One-Electron Oxidation in Aromatic Hydrocarbon Carcinogenesis

11

Downloaded by PENNSYLVANIA STATE UNIV on May 16, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch011

One-Electron Oxidation in Aromatic Hydrocarbon Carcinogenesis ERCOLE L. CAVALIERI and ELEANOR G. ROGAN Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, NE 68105

Two main pathways are involved in the carcinogenic activation of polycyclic aromatic hydrocarbons (PAH): one-electron oxidation and monooxygenation. One-electron oxidation produces PAH radical cations, which can react with cellular nucleophiles. Biochemical and biological data indicate that only PAH with relatively low ionization potentials (below ca. 7.35 eV) can be activated by one-electron oxidation. Furthermore, a carcinogenic PAH must have a relatively high charge localization in its radical cation to react effectively with target cellular macromolecules. Binding of benzo[a]pyrene (BP) to DNA in vitro and in vivo occurs predominantly at C-6, the position of highest charge density in the BP radical cation, and binding of 6-methylBP to mouse skin DNA yields a major adduct in which the 6-methyl is bound to the 2-amino of deoxyguanosine. PAH radical cations are also involved in the metabolic conversion of PAH to PAH diones. Carcinogenicity studies of PAH in rat mammary gland indicate that only PAH with ionization potential low enough for activation by one-electron oxidation induce tumors in this target organ. These results and others indicate that one-electron oxidation of PAH is involved in their tumor initiation process. C o v a l e n t b i n d i n g o f chemical c a r c i n o g e n s t o c e l l u l a r m a c r o m o l e c u l e s , DNA, RNA and p r o t e i n , i s w e l 1 - a c c e p t e d t o be t h e f i r s t s t e p i n t h e tumor i n i t i a t i o n p r o c e s s (_1,_2). Most c a r c i n o g e n s , i n c l u d i n g p o l y c y c l i c a r o m a t i c hydrocarbons (PAH), r e q u i r e m e t a b o l i c a c t i v a t i o n t o produce t h e u l t i m a t e e l e c t r o p h i 1 i c s p e c i e s which r e a c t w i t h c e l l u l a r macromolecules. U n d e r s t a n d i n g t h e mechanisms o f a c t i v a t i o n and t h e enzymes which c a t a l y z e them i s c r i t i c a l t o e l u c i d a t i n g t h e tumor i n i t i a t i o n process. H i s t o r i c a l l y t h e p r o c e s s o f a c t i v a t i o n has almost e x c l u s i v e l y been s t u d i e d by m e t a b o l i z i n g compounds w i t h l i v e r p r e p a r a t i o n s , l e a d i n g most i n v e s t i g a t o r s i n chemical c a r c i n o g e n e s i s t o t h i n k t h a t

0097-6156/85/0283-0289S06.00/0 © 1985 American Chemical Society

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


290

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

o x y g e n a t i o n i s t h e c r i t i c a l s t e p t o produce p r o x i m a t e a n d / o r u l t i mate c a r c i n o g e n s . T h i s emphasis has i n d e e d been predominant f o r PAH, i n which f o r m a t i o n o f b a y - r e g i o n v i c i n a l d i o l e p o x i d e s has been d e s c r i b e d t o be t h e most i m p o r t a n t , i f not e x c l u s i v e , pathway o f a c t i v a t i o n (2-5.)* At p r e s e n t a v a r i e t y o f s t u d i e s w i t h PAH, as w e l l as o t h e r c h e m i c a l s , s u g g e s t t h a t m e t a b o l i c a c t i v a t i o n i n t a r g e t t i s s u e s can o c c u r by o n e - e l e c t r o n o x i d a t i o n (6^,7). The e l e c t r o p h i l i c i n t e r m e d i a t e r a d i c a l c a t i o n s g e n e r a t e d by t h T s mechanism can r e a c t d i r e c t l y with various c e l l u l a r n u c l e o p h i l e s . In t h i s p a p e r , we w i l l d i s c u s s c h e m i c a l , b i o c h e m i c a l and b i o l o g i c a l e v i d e n c e which i n d i c a t e s t h a t o n e - e l e c t r o n o x i d a t i o n p l a y s an i m p o r t a n t r o l e i n 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. Chemical

Properties of

PAH R a d i c a l

Cations

N u c l e o p h i l i c Trapping of Radical Cations. To i n v e s t i g a t e some o f the p r o p e r t i e s o f PAH r a d i c a l c a t i o n s t h e s e i n t e r m e d i a t e s have been g e n e r a t e d i n two o n e - e l e c t r o n o x i d a n t s y s t e m s . The f i r s t c o n t a i n s i o d i n e as o x i d a n t and p y r i d i n e as n u c l e o p h i l e and s o l v e n t ( 8 - 1 0 ) , w h i l e t h e second c o n t a i n s M n ( 0 A c ) i n a c e t i c a c i d ( 1 0 , 1 1 ) . Studies w i t h a number o f PAH i n d i c a t e t h a t t h e f o r m a t i o n o f p y n d i n i u m - P A H o r acetoxy-PAH by o n e - e l e c t r o n o x i d a t i o n w i t h M n ( 0 A c ) o r i o d i n e , r e s p e c t i v e l y , i s r e l a t e d t o t h e i o n i z a t i o n p o t e n t i a l (IP) o f t h e PAH. For PAH w i t h r e l a t i v e l y h i g h IP, such as p h e n a n t h r e n e , c h r y s e n e , 5 - m e t h y l c h r y s e n e and d i b e n z [ a , h ] a n t h r a c e n e , no r e a c t i o n o c c u r s w i t h t h e s e two o x i d a n t s y s t e m s . Another i m p o r t a n t f a c t o r i n f l u e n c i n g t h e s p e c i f i c r e a c t i v i t y o f PAH r a d i c a l c a t i o n s w i t h n u c l e o p h i l e s i s l o c a l i z a t i o n o f t h e p o s i t i v e c h a r g e at one o r a few carbon atoms i n t h e r a d i c a l c a t i o n . 3

