r o b l a s t c u l t u r e s from spontaneously aborted embryos w i t h abnormal karyotypes. The l a s t sample c o n s i s t e d of c e l l s shedding r u b e l l a v i r u s . Figure 7. Amount of H-TdR i n c o r p o r a t i o n and DNA content c e l l s . Syrian-hamster c e l l s were p r e t r e a t e d w i t h ADM f o r 3 days and then exposed to 4NQ0 (4x10"6M) and 3H-TdR f o r \\ hours. Autoradiographs. The small nucleus i s probably i n the phase and the l a r g e r one i n Q>2 phase. Fi g u r e 8. 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 amounts of DNA r e p a i r s y n t h e s i s . C u ltured Syrian-hamster c e l l s were kept i n ADM f o r 3 days to block DNA r e p l i c a t i o n and m i t o s i s , p r i o r to a p p l i c a t i o n of 4NQ0 (4xl0"6M) and H-TdR f o r 1% hours. Autoradiography, g r a i n counts, (Measurements = Number of N u c l e i ) . MEASUREMENTS co To c a r r y out a comparative q u a n t i t a t i v e study of DNA r e p a i r s y n t h e s i s a f t e r treatment w i t h v a r i o u s mutagens, i t i s thus important to s e l e c t c e l l s of the same p l o i d y . DNA Repair Synthesis and C a r c i n o g e n i c i t y of a Compound The two core chemicals i n t h i s study are 4 - n i t r o q u i n o l i n e 1-oxide (4NQ0) and 4 - n i t r o p y r i d i n e 1-oxide (4NP0). D e r i v a t i v e s of these two compounds have been found to possess v a r y i n g oncogenic and mutagenic c a p a c i t y5>5 4>5 5 > 5 6»8 3>8 4. The question must be r a i s e d whether there i s a c o r r e l a t i o n between the oncog e n i c i t y and mutagenicity of a compound and i t s c a p a c i t y to induce l e s i o n s i n the DNA which i n turn t r i g g e r a DNA r e p a i r s y n t h e s i s . Twenty-five 4NQ0 d e r i v a t i v e s and f i v e r e l a t e d compounds of 4NP0 were t e s t e d f o r the i n d u c t i o n of DNA r e p a i r s y n t h e s i s and chromosome a b e r r a t i o n s . The oncog e n i c i t y data are based on i n v i v o experiments done by p a i n t i n g on the s k i n or i n j e c t i o n £ „ . „ _ , . 5,54,55,56,83,84 „ , ^ „ , of the agents i n t o r a t s and mice ' ' ' ' ' . The s t r u c t u r a l formula of 4NQ0 and the l i s t of d e r i v a t i v e s are shown i n Fi g u r e 9. Group I comprises isomers of the parent compound 4NQ0. They d i f f e r by having the n i t r o group l o c a t e d at d i f f e r e n t p o s i t i o n s on the molecule. I t i s i n t e r e s t i n g to note that of the s i x isomers, only 4NQ0 has been found to e x h i b i t c a r c i n o g e n i c a c t i v i t y . The presence of the n i t r o group at the 4 - p o s i t i o n of the q u i n o l i n e and an N-oxide group on the r i n g n i t r o g e n were thought to be a s t r u c t u r a l requirement f o r 4NQ0 carcin o g e n e s i s . The production of a charge t r a n s f e r complex Group I 4NQ0 Isomers Group I I I QO D e r i v a t i v e s 3NQ0 4HAQ0 5NQ0 4-NH2-Q0 6NQ0 4-OH2-QO 7NQ0 4-phenylsulfonyl-QO 8NQ0 QO 4 N Q O Group I I Group IV A l k y l and Halogeno S u b s t i t u t e d 4NQ0 Qu i n o l i n e D e r i v a t i v e s 2- methyl-4NQ0 6-n-butyl-4NQ0 4NQ 3- methyl-4NQO 6-t-butyl~4NQ0 4HAQ 5- methyl-4NQ0 6-n-hexy1-4NQ0 4-NH2"Q 6- methyl-4NQO 3-fluoro-4NQ0 7- methyl-4NQO 6-chloro-4NQ0 8- methyl-4NQ0 6-carboxy-4NQ0 Figure 9. 4 - N i t r o q u i n o l i n e 1-oxide (4NQ0) and r e l a t e d compounds. NQO: n i t r o q u i n o l i n e 1-oxide 4HAQ0: 4-hydroxyaminoquinoline 1-oxide QO: q u i n o l i n e 1-oxide 4NQ: 4 - n i t r o q u i n o l i n e 4HAQ: 4-hydroxyaminoquinoline Q: q u i n o l i n e has been i m p l i c a t e d i n the mode of a c t i o n of 4NQ0 on DNA0 0>°y' . Perhaps t h i s arrangement of the n i t r o and N-oxide group at the 1 and 4 p o s i t i o n f a c i l i t a t e s the formation of such a complex w i t h DNA. A l l of the a l k y l and halogeno s u b s t i t u t e d 4NQ0 d e r i v a t i v e s l i s t e d i n Group I I are oncogenic. One feat u r e worthy of note i s that a l l the d e r i v a t i v e s have the i n t a c t 4NQ0 parent s t r u c t u r e . I f the charge t r a n s f e r hypothesis i s c o r r e c t , one may e x p l a i n the v a r y i n g degree of oncogenicity by the ease w i t h which a charge t r a n s f e r complex w i t h DNA may be formed. In t h i s r e s p e c t , the presence of an a l k y l or halogeno group at a p a r t i c u l a r p o s i t i o n may f a c i l i t a t e or impede a charge t r a n s f e r . Q u i n o l i n e 1-oxide and four of i t s d e r i v a t i v e s are in c l u d e d i n Group I I I . Of p a r t i c u l a r i n t e r e s t to note i s 4HAQ0, the only . , . . „ u . 30,112 _ „ . , • . oncogenic d e r i v a t i v e i n t h i s group . I t i s a r e d u c t i v e m e t a b o l i t e of 4NQ0 and b e l i e v e d to represent the proximate carcinogen"*6. The q u i n o l i n e compounds i n Group IV are c h a r a c t e r i s e d by the absence of an N-oxide i n t h e i r s t r u c t u r e . Except f o r 4 - n i t r o q u i n o l i n e , the other two d e r i v a t i v e s have not been found to e x h i b i t any ca r c i n o g e n i c a c t i v i t y _in v i v o ^ . F i g u re 10 d e p i c t s 4 - n i t r o p y r i d i n e 1-oxide (4NP0), 4-hydroxyaminopyridine 1-oxide (4HAP0) and four d e r i v a t i v e s w i t h one or two a l k y l s u b s t i t u t e d groups. The c a r c i n o g e n i c i t y data on these chemical agents are scanty, w i t h only 4NP0, 3-methyl-4NP0 and 2,3-dimethyl-4NPCi being t e s t e d to d a t e 5 ' 5 ^ . They are NO. 4 N PO 4NP0 3-methyl-4NP0 2,3-dimethy1-4NP0 4HAP0 3-methyl-4HAP0 2,3-d ime thy1-4HAP0 Figure 10. 4 - N i t r o p y r i d i n e 1-oxide (4NP0) and d e r i v a t i v e s . 4NP0: 4 - n i t r o p y r i d i n e 1-oxide 4HAP0: 4-hydroxyaminopyridine 1-oxide very weak carcinogens compared w i t h the c a r c i n o g e n i c q u i n o l i n e d e r i v a t i v e s . 4NP0 and 3-methy1-4NP0 have been shown to induce s i n g l e strand breaks i n DNA-3^. Such r e s u l t s were not observed a f t e r treatment w i t h 2,3-dimethyl-4NP05\ To screen f o r the 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 by the 4NQ0 and 4NP0 d e r i v a t i v e s , the f o l l o w i n g experimental p r o t o c o l was used r o u t i n e l y : c u l t u r e d Syrian-hamster c e l l s from the t h i r d t r a n s f e r passage were kept i n ADM f o r three days to block c e l l p r o l i f e r a t i o n and i n h i b i t DNA r e p l i c a t i o n . Various concentrations of the h i g h l y , weakly or non-mutagenic 4NQ0 and 4NP0 d e r i v a t i v e s were d i s s o l v e d i n ADM and added to the c e l l c u l t u r e s . A f t e r treatment w i t h the v a r i o u s chemicals f o r 1% o hours, JH-TdR was a p p l i e d f o r 1-g hours (10 /iCi/ml i n ADM). The samples were then prepared f o r autoradiography. The number of g r a i n s per nucleus of c e l l s exposed to the v a r i o u s 4NQ0 d e r i v a t i v e s was counted. The r e s u l t s are summarized i n Tables I I , I I I and IV. Among the isomeric q u i n o l i n e 1-oxides only the h i g h l y oncogenic 4NQ0 induced c e l l l e s i o n s r e s u l t i n g i n an extensive DNA r e p a i r s y n t h e