Effects of Methyl and Fluorine Substitution on the Metabolic Activation

5

Downloaded by UNIV OF MICHIGAN ANN ARBOR on May 16, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch005

Effects of Methyl and Fluorine Substitution on the Metabolic Activation and Tumorigenicity of Polycyclic Aromatic Hydrocarbons STEPHEN S. HECHT, SHANTU AMIN, ASSIEH A. MELIKIAN, E D M O N D DIETRICH H O F F M A N N

J. LAVOIE,

and

Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595

The effects of methyl and fluorine substitution on the metabolic activation and tumorigenicity of polycyclic aromatic hydrocarbons (PAH) are reviewed. The struc tural requirements favoring tumorigenicity of methyl ated PAH are a bay region methyl group and a free peri position, both adjacent to an unsubstituted angular ring. The enhancing effect of a bay region methyl group on PAH tumorigenicity appears to be due to the relatively high reactivity with DNA and exceptional tumorigenicity of a dihydrodiol epoxide metabolite having a methyl group and epoxide ring i n the same bay region. The inhibiting effect of a peri methyl substituent on tumorigenicity can be due to either the diaxial conformation of the trans dihydrodiol at the adjacent double bond, or to inhibition of dihydrodiol formation. Substitution of fluorine i n the angular ring of PAH can prevent bay region dihydrodiol epoxide formation and can thereby diminish tumorigenicity. Fluorine substitution at the peri position adjacent to the angular ring also inhibits tumorigenicity, by mechanisms similar to those observed i n peri-methyl substituted PAH. Human exposure to complex mixtures of polycyclic aromatic hydrocar bons (PAH) occurs through inhalation of tobacco smoke and polluted indoor or outdoor a i r , through ingestion of certain foods and pollut ed water, and by dermal contact with soots, tars, and oils (1_). Methylated PAH are always components of these mixtures and i n some cases, as i n tobacco smoke and i n emissions from certain fuel proc esses, their concentrations can be i n the same range as some unsub stituted PAH. The estimated emission of methylated PAH from mobile sources i n the U.S. i n 1979 was approximately 1700 metric tons (2). The occurrence of methylated and unsubstituted PAH has been recently reviewed 0^.2 )• In addition to their environmental occurrence, methylated PAH are among the most important model compounds i n experimental carcinogenesis. 7,12-Dimethylbenz[ajanthracene, one of 0097-6156/85/0283-0085$06.00/0 © 1985 American Chemical Society

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


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POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

the most p o t e n t known PAH c a r c i n o g e n s , i s e x t e n s i v e l y used f o r the i n d u c t i o n of b r e a s t tumors i n Sprague-Dawley r a t s . 7,12-Dimethylb e n z [ a j a n t h r a c e n e i s an e x c e l l e n t example of a s t r o n g c a r c i n o g e n , formed by m e t h y l s u b s t i t u t i o n of an e s s e n t i a l l y i n a c t i v e p a r e n t com pound, b e n z [ a ] a n t h r a c e n e . Many examples a r e a v a i l a b l e of s t r i k i n g d i f f e r e n c e s i n t u m o r i g e n i c a c t i v i t y between m e t h y l s u b s t i t u t e d PAH and t h e i r p a r e n t compounds o r between d i f f e r e n t m e t h y l isomers i n the same s e r i e s ( 3 ) . I n t h i s r e v i e w , we w i l l d i s c u s s the s t r u c t u r a l f e a t u r e s t h a t f a v o r t u m o r i g e n i c i t y among th e m e t h y l a t e d PAH and w i l l c o n s i d e r the m e c h a n i s t i c b a s i s f o r these o b s e r v a t i o n s . F l u o r i n a t e d PAH have been used e x c l u s i v e l y as model compounds t o probe the mechanisms of PAH m e t a b o l i c a c t i v a t i o n . I t has g e n e r a l l y been assumed t h a t s u b s t i t u t i o n of a s m a l l , e l e c t r o n e g a t i v e f l u o r i n e atom a t a p a r t i c u l a r p o s i t i o n i n a PAH r i n g system w i l l b l o c k enzy matic oxidation at that p o s i t i o n . Thus, i f a f l u o r i n a t e d PAH were l e s s t u m o r i g e n i c than i t s p a r e n t compound, i t c o u l d be c o n c l u d e d t h a t the carbon b e a r i n g the f l u o r i n e atom was i n v o l v e d i n m e t a b o l i c a c t i v a t i o n of the p a r e n t PAH. T h i s " f l u o r i n e - p r o b e approach" has been u s e f u l and i n many cases the t u m o r i g e n i c i t y d a t a have been s u p p o r t e d by metabolism s t u d i e s . The e f f e c t s of f l u o r i n e s u b s t i t u t i o n on PAH t u m o r i g e n i c i t y and m e t a b o l i c a c t i v a t i o n w i l l be r e v i e w e d . I n some ways, the e f f e c t s of f l u o r i n e o r m e t h y l s u b s t i t u t i o n on PAH t u m o r i g e n i c i t y are s i m i l a r . S t r u c t u r a l Requirements

F a v o r i n g T u m o r i g e n i c i t y of M e t h y l a t e d

PAH

I n 1979, based on our work on m e t h y l c h r y s e n e s and on l i t e r a t u r e d a t a on o t h e r m e t h y l a t e d PAH, we suggested t h a t the s t r u c t u r a l r e q u i r e ments f a v o r i n g t u m o r i g e n i c i t y of m e t h y l a t e d PAH were a bay r e g i o n

bay

region

peri position I

angular ring bay

region

peri position

m e t h y l group and a f r e e p e r i p o s i t i o n , b o t h a d j a c e n t t o an u n s u b s t i t u t e d a n g u l a r r i n g ( 4 ) . The bay r e g i o n s , p e r i p o s i t i o n s , and a n g u l a r r i n g s of chrysene a r e i n d i c a t e d . Examples o f h i g h l y t u m o r i g e n i c m e t h y l a t e d PAH h a v i n g a bay r e g i o n m e t h y l group a d j a c e n t t o an unsub s t i t u t e d a n g u l a r r i n g a r e i l l u s t r a t e d i n F i g u r e 1. A l l of t h e s e com pounds a r e more t u m o r i g e n i c than the c o r r e s p o n d i n g u n s u b s t i t u t e d PAH. 1,4-Dimethylphenanthrene and 4,10-dimethylphenanthrene a r e more t u m o r i g e n i c t h a n s e v e r a l o t h e r dimethylphenanthrenes ( 6 ) . However, 4-methylphenanthrene, w h i c h has a bay r e g i o n m e t h y l group a d j a c e n t t o an u n s u b s t i t u t e d a n g u l a r r i n g , i s i n a c t i v e (_5). T h i s e x c e p t i o n w i l l be d i s c u s s e d f u r t h e r below. 7,12-Dimethylbenz[ajanthracene i s the most p o t e n t of the d i m e t h y l b e n z [ a j a n t h r a c e n e s ( 3 ) . 1 2 - M e t h y l b e n z [ a j a n t h r a c e n e i s more t u m o r i g e n i c t h a n b e n z [ a j a n t h r a c e n e and l - , 2 - , 3 - ,


5.

