The Intercalation of Benzo[a]pyrene and 7,12-Dimethylbenz[a

9

Downloaded by UNIV OF MISSOURI COLUMBIA on May 16, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch009

The Intercalation of Benzo[a]pyrene and 7,12-Dimethylbenz[a]anthracene Metabolites and Metabolite Model Compounds into DNA P. R. LEBRETON Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60680

Carcinogenic metabolites and metabolite model com pounds of benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene intercalate into DNA with physical binding constants in the range 10 -10 M . The association constants for hydrocarbon-base stack ing are similar in magnitude to those for base -base stacking and are much smaller than those of intercalating drugs such as ethidium bromide which has a value >10 M . The intercalation of these molecules strongly depends on DNA structure and environment. Increases in solvent polarity en hance intercalation. The DNA stabilizers Mg and polyamines inhibit intercalation. Hydrocarbon -base π interactions are much weaker in heat dena tured DNA than in native double-stranded DNA. For metabolites and metabolite models for which com parisons were made, binding to native single -stranded DNA is favored over binding to circular double-stranded DNA, and binding to poly(dA-dT) is favored over binding to other synthetic polynu cleotides. In studies of nonreactive model com pounds which have steric and electronic properties similar to those of reactive metabolites of 7,12dimethylbenz [a] anthracene and benzo[a]pyrene, i t is found that analogs of bay region epoxides are better intercalating agents than those of less carcinogenic epoxides. 3

6

4

-1

- 1

+2

M e t a b o l i t e s o f c a r c i n o g e n i c p o l y c y c l i c a r o m a t i c hydrocarbons (PAH) such as (±) t r a n s - 7 , 8 - d i h y d r o x y - a n t i - 9 , 1 0 - e p o x y - 7 , 8 , 9 , 1 0 - t e t r a h y drobenzo[a]pyrene (BPDE) and 7 , 8 , 9 , 1 0 - t e t r a h y d r o x y t e t r a h y d r o b e n zo [a]pyrene (BPT) p a r t i c i p a t e i n TT b i n d i n g i n t e r a c t i o n s w i t h nu c l e o t i d e bases (1-19) which l e a d t o t h e r e v e r s i b l e f o r m a t i o n o f p h y s i c a l complexes. Almost a l l t h e p h y s i c a l l y bound hydrocarbon m e t a b o l i t e s a r e i n t e r c a l a t e d between the n u c l e o t i d e bases. A g r e a t d e a l o f i n f o r m a t i o n about events i m p o r t a n t t o c h e m i c a l c a r 0097-6156/ 85/ 0283-0209$08.25/ 0 © 1985 American Chemical Society In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.


210

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

c i n o g e n e s i s has r e c e n t l y come t o l i g h t as a r e s u l t of t h e study of r e a c t i v e i n t e r a c t i o n s o c u r r i n g between epoxide c o n t a i n i n g metabo l i t e s o f PAH and DNA (20-34). Much l e s s i s known about the n a t u r e and t h e s i g n i f i c a n c e o f r e v e r s i b l e p h y s i c a l i n t e r a c t i o n s between h y d r o c a r b o n m e t a b o l i t e s and DNA. The p o t e n t i a l importance o f hydrocarbon-nucleotide p h y s i c a l b i n d i n g t o the mechanisms o f PAH c a r c i n o g e n e s i s i s shown by t h e data g i v e n i n Table I which g i v e s b i n d i n g c o n s t a n t s f o r s t a c k e d complexes formed from the i n t e r a c t i o n o f n u c l e o t i d e bases w i t h pyrene ( 3 5 ) , BPDE ( 3 ) and e t h i d i u m bromide ( 3 6 ) . Table I a l s o c o n t a i n s b i n d i n g c o n s t a n t s f o r s t a c k e d complexes formed from t h e s e l f - a s s o c i a t i o n o f n u c l e o s i d e s ( 3 7 ) . A comparison of t h e DNA b i n d i n g c o n s t a n t s i n Table I i n d i c a t e s t h a t t h e TT s t a c k i n g i n t e r a c t i o n s of hydrocarbon m e t a b o l i t e s such as BPDE a r e much weaker than those o f s t r o n g i n t e r c a l a t i n g drugs such as e t h i d i u m bromide. The b i n d i n g c o n s t a n t f o r BPDE i n t e r c a l a t i o n i n t o DNA i s s i m i l a r i n magnitude t o t h e b i n d i n g con s t a n t s f o r the hydrogen bonding o f base p a i r s . In organic s o l v e n t s (CDCl^ and CCl^) a t 25° C a s s o c i a t i o n c o n s t a n t s f o r basep a i r hydrogen bonding t y p i c a l l y l i e i n the range 10^-10 M~* ( 3 7 ) . An e x a m i n a t i o n o f t h e n u c l e o s i d e and n u c l e o t i d e b i n d i n g d a t a i n Table I a l s o shows t h a t the f o r c e s r e s p o n s i b l e f o r t h e s t a c k e d a s s o c i a t i o n o f n u c l e o s i d e s a r e s i m i l a r i n magnitude t o those l e a d i n g t o b i n d i n g o f pyrene t o m o n o n u c l e o t i d e s . I n v i v o t h e r e v e r s i b l e p h y s i c a l b i n d i n g o f n u c l e o t i d e bases t o one another v i a base s t a c k i n g and hydrogen bonding i s r e s p o n s i b l e f o r the s t o r a g e and t r a n s m i s s i o n o f g e n e t i c i n f o r m a t i o n and p l a y s an important r o l e i n d e t e r m i n i n g the s t r u c t u r e and s t a b i l i t y o f d o u b l e - s t r a n d e d h e l i c a l DNA. The s i m i l a r i t y between t h e p h y s i c a l i n t e r a c t i o n s of bases w i t h one another and the p h y s i c a l i n t e r a c t i o n s o f bases w i t h h y d r o c a r b o n s and hydrocarbon m e t a b o l i t e s may be i m p o r t a n t t o mecha nisms o f PAH c a r c i n o g e n e s i s . Recent s t r u c t u r e - a c t i v i t y s t u d i e s o f l - a l k y l b e n z o [ a ] p y r e n e s a l s o suggest t h a t DNA i n t e r c a l a t i o n o f benzo[a]pyrene (BP) metabo l i t e s p l a y s a r o l e i n t h e mechanism of BP c a r c i n o g e n e s i s ( 3 8 ) . The a d d i t i o n o f b u l k y a l k y l groups a t t h e 1 - p o s i t i o n o f BP, w h i c h i n h i b i t DNA i n t e r c a l a t i o n o f 1 - a l k y l - B P m e t a b o l i t e s ( 1 9 ) , causes a reduction i n carcinogenic a c t i v i t y . In the e a r l i e s t s t u d i e s of the p h y s i c a l b i n d i n g of carcinoge n i c hydrocarbons w i t h DNA t h e e f f e c t s o f DNA upon the s o l u b i l i t y of pyrene and benzo[a]pyrene were examined. I n a s o l u t i o n o f DNA (0.05% by w e i g h t ) the s o l u b i l i t y of these hydrocarbons i s i n c r e a s ed as much as 70 times (39, 4 0 ) . The b i o c h e m i c a l s i g n i f i c a n c e of these e a r l y s t u d i e s has been q u e s t i o n e d ( 4 1 ) , and i t has been a r gued t h a t mechanisms o f h y d r o c a r b o n c a r c i n o g e n e s i s depend much more upon t h e i n t e r a c t i o n o f parent hydrocarbons w i t h p r o t e i n s than w i t h DNA. T h i s c r i t i c i s m i s supported by c u r r e n t s t u d i e s o f hydrocarbon c a r c i n o g e n e s i s w h i c h p o i n t t o the i m p o r t a n t r o l e t h a t a c t i v a t i o n o f parent hydrocarbons by microsomal enzyme systems c o n t a i n i n g cytochrome P-450 p l a y s i n t h e f o r m a t i o n o f u l t i m a t e r e a c t i v e carcinogens (42). Most r e c e n t s t u d i e s o f hydrocarbon i n t e r a c t i o n s w i t h DNA have f o c u s s e d on t h e b i n d i n g o f hydrocarbon m e t a b o l i t e s r a t h e r than on the b i n d i n g o f the parent h y d r o c a r b o n s . Much o f t h i s work d e a l s

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


9. LEBRETON

211

Intercalation of Metabolite Model Compounds into DNA

T a b l e I . Comparison of B i n d i n g C o n s t a n t s f o r Stacked Complexes a t 23-25 °C

1

KCM' )

Complex a b Nucleoside-Nucleoside ' Pyrene-Nucleotide BPDE-DNA E t h i d i u m Bromide-DNA » 0

a

a b c d e

e

-1.0 x 1 0 -3.0 x 1 0 6.5 x 1 0 >10 6

In H 0 Taken from r e f . 37. I n H 0 + 5% M e t h a n o l . Taken from r e f . 35. I n H 0 + 2% E t h a n o l . Taken from r e f . 3. Taken from r e f . 36. 2

