Drug Metabolism Concepts

6 Role of Purified Cytochrome P-448 and Epoxide Hydrase in the Activation and Detoxification of Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 23, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

Benzo[α]pyrene W. LEVIN, A. W. WOOD, A. Y. H. LU, D. RYAN, S. WEST, and A. H. CONNEY Department of Biochemistry and Drug Metabolism, Hoffman-La Roche Inc., Nutley, NJ 07110 D. R. THAKKER, H. YAGI, and D. M. JERINA National Institute of Arthritis, Metabolism and Digestive Disesases, National Institutes of Health, Bethesda, MD 20014

The liver microsomal monoxygenase system is a membranebound, multicomponent electron transport system which is responsible for the oxidative metabolism of a variety of endogenous and exogenous substrates such as steroids, fatty acids, drugs, insecticides and chemical carcinogens (1). Of the three components involved in microsomal drug metabolism (cytochrome P-450, NADPH-cytochrome c reductase and phosphatidylcholine), cytochrome P-450 is undoubtedly the most important because of its vital role in oxygen activation, substrate binding and in determining the overall substrate specificity of the enzyme system (2,3). The rate at which various compounds are metabolized by this enzyme system varies widely and depends on the species, strain, age, tissue and pretreatment of the animal (1). Over the last decade, numerous studies have suggested that different forms of cytochrome P-450 exist in liver microsomes, and, more recently, the purification and reconstitution of the monoxygenase system have established the existance of multiple forms of cytochrome P-450 having different substrate specificities. The various purified forms of cytochrome P-450 differ from one another not only in their substrate specificity but also in their spectral and immunological properties as well as in their minimum molecular weights as determined by SDS-gel electrophoresis (4-9). The importance of this rather versatile enzyme system has become increasingly apparent during the last 10 years. Today we live in a society that has become increasingly aware of the potential dangers of environmental pollutants which include chemical carcinogens. It has been estimated that 60-80% of a l l human cancers are caused by environmental factors (10-12), and many chemicals in our environment are metabolically activated to ultimate carcinogens by the microsomal monoxygenase system (13). One 99 Jerina; Drug Metabolism Concepts ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

100

DRUG M E T A B O L I S M C O N C E P T S

of these environmental p o l l u t a n t s , the p o l y c y c l i c aromatic hydro carbon benzolajpyrene (BP), may b e o n e o f t h e m o s t p r e v a l e n t c h e m i c a l c a r c i n o g e n s t o w h i c h man i s e x p o s e d (14).

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on February 23, 2018 | https://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0044.ch006