HECHT ET AL.

87

Effects of Methyl and Fluorine Substitution

CH3


8

9

1,4- DIMETHYLPHENANTHRENE

5,11-DIMETHYLCHRYSENE

II - METHYLBEN 20(o) PYRENE

7,12- DIMETHYLBENZ(a) ANTHRACENE

5-METHYLCHRYSENE (3M«C)

3 , « - DIMETHYLCHOLANTHRENE

l2-METHYLBENZ(o) ANTHRACENE

15,16- DIHY0R0 - II- METHYLCYCLO PENTA(o) PHENANTHREN-17-ONE

7,14- OIMETHYLDIBENZ (o,h) ANTHRACENE

7,14 • DIMETHYLMBENZ (§, )) ANTHRACENE

F i g u r e 1. H i g h l y t u m o r i g e n i c m e t h y l a t e d PAH h a v i n g a bay r e g i o n m e t h y l group a d j a c e n t t o a n u n s u b s t i t u t e d a n g u l a r r i n g . A l l compounds a r e more t u m o r i g e n i c than t h e i r p a r e n t PAH. W i t h t h e e x c e p t i o n o f 12m e t h y l b e n z [ a ] a n t h r a c e n e , a l l compounds shown a r e a l s o more t u m o r i genic than any o t h e r assayed methyl or dimethyl isomers. R e f e r e n c e s : 1,4-dimethylphenanthrene (5,6.); 7,12-dimethylbenz [ a j anthracene ( 3 ) ; 12-me thy l b e n z [ a j a n t h r a c e n e (3,7.); 5,11-dimethy1chrysene ( 4 ) ; 5-methylchrysene (8); 15,16-dihydro-ll-methylcyclopenta[a]phenanthren-17-one ( 9 ) ; 11-methylbenzo[a]pyrene ( 1 0 ) ; 3,6-dimethylcholanthrene (11,12); 7,14-dimethyldibenz[a,h]anthra cene ( 1 1 , 1 3 ) ; 7 , 1 4 - d i m e t h y l d i b e n z [ a , j j a n t h r a c e n e ( 1 1 ) .



88


4-,5-,9-,10-, and 1 1 - m e t h y l b e n z [ a j a n t h r a c e n e 03>Z)' However, i t i s l e s s t u m o r i g e n i c than 7 - m e t h y l b e n z [ a j a n t h r a c e n e and has a c t i v i t y s i m i l a r t o those of 6-methylbenz[ajanthracene and 8-methylbenz[aj a n t h r a c e n e 03,_7)» 5,11-Dlmethylchrysene i s the most p o t e n t d i m e t h y l chrysene t e s t e d , w i t h t u m o r i g e n i c a c t i v i t y e x c e e d i n g t h a t o f 5m e t h y l c h r y s e n e (5-MeC) ( 4 ) . 5-MeC i s more t u m o r i g e n i c than c h r y s e n e o r any o f t h e o t h e r m e t h y l c h r y s e n e s ( 8 ) . 1 5 , 1 6 - D i h y d r o - l l - m e t h y l c y c l o p e n t a [ a ] p h e n a n t h r e n e - 1 7 - o n e i s more t u m o r i g e n i c than t h e p a r e n t compound o r o t h e r raonomethyl isomers i n t h i s system ( 9 ) . 1 1 - M e t h y l b e n z o [ a ] p y r e n e i s more t u m o r i g e n i c t h a n b e n z o [ a j p y r e n e o r any o f t h e o t h e r monomethylbenzo[a]pyrenes ( 1 0 ) . 3,6-Dimethylcholanthrene i s more t u m o r i g e n i c t h a n c h o l a n t h r e n e o r 3 - m e t h y l c h o l a n t h r e n e ( 1 1 , 1 2 ) . 7,14-Dimethyldibenz[a,h]anthracene and 7,14-dimethyldibenz[a,j ] a n t h r a c e n e a r e more t u m o r i g e n i c than t h e p a r e n t h y d r o c a r b o n s o r t h e 7-methyl analogues (11,13). These r e s u l t s f i r m l y e s t a b l i s h t h e enhancing e f f e c t on t u m o r i g e n i c i t y o f a bay r e g i o n m e t h y l group adjacent to an unsubstituted angular r i n g . However, t h e p e r i p o s i t i o n a d j a c e n t t o t h e a n g u l a r r i n g must a l s o be u n s u b s t i t u t e d f o r maximum a c t i v i t y t o be o b s e r v e d . Examples of i n h i b i t i o n o f t u m o r i g e n e s i s by m e t h y l s u b s t i t u t i o n a t t h e p e r i p o s i t i o n a r e i l l u s t r a t e d i n F i g u r e 2. The c o m p a r a t i v e c a r c i n o g e n i c a c t i v i t i e s on mouse s k i n o f 5 , 1 2 - d i m e t h y l c h r y s e n e , 5 , 1 1 - d i m e t h y l c h r y sene, and 5-MeC p r o v i d e a c l e a r example, a s i l l u s t r a t e d i n F i g u r e 3. The r e q u i r e m e n t f o r a f r e e p e r i p o s i t i o n c a n a l s o be seen by t h e i n a c t i v i t y o f 9 , 1 4 - d i b e n z [ a , c ] a n t h r a c e n e , w h i c h h a s a bay r e g i o n m e t h y l group b u t no f r e e p e r i p o s i t i o n a d j a c e n t t o t h e a n g u l a r r i n g

en).