2

2


1

1

3


212


w i t h t h e r e a c t i o n p r o p e r t i e s of c a r c i n o g e n i c e p o x i d e s of BP and 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e (DMBA). There i s c o n s i d e r a b l e e v i dence s u g g e s t i n g t h a t DNA l e s i o n s r e s u l t i n g from r e a c t i o n s w i t h those and o t h e r c a r c i n o g e n s a r e an e s s e n t i a l s t e p i n tumor i n i t i a t i o n (43,44). I t i s found t h a t i n a s i g n i f i c a n t number of cases m e t a b o l i t e r e a c t i v i t y p r o v i d e s a good i n d e x f o r p r e d i c t i n g PAH c a r c i n o g e n i c potency (45, 4 6 ) . F o r example, bay r e g i o n epoxides and p r e c u r s o r s of bay r e g i o n e p o x i d e s o f BP and DMBA a r e more mutagenic and c a r c i n o g e n i c than l e s s r e a c t i v e e p o x i d e s , such as K - r e g i o n e p o x i d e s , d e r i v e d from t h e same p a r e n t hydrocarbons ( 4 5 - 5 3 ) . F i g u r e s 1 and 2 show bay r e g i o n e p o x i d e s of BP and DMBA along w i t h s t r u c t u r e s of less a c t i v e epoxides. In v i t r o s t u d i e s of DNA i n t e r a c t i o n s w i t h t h e r e a c t i v e ben z o y l pyrene e p o x i d e BPDE i n d i c a t e t h a t p h y s i c a l b i n d i n g of BPDE o c c u r s r a p i d l y on a m i l l i s e c o n d time s c a l e f o r m i n g a complex t h a t then r e a c t s much more s l o w l y on a time s c a l e o f minutes ( 1 7 ) . Se v e r a l r e a c t i v e events f o l l o w f o r m a t i o n of the p h y s i c a l complex. The most f a v o r a b l e r e a c t i o n i s the DNA c a t a l y z e d h y d r o l y s i s o f BPDE t o t h e t e t r o l , BPT (3,5,6,8,17). At 25°C and pH=7.0, the hy d r o l y s i s o f BPDE t o BPT i n DNA i s as much as 80 times f a s t e r than h y d r o l y s i s w i t h o u t DNA ( 8 ) . Other r e a c t i o n s which f o l l o w forma t i o n o f p h y s i c a l complexes i n c l u d e those i n v o l v i n g the n u c l e o t i d e bases and p o s s i b l y t h e p h o s p h o d i e s t e r backbone. These can l e a d t o DNA s t r a n d s c i s s i o n (9> 34, 54-56) and t o the f o r m a t i o n of s t a b l e BPDE-DNA a d d u c t s . Adduct f o r m a t i o n o c c u r s a t t h e e x o c y c l i c amino groups on t h e n u c l e o t i d e bases and a t o t h e r s i t e s (1,2,9,17,20, 28,33,34,57,58). The pathway which leads t o h y d r o c a r b o n adducts c o v a l e n t l y bound t o t h e 2-amino group of guanine has been t h e most widely studied. S e v e r a l l a b o r a t o r i e s have examined whether BPDE c o v a l e n t l y bound t o DNA assumes an i n t e r c a l a t e d c o n f o r m a t i o n o r i s e x t e r n a l l y bound. Different groups have reported different results (5,6,8,20,34,59-69). M o b i l i t y studies using relaxed circular pBR322 DNA i n d i c a t e t h a t r e a c t i o n w i t h BPDE g i v e s r i s e t o r a p i d p o s i t i v e s u p e r c o i l i n g which i s s u g g e s t i v e of a c o n f o r m a t i o n i n which t h e h y d r o c a r b o n o c c u p i e s an i n t e r n a l s i t e i n t h e DNA ( 3 4 ) . On t h e o t h e r hand, from r e s u l t s of the most r e c e n t s p e c t r o s c o p i c s t u d i e s i t i s concluded t h a t t h e c o v a l e n t adduct formed from t h e more c a r c i n o g e n i c (+) enantiomer of BPDE i s i n an e x t e r n a l c o n f o r mation ( 6 8 , 6 9 ) . On t h e b a s i s o f PAH p h y s i c a l b i n d i n g s t u d i e s (18) i t has been p r e v i o u s l y suggested t h a t BPDE adduct c o n f o r m a t i o n s a r e s t r o n g l y dependent upon t h e DNA environment and t h a t t h i s may be p l a y i n g a r o l e i n t h e v a r y i n g r e s u l t s r e p o r t e d by d i f f e r e n t l a b o r a t o r i e s . A mechanism f o r r e a c t i o n has been proposed ( 2 ) which i n v o l v e s i n i t i a l i n t e r c a l a t i o n o f BPDE f o l l o w e d by r e a c t i o n which can l e a d u l t i m a t e l y t o n o n i n t e r c a l a t e d complexes. T h i s mechanism i s s u p p o r t ed by r e c e n t k i n e t i c f l o w d i c h r o i s m s t u d i e s ( 6 9 ) . Two s p e c i f i c s u g g e s t i o n s c o n c e r n i n g t h e r o l e t h a t t h e r e v e r s i b l e p h y s i c a l b i n d i n g of proximate and u l t i m a t e c a r c i n o g e n s de r i v e d from BP p l a y i n c a r c i n o g e n e s i s have been made. The f i r s t i s based on r e c o g n i t i o n t h a t DNA-BPDE complex f o r m a t i o n precedes r e -


9.

LEBRETON



Reactive

Metabolites

213

M e t a b o l i t e Model Compounds

Bay Region Epoxide

7,8,9,10-tetrahydroxy tetrahydro-BP

BPDE

7-hydroxy-7,8,9,10-tetrahydro-BP

Less C a r c i n o g e n i c

Epoxides 7,8-dihydroxy-7,8,9,10-tetrahydro-BP

HO.

trans-9,10- di hydroxy-anU-7,8epoxy-7,8,9,10-tetrahydro-BP

•OH BP-4,5-oxide trans - 4,5 - dihydroxy - 4,5 - dihydro-BP

F i g u r e 1. M e t a b o l i t e s and m e t a b o l i t e model compounds d e r i v e d from benzo[a]pyrene•



214


Less C a r c i n o g e n i c Epoxides

CH

5,6-dihydro-BA

3

DMBA-5,6-oxide

HO* OH

CH

3

8,9,10,11-tetrahydro-BA

8,9-dihydroxy-10,ll-epoxy8,9,10,11-tetrahydro-DMBA

F i g u r e 2.

R e a c t i v e m e t a b o l i t e s and m e t a b o l i t e model compounds de r i v e d from 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e .



9. LEBRETON


215

action. T h i s has l e d t o s p e c u l a t i o n (1) t h a t t h e s t e r e o c h e m i s t r y a s s o c i a t e d w i t h t h e f o r m a t i o n o f p r e r e a c t i o n i n t e r c a l a t e d com p l e x e s w i t h DNA i s r e l a t e d t o the v a r y i n g r e a c t i v e and c a r c i n o g e n i c p r o p e r t i e s o f d i f f e r e n t s t e r e o i s o m e r s o f BP e p o x i d e s . I t has a l s o been suggested ( 9 ) t h a t s i t e s f o r s t r a n d s c i s s i o n a r e d e t e r mined by t h e f o r m a t i o n of i n t e r c a l a t e d complexes b e f o r e r e a c t i o n . The second i s based on t h e o b s e r v a t i o n (18) t h a t t h e nonreact i v e proximate c a r c i n o g e n _trans_-7 ,8-dihydroxy-7,8-dihydro-BP binds f a v o r a b l y t o DNA. T h i s b i n d i n g may l e a d t o i n c r e a s e d i n v i v o n u c l e a r c o n c e n t r a t i o n s of t h e d i o l . N u c l e a r membrane bound c y t o chrome P-450 (70,71) i s a v a i l a b l e f o r f u r t h e r a c t i v i a t i o n o f t h e d i o l , and p o o l i n g o f t h e proximate c a r c i n o g e n i n the n u c l e u s and subsequent metabolism t o BPDE may be i m p o r t a n t . T h i s would p r o v i d e an e f f i c i e n t mechanism by which b i o l o g i c a l l y s i g n i f i c a n t l e v e l s of unstable r e a c t i v e metabolites reach nuclear target s i t e s . S e v e r a l groups have i n v e s t i g a t e d t h e DNA p h y s i c a l b i n d i n g p r o p e r t i e s o f BP (39,40,72), of n o n r e a c t i v e BP m e t a b o l i t e s and o f r e a c t i v e BP m e t a b o l i t e s (1-6,8,11). Nonreactive metabolites which have been studied are trans-7,8-dihydroxy-7,8-dihydro-BP (15,16,18,19), BPT (3-5,7,10,18) and t r a n s - 4 , 5 - d i h y d r o x y - 4 , 5 - d i h y dro-BP ( 1 5 , 1 8 ) . The f i r s t o f these molecules i s a proximate c a r cinogen and a m e t a b o l i c p r e c u r s o r o f BPDE. The second and t h i r d m o l e c u l e s a r e t h e h y d r o l y s i s p r o d u c t s o f BPDE and o f t h e l e s s c a r c i n o g e n i c K-region epoxide, benzo[a]pyrene-4,5-oxide, r e s p e c t i v e ly. Studies of the p h y s i c a l b i n d i n g of r e a c t i v e metabolites of benzo[a]pyrene have f o c u s s e d on BPDE and on t r a n s - 9 , 1 0 - d i h y d r o x y anti-7,8-epoxy-7 ,8,9,10-tetrahydro-BP (J_-4_, _6, JJL_) • These i n v e s t i g a t i o n s of the noncovalent b i n d i n g of r e a c t i v e metabolites t o DNA a r e made d i f f i c u l t by t h e r e a c t i o n s , e s p e c i a l l y h y d r o l y s i s , w h i c h f o l l o w the f o r m a t i o n o f a p h y s i c a l complex. In o r d e r t o g a i n more d e t a i l e d i n f o r m a t i o n about the p h y s i c a l b i n d i n g o f hydrocarbon m e t a b o l i t e s t o DNA, s t u d i e s have a l s o been c a r r i e d o u t w i t h model compounds which have many o f t h e s t e r i c and e l e c t r o n i c p r o p e r t i e s o f c a r c i n o g e n i c epoxides b u t no r e a c t i v e e p o x i d e group. The use o f n o n r e a c t i v e model compounds p e r m i t s t h e c l e a r s e p a r a t i o n o f p h y s i c a l b i n d i n g i n t e r a c t i o n s from r e a c t i v e interactions. Benzo[a]pyrene m e t a b o l i t e model compounds which have been examined i n c l u d e 7-hydroxy-7,8,9,10-tetrahydro-BP (_4) , and c i s ( 4 ) and t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 , 9 , 1 0 - t e t r a h y d r o - B P ( 9 ) . The DMBA m e t a b o l i t e model compounds which have been examined i n c l u d e t h e b e n z [ a ] a n t h r a c e n e (BA) d e r i v a t i v e s , 1,2,3,4-tetrahydroBA ( 1 2 , L 3 ) , 5,6-dihydro-BA ( 1 2 ) , and 8,9,10,11-tetrahydro-BA ( 1 2 , 13, 1 4 ) , as w e l l as anthracene ( 1 2 ) and 9,10-dimethylanthracene (DMA) ( 1 4 ) . F i g u r e s 1 and 2 show s t r u c t u r e s o f n o n r e a c t i v e meta b o l i t e s and m e t a b o l i t e model compounds d e r i v e d from BP and DMBA f o r which DNA p h y s i c a l b i n d i n g s t u d i e s have been c a r r i e d o u t . The major g o a l s o f r e c e n t s t u d i e s o f t h e p h y s i c a l b i n d i n g t o DNA o f BP and DMBA m e t a b o l i t e s and m e t a b o l i t e models a r e t o d e t e r mine: ( 1 ) t h e magnitudes o f t h e b i n d i n g c o n s t a n t s , (2) t h e con f o r m a t i o n s o f p h y s i c a l complexes w h i c h a r e formed and t h e n a t u r e of DNA b i n d i n g s i t e s , (3) how DNA s t r u c t u r e and environment i n f l u ence p h y s i c a l b i n d i n g , ( 4 ) how t h e s t r u c t u r e o f hydrocarbon meta b o l i t e s i n f l u e n c e s p h y s i c a l b i n d i n g p r o p e r t i e s , ( 5 ) whether t h e


216


p h y s i c a l b i n d i n g of h y d r o c a r b o n m e t a b o l i t e s e x h i b i t s s p e c i f i c i t y f o r c e r t a i n base sequences i n DNA, and (6) whether d i f f e r e n t meta b o l i t e s d e r i v e d from t h e same parent hydrocarbon but w i t h v a r y i n g c a r c i n o g e n i c potency e x h i b i t d i f f e r e n t DNA p h y s i c a l b i n d i n g p r o perties.