3

For u n s u b s t i t u t e d PAH, such as b e n z o [ a ] p y r e n e ( B P ) , p y r i d i n i u m o r a c e t o x y d e r i v a t i v e s a r e formed by d i r e c t a t t a c k o f p y r i d i n e o r a c e t a t e i o n , r e s p e c t i v e l y , on t h e r a d i c a l c a t i o n at C - 6 , t h e p o s i t i o n o f maximum c h a r g e d e n s i t y (Scheme 1 ) . T h i s i s f o l l o w e d by a second o n e - e l e c t r o n o x i d a t i o n o f t h e r e s u l t i n g r a d i c a l and l o s s o f a proton t o y i e l d the 6 - s u b s t i t u t e d d e r i v a t i v e . For m e t h y l - s u b s t i t u t e d PAH i n which t h e maximum c h a r g e d e n s i t y o f t h e r a d i c a l c a t i o n a d j a c e n t t o t h e methyl group i s a p p r e c i a b l e , as i n 6 - m e t h y l b e n z o [ a ] pyrene (6-methylBP) (Scheme 2 ) , l o s s o f a methyl p r o t o n y i e l d s a benzylic T h i s r e a c t i v e s p e c i e s i s r a p i d l y o x i d i z e d by i o d i n e o r Mn t o a b e n z y l i c carbonium i o n w i t h subsequent t r a p p i n g by p y r i d i n e o r a c e t a t e i o n , r e s p e c t i v e l y . For a c t i v a t i o n by o n e - e l e c t r o n o x i d a t i o n , t h e s e p r o p e r t i e s o f PAH r a d i c a l c a t i o n s e n a b l e us t o p r e d i c t t h e p o s i t i o n ( s ) at which c o v a l e n t b i n d i n g o f PAH t o c e l l u l a r t a r g e t s may o c c u r .

radical.

S y n t h e s i s o f R a d i c a l C a t i o n P e r c h l o r a t e s and Subsequent C o u p l i n g with NucleophilesT Syntheses o f t h e r a d i c a l c a t i o n p e r c h l o r a t e s o f BP and 6-methylBP (12) were a c c o m p l i s h e d by t h e method r e p o r t e d e a r l i e r f o r the preparation of the perylene r a d i c a l c a t i o n (13,14). More r e c e n t l y we have a l s o s y n t h e s i z e d t h e r a d i c a l c a t i o n p e r c h l o r ate of 6-fluoroBP (15). O x i d a t i o n o f t h e PAH w i t h i o d i n e i n benzene i n t h e p r e s e n c e o f AgClO. i n s t a n t a n e o u s l y produces a b l a c k p r e c i p i t a t e c o n t a i n i n g t h e r a d i c a l c a t i o n p e r c h l o r a t e adsorbed on A g l w i t h


291

One-Electron Oxidation


11. CAVALIER I AND ROGAN

Scheme

2.

Stepwise

sequent t r a p p i n g

one-electron

oxidation

of

6-methylBP

and

by a n u c l e o p h i l e (Nu).


sub-

292


y i e . l d s _ o f 28, 28 and 39% f o r BP*C10~ 6 - m e t h y l B P » C 1 OT and 6 - f l u o r o BP'CIOT, r e s p e c t i v e l y . The BP and 8-methylBP r a d i c a l c a t i o n s have been c h a r a c t e r i z e d by e l e c t r o n s p i n resonance s p e c t r o s c o p y (12) and by t r a p p i n g w i t h s t r o n g n u c l e o p h i l e s . R e a c t i o n o f t h e BP r a d i c a l c a t i o n w i t h t h e two s t r o n g n u c l e o p h i l e s NaSCN and NaN0 y i e l d s 6 - t h i o c y a n o - and 6 - n i t r o B P , but a l s o d e r i v a t i v e s at C - i . Incident a l l y , i n t h e BP r a d i c a l c a t i o n , C-6 i s t h e p o s i t i o n o f h i g h e s t c h a r g e d e n s i t y , f o l l o w e d by C - l and C - 3 . When t h e 6-methylBP and 6 - f l u o r o B P r a d i c a l c a t i o n s r e a c t w i t h NaNOp and NaSCN, o n l y d e r i v a t i v e s at t h e 1 a n d / o r 3 - p o s i t i o n a r e o b t a i n e d . Neither s u b s t i t u t i o n at t h e 6-methyl group nor d i s p l a c e m e n t o f t h e f l u o r i n e atom i s o b served. These r e s u l t s g e n e r a l l y i n d i c a t e t h a t s t r o n g n u c l e o p h i l e s d i s p l a y low s e l e c t i v i t y toward t h e p o s i t i o n i n which the p o s i t i v e c h a r g e i s b e t t e r l o c a l i z e d . R e a c t i o n o f BP and 6 - f l u o r o B P r a d i c a l c a t i o n s w i t h t h e weak n u c l e o p h i l e H 0 a f f o r d s a m i x t u r e o f B P - 1 , 6 - , - 3 , 6 - and - 6 , 1 2 - d i o n e . These p r o d u c t s a r e t h e r e s u l t o f an i n i t i a l a t t a c k o f FLO at C - 6 .


2

?

+

BP*C107

When t h e weak n u c l e o p h i l e a c e t a t e i o n i n water i s u s e d , y i e l d s s p e c i f i c a l l y 6-acetoxyBP and t h e t h r e e d i o n e s , which a r e t h e r e s u l t o f R e a c t i o n o f t h e r a d i c a l c a t i o n w i t h FLO. In t h e c a s e o f 6-fluoroBP#C10T, BP d i o n e s a r e t h e predominant p r o d u c t s , whereas o n l y t r a c e s o f 6-acetoxyBP a r e o b t a i n e d . This i n d i c a t e s that the a t t a c k by t h e a c e t a t e i o n i s s t e r i c a l l y h i n d e r e d at t h e 6 - p o s i t i o n in the 6-fluoroBP*ClOT. The o v e r a l l c o n c l u s i o n from t h e r e a c t i o n o f BP and 6 - s u b s t i t u t e d BP r a d i c a l c a t i o n s w i t h n u c l e o p h i l e s o f v a r i o u s s t r e n g t h s i s t h a t weak n u c l e o p h i l e s d i s p l a y h i g h e r s e l e c t i v i t y toward t h e p o s i t i o n o f highest charge l o c a l i z a t i o n . Thus a n o t h e r i m p o r t a n t f a c t o r i n t h e chemical r e a c t i v i t y o f r a d i c a l c a t i o n s i s r e p r e s e n t e d by t h e strength of the n u c l e o p h i l e . Ionization Cations