9,14-dimethyldibenz(a,c)anthracene

As d i s c u s s e d i n p r e v i o u s c h a p t e r s , t h e m e t a b o l i c a c t i v a t i o n o f m e t h y l a t e d PAH i n v o l v e s f o r m a t i o n o f d i h y d r o d i o l e p o x i d e s i n t h e a n g u l a r r i n g , a s i n t h e u n s u b s t i t u t e d PAH. T h i s p r o c e s s c a n be p a r t i a l l y i n h i b i t e d by s u b s t i t u t i o n i n t h e a n g u l a r r i n g . Metabolic s t u d i e s on 7-methylbenzo[a]pyrene have shown t h a t 7,8-dihydro-7,8d i h y d r o x y - 7 - m e t h y l b e n z o [ a ] p y r e n e i s formed, b u t t o a l e s s e r e x t e n t than i n t h e metabolism o f benzo[a]pyrene (20,21). Therefore, i t i s not s u r p r i s i n g t h a t 7 - m e t h y l b e n z o [ a j p y r e n e , as w e l l a s 8-,9-,and 10methylbenzo[a]pyrene a r e l e s s tumorigenic than benzo[a]pyrene. In the b e n z [ a j a n t h r a c e n e s e r i e s , 1 , 7 , 1 2 - t r i m e t h y l b e n z [ a j a n t h r a c e n e and 2,7,12-trimethylbenz[a]anthracene a r e l e s s a c t i v e than 7,12-dimethyl benz [ a j a n t h r a c e n e ( 2 2 ) , p r o b a b l y a s a r e s u l t o f i n h i b i t i o n o f 3,4d i h y d r o d i o l - 1 , 2 - e p o x i d e f o r m a t i o n . W h i l e t h e r e a r e many examples o f m e t h y l a t e d PAH w h i c h have t u m o r i g e n i c a c t i v i t i e s i n agreement w i t h the s t r u c t u r a l r e q u i r e m e n t s d i s c u s s e d above, t h e r e a r e a l s o some n o t a b l e e x c e p t i o n s . These i n c l u d e 6-,7-, and 8 - m e t h y l b e n z [ a j a n t h r a cene. None o f t h e s e compounds have bay r e g i o n m e t h y l groups y e t a l l



3

3

7,14-DIMETHYLDIBENZ(o,h) PYRENE

CH

5,7,12-TRIMETHYLBENZ (o) ANTHRACENE

3,11 - DIMETHYLCHOLANTHRENE

F i g u r e 2. I n h i b i t i o n o f t u m o r i g e n i c i t y by p e r i - m e t h y l s u b s t i t u t i o n . The m e t h y l a t e d PAH shown a r e l e s s t u m o r i g e n i c t h a n o t h e r m e t h y l isomers i n t h e same s e r i e s o r t h a n t h e i r p a r e n t compounds. R e f e r e n c e s : 5,12-dimethylchrysene ( 1 4 ) ; 4,9-dimethylphenanthrene ( 6 ) ; 6-methylbenzo[a]pyrene (10,15); 5 , 7 - d i m e t h y l b e n z [ a j a n t h r a c e n e ( 1 6 ) ; 5 , 7 , 1 2 - t r i m e t h y l b e n z [ a ] a n t h r a c e n e ( 1 7 ) ; 3,11-dimethy1cholanthrene (18); 5,8-dimethyldibenz[a,i]pyrene ( 1 9 ) ; 7,14-dimethyldibenz[a,h]pyrene (19).

5,8 -OIMETHYLOIBENZ(o,i) PYRENE

CH

5,7-DIMETHYLBENZ(o) ANTHRACENE


go

§



90



5.

HECHT ET AL.


91

are s t r o n g tumorigens. 7-Methylbenz[ajanthracene i s the strongest tumorigen among t h e monomethylbenz [ a j a n t h r a c e n e s ( 3 , j O . The reasons f o r t h e unique a c t i v i t y o f 7-methylbenz[a]anthracene a r e n o t known. 4-Methylphenanthrene f u l f i l l s t h e s t r u c t u r a l r e q u i r e m e n t s b u t , as i n t h e case o f t h e o t h e r monomethylphenanthracenes, i s i n a c t i v e a s a tumor i n i t i a t o r on mouse s k i n (_5). T h i s seems t o be due t o f a c i l e m e t a b o l i c d e t o x i f i c a t i o n by f o r m a t i o n o f t h e 9 , 1 0 - d i h y d r o d i o l , a p r o cess which i s b l o c k e d i n t h e t u m o r i g e n i c isomers 1,4- and 4 , 1 0 - d i methylphenanthrene 0,6). Among t h e m e t h y l a t e d benzo[clphenant h r e n e s , t h e 3-,4-,5-, and 6-methyl isomers a r e t h e most t u m o r i g e n i c . The 1-methyl i s o m e r , i n which t h e m e t h y l group i s p r e s e n t i n a 4 - s i d e d " f j o r d " , i s o n l y weakly a c t i v e l i k e t h e p a r e n t h y d r o c a r b o n (23). M e c h a n i s t i c B a s i s f o r t h e Enhancing E f f e c t on T u m o r i g e n i c i t y o f a Bay R e g i o n M e t h y l Group 5-MeC i s an e x c e l l e n t model compound f o r s t u d y i n g t h e enhancing e f f e c t on t u m o r i g e n i c i t y o f a bay r e g i o n m e t h y l group because i t has two bay r e g i o n s , one o f w h i c h has t h e m e t h y l group a d j a c e n t t o a n u n s u b s t i t u t e d a n g u l a r r i n g . B i o a s s a y s o f 5-MeC m e t a b o l i t e s f o r tumor i n i t i a t i n g a c t i v i t y on mouse s k i n demonstrated t h a t 1,-2-,3-,7-,8and 9-hydroxy-5-MeC were l e s s t u m o r i g e n i c than was 5-MeC. 5-Hydroxym e t h y l c h r y s e n e had a c t i v i t y comparable t o t h a t o f 5-MeC ( 2 4 ) . Among t h e d i h y d r o d i o l m e t a b o l i t e s , l , 2 - d i h y d r o - l , 2 - d i h y d r o x y - 5 - M e C (5-MeC1 , 2 - d i o l , F i g u r e 4 ) was more t u m o r i g e n i c than was 5-MeC whereas 5-MeC-7,8-diol was l e s s t u m o r i g e n i c t h a n 5-MeC. 5-MeC-9,10-diol was i n a c t i v e ( 2 5 ) . B o t h 5 - M e C - l , 2 - d i o l and 5-MeC-7,8-diol c o u l d form bay r e g i o n d i h y d r o d i o l e p o x i d e s and, by a n a l o g y t o u n s u b s t i t u t e d PAH, t h e s e m e t a b o l i t e s might be expected t o be u l t i m a t e c a r c i n o g e n s . However, t h e r e l a t i v e l y h i g h a c t i v i t y o f 5 - M e C - l , 2 - d i o l compared t o 5-MeC and 5-MeC-7,8-diol suggested t h a t t h e m e t h y l group i n t h e bay r e g i o n h a d a s p e c i a l e f f e c t on d i h y d r o d i o l e p o x i d e t u m o r i g e n i c i t y . T h i s was found t o be t h e case when t h e t u m o r i g e n i c a c t i v i t i e s o f anti-1,2-dihydroxy-3,4-epoxy-l,2,3,4-tetrahydro-5-MeC (anti-DE-I, F i g u r e 4 ) , syn-DE-I, and a n t i - D E - I I were compared i n newborn mice and on mouse s k i n . The r e s u l t s o f t h e newborn mouse experiment which a r e summarized i n T a b l e I c l e a r l y show t h a t a n t i - D E - I has e x c e p t i o n a l t u m o r i g e n i c a c t i v i t y and i s a major u l t i m a t e c a r c i n o g e n o f 5-MeC. A n t i - D E - I i s t h e o n l y example o f an u l t i m a t e c a r c i n o g e n o f a m e t h y l a t e d PAH t o be e s t a b l i s h e d i n a b i o a s s a y . A n t i - D E - I was a l s o more t u m o r i g e n i c than a n t i - D E - I I on mouse s k i n , b u t was l e s s a c t i v e t h a n 5-MeC ( T a b l e I I ) . These r e s u l t s demonstrate t h a t a bay r e g i o n m e t h y l group c a n enhance t h e t u m o r i g e n i c i t y of a d i h y d r o d i o l e p o x i d e metabo l i t e a s i n t h e case o f a n t i - D E - I . Thus, t h e enhancing e f f e c t o f t h e bay r e g i o n m e t h y l group on PAH t u m o r i g e n i c i t y would appear t o r e s u l t from t h e unique a c t i v i t y o f a bay r e g i o n d i h y d r o d i o l epoxide metabo l i t e , h a v i n g t h e m e t h y l group and epoxide r i n g i n t h e same bay region. Experiments on t h e m e t a b o l i c a c t i v a t i o n o f 5-MeC i n mouse s k i n are i n agreement w i t h t h e p i v o t a l r o l e o f a n t i - D E - I i n e x p r e s s i n g i t s tumorigenicity. The major DNA adduct formed i n mouse s k i n t r e a t e d w i t h [ H]5-MeC has t h e s t r u c t u r e i n d i c a t e d i n F i g u r e 5, r e s u l t i n g from a d d i t i o n o f t h e e x o c y c l i c amino group o f deoxyguanosine t o c a r 3