S p e c t r o s c o p i c Probes of P h y s i c a l B i n d i n g P r o p e r t i e s Most s t u d i e s o f t h e p h y s i c a l b i n d i n g o f h y d r o c a r b o n m e t a b o l i t e s and m e t a b o l i t e model compounds have measured t h e e f f e c t o f DNA b i n d i n g on h y d r o c a r b o n f l u o r e s c e n c e intensities, fluorescence l i f e t i m e s and UV a b s o r p t i o n s p e c t r a . R a d i o a c t i v e l a b e l l i n g has a l s o been used, but l e s s f r e q u e n t l y . S p e c t r o s c o p i c methods a r e p a r t i c u l a r l y convenient. These methods, e s p e c i a l l y f l u o r e s c e n c e methods, a r e a l s o v e r y s e n s i t i v e . A l l o f t h e hydrocarbons i n F i g u r e 1 e x c e p t t h e e p o x i d e s have h i g h f l u o r e s c e n c e quantum y i e l d s , which permit r o u t i n e d e t e c t i o n i n t h e 10~^-10 M concen t r a t i o n range. W i t h s p e c t r o s c o p i c methods i t i s p o s s i b l e t o o b t a i n i n f o r m a t i o n about t h e c o n f o r m a t i o n of hydrocarbon-DNA complexes. The f l u o r e s c e n c e quantum y i e l d s o f a r o m a t i c hydrocarbons a r e g r e a t l y reduced when they b i n d t o DNA i n i n t e r c a l a t e d c o n f o r m a t i o n s . F i g u r e 3 shows how t h e i n t e n s i t y of t h e e m i s s i o n spectrum o f DMA d e c r e a s e s w i t h i n c r e a s i n g c o n c e n t r a t i o n s of DNA i n 15% methanol. ( I n F i g u r e 3 and throughout t h i s d i s c u s s i o n DNA c o n c e n t r a t i o n s and a s s o c i a t i o n c o n s t a n t s have been r e p o r t e d i n terms o f P0^~ m o l a r i t y u n l e s s o t h e r w i s e i n d i c a t e d . The s o l u t i o n content o f o r g a n i c s o l v e n t s i s g i v e n i n percent volume.) The mechanism o f t h e f l u o r e s c e n c e quenching p r o c e s s which a c companies h y d r o c a r b o n i n t e r c a l a t i o n i s n o t t h o r o u g h l y under stood. However, i n s t u d i e s o f 9-methylanthracene and phenan t h r e n e , which have p r o p e r t i e s s i m i l a r t o the m o l e c u l e s c o n s i d e r e d h e r e , i t i s found t h a t s m a l l p e r t u r b a t i o n s such as those a r i s i n g from temperature v a r i a t i o n (73) o r from d e u t e r i u m s u b s t i t u t i o n f o r hydrogen a t s p e c i f i c p o s i t i o n s (74) can s t r o n g l y a l t e r f l u o r escence quantum y i e l d s . These changes i n quantum y i e l d s a r e due almost e x c l u s i v e l y t o changes i n t h e r a t e of i n t e r s y s t e m c r o s s ing. I t i s r e a s o n a b l e t o e x p e c t t h a t quenching due t o DNA i n t e r c a l a t i o n a l s o i n v o l v e s an i n c r e a s e i n t h e r a t e o f i n t e r s y s t e m c r o s s i n g which accompanies b i n d i n g . T h i s c o n c l u s i o n i s supported by t h e o b s e r v a t i o n t h a t t h e r e i s n e a r l y a 1:1 correspondence be tween the d i s a p p e a r a n c e of s i n g l e t e x c i t e d s t a t e s and the appear ance o f t r i p l e t s i n i n t e r c a l a t e d DNA complexes formed from p o l y c y c l i c a r o m a t i c hydrocarbons ( 7 2 , 7 5 ) . I n some cases i t i s p o s s i b l e t o o b t a i n a measure of the a s s o c i a t i o n c o n s t a n t f o r i n t e r c a l a t i o n d i r e c t l y from f l u o r e s c e n c e quenching d a t a . T h i s method i s a p p l i c a b l e when t h e dynamic quenching of t h e hydrocarbon f l u o r e s c e n c e by DNA i s s m a l l and when the i n t e r c a l a t e d h y d r o c a r b o n has a n e g l i g i b l e f l u o r e s c e n c e quantum y i e l d compared t o t h a t of t h e f r e e h y d r o c a r b o n . I f these c o n d i t i o n s a r e met, t h e a s s o c i a t i o n c o n s t a n t f o r i n t e r c a l a t i o n , KQ, i s e q u a l t o the Stern-Volmer quenching c o n s t a n t Kgy (76) and i s g i v e n by E q u a t i o n 1.



9. LEBRETON


390

410 430 450 470 490

510

X (nm)

Figure

3. U n c o r r e c t e d e m i s s i o n s p e c t r a o f DMA i n 15% methanol measured a t v a r y i n g c a l f thymus DNA c o n c e n t r a t i o n s . The s p e c t r a were measured under the c o n d i t i o n s d e s c r i b ed i n r e f . 14.


217

218



K

Q

- K

g v

- [DNA ]

1

[ ^ - - 1]

(1)

I n E q u a t i o n 1, IQ i s t h e f l u o r e s c e n c e i n t e n s i t y o f t h e hydrocarbon measured w i t h o u t DNA, and I i s the i n t e n s i t y measured w i t h DNA. F i g u r e 4 shows Stern-Volmer p l o t s measured f o r t h e DNA quenching of DMA, 1,2,3,4-tetrahydro-BA, 5,6-dihydro-BA, 8,9,10,11-tetrahydro-BA and a n t h r a c e n e . F i g u r e 5 shows s i m i l a r p l o t s f o r t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o - B P and t r a n s - 4 , 5 - d i h y doxy-4,5-dihydro-BP. F i g u r e s 4 and 5 c o n t a i n d a t a o b t a i n e d b o t h w i t h n a t i v e DNA and w i t h h e a t - d e n a t u r e d DNA. Both f i g u r e s show t h a t t h e s i g n i f i c a n t f l u o r e s c e n c e quenching which i s observed i n n a t i v e DNA i s g r e a t l y d i m i n i s h e d i n denatured DNA. This strong dependence o f the s p e c t r o s c o p i c p u r t u r b a t i o n due t o b i n d i n g , on DNA secondary s t r u c t u r e i s i n d i c a t i v e o f an i n t e r c a l a t i v e b i n d i n g process (36). Measurements o f t h e h y d r o c a r b o n f l u o r e s c e n c e l i f e t i m e s p r o vide important i n f o r m a t i o n which i s u s e f u l i n i n t e r p r e t i n g the Stern-Volmer p l o t s . I n cases where E q u a t i o n 1 i s v a l i d , the hy d r o c a r b o n f l u o r e s c e n c e decay p r o f i l e s must be the same w i t h and w i t h o u t DNA. I n some c a s e s , BP f o r example, t h i s i s n o t t h e case. F o r BP t h e observed decay p r o f i l e changes s i g n i f i c a n t l y when DNA i s added ( 7 2 ) . However f o r s e v e r a l o f t h e m o l e c u l e s shown i n F i g u r e s 1 and 2, DNA has o n l y a s m a l l e f f e c t on the observed f l u o r e s c e n c e l i f e time. These m o l e c u l e s i n c l u d e t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o - B P (15,18,19), trans-4,5-dlhydroxy-4,5-dihydro-BP (15,18), BPT ( 7 , 1 8 ) , 1,2,3,4-tetrahydro-BA ( 1 2 ) , 8,9,10,11-tetrahydro-BA ( 1 4 ) , 5,6-dihydro-BA ( 1 2 ) , anthracene (12) and DMA ( 1 4 ) . T y p i c a l decay p r o f i l e s o b t a i n e d i n f l u o r e s c e n c e l i f e t i m e measurements of t r a n s 7,8-dihydroxy-7,8-dihydro-BP and o f 8,9,10,11-tetrahydro-BA a r e shown i n F i g u r e 6. The l i f e t i m e s e x t r a c t e d from t h e decay p r o f i l e s shown here have been o b t a i n e d by u s i n g a l e a s t - s q u a r e s dec o n v o l u t i o n procedure which c o r r e c t s f o r t h e f i n i t e d u r a t i o n o f the e x c i t a t i o n lamp p u l s e ( 7 7 ) . F o r 8,9,10,11-tetrahydro-BA t h e l i f e t i m e s measured w i t h and w i t h o u t DNA a r e t h e same w i t h i n e x p e r i m e n t a l e r r o r (± 2 n s e c ) . Without DNA t h e decay p r o f i l e o f t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o BP f o l l o w s a s i n g l e - e x p o n e n t i a l decay l a w . W i t h DNA the decay p r o f i l e has a s m a l l c o n t r i b u t i o n from a s h o r t - l i v e d component ( x = 5 nsec) which a r i s e s from DNA complexes. This i n d i c a t e s that E q u a t i o n 1 i s not s t r i c t l y v a l i d . However, the a n a l y s i s o f the decay p r o f i l e w i t h DNA a l s o i n d i c a t e s t h a t t h e s h o r t l i f e t i m e com ponent c o n t r i b u t e s l e s s than 11% t o t h e t o t a l e m i s s i o n observed a t [ P O a ~ ] » 5 x 10~* M. Under these c o n d i t i o n s E q u a t i o n 1 s t i l l y i e l d s a good approximate v a l u e t o t h e a s s o c i a t i o n c o n s t a n t f o r intercalation. F o r BP m e t a b o l i t e s and m e t a b o l i t e model compounds UV a b s o r p t i o n e x p e r i m e n t s p r o v i d e an independent means by which b i n d i n g c o n s t a n t s f o r hydrocarbon i n t e r c a l a t i o n i n t o DNA can be measur ed. I n t e r c a l a t i v e b i n d i n g g i v e s r i s e t o a r e d s h i f t (~ 10 nm) i n the hydrocarbon UV a b s o r p t i o n spectrum of PAH. F i g u r e 7 shows ab s o r p t i o n spectra of trans-7,8-dihydroxy-7,8-dihydro-BP at varying



9.

