The Mutational Consequences of DNA Damage Induced by Benzo[a

13 The Mutational Consequences of DNA Damage Induced by Benzo[a]pyrene Downloaded by PENNSYLVANIA STATE UNIV on May 16, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

ERIC EISENSTADT Department of Cancer Biology and Laboratory of Toxicology, Harvard School of Public Health, Boston, MA 02115 Induced mutagenesis i n Escherichia c o l i i s an active process involving proteins with DNA replication, re pair, and recombination functions. The available evi dence suggests that mutations are generated at sites where DNA has been damaged and that they arise v i a an error-prone repair activity. In an attempt to under stand what specific contributions to mutagenesis are made by DNA lesions, we have studied the mutational specificity of some carcinogens, such as benzo[a]pyrene and aflatoxin B1, whose chemical reactions with DNA are well-studied. Our results, obtained by monitoring the distribution of lacI nonsense mutations i n E. coli, suggest that the major mutational events induced by benzo[a]pyrene and aflatoxin B are base substitutions. The base substitutions are primarily transversions at G:C base pairs and the available evidence suggests that these mutations are induced by apurinic sites which are generated as secondary consequences of the initial al kylation event. The significance of these results i n the context of carcinogenesis i s briefly considered. 1

The h i g h f i d e l i t y w i t h w h i c h genomes a r e r e p l i c a t e d in v i v o and passed on t o daughter c e l l s i s a c h i e v e d by a r e p e r t o i r e o f a c t i v i t i e s which f u n c t i o n d u r i n g r e p l i c a t i o n , r e p a i r , and r e c o m b i n a t i o n (1^,2). These a c t i v i t i e s , which c o l l e c t i v e l y m a i n t a i n the s t r u c t u r a l and i n f o r m a t i o n a l i n t e g r i t y o f the DNA m o l e c u l e , a r e s e v e r e l y t e s t e d when the DNA t e m p l a t e i s damaged and becomes n o n - r e p l i c a b l e . Under these c i r c u m s t a n c e s , which o b t a i n , f o r example, when c e l l s a r e exposed t o such human c a r c i n o g e n s as U V - l i g h t (3) o r p o l y c y c l i c a r o m a t i c h y d r o carbons ( 4 ) , i t i s commonly observed t h a t the f r e q u e n c y o f m u t a t i o n i s enhanced by many o r d e r s o f magnitude. The c o r r e l a t i o n between t h e mutagenic and c a r c i n o g e n i c a c t i v i t y o f many p h y s i c a l and c h e m i c a l agents has been well-documented (_5). Recent o b s e r v a t i o n s even sug g e s t the p o s s i b i l i t y t h a t one s t e p i n t u m o r i g e n e s i s might l i t e r a l l y i n v o l v e the m u t a t i o n a l a l t e r a t i o n o f s p e c i f i c chromosomal genes (6-8). 0097-6156/ 85/ 0283-0327506.00/ 0 © 1985 American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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I n t h i s c h a p t e r I w i l l r e v i e w some a s p e c t s o f mutagenesis mecha nisms and t h e m u t a t i o n a l consequences o f DNA damage g e n e r a t e d by ben zo [a] p y r e n e . The f o c u s w i l l be on knowledge d e r i v e d from i n v e s t i g a t i o n s i n v o l v i n g the bacterium E s c h e r i c h i a c o l i .

Downloaded by PENNSYLVANIA STATE UNIV on May 16, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch013