Potential

of

PAH and Charge L o c a l i z a t i o n i n

Radical

From knowledge p r e s e n t l y a v a i l a b l e , t h e a b i l i t y o f PAH t o b i n d c o v a l e n t l y t o c e l l u l a r macromolecules appears t o depend m a i n l y on two factors: t h e ease o f f o r m a t i o n o f PAH r a d i c a l c a t i o n s , which i s measured by t h e i r IP, and l o c a l i z a t i o n o f p o s i t i v e c h a r g e i n t h e radical cation. The IP o f numerous PAH have been d e t e r m i n e d and compared t o a q u a l i t a t i v e measure o f t h e i r c a r c i n o g e n i c i t y ( 1 6 ) . Some o f t h e most r e p r e s e n t a t i v e PAH w i t h h i g h and low IP a r e p r e s e n t e d i n T a b l e I. Only PAH w i t h r e l a t i v e l y low IP (below c a . 7.35 eV) can be b i o l o g i c a l l y a c t i v a t e d by o n e - e l e c t r o n o x i d a t i o n ( 1 6 ) . T h i s has been o b s e r v e d i n s t u d i e s o f r a t mammary g l a n d c a r c i n o g e n e s i s ( 1 0 , 1 7 , 1 8 ) , i n which t h e r e s u l t s from d i r e c t a p p l i c a t i o n o f PAH i n d i c a t e t h a t o n l y PAH w i t h low IP i n d u c e tumors i n t h i s t a r g e t organ (see b e l o w ) . In a d d i t i o n when t h e b i n d i n g o f PAH t o DNA i s s t u d i e d u s i n g h o r s e r a d i s h p e r o x i d a s e / H 0 , a system which c a t a l y z e s o n e - e l e c t r o n o x i d a t i o n o f a v a r i e t y o f c n e m i c a l s , o n l y t h o s e PAH w i t h IP < ca 7.35 eV a r e s i g n i f i c a n t l y bound ( 1 6 ) . The c a r c i n o g e n i c i t y o f PAH w i t h r e l a t i v e l y h i g h IP, such as benzo[c]phenanthrene, benz[a]anthracene, chrysene, 5-methylchrysene and d i b e n z [ a , h ] a n t h r a c e n e ( T a b l e I ) , can be r e l a t e d t o t h e f o r m a t i o n o f b a y - r e g i o n d i o l e p o x i d e s c a t a l y z e d by monooxygenase enzymes (j>). However, t h e most p o t e n t c a r c i n o g e n i c PAH have IP < c a . 7.35 eV. 2

2


11.

CAVALIERI AND ROGAN

Table

I.

Structure,

Compound


Ionization Potential, S e l e c t e d PAH

Structure

and C a r c i n o g e n i c i t y

Ionization potential ieVj

of

8

. Carcinogenicity 1

Phenanthrene


Benzo[c]phenanthrene

Chrysene

5-Methylchrysene

Benzo[e]pyrene

Dibenz[a,h]anthracene

Benz[a]anthracene

Pyrene

Anthracene

7-Methylbenz[a]anthracene

C o n t i n u e d on next


page.


T a b l e I.


Compound

Continued. Ionization* potential (eV)

Carcinogenicity

Dibenzo[a,e]pyrene

7.35

+++

Dibenzo[a,l]pyrene

7.26

+++

D1benzo[a,i]pyrene

7.25

++ + +

Benzo[a]pyrene

7.23

++ + +

6-FluorobenzoLajpyrene

7.23

-I- +

7,12-D1methy1benz[a]anthracene

7.22

- H - + +

3-Methylcholanthrene

7.12

+ ++ +

6-Methylbenzo:a]pyrene

7.08

Peryle

+

+ ++

7.06

C o n t i n u e d on next


page.


11. CAVALIERI AND ROGAN

295


T a b l e I.

Continued. Ionization P o t e n t i a l (eV) C a r c i n o g e n i c i t y 9

Compound

Structure

Dibenzo[a.hjpyrene

6.97

+++ +

Anthanthrene

6.96

+

Determined from a b s o r p t i o n maximum o f t h e c h a r g e - t r a n s f e r complex o f each compound w i t h c h l o r a n i l , w i t h t h e e x c e p t i o n o f d i b e n z [ a , h ] a n t h r a c e n e d e t e r m i n e d by p o l a r o g r a p h i c o x i d a t i o n ( 2 4 ) . E x t r e m e l y a c t i v e , +++++; v e r y a c t i v e , ++++; a c t i v e , +++; m o d e r a t e l y a c t i v e , ++; weakly a c t i v e , +; v e r y weakly a c t i v e , +.; and i n a c t i v e , — .

b



296


T h i s l i s t i n c l u d e s BP, 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 , 3 - m e t h y l c h o l a n t h r e n e , d i b e n z o [ a , i ] p y r e n e and d i b e n z o [ a , h ] p y r e n e . These PAH can be a c t i v a t e d both by o n e - e l e c t r o n o x i d a t i o n a n d / o r monooxygenation. There a r e a few PAH w i t h low IP which a r e i n a c t i v e ( T a b l e I ) , such as p e r y l e n e , o r weakly a c t i v e , such as a n t h a n t h r e n e . This i n d i c a t e s t h a t low IP i s a n e c e s s a r y , but not s u f f i c i e n t f a c t o r f o r d e t e r m i n i n g c a r c i n o g e n i c a c t i v i t y by o n e - e l e c t r o n o x i d a t i o n . These i n a c t i v e o r weakly a c t i v e PAH have t h e h i g h e s t d e n s i t y o f p o s i t i v e charge d e l o c a l i z e d o v e r s e v e r a l a r o m a t i c carbon atoms i n t h e i r r a d i c a l c a t i o n s , whereas t h e a c t i v e PAH w i t h low IP have c h a r g e m a i n l y l o c a l i z e d on one o r a few carbon atoms i n t h e i r r a d i c a l c a t i o n s . These o b s e r v a t i o n s l e a d us t o suggest t h a t t h e second c r i t i c a l f a c t o r i n b i n d i n g o f PAH r a d i c a l c a t i o n s i s t h a t t h e c a r c i n o g e n i c PAH must have r e l a t i v e l y h i g h c h a r g e l o c a l i z a t i o n i n t h e i r r a d i c a l c a t i o n s t o g i v e them s u f f i c i e n t r e a c t i v i t y t o b i n d w i t h c e l l u l a r n u c l e o p h i l e s (6.,_7)E v i d e n c e on t h i s p o i n t has been o b t a i n e d by o n e - e l e c t r o n o x i d a t i o n o f PAH w i t h i o d i n e (8-10) and Mn(0Ac)~ ( 1 0 , 1 1 ) , a l t h o u g h t h i s concept o f charge locaTTzation r e q u i r e s f u r t h e r s t u d y by more q u a n t i t a t i v e a p p r o a c h e s . Metabolic o f BP