92


5-MeC-l,2-DI0L

F i g u r e 4. S t r u c t u r e s o f 5-MeC bay r e g i o n precursor dihydrodiols.

5-MeC-7,8-DI0L

dihydrodiol

epoxides

and t h e i r


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

100

100

100

100

100

100

5-MeC-l,2-diol

5-MeC-7,8-diol .

Anti-DE-I

Syn-DE-I

Anti-DE-II

DMSO

41 48 89

50 49 99

41 49 90

48 38 86

45 46 91

43 44 87

48 35 83

7 4 6

6 18 12

29 6 17

81 82 81

18 13 15

12 11 12

21 20 21

0.29 0.43 0.34 0.23 0.52 0.38 0.49 0.02 0.25 0.13 2.6 1.2 0.46 0.20 0.37 0.04 0.02 0.03 0.02 0.04 0.03

12 23 17 7 25 16 11 2 7 4 34 19 7 14 11 4 2 3 2 2 2

0.14 0.18 0.16 0.24 0.13 0.19 5.6 3.3 4.6 0.34 0.06 0.17 0.06 0.18 0.12 0.07 0.04 0.06

Hepatic tumors tumors/ % tumor animal bearing animals

0.25 0.26 0.25

Pulmonary tumors % tumor tumors/ bearing animals animal

Note: Ha/ICR mice were g i v e n i . p . i n j e c t i o n s o f each compound ( t o t a l dose, 56 nmol) i n DMSO on t h e 1 s t , 8 t h , and 1 5 t h days o f l i f e . Mice were weaned a t age 21 days, s e p a r a t e d by s e x , and s a c r i f i c e d a t age 35 weeks.

Female Male Total

E f f e c t i v e no of mice

100

No of mice injected

T u m o r i g e n i c i t y o f 5-MeC M e t a b o l i t e s i n Newborn Mice

5-MeC

Compound

T a b l e I.


w H >

X H

o

m

X

L/1

SO

thyl and Fluorine Substitutic

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985. 85 70 10 5 60 35 0 0 0

100 33 100 33 100 33 100 33

-

5-MeC-l,2-diol

5-MeC-7,8-diol

Anti-DE-I

Anti-DE-II

10

0 5

80 65

75 30

100 85

90 80

0

0 0

1.8 0.4

0.1 0.1

4.3 2.4

1.2 1.1

0.1

0 0.1

4.4 1.3

1.3 0.3

12.7 9.9

5.2 3.9

a

Weeks o f treatment

with

tetradecanoyl phorbol

acetate.

Note: Groups o f 20 female CD-1 mice (age 50-55 days) were shaved and t r e a t e d w i t h a s i n g l e dose o f each compound i n 0.1 ml acetone. Ten days l a t e r , each group was t r e a t e d 3 times weekly w i t h 2 .5 ug of t e t r a d e c a n o y l p h o r b o l a c e t a t e i n 0.1 ml acetone f o r 25 weeks.

Acetone

50 45

3

Tumors P e r Animal 15 weeks 25 weeks

o f 5-MeC M e t a b o l i t e s on Mouse S k i n

P e r c e n t Tumor Bearing Animals 15 w e e k s 25 weeks

100 33

Dose (nmol)

Tumor I n i t i a t i n g A c t i v i t y

5-MeC

Table I I .



5.

HECHT ET AL.


95

DE-n-dG F i g u r e 5. S t r u c t u r e s o f the major adducts formed upon r e a c t i o n o f ( A ) a n t i DE-I and ( B ) a n t i - D E - I I w i t h DNA i n v i t r o .