Mutagenesis i s an a c t i v e p r o c e s s 12. c o l i and e u k a r y o t i c c e l l s can respond t o DNA damage by i n d u c i n g the s y n t h e s i s o f s p e c i f i c gene p r o d u c t s ( 9 - 1 2 ) . The phenomenon o f gene i n d u c t i o n by DNA damage has been most t h o r o u g h l y d e s c r i b e d f o r 12. c o l i and has r e c e n t l y been reviewed by Walker ( 9 ) . Among t h e i n d u c i b l e r e s p o n s e s t o DNA damage i s the mutagenic r e p a i r p r o c e s s , whose e x i s t e n c e was f i r s t suggested o v e r 30 y e a r s ago by t h e e x p e r i ments o f W e i g l e ( 1 3 ) . W e i g l e showed t h a t U V - l i g h t was mutagenic t o b a c t e r i o p h a g e lamb da o n l y i f t h e U V - i r r a d i a t e d lambda were grown on b a c t e r i a w h i c h had a l s o been i r r a d i a t e d w i t h U V - l i g h t . I n o t h e r words, t h e UV treatment was n o t mutagenic p e r s e . F u r t h e r m o r e , he demonstrated t h a t i r r a d i ated lambda phage c o u l d be r e a c t i v a t e d by growing t h e phage on p r e i r r a d i a t e d b a c t e r i a . H i s r e s u l t s suggested t h e p o s s i b i l i t y t h a t bac t e r i a had an i n d u c i b l e system f o r DNA r e p a i r and mutagenesis w h i c h a c t e d on U V - i r r a d i a t e d lambda phage. The g e n e t i c s o f what i s now c a l l e d W e i g l e o r W - r e a c t i v a t i o n and W-mutagenesis i s now v e r y w e l l understood. Some twenty genes i n J2. c o l i — known c o l l e c t i v e l y as d i n genes (damage i n d u c i b l e ; 9,14) a r e c o o r d i n a t e l y r e g u l a t e d by t h e p r o d u c t s o f t h e genes recA and l e x A . The LexA p r o t e i n r e p r e s s e s d i n gene e x p r e s s i o n by b i n d i n g t o t h e o p e r a t o r r e g i o n o f each gene and p r e v e n t i n g i t s t r a n s c r i p t i o n i n t o RNA by RNA polymerase. Treatments w h i c h damage t h e c e l l ' s DNA o r o t h e r w i s e i n t e r f e r e w i t h DNA s y n t h e s i s , a c t i v a t e t h e RecA p r o t e i n ; a c t i v a t e d RecA p r o t e i n t h e n promotes t h e p r o t e o l y t i c i n a c t i v a t i o n o f LexA r e p r e s s o r ( 1 5 ) . Genes whose t r a n s c r i p t i o n had been r e p r e s s e d by LexA p r o t e i n c a n now be t r a n s c r i b e d and new p r o t e i n s c a n be s y n t h e s i z e d . The o v e r a l l response o f 12. c o l i t o DNA damage, w h i c h i s g e n e t i c a l l y r e g u l a t e d by t h e r e c A and l e x A l o c i , i s known as t h e SOS-response ( 1 6 , 1 7 ) . M u t a t i o n s i n e i t h e r recA o r l e x A c a n a b o l i s h t h e SOS-response and e l i m i n a t e b o t h W - r e a c t i v a t i o n and W-mutagenesis. These m u t a t i o n s a l s o e l i m i n a t e t h e m u t a b i l i t y o f t h e b a c t e r i a by U V - i r r a d i a t i o n ( 1 6 ) . The o b s e r v a t i o n t h a t UV mutagenesis depended on t h e SOS-response e s t a b l i s h e d t h a t m u t a t i o n s were n o t i n e v i t a b l e outcomes o f DNA damage and t h a t DNA damage r e q u i r e d p r o c e s s i n g by c e l l u l a r mechanisms i n o r der f o r m u t a t i o n s t o be r e c o v e r e d . What s p e c i f i c p r o c e s s e s r e g u l a t e d by t h e SOS-response a r e r e s p o n s i b l e f o r mutagenesis? A major c o n t r i b u t i o n towards answering t h i s q u e s t i o n was made by the i s o l a t i o n o f m u t a t i o n s w h i c h s p e c i f i c a l l y e l i m i n a t e d t h e m u t a b i l i t y o f 12. c o l i w i t h o u t a f f e c t i n g any o f t h e o t h e r components o f t h e SOS-response. M u t a t i o n s a t t h e umuDC l o c u s were i n d e p e n d e n t l y d i s covered by Kato and S h i n o u r a (18) and S t e i n b o r n (19) t o a b o l i s h t h e m u t a t i o n a l a f f e c t s o f DNA damage by U V - i r r a d i a t i o n . These mutants were a l s o shown t o be d e f e c t i v e i n W-mutagenesis (18,20) and W-react i v a t i o n (18,20). The b i o c h e m i c a l n a t u r e o f t h e a c t i v i t y performed by the umuDC gene p r o d u c t s i s n o t known. However, s e v e r a l o b s e r v a t i o n s suggest t h a t t h e f u n c t i o n o f t h e umuDC p r o t e i n s i s t o enhance some mode o f DNA r e p a i r , e i t h e r d i r e c t l y o r i n d i r e c t l y : 1) J2. c o l i c a r r y -


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i n g t h e umuC36 a l l e l e i s more s e n s i t i v e t o t h e l e t h a l e f f e c t s o f UVi r r a d i a t i o n (18) and a n g e l i c i n p l u s near-UV ( 2 1 ) ; 2) P l a s m i d borne a n a l o g s o f the umuDC l o c u s (mucAB; 12) enhance W - r e a c t i v a t i o n and t h e resistance of b a c t e r i a to the l e t h a l e f f e c t s of U V - i r r a d i a t i o n (23); 3) As p r e v i o u s l y n o t e d , W - r e a c t i v a t i o n i n U v r b a c t e r i a i s e l i m i n a t e d by m u t a t i o n s a t the umuDC l o c u s ( 1 8 ) . Of t h e a p p r o x i m a t e l y twenty genes induced by DNA damage, the umuDC genes and t h e i r p r o d u c t s a r e the b e s t c a n d i d a t e s f o r d i r e c t p a r t i c i p a n t s i n the b i o c h e m i c a l p r o c e s s i n g o f DNA l e s i o n s t o m u t a t i o n s . The p r o c e s s i n g o f DNA damage i n °li umuDC gene p r o d u c t s and t h e a s s o c i a t e d p r o t e i n s r e g u l a t e d by t h e SOS-response i s c a l l e d SOS-processing (9) o r , sometimes, e r r o r - p r o n e r e p a i r ( 1 6 , 1 7 ) . Mutagenesis i n IS. c o l i , t h e r e f o r e , a p pears t o be a g e n e t i c a l l y and b i o c h e m i c a l l y a c t i v e p r o c e s s r e q u i r i n g the p a r t i c i p a t i o n o f i n d u c i b l e p r o t e i n s . Not a l l mutagenesis i n IS. c o l l i s dependent on S O S - p r o c e s s i n g . M u t a t i o n s may a r i s e q u i t e s i m p l y d u r i n g DNA r e p l i c a t i o n i f a base i s s u b s t i t u t e d by o r c o n v e r t e d t o a n o t h e r , i n c o r r e c t , base. Consider the consequence o f o x i d a t i v e d e a m i n a t i o n o f t h e base 5 - m e t h y l c y t o s i n e to thymine. R e p l i c a t i o n f o l l o w e d by daughter s t r a n d s e g r e g a t i o n w i l l r e s u l t i n a G:C base p a i r h a v i n g been mutated t o an A:T base p a i r . S i t e s c o n t a i n i n g 5 - m e t h y l c y t o s i n e a r e h o t s p o t s f o r G:C t o A:T t r a n s i t i o n s i n E. c o l i ( 2 4 ) . A l k y l a t i o n o f some bases a t the e x o c y c l i c oxygen atoms c a n l e a d to c h e m i c a l l y s t a b l e a l t e r a t i o n s i n t h e base p a i r i n g p r o p e r t i e s o f a base and, t h e r e b y , d i r e c t l y induce base m i s - p a i r i n g by DNA polymer a s e . A w e l l - s t u d i e d example o f t h i s i s t h e consequence o f a l k y l a t i n g guanine a t the 0-6 p o s i t i o n ( 2 5 - 2 7 ) . T h i s has t h e e f f e c t o f f r e e z i n g guanine i n i t s ( r a r e ) e n o l tautomer p e r m i t t i n g the G:T mismatch t o form i n p l a c e o f the u s u a l G:C base p a i r . A subsequent round o f DNA r e p l i c a t i o n l e a d s t o the g e n e r a t i o n o f a G:C t o A:T t r a n s i t i o n muta t i o n . These e x c e p t i o n s n o t w i t h s t a n d i n g , most DNA damaging agents i n duce mutations i n IS. c o l i v i a SOS-processing.