Formation o f Quinones by an I n i t i a l

One-Electron

Oxidation

Metabolism o f BP mediated by t h e cytochrome P-450 monooxygenase system forms t h r e e c l a s s e s o f p r o d u c t s : p h e n o l s , d i h y d r o d i o l s and quinones. Formation o f p h e n o l s and d i h y d r o d i o l s i s o b t a i n e d by an i n i t i a l e l e c t r o p h i l i c a t t a c k o f an enzyme-generated oxygen atom. The same pathway o f a c t i v a t i o n has been p o s t u l a t e d i n t h e f o r m a t i o n o f q u i n o n e s , a l t h o u g h t h e p u t a t i v e 6-hydroxyBP p r e c u r s o r has never been i s o l a t e d ( 1 9 , 2 0 ) . In t h i s mechanism, f o r m a t i o n o f quinones would proceed by a u t o x i d a t i o n o f 6-hydroxyBP ( 2 0 ) . However, s u b s t a n t i a l evidence i n d i c a t e s that the f i r s t step in formation of quinones does not i n v o l v e t h e t y p i c a l a t t a c k o f t h e e l e c t r o p h i l i c a c t i v e oxygen t o y i e l d 6-hydroxyBP, but i n s t e a d c o n s i s t s o f t h e l o s s o f one e l e c t r o n from BP t o produce t h e r a d i c a l c a t i o n . The f i r s t l i n e o f e v i d e n c e d e r i v e s from t h e predominant f o r m a t i o n o f quinones when metabolism o f BP i s conducted under p e r o x i d a s e c o n d i t i o n s , namely by p r o s t a g l a n d i n H s y n t h a s e (21) or by cytochrome P-450 w i t h cumene h y d r o p e r o x i d e as c o f a c t o r ~ T 2 2 ) • Under these metabolic c o n d i t i o n s o n e - e l e c t r o n o x i d a t i o n i s the prepond e r a n t mechanism o f a c t i v a t i o n . Second, metabolism o f 6 - f l u o r o B P by r a t l i v e r microsomes y i e l d s the same BP quinones o b t a i n e d i n t h e metabolism o f BP ( 2 3 ) . T h i s s u g g e s t s t h a t t h e s e p r o d u c t s a r e formed by an i n i t i a l a t t a c k o f a n u c l e o p h i l i c oxygen atom at C-6 i n t h e 6 - f l u o r o B P r a d i c a l c a t i o n w i t h d i s p l a c e m e n t o f t h e f l u o r o atom. In f a c t , when 6 - f l u o r o B P i s t r e a t e d w i t h t h e o n e - e l e c t r o n o x i d a n t M n ( 0 A c ) , t h e major p r o d u c t s o b t a i n e d are 6-acetoxyBP and a m i x t u r e o f 1,6- and 3 , 6 - d i a c e t o x y B P ( 1 5 ) , i n d i c a t i n g t h a t r e a c t i o n o c c u r s v i a an i n i t i a l a t t a c k o f a c e t a t e i o n at C-6 o f t h e 6 - f l u o r o B P r a d i c a l c a t i o n . On t h e o t h e r hand e l e c t r o p h i l i c s u b s t i t u t i o n o f 6 - f l u o r o B P w i t h bromine o r d e u t e r i u m i o n shows no d i s p l a c e m e n t o f f l u o r i n e at C - 6 , a l t h o u g h i n both c a s e s s u b s t i t u t i o n o c c u r s at C - l a n d / o r C - 3 . These r e s u l t s i n d i c a t e t h a t 3


11.

CAVALIERI AND ROGAN

297


t h e o n l y p l a u s i b l e c h e m i s t r y i n t h e m e t a b o l i c f o r m a t i o n o f quinones from 6 - f l u o r o B P i s c o n s i s t e n t w i t h a n i n i t i a l o n e - e l e c t r o n o x i d a t i o n o f t h e compound t o form 6 - f l u o r o B P » . F i n a l l y , we have s t u d i e d t h e metabolism o f a s e r i e s o f PAH w i t h d e c r e a s i n g IP. In t h e s e m e t a b o l i c s t u d i e s w i t h A r o c l o r - i n d u c e d r a t l i v e r microsomes, t h e f o r m a t i o n o f quinones was measured i n t h e p r e sence o f NADPH o r cumene h y d r o p e r o x i d e as c o f a c t o r . As p r e s e n t e d i n T a b l e II, no quinones a r e o b t a i n e d w i t h NADPH f o r d i b e n z [ a , h ] a n t h r a c e n e and b e n z [ a ] a n t h r a c e n e , whereas w i t h cumene h y d r o p e r o x i d e a t r a c e amount o f b e n z [ a ] a n t h r a c e n e quinone i s o b served. For t h e PAH w i t h low IP, quinones a r e formed i n t h e p r e sence o f both c o f a c t o r s . The r e l a t i o n s h i p between IP and f o r m a t i o n o f quinones c o n s t i t u t e s f u r t h e r e v i d e n c e t h a t t h e s e m e t a b o l i t e s a r e o b t a i n e d by an i n i t i a l o n e - e l e c t r o n o x i d a t i o n o f t h e PAH w i t h f o r m a tion of i t s radical cation.