1

S i n c e p o l y c y c l i c h y d r o c a r b o n s s u c h a s BP a r e c h e m i c a l l y i n e r t , t h e i r c a r c i n o g e n i c i t y i s thought to r e s u l t from metabolic a c t i v a t i o n by t h e m i c r o s o m a l monoxygenase system t o a c h e m i c a l l y reactive iηtermediate(s)(13,15-17). The o x i d a t i v e m e t a b o l i s m o f BP p r o c e e d s i n i t i a l l y t h r o u g h t h e f o r m a t i o n o f r e a c t i v e a r e n e oxides which spontaneously isomerize to phenols, are hydrated t o d i h y d r o d i o l s by microsomal epoxide hydrase or are c o n j u g a t e d with glutathione v i a the soluble glutathione S-transferases (ljj>« 16,18). In o r d e r t o e l u c i d a t e t h e r o l e o f m e t a b o l i s m i n t h e m u t a g e n i c i t y and c a r c i n o g e n i c i t y o f BP, a b a s i c understanding o f t h e p r o p e r t i e s and mechanism o f a c t i o n o f t h e enzymes involved i n t h e a c t i v a t i o n a n d i n a c t i v a t i o n o f BP i s e s s e n t i a l . Thus, t h e p u r i f i c a t i o n and r e c o n s t i t u t i o n o f t h e monoxygenase s y s t e m (19) i n t h e p r e s e n c e o r absence o f p u r i f i e d e p o x i d e hydrase (20) has e n a b l e d us t o s t u d y t h e r o l e o f t h e s e enzymes i n t h e m e t a b o l i s m o f BP a n d B P d e r i v a t i v e s a n d t o m a n i p u l a t e t h e s o u r c e o f t h e p u r i f i e d c y t o c h r o m e P-450 as w e l l as t h e l e v e l o f e p o x i d e hydrase to generate mutagenic metabolites of t h i s p o l y c y c l i c aromatic hydrocarbon. F i n a l l y , the synthesis of approximately t h i r t y BP d e r i v a t i v e s a n d m e t a b o l i t e s ( 2 1 ) h a s p e r m i t t e d u s t o u t i l i z e t h e s e compounds as s u b s t r a t e s f o r t h e p u r i f i e d monoxy g e n a s e s y s t e m i n an e f f o r t t o i d e n t i f y t h e b i o a c t i v a t e d m e t a b o l i t e s o f BP. M e t a b o l i s m o f B e n z o l a j p y r e n e and B e n z o l a j p y r e n e A r e n e O x i d e s b £ t h e P u r i f i e ? Wônoxygenase System and E p o x i d e H y d r a s e . Tïïë p u r i f i e d , r e c o n s t i t u t e d monoxygenase system w i t h and w i t h o u t a d d i t i o n o f p u r i f i e d e p o x i d e hydrase has been u t i l i z e d t o s t u d y the metabolism o f L CJ-benzolajpyrene (Table I). Although high pressure l i q u i d chromatography i s h i g h l y e f f i c i e n t f o r the separ a t i o n and q u a n t i t a t i o n o f d i h y d r o d i o l s , p h e n o l s and q u i n o n e s formed from BP, a l l p o t e n t i a l m e t a b o l i t e s w i t h i n each group have not been i d e n t i f i e d i n t h e s e s t u d i e s ( 2 2 J . Thus, d i o l fractions 1 , 2 a n d 3 c o r r e s p o n d t o BP 9 , 1 0 - , 4 , 5 - a n d 7,8-dihydrodiols, q u i n o n e f r a c t i o n 1 c o r r e s p o n d s t o BP 1 , 6 - , 3 , 6 - and 4,5-quinones, q u i n o n e f r a c t i o n 2 c o r r e s p o n d s t o BP 1 1 , 1 2 - a n d 6 , 1 2 - q u i n o n e s a n d BP 4 , 5 - o x i d e , p h e n o l f r a c t i o n 1 c o r r e s p o n d s t o 2 - , 6 - , 8and 9-HOBP and p h e n o l f r a c t i o n 2 c o r r e s p o n d s t o t h e o t h e r 8 i s o m e r i c p h e n o l s o f BP ( 1 - , 3 - , 4 - , 5 - , 7 - , 1 0 - , 1 1 - a n d 1 2 - H O B P ) . In t h e a b s e n c e o f e p o x i d e h y d r a s e , t h e r e c o n s t i t u t e d c y t o c h r o m e P - 4 4 8 c o n t a i n i n g m o n o x y g e n a s e s y s t e m m e t a b o l i z e s BP t o p h e n o l s and q u i n o n e s ( T a b l e I ) . Upon a d d i t i o n o f p u r i f i e d e p o x i d e h y d r a s e , t h e r a t e o f t o t a l BP m e t a b o l i s m i s u n c h a n g e d , b u t s i g n i f i c a n t amounts o f d i h y d r o d i o l s a r e p r o d u c e d a t t h e expense o f phenols. F o r m a t i o n o f d i o l f r a c t i o n s 2 and 3 (BP 4 , 5 - and 7 , 8 d i h y d r o d i o l s , r e s p e c t i v e l y ) r e a c h e d a maximum l e v e l w i t h t h e a d d i t i o n of 5 u n i t s of epoxide hydrase whereas f u r t h e r a d d i t i o n

Jerina; Drug Metabolism Concepts ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

6.

LEVIN E T A L .