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The dependence o f m u t a t i o n on f u n c t i o n s i n v o l v i n g DNA r e p a i r seems t o be widespread among organisms. Many p r o k a r y o t i c s p e c i e s a r e i n h e r e n t l y non-mutable by U V - l i g h t b u t become mutable when p l a s m i d s encod i n g f o r f u n c t i o n s analogous t o the umuDC f u n c t i o n s a r e i n t r o d u c e d (e.g. 2 8 ) . Non-mutable mutants o f the y e a s t Saccharomyces c e r e v i s i a e have been i s o l a t e d and shown t o possess d e f e c t s which i m p l i c a t e DNA r e p a i r and r e c o m b i n a t i o n p r o c e s s e s ( s e e 29^ f o r a r e c e n t r e v i e w ) . F u r t h e r m o r e , t h e r e a r e many examples o f DNA r e p a i r s t r a t e g i e s which a r e common t o p r o k a r y o t i c and e u k a r y o t i c organisms ( n u c l e o t i d e e x c i s i o n r e p a i r , DNA g l y c o s y l a s e s , a p u r i n i c / a p y r i m i d i n i c e n d o n u c l e a s e s , 0 -methylguanine-DNA-methyl t r a n s f e r a s e ; 2,30). P u r i f i e d DNA p o l y merases from mammalian c e l l s and v i r u s e s behave s i m i l a r l y _in v i t r o t o IS. c o l i DNA polymerase when DNA damage i s encountered — r e p l i c a t i o n ceases a t t h e s i t e o f t h e l e s i o n ( 3 1 ) . Of c o u r s e , even i f ana logues o f SOS-processing a r e i d e n t i f i e d i n e u k a r y o t e s , t h e r e g u l a t i o n of these a c t i v i t i e s might d i f f e r i n d e t a i l from the scheme which ob t a i n s i n IS. c o l i (e.£. analogous f u n c t i o n s may be c o n s t i t u t i v e l y ex p r e s s e d ) . N o n e t h e l e s s , Ruby and S z o s t a k (10) have demonstrated t h e e x i s t e n c e o f DNA damage i n d u c i b l e l o c i i n j>. c e r e v i s i a e and e s t i m a t e


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t h a t t h e r e may e x i s t as many as 80 such genes ( 1 1 ) . Shorpp e t a l . (12) have r e c e n t l y r e p o r t e d t h a t UV l i g h t enhances t h e s y n t h e s i s o f a t l e a s t e i g h t p r o t e i n s i n human f i b r o b l a s t c e l l s .


Are m u t a t i o n s d i s t r i b u t e d a t s i t e s o f DNA damage? The dependence o f mutagenesis on SOS p r o c e s s i n g r a i s e d q u e s t i o n s about t h e r o l e ( s ) p l a y e d by DNA l e s i o n s i n mutagenesis. Do DNA l e s i o n s s i m p l y t r i g g e r the SOS response by i n t e r f e r i n g w i t h DNA r e p l i c a t i o n t h e r e b y g e n e r a t i n g m u t a t i o n s i n d i r e c t l y v i a an e r r o r - p r o n e form o f DNA r e p l i c a t i o n ? Or do m u t a t i o n s a r i s e d i r e c t l y a t t h e s i t e s i n DNA where damage has been generated? The o b s e r v a t i o n s t h a t m u t a t i o n f r e q u e n c i e s a r e e l e v a t e d s e v e r a l f o l d above normal l e v e l s i n mutants w h i c h c o n s t i t u t i v e l y e x p r e s s t h e i r S O S - f u n c t i o n s (32) and t h a t the m u t a t i o n f r e q u e n c y o f u n i r r a d i ated phage i s e l e v a t e d by growing them on i r r a d i a t e d ( i . ^ e . SOS-in duced) b a c t e r i a ( 3 3 ) , have been i n v o k e d t o argue f o r the n o t i o n t h a t mutagenesis v i a SOS-processing may be i n d i r e c t . On the o t h e r hand, the o b s e r v a t i o n t h a t 95% o f the UV induced base s u b s t i t u t i o n m u t a t i o n s a r o s e a t the v e r y s i t e s ( p y r i m i d i n e - p y r i midine sequences) where the major f r a c t i o n o f UV damage i s d e p o s i t ed suggested t h a t a t l e a s t the UV i n d u c e d m u t a t i o n s were t a r g e t e d (24). Drake and B a l t z (34) and W i t k i n and Wermundsen (35) p r e s e n t e d arguments i n f a v o r o f the n o t i o n t h a t , f o r the most p a r t , SOS muta g e n e s i s was o c c u r r i n g a t s i t e s o f DNA damage. More r e c e n t e v i d e n c e , based on a n a l y z i n g the d i s t r i b u t i o n o f m u t a t i o n s w i t h i n the l a d gene of _E. c o l i , s t r o n g l y s u g g e s t s t h a t m u t a t i o n s a r i s i n g v i a SOS-process i n g a r e o c c u r r i n g a t the s i t e s o f DNA damage ( 3 6 , 3 7 ) . B r i e f l y , when one examines t h e spectrum o f m u t a t i o n s i n d u c e d by a v a r i e t y o f muta gens whose a c t i v i t y i s dependent on S O S - p r o c e s s i n g , one f i n d s t h a t both where the m u t a t i o n s a r e i n d u c e d and w h i c h m u t a t i o n s a r e induced depends on the mutagen. The observed d i f f e r e n c e s among mutagens a p p l y b o t h t o the m u t a t i o n a l e v e n t s t h a t a r e d i s t r i b u t e d non-randomly at o n l y a few s i t e s ( h o t s p o t s ) and t o e v e n t s t h a t a r e d i s t r i b u t e d randomly a t many d i f f e r e n t s i t e s w i t h i n t h e gene ( l o w f r e q u e n c y o c c u r r e n c e s o r LFO e v e n t s ) ( 3 6 ) . S i n c e each mutagenic t r e a t m e n t l e a v e s behind i t s own c h a r a c t e r i s t i c d i s t r i b u t i o n o f m u t a t i o n s w i t h i n t h e gene ( 3 7 ) , m u t a t i o n s g e n e r a t e d b y SOS-processing o f damaged DNA must be o c c u r r i n g a t the s i t e s o f damage. F u r t h e r m o r e , t h e r e c e n t s t u d y by M i l l e r and Low (38) on the d i s t r i b u t i o n o f m u t a t i o n s g e n e r a t e d by t u r n i n g on the SOS-response w i t h o u t DNA-damaging t r e a t m e n t s shows t h a t even these m u t a t i o n s a r e g e n e r a t e d a t s p e c i f i c s i t e s i n a gene as i f t h e y arose a t s i t e s where s p o n t a n e o u s l y g e n e r a t e d l e s i o n s o c c u r with a h i g h frequency. The w e l l c h a r a c t e r i z e d r e a c t i o n s o f c a r c i n o g e n s such as benzo[a]pyrene and a f l a t o x i n B^ w i t h DNA (39-50) suggested t o us t h a t an a n a l y s i s o f the k i n d s o f m u t a t i o n s t h e s e agents i n d u c e d c o u l d shed l i g h t on the c o n t r i b u t i o n o f s p e c i f i c DNA l e s i o n s t o mutagenesis. Such an a n a l y s i s c o u l d , i n t u r n , p r o v i d e c l u e s as t o w h i c h s p e c i f i c DNA l e s i o n s g e n e r a t e d by t h e s e agents were mutagenic. I would l i k e t o d e s c r i b e o u r i n v e s t i g a t i o n s , performed i n c o l l a b o r a t i o n w i t h J e f f r e y M i l l e r . To b e g i n , I w i l l b r i e f l y o u t l i n e t h e g e n e t i c system d e v e l o p e d by M i l l e r w h i c h p e r m i t s a r a p i d , r i g o r o u s d e t e r m i n a t i o n o f t h e p o s i t i o n and k i n d s o f mutants i n d u c e d i n a p a r t i c u l a r gene by DNA. damaging a g e n t s *