+

Table

II.

M e t a b o l i c Formation o f Quinones Various I o n i z a t i o n P o t e n t i a l s

Ionization Potential

(eV)*

Compound

f o r PAH o f

Formation o f Quinone by A r o c l o r - i n d u c e d Rat L i v e r Microsomes w i t h Cumene Hydroperoxide NADPH

Dibenz[a,h]anthracene

7.57

-

Benz[a]anthracene

7.54

-

Benzo[a]pyrene

7.23

+

+

Dibenzo[a,i]pyrene

7.20

+

+

Dibenzo[a,h]pyrene

6.97

+

+

Anthanthrene

6.96

+

+

-

Determined from a b s o r p t i o n maximum o f t h e c h a r g e - t r a n s f e r complex o f each compound w i t h c h l o r a n i l , w i t h t h e e x c e p t i o n o f d i b e n z [ a . h ) ] a n t h r a c e n e . which was d e t e r m i n e d by p o l a r o graphic oxidation (24). +_ i n d i c a t e s

f o r m a t i o n o f a t r a c e amount o f q u i n o n e .

We propose t h a t t h e f i r s t s t e p i n t h e f o r m a t i o n o f q u i n o n e s , as shown i n Scheme 3 f o r BP, i n v o l v e s an e l e c t r o n t r a n s f e r from t h e hydrocarbon t o t h e a c t i v a t e d cytochrome P - 4 5 0 - i r o n - o x y g e n complex. The g e n e r a t e ^ n u c l e o p h i l i c oxygen atom o f t h i s complex would r e a c t at C-6 o f BP* i n which t h e p o s i t i v e c h a r g e i s a p p r e c i a b l y l o c a l i z e d . The 6-oxy-BP r a d i c a l formed would then d i s s o c i a t e t o l e a v e t h e i r o n o f cytochrome P-450 i n t h e normal f e r r i c s t a t e . Autoxidation of the 6-oxy-BP r a d i c a l i n which t h e s p i n d e n s i t y i s l o c a l i z e d m a i n l y on t h e o x y g e n , C - l , C-3 and C-12 U 9 , 2 0 ) would produce t h e t h r e e BP diones.




298


CAVALIERI AND ROGAN



11.


299

300


Binding

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS of

PAH t o

DNA i n v i t r o and i n

vivo

While most r e s e a r c h on t h e e n z y m a t i c a c t i v a t i o n o f c h e m i c a l c a r c i n o gens has f o c u s e d on monooxygenation by cytochrome P-450, i t has become i n c r e a s i n g l y c l e a r t h a t a c t i v a t i o n by c e l l u l a r p e r o x i d a s e s , i n c l u d i n g t h e p r o s t a g l a n d i n H s y n t h a s e c o m p l e x , p l a y s an important r o l e i n t h e a c t i v a t i o n o f many c a r c i n o g e n s ( 2 5 ) . The model h o r s e r a d i s h peroxidase/HgOp system has been found t o m e t a b o l i z e N-hyd r o x y - 2 - a c e t y l a m i n o f l u o r e n e ( 2 6 , 2 7 ) , d i e t h y l s t i l b e s t r o l ( 2 8 ) , phenol ( 2 9 ) , a m i n o p y r i n e ( 3 0 ) , benzicTTne and d e r i v a t i v e s (3U, ^ 2 7 7 t e t r a methyl h y d r a z i n e (33T"~and BP (34) by o n e - e l e c t r o n o x i d a t i o n . Mamm a l i a n p e r o x i d a s e s a l s o f o l l o w t h i s mechanism: f o r example, mouse u t e r i n e p e r o x i d a s e and r a t bone marrow p e r o x i d a s e w i t h d i e t h y l s t i l b e s t r o l (28) and phenol ( 2 9 ) , r e s p e c t i v e l y . Furthermore p r o s t a g l a n d i n H s y n t h a s e has been proposed t o a c t i v a t e b e n z i d i n e i n k i d n e y c a r c i n o g e n e s i s (35, 3 6 ) , N - h y d r o x y - 2 - a c e t y l a m i n o f l u o r e n e i n mammary c e l l s ( 3 7 ) , t e t r a m e t h y l h y d r a z i n e (38) and d i e t h y l s t i l b e s t r o l (39), a p p a r e n t l y by o n e - e l e c t r o n o x i d a t i o n . Both h o r s e r a d i s h p e r o x i d a s e and p r o s t a g l a n d i n H s y n t h a s e e f f i c i e n t l y c a t a l y z e t h e b i n d i n g o f BP t o DNA in_ v i t r o , y i e l d i n g 89 +^ 5 and 310 + 64 y m o l e BP bound/mole DNA-P, r e s p e c t i v e l y . Horseradish p e r o x i d a s e has a l r e a d y been seen t o b i n d o t h e r PAH w i t h r e l a t i v e l y low IP t o DNA ( 1 6 ) . For both BP (34) and 6-methyl BP ( 4 0 ) , we have o b t a i n e d c l e a r e v i d e n c e c o n f i r m i n g o n e - e l e c t r o n o x i d a t i o n as t h e mechanism o f a c t i v a t i o n . In t h e c a s e o f 6-methylBP we have i d e n t i f i e d a DNA adduct i n which t h e 6-methyl group i s c o v a l e n t l y bound t o the 2-amino group o f deoxyguanosine ( 4 0 ) . T h i s DNA adduct i s a l s o p r e s e n t i n mouse s k i n t r e a t e d w i t h racTTolabeled 6-methyl BP, p r o v i d i n g t h e f i r s t e v i d e n c e f o r a c t i v a t i o n o f a PAH i n a t a r g e t t i s s u e by o n e - e l e c t r o n o x i d a t i o n ( 4 0 ) . We have begun t o examine BP-DNA adducts formed i n mouse s k i n u s i n g h i g h p r e s s u r e l i q u i d c h r o m a t o g r a phy a f t e r enzymic d i g e s t i o n o f t h e p u r i f i e d DNA t o m o n o n u c l e o s i d e s . In a d d i t i o n t o BP d i o l e p o x i d e a d d u c t ( s ) , we o b s e r v e an adduct p r o f i l e which i s q u a l i t i a t i v e l y s i m i l a r t o t h e adduct p r o f i l e s o b t a i n e d from DNA w i t h BP bound by i n c u b a t i o n w i t h h o r s e r a d i s h p e r o x i d a s e / H p 0 and from BP r a d i c a l c a t i o n bound t o d e o x y g u a n o s i n e . We are c u r r e n t l y i d e n t i f y i n g t h e s t r u c t u r e o f t h e common adducts o b t a i n e d on t h e ^ k i n , w i t h h o r s e r a d i s h p e r o x i d a s e a c t i v a t i o n and by r e a c t i o n o f BP w i t h d e o x y g u a n o s i n e . I d e n t i f i c a t i o n o f DNA adducts formed by o n e - e l e c t r o n o x i d a t i o n can p r o v i d e e v i d e n c e t h a t t h i s mechanism o f a c t i v a t i o n i s o p e r a t i v e i n t a r g e t t i s s u e s , a l t h o u g h t h i s does not prove t h a t i t i s r e s p o n s i b l e f o r i n i t i a t i n g t h e tumor process. 2