96


bon 4 o f a n t i - D E - I . A s t r u c t u r a l l y s i m i l a r major adduct i s formed from a n t i - D E - I I ( 2 6 ) . However, a n t i - D E - I adducts exceed a n t i - D E - I I adducts by 2-3 f o l d i n mouse s k i n , 4-48 h r a f t e r t r e a t m e n t w i t h [ % ] 5-MeC ( 2 7 ) . The predominance o f a n t i - D E - I adducts over a n t i - D E - I I adducts i n mouse s k i n i s n o t due t o d i f f e r e n c e s i n e x t e n t s o f forma t i o n of 5 - M e C - l , 2 - d i o l and 5-MeC-7,8-diol s i n c e t h e l e v e l s o f t h e s e m e t a b o l i t e s i n mouse e p i d e r m i s a r e t h e same from 0.33-4 h r a f t e r t r e a t m e n t w i t h [ H]5-MeC ( 2 7 ) . The r a t e s o f h y d r o l y s i s and b i n d i n g t o DNA o f a n t i - D E - I , syn-DEI , a n t i - D E - I I , s y n - D E - I I , and a n t i - 1 , 2 - d i h y d r o x y - 3 , 4 - e p o x y - l , 2 , 3 , 4 t e t r a h y d r o c h r y s e n e ( a n t i - c h r y s e n e - D E ) were s t u d i e d i n o r d e r t o r e l a t e the c h e m i c a l r e a c t i v i t y o f t h e s e d i h y d r o d i o l e p o x i d e s t o t h e i r b i o logical activities. The h a l f - l i v e s o f t h e d i h y d r o d i o l e p o x i d e s i n c a c o d y l a t e b u f f e r a t pH 7.0 and 37°C a r e summarized i n T a b l e I I I and t h e i r r e l a t i v e e x t e n t s o f b i n d i n g t o DNA i n T a b l e I V . I t i s c l e a r t h a t t h e r a t e s o f h y d r o l y s i s o f t h e d i h y d r o d i o l e p o x i d e s do n o t c o r r e l a t e w i t h t h e i r DNA b i n d i n g p r o p e r t i e s .


3

Table I I I . H a l f - L i v e s o f D i h y d r o d i o l Epoxides o f 5-MeC and Chrysene at pH 7.0 and 37 °C i n the Absence and Presence o f N a t i v e and Denatured C a l f Thymus DNA t^/2 Compound

Buffer Solution Only

(minutes) Denatured DNA

Native DNA

Anti-DE-I

59

24

Syn-DE-I

62

48

22

Anti-DE-II

17.5

9

2

4.9

2.8

Syn-DE-II Anti-chrysene-DE

5.4 104

77

3.5

21

When t h e h y d r o l y s e s were c a r r i e d o u t i n t h e p r e s e n c e o f denatured DNA, a r a t e enhancement o f 1.1 t o 2.5 f o l d was o b s e r v e d w h i l e i n t h e p r e s e n c e o f n a t i v e DNA t h e enhancement was 2 t o 17 f o l d ( s e e T a b l e III). The r a t i o s o f t h e r a t e s o f h y d r o l y s i s i n t h e presence o f n a t i v e DNA t o t h e r a t e s o f h y d r o l y s i s i n t h e presence o f denatured DNA d i d c o r r e l a t e w i t h t h e e x t e n t s o f b i n d i n g t o DNA as i l l u s t r a t e d i n F i g u r e 6. Our i n t e r p r e t a t i o n o f t h e s e r e s u l t s i s t h a t i n t e r c a l a t i o n o f t h e d i h y d r o d i o l e p o x i d e i n DNA precedes r e a c t i o n , as has been observed w i t h b e n z o [ a ] p y r e n e - 7 , 8 - d i h y d r o d i o l - 9 , 1 0 - e p o x i d e s (28-30). T h i s may be t h e k e y f a c t o r i n d e t e r m i n i n g e x t e n t s o f b i n d i n g o f d i h y d r o d i o l e p o x i d e s t o DNA i n v i t r o . I t i s of i n t e r e s t that the g r e a t e s t r a t e enhancement and h i g h e s t e x t e n t o f b i n d i n g were o b s e r v e d f o r a n t i - D E - I , w h i c h a l s o b i n d s t o DNA t o a g r e a t e r e x t e n t t h a n a n t i DE-II i n v i v o and i s t h e most t u m o r i g e n i c o f t h e m e t h y l a t e d bay r e g i o n d i h y d r o d i o l e p o x i d e s t e s t e d . W h i l e t h e s e r e s u l t s suggest t h a t


5.

HECHT ET AL.

97



Table IV. R e l a t i v e E x t e n t s o f B i n d i n g o f D i h y d r o d i o l Epoxides o f 5-MeC and Chrysene t o N a t i v e Calf-Thymus DNA a t pH 7.0 and 37 °C