s i s . A l l the non-oncogenic 4NQ0 isomers ( f i r s t group i n Table I I ) f a i l e d to t r i g g e r a DNA r e p a i r s y n t h e s i s . However, v i r t u a l l y a l l the s u b s t i t u t e d 4NQ0 d e r i v a t i v e s examined e l i c i t e d to v a r i o u s degrees an unscheduled i n c o r p o r a t i o n of 3H-TdR (second group i n Table I I ) . The same c o r r e l a t i o n between c a r c i n o g e n i c i t y and DNA r e p a i r i s again s u b s t a n t i a t e d among the q u i n o l i n e 1-oxides Table I I . DNA Repair Synthesis i n Syrian-Hamster C e l l s Exposed to Various 4NQ0 D e r i v a t i v e s ===================== ========== ============ =========== ============= ============ ======== D e r i v a t i v e s3 Oncogenicity Grains/nucleus at concentrations 5x10"5M 8x10"6M 4x10"6M 2x10"6M 1x10"6M 5x10"7M 3NQ0 0 0 0 0 0 0 4NQ0 ++ 14 101 66 51 43 19 5NQ0 - 0 0 0 0 0 0 6NQ0 - 0 0 0 0 0 0 7NQ0 - 0 0 0 0 0 0 8NQ0 - 0 0 0 0 0 0 2-methyl-4NQ0 -H- 59 88 63 36 25 10 3-methyl-4NQ0 + 7 2 1 0 0 0 5-methyl-4NQ0 ++ 15 44 21 12 7 3 6-methyl-4NOO ++ 14 63 39 29 15 7 ?-methyl-4NQ0 ++ 41 57 41 9 8-methyl-4NQ0 + 21 5 0 0 0 0 6-n-butyl-4NQO ++ 13 52 20 4 6-t-butyl-4NQO + 32 14 4 0 0 0 6-n-hexyl-4NQ0 + 0 0 0 0 0 0 3-fluoro-4NQ0 + 3 4 1 3 0 0 6-chloro-4NQ0 -H- 10 42 51 38 26 12 6-carboxy-4NQO + 16 2 1 0 0 0 4HAQ0 ++ 90 51 5 0 0 0 4-NH2-QO - 0 0 0 0 0 0 4-0H2-Q0 - 0 0 0 0 0 0 4-phenylsulphonyl-QO - 0 0 0 0 0 0 QO - 0 0 0 0 •, 0 0 4NQ + 28 9 4 0 0 0 4HAQ 0 0 0 0 0 0 4-NH2~Q - 0 0 0 0 0 0 a NQO: n i t r o q u i n o l i n e 1-oxide; 4HAQ0: 4-hydroxyaminoquinoline 1-oxide; QO: q u i n o l i n e 1-oxide; 4HAQ: 4-hydroxyaminoquinoline; Q: q u i n o l i n e . b ++: Stro n g l y oncogenic; +: Weakly oncogenic; -: Tested but not proved to be oncogenic; ?: Not t e s t e d . Table I I I . C o r r e l a t i o n between C a r c i n o g e n i c i t y of Some 4NQ0 D e r i v a t i v e s and Induced DNA Repair Synthesis i n Syrian-Hamster C e l l s (A Summary). G R A I N S / N U C L E U S 0 30 40 60 80 100 4NQO 2-methvl-4NQO 5- methyl-4NQO 6- mcthyl-4NQO 7- methyl-4NQO 6-n-bulyl-4NQO 6-chloro-4NQO 3-melhyl-4NQO 8-melhyl-4NQO 3-fluoro-4NQO 6-carboxy-4NQO 3NQO 5NQO 6NQO 7NQO 8N00 4-NHj-OO 4-OHj-OO 4-phenylsulphonyl-QO QO This Table shows the maximum g r a i n count obtained w i t h i n the con c e n t r a t i o n range 5x10""*M to 5x10" 7M. F i r s t Group: Strongly c a r c i n o g e n i c . Second Group: Weakly c a r c i n o g e n i c . T h i r d Group: Non-carcinogenic. Fourth Group: Non-carcinogenic. Figure 11. DNA r e p a i r s y n t h e s i s i n ADM-arrested Syrian-hamster c e l l s exposed to 4NQ0 d e r i v a t i v e s (lxlO"5M) and 3H-TdR f o r 1% hours. Autoradiographs. (a) 2-methyl-4NQO (c a r c i n o g e n i c ) (b) 8-methyl-4NQO (weakly c a r c i n o g e n i c ) (c) 4AQ0 (non-carcinogenic) grouped i n the t h i r d paragraph of Table I I . DNA r e p a i r s y n t h e s i s i s e l i c i t e d by 4HAQ0, the only oncogenic compound i n t h i s group. 4NP0 and i t s r e d u c t i o n product, 4HAP0, c l o s e l y resemble s t r u c t u r a l l y t h e i r corresponding q u i n o l i n e d e r i v a t i v e s (Figures 9 and 10). Because of t h i s s i m i l a r i t y i n s t r u c t u r e and chemical r e a c t i v i t y , they have been expected i n the past to possess oncogenic c a p a c i t y . Y e t , they do not induce neoplasms nor e l i c i t any dete c t a b l e DNA r e p a i r s y n t h e s i s . However, the 3-methyl-substituted d e r i v a t i v e , which i s c a r c i n o g e n i c , and the other a l k y l s u b s t i t u t e d d e r i v a t i v e s a l l induced a v a r y i n g degree of DNA r e p a i r , s y n t h e s i s i n t r e a t e d c e l l s (Table I V ) . There are three s i g n i f i c a n t f e a t u r e s of the c e l l u l a r response w i t h respect to DNA r e p a i r s y n t h e s i s : ( i ) only c e l l s exposed to oncogenic compounds e x h i b i t DNA r e p a i r s y n t h e s i s (Figure 11 and Table I I I ) ; ( i i ) H-TdR i n c o r p o r a t i o n can be detected at co n s i d e r a b l y lower doses a f t e r treatment w i t h h i g h l y oncogenic d e r i v a t i v e s than w i t h weak ones; ( i i i ) the maximum g r a i n count a f t e r exposure to weakly oncogenic d e r i v a t i v e s i s always lower than i n c e l l s t r e a t e d w i t h h i g h l y oncogenic compounds. The observations s t r o n g l y suggest a l i n k between onc o g e n i c i t y of a compound and i t s c a p a c i t y to induce DNA l e s i o n s r e s u l t i n g i n DNA r e p a i r . The powerful mutagen1,7''" and c a r c i n o g e n1 0 6'1 2^ , N-methyl-N ' - n i t r o - n i t r o s o g u a n i d i n e (MNNG) has been shown to t r i g g e r a non-conservative mode of DNA r e p a i r r e p l i c a t i o n i n E s c h e r i c h i a  c o l i * " * . I t was included i n the present study to r e i n f o r c e Table IV. DNA Repair Synthesis i n Syrian-Hamster (4NP0) D e r i v a t i v e s . D e r i v a t i v e s3 Oncogenicity 3xl0'4M 4NPO ± 0 4HAPO - 0 3:methyl-4NP0 ? 6 3-methyl-4HAP0 ? 21 2,3-dimethyl-4NPO - 20 2,3-dimethyl-4HAPO ? 20 C e l l s Exposed to Various 4 - n i t r o p y r i d i n e 1-oxide Grains/Nucleus at Concentrations l x l 0 "4M 3x10"5M 1x10"5M 3x10"6M 1x10 0 0 0 0 0 0 18 9 2 1 0 14 3 0 0 0 4 1 0 0 0 14 11 5 0 0 a 4NP0: 4 - N i t r o p y r i d i n e 1-oxide; 4HAP0: 4-Hydroxyaminopyridine 1-oxide. b +: Oncogenic; -: Tested but not proved to be oncogenic; ?: Not t e s t e d . the b e l i e f t h a t the unscheduled H-TdR i n c o r p o r a t i o n was i n d i c a t i v e of DNA r e p a i r s y n t h e s i s and that an e r r o r - c o r r e c t i n g mechanism e x i s t s i n mammalian c e l l s to r e p a i r d i f f e r e n t types of DNA damage. Appropriate d i l u t i o n s of MNNG i n t i s s u e c u l t u r e medium were added to the c e l l c u l t u r e s f o r three hours, followed by 3 the usual p u l s e - l a b e l l i n g w i t h H-TdR and autoradiographic procedure. The g r a i n count e x h i b i t e d a dose dependence, reaching a maximum of 46 g r a i n s per nucleus at a co n c e n t r a t i o n of 5xlO"^M (Table V ) . Table V. DNA Repair Synthesis i n Syrian-Hamster C e l l s Exposed to N-Methyl-N1-Nitro-Nitrosoguanidine (MNNG) and 4 - N i t r o q u i n o l i n e 1-Oxide (4NQ0) Grains/Nucleus at Concentration 5xlO~5M 8xlO~6M 4x10"6M 2 x l 0_ 6M MNNG 46 18 11 7 4NQ0 14 101 66 51 Wi t h i n a c e r t a i n c o n c e n t r a t i o n range, the amount of H-TdR i n c o r p o r a t i o n was s t r i c t l y dependent upon the dose to which c u l t u r e d c e l l s were exposed. Thus, f o r the h i g h l y oncogenic 4NQ0 d e r i v a t i v e s used i n t h i s study, the g r a i n count per nucleus increased as the co n c e n t r a t i o n was r a i s e d from 5x10"7M to 8xl0~6M (Table I I ) . With the exception of 4HA00, the induced DNA r e p a i r s y n t h e s i s d e c l i n e d when concentrations exceeding 10"^ M were used. This reduced DNA r e p a i r of high c o n c e n t r a t i o n s of the N-oxides may be a t t r i b u t e d to a general t o x i c i t y as evidenced by the low clone forming c a p a c i t y (Table V I I ) . The DNA damage induced by such