LEBRETON


219

[P0 ~] X I 0 M 4

F i g u r e 4.

4

Stern-Volmer p l o t s and quenching c o n s t a n t s d e r i v e d from the f l u o r e s c e n c e quenching o f DMA ( T ) , 1 , 2 , 3 , 4 - t e t r a hydro-BA ( • ) , 5,6-dihydro-BA ( A ) , 8 , 9 , 1 0 , 1 1 - t e t r a h y d r o BA (•) and anthracene (•) by DNA i n 15% methanol a t 23° C. E m i s s i o n and e x c i t a t i o n wavelengths and d e t a i l s concerning the experimental conditions are given i n r e f s , 12 and 14. The open symbols, o and V, show I ^ / I f o r 1,2,3,4-tetrahydro-BA and DMA r e s p e c t i v e l y i n dena t u r e d DNA([PO "] = 4.4 x 1 0 ~ M). 4



220


F i g u r e 5.

Stern-Volmer p l o t s and quenching c o n s t a n t s d e r i v e d from the f l u o r e s c e n c e quenching o f _trans_-7,8-dihydroxy-7,8dihydro-BP and t r a n s - 4 , 5 - d i h y d r o x y - 4 , 5 - d i h y d r o - B P i n n a t i v e DNA ( c l o s e d symbols) and i n denatured DNA (open symbols) i n 15% methanol a t 23° C. D e t a i l s about t h e e x p e r i m e n t a l c o n d i t i o n s a r e g i v e n i n r e f . 15.



9.


LEBRETON

221

CO

6

5

15 25 35 4 5 5 5 6 5

5 15 2 5 35 4 5 5 5 6 5

t (nanoseconds) F i g u r e 6. F l u o r e s c e n c e decay p r o f i l e s o f trans_-7,8-dihydroxy-7,8dihydro-BP and 8,9,10,11-tetrahydro-BA measured a t 23 °C w i t h and w i t h o u t n a t i v e DNA. Taken from r e f s . 14 and 15. The upper l e f t - h a n d c o r n e r c o n t a i n s an i n strument response p r o f i l e . E m i s s i o n and e x c i t a t i o n w a v e l e n g t h s , l i f e t i m e s , and v a l u e s o f x o b t a i n e d from d e c o n v o l u t i o n o f the l i f e t i m e d a t a are a l s o g i v e n . 2



222


350

360

370

380

390

WAVELENGTH (nm)

Figure

7. A b s o r p t i o n s p e c t r a i n 15% methanol a t 23°C o f t r a n s 7,8-dihydroxy-7,8-dihydrobenzo[5]pyrene i n n a t i v e DNA a t c o n c e n t r a t i o n s ^ o f 0.0, 8.0 x _ l O , 1.6 x 10 **, 2.4 x lo""4, 3.2 x lo"*4 and 4.0 x lo"*4 M. The broken l i n e shows a spectrum i n t h e presence o f 3.2 x 10 M DNA and 3.2 x 10 M spermine. (Reproduced w i t h p e r m i s s i o n from R e f . 15. C o p y r i g h t 1985, A l a n R. L i s s . ) 5



9. LEBRETON


223

DNA c o n c e n t r a t i o n s . The appearance of t h e r e d - s h i f t e d UV a b s o r p t i o n spectrum i s s t r o n g l y dependent upon DNA secondary s t r u c t u r e and i s g r e a t l y reduced i n denatured DNA. L i k e t h e f l u o r e s c e n c e quenching t h e r e d - s h i f t e d a b s o r p t i o n spectrum i s due t o h y d r o c a r bon i n t e r c a l a t i o n ( 1 5 , 1 8 ) . Standard methods f i r s t d e r i v e d by B e n e s i and H i l d e b r a n d (78) and l a t e r m o d i f i e d (_3, 4_, 9_, 79) have been used t o a n a l y z e t h e UV d a t a . Table I I contains a s s o c i a t i o n c o n s t a n t s o b t a i n e d from Stern-Volmer quenching d a t a a l o n g w i t h a s s o c i a t i o n c o n s t a n t s o b t a i n e d from UV b i n d i n g s t u d i e s o f BP metabo l i t e s and m e t a b o l i t e models. F o r a l l t h r e e o f t h e m o l e c u l e s con t a i n e d i n Table I I , f l u o r e s c e n c e l i f e t i m e s t u d i e s (9_, 15) i n d i c a t e t h a t decay p r o f i l e s measured w i t h and w i t h o u t DNA a r e v e r y s i m i lar. I n each case t h e f l u o r e s c e n c e s p e c t r a and t h e a b s o r p t i o n s p e c t r a were measured under i d e n t i c a l c o n d i t i o n s . A comparison o f the r e s u l t s o b t a i n e d from t h e two d i f f e r e n t methods i n d i c a t e s t h a t the agreement i n v a l u e s f o r t h e i n t e r c a l a t i o n b i n d i n g c o n s t a n t s i s good. For the DMBA m e t a b o l i t e models s t u d i e d t o date a s i m i l a r com p a r i s o n of r e s u l t s from f l u o r e s c e n c e and a b s o r p t i o n s t u d i e s has not been c a r r i e d o u t . I n these m o l e c u l e s a l l o f t h e more i n t e n s e a b s o r p t i o n bands occur a t wavelengths below 300nm where DNA ab s o r p t i o n i n t e r f e r e s . T h i s makes i t d i f f i c u l t t o determine t h e DNA b i n d i n g c o n s t a n t s u s i n g a b s o r p t i o n measurements. However t h e good agreement between t h e a b s o r p t i o n and f l u o r e s c e n c e r e s u l t s f o r t h e BP d e r i v a t i v e s supports t h e c o n c l u s i o n t h a t when t h e hydrocarbon f l u o r e s c e n c e decay p r o f i l e measured w i t h o u t DNA i s s i m i l a r t o t h a t w i t h DNA, the Stern-Volmer quenching c o n s t a n t p r o v i d e s a good mea sure o f the a s s o c i a t i o n c o n s t a n t f o r PAH i n t e r c a l a t i o n . Review Of R e s u l t s I n t e r c a l a t i o n o f BPDE. S e v e r a l groups have s t u d i e d t h e r e v e r s i b l e i n t e r c a l a t i v e b i n d i n g o f BPDE t o DNA. The f l u o r e s c e n c e quantum y i e l d o f BPDE i s much lower than t h a t o f BP d e r i v a t i v e s which do not c o n t a i n an epoxide group and f l u o r e s c e n c e t e c h n i q u e s have n o t been w i d e l y used t o study BPDE p h y s i c a l b i n d i n g t o DNA (A_). A s s o c i a t i o n c o n s t a n t s f o r the DNA i n t e r c a l a t i o n o f BPDE have been ob t a i n e d by measuring r e d s h i f t s i n t h e UV a b s o r p t i o n s p e c t r a o f BPDE which o c c u r upon t h e f o r m a t i o n of i n t e r c a l a t e d complexes (3_»A.».§.»JD and from f l u o r e s c e n c e s t u d i e s ( 8 ) o f the k i n e t i c s of DNA c a t a l y z e d h y d r o l y s i s o f BPDE. The h y d r o l y s i s r e a c t i o n I s c o n v e n i e n t l y m o n i t o r e d by f o l l o w i n g t h e f l u o r e s c e n c e o f t h e h y d r o l y s i s p r o d u c t , BPT, which has a quantum y i e l d many times g r e a t e r than BPDE. A summary o f BPDE a s s o c i a t i o n c o n s t a n t s f o r i n t e r c a l a t i o n ob t a i n e d from d i f f e r e n t s t u d i e s i s g i v e n i n Table I I I . The wide v a r i a t i o n i n r e p o r t e d a s s o c i a t i o n c o n s t a n t s can be a t t r i b u t e d i n p a r t t o d i f f e r e n c e s i n s o l v e n t c o n d i t i o n s . The low b i n d i n g c o n s t a n t o b t a i n e d i n r e f . ^_ i s due t o t h e h i g h i o n i c s t r e n g t h and h i g h c o n c e n t r a t i o n o f o r g a n i c s o l v e n t employed i n t h e e x p e r i ments. The d i f f e r e n c e i n t h e v a l u e s o f a s s o c i a t i o n c o n s t a n t s r e p o r t e d i n r e f s . 3_ and 8_ i s most l i k e l y due t o t h e d i f f e r e n c e i n the o r g a n i c content o f t h e s o l u t i o n s used.


224


Table I I . I n t e r c a l a t i o n A s s o c i a t i o n Constants and Stern-Volmer Quenching Constants f o r Benzo[a]pyrene M e t a b o l i t e s and M e t a b o l i t e Model Compounds w i t h C a l f Thymus DNA 3

KpV" )

KsV


1

trans-7,8-dihydroxy7,8-dihydro-BP trans-4,5-dlhydroxy4,5-dihydro-BP trans-7,8-dihydroxy7,8,9,10-tetrahydro-

C(!rl)

5400

6100

1900

2100

750-910

740

d

d

The e s t i m a t e d u n c e r t a i n t y i n t h e a s s o c i a t i o n c o n s t a n t s and quenching c o n s t a n t s i s ± 10%. A s s o c i a t i o n c o n s t a n t s from UV a b s o r p t i o n s t u d i e s . *T Stern-Volmer Quenching C o n s t a n t s . Measured i n 15% methanol. Taken from r e f s . 15 and 18. Measured i n 2.5% DMSO. Taken from r e f . 9. d

e

Table I I I . DNA I n t e r c a l a t i o n A s s o c i a t i o n Constants Reported f o r trans-7,8-Dihydroxy-ant i-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene Type o f DNA

Buffer (pH)

Salmon Testes (sheared) Calf Thymus (sonicated) C a l f Thymus

a,b

a , C

Additional Ions

20 mM Tris«HCl (7.4) 10 mM NaHCOo (9.0) 5 mM Sodium Cacodylate (7.1)

50 mM NaCl

Organic Solvent

T(°C) K(M"