Carcinogenicity

Studies

i n Two T a r g e t

Organs

The c a r c i n o g e n i c i t y o f a s e r i e s o f PAH i n t h e mammary g l a n d has been examined i n 5 0 - d a y - o l d f e m a l e Sprague-Dawley r a t s u s i n g d i r e c t a p p l i c a t i o n o f t h e compound t o t h e mammary t i s s u e ( 1 0 , 17, 1 8 ) . The r e s u l t s o f t h e s e e x p e r i m e n t s , p r e s e n t e d i n T a b l e III, a r e compared t o t h e c a r c i n o g e n i c i t y r e s u l t s i n mouse s k i n from r e p e a t e d a p p l i c a t i o n o b t a i n e d i n our l a b o r a t o r y and o t h e r s . PAH were s e l e c t e d b e cause t h e y were o r were not e x p e c t e d t o be a c t i v a t e d by o n e - e l e c t r o n o x i d a t i o n , based on t h e h y p o t h e s i s t h a t compounds w i t h r e l a t i v e l y h i g h IP cannot be a c t i v a t e d by t h i s mechanism. F u r t h e r m o r e , some


11.

Table

III.

Comparative

C a r c i n o g e n i c i t y o f PAH i n Mouse Skin and Rat Mammary Gland

Ionization Potential (eV)


Compound Cyclopenta[cd]pyrene Benzo[a]pyrene dihydrodiol

301


CAVALIER I AND ROGAN

7,8-

3

Carcinogenicity in: Rat Mammary Gland Mouse Skin

++

-

+ + ++

-

+ ++

Di b e n z [ a , h ] a n t h r a c e n e

7.57

+ ++

Benz[a]anthracene

7.54

+

-

7-Methylbenz[a]anthracene

7.37

+ ++

+

Benzo[a]pyrene

7.23

+ + ++

+ ++

7,12-Dimethylbenz[a]anthracene

7.22

+ + + ++

+ + ++

10-F1uoro-3-methylcholanthrene

7.17

N.T.

++

1,3-Dimethylcholanthrene

7.15

8-F1uoro-3-methylcholanthrene

7.14

N.T.

++

2,3-Dimethylcholanthrene

7.13

N.T.

++

3-Methylcholanthrene

7.12

+ + ++

+ + ++

6-Methylbenzo[a]pyrene

7.08

+ ++

+

5-Methylchrysene

ca.

7.7

C

-

++

Determined from a b s o r p t i o n maximum o f t h e c h a r g e - t r a n s f e r complex o f each compound w i t h c h l o r a n i l , with t h e e x c e p t i o n o f dibenzC&J}.]a n t h r a c e n e determined by p o l a r o g r a p h i c o x i d a t i o n ( 2 4 ) . Extremely a c t i v e , + + + + + ; v e r y a c t i v e , + + + +; a c t i v e , + + +, m o d e r a t e l y a c t i v e , + +; weakly a c t i v e , +; v e r y weakly a c t i v e , HH; inactive, C

N.T.

= not t e s t e d .