concentrations may be so extensive as to suppress many c e l l u l a r r e a c t i o n s i n c l u d i n g the r e p a i r mechanism. A l t e r n a t i v e l y , the i n c l u s i o n of dying c e l l s i n the examination of the autoradiographic preparations may account 3 f o r the observed decrease i n H-TdR i n c o r p o r a t i o n at h i g h c o n c e n t r a t i o n of mutagens. Chromosome A b e r r a t i o n s and C a r c i n o g e n i c i t y of a Compound i n C e l l s Exposed to 4NQ0 D e r i v a t i v e s Before embarking on a d e s c r i p t i o n of the work i n t h i s s e c t i o n , i t may be worthwhile to mention some of the p r e l i m i n a r y chromosomal s t u d i e s w i t h 4NQ0*'''''. Three sets of experiments were done. In one s e t , c e l l s at the l o g a r i t h m i c growth phase were exposed to a 90-minute p e r i o d of 4NQ0. In another s e t , the p r o l i f e r a t i o n of c e l l s was a r r e s t e d by keeping the c e l l c u l t u r e s i n ADM f o r two' to three days, then they were t r e a t e d with 4NQ0 and t r a n s f e r r e d back i n t o MEM to induce c e l l d i v i s i o n . The types of observable chromosome a b e r r a t i o n s c o n s i s t e d of s i n g l e and m u l t i p l e chromatid breaks and s i n g l e and m u l t i p l e chromatid exchanges (Figure 12). I t i s of i n t e r e s t to note that the frequency of such a b e r r a t i o n s observed i n both sets of experiments d i d not d i f f e r ' s i g n i f i c a n t l y . There was no increa s e Figure 12. Chromosome a b e r r a t i o n s i n Syrian-hamster c e l l s a f t e r exposure to 4NQ0, The c e l l c u l t u r e s were a r r e s t e d at — ft G]_ by a r g i n i n e d e p r i v a t i o n (3 days ) , exposed to 4NQ0 (2x10 M, 1% h o u r s ) , t r a n s f e r r e d i n t o MEM (18 to 24 h o u r s ) , t r e a t e d w i t h c o l c h i c i n e (4 hours) and ha r v e s t e d . Ac e t o - o r c e i n s t a i n i n g . (a) Part of a normal Syrian-hamster chromosome complement. No 4NQ0 treatment. (b) Part of a metaphase p l a t e showing s i n g l e chromatid break and exchange. (c) Part of a metaphase p l a t e showing chromatid breaks, ; chromatid fragments and m u l t i p l e chromatid exchanges. (d) and (e) E f f e c t of 4NQ0 on metaphase chromosomes. C o l c h i c i n e - b l o c k e d Syrian-hamster c e l l s (4 hours) were exposed to 4NQ0 (2x10"6M, Ik hours) and ha r v e s t e d . Note u n c o i l i n g of chromosomes. 38 i n the incidence of chromatid breaks and exchanges as a r e s u l t of two to three days of a r g i n i n e d e p r i v a t i o n1 1 5. The e f f e c t of 4NQ0 on metaphase chromosomes was s t u d i e d by b l o c k i n g w i t h c o l c h i c i n e p r i o r to chemical treatment. With i n c r e a s i n g 4NQ0 c o n c e n t r a t i o n , p r o g r e s s i v e u n c o i l i n g of the metaphase chromosomes was o b s e r v e d1 1 7 (Figure 12). In screening f o r 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 exposed to 4NQ0 and r e l a t e d compounds, the f o l l o w i n g standard procedure was adopted. C e l l p r o l i f e r a t i o n was a r r e s t e d by keeping the c e l l c u l t u r e s i n ADM f o r two to three days. They were then exposed to 4NQ0 or i t s d e r i v a t i v e s f o r 1\ hours. A f t e r chemical treatment, the c e l l s were placed back i n t o MEM to t r i g g e r them i n t o d i v i s i o n . The f i r s t m i t o t i c wave u s u a l l y occurred at about 18 to 22 hours a f t e r the t r a n s f e r i n t o MEM, This might be delayed i n the case of c e l l s exposed to hi g h e r c o n c e n t r a t i o n s of the chem i c a l . In some i n s t a n c e s , the f i r s t m i t o s i s d i d not appear u n t i l 30 to 40 hours post-treatment. The d u r a t i o n of previous a r g i n i n e d e p r i v a t i o n a l s o played a r o l e . The longer the c e l l s were i n a r g i n i n e d e f i c i e n t medium, the greater would be the delay of m i t o s i s . C e l l s at metaphase can be r e a d i l y observed under an i n v e r t e d microscope: they are rounded, as opposed to the pseudopodial appearance assumed by interphase c e l l s . Judging from the abundance of metaphases, c o l c h i c i n e was administered at the onset of the f i r s t m i t o t i c , wave. The c o l c h i c i n e treatment was maintained f o r four hours. C e l l c u l t u r e s were then subjected to hypotonic treatment (17° sodium c i t r a t e ) f o r 15 minutes, f i x e d w i t h Carnoy's (10 m i n u t e s ) , a i r d r i e d , s t a i n e d w i t h 2% a c e t o - o r c e i n , dehydrated and mounted i n Permount ( F i s h e r S c i e n t i f i c Co.). Among the s e l e c t e d compounds t e s t e d , we in c l u d e d h i g h l y , weakly and non-oncogenic 4NQ0 d e r i v a t i v e s . A c o r r e l a t i o n was observed between the oncog e n i c i t y o f a compound and i t s c a p a c i t y to induce chromosome a b e r r a t i o n s . W i t h i n the range of concentrations used, the frequency of c e l l s w i t h chromosome ab e r r a t i o n s increased w i t h the co n c e n t r a t i o n of 4NQ0 (Table V I ) . Absence of any metaphase p l a t e s a f t e r exposure to 2.5x10" M might be a t t r i b u t e d to c e l l t o x i c i t y . The non-oncogenic isomer, 6NQ0, on the other hand, f a i l e d to e l i c i t any chromosomal abnormality even at concentrations 40 f o l d h i g h e r . A l l four of the mutagenic s u b s t i t u t e d 4NQ0 d e r i v a t i v e s t e s t e d produced a p o s i t i v e response, a l b e i t the dose at which chromosomal a b e r r a t i o n s were observed v a r i e d as the c a r c i n o g e n i c i t y . With the h i g h l y oncogenic b-methyl 4NQ0, 83.3% of the metaphases screened had a b e r r a t i o n s a f t e r a dose of 2.5x10" M. T o x i c i t y , as evidenced by the l a c k of metaphase p l a t e s i n the p r e p a r a t i o n , set i n at concentrations'exceeding 5x10"6M. With 3-methyl 4NQ0, a weak carcinogen by comparison, a b e r r a t i o n s were not detected at doses lower than 1x10"5M (beyond the t o x i c range f o r 6-methyl 4NQ0). The r e l a t i v e l y low incidence of a b e r r a t i o n s observed a f t e r treatment w i t h 6 - n i t r o 4NQ0 may be accounted f o r by i t s poor s o l u b i l i t y i n the 'culture medium. Table V I . Frequency of Chromosome Breaks and Exchanges i n Syrian-Hamster C e l l s Exposed to Various 4NQ0 and 4NP0 D e r i v a t i v e s . Frequency of metaphase p l a t e s w i t h chromosome a u a b e r r a t i o n s at conc e n t r a t i o n 0 » (M) D e r i v a t i v e s Oncogenicity 10"4 5x l O "5 2.5x 1 0 ° l x l O "5 5x I 0 "6 2.5x 10" 6 l x 10"6 5x 10" 7 2.5x lO" 7 l x 10" 7 4NQ0 .