Compound

R e l a t i v e Extents of Binding

Anti-DE-I

4.9

Syn-DE-I

2.0

Anti-DE-II

2.6

Syn-DE-II

1

Anti-chrysene-DE

2.4

e x t e n t s o f DNA b i n d i n g o f t h e d i h y d r o d i o l e p o x i d e s a r e a d e t e r m i n a n t of t u m o r i g e n i c a c t i v i t y , t h e e x c e p t i o n a l t u m o r i g e n i c i t y o f a n t i - D E - I can p r o b a b l y n o t be e x p l a i n e d on t h i s b a s i s a l o n e . The r e s u l t s o f t h e s e s t u d i e s demonstrate t h a t t h e enhancing e f f e c t o f a bay r e g i o n m e t h y l group on t u m o r i g e n i c i t y , as i n 5-MeC., i s due t o t h e e x c e p t i o n a l t u m o r i g e n i c i t y and r e l a t i v e l y h i g h r e a c t i v i t y w i t h DNA o f a d i h y d r o d i o l e p o x i d e m e t a b o l i t e , anti-DE-I, h a v i n g a m e t h y l group and a n e p o x i d e r i n g i n t h e same bay r e g i o n . Among t h e monoraethylchrysenes, o n l y 5-MeC c a n form such a metabo lite. T h i s p a r t i a l l y e x p l a i n s i t s unique a c t i v i t y . S t u d i e s on t h e metabolic a c t i v a t i o n of 15,16-dihydro-ll-methylcyclopenta[a]phenanthrene-17-one and 7 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a c e n e have shown t h a t bay r e g i o n d i h y d r o d i o l e p o x i d e s a r e l i k e l y u l t i m a t e carcinogens (31,32). I t appears l i k e l y t h a t t h e enhancing e f f e c t o f a bay r e g i o n m e t h y l group on t u m o r i g e n i c i t y i n these systems i s a l s o a r e s u l t o f the e x c e p t i o n a l t u m o r i g e n i c i t y o f t h e s e d i h y d r o d i o l e p o x i d e metabo l i t e s h a v i n g a m e t h y l group and epoxide r i n g i n t h e same bay r e g i o n . I n t h e case o f 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 , i t has been proposed t h a t t h e bay r e g i o n s y n - d i h y d r o d i o l e p o x i d e may be i m p o r t a n t i n i t s metabolic a c t i v a t i o n (33). The l o w t u m o r i g e n i c i t y o f syn-DE-I sug g e s t s , however, t h a t t h i s may n o t be t h e case and i n d i c a t e s t h e importance of s t e r i c f a c t o r s i n determining d i h y d r o d i o l epoxide t u m o r i g e n i c i t y , a s r e p o r t e d f o r u n s u b s t i t u t e d PAH. An i m p o r t a n t s t r u c t u r a l f e a t u r e o f the m e t h y l a t e d PAH w i t h a bay r e g i o n m e t h y l group i s t h e i r n o n - p l a n a r i t y . S t e r i c h i n d r a n c e between the m e t h y l group and t h e a d j a c e n t bay r e g i o n hydrogen causes d i s t o r t i o n and d e v i a t i o n from p l a n a r i t y i n 7 , 1 2 - d i m e t h y l b e n z [ a j a n t h r a cene and 5-MeC (34,35). I t has been s u g g e s t e d t h a t n o n - p l a n a r i t y may p l a y a r o l e i n t h e h i g h t u m o r i g e n i c i t y o f t h e s e compounds. I n view of t h e r e s u l t s d i s c u s s e d above, i t would appear t h a t t h i s e f f e c t would have t o o p e r a t e a t t h e l e v e l o f t h e d i h y d r o d i o l e p o x i d e metabo lites. I t would be u s e f u l t o determine t h e x - r a y c r y s t a l s t r u c t u r e s of a n t i - D E - I and a n t i - D E - I I i n o r d e r t o e s t a b l i s h whether d i f f e r e n c e s i n p l a n a r i t y o f t h e s e m e t a b o l i t e s , i f any, c o u l d c o n t r i b u t e t o t h e i r d i f f e r i n g e x t e n t s o f r e a c t i o n w i t h DNA.




98

0

1

2

3

4

5

RELATIVE E X T E N T OF BINDING O F DIHYDRODIOL EPOXIDES TO DNA

F i g u r e 6, P l o t of the r a t i o s of the h a l f - l i v e s of d i h y d r o d i o l epoxides i n the p r e s e n c e of d e n a t u r e d DNA t o t h o s e i n t h e presence o f n a t i v e DNA v s . e x t e n t s o f DNA b i n d i n g i n v i t r o .


5.

HECHT ET AL.



M e c h a n i s t i c B a s i s f o r the I n h i b i t o r y E f f e c t P e r i M e t h y l Group

99

on T u m o r i g e n i c i t y o f a

Unhindered a n g u l a r r i n g t r a n s - d i h y d r o d i o l s such as t r a n s - 7 , 8 - d i h y d r o 7,8-dihydroxybenzo[a]pyrene e x i s t p r e f e r e n t i a l l y i n the pseudo d i e q u a t o r i a l c o n f o r m a t i o n and can u s u a l l y be o x i d i z e d e n z y m a t i c a l l y t o the c o r r e s p o n d i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s . However, a p e r i m e t h y l group causes crowding and as a r e s u l t the c o n f o r m a t i o n o f a t r a n s - d i h y d r o d i o l i n the a d j a c e n t a n g u l a r r i n g w i l l be p r e f e r e n t i a l l y diaxial. T h i s phenomenon has been n o t e d by s e v e r a l groups and h a s been p r e v i o u s l y r e v i e w e d (36, 3 7 ) . I t has been suggested t h a t the i n h i b i t o r y e f f e c t o f a p e r i m e t h y l group on t u m o r i g e n i c i t y i s due t o the r e l a t i v e d i f f i c u l t y o f enzymatic c o n v e r s i o n o f d i a x i a l d i h y d r o d i o l s t o t h e i r corresponding d i h y d r o d i o l epoxides (36,37). Some experimental evidence supports t h i s suggestion although d e t a i l e d m e t a b o l i c s t u d i e s on compounds such a s 6-methylbenzo[ajpyrene, 3,11d i m e t h y l c h o l a n t h r e n e , and 5 , 7 , 1 2 - t r i m e t h y l b e n z [ a ] a n t h r a c e n e have not been r e p o r t e d . Whereas p e r i m e t h y l s u b s t i t u t i o n does n o t b l o c k d i h y d r o d i o l f o r m a t i o n i n t h e a d j a c e n t r i n g i n t h e b e n z [ a j a n t h r a c e n e system ( 3 8 , 3 9 ) , i t a p p a r e n t l y does so i n the c h r y s e n e system. 7,8-Dihydro7 , 8 - d i h y d r o x y - 5 , 1 2 - d i m e t h y l c h r y s e n e was a major m e t a b o l i t e o f 5,12d i m e t h y l c h r y s e n e i n r a t and mouse h e p a t i c 9000 x g s u p e r n a t a n t , b u t 1,2-dihydro-l,2-dihydroxy-5,12-diraethylchrysene c o u l d n o t be d e tected. S i m i l a r l y , the r a t i o o f 7-hydroxy-5,12-dimethylchrysene t o 1-hydroxy-5,12-dimethylchrysene was about 100 t o 1 i n l i v e r s u p e r n a t a n t s from 3 - m e t h y l c h o l a n t h r e n e p r e t r e a t e d mice and r a t s . In c o n t r a s t , 1 , 2 - d i h y d r o - 1 , 2 - d i h y d r o x y - 5 , 1 1 - d i m e t h y l c h r y s e n e was a major m e t a b o l i t e of 5,11-dimethylchrysene ( 4 0 ) . These r e s u l t s suggest t h a t the low t u m o r i g e n i c i t y o f 5,12-dimethylchrysene i s due t o i n h i b i t i o n of f o r m a t i o n o f i t s l i k e l y major p r o x i m a t e c a r c i n o g e n , 1,2-dihydro1,2-dihydroxy-5,12-dimethylchrysene• E f f e c t s of F l u o r i n e S u b s t i t u t i o n on the T u m o r i g e n i c i t y o f PAH T a b l e V summarizes l i t e r a t u r e on the t u m o r i g e n i c a c t i v i t i e s of f l u o r i n a t e d PAH, t e s t e d e i t h e r as tumor i n i t i a t o r s o r complete c a r c i n o g e n s on mouse s k i n . I n g e n e r a l , the r e s u l t s a r e c o n s i s t e n t w i t h the h y pothesis that f l u o r i n e s u b s t i t u t i o n could block the formation of a n g u l a r r i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s . Thus, d e c r e a s e d tumor i g e n i c i t y was o b s e r v e d upon s u b s t i t u t i o n o f f l u o r i n e i n t h e a n g u l a r r i n g s o f 7-methylbenz[ajanthracene, 7,12-dimethylbenz[ajanthracene, b e n z o [ a ] p y r e n e , d i b e n z o [ a , 1 ] p y r e n e , and d i b e n z o [ a , h ] p y r e n e . I n the case o f 5-methylchrysene and 5-hydroxymethylchrysene w h i c h each have 2 a n g u l a r r i n g s and 2 bay r e g i o n s , d e c r e a s e d t u m o r i g e n i c i t y was observed o n l y upon f l u o r i n e s u b s t i t u t i o n i n the 1-4 r i n g which i s t h e major s i t e o f m e t a b o l i c a c t i v a t i o n a s d i s c u s s e d above. Decreased t u m o r i g e n i c i t y was a l s o observed upon f l u o r i n e s u b s t i t u t i o n a t t h e p e r i - p o s i t i o n s a d j a c e n t t o the a n g u l a r r i n g s i n v o l v e d i n m e t a b o l i c a c t i v a t i o n , as seen w i t h the m e t h y l a t e d PAH. Increases i n tumorigen i c i t y were observed upon s u b s t i t u t i o n o f f l u o r i n e a t t h e 7- and 1 2 - p o s i t i o n s of b e n z [ a j a n t h r a c e n e d e r i v a t i v e s , a l t h o u g h the i n c r e a s e s were much l e s s t h a n observed upon s u b s t i t u t i o n o f m e t h y l groups a t those p o s i t i o n s . Whereas the e f f e c t s o f f l u o r i n e s u b s t i t u t i o n i n t h e a n g u l a r r i n g s and p e r i - p o s i t i o n s a r e r e a s o n a b l y w e l l u n d e r s t o o d as