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l a d system f o r a n a l y z i n g nonsense m u t a t i o n s I n E. c o l i

331

The l a d system has been d e s c r i b e d i n d e t a i l by M i l l e r ( 5 1 ) . The l a d gene product i s the r e p r e s s o r o f t h e l a c operon. C e l l s w h i c h have normal r e p r e s s o r a c t i v i t y a r e r e p r e s s e d f o r t h e s y n t h e s i s o f the l a c Z gene p r o d u c t , 3 - g a l a c t o s i d a s e , and t h e o t h e r p r o d u c t s o f t h e l a c operon. C e l l s w h i c h c a r r y m u t a t i o n s i n l a d w h i c h l e a d t o s y n t h e s i s of a d e f e c t i v e r e p r e s s o r p r o t e i n w i l l c o n s t i t u t i v e l y s y n t h e s i z e 3g a l a c t o s i d a s e . Such mutants can be s e l e c t e d by demanding growth o f b a c t e r i a on a medium c o n t a i n i n g a g a l a c t o s i d e a n a l o g such a s p h e n y l 0 - D - g a l a c t o s i d e ( P - g a l ) . P - g a l i s n o t i t s e l f an i n d u c e r o f t h e l a c operon. Thus, i t i s a s i m p l e m a t t e r t o t r e a t a p o p u l a t i o n o f b a c t e r i a l c e l l s w i t h a DNA damaging a g e n t , grow them o u t n o n - s e l e c t i v e l y t o p e r m i t p r o c e s s i n g o f DNA damage and p h e n o t y p i c e x p r e s s i o n , and t h e n p l a t e them on P - g a l t o s e l e c t f o r c e l l s c a r r y i n g m u t a t i o n s i n l a c l . A l a r g e c l a s s o f base s u b s t i t u t i o n mutants can be a n a l y z e d d i r e c t l y by s c r e e n i n g f o r s u p p r e s s i b l e m u t a t i o n s among t h e c o l l e c t i o n of l a c l mutants. The s u p p r e s s i b l e m u t a t i o n s a r e due t o w i l d - t y p e codons h a v i n g been mutated t o TAA, TAG, o r TGA. These nonsense c o dons a r e n o r m a l l y s i g n a l s f o r t h e t e r m i n a t i o n o f p r o t e i n s y n t h e s i s by ribosomes and c a n a r i s e v i a a l l s i n g l e base p a i r s u b s t i t u t i o n muta t i o n s w i t h t h e e x c e p t i o n o f t h e A:T t o G:C t r a n s i t i o n . Thus, a l l base p a i r s u b s t i t u t i o n s , e x c e p t f o r t h e one t r a n s i t i o n , can be moni t o r e d by c o l l e c t i n g nonsense m u t a t i o n s i n l a c l . There a r e o v e r 60 s i t e s i n l a c l a t w h i c h a s i n g l e base p a i r s u b s t i t u t i o n w i l l g e n e r a t e a nonsense codon. L a c l nonsense mutants can be i d e n t i f i e d u s i n g c l a s s i c a l b a c t e r i a l g e n e t i c methods. The e n t i r e gene has been sequenced ( 5 2 ) . The s i t e , and t h e r e f o r e , t h e base p a i r w h i c h has been mutated c a n be in dent i f l e d s i m p l y by mapping t h e p o s i t i o n o f t h e nonsense m u t a t i o n . T h i s can be a c c o m p l i s h e d by u s i n g an e x t e n s i v e s e t o f l a c l d e l e t i o n mutants ( 5 3 ) . Mapping t h e m u t a t i o n a l l o w s one t o determine w h i c h base p a i r s u b s t i t u t i o n has been g e n e r a t e d by a p a r t i c u l a r t r e a t m e n t . Thus, by i d e n t i f y i n g many nonsense m u t a t i o n s induced by a mutagen, a p i c t u r e emerges o f b o t h t h e mutagens s i t e s p e c i f i c i t y (where, w i t h i n the gene t h e m u t a t i o n s a r i s e ) and i t s mutagenic s p e c i f i c i t y (which p a r t i c u l a r base s u b s t i t u t i o n s a r e g e n e r a t e d ) . To d e t e r m i n e c l a s s e s of m u t a t i o n o t h e r than base p a i r s u b s t i t u t i o n s , i t i s p o s s i b l e t o g e n e t i c a l l y c r o s s a g i v e n l a c l a l l e l e o n t o s m a l l p l a s m i d o r phage m o l e c u l e s and d e t e r m i n e t h e sequence o f t h e mutant a l l e l e (54,55)• We have a p p l i e d t h e g e n e t i c system f o r a n a l y z i n g l a c l nonsense mutants t o t h e i n v e s t i g a t i o n o f t h e mutagenic s p e c i f i c i t y o f benzo[a]pyrene (56) and a f l a t o x i n B. ( 5 7 ) . The r e s u l t s o f our s t u d i e s have p r o v i d e d some i m p o r t a n t c l u e s as t o t h e c h e m i c a l n a t u r e o f t h e mutagenic l e s i o n s induced by b e n z o [ a ] p y r e n e . B e f o r e I d i s c u s s these r e s u l t s , I w i l l b r i e f l y summarize p r e v i o u s i n v e s t i g a t i o n s on the mu t a g e n i c i t y o f BPDE. The m u t a g e n i c i t y inves t igat ions