Activation

and Detoxification

of Benzolajpyrene

101

o f t h e enzyme r e s u l t e d i n a c o n t i n u e d i n c r e a s e i n d i o l f r a c t i o n 1 (BP 9 , 1 0 - d i h y d r o d i o l ) , p r o b a b l y as a r e s u l t o f t h e marked i n s t a b i l i t y o f BP 9 , 1 0 - o x i d e ( t , < 2 m i n u t e s a t 3 7 ° i n 1 0 0 mM p o t a s s i u m p h o s p h a t e b u f f e r ) c o m p a r e d t o BP 4 , 5 - a n d 7 , 8 - o x i d e s . T h u s , h i g h e r amounts o f e p o x i d e h y d r a s e w o u l d be r e q u i r e d t o


/

2

Table

I

Metabolism o f B e n z o l a j p y r e n e b y a P u r i f i e d Cytochrome P-448 Dependent Monoxygenase System and Epoxide Hydrase

Epoxide Hydrase

Diol 1

(units)

None

2

3

(nmol

product

1

Quinone 2

formed/nmol

0.06

0.05

0.07

1.54

5

0.59

0.41

0.57

1.66

15

0.79

0.47

0.61

50

1.19

0.48

0.56

0.52

1

Phenol 2

Total

hemeprotein/min)

0.87

1.31

4.42

-

0.63

1.00

4.86

1.62

-

0.35

0.90

4.74

1.39

-

0.13

0.74

4.49

I n c u b a t i o n m i x t u r e s c o n t a i n e d 0 . 2 nmol c y t o c h r o m e P - 4 4 8 , 120 u n i t s o f NADPH c y t o c h r o m e c r e d u c t a s e , 0 . 1 mg o f l i p i d , 0 . 5 p m o l o f NADPH, 3 y m o l o f M g C l , 100 y m o l o f p o t a s s i u m p h o s p h a t e b u f f e r (pH 6.a) a n d 95 nmol o f ( ^ C J - B P i n a f i n a l v o l u m e o f 1 m l . One u n i t o f e p o x i d e h y d r a s e p r o d u c e s 1 nmol o f s t y r e n e g l y c o l p e r m i n from styrene oxide. M e t a b o l i t e s o f BP w e r e a n a l y z e d b y 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 p h y as d e s c r i b e d b y H o l d e r e t jrt. ( 2 2 ) . 2