++ X X X X X X 91.7 63.1 25.8 11.1 4HAQ0 ++ x X X X 84.0 19.6 5.8 4.7 3.8 2.4 6-methyl-4NQO ++ X X X X X 83.3 34.8 11.6 1.3 1.5 3-methyl-4NQ0 + 83.9 52.6 12.0 1.6 0.5 o.o 0.3 0.4 - -6-nitro-4NQO + 18.9 4.7 3.8 2.1 1.9 1.9 0.4 0.3 - -6-carboxy-4NQ0 29.6 1.2 0.2 0.7 0.0 0.4 0.4 0 o0 - -6-NQ0 - 0.2 0.0 0.7 0.9 0.1 - - - - -4-HAQ - 0.9 0.7 0.2 0.3 o.o - - - - -QO - 1.1 0.5 0.1 0.4 0.5 - - - - -4NP0 ± X 0.0 0.0 0.0 0.0 • - - - - . -2,3-dimethyl-4NPO - 16.4 7.4 4.0 3.4 2.6 - - - -2,3-dime thy1-4HAP0 1 . - 63.8 26.2 24.2 9.4 - - - - -a NQO: n i t r o q u i n o l i n e 1-oxide; 4HAQ0: 4-hydroxyaminoquinoline 1-oxide; Q0: q u i n o l i n e 1-oxide; 4NQ: 4- n . i t r o q u i n o l i n e ; 4HAQ: 4-hydroxyaminoquinoline; 4NP0: 4 - n i t r o p y r i d i n e 1-oxide; 4HAP0: 4-hydroxyaminopyridine 1-oxide; Q: q u i n o l i n e . b +•(-: Strongly oncogenic; +: Weakly oncogenic; -: Tested but not proved to be oncogenic; ?: Not t e s t e d , c The symbol x i n d i c a t e s a l e t h a l e f f e c t . d The symbol - i s used to show that the conc e n t r a t i o n has not been used. Anomalies at the chromosomal l e v e l were a l s o e l i c i t e d by the proximate c a r c i n o g e n , 4HAQ0, whereas the e f f e c t was not apparent w i t h the non-oncogenic QO and 4HAQ. 4NP0 has not been found to possess any c a r c i n o g e n i c a c t i v i t y at the time of t h i s i n v e s t i g a t i o n and no DNA r e p a i r s y n t h e s i s was d e t e c t a b l e i n c e l l s exposed to t h i s c h e m i c a l . However, aberrant chromosomes have been observed i n one instance a f t e r 4NP0 treatment. There was some i n d i c a t i o n from recent in v i v o experiments that 4NP0 i s weakly c a r c i n o g e n i c5 4. Attempts to reproduce the chromosome data were not s u c c e s s f u l though the d e r i v a t i v e s t i l l f a i l e d to evoke any DNA r e p a i r s y n t h e s i s . This chemical deserves f u r t h e r i n v e s t i g a t i o n . With 2,3-diraethyl-4NP0 and 2,3-dimethyl-4HAP0, chromosomal a b e r r a t i o n s were observed. A b e r r a t i o n s i n metaphase chromosomes obtained a f t e r exposure to v a r i o u s 4NP0 d e r i v a t i v e s were s i m i l a r to those obtained a f t e r 4NQ0 treatment. These included s i n g l e and m u l t i p l e chromatid breaks and s i n g l e and m u l t i p l e chromatid exchanges. Since the aim was to compare the frequency of c e l l s w i t h aberrant chromosomes, the d i f f e r e n t types of anomalies were not scored s e p a r a t e l y . Clone Forming Capacity of C e l l s and C a r c i n o g e n i c i t y of a Compound The s u r v i v a l of c e l l s a f t e r exposure to v a r i o u s 4NQ0 d e r i v a t i v e s was estimated from t h e i r c a p a c i t y to form clones f o l l o w i n g chemical treatment. For comparative purposes, only c l o n e s c o m p r i s i n g about 60 o r more c e l l s were c o u n t e d . The r e s u l t s a r e d e p i c t e d i n F i g u r e 13 and T a b l e V I I . There was a r e l a t i o n s h i p between the t u m o r i g e n i c i t y o f a compound and i t s e f f e c t on the c l o n e f o r m i n g a b i l i t y o f c e l l s . Treatment w i t h a non-oncogenic d e r i v a t i v e d i d n o t seem to reduce the c l o n i n g e f f i c i e n c y o f c e l l s . A t the o t h e r extreme, " c e l l s exposed t o o n c o g e n i c d e r i v a t i v e s showed a d r a s t i c d e c r e a s e i n the number o f c l o n e s formed. I n t e r m e d i a t e r e s u l t s were o b t a i n e d w i t h weakly c a r c i n o g e n i c compounds. Figure 13. Clone forming capacity of Syrian-hamster c e l l s exposed to various 4NQ0 d e r i v a t i v e s . Table V I I . Clone Forming Capacity of Syrian-Hamster C e l l s A f t e r Exposure to Various 4NQ0 and 4NP0 D e r i v a t i v e s . D e r i v a t i v e s3 Oncogenicity'3 Clones (%) at_ • • . - • '- -•• 2x10"6M 5 x l 0_ /M 3NQ0 _ 94 109 4NQ0 ++ 0.5 3 6NQ0 - 103 98 8NQ0 - 100 103 2-methyl-4NQO -H- 0.8 5 3-methyl-4NQ0 + 57 75 6-methyl-4NQ0 ++ 1 15 7-methyl-4NQO -H- 0.6 7 8-methyl-4NQ0 + 43 72 3-fluoro-4NQO + 40 65 6-carboxy-4NQ0 + ' 38 68 4HAQ0 ++ 32 45 4-0H-Q0 - 103 100 4NQ + 75 87 4HAQ 101 96 4NP0 ± 97 96 4HAP0 - 99 102 3-methyl-4NPO ? 33 43 2,3-dimethyl-4NP0 - 80 88 Co n t r o l (no d e r i v a t i v e ) 100 100 a NQO: n i t r o q u i n o l i n e 1-oxide; 4HAQ0: 4-hydroxyaminoquinoline 1-oxide; Q0: q u i n o l i n e 1-oxide; 4NQ: 4 - n i t r o q u i n o l i n e ; 4HAQ: 4-hydroxyaminoquinoline; 4NP0: 4 - n i t r o p y r i d i n e 1-oxide 4HAP0: 4-hydroxyaminopyridine 1-oxide; Q: q u i n o l i n e . b ++: Strongly oncogenic; +: Weakly oncogenic; -: Tested but not proved to be oncogenic; ?: Not t e s t e d . DISCUSSION General Comments The present study of a s e r i e s of N-oxides revealed (1) a l i n k between the degree of c a r c i n o g e n i c i t y of a compound and i t s c a p a c i t y to induce a DNA damage which r e s u l t s i n a de t e c t a b l e DNA r e p a i r s y n t h e s i s ; (2) a l i n k between the degree of c a r c i n o -g e n i c i t y of a compound and major chromosome a b e r r a t i o n s d e t e c t -able at the l i g h t m i croscopic l e v e l , and (3) a l i n k between the degree of c a r c i n o g e n i c i t y of a compound and i t s c y t o t o x i c e f f e c t (Table V I I I ) . Table V I I I . Response of Syrian-Hamster C e l l s to S t r o n g l y , Weakly and Non-Carcinogenic D e r i v a t i v e s of 4NQ0 and 4NP0 Degree of Leve l of Frequency of Clone C a r c i n o g e n i c i t y DNA Repair Chromosome Forming A b e r r a t i o n s A b i l i t y + +• + + + + + + + + + _ + + The h i g h l y c a r c i n o g e n i c n i t r o q u i n o l i n e N-oxides e l i c i t e d 3 an a p p r e c i a b l e amount of unscheduled H-TdR i n c o r p o r a t i o n and an e l e v a t e d f r e q u e n c y o f chromosome a b e r r a t i o n s . W i t h the weakly c a r c i n o g e n i c ANQO d e r i v a t i v e s , t h e s e phenomena o c c u r r e d t o a s m a l l e r degree. The non-oncogenic N - o x i d e s f a i l e d t o produce any d e t e c t a b l e l e s i o n s a t the DNA and chromosome l e v e l . The e x t e n t o f c e l l s u r v i v a l , as measured by the c l o n e f o r m i n g a b i l i t y o f t r e a t e d c e l l s , a l s o seemed to be r e l a t e d t o the o n c o g e n i c c a p a c i t y o f a compound. P o t e n t c a r c i n o g e n s a r e h i g h l y c y t o t o x i c , whereas weakly c a r c i n o g e n i c compounds show o n l y a s l i g h t l e t h a l e f f e c t and non-oncogenic d e r i v a t i v e s do not a f f e c t the c l o n e f o r m i n g c a p a c i t y o f c u l t u r e d mammalian c e l l s . Whether these phenomena a r e c a u s a l l y l i n k e d i s d i f f i c u l t t o a s s e s s . A d e t a i l e d and c r i t i c a l d i s c u s s i o n o f the above proposed r e l a t i o n s h i p i s g i v e n i n the f o l l o w i n g c h a p t e r s . C a r c i n o g e n i c i t y and DNA R e p a i r One b a s i c assumption i n the p r e s e n t study i s t h a t the 3 u n s c h e d u l e d uptake o f H-TdR i n c u l t u r e d mammalian c e l l s exposed t o a c a r c i n o g e n i n d i c a t e s DNA e x c i s i o n r e p a i r . C o n v e r s e l y , 3 the absence o f any d e t e c t a b l e H-TdR i n c o r p o r a t i o n a f t e r t r e a t m e n t w i t h n o n - c a r c i n o g e n i c 4NQ0 d e r i v a t i v e s was t a ken to mean t h a t no DNA a l t e r a t i o n s d i d o c c u r and, t h e r e f o r e , no DNA r e p a i r s y n t h e s i s has taken p l a c e . However, s e v e r a l arguments c o u l d be r a i s e d a g a i n s t t h i s i n t e r p r