of benzo[a]pyrene d i o l e p o x i d e —

previous

C a r c i n o g e n s f i r s t began t o be e v a l u a t e d d i r e c t l y f o r mutagenic a c -



332


t i v i t y a g a i n s t microorganisms 35 y e a r s ago by B a r r e t t and Tatum ( 5 8 ) . However, the s y s t e m a t i c use of s e n s i t i v e m i c r o b i a l m u t a t i o n a s s a y s t o m o n i t o r the b i o l o g i c a l a c t i v i t y o f c a r c i n o g e n s was not a c h i e v e d u n t i l the r e a l i z a t i o n t h a t m e t a b o l i c a c t i v a t i o n o f c a r c i n o g e n s was e s s e n t i a l ( r e v i e w e d i n 5 9 ) . By the use of s u b - c e l l u l a r f r a c t i o n s d e r i v e d from l i v e r homogenates, i t became p o s s i b l e t o d e t e c t the mutagenic a c t i v i t y of benzo[a]pyrene and many o t h e r p o l y c y c l i c a r o m a t i c h y d r o carbons • The m u t a g e n i c i t y of benzo[a]pyrene f o r b a c t e r i a was demonstrated by Ames e t a l . ( 6 0 ) . They found t h a t i n the presence o f r a t l i v e r homogenates benzo[a]pyrene i n d u c e d b o t h f r a m e s h i f t and b a s e - p a i r sub s t i t u t i o n m u t a t i o n s . When the c h e m i s t r y of benzo[a]pyrene a c t i v a t i o n had been worked out and t h e u l t i m a t e c a r c i n o g e n i c form i d e n t i f i e d as a d i o l e p o x i d e , BPDE ( r e v i e w e d i n 61-62), s e v e r a l i n v e s t i g a t o r s ( 6 3 66) showed t h a t BPDE was an e x t r e m e l y p o t e n t mutagen, a l s o c a p a b l e o f i n d u c i n g b o t h f r a m e s h i f t and b a s e - p a i r s u b s t i t u t i o n m u t a t i o n s . McCann e t a l . (67) had shown t h a t benzo[a]pyrene was mutagenic f o r J5. typhimurium o n l y i f the b a c t e r i a c a r r i e d the m u t a t i o n enhancing p l a s mid pKMlOl whose a c t i v i t y was l a t e r shown by Walker (23) t o be en t i r e l y dependent on b a c t e r i a l r e c A and l e x A c o n t r o l l e d f u n c t i o n s . T h i s p r o v i d e d e a r l y e v i d e n c e t h a t the m u t a g e n i c i t y o f c a r c i n o g e n s such as b e n z o [ a ] p y r e n e was dependent on S O S - r e p a i r . L a t e r , I v a n o v i c and W e i n s t e i n (68) d i r e c t l y showed t h a t benzo[a]pyrene was mutagenic +

f o r JE. c o l i o n l y i f the b a c t e r i a were b o t h r e c A and l e x A * . Two q u e s t i o n s t h a t a r e r a i s e d by t h e s e o b s e r v a t i o n s a r e : 1) what i s the mutagenic s p e c i f i c i t y o f BPDE, i_.je. what k i n d s o f m u t a t i o n s are i n d u c e d by t r e a t i n g c e l l s w i t h BPDE? 2) what i s ( a r e ) the p r e - m u t a t i o n a l l e s i o n ( s ) g e n e r a t e d by BPDE which i s ( a r e ) r e s p o n s i b l e f o r mutations? The mutagenic