102


c o m p l e t e l y c o n v e r t BP 9 , 1 0 - o x i d e t o t h e c o r r e s p o n d i n g dihydrodiol. A g o o d s t o i c h i o m e t r i c r e l a t i o n s h i p was f o u n d b e t w e e n t h e i n c r e a s e i n t h e d i o l 2 f r a c t i o n and t h e d e c r e a s e i n q u i n o n e f r a c t i o n 2 w h i c h c o n t a i n s m a i n l y BP 4 , 5 - o x i d e . Direct confirm a t i o n t h a t a r e n e o x i d e s o f BP a r e s u b s t r a t e s f o r t h e p u r i f i e d e p o x i d e h y d r a s e i s shown i n T a b l e I I . BP 4 , 5 - , 7,8and 9 , 1 0 oxides are a l l metabolized to the corresponding dihydrodiols a t c o m p a r a b l e r a t e s u s i n g t h e p u r i f i e d e n z y m e w h i l e BP 1 1 , 1 2 o x i d e i s a r e l a t i v e l y poor s u b s t r a t e f o r t h e enzyme. No d e t e c t a b l e BP 1 1 , 1 2 - d i h y d r o d i o l i s formed by t h e p u r i f i e d cytochrome P-448 c o n t a i n i n g monoxygenase system i n the presence of epoxide hydrase. T h e r e s u l t s o f o u r s t u d i e s o n t h e m e t a b o l i s m o f BP i n the presence or absence of epoxide hydrase demonstrate t h a t t h e d i h y d r o d i o l s a n d p h e n o l i c m e t a b o l i t e s o f BP s h a r e a r e n e o x i d e s a s common precursors. Requirements f o r t h e M e t a b o l i c A c t i v a t i o n o f Benzol a Ipyrene t o M u t a g e n i c P r o d u c t s by a P u r i f i e d Monoxygenase System. Mutag e n i c i t y t e s t s u t i l i z i n g m i c r o o r g a n i s m s o r c u l t u r e d mammalian c e l l s have been used w i t h i n c r e a s i n g f r e q u e n c y t o i d e n t i f y b i o activated metabolites of carcinogens. Metabolic activation of c h e m i c a l s t o m u t a g e n i c m e t a b o l i t e s has been most commonly p e r f o r m e d b y a p r o c e d u r e d e v e l o p e d b y Ames a n d h i s a s s o c i a t e s (23, 24). G e n e r a l l y , t h e c h e m i c a l , b a c t e r i a and a p p r o p r i a t e c o f a c E o r s are i n c u b a t e d w i t h microsomes or a 9000 xg s u p e r n a t a n t f r a c t i o n o f l i v e r i n a s e m i s o l i d agar g e l f o r 48 h o u r s . When l i t t l e is known a b o u t t h e m e t a b o l i s m o f a p a r t i c u l a r compound, o r a l a r g e number o f d i v e r s e c h e m i c a l s a r e b e i n g e v a l u a t e d f o r m u t a g e n i c a c t i v i t y , r e l a t i v e l y c r u d e t i s s u e homogenates s h o u l d be used as t h e s o u r c e o f enzymes t o e n s u r e t h a t a l l p o s s i b l e m e t a b o l i c pathways are being e v a l u a t e d . W h i l e t h i s p r o c e d u r e has been u s e d s u c c e s s f u l l y t o a c t i v a t e a number o f c a r c i n o g e n s t o b a c t e r i a l mutagens, i t has l i m i t a t i o n s f o r i d e n t i f y i n g t h e m u t a g e n i c m e t a b o l i t e s f o r m e d f r o m a compound w h i c h u n d e r g o e s oxidation v i a m u l t i p l e pathways. The 9000 xg s u p e r n a t a n t f r a c t i o n i s r e l a t i v e l y c r u d e and c o n t a i n s many e n z y m a t i c and s t r u c t u r a l p r o t e i n s , n u c l e i c a c i d s and numerous n u c l e o p h i l i c and e l e c t r o p h i l i c groups which could i n t e r a c t with the b i o a c t i v a t e d metabolites before they reach the b a c t e r i a . Moreover, regardless of the source of the monoxygenase a c t i v i t y , long i n c u b a t i o n times can r e s u l t i n spontaneous or enzymatic breakdown o f p r i m a r y m e t a b o l i t e s t o as y e t i l l - d e f i n e d p r o d u c t s ( 2 5 , 2 6 ) . An e x a m i n a t i o n o f t h e m e t a b o l i t e p r o f i l e o b t a i n e d f r o m BP w h e n l i v e r m i c r o s o m e s a r e i n c u b a t e d f o r 30 m i n u t e s w i t h l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n s r e v e a l e d t h a t a l l p r i m a r y o x i d a t i v e p r o d u c t s o f BP ( d i h y d r o d i o l s , q u i n o n e s and p h e n o l s ) undergo e x t e n s i v e s e c o n d a r y m e t a b o l i s m by t h e monoxygenase system (26). Thus, use of prolonged i n c u b a t i o n times f o r m e t a b o l i c a c t i v a t i o n s t u d i e s i n agar gel would a l l but e l i m i n a t e the p o s s i b i l i t y of o b t a i n i n g a p r o f i l e of the m e t a b o l i t e s formed under t h e c o n d i t i o n s which induce mu-


6.

LEVIN ET A L .

Activation

and Detoxification

Table


Oxides

445

BP

321

BP 9 , 1 0 - o x i d e

BP

11,12-oxide

by P u r i f i e d

Epoxide

D i h y d r o d i o l Formed (nmol/mg p r o t e i n / m i n )

BP 4 , 5 - o x i d e

7,8-oxide

103

II

Metabolism o f Benzolajpyrene Arene "Hydrase

Substrate

of Benzolajpyrene

390

31

Incubation m i x t u r e s c o n t a i n e d 2-6 y g o f p u r i f i e d epoxide hydrase, 30 y g o f p h o s p h a t i d y l c h o l i n e , 12.5 ymol o f T r i s b u f f e r and 10-25 nmol o f t r i t i u m l a b e l l e d s u b s t r a t e i n a f i n a l v o l u m e o f 8 0 y l .