e t a t i o n . C h e m i c a l agents might i n d u c e membrane changes to bar the e n t r a n c e o f the r a d i o a c t i v e i s o t o p e which i s used to measure DNA r e p a i r s y n t h e s i s . T h i s e x p l a n a t i o n i s u n l i k e l y besause the S-phase n u c l e i o f the t r e a t e d c e l l p o p u l a t i o n were h e a v i l y l a b e l l e d w i t h JH-TdR. Another p o s s i b l e source of e r r o r may a r i s e from the presence of c e l l mutants d e f i c i e n t i n DNA r e p a i r s y n t h e s i s or l a c k i n g the c a p a c i t y of H-TdR uptake from the c u l t u r e medium. However, 2 x l 06 c e l l s have been screened and only 1 i n about 150,000 n u c l e i showed no i n c o r p o r a t i o n of H-TdR f o l l o w i n g exposure - f% to 4x10 M 4NQ0 f o r 1% hours. These n u c l e i were u s u a l l y small and s t a i n e d h e a v i l y w i t h o r c e i n . They probably represent py c n o t i c n u c l e i of dying or dead c e l l s . I f v i a b l e c e l l s d e f i c i e n t i n JH-TdR uptake or i n DNA r e p a i r s y n t h e s i s e x i s t at a l l i n our p r e p a r a t i o n s , t h e i r frequency would be too low to o f f s e t the average g r a i n count per nucleus of the c e l l p o p u l a t i o n as a whole. The l a c k of a DNA r e p a i r s y n t h e s i s could a l s o r e s u l t from the f a i l u r e of a mutagen or .carcinogen to reach the chromosomes of a c e l l . This idea can be discarded because the uptake of l a b e l l e d oncogenic 4NQ0 compounds i s comparable to that of the non-oncogenic d e r i v a t i v e s5 4. A l t e r n a t i v e l y , the non-active 4NQ0 d e r i v a t i v e s may be degraded w i t h i n the c e l l c r they may r e q u i r e an enzymatic 77 7P a c t i v a t i o n . Metabolic conversion of 2-acetylaminofluorene ' , V d i b e n z ( a ) a n t h r a c e n e1 0 8 and the n i t r o s a m i n e s7 0 to t h e i r proximate c a r c i n o g e n i c forms are w e l l known instances of t h i s requirement f o r a c t i v a t i o n . The r e d u c t i o n of 4NQ0 to 4HAQ0 i s a l s o i m p l i c a t e d 32 125 126 i n 4NQ0 carci n o g e n e s i s ' ' . I f t h i s a c t i v a t i o n process i s slow, then very l i t t l e of the agent would be rendered a c t i v e w i t h i n a u n i t time and only a l i m i t e d damage would be i n f l i c t e d on the DNA p r i o r to the onset of our t e s t . Exposure of c u l t u r e d c e l l s to non-oncogenic 4NQ0 compounds f o r d i f f e r e n t periods of time (1% to 12 hours) w i t h concomitant H-TdR l a b e l l i n g f a i l e d to show any DNA r e p a i r s y n t h e s i s . Therefore, any enzymatic a c t i v a t i o n of the 4NQ0 d e r i v a t i v e s , i f at a l l r e q u i r e d , would not be a l i m i t i n g f a c t o r i n the assay f o r DNA r e p a i r . I n s t a b i l i t y of the chemical agent i n s o l u t i o n poses another problem. This i s perhaps not the case f o r the 4NQ0 d e r i v a t i v e s t e s t e d because unscheduled H-TdR uptake could s t i l l be detected i n c u l t u r e d c e l l s t r e a t e d w i t h two-month o l d s o l u t i o n s of 4NQ06 1. I t may be argued that not a l l of the examined 4NQ0 d e r i v a t i v e s ( e s p e c i a l l y the non-oncogenic ones) are s t a b l e i n s o l u t i o n . This d i f f i c u l t y can be obviated by 3 concomitant a p p l i c a t i o n of the chemical and H-TdR so that any DNA r e p a i r s y n t h e s i s o c c u r r i n g may be followed i n s t a n t a n e o u s l y Using t h i s technique, i t was s t i l l not p o s s i b l e to detect any unscheduled i n c o r p o r a t i o n of H-TdR. Thus, i t i s not l i k e l y that the chemicals were unstable i n s o l u t i o n . Another argument against the l a c k of DNA r e p a i r s y n t h e s i s i s that the concentrations used were not hig h enough, or the treatment was too s h o r t . Exposure of hamster c e l l s to longer _ 3 (up to 12 hours) and higher doses (up to 10 M) of non-oncogenic d e r i v a t i v e s f a i l e d to show any v i s i b l e DNA r e p a i r s y n t h e s i s . 3 I t was p o s s i b l e that the absence of any H-TdR i n c o r p o r a t i o n i n c e l l s t r e a t e d w i t h the hon-oncogenic 4NQ0 d e r i v a t i v e s may have r e s u l t e d from i n h i b i t i o n of the r e p a i r mechanism by the compound. To determine whether such an a c t i v i t y was present w i t h non-oncogenic d e r i v a t i v e s , c u l t u r e d hamster c e l l s were i r r a d i a t e d 9 w i t h a s i n g l e dose of UV l i g h t (900 ergs/mm ) to induce DNA r e p a i r and then exposed f o r 1% hours to the non-oncogenic 8NQ0 and H-TdR. Treatment w i t h 8NQ0 d i d not produce any s i g n i f i c a n t r e d u c t i o n i n DNA r e p a i r s y n t h e s i s i n the UV i r r a d i a t e d c e l l s . L i k e w i s e , pretreatment w i t h the h i g h l y potent 4NQ0 (4x10 M; \\ hours) followed by 8NQ0 ( l x l O "4M ; \\ hours) gave only a s l i g h t l y diminished g r a i n count compared to that i n c e l l s t r e a t e d w i t h 4NQ0 a l o n e1 2 1 (Table I X ) . Table IX. E f f e c t of 8NQ0 (A Non-Carcinogenic Compound) on DNA Repair Synthesis Grains/Nucleus 8NQ0 (lxlO'^M) 0 UV (900 ergs/mm2) 39.7 UV + 8NQ0 34.5 4NQ0 (4x10" 6M) 44.2 4NQ0 + 8NQ0 37.2 Both in v i v o and in v i t r o s t u d i e s have been done on the 51 57 73 i n t e r a c t i o n of 4NQ0 d e r i v a t i v e s w i t h DNA ' ' . By means of r a d i o a c t i v e t r a c e r s or simply u s i n g fluorescence microscopy, i t has been shown that c a r c i n o g e n i c d e r i v a t i v e s of 4NQ0 bind strongly to DNA whereas the nori-carcinogenic derivatives do not. This relationship between oncogenicity of a compound and i t s capacity to bind in vivo and in vitro to DNA supports the conclusion that DNA alterations are involved in the process of neoplastic transformation. Carcinogenicity and Chromosome Aberrations The present study reveals a parallelism between the carcinogenicity of 4NQ0 derivatives and their capacity to induce chromosome aberrations in cultured mammalian c e l l s . Several other, i f not a l l , chemical carcinogens are also known to induce changes at the chromosome l e v e l 7 ' 4 3 ' 9 8 . This invariably raises the following questions: (1) Are chromosome aberrations and neoplasm causally linked? (2) If carcinogenesis is under chromosomal control, could we expect people with constitutional chromosome aberrations to have an unusual l i a b i l i t y to develop cancer? To answer the f i r s t question, let us take a look at the available evidence from "spontaneous" sarcomas and carcinomas of man as well as from experimentally induced tumors of rodents. With few exceptions (such as acute leukemias and childhood cancers) abnormal chromosome complements are a characteristic feature in human tumor cells . In most instances, there is one or more marker chromosomes accompanied by the loss, or more often gain, of chromosomes6. A single injection of the carcinogen, 7,12-dimethy1benz(a)anthracene, i n t o r a t s induced chromosome a b e r r a t i o n s i n the bone marrow. A f t e r m u l t i p l e 98 i n j e c t i o n s of