s p e c i f i c i t y o f BPDE

We have o b t a i n e d i m p o r t a n t c l u e s t o t h e s e q u e s t i o n s by d e t e r m i n i n g the spectrum of 185 nonsense m u t a t i o n s induced i n the l a c l gene o f IS. c o l i by BPDE. The r e s u l t s o f t h i s i n v e s t i g a t i o n (56) a r e summarized i n T a b l e s I t o I I I and i n F i g u r e 1. The r e s u l t s were s t r i k i n g . They showed t h a t t r a n s v e r s i o n m u t a t i o n s a t G:C base p a i r s were the domi nant i n d u c e d e v e n t , a l t h o u g h , o t h e r s u b s t i t u t i o n s , i n p a r t i c u l a r A:T to T:A t r a n s v e r s i o n were c l e a r l y i n d u c e d , but a t l o w e r f r e q u e n c i e s . The s p e c i f i c i t y of i n d u c t i o n o f G:C t o T:A was most c l e a r l y seen by examining the m u t a t i o n s o c c u r r i n g a t the TAC codons f o r t y r o s i n e ( T a ble I I I ) . A t t h e s e s i t e s , b o t h G:C t o T:A m u t a t i o n s ( y i e l d i n g TAA, ochre nonsense mutants) and G:C t o C:G ( y i e l d i n g TAG, amber nonsense mutants) t r a n s v e r s i o n s are m o n i t o r a b l e . T a b l e I I I c l e a r l y shows t h a t at the two t y r o s i n e codons where m u t a t i o n s were w e l l - i n d u c e d t h e r e i s a s t r i k i n g p r e f e r e n c e f o r one m u t a t i o n a l event over the o t h e r . We have not d i r e c t l y determined t h e r e l a t i v e f r e q u e n c i e s o f f r a m e s h i f t m u t a t i o n s and o t h e r m u t a t i o n a l e v e n t s i n comparison t o the base s u b s t i t u t i o n m u t a t i o n s . However, based on the h i g h f r e q u e n c y o f nonsense m u t a t i o n s (11%) among a l l l a c l mutants induced by BPDE and because nonsense m u t a t i o n s a r e m o n i t o r a b l e a t l e s s than o n e - f i f t h o f the l a c l codons and, even t h e n , o n l y v i a c e r t a i n base p a i r s u b s t i t u t i o n s , we b e l i e v e t h a t base s u b s t i t u t i o n s account f o r a major f r a c t i o n o f m u t a t i o n s induced by BPDE.


13.

EISENSTADT


333

T a b l e I . Summary of Base S u b s t i t u t i o n Events Generated by BPDE


Substitution

No. o f Available Sites

No. o f Sites Found

T o t a l No. o f Occurrences

% of Analyzed Mutations

G:C t o A:T

26

12

22

12

G:C t o T:A

23

21

123

66

A:T t o T:A

15

33

18

9

A:T t o C:G

5

3

4

2

G:C t o C:G

3

2

3

2

Total

72

47

185

Amber

36

26

96

Ochre

36

21

89

What i s ( a r e ) the p r e - m u t a t i o n a l

l e s i o n ( s ) i n d u c e d by BPDE?

BPDE r e a c t s a t s e v e r a l d i f f e r e n t s i t e s on DNA t o g e n e r a t e s e v e r a l k i n d s o f l e s i o n s a t t h e N2 (43-45) and N7 (46,47) p o s i t i o n s o f guan i n e , a p u r i n i c s i t e s ( 4 8 , 4 9 ) , and s t r a n d b r e a k s ( 5 0 ) . Which o f t h e s e l e s i o n s a r e r e s p o n s i b l e f o r t h e t r a n s v e r s i o n m u t a t i o n s a t G:C s i t e s ? E v i d e n c e d e r i v e d from a number o f e x p e r i m e n t s s u g g e s t s t h e h y p o t h e s i s t h a t a p u r i n i c s i t e s g e n e r a t e d by BPDE r e a c t i o n s w i t h DNA a r e r e s p o n s i b l e f o r the transversion mutations: 1. When we examined t h e mutagenic s p e c i f i c i t y a f l a t o x i n B^, a c a r c i n o g e n w h i c h s p e c i f i c a l l y r e a c t s w i t h t h e N7 atom o f guanine ( 3 9 4 2 ) , we found v i r t u a l l y o n l y G:C t o T:A t r a n s v e r s i o n s were i n d u c e d ( 5 7 ) ; N7 p u r i n e adducts can i n d u c e d e p u r i n a t i o n by d e s t a b i l i z i n g t h e N - g l y c o s y l i c bond ( 6 9 ) . 2. The work o f Loeb and K u n k e l and t h e i r c o l l e a g u e s (70-72) h a s c l e a r l y e s t a b l i s h e d t h a t a p u r i n i c s i t e s i n DNA a r e mutageniC.; t h e y s p e c i f i c a l l y cause t r a n s v e r s i o n m u t a t i o n s , due t o a s t r o n g p r e f e r e n c e f o r t h e i n c o r p o r a t i o n o f adenine r e s i d u e s d u r i n g bypass o f a p u r i n i c s i t e s i n t e m p l a t e DNA. Thus, A:T t o T:A and G:C t o T:A t r a n s v e r s i o n s a r e t h e major mutagenic outcome g e n e r a t e d by d e p u r i n a t i o n o f DNA. 3. R e c e n t l y , Sage and H a s e l t i n e (49) have q u a n t i t a t i v e l y d e t e r mined t h e spectrum o f DNA l e s i o n s i n d u c e d by r e a c t i o n s o f BPDE w i t h DNA. They found t h a t a l k a l i - l a b i l e l e s i o n s account f o r about 40% o f the DNA a d d u c t s . There was a s t r i k i n g c o r r e l a t i o n between t h e muta t i o n f r e q u e n c i e s induced by BPDE i n l a c l and t h e f r e q u e n c i e s o f a l k a l i s e n s i t i v e l e s i o n s a t G, A, and C r e s i d u e s . Apurinic/apyrimidinic s i t e s a r e common a l k a l i - s e n s i t i v e l e s i o n s . E a r l i e r work by D r i n k w a t e r e t a l . ( 4 8 ) had a l s o shown t h a t t r e a t m e n t o f DNA w i t h BPDE gen erated a p u r i n i c / a p y r i m i d i n i c s i t e s .


334

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS Table I I .