104


tations. F i n a l l y , t h e a b i l i t y t o m a n i p u l a t e t h e amount o f v a r i o u s BP m e t a b o l i t e s f o r m e d t h r o u g h a l t e r a t i o n o f t h e r a t i o o f e p o x i d e hydrase t o t h e monoxygenase system ( c f T a b l e I) should be o f v a l u e i n d e t e r m i n i n g t h e n a t u r e o f t h e b i o a c t i v a t e d m e t a b o l i t e s formed from BP. We, t h e r e f o r e , s o u g h t t o d e v e l o p an e n z y m a t i c a l l y w e l l - d e f i n e d monoxygenase system which would m e t a b o l i z e BP a n d BP d e r i v a t i v e s t o m u t a g e n i c p r o d u c t s u n d e r c o n d i t i o n s w h i c h w o u l d p e r m i t t h e a n a l y s i s and i d e n t i f i c a t i o n o f the metabolites (27). Except f o r tRê presence of b a c t e r i a (Salmonella typhimurium s t r a i n T A 9 8 ) i n t h e r e a c t i o n m i x t u r e , t h e m e t a b o l i s m o f BP t o m u t a g e n i c p r o d u c t s by t h e r e c o n s t i t u t e d s y s t e m was p e r f o r m e d e s s e n t i a l l y as d e s c r i b e d f o r m e t a b o l i t e i d e n t i f i c a t i o n . Bacteria (2 χ 1 0 c e l l s ) were suspended i n a t o t a l i n c u b a t i o n volume o f 0 . 5 ml c o n t a i n i n g 2 . 5 y m o l o f s o d i u m p h o s p h a t e , 75 y m o l o f sodium c h l o r i d e , 0 . 0 8 y m o l (50 y g ) o f p h o s p h a t i d y l c h o l i n e , 150 u n i t s o f N A D P H - c y t o c h r o m e c r e d u c t a s e , 0 . 0 2 - 0 . 2 nmol o f c y t o c h r o m e P - 4 5 0 o r P - 4 4 8 , 25 nmol o f BP ( i n 1 2 . 5 y 1 a c e t o n e ) a n d 0 . 1 y m o l o f NADPH. T h e f i n a l pH o f t h e i n c u b a t i o n m i x t u r e was 6 . 8 . After i n c u b a t i o n a t 37 f o r 5 m i n u t e s , 9 nmol o f m e n a d i o n e was a d d e d t o s t o p t h e r e a c t i o n f o l l o w e d i m m e d i a t e l y by 2.0 ml o f m o l t e n t o p a g a r , and t h e s t a n d a r d Ames p o u r p l a t e p r o c e d u r e ( 2 4 ) was performed. S t u d i e s on t h e r e q u i r e m e n t s f o r t h e enzymaTTc acti v a t i o n o f BP b y t h e r e c o n s t i t u t e d c y t o c h r o m e P - 4 4 8 s y s t e m i n dicated that optimal metabolic a c t i v a t i o n to mutagenic metabolites r e q u i r e d t h e p r e s e n c e o f NADPH, N A D P H - c y t o c h r o m e c r e d u c t a s e , c y t o c h r o m e P-448 and p h o s p h a t i d y l c h o l i n e ( T a b l e I I I ) . A 5 minute i n c u b a t i o n w i t h 0.1 nmol o f c y t o c h r o m e P - 4 4 8 and s a t u r a t i n g a m o u n t s o f N A D P H - c y t o c h r o m e c r e d u c t a s e , p h o s p h o l i p i d , NADPH a n d BP i n d u c e d a p p r o x i m a t e l y a 1 5 - f o l d i n c r e a s e i n h i s t i d i n e i n d e p e n d e n t c o l o n i e s i n s t r a i n TA 9 8 . An a b s o l u t e r e q u i r e m e n t was o b s e r v e d f o r a l l c o m p o n e n t s o f t h e s y s t e m e x c e p t p h o s p h a t i d y l c h o l i n e which i s i n agreement w i t h t h e r e q u i r e d components for t h e m e t a b o l i s m o f BP t o p h e n o l i c m e t a b o l i t e s ( 1 9 ) . In o t h e r e x p e r i m e n t s ( 2 7 ) , i t was e s t a b l i s h e d t h a t t h e number o f m u t a t i o n s i n d u c e d i n S a l m o n e l l a t y p h i m u r i u m s t r a i n TA 9 8 was p r o p o r t i o n a l t o t h e amount o f added c y t o c h r o m e P - 4 4 8 and t o t h e t i m e o f i n cubation (Figure 1). B