302


PAH were chosen i n which a c t i v a t i o n by monooxygenation o r o n e - e l e c t r o n o x i d a t i o n was b l o c k e d . Compounds which have low IP and s u f f i c i e n t c h a r g e l o c a l i z a t i o n i n t h e r a d i c a l c a t i o n , namely 7 - m e t h y l b e n z [ a ] a n t h r a c e n e , BP, 7,12-dimethylbenz[a]anthracene, 10-f1uoro-3-methylcholanthrene, 8-fluoro-3-methylcholanthrene, 2,3-dimethylcholanthrene, 3-methylc h o l a n t h r e n e , and 6-methylBP, a r e g e n e r a l l y c a r c i n o g e n i c , both i n mouse s k i n and r a t mammary g l a n d . However, 1 , 3 - d i m e t h y l c h o l a n t h r e n e , which has a low IP, i s i n a c t i v e i n r a t mammary g l a n d and a c t i v e i n mouse s k i n . T h i s i s presumably due t o s t e r i c h i n d r a n c e at C - l , the p o s i t i o n of n u c l e o p h i l i c s u b s t i t u t i o n in the 3-methylcholanthrene r a d i c a l c a t i o n . Its c a r c i n o g e n i c a c t i v i t y i n mouse s k i n can be a t t r i b u t e d t o a c t i v a t i o n by monooxygenation. In c o n t r a s t 2 , 3 - d i m e t h y l c h o l a n t h r e n e , i n which t h e methyl s u b s t i t u e n t at C-2 does not p r e v e n t n u c l e o p h i l i c s u b s t i t u t i o n at C - l i n t h e r a d i c a l cation, is carcinogenic. PAH w i t h r e l a t i v e l y h i g h IP, such as d i b e n z [ a , h ] a n t h r a c e n e and 5 - m e t h y l c h r y s e n e , are not a c t i v e when d i r e c t l y a p p l i e d t o t h e mammary g l a n d . The c a r c i n o g e n i c i t y o f 5 - m e t h y l c h r y s e n e i n mouse s k i n has been demonstrated t o o c c u r v i a a d i o l e p o x i d e mechanism ( 4 1 ) , and t h e p o t e n t a c t i v i t y o f d i b e n z [ a , h ] a n t h r a c e n e i s presumably induced by t h e same mechanism ( 5 ) . The i n a c t i v i t y o f t h e s e two s k i n c a r c i n o g e n s s u g g e s t s t h a t dTol e p o x i d e s are not formed i n t h e mammary g l a n d . No c a r c i n o g e n i c a c t i v i t y i s o b s e r v e d i n t h i s t a r g e t organ f o r t h e two mouse s k i n c a r c i n o g e n s BP 7 , 8 - d i h y d r o d i o l (5^) and c y c l o p e n t a [ c d ] p y r e n e ( 4 2 ) , both o f which r e q u i r e a s i m p l e e p o x i d a t i o n t o become a c t i v e . From t h e s e experiments we can draw t h r e e main c o n c l u s i o n s : 1) o x y g e n a t i o n o f PAH by cytochrome P-450 monooxygenase enzymes does not seem t o p l a y a r o l e i n e l i c i t i n g c a r c i n o g e n i c i t y i n r a t mammary g l a n d ; 2) t h e r e s u l t s i n t h e mammary e x p e r i m e n t s s u p p o r t t h e h y p o t h e s i s t h a t o n e - e l e c t r o n o x i d a t i o n might be t h e predominant mechanism o f a c t i v a t i o n i n t h i s t a r g e t o r g a n ; and 3) m u l t i p l e mechanisms o f a c t i v a t i o n appear t o o c c u r i n mouse s k i n , a l t h o u g h t h e s e e x p e r i ments do not p r o v i d e d i r e c t e v i d e n c e on t h i s p o i n t . Conclusions Based on p r e s e n t knowledge t h e c a r c i n o g e n i c i t y o f PAH i s best u n d e r s t o o d i n terms o f two major mechanisms o f a c t i v a t i o n : one-electron o x i d a t i o n and monooxygenation. The b a y - r e g i o n d i o l e p o x i d e s can be c o n s i d e r e d major u l t i m a t e c a r c i n o g e n i c i n t e r m e d i a t e s when a c t i v a t i o n o c c u r s by monooxygenation (2-5.) • O n e - e l e c t r o n o x i d a t i o n o f PAH w i t h f o r m a t i o n o f r a d i c a l c a t i o n s can o n l y p l a y a r o l e i n b i o l o g i c a l systems when PAH have an IP below c a . 7.35 eV ( T a b l e I) (6,_7). Thus c a r c i n o g e n i c i t y o f compounds w i t h r e l a t i v e l y h i g h IP (TabTe I ) , such as b e n z o [ c ] p h e n a n t h r e n e , c h r y s e n e , 5 - m e t h y l c h r y s e n e and d i b e n z [ a , h ] a n t h r a c e n e , can be a t t r i b u t e d t o monooxygenation w i t h f o r m a t i o n o f bay-region diol epoxides. Most o f t h e p o t e n t PAH, however, have IP below c a . 7.35 eV. T h i s l i s t i n c l u d e s BP, 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 , 3 - m e t h y l c h o l a n t h r e n e , d i b e n z o [ a , i ] p y r e n e and d i b e n z o [ a , h j pyrene. These PAH can be a c t i v a t e d by both o n e - e l e c t r o n o x i d a t i o n and monooxygenation, depending on t h e enzymes p r e s e n t i n t h e t a r g e t o r g a n . The u b i q u i t y o f p e r o x i d a s e s , i n p a r t i c u l a r p r o s t a g l a n d i n H s y n t h a s e , i n e x t r a h e p a t i c t i s s u e s which are r e s p o n s i v e t o PAH l e a d s us t o s u g g e s t t h a t o n e - e l e c t r o n o x i d a t i o n may be a major pathway o f




303

a c t i v a t i o n i n most t a r g e t t i s s u e s . Combined s t u d i e s o f enzymology, c a r c i n o g e n i c i t y and b i n d i n g t o c e l l u l a r macromolecules s h o u l d p r o vide the information necessary to determine the r o l e of the d i f f e r e n t mechanisms o f PAH a c t i v a t i o n r e s p o n s i b l e f o r i n i t i a t i o n o f the cancer process in a c e r t a i n target organ.


Acknowl edgments We a p p r e c i a t e t h e v a l u a b l e c o l l a b o r a t i o n o f D r s . C. Warner, P. Cremonesi and A. Wong and o f Mr. S. T i b b e l s . We a r e a l s o g r a t e f u l t o Ms. M. Susman f o r e x c e l l e n t e d i t o r i a l a s s i s t a n c e . F i n a l l y we thank t h e N a t i o n a l I n s t i t u t e s o f H e a l t h f o r s u p p o r t i n g t h i s r e s e a r c h t h r o u g h g r a n t s R01 CA25176, R01 CA32376 and R01 ES02145.