•1

2% E t h a n o l

21

6,580

10% Acetone

25

377

0.2% T e t r a hydrofuran

25

12,000

^Based on UV a b s o r p t i o n measurements. Taken from r e f 3. R e s u l t s w i t h c a l f thymus DNA are r e p o r t e d t o be s i m i l a r t o those w i t h salmon t e s t e s DNA. T a k e n from r e f . 4. Based on UV a b s o r p t i o n s t u d i e s and on f l u o r e s c e n c e s t u d i e s of t h e k i n e t i c s o f DNA c a t a l y z e d BPDE h y d r o l y s i s . Taken from r e f . 8. c



9. LEBRETON


225

The d e r i v a t i o n of an i n t e r c a l a t i o n a s s o c i a t i o n c o n s t a n t from k i n e t i c s t u d i e s o f BPDE h y d r o l y s i s presumes t h a t t h e r e a c t i o n p r o ceeds v i a an i n t e r c a l a t e d complex. T h i s mechanism i s s u p p o r t e d by the o b s e r v a t i o n s t h a t the c a t a l y t i c a c t i v i t y o f denatured DNA i s lower than t h a t o f n a t i v e DNA ( 8 ) , t h a t c a t a l y s i s i s i n h i b i t e d a t h i g h i o n i c s t r e n g t h s (_3, 8^, 1 7 ) , and t h a t mononucleotides such as GMP e x h i b i t much g r e a t e r c a t a l y t i c a c t i v i t y than does f r e e phos phate ( 8 0 ) . I n a d d i t i o n t o UV b i n d i n g s t u d i e s o f BPDE, one o t h e r l e s s carcinogenic epoxide, (±) trans-9,10-dihydroxy-anti-7,8-epoxy7,8,9,10-tetrahydro-BP, was examined. Table IV which c o n t a i n s r e s u l t s o b t a i n e d i n t h i s study i n d i c a t e s t h a t t h e i n t e r c a l a t i o n b i n d i n g c o n s t a n t i s 23% lower than t h a t o f BPDE ( 3 ) . T a b l e IV a l s o c o n t a i n s r e s u l t s o f UV a b s o r p t i o n s t u d i e s o f h y d r o x y l a t i o n e f f e c t s on t h e DNA i n t e r c a l a t i v e b i n d i n g o f ben zo [a] pyrene m e t a b o l i t e s and m e t a b o l i t e model compounds. The most i m p o r t a n t f e a t u r e o f these r e s u l t s i s t h a t h y d r o l y s i s o f BPDE t o BPT causes a f o u r - f o l d r e d u c t i o n i n the i n t e r c a l a t i o n a s s o c i a t i o n constant. Of a l l t h e BP d e r i v a t i v e s s t u d i e d , t h e t e t r o l has t h e lowest b i n d i n g c o n s t a n t f o r i n t e r c a l a t i o n . The s m a l l b i n d i n g con s t a n t o f t h e t e t r o l compared w i t h BPDE, coupled w i t h t h e DNA c a t a l y z e d h y d r o l y s i s o f BPDE t o t h e t e t r o l may p r o v i d e a d e t o x i f i c a t i o n pathway f o r removal o f a p o r t i o n o f u n r e a c t e d i n t e r c a l a t e d BPDE. P h y s i c a l B i n d i n g o f M e t a b o l i t e s and M e t a b o l i t e Model Compounds t o Secondary S i t e s on DNAT F o r BPT a secondary DNA b i n d i n g s i t e has been r e p o r t e d on t h e b a s i s of d i a l y s i s experiments (_7). I n f l u o r escence l i f e t i m e s t u d i e s i t was found t h a t t h e f l u o r e s c e n c e decay p r o f i l e o f BPT bound a t the secondary s i t e i s very s i m i l a r t o t h a t of t h e unbound m e t a b o l i t e . T h i s suggests t h a t t h e secondary s i t e o c c u r s on t h e o u t s i d e o f DNA. I n i t i a l r e s u l t s from t h e d i a l y s i s e x p e r i m e n t s i n d i c a t e d t h a t f o r BPT t h e b i n d i n g c o n s t a n t f o r e x t e r n a l b i n d i n g i s 2 t o 4 times lower than t h a t f o r i n t e r c a l a t i o n . More r e c e n t s t u d i e s (75,81) i n d i c a t e t h a t t h e b i n d i n g c o n s t a n t f o r complex f o r m a t i o n a t secondary s i t e s i s a t l e a s t 2 times s m a l l e r than that o r i g i n a l l y reported. D i a l y s i s experiments c a r r i e d out on t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o - B P , trans-4,5-dihydroxy-4,5-dihydro-BP and pyrene i n d i c a t e t h a t f o r these m o l e c u l e s b i n d i n g a t secondary s i t e s o c c u r s w i t h b i n d i n g c o n s t a n t s which a r e about 9 times lower than those f o r i n t e r c a l a t i o n ( 1 8 ) . F o r a l l these mol e c u l e s i n t e r c a l a t i o n i s by f a r t h e most important b i n d i n g mode. Base S p e c i f i c i t y o f P h y s i c a l B i n d i n g . To determine whether t h e p h y s i c a l b i n d i n g o f hydrocarbon m e t a b o l i t e s t o DNA e x h i b i t s base s p e c i f i c i t y , the binding of trans-7,8-dihydroxy-7,8,9,10-tetrahydro-BP was examined u s i n g f l u o r e s c e n c e and a b s o r p t i o n techniques (9) . A comparison was a l s o made o f t h e v a r y i n g degrees t o which d i f f e r e n t s y n t h e t i c p o l y n u c l e o t i d e s a r e a b l e t o s o l u b i l i z e BPT (10) . Results with trans-7,8-dihydroxy-7,8,9,10-tetrahydro-BP i n d i c a t e t h a t i n 100 mM NaCl and 2.5% DMSO a t pH 7.0 t h e a s s o c i a t i o n c o n s t a n t f o r i n t e r c a l a t i o n i n t o poly(dA-dT) i s more than 5 times



226


Table IV. Comparison of R e s u l t s from UV A b s o r p t i o n S t u d i e s o f the I n t e r c a l a t i o n B i n d i n g C o n s t a n t s f o r Benzo[a]pyrene M e t a b o l i t e s and M e t a b o l i t e Model Compounds i n t o DNA Compound BPDE

K(M

a

)

6580

±trans-9,10-dihydroxy-anti-7,8-epoxy7,8,9,10-tetrahydro-BP

5080

a

7,8,9,10-tetrahydroxytetrahydro-BP ibid,

a

1650

b

44

7-hydroxy-7,8,9,10-tetrahydro-BP

b

160

trans-9,10-dihydroxy-7,8,9,10-tetrahydro-BP cis-7,8-dihydroxy-7,8,9,10-tetrahydro-BP

b

b

177 257

a

Measured w i t h sheared salmon t e s t e s DNA a t 21 °C i n 2% e t h a n o l b u f f e r e d t o a pH o f 7.4 w i t h 20 mM t r i s # H C l . Taken from r e f . 3.

b

Measured w i t h s o n i c a t e d c a l f thymus DNA i n 200 mM N a C l , 2 mM Mg and 10% acetone b u f f e r e d t o a pH o f 9.0 w i t h 10 mM NaHC0 . Taken from r e f . 4. o


9. LEBRETON


227


g r e a t e r than t h e a s s o c i a t i o n c o n s t a n t s f o r i n t e r c a l a t i o n i n t o pol y ( d G - d C ) , p o l y d G r p o l y d C , p o l y d A r p o l y d T o r c a l f thymus DNA ( 9 ) . S o l u b i l i z a t i o n s t u d i e s w i t h BPT y i e l d e d s i m i l a r r e s u l t s ( 1 0 ) . F o r BPT i n 10 mM sodium phosphate, 10 mM NaCl and 1 mM EDTA a t pH 7.0 the s o l u b i l i z i n g a c t i v i t y o f the p o l y n u c l e o t i d e s s t u d i e d i n c r e a s e s i n the order polydArpolydT * polydGrpolydC < poly(dA-dC)rpoly(dGdT) « poly(dG-dC) < p o l y ( d A - d T ) . T h i s base s p e c i f i c i t y a l s o e x h i b i t s a pH dependence. When t h e p o l y n u c l e o t i d e s a r e p r o t o n a t e d i n 10 mM sodium c i t r a t e (pH=3.8) t h e s o l u b i l i z i n g a c t i v i t y o f p o l y ( d A - d T ) , poly(dG-dC) and p o l y ( d A - d C ) r p o l y ( d G - d T ) a r e a p p r o x i mately e q u i v a l e n t . Comparison o f The P h y s i c a l B i n d i n g P r o p e r t i e s o f BP and DMBA Meta b o l i t e Model Compounds. The r e s u l t s i n F i g u r e s 4 and 5 show how s t r u c t u r a l d i f f e r e n c e s t h a t occur i n d i f f e r e n t m e t a b o l i t e s d e r i v e d from BP and DMBA i n f l u e n c e p h y s i c a l b i n d i n g t o DNA. The r e s u l t s i n d i c a t e that both trans-7,8-dihydroxy-7,8-dihydro-BP and t r a n s 4 , 5 - d i h y d r o x y - 4 , 5 - d i h y d r o - B P a r e b e t t e r i n t e r c a l a t i n g agents than the DMBA m e t a b o l i t e model compounds. W h i l e no b i n d i n g s t u d i e s o f the bay r e g i o n d i o l epoxide o f DMBA have yet been c a r r i e d o u t , t h e model compound s t u d i e s suggest t h a t BPDE i s the b e t t e r i n t e r c a l a t i n g agent. The r e s u l t s i n F i g u r e s 4 and 5 a l s o show t h a t f o r a g i v e n p a r e n t h y d r o c a r b o n , bay r e g i o n m e t a b o l i t e model compounds are b e t t e r i n t e r c a l a t i n g agents than model compounds o f l e s s c a r c i n o g e n i c me tabolites. F o r example, t h e i n t e r c a l a t i o n b i n d i n g c o n s t a n t s f o r 1,2,3,4-tetrahydro-BA and DMA are more than 4.4 times g r e a t e r than those f o r 5,6-dihydro-BA, 8,9,10,11-tetrahydro-BA and anthracene ( 1 2 , 14). The i n t e r c a l a t i o n b i n d i n g c o n s t a n t f o r t r a n s - 7 , 8 - d i h y droxy-7,8-dihydro-BP i s 2.9 times g r e a t e r than t h a t f o r t r a n s - 4 , 5 dihydroxy-4,5-dihydro-BP (15). E l e c t r o n i c I n f l u e n c e s on S t a c k i n g I n t e r a c t i o n s . The r e s u l t s o f r e c e n t s t u d i e s o f t h e i n t e r c a l a t i v e b i n d i n g of hydrocarbon metabo l i t e models t o DNA show how e l e c t r o n i c f a c t o r s i n f l u e n c e TT b i n d i n g i n t e r a c t i o n s between n u c l e o t i d e s and p o l y c y c l i c a r o m a t i c h y d r o c a r bons. I n some s t a c k e d complexes i n v o l v i n g DNA and RNA b a s e s , a s s o c i a t i o n c o n s t a n t s i n c r e a s e as t h e TT i o n i z a t i o n p o t e n t i a l s o f t h e bases decrease ( 8 2 , 8 3 ) . T h i s o c c u r s when charge t r a n s f e r o r d i s p e r s i o n f o r c e s between t h e i n t e r a c t i n g p a r t n e r s a r e important (37,82,84,). F i g u r e 8 shows how a s s o c i a t i o n c o n s t a n t s f o r TT s t a c k i n g v a r y w i t h n u c l e o s i d e i o n i z a t i o n p o t e n t i a l s i n complexes formed from the s e l f a s s o c i a t i o n o f n u c l e o s i d e s and from the b i n d ing of r i b o f l a v i n to nucleosides. I n b o t h examples t h e a s s o c i a t i o n c o n s t a n t s i n c r e a s e as t h e n u c l e o s i d e i o n i z a t i o n p o t e n t i a l s decrease. This r e l a t i o n s h i p i s u s e f u l f o r understanding the d i f f e r e n t i n t e r c a l a t i o n b i n d i n g c o n s t a n t s o f hydrocarbons w i t h s i m i l a r TT systems. T h i s i s i n d i c a t e d i n F i g u r e 4, by a comparison o f d a t a f o r 1,2,3,4-tetrahydro-BA, DMA and a n t h r a c e n e . F o r 1 , 2 , 3 , 4 - t e t r a hydro-BA the presence o f a n o n p l a n a r a l i c y c l i c group i s e x p e c t e d to s t e r i c a l l y i n h i b i t i n t e r c a l a t i o n . The n o n p l a n a r m e t h y l groups of DMA p l a y a s i m i l a r s t e r i c r o l e . However f o r b o t h 1,2,3,4-te-