Base Substitutions

D i s t r i b u t i o n o f l a c l Nonsense M u t a t i o n s Induced by BPDE

Site

No. Independent Occurrences

Site

No. Independent Occurrences 0 0 0 0 0 3 3 2 1 1 0 0

G:C - A:T

A5 *A6 A9 *A15 A16 A19 A21 A23 A24 A26 A31 A33 *A34 A35

0 5 0 0 0 1 2 0 1 0 1 1 1 0

09 010 Oil 013 017 021 024 027 028 029 034 035

G:C - T:A„ G:C - C:G

A2 A7 A10

7 5 5

A12 A13 A17 A20 A25 A27 A28

18 5 5 5 4 5 3

03,A1 06 07 „ 08, A8 014 015 019 020 025 026 030,A29 031 032 036

#

12,1 0 0 4,0 2 7 4 12 0 4 1,2 2 1 12

#

(13)

(4)

(3)

2 5 0 (0) 0*>A3^ 05,A4 2 (2) 0 012 6 0 018,A14 1 (1) 4 A 023,A22^ 0 (0) 033,A30 1 (1) S i t e s a t w h i c h nonsense m u t a t i o n s a r e d e t e c t e d a r e i d e n t i f i e d by t h e i r amber (A) o r o c h r e (0) a l l e l e s ( C o u l o n d r e and M i l l e r , 1977). The 8 t y r o s i n e codons i n l a c l each have two nonsense a l l e l e s , one amber and one o c h r e . The amber a l l e l e s a t t h e s e s i t e s a r e marked by the symbols # and @. * S i t e s c o n t a i n i n g 5 - m e t h y l c y t o s i n e s (CCAGG). These a r e spontaneous l a c l hotspots.

A:T - T:A A:T - C:G

0

All A18 A32 A36

2 4 1 8

01

&

0 2

L

0 1 6

L

0

2

2

L


13.

EISENSTADT

Table I I I .


Site

BPDE-Induced M u t a t i o n s a t the Three TAC T y r o s i n e Codons i n the l a c l Gene

coding p o s i t i o n

^,03

tyr 7

A8,08 A29,030 Total

335


# o f independent o c c u r r e n c e s o f : TAC - TAA TAC - TAA (GC - CG) (GC - TA)

12

1

t y r 47

4

0

tyr

1

2

17

3

273

Both amber and o c h r e m u t a t i o n s can be g e n e r a t e d a t t h e s e s i t e s , a l l o w i n g b o t h G:C t o T:A and G:C t o C:G t r a n s v e r s i o n s t o be monitored.

Thus, w h i l e BPDE and a f l a t o x i n might g e n e r a t e G:C t o T:A t r a n s v e r s i o n s v i a d i f f e r e n t pathways, i t i s r e a s o n a b l e t o c o n s i d e r the h y p o t h e s i s t h a t t h e r e i s a common mechanism by w h i c h t h e y i n d u c e t h i s m u t a t i o n and t h a t , t h e r e f o r e the t r a n s v e r s i o n m u t a t i o n s induced by BPDE r e s u l t , not from the major adduct t o the N2 atom o f guanine but from the g e n e r a t i o n o f a p u r i n i c s i t e s i n DNA. These secondary l e s i o n s might be g e n e r a t e d s p o n t a n e o u s l y o r v i a the a c t i v i t y of DNA g l y c o s y l a s e s (2_, 3 0 ) . An a l t e r n a t i v e h y p o t h e s i s i s t h a t b u l k y l e s i o n s i n g e n e r a l , the N2 adduct among them, may be n o n i n f o r m a t i o n a l s i t e s o p p o s i t e w h i c h adenines a r e p r e f e r e n t i a l l y i n s e r t e d d u r i n g r e p l i c a t i o n a f t e r DNA damage. Other m o l e c u l a r g e n e t i c s t u d i e s on the m u t a g e n i c i t y o f BPDE The g e n e t i c system we used t o s t u d y the mutagenic s p e c i f i c i t y o f BPDE l i m i t s one t o a n a l y z i n g base s u b s t i t u t i o n m u t a t i o n s . What i s known about the a b i l i t y o f BPDE t o i n d u c e o t h e r c a t e g o r i e s o f m u t a t i o n ? As mentioned above, r e s u l t s from the Ames t e s t r e v e a l e d t h a t b e n z o [ a ] p y rene and i t s d i o l e p o x i d e were c a p a b l e o f i n d u c i n g f r a m e s h i f t muta t i o n s (60,63,64). More r e c e n t l y , Mizusawa and co-workers (73-76) have i n v e s t i g a t e d the m u t a t i o n a l consequences o f m o d i f y i n g p l a s m i d DNA i n v i t r o w i t h BPDE. I n a s e r i e s o f s t u d i e s t h e y have shown t h a t : _ 1. p l a s m i d m o l e c u l e s a r e i n a c t i v a t e d (become n o n - r e p l i c a b l e ) i n Uvr b a c t e r i a by 1 c o v a l e n t adduct (73,76) per m o l e c u l e ; t h i s r e s u l t i s i n p e r f e c t agreement w i t h an e a r l i e r r e p o r t by Hsu eit a l . (77) w h i c h had demonstrated t h a t one m o l e c u l e o f bound BPDE was s u f f i c i e n t




GC-^AT

10-,

I

I

20p

GC-^TA

10 +

A

I II I ,l I ll GC-^CG

AT-^TA

IX AT-^CG

10r

oL 100

200

300

AMINO ACID RESIDUE F i g u r e 1. The f r e q u e n c i e s o f amber m u t a t i o n s i n t h e l a c l gene i n d u c e d by BPDE. S o l i d b a r s , i n d i v i d u a l s i t e s a t w h i c h we d e t e c t e d m u t a t i o n s ; open b a r s , s i t e s a t w h i c h we d i d not detect mutations; a s t e r i s k s ( i n a ) , s i t e s a t which the t a r g e t codon c o n t a i n s 5 - m e t h y l c y t o s i n e .



13.