the carcinogen, the r a t s developed leukemia The i n d u c t i o n of chromosome changes and the development of leukemia i n a s e q u e n t i a l f a s h i o n s t r o n g l y suggests a c e r t a i n , a l b e i t unknown, r o l e played by chromosomes i n c a r c i n o g e n e s i s . Fibrosarcomas induced i n Chinese hamsters and r a t s by Rous sarcoma v i r u s or 7,12-dimethyibenz(a)anthracene (DMBA) 80 are a s s o c i a t e d w i t h non-random chromosome v a r i a t i o n . Golden hamster c e l l s transformed a f t e r treatment w i t h a v i r a l (polyoma)4 6 or chemical (dimethylnitrosamine) carcinogen revealed a p r e f e r e n t i a l increase i n the number of chromosomes i n group 5. These transformed c e l l s were able to form tumors _in v i v o . However, r e v e r t a n t s from these c e l l s l o s t the tumor fonaing a b i l i t y and the _in v i t r o transformed p r o p e r t i e s . I t i s i n t e r e s t i n g to note that r e v e r s i o n of the transformed c e l l s were a s s o c i a t e d w i t h the p r e f e r e n t i a l l o s s of group 5 chromosomes. This suggests that the expression of malignancy can be r e g u l a t e d by the balance between c e r t a i n chromosomes. To answer the question whether people w i t h c o n s t i t u t i o n a l chromosome a b e r r a t i o n s are more l i a b l e to develop cancer, one can c i t e the well-known example of Down's syndrome. P a t i e n t s a f f l i c t e d w i t h t h i s disease c o n d i t i o n have shown an unusually 113 high incidence of leukemia . In a recent study, the number of cancer cases i n 2706 people w i t h an abnormal karyotype was very small4"*. I t may be argued that w h i l e the c o r r e l a t i o n may be true for Down's syndrome, the same may not be true for other types of malignant disease. Carcinogenicity and Clone Forming A b i l i t y The present study indicates a. 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 of a chemical agent and the clone forming capacity i n treated c e l l s . Potent carcinogens cause high c e l l l e t h a l i t y , whereas weakly carcinogenic compounds are only s l i g h t l y c y t o t o x i c . Non-carcinogenic compounds do not a f f e c t the clone forming capacity of cultured mammalian c e l l s . Since a high l e v e l of DNA r e p a i r synthesis and a low clone forming capacity was observed i n c e l l s a f t e r exposure to a carcinogen, the question must be r a i s e d whether c e l l t o x i c i t y i s a d i r e c t r e s u l t of a DNA damage. An incomplete r e p a i r of carcinogen-induced DNA a l t e r a t i o n s before the c e l l enters d i v i s i o n may lead to l e t h a l i t y . On the other hand, a resynthesis of the excised damaged DNA segments does not n e c e s s a r i l y mean that the DNA l e s i o n has been corrected in a functional manner. Indeed, there i s evidence that a f t e r X- or UV i r r a d i a t i o n , 29 1 c e l l s u r v i v a l does not correspond to the extent of DNA r e p a i r ' It follows that r e s i d u a l base a l t e r a t i o n or a change in base sequence, which could a l t e r the coding properties and/or a f f e c t the secondary or t e r t i a r y structure of the DNA h e l i x could be present even though DNA breaks are rejoined. One c r i t i c i s m of the cloning technique i s whether i t furnishes a v a l i d representation of c e l l s u r v i v a l or merely a measure o f the p r o l i f e r a t i v e a b i l i t y o f the c e l l s a f t e r exposure t o a c h e m i c a l agent. A f r a c t i o n o f the c e l l s t h a t s u r v i v e d the c h e m i c a l t r e a t m e n t may n o t e n t e r d i v i s i o n o r perhaps d i v i d e o n l y a few t i m e s . A p a r a l l e l assay w i t h the use o f v i t a l s t a i n i n g seems an a d v i s a b l e supplement. DNA R e p a i r 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 o f Mammalian C e l l s Many, i f not a l l , c h e m i c a l c a r c i n o g e n s a r e a l s o p o t e n t mutagens when t e s t e d on s e v e r a l m i c r o b i a l systems , J » , D r o s o p h i l a 3 6 ' 3 7 o r mammalian c e l l s 1 6 , 3 4 , 5 0 . T h i s phenomenon i s not i n c o n t r a d i c t i o n t o the somatic m u t a t i o n t h e o r y o f c a r c i n o g e n e s i s 1 0 . In a s i m p l i s t i c way, the c o r r e l a t i o n between c a r c i n o g e n i c i t y and m u t a g e n i c i t y may be m e d i a t e d through the e x t e n t o f DNA r e p a i r i n the exposed c e l l s . A p e r f e c t o p e r a t i n g r e p a i r w i l l l e a v e none o f the DNA damage " u n c o r r e c t e d " . The t r e a t e d c e l l s w i l l thus s u r v i v e and w i l l show no g e n e t i c a l t e r a t i o n . On the o t h e r hand, a s e v e r e l y d e f i c i e n t or d e f e c t i v e r e p a i r w i l l l e a v e the c e l l w i t h so many damaged DNA m o l e c u l e s when i t e n t e r s d i v i s i o n t h a t death i s imminent. Only when the DNA r e p a i r i s m o d e r a t e l y d e f i c i e n t o r d e f e c t i v e w i l l a v i a b l e mutated c e l l r e s u l t . The few u n r e p a i r e d segments i n the DNA may l e a d t o copy e r r o r s d u r i n g DNA r e p l i c a t i o n . S e v e r a l l i n e s o f e v i d e n c e seem t o i n d i c a t e the i n v o l v e m e n t o f a d e f e c t i v e DNA r e p a i r i n n e o p l a s t i c t r a n s f o r m a t i o n . U s i n g e x c i s i o n r e p a i r i n h i b i t o r s , i t has been p o s s i b l e to enhance 131 tumor response t o X-ray and a l k y l a t i n g agents . The case o f the h e r e d i t a r y d i s e a s e Xeroderma pigmentosum lends support t o t h i s h y p o t h e s i s . Persons who are homozygous f o r the autosomal r e c e s s i v e XP mutants are h y p e r s e n s i t i v e to s u n l i g h t ; they develop m u l t i p l e tumors i n t h e i r sun-exposed s k i n a r e a s . P a t i e n t s w i t h hundreds of s k i n tumors were r e p e a t e d l y r e p o r t e d 9 7 . F i b r o b l a s t s of most XP p a t i e n t s e x h i b i t e d a reduced l e v e l o f unscheduled 3 H-TdR i n c o r p o r a t i o n a f t e r exposure to UV i r r a d i a t i o n 9 ' 1 4 ' 2 0 ' 3 3 ' 101,111,118 o r c e r t a ; [ n groups of chemica l carc inogens and m u t a g e n s 1 1 0 ' 1 1 8 ' 1 1 9 ' 1 2 2 ' 1 2 3 . The DNA r e p a i r s y n t h e s i s appeared IH 58 normal a f t e r treatment w i t h X - r a y ' , N - m e t h y l - N ' - n i t r o -122 120 n i t r o s o g u a n i d i n e (MNNG) and a l k y l a t i n g agents ( e . g . MMS) The XP c e l l s are not d e f i c i e n t i n the r e p a i r of a DNA damage when DNA s t rands are d i r e c t l y broken by the mutagen. However, the XP c e l l s show a reduced r e p a i r of a damage i n f l i c t e d by agents not c a u s i n g d i r e c t s t r a n d breaks but on ly a f f e c t i n g the t h r e e - d i m e n s i o n a l DNA p a t t e r n . A l t h o u g h there i s a grea t Q 11 111 v a r i a t i o n i n the l e v e l of DNA r e p a i r from, p a t i e n t to p a t i e n t ^ ' , the p o s s i b i l i t y of a l i n k between d e f i c i e n t DNA r e p a i r 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 