100

T a b l e V.

T u m o r i g e n i c i t y o f F l u o r i n a t e d PAH on Mouse +


Benz[ajanthracene

7-methylbenz[a]anthracene

b

7,12-dimethylbenz[ajanthracene

4F (41) 5F

12F (_16)

6F (41) 9F 10F

3F (16,41-43) 4F 5F 5F (16)

11F (17,44)

6F (45)

5-hydroxymethylchrysene

a

-

7F (16) 0

5-methylchrysene

benzo[a]pyrene

NC

7F (16) 12F 7,12diF

12-methylbenz[ajanthracene

Skin

IF (17,44) 2F 5F

6F (45,46) 7F 9F 11F

IF (45,46) 3F 12F

7F (24)

3F (24)

d

6F (47-49) 7F 8F 9F 10F

dibenzo[a,i]pyrene

e

2F (50,_51) 3F 2,10-diF

dibenzo[a,h]pyrene

3,10-diF (52)

a

+; more a c t i v e

b

When t e s t e d by s.c i n j e c t i o n i n r a t s , 6F was + (41,53); 2F, 3F, 5F, 9F, 10F were (22^41-43). when t e s t e d by s . c . i n j e c t i o n i n mice, 3F, 6F were + (41_); 5F, 9F, 10F were - (41-43).

c

When t e s t e d (22,53).

d

When t e s t e d f o r i n d u c t i o n o f l u n g adenomas i n mice o r by s . c . i n j e c t i o n i n r a t s , 6F was NC C49).

e

When t e s t e d by s . c . i n j e c t i o n

t h a n p a r e n t hydrocarbon; NC., no change; -, l e s s

by s . c . i n j e c t i o n

i n rats

I F , 2F, 4F were

-

active.

and 8F, 11F were

i n mice 3F and 2,10-diF were - ( 5 4 ) .


NC

5.

HECHT ET AL.

101


d e s c r i b e d below, t h e reasons f o r t h e i n c r e a s e s i n t u m o r i g e n i c i t y upon s u b s t i t u t i o n a t t h e 7- and 1 2 - p o s i t i o n s o f b e n z [ a j a n t h r a c e n e a r e obscure.