EISENSTADT


337

to i n h i b i t t h e r e p l i c a t i o n o f s i n g l e m o l e c u l e o f t h e s i n g l e s t r a n d e d b a c t e r i o p h a g e 0X174. 2. BPDE-induced m u t a t i o n s i n p l a s m i d borne genes c a n be depen dent on umuC ( 7 5 ) ; 3. m u t a t i o n s induced by BPDE i n c l u d e t r a n s v e r s i o n , t r a n s i t i o n , and f r a m e s h i f t m u t a t i o n s ( 7 4 , 7 6 ) . Wei e t a l . ( 7 8 ) c h a r a c t e r i z e d m u t a t i o n s r e s u l t i n g from a l k y l a t i o n o f a 10-base p a i r o l i g o n u c l e o t i d e w i t h BPDE. D e l e t i o n m u t a t i o n s were t h e major m u t a t i o n a l event d e t e c t e d . The number o f m u t a t i o n s a n a l y z e d i n each o f t h e s e i n v e s t i g a t i o n s was t o o s m a l l ( o n l y 7 t o 8) t o p e r m i t drawing f i r m c o n c l u s i o n s about m u t a t i o n a l and s i t e s p e c i f i c i t i e s . However, t h e r e s u l t s suggest t h a t , under some c i r c u m s t a n c e s , BPDE c a n i n d u c e many d i f f e r e n t k i n d s of m u t a t i o n s . F u t u r e s t u d i e s on t h e g e n e t i c e f f e c t s o f BPDE To r i g o u r o u s l y e s t a b l i s h t h e g e n e t i c consequences r e s u l t i n g from BP adduct t o t h e N2 p o s i t i o n o f g u a n i n e , t h e approach t a k e n by Essigman and h i s c o l l e a g u e s (79,80) w i l l be r e q u i r e d . They have been d e v e l o p i n g t e c h n i q u e s f o r p l a c i n g d e f i n e d c h e m i c a l l e s i o n s i n t o p l a s m i d DNA at pre-determined s i t e s a t which m u t a t i o n s c a n be m o n i t o r e d . If a BP adduct can be " b u i l t " i n t o DNA a t t h e N2 o f g u a n i n e , i t s b i o l o g i c a l and g e n e t i c e f f e c t s can be d e t e r m i n e d . I t would be i n t e r e s t i n g t o know i f t h e m u t a t i o n a l consequences of DNA l e s i o n s i n mammalian c e l l s were t h e same as t h o s e w h i c h o b t a i n i n b a c t e r i a . Methods f o r r e t r i e v i n g and sequencing m u t a t i o n s i n mam m a l i a n c e l l s and t h e i r v i r u s e s a r e now b e i n g developed ( 8 1 - 8 3 ) . I f y e a s t , a e u k a r y o t i c m i c r o o r g a n i s m , c a n be c o n s i d e r e d r e p r e s e n t a t i v e of h i g h e r e u k a r y o t e s , then j u d g i n g from t h e o b s e r v a t i o n s t h a t t h e m u t a t i o n a l s p e c t r a f o r U V - i r r a d i a t i o n and 4 - n i t r o q u i n o l i n e - l - o x i d e t r e a t m e n t a r e i d e n t i c a l f o r y e a s t (84) and b a c t e r i a ( 8 5 ) , t h e spec trum o f m u t a t i o n s induced by BPDE i n mammalian c e l l s c o u l d w e l l r e semble those induced i n IS. c o l i . I s t h e m u t a g e n i c i t y o f BPDE d i r e c t l y r e s p o n s i b l e f o r i t s carcinogenicity? Though one i s f a r from b e i n g a b l e t o make a d e f i n i t i v e s t a t e m e n t , t h e r e a r e some i n d i c a t i o n s t h a t t h e answer t o t h i s q u e s t i o n might be no. Recent experiments w i t h mammalian c e l l c u l t u r e systems, w h i c h a l l o w one t o s t u d y t h e p r o g r e s s i o n o f c e l l s from a s t a t e where t h e i r growth i s n o r m a l l y r e g u l a t e d t o a t u m o r i g e n i c s t a t e have r e v e a l e d t h a t t h e t r a n s f o r m a t i o n p r o c e s s r e q u i r e s a t l e a s t two s t e p s ( 8 6 - 8 8 ) • The f i r s t s t e p , which i s induced f o l l o w i n g exposure t o a c a r c i n o g e n ( X - r a y s : 86,88; 3 - m e t h y l c h o l a n t h r e n e : 8 7 ) , o c c u r s w i t h a v e r y h i g h f r e q u e n c y and s e e m i n g l y a f f e c t s e v e r y exposed c e l l i n t h e t r e a t e d p o p u l a t i o n . The f r e q u e n c y o f t h e i n i t i a l event i m p l i e s t h a t i t i s not a m u t a t i o n a l event b u t r a t h e r an e p i g e n e t i c one r e l a t e d , perhaps, to t h e responses i n d u c e d by DNA damage i n IS. c o l i and S* c e r e v i s i a e . The second event i s a v e r y r a r e event c o n s i s t e n t w i t h t h e p o s s i b i l i t y t h a t i t might be m u t a t i o n a l i n n a t u r e . However, s i n c e t h e second event o c c u r s many c e l l g e n e r a t i o n s a f t e r t h e exposure t o DNA damaging a g e n t s , i t seems h i g h l y improbable t h a t i t o c c u r s as a d i r e c t conse quence o f r e p a i r i n g t h e i n i t i a l DNA damage. Thus, t h e l i n k between


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c a r c i n o g e n e s i s and mutagenesis may s i m p l y be t h a t t h e two p r o c e s s e s o r i g i n a t e from t h e same s t a r t i n g p o i n t , namely DNA damage. Acknowledgments


Work i n my l a b o r a t o r y has been supported by g r a n t s from t h e NIH. I am i n d e b t e d t o many o f my p r e s e n t and former c o l l e a g u e s , i n p a r t i c u l a r t o D r s . A . J . Warren and P.L. F o s t e r f o r t h e i r work on the muta g e n i c s p e c i f i c i t y o f c h e m i c a l c a r c i n o g e n s and t o D r . J.H. M i l l e r f o r h i s c o l l a b o r a t i v e e f f o r t i n s t u d y i n g t h e mutagenic s p e c i f i c i t y o f b e n z o [ a ] p y r e n e and a f l a t o x i n B^.

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

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