cannot be so e a s i l y d i s m i s s e d . Normal unscheduled DNA s y n t h e s i s has been observed i n some spontaneous human tumors, HeLa c e l l s and i n p a t i e n t s s u f f e r i n g from Rothmund-Thomson d i s e a s e (h igh i n c i d e n c e o f s k i n t u m o r s ) 8 6 . This suggests that muta t ions l e a d i n g to reduced DNA r e p a i r s y n t h e s i s i n XP are not commonly a s s o c i a t e d w i t h tumors i n man. R e c e n t l y , there were r e p o r t s from in v i v o experiments that4NP0 was weakly carcinogenic"* 4, though repeated t r i a l s for DNA repair synthesis failed to show any detectable 3H-TdR incorporation 1 2 0. Among the 4NP0 derivatives tested, this is the only one for which a correlation between carcinogenicity and induction of DNA repair synthesis cannot be established. If the conflicting observations should stand, then the mechanism by which 4NP0 brings about neoplastic transformation must diff e r from that previously proposed. Preliminary studies have shown an inhibitory effect of 4NP0 on DNA repair synthesis. 4NP0 could prove to be oncogenic by virtue of suppressing DNA repair, thus increasing the "spontaneous" mutation rate. If this is correct, then chemical agents which induce alterations in the DNA and simultaneously inhibits DNA repair should be more hazardous than those which exhibit only one of these two a c t i v i t i e s . Outlook A correlation between the carcinogenicity of a compound, it s mutagenic property and i t s capacity to induce DNA lesions resulting in a DNA repair synthesis has been established for several compounds belonging to different chemical groups (e.g. methylrnethane sulfonate. 1 7 , 4' 1' 6 6'' 1" 2 0, nitrogen mustard 1^' 6 6, t-butyl-hydroperoxide 1 2 0, f$-propiolactone66, nitrosomethyl u r e a 1 2 0 , 1 5 1 2 2 N-methyl-N'-nitro-N-nitrosoguanidine ' , acetylaminofluorene d e r i v a t i v e s 6 6 ' 1 1 0 ' 1 2 3 and benz(a)anthracene d e r i v a t i v e s 1 1 9 . Pending successful t r i a l s V i t h key compounds of other categories ( e . g . a n t i b i o t i c s , i i a f l a t o x i n s ) , the d e s c r i b e d assay f o r DNA r e p a i r s y n t h e s i s may prove t o be an economic and r e l e v a n t t o o l f o r i d e n t i f y i n g p o t e n t i a l l y hazardous c h e m i c a l s i n man's environment. T e s t s f o r c a r c i n o g e n i c a c t i v i t y o f c h e m i c a l agents i n l a b o r a t o r y a n i m a l s (mice, r a t s o r hamsters) r e q u i r e a c o n s i d e r a b l e amount o f space, i n t e n s i v e a f t e r - c a r e and a -large f i n a n c i a l s u p p o r t . F u r t h e r m o r e , the experi m e n t s take as l o n g as two y e a r s t o complete. W i t h the use o f m i c r o b i a l assay systems, the problem o f r u n n i n g c o s t and time i s v e r y much re d u c e d . However, t h e r e w i l l always remain the q u e s t i o n whether the e x p e r i m e n t a l f i n d i n g s can be e x t r a p o l a t e d from lower organisms t o man. In t h i s r e s p e c t , the use o f human t i s s u e as a t e s t s u b j e c t i s more r e l e v a n t . F i b r o b l a s t s from s k i n b i o p s i e s can be r e a d i l y put i n t o c u l t u r e and p r e p a r e d f o r use i n one o r two weeks. More c o n v e n i e n t l y , lymphocytes may be e m p l o y e d 1 7 ' 6 6 . The a u t o r a d i o g r a p h i c p r o c e d u r e i n the p r e s e n t study r e q u i r e s about two weeks to complete. However, by p l a c i n g the a u t o r a d i o g r a m s i n t o c o n t a c t w i t h s c i n t i l l a t i o n f l u i d , t h e r e b y a m p l i f y i n g the "photodynamic" r e a c t i o n , the whole p r o c e d u r e can 129 be s h o r t e n e d t o c o m p l e t i o n w i t h i n a day . The assay f o r 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 a b i l i t y a r e a l s o f a v o u r a b l e c a n d i d a t e s f o r use as s c r e e n i n g t o o l s , though which o f t h e s e would be the most s u i t a b l e i n a l a r g e s c a l e s c r e e n i n g program o f c h e m i c a l s i s d i f f i c u l t t o a s s e s s a t the moment. One f i n a l q u e s t i o n i s ' t h e u n i v e r s a l i t y o f DNA r e p a i r s y n t h e s i s a f t e r t r e a t m e n t o f c e l l s w i t h c h e m i c a l mutagens and c a r c i n o g e n s . S e v e r a l compounds d i d not e l i c i t d e t e c t a b l e l e v e l s 3 o f an u n s c h e d u l e d H-TdR i n c o r p o r a t i o n ( e . g . b e n z a n t h r a c e n e , m e t h y l c h o l a n t h r e n e , d i m e t h y l n i t r o s a m i n e and the my c o t o x i n l u t e o s k y r i n ) . I t i s l i k e l y t h a t t h ese compounds r e q u i r e a c t i v a t i o n i n t o n u c l e o p h i l i c forms by s p e c i f i c enzymes which are p r e s e n t i n the l i v e r , but absent i n f i b r o b l a s t s . T h i s would l i m i t the use o f f i b r o b l a s t s i n b i o a s s a y s . The enzymatic a c t i v a t i o n o f d i m e t h y l n i t r o s a m i n e has r e c e n t l y been s u c c e s s f u l l y a c c o m p l i s h e d ^ 2 . By the c o n c o m i t a n t a p p l i c a t i o n o f t h i s c a r c i n o g e n and mouse l i v e r microsomes t o c u l t u r e d human c e l l s , i t has been p o s s i b l e t o d e t e c t an u n s c h e d u l e d uptake o f H-TdR. With the development o f new t e c h n i q u e s f o r the c u l t u r e o f s p e c i f i c t i s s u e s , DNA r e p a i r s y n t h e s i s may be used t o s c r e e n a w i d e r spectrum o f c h e m i c a l s f o r c a r c i n o g e n i c a c t i v i t y . SUMMARY 1. The aim of t h i s study has been to demonstrate a l i n k between the oncogenic a c t i v i t y of a chemical compound and i t s c a p a c i t y to induce DNA damage and chromosome a b e r r a t i o n s which may r e s u l t i n mutations and/or n e o p l a s t i c t r a n s f o r m a t i o n . 2. Twenty-five 4 - n i t r o q u i n o l i n e 1-oxide (4NQ0) d e r i v a t i v e s and f i v e r e l a t e d compounds of 4 - n i t r o p y r i d i n e 1-oxide (4NP0) of v a r y i n g c a r c i n o g e n i c i t y were examined. These i n c l u d e : (a) isomers of 4NQ0; (b) a l k y l - and h a l o g e n o - s u b s t i t u t e d q u i n o l i n e and p y r i d i n e N-oxides; (c) amino- and hydroxyamino- d e r i v a t i v e s of q u i n o l i n e , p y r i d i n e and t h e i r N-oxides. 3. Monolayer c u l t u r e s of embryonal Syrian-hamster c e l l s and an e s t a b l i s h e d l i n e of baby hamster kidney c e l l s (BHK-21) were employed. 4. DNA r e p a i r s y n t h e s i s was assayed a u t o r a d i o g r a p h i c a l l y from the unscheduled uptake of H-TdR i n c e l l s exposed to the chemical compounds. 5. To d i s t i n g u i s h DNA r e p a i r s y n t h e s i s from DNA r e p l i c a t i o n at S-phase, the c u l t u r e d c e l l s were a r r e s t e d at G^ by keeping them i n an a r g i n i n e d e f i c i e n t medium f o r 2-3 days p r i o r to a p p l i c a t i o n of the chemical carcinogens. 