228

8.0

8.5

9.0

IONIZATION POTENTIAL (eV)

8.0

8.5

9.0

IONIZATION POTENTIAL (eV)

F i g u r e 8. Dependence o f TT complex b i n d i n g c o n s t a n t s upon n u c l e o s i d e i o n i z a t i o n p o t e n t i a l s f o r (•) u r i d i n e , (») t h y m i d i n e , (A) c y t i d i n e , ( o ) adenosine, ( D ) guanosine, and (A) N,N,-dimethyladenosine. P a n e l A shows a s s o c i a t i o n constants f o r the binding of nucleosides t o r i b o f l a vin. P a n e l B shows a s s o c i a t i o n c o n s t a n t s f o r t h e s e l f a s s o c i a t i o n of n u c l e o s i d e s . (Reproduced from R e f . 82. C o p y r i g h t 1981, American Chemical S o c i e t y . )



9.

LEBRETON


229

t r a h y d r o - B A and DMA the a s s o c i a t i o n c o n s t a n t s f o r i n t e r c a l a t i o n are g r e a t e r than t h a t f o r a n t h r a c e n e . F o r 1,2,3,4-tetrahydro-BA the enhanced b i n d i n g i s accompanied by a decrease i n the i o n i z a t i o n p o t e n t i a l s o f the f i v e h i g h e s t o c c u p i e d ir o r b i t a l s compared t o c o r r e s p o n d i n g o r b i t a l s i n anthracene ( 8 5 ) . T h i s i s shown i n F i g u r e 9 which g i v e s the p h o t o e l e c t r o n spectrum o f 1 , 2 , 3 , 4 - t e t r a hydro-BA. Previous photoelectron studies of p o l y c y c l i c aromatic hydrocarbons i n d i c a t e t h a t the methyl groups p l a y a s i m i l a r r o l e i n d e s t a b i l i z i n g the m a n i f o l d o f upper o c c u p i e d TT o r b i t a l s i n DMA (85-87). For b o t h 1,2,3,4-tetrahydro-BA and DMA e l e c t r o n i c e f f e c t s which accompany the a d d i t i o n o f a l i c y c l i c groups and m e t h y l groups enhance i n t e r c a l a t i o n . These e f f e c t s are more i m p o r t a n t t h a n s t e r i c e f f e c t s , which i n h i b i t i n t e r c a l a t i o n . The I n f l u e n c e o f DNA S t r u c t u r e and Environment on the I n t e r c a l a t i o n o f Hydrocarbon M e t a b o l i t e s and M e t a b o l i t e Model Compounds. The p h y s i c a l b i n d i n g o f hydrocarbon m e t a b o l i t e s t o DNA i s v e r y s e n s i t i v e t o DNA s t r u c t u r e and environment. T h i s i s demonstrated by the d a t a i n F i g u r e s 4 and 5, which show how heat d e n a t u r a t i o n of DNA i n h i b i t s h y d r o c a r b o n quenching. These r e s u l t s are c o n s i s t e n t w i t h e a r l y s t u d i e s which i n d i c a t e t h a t the a b i l i t y o f n a t i v e DNA t o s o l u b i l i z e pyrene and BP i s much g r e a t e r than t h a t o f dena t u r e d DNA ( 4 0 ) . I n a d d i t i o n t o d e n a t u r a t i o n , hydrocarbon p h y s i c a l b i n d i n g i s s e n s i t i v e t o o t h e r DNA s t r u c t u r a l changes. Radioactive l a b e l l i n g s t u d i e s have been c a r r i e d out t o compare the b i n d i n g of t r a n s - 7 , 8 d i h y d r o x y - 7 , 8 - d i h y d r o - B P t o d o u b l e - s t r a n d e d DNA v e r s u s s i n g l e s t r a n d e d DNA ( 1 6 ) . The r e s u l t s show t h a t b i n d i n g t o b a c t e r i o p h a g e s i n g l e - s t r a n d e d x 174 DNA i s more than 4.9 times s t r o n g e r than b i n d i n g t o a r e p l i c a t i v e form d o u b l e - s t r a n d e d c i r c u l a r X DNA. The a b i l i t y of hydrocarbon m e t a b o l i t e s t o i n t e r c a l a t e i n t o DNA i s s t r o n g l y dependent on DNA environment as w e l l as on DNA s t r u c t u r e , as demonstrated i n F i g u r e 10. The top p a n e l o f F i g u r e 10 shows the e f f e c t o f methanol on the f l u o r e s c e n c e quenching o f DMA by DNA. The r e d u c t i o n o f DMA b i n d i n g a t i n c r e a s i n g methanol c o n c e n t r a t i o n i s due t o the decrease i n s o l v e n t p o l a r i t y . The bottom p a n e l o f F i g u r e 10 shows how DMA quenching i s g r e a t l y r e duced by the a d d i t i o n of the DNA s t a b i l i z e r Mg - S i m i l a r r e s u l t s have been o b t a i n e d f o r BPDE (3,4,8,11,63). I t has a l s o been shown t h a t i n c r e a s i n g i o n i c s t r e n g t h by a d d i n g NaCl i n h i b i t s i n t e r c a l a t i v e b i n d i n g o f BPDE ( 4 , 6 3 ) . Polyamine DNA s t a b i l i z e r s such as spermine have the same e f f e c t on the i n t e r c a l a t i o n o f b o t h BPDE and on t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o - B P ( 4 , 1 1 , 1 5 ) . T h i s i s i n d i c a t e d by F i g u r e 7, which shows how the i n t e n s i t y o f the r e d s h i f t e d band a r i s i n g from DNA complexes w i t h t r a n s - 7 , 8 - d i h y d r o x y 7,8-dihydro-BP i s reduced when spermine i s added. For BPDE (4,63) the r e l a t i v e e f f i c i e n c y of DNA s t a b i l i z e r s f o r i n h i b i t i n g i n t e r c a l a t i o n i n c r e a s e s i n the o r d e r NaCl < M g C l < s p e r m i n e . I t i s i m p o r t a n t t o note t h a t i n v i v o polyamine and Mg c o n c e n t r a t i o n s a r e i n the m i l l i m o l a r range ( 8 8 , 8 9 ) . I t i s expected that a t these l e v e l s they w i l l e f f i c i e n t l y p r o t e c t DNA a g a i n s t h y d r o c a r b o n i n tercalation. The dependence o f hydrocarbon i n t e r c a l a t i o n on DNA conforma2

2

+

2




230

10.10

6.0

7.0

8.0

9.0

Ionization Potential

Figure

10.0 (eV)

9. H e ( I ) p h o t o e l e c t r o n s p e c t r a o f 1,2,3,4-tetrahydro-BA. Assignments a r e g i v e n a l o n g w i t h probe t e m p e r a t u r e s . Numbers i n p a r e n t h e s e s a r e i o n i z a t i o n p o t e n t i a l s f o r c o r r e s p o n d i n g o r b i t a l s i n a n t h r a c e n e . (Reproduced w i t h p e r m i s s i o n from Ref. 12. C o p y r i g h t 1983, Academic.)