M e t a b o l i s m o f Benzol a j p y r e n e t o M u t a g e n i c M e t a b o l i t e s by Various Pur i f i e d T o r m s of Cytochrome P-450. Γη t h e l a s t d e c a d e , numerous l a b o r a t o r i e s have i n v e s t i g a t e d t h e p o s s i b i l i t y t h a t m u l t i p l e hydroxylase systems e x i s t i n l i v e r microsomes. The p u r i f i c a t i o n and r e c o n s t i t u t i o n o f t h i s enzyme s y s t e m has p r o v i d e d t h e means t o s t u d y i n d e t a i l t h e p h y s i c a l p r o p e r t i e s o f e a c h o f t h e components and t h e c a t a l y t i c a c t i v i t y o f c y t o c h r o m e P-450. These s t u d i e s have p r o v i d e d d i r e c t e v i d e n c e f o r t h e e x i s t ance of m u l t i p l e forms of cytochrome P-450, each of which have d i f f e r e n t , but o v e r l a p p i n g , s u b s t r a t e s p e c i f i c i t i e s ( 4 - 7 ) . T a b l e I V s h o w s a c o m p a r i s o n o f t h e m e t a b o l i s m o f BP t o m u t a g e n i c


LEVIN E T A L .

6.

Activation

and Detoxification

Table

of Benzo[a]pyrene

105

III


Requirements f o r t h e M e t a b o l i c A c t i v a t i o n o f BenzoI a)pyrene t o mutagenic"ProducTs i n Salmonella Typïïimuriûm"Strain TA""98

Addition

His

Revertants/Plate

None

36

BP

37

Benzolajpyrene Metabolism (% A c t i v i t y )

-

440

100

30

0

32

1

32

2

-Phosphatidylcholine

74

18

-NADPH

34

0

Complete Monoxygenase

System

-BP -NADPH-cytochrome -Cytochrome

c

P-448

reductase

The c o m p l e t e monoxygenase system c o n s i s t e d o f 50 y g o f p h o s p h a t i d y c h o l i n e , 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 0 . 1 nmol o f c y t o c h r o m e P - 4 4 8 , 0 . 1 y m o l o f NADPH a n d 2 5 n m o l o f B P i n a f i n a l volume o f 0 . 5 ml c o n t a i n i n g 2 χ 1 0 b a c t e r i a . Incubations were a t 3 7 ° f o r 5 m i n . 8



106

Table

Metabolism

of

Benzolajpyrene


Purified

Forms

to Mutagenic of

Source of Cytochrome P-450 Species Pretreatment of Animals

Rat

Rabbit

Products

by

Various

P-450

BenzolaJpyrene Metabolism (pmol f o r m e d / pmol h e m e p r o t e i n )

Mutations in S t r a i n TA 9 8 (His Revertants/pmol hemeprotein) +

38.0

1254

13.0

26.5

Phénobarbital

0.7

Aroclor

Rat

Cytochrome

15.0

3-Methylcholanthrene

Rat

IV

0.55

3-Methylcholanthrene

0.7

0.25

Phénobarbital,

0.5

0.05

0.1

0.05

Mouse

Fraction Mouse

A

2