Literature Cited 1. Miller, J.A. Cancer Res. 1970, 30, 559-76. 2. Miller, E.C.; Miller J.A. Cancer, 1981, 47, 2327-45. 3. Nordqvist, 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"; Bhatnager, R.S., Ed.; Ann Arbor Science Publishers: Ann Arbor, Mich., 1979; pp. 329-357. 4. Sims, P.; Grover, P.L. In "Polycyclic Hydrocarbons and Cancer"; Gelboin, H.V.; Ts'o, P.O.P, Ed.; Academic: New York, 1978; Vol. 1, pp. 117-81. 5. Conney, A.H. Cancer Res. 1982, 42, 4875-917. 6. Cavalieri, E.L.; Rogan, E.G. In "Free Radicals in Biology"; Pryor, W.A., Ed.; Academic: New York, 1984; Vol. VI, pp. 323-369. 7. Cavalieri, E.; Rogan, E. In "Chemical Induction of Cancer"; by Woo, Y.-T; Lai, D.Y., Arcos, J.C.; Argus, M.F.; ; Academic: New York, 1984; in press. 8. Cavalieri, E.; Roth, R. J. Org. Chem., 1976, 41, 2679-84. 9. Cavalieri, E.; Roth, R.; Rogan, E.G. In "Polynuclear Aromatic Hydrocarbons: Chemistry, Metabolism and Carcinogenesis"; Freudenthal, R.I.; Jones, P.W., Eds.; Raven: New York, 1976; Vol. 1, pp. 181-190. 10. Cavalieri, E.; Rogan, E. In "Polynuclear Aromatic Hydrocarbons: Formation, Metabolism and Measurement"; Cooke, M.; Dennis, A.J., Eds.; Battelle Press, Columbus, Ohio, 1983; pp. 1-26. 11. Rogan, E.G.; Roth, R.; Cavalieri, E. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjørseth, A.; Dennis, A.J., Eds.; Battelle Press: Columbus, Ohio, 1980; pp. 259-265. 12. Cavalieri, E.; Rogan, E.; Warner, C.; Bobst, A. In "Polynuclear Aromatic Hydrocarbons: Mechanisms, Methods and Metabolism"; Cooke, M.; Dennis, A.J., Eds.; Battelle Press, Columbus, Ohio, in press. 13. Sato, Y.; Kinoshita, M.; Sano, M.; Akamatu, H. Bull. Chem. Soc. Jap., 1969, 42, 3051-5. 14. Ristagno, C.V.; Shine, H.J. J. Org. Chem., 1971, 36, 4050-5. 15. Cavalieri, E.; Cremonesi, P.; Warner, C.; Tibbels, S.; Rogan, E. Proc. Am. Assoc. Cancer Res., 1984, 25, 124. 16. Cavalieri, E.L.; Rogan, E.G.; Roth, R.W.; Saugier, R.K.; Hakam, A. Chem.-Biol. Interact. 1983, 47, 87-109.



304


17. Cavalieri, E.; Sinha, D.; Rogan, E. In "Polynuclear Aromatic Hydrocarbons: Chemistry and Biological Effects"; Bjørseth, A.; Dennis, A.J., Eds.; Battelle Press: Columbus, Ohio, 1980; pp. 215-231. 18. Cavalieri, E.; Rogan, E. In "Polynuclear Aromatic Hydrocarbons: Physical and Biological Chemistry"; Cooke, M.; Dennis, A.J.; Fisher, G.L., Eds.; Battelle Press: Columbus, Ohio, 1982; pp. 145-155. 19. Nagata, C.; Kodama, M.; Ioki, Y.; Kimura, T. In "Free Radicals and Cancer"; Floyd, R.A., Ed.; Marcel Dekker: New York, 1982; pp. 1-62. 20. Lorentzen, R.J.: Caspary, W.J.; Lesko, S.A.; Ts'o, P.O.P. Biochemistry, 1975, 14, 3970-7. 21. Marnett, L.J.; Reed, G.A. Biochemistry, 1979, 18, 2923-9. 22. Renneberg, R.; Capdevila, J.; Chacos, H.; Estabrook, R.W.; Prough, R.A. Biochem. Pharmacol., 1981, 30, 843-8. 23. Buhler, D.R.; Unlü, F.; Thakker, D.R.; Slaga, T.J.; Conney, A.H.; Wood, A.W.; Chang, R.L.; Levin, W.; Jerina, D.M. Cancer Res., 1983, 43, 1541-9. 24. Pish, E.S.; Yang, N.C. J. Am. Chem.Soc.,1963, 85, 2124-30. 25. Eling, T.; Boyd, J.; Reed, G.; Mason, R.; Sivarajah, K. Drug Metab. Rev., 1983, 14, 1023-53. 26. Bartsch, H.; Hecker, E. Biochim. Biophys. Acta, 1971, 237, 567-78. 27. Floyd, R.A.; Soong, L.M.; Culver, P.L. Cancer Res., 1976, 36, 1510-9. 28. Metzler, M.; McLachlan, J.A. Biochem. Biophys. Res. Comm., 1978, 85, 874-88. 29. Sawahata, T.; Neal, R.A. Biochem. Biophys. Res. Comm., 1982, 109, 988-94. 30. Griffith, B.W.; Ting, P.L. Biochemistry, 1978, 17, 2206-11. 31. Josephy, P.D.; Eling, T.; Mason, R.P. J. Biol. Chem., 1982, 257, 3669-75. 32. Josephy, P.D.; Mason, R.P.; Eling, T. Carcinogenesis, 1982, 3, 1227-30. 33. Kalyanaraman, B.; Mason, R.P. Biochem. Biophys. Res. Comm., 1982, 105, 217-24. 34. Rogan, E.G.; Katomski, P.A.; Roth, R.W.; Cavalieri, E.L. J. Biol. Chem., 1979, 254, 7055-9. 35. Mattammal, M.B.; Zenser, T.V.; Davis, B.B. Cancer Res., 1981, 41, 4961-6. 36. Josephy, P.D.; Eling, T.E.; Mason, R.P. J. Biol. Chem., 1983, 258, 5561-9. 37. Wong, P.K.; Hampton, M.J.; Floyd, R.A. In "Prostaglandins and Cancer: First International Conference", Powles, T.J.; Bockman, R.S.; Honn, K.V.; Ramwell, P., Eds.; Alan R. Liss: New York, 1982; pp. 167-179. 38. Kalyanaraman, B.; Sivarajah, K.; Eling, T.E.; Mason, R.P. Carcinogenesis, 1983, 4, 1341-3. 39. Degen, G.H.; Eling, T.E.; McLachlan, J.A. Cancer Res., 1982, 42, 919-23. 40. Rogan, E.G.; Hakam, A.; Cavalieri, E.L. Chem.-Biol. Interact., 1983, 47, 111-22.




305

41. Hecht, S.S.; Mazzarese, R.; Amin, S.; LaVoie, E.; Hoffmann, D. In "Polynuclear Aromatic Hydrocarbons. Third International Symposium on Chemistry and Biology—Carcinogenesis and Mutagenesis"; Ann Arbor Science Publishers: Ann Arbor, Mich., 1979; pp. 733-52. 42. Cavalieri, E.; Rogan, E.; Toth, B.; Munhall, A. Carcinogenesis, 1981, 2, 277-81.


RECEIVED March 5, 1985


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