9. LEBRETON

231

5.0 r 4.0

\

CH


- \

I

3.0

~

3

\

X

i

CH

3

2.0 , 1.0 0

20

40 60 % Me OH

80

B 2.5

iCH

I

3

2.0 ^

1.5

1.0

CH

3

r-\

1.0

2.0

3.0

4.0

r = [Mg* ]/[Po ] 2

4

Figure

10. A. E f f e c t s o f v a r y i n g methanol l e v e l s on the f l u o r e s cence quenching o f 9,10-dimethylanthracene and 8,9,10,11-tetrahydro-BA by n a t i v e DNA ( [ p O i / ] - 5.0+x 10 M). B. E f f e c t s o f Mg on h y d r o c a r b o n f l u o r e s c e n c e quench i n g by n a t i v e DNA ([po ] 5.0 x io" * M) • (Reproduced w i t h p e r m i s s i o n from Ref. 14. Copyright 1984, Adenine.) 2

s

1

u



232


t i o n , on t h e p o l a r i t y of t h e DNA environment, and on t h e presence of DNA s t a b i l i z e r s i n d i c a t e s t h a t ±n_ v i v o hydrocarbon i n t e r c a l a t i o n i n t o DNA i s a p r o c e s s which can o c c u r t o d i f f e r e n t degrees as DNA f u n c t i o n changes d u r i n g t h e course of a c e l l c y c l e . I t i s i n t e r e s t i n g t h a t t r a n s - 7 , 8 - d i h y d r o x y - 7 , 8 - d i h y d r o - B P ex h i b i t s l i t t l e f l u o r e s c e n c e quenching w i t h h e a t - d e n a t u r e d c a l f t h y mus DNA b u t b i n d s s t r o n g l y t o s i n g l e - s t r a n d e d X 174 DNA. A t p r e sent t h e r e i s no i n f o r m a t i o n c o n c e r n i n g t h e nature of t h e b i n d i n g t o s i n g l e - s t r a n d e d DNA. However, n a t i v e s i n g l e - s t r a n d e d DNA i s l e s s s t e r i c a l l y crowded than d o u b l e - s t r a n d e d DNA and has secondary s t r u c t u r e t h a t i n v o l v e s r e g i o n s w i t h a s i g n i f i c a n t degree o f base stacking (90). The b i n d i n g s t u d i e s w i t h trans_-7,8-dihydroxy-7,8d i h y d r o - B P i n d i c a t e t h a t t h i s s t r u c t u r e may p r o v i d e a v e r y f a v o r able host f o r hydrocarbon metabolite i n t e r c a l a t i o n . The g r e a t e r a f f i n i t y of trans-7,8-dihydroxy-7,8-dihydro-BP t o s i n g l e - s t r a n d e d DNA than t o d o u b l e - s t r a n d e d DNA and the e f f e c t i v e i n h i b i t i o n o f h y d r o c a r b o n i n t e r c a l a t i o n by DNA s t a b i l i z e r s has l e d t o s p e c u l a t i o n t h a t h y d r o c a r b o n i n t e r c a l a t i o n i n t o d o u b l e - s t r a n d e d DNA may be most f a v o r a b l e when DNA i s p a r t i a l l y unwound (14, 1 6 ) . Such c o n f o r m a t i o n s a r e thought t o o c c u r when DNA i s u n d e r g o i n g r e p l i c a t i o n and when gene e x p r e s s i o n i s t a k i n g p l a c e ( 9 1 ) . Summary Benzo[a]pyrene and 7 , 1 2 - d i m e t h y l b e n z [ a ] a n t h r a c e n e metabolites which a r e u l t i m a t e c a r c i n o g e n s and bay r e g i o n m e t a b o l i t e model compounds which s i m u l a t e t h e IT s t r u c t u r e o f u l t i m a t e c a r c i n o g e n s are good i n t e r c a l a t i n g a g e n t s . Bay r e g i o n model compounds of BP m e t a b o l i t e s a r e b e t t e r i n t e r c a l a t i n g agents than comparable model compounds o f DMBA m e t a b o l i t e s . F o r a g i v e n p a r e n t h y d r o c a r b o n (BP or DMBA) bay r e g i o n m e t a b o l i t e model compounds which have been s t u d i e d t o date a r e b e t t e r i n t e r c a l a t i n g agents than model com pounds of l e s s c a r c i n o g e n i c m e t a b o l i t e s examined under i d e n t i c a l conditions. The i n t e r c a l a t i o n o f a r o m a t i c hydrocarbons i n t o DNA i s h i g h l y dependent upon t h e c o n f o r m a t i o n and t h e environment o f the DNA. There a r e a l a r g e number o f p o t e n t i a l l y i m p o r t a n t ways i n w h i c h p h y s i c a l b i n d i n g o f hydrocarbon m e t a b o l i t e s t o DNA can i n fluence carcinogenic a c t i v i t y . The p h y s i c a l b i n d i n g o f nonreac t i v e p r o x i m a t e c a r c i n o g e n s t o DNA may l e a d t o t h e p o o l i n g o f t h e s e molecules at u l t i m a t e t a r g e t s i t e s . Current r e s u l t s i n d i c a t e that p h y s i c a l b i n d i n g o f r e a c t i v e hydrocarbon m e t a b o l i t e s t o DNA p r e cedes r e a c t i o n and enhances m e t a b o l i t e r e a c t i v i t y . Following the f o r m a t i o n o f p h y s i c a l complexes o f r e a c t i v e m e t a b o l i t e s w i t h DNA, r e a c t i o n s o c c u r which l e a d t o epoxide h y d r o l y s i s and t o DNA modi f i c a t i o n . The base s p e c i f i c i t y and t h e s t e r e o c h e m i s t r y a s s o c i a t e d w i t h i n t e r c a l a t e d complexes may i n f l u e n c e these r e a c t i o n s . In a d d i t i o n t o i n f l u e n c i n g hydrocarbon metabolite-DNA r e a c t i o n s , the p h y s i c a l b i n d i n g p r o p e r t i e s of hydrocarbon metabolites c o v a l e n t l y bound t o DNA may a l s o be i m p o r t a n t t o c a r c i n o g e n i c a c tivity. The c o v a l e n t b i n d i n g o f u l t i m a t e c a r c i n o g e n s d e r i v e d from BP and DMBA t o DNA produces adducts w i t h IT b i n d i n g p r o p e r t i e s s i m i l a r t o those o f n a t u r a l l y o c c u r r i n g n u c l e o t i d e s . These adducts


9. LEBRETON Intercalation of Metabolite Model Compounds into DNA 233

can p a r t i c i p a t e i n TT b i n d i n g i n t e r a c t i o n s w i t h DNA, w i t h RNA and w i t h DNA r e g u l a t i n g enzymes.

other

s t r a n d s of

Acknowledgments


Support of t h i s work by t h e N a t i o n a l I n s t i t u t e s of H e a l t h , the American Cancer S o c i e t y and the R e s e a r c h Board o f the U n i v e r s i t y of I l l i n o i s i s g r a t e f u l l y acknowledged. The a u t h o r a l s o w i s h e s t o thank P r o f e s s o r s Ronald G. Harvey, N i c h o l a s G e a c i n t o v , M i c h a e l MacLeod and Nien-chu Yang f o r h e l p f u l d i s c u s s i o n s and P a t r i c i a Campbell and R e g i n a G i e r l o w s k i f o r p r e p a r a t i o n of t h i s m a n u s c r i p t .

Literature Cited 1.

Meehan, T.; Straub, K. Nature 1979, 277, 410-412.

2.

Lin, J.-H.; LeBreton, P. R.; Shipman, L. L. J. Phys. Chem. 1980, 84, 642-649.

3.

MacLeod, M. C.; Selkirk, J. K. Carcinogenesis 1982, 3, 287292.

4.

Meehan, T.; Gamper, H.; Becker, J. F. J. Biol. Chem. 1982, 257, 10479-10485.

5.

Geacintov, N. E.; Ibanez, V.; Gagliano, A. G.; Yoshida, H.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1980, 92, 13351342.

6.

Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Harvey, R. G. Bio chem. Biophys. Res. Commun. 1981, 100, 1569-1577.

7.

Ibanez, V.; Geacintov, N. E.; Gagliano, A. G.; Brandimarte, S.; Harvey, R. G. J. Am. Chem. Soc. 1980, 102, 5661-5666.

8.

Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Harvey, R. G. Bio chemistry 1982, 21, 1864-1869.

9.

Yang, N. C.; Hrinyo, T. P.; Petrich, J. W.; Yang, D.-D. H. Biochem. Biophys. Res. Commun. 1983, 114, 8-13.

10. Chen, F.-M. Carcinogenesis 1984, 5, 753-758. 11. Meehan, T.; Becker, J. F.; Gamper, H. Proc. Amer. Assoc. Can cer Res. 1981, 22, 92. 12. Shahbaz, M.; Harvey, R. G.; Prakash, A. S.; Boal, T. R.; Zegar, I. S.; LeBreton, P. R. Biochem. Biophys. Res. Commun. 1983, 112, 1-7. 13. Prakash, A. S.; Zegar I. S.; Shahbaz, M.; LeBreton, P. R. Int. J. Quant. Chem. Quantum Biology Symposium 1983, 10, 349356.


234


14. Zegar, I. S.; Prakash, A. S.; LeBreton, P. R. J. Biomol. Struct. Dynamics 1984, 2, 531-542. 15. Abramovich, M.; Zegar, I. S.; Prakash, A.S.; Harvey, R. G.; LeBreton, P. R. in "The Molecular Basis of Cancer, Part A; Macromolecular Structure Carcinogens, and Oncogenes"; Rein, R. Ed.; Alan R. Liss: New York, 1985, pp. 217-225.


16. Hsu, W.-T.; Harvey, R. G.; Weiss, S. B. Biochem. Biophys. Res. Commun. 1981, 101, 317-325. 17. Geacintov, N. E.; Hibshoosh, H.; Ibanez, V.; Benjamin, M. J.; Harvey, R. G. Biophysical Chemistry 1984, 20, 121-133. 18. Abramovich, M.; Prakash, A. S.; Harvey, R. G.; Zegar, I. S.; LeBreton, P. R. Chem.-Biol. Interactions submitted. 19. Paulius, D. E.; Prakash, A. S.; Harvey, R. G., Abramovich, M.; LeBreton, P. R. in "Proceedings of the Ninth Internation al Symposium on Polynuclear Aromatic Hydrocarbons, Polynucle ar Aromatic Hydrocarbons: Chemistry, Characterization and Carcinogenesis"; Cooke, M.; Dennis, A. G. Eds.; Battelle: Columbus, Ohio, 1985, in press. 20. Harvey, R. G. Acc. Chem. Res. 1981, 14, 218-226. 21. Remsen, J.; Jerina, D.; Yagi, H.; Cerrutti, P. Biochem. Bio phys. Res. Commun. 1977, 74, 934-940. 22. Sims, P.; Grover, P. L. in "Advances in Cancer Research"; Klein, G.; Weinhouse, S., Eds.; Academic: New York, 1974 pp. 165-274. 23. Sims, P.; Grover, P. L.; Swaisland, A.; Pal, K.; Hewer, A. Nature 1974, 252, 326-328. 24. Kootstra A.; Haas, B. L.; Slaga, T. J. Biochem. Biophys. Res. Commun. 1980, 94, 1432-1438. 25. Pelling, J. C.; Slaga, T. J. Carcinogenesis 1982, 3, 11351141. 26. Kootstra, A. Carcinogenesis 1982, 3, 953-955. 27. Ivanovic, V.; Geacintov, N. E.; Jeffrey, A. M.; Fu, P. P.; Harvey, R. G.; Weinstein, I. B. Cancer Lett. 1978, 4, 131140. 28. Weinstein, I. B.; Jeffrey, A. M. Jennette, K. W.; Blobstein, S. H.; Harvey, R. G.; Harris, C. Autrup, H.; Kasai, H.; Na kanishi, K. Science 1976, 193, 592-595. 29. Grover, P. L. in "Drug Metabolism-From Microbe to Man"; Parke, D. V.; Smith, R. L., Eds.; Taylor and Francis: London, 1977 pp. 105-122.