E f f e c t s o f F l o r i n e S u b s t i t u t i o n on t h e M e t a b o l i c A c t i v a t i o n o f PAH M e t a b o l i s m s t u d i e s on f l u o r i n a t e d d e r i v a t i v e s o f 5-MeC., 5-hydroxym e t h y l c h r y s e n e , b e n z o [ a j p y r e n e and d i b e n z o [ a , i j p y r e n e have a l l shown t h a t f l u o r i n e e f f e c t i v e l y b l o c k s o x i d a t i o n o f a PAH a t t h e f o r m a l double bond t o w h i c h t h e f l u o r i n e i s a t t a c h e d (24,45,47,50). Only one example o f m e t a b o l i c l o s s o f f l u o r i n e has been r e p o r t e d ; 6f l u o r o b e n z o [ a ] p y r e n e was p a r t i a l l y c o n v e r t e d t o t h e 1,6- and 3,6quinones ( 4 8 ) . S i n c e d i h y d r o d i o l epoxide f o r m a t i o n r e q u i r e s s u c c e s s i v e o x i d a t i o n i n t h e same r i n g , a s i n g l e f l u o r i n e atom i n a n a n g u l a r r i n g w i l l i n h i b i t t h i s pathway o f m e t a b o l i c a c t i v a t i o n . Thus, t h e DNA b i n d i n g ( 5 5 ) and c a r c i n o g e n i c i t y o f t h e a n g u l a r r i n g f l u o r i n a t e d compounds a r e lower t h a n those o f t h e c o r r e s p o n d i n g hydrocarbons (24,45,47,50). The e f f e c t s o f f l u o r i n e s u b s t i t u t i o n a t t h e p e r i - p o s i t i o n s a d j a c e n t t o a n g u l a r r i n g s o f PAH a r e n o t a s s t r a i g h t f o r w a r d . I t has been shown t h a t t h e t r a n s - d i h y d r o d i o l s a d j a c e n t t o p e r i f l u o r i n e subs t i t u e n t s adopt t h e p s e u d o - d i a x i a l c o n f o r m a t i o n , a s i n t h e case o f peri-methyl substitution. Thus, NMR e x p e r i m e n t s demonstrated t h a t the 5,6- and 8 , 9 - d i h y d r o d i o l s o f 7 - f l u o r o b e n z [ a j a n t h r a c e n e and t h e 7,8-dihydrodiol of 6-fluorobenzo[a]pyrene e x i s t i n the p s e u d o - d i a x i a l c o n f o r m a t i o n ( 4 8 , 5 6 ) . I t was suggested t h a t t h e i n h i b i t o r y e f f e c t o f a p e r i - f l u o r i n e s u b s t i t u e n t on t u m o r i g e n i c i t y , as observed f o r 5F-7methylbenz[ajanthracene, 5F-12-methylbenz[ajanthracene, 5F-7,12-di methylbenz [a] a n t h r a c e n e , 6F-benzo[a]pyrene, and 12F-5-methylchrysene, might be due e i t h e r t o a low r a t e o f c o n v e r s i o n o f t h e d i a x i a l d i h y d r o d i o l s t o t h e c o r r e s p o n d i n g bay r e g i o n d i h y d r o d i o l e p o x i d e s o r t o the i n h e r e n t l y lower t u m o r i g e n i c i t y o f t h e d i h y d r o d i o l e p o x i d e metab o l i t e (56). F o r 6F-benzo[a]pyrene, t h i s e x p l a n a t i o n appears t o be c o r r e c t ( 4 8 ) . Thus, t h e 7 , 8 - d i h y d r o d i o l i s formed m e t a b o l i c a l l y a t s i m i l a r r a t e s from b o t h 6F-benzo[ajpyrene and b e n z o [ a ] p y r e n e . Both d i h y d r o d i o l s have t h e same a b s o l u t e c o n f i g u r a t i o n , b u t t h e 7,8-dihy d r o d i o l o f 6F-benzo[a]pyrene i s d i a x i a l and i s n o t a p p r e c i a b l y muta g e n i c toward C h i n e s e hamster V79 c e l l s , i n c o n t r a s t t o b e n z o [ a ] pyrene-7,8-dihydrodiol. I n c o n t r a s t , t h e f l u o r i n e atom a t t h e p e r i - p o s i t i o n o f 12F-5m e t h y l c h r y s e n e i n f l u e n c e s d i h y d r o d i o l f o r m a t i o n i n t h e a d j a c e n t angu lar ring. Whereas t h e r a t i o o f 5-MeC-7,8-diol t o 5 - M e C - l , 2 - d i o l i n mouse e p i d e r m i s was 1:1, 2 h r a f t e r t o p i c a l a p p l i c a t i o n o f [ H]5-MeC., the r a t i o o f 1 2 F - 5 - m e t h y l c h r y s e n e - 7 , 8 - d i o l t o 12F-5-methylchrysene1 , 2 - d i o l was 68:1. I n c o n t r a s t t o 5-MeC., t h e m e t a b o l i t e s formed from 12F-5-methylchrysene i n mouse s k i n r e s u l t e d a l m o s t e x c l u s i v e l y from o x i d a t i o n a t t h e 7,8-bond ( 5 7 ) . Thus, m e t a b o l i c s w i t c h i n g t o t h e l e s s t u m o r i g e n i c 7 , 8 - d i h y d r o d i o l appears t o be t h e b a s i s f o r t h e lower t u m o r i g e n i c i t y o f 12F-5-methylchrysene compared t o 5-MeC. 3

Prospects f o r F u r t h e r Research W i t h some e x c e p t i o n s , t h e r e l a t i o n s h i p between s t r u c t u r e and a c t i v i t y of v a r i o u s m e t h y l a t e d and f l u o r i n a t e d PAH i s now r e a s o n a b l y w e l l understood. Important e x c e p t i o n s a r e 6-,7-, and 8 - m e t h y l b e n z [ a ] -



102


anthracene and 7,12-dimethylbenz[ajanthracene. There i s presently no satisfactory explanation for the potent tumorigenicity of these com pounds compared to other monomethyl or dimethylbenz[a]anthracenes. Nevertheless, current knowledge of structure-tumorigenicity relation ships should allow accurate prediction of the tumorigenic activities on mouse skin of untested methylated PAH. Extension of methylated PAH testing to other bioassay systems would be desirable. L i t t l e i s known, for example, about the carcinogenicity of methylated PAH administered by inhalation or i n the diet. In addition, the carcino genic activities of mixtures of PAH should be more extensively investigated since human exposure i s always to mixtures. Whereas bay region dihydrodiol epoxides appear to be major u l t i mate carcinogens of a number of methylated PAH, the stereochemical aspects of dihydrodiol epoxide reactions with DNA and tumorigenicity require further investigation. Among the unsubstituted PAH, i t is known that the absolute configuration of dihydrodiol epoxide metabo l i t e s i s a key feature in their tumorigenic a c t i v i t i e s . Whether such stereochemical subtleties operate for the bay region dihydrodiol epoxides having a methyl group and epoxide ring i n the same bay region i s unknown, but appears likely based on the differences i n tumorigenicity between anti-DE-I and syn-DE-I. Since the general features of methylated PAH metabolic activa tion are known, i t should now be possible to design effective chemopreventive strategies. A key to this approach i s a better under standing of the a b i l i t y of the organism to detoxify dihydrodiol epox ide metabolites by conjugation with glutathione. If glutathione con jugates of methylated PAH dihydrodiol epoxides are formed, i t may be possible to enhance their rates of formation by various pretreatments. In addition, i t w i l l be important to identify naturally occurring or synthetic compounds that can prevent dihydrodiol epoxide formation or reaction with DNA i n vivo. Finally, i t i s important that further research be carried out on the identification and mechanism of action of environmental cocarcinogens and tumor promoters which can enhance the carcinogenicity of PAH. Human exposure to at least trace amounts of PAH i s unavoida ble. The probability of eventual tumor development may be controlled primarily by repeated exposure to cocarcinogens or promoters. Acknowle dgment s Our research on methylated PAH i s supported by Grant CA-32242 from The National Cancer Institute. Literature Cited 1. 2.

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