6. W i t h i n the range of c o n c e n t r a t i o n s used, there were three s i g n i f i c a n t features* of the c e l l u l a r response w i t h respect to DNA r e p a i r s y n t h e s i s ; (a) only c e l l s exposed to oncogenic compounds e x h i b i t e d DNA r e p a i r s y n t h e s i s ; 3 (b) H-TdR i n c o r p o r a t i o n could be detected at con s i d e r a b l y lower doses a f t e r treatment w i t h h i g h l y oncogenic d e r i v a t i v e s than w i t h weak ones; (c) the maximum g r a i n count a f t e r exposure to weakly oncogenic d e r i v a t i v e s was always lower than i n c e l l s t r e a t e d w i t h h i g h l y oncogenic compounds. 7. Chromosome a b e r r a t i o n s were st u d i e d i n ADM-arrested c e l l s which were exposed to the v a r i o u s compounds and then t r i g g e r e d i n t o d i v i s i o n by t r a n s f e r r i n g them i n t o the r e g u l a r growth medium. The types of observable chromosome a b e r r a t i o n s i n c l u d e d s i n g l e and m u l t i p l e chromatid breaks and s i n g l e and m u l t i p l e chromatid exchanges. A d i r e c t p r o p o r t i o n a l i t y was observed between the degree of c a r c i n o g e n i c i t y of a chemical compound and the frequency of induced chromosome a b e r r a t i o n s . 8. The clone forming c a p a c i t y of c u l t u r e d c e l l s t r e a t e d w i t h ANQO and 4NP0 d e r i v a t i v e s v a r i e d as the degree of o n c o g e n i c i t y of the compound. Strongly c a r c i n o g e n i c d e r i v a t i v e s were c y t o t o x i c ; weak carcinogens showed only a s l i g h t l e t h a l e f f e c t and the non-carcinogenic compounds d i d not 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 c e l l s . 9. The unscheduled 3H-TdR uptake i n c u l t u r e d c e l l s exposed to a carcinogen was assumed to i n d i c a t e DNA e x c i s i o n r e p a i r . Conversely, the absence of1 any d e t e c t a b l e 3H-TdR i n c o r p o r a t i o n a f t e r treatment w i t h non-carcinogenic 4NQ0 d e r i v a t i v e s was taken to mean that no DNA a l t e r a t i o n s d i d occur and, t h e r e f o r e , no DNA r e p a i r s y n t h e s i s has taken p l a c e . Other p o s s i b i l i t i e s 3 that could e x p l a i n the absence of any H-TdR uptake were d i s c u s s e d . These i n c l u d e : (a) i n s t a b i l i t y of the compound i n s o l u t i o n ; (b) f a i l u r e of a mutagen or carcinogen to reach the chromosomes of a c e l l ; (c) requirement f o r a c t i v a t i o n of the carcinogen; (d) amount of DNA damage and r e p a i r s y n t h e s i s too low to be detected w i t h the present assay system (concentrations too low or exposure too s h o r t ) ; (e) i n h i b i t o r y e f f e c t exerted by the carcinogen on the r e p a i r mechanism; ( f ) presence of c e l l mutants d e f i c i e n t i n DNA r e p a i r 3 s y n t h e s i s or l a c k i n g the c a p a c i t y of H-TdR uptake from the c u l t u r e medium. 10. The p o s s i b l e r o l e of DNA and chromosome damage i n n e o p l a s t i c transformation was d i s c u s s e d . 11. The use of DNA r e p a i r s y n t h e s i s as an economic and r e l e v a n t t o o l f o r i d e n t i f y i n g mutagens and/or carcinogens has been suggested. REFERENCES 1. 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Comm. 29: 469-477, 1967. 80 128. Takayama, S, , Kuwabara, N., Azama, Y. , and Sugimura, T. Skin tumors i n mice painted w i t h N-methyl-N'-nitro-N-n i t r o s o g u a n i d i n e . J . Nat. Cancer I n s t . 46: 973-980, 1971. 129. Weingrad, D., Folkman, J . , and Sade, R.M. A method f o r r a p i d autoradiography. Exp. C e l l Res., In p r e s s , 1971. 130. W i t k i n , E.M. U l t r a v i o l e t - i n d u c e d mutations and DNA r e p a i r . Ann. Rev. Genetics 3: 525-552, 1969. 131. Y i e l d i n g , K.L., and Gaudin, D. Enhancement of tumour response to X-ray and a l k y l a t i n g agents by the e x c i s i o n r e p a i r i n h i b i t o r s , c a f f e i n e and c h l o r o q u i n e , i n "Genetic Concepts and N e o p l a s i a " , 23rd Ann. Symp. Fund. Cancer Res. 523-527, 1969. 132. Yoshida, T.H., K u r i t a , Y. and M o r i w a k i , K. Chromosomal a b e r r a t i o n s i n Yoshida sarcoma c e l l s t r e a t e d w i t h 4 - n i t r o -q u i n o l i n e 1-oxide. Gann 56: 523-528, 1965. 81 APPENDIX 1 Hank's Balanced S a l t S o l u t i o n (BSS). To prepare 1 l i t r e of lOx stock s o l u t i o n : -1. Sodium C h l o r i d e (NaCl) 80 g. Potassium C h l o r i d e (KC1) 4 g. Magnesium Sulphate (MgS0^.7H20) 1 g. Sodium Phosphate, d i b a s i c (Na2HP0^) 0.48 g. Potassium Phosphate, monobasic (KH2PO4) 0.6 g. Glucose 10.0 g. (D i s s o l v e d i n 800 ml of d i s t i l l e d water) 2. Calcium C h l o r i d e ( C a C l J 1.4 g. (Dissolved i n 100 ml of d i s t i l l e d water) 3. Phenol Red 0.1 g. (D i s s o l v e d 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 m l , the pH has to be adjusted to 7.0 w i t h ' 0.05 N NaOH) 4. M i x i n g of the above three s o l u t i o n s gives 1 l i t r e of lOx stock Hank's BSS. APPENDIX 2 A r g i n i n e D e f i c i e n t Medium (ADM) To prepare 10 l i t r e s of ADM: -1. Hank's BSS„ 1 l i t r e of lOx stock s o l u t i o n (prepared as described i n Appendix 1 ) . P o r t i o n s of t h i s may be d i l u t e d w i t h d i s t i l l e d water to give l x stock f o r preparing s o l u t i o n s of the amino aci d s and v i t a m i n s . 2. E s s e n t i a l Amino Acid s L - H i s t i d i n e 310 mg. L-Leucine 520 mg. L-Lysine 580 mg. L - I s o l e u c i n e 520 mg. L-Methionine 150 mg. L-Phenylalanine 320 mg. L-Threonine 480 mg. L-Tryptophan 100 mg. L-V a l i n e 460 mg. (Di s s o l v e d i n 100 ml 1 x Hank's BSS) L-Tyrosine L-Cystine L-Glutamine 360 mg. (Dissolved i n 100 ml 0.1 N HCl) 240 mg. ( D i s s o l v e d i n 100 ml 0.1 N HCl) 2.92 g. (Dissolved i n 100 ml l x Hank's BSS) 3. Non-Essential Amino Acids L-Alanine 89 mg. L-Asparagine 150 mg. L - A s p a r t i c A c i d 133 mg. L-Glutamic A c i d 147 mg. L - P r o l i n e 115 mg. L-Serine 105 mg. Gly c i n e 75 mg. (Di s s o l v e d i n 100 ml l x Hank's BSS) 4. Vitamins Choline C h l o r i d e 100 mg. Nicotinamide 100 mg. i - I n o s i t o l 200 mg. P y r i d o x a l 100 mg. R i b o f l a v i n 10 mg. D-Ca-Pantothenate 100 mg. Thiamine HCl 100 mg. (Di s s o l v e d i n 100 ml l x BSS) F o l i c A c i d 10 mg. (Dis s o l v e d i n 100 ml l x BSS) The s o l u t i o n s 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 b r i n g the f i n a l volume to 10 l i t r e s . The c u l t u r e 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 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 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 c u l t u r e j u s t 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 v i t a m i n s and n o n - e s s e n t i a l amino aci d s are ob t a i n a b l e i n the form of lOOx concentrated mixture from Flow L a b o r a t o r i e s , I n c . (Ingle-wood, C a l i f o r n i a ) . 84 APPENDIX 3 . - * Phosphate Buffered S a l i n e (PBS, without calcium and magnesium) NaCl 8.0 g. KC1 0.2 g. Na 2HP0 4 1.15 g. KH 2P0 4 0.2 g. (Di s s o l v e d i n d i s t i l l e d water to make 1 l i t r e of l x stock s o l u t i o n ) . 

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