9. LEBRETON

235


30. Leffler, S.; Pulkrabek, P.; Grunberger, D.; Weinstein, I. B. Biochemistry 1977, 16, 3133-3136. 31. Pulkrabek, P.; Leffler, S.; Grunberger, D.; Weinstein, I. B. Biochemistry 1979, 18, 5128-5134.


32. King, H. W. S.; Osborne, M. R.; Beland, F. A.; Harvey, R. G.; Brookes, P. Proc. Natl. Acad. Sci. USA 1976, 73, 2679-2681. 33. Brookes, P.; Osborne, M. R. Carcinogenesis 1982, 3, 12231226. 34. Agarwal, K. L.; Hrinyo, T. P.; Yang, N. C. Biochem. Biophys. Res. Commun. 1983, 114, 14-19. 35. Lianos, P.; Georghiou, S. Photochem. and Photobiol. 29, 13-21.

1979,

36. LePecq, J.-B.; Paoletti, C. J. Mol. Biol. 1967 27, 87-106. 37. Ts'o, P. O. P. in "Basic Principles in Nucleic Acid Chemis try"; Ts'o, P. O. P., Ed.; Academic: New York, 1974; Vol. I, pp. 453-584. 38. Osborne, M. R.; Connell, J. R.; Venitt, S.; Crofton-Sleigh, C.; Brookes, P.; DiGiovanni, J.; Potaki, J.; Harvey, R. G. in "The Role of Chemicals and Radiation in the Etiology of Can cer"; Huberman, E., Ed.; Raven: New York, 1985, in press. 39. Boyland, E.; Green, B. Brit. J. Cancer 1962, 16, 507-517. 40. Boyland, E.; Green, B.; Liu, S.-L. Biochim. Biophys. Acta 1964, 87, 653-663. 41. Giovanella, B. C.; McKinney, L. E.; Heidelberger, C. J. Mol. Biol. 1964, 8, 20-27. 42. Yang, S. K.; Deutsch, J.; Gelboin, H. V. in "Polycyclic Hy drocarbons and Cancer"; Gelboin, H. V.; Ts'o, P. O. P., Eds.; Academic: New York, 1978; Vol. I, pp. 205-231. 43. Weinstein, I. B. J. Supramol. Struct, and Cell. Biochem. 1981, 17, 99-120. 44. Trosko, J. E.; Chang, C. C. in "Chemical Carcinogens and DNA"; Grover, P. L.; Ed.; CRC Press: Boca Raton, 1979; Vol. II, pp. 181-200. 45. Jerina, D. M.; Lehr, R. E. in "Microsomes and Drug Oxida tions"; Ullrich, V.; Roots, I.; Hildebrandt, A.; Estabrook, R. W.; Conney, A. H. Eds.; Pergamon: Oxford, 1977; pp. 709720. 46. Nordqvist, M. M.; Thakker, D. R.; Yagi H.; Lehr, R. E.; Wood, A. W.; Levin, W.; Conney, A. H.; Jerina, D. M. in "Molecular Basis of Environmental Toxicity"; Bhatnagar, R. S. Ed.; Ann Arbor Science Publishers: Ann Arbor, 1980 pp. 329-357. In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

236


47. Huberman, E.; Chou, M. W.; Yang, S. K. Proc. Natl. Acad. Sci. USA 1979, 76, 862-866. 48. Flesher, J. W.; Harvey, R. G.; Syndor, K. L. Int. J. Cancer 1976, 18, 351-353. 49. Slaga, T. J.; Gleason, G. L.; DiGiovanni, J.; Sukuraaran, K. B.; Harvey, R. G. Cancer Res. 1979, 39, 1934-1936.


50. Dipple, A.; Pigott, M.; Moschel; R. C.; Costantino, N. Cancer Res. 1983, 43, 4132-4135. 51. Vigny, P.; Kindts, M.; Cooper, C. S.; Grover P. L.; Sims, P. Carcinogenesis 1981, 2, 115-119. 52. Slaga, T. J.; Gleason, G. L.; DiGiovanni, J.; Berry, D. L.; Juchau, M. R.; Fu, P. P.; Sukumaran, K. B.; Harvey, R. G. in "Polynuclear Aromatic Hydrocarbons"; Jones, P. W.; Leber, P., Eds.; Ann Arbor Science: Ann Arbor, 1979; pp. 753-764. 53. Slaga, T. J.; Bracken, W. M.; Viaje, A.; Levin, W.; Yagi, H.; Jerina, D. M.; Conney, A. H. Cancer Res. 1977, 37, 4130-4133. 54. Kakefuda, T.; Yamamoto, H. in "Polycyclic Hydrocarbons and Cancer"; Gelboin, H.; Ts'o, P.O.P. Eds.; Academic: New York, 1978; Vol. II, 63-74. 55. Drinkwater, N. R.; Miller, E. C.; Miller, A. J. Biochemistry 1980, 19, 5087-5092. 56. Gamper, H. B.; Bartholomew, J. C.; Calvin, M. Biochemisty 1980, 19, 3948-3956. 57. Jeffrey, A. M.; Weinstein, I. B.; Jennette, K. W.; Grzeskowiak, K.; Nakanishi, K.; Harvey, R. G.; Autrup, H.; Harris, C. Nature 1977, 269, 348-350. 58. Ivanovic, V.; Geacintov, N. E.; Yamasaki, H.; Weinstein, I. B. Biochemistry 1978, 17, 1597-1603. 59. Geacintov, N. E.; Gagliano, A.; Ivanovic, V.; Weinstein, I. B. Biochemistry 1978, 17, 5256-5262. 60. Yang, N. C.; Ng, L.-K.; Neoh, S. B.; Leonov, D. Biochem. Bio phys. Res. Commun. 1978, 82, 929-934. 61. Undeman, O.; Lycksell, P.-O.; Graslund, A.; Astlind, T.; Ehrenberg, A.; Jernstro'm, B.; Tjerneld, F.; Norden, B. Cancer Res. 1983, 43, 1851-1860. 62. Prusik, T.; Geacintov, N. E. Biochem. Biophys. Res. Commun. 1979, 88, 782-790. 63. Gamper, H. B.; Straub, K.; Calvin, M.; Bartholomew, J. C. Proc. Natl. Acad. Sci. USA 1980, 77, 2000-2004.


9. LEBRETON Intercalation of Metabolite Model Compounds into DNA

237

64. Hogan, M. E. Dattagupta, N.; Whitlock, J. P. J. Biol. Chem. 1981, 256, 4504-4513. 65. Geacintov, N. E.; Gagliano, A. G.; Ibanez, V.; Harvey, R. G. Carcinogenesis 1982, 3, 247-253. 66. MacLeod, M. C.; Mansfield, B. K.; Selkirk, J. K. Carcinogene sis 1982, 3, 1031-1037.


67. Undeman, O.; Sahlin, M.; Graslund, A.; Ehrenberg, A. Biochem. Biophys. Res. Commun. 1980, 94, 458-465. 68. Geacintov, N. E.; Ibanez, V.; Gagliano, A. G.; Jacobs, S. A.; Harvey, R. G. J. Biomol. Struct. Dyanmics 1984, 1, 14731484. 69. Geacintov, N. E.; Yoshida, H.; Ibanez, V.; Jacobs, S. A.; Harvey, R. G. Biochem. Biophys. Res. Commun. 1984, 122, 3339. 70. Pezutto, J. M.; Yang, C. S.; Yang, S. K.; McCourt, D. W.; Gelboin, H. V. Cancer Res. 1978, 38, 1241-1245. 71.

Chou, M. W.; Yang, S. K.; Sydor, W.; Yang, C. S. Cancer Res. 1981, 41, 1559-1564.

72. Geacintov, N. E.; Prusik, T.; Khosrofian, J. M. J. Am. Chem. Soc. 1976, 98, 6444-6452. 73. Bennett, R. G.; McMartin, P. J. J. Chem. Phys. 1966, 44, 1969-1972. 74. O'Brien, J. J.; Henry, B. R.; Selinger, B. K. Chem. Phys. Lett. 1977, 46, 271-274. 75. Geacintov, N. E. private communication. 76. Moon, A. Y.; Poland, D. C.; Scheraga, H. A. J. Phys. Chem. 1965, 69, 2960-2966. 77. Hui, M.-H.; Ware, W. R. J. Am. Chem. Soc. 1976, 98, 47184727. 78. Benesi, H. A.; Hildebrand, J. H. J. Am. Chem. Soc. 1949, 71, 2703-2707. 79. Schmechel, D. E. V.; Crothers, D. M. Biopolymers 1971, 10, 465-480. 80. Gupta, S. C.; Pohl, T. M.; Friedman, S. L.; Whalen, D. L.; Yagi, H.; Jerina, D. M. J. Am. Chem. Soc. 1982, 104, 31013104. 81. MacLeod, M. C. private communication.


238 POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

82. Yu, C.; O'Donnell, T. J.; LeBreton, P. R. J. Phys. Chem. 1981, 85, 3851-3855. 83. Hush, N. S.; Cheung, A. S. Chem. Phys. Lett. 1975, 34, 11-13. 84. Peng, S.; Padva, A.; LeBreton, P. R.; Proc. Natl. Acad. Sci. USA 1976, 73, 2966-2968.


85. Boschi, R.; Clar, E.; Schmidt, W. J. Chem. Phys. 1974, 60, 4406-4418. 86. Akiyama, I.; Harvey, R. G.; LeBreton, P. R. J. Am. Chem. Soc. 1981, 103, 6330-6332. 87. Shahbaz, M.; Akiyama, I.; LeBreton, P. R. Biochem. Biophys. Res. Commun. 1981, 103, 25-30. 88. Cohen, S. S. in "Introduction to the Polyamines"; Prentice -Hall: Englewood Cliffs, 1971; pp. 29-63. 89. Hughes, M. N. in "The Inorganic Chemistry of Biological Pro cesses"; John Wiley: New York, 1981, p. 258. 90. Freifelder, D. in "The DNA Molecule Structure and Proper ties"; W. H. Freeman: San Francisco, 1978 pp. 268-270. 91. Lewin, B. in "Genes"; John Wiley: New York, 1983; pp. 503536. RECEIVED May 13, 1985


The Intercalation of Benzo[a]pyrene and 7,12-Dimethylbenz[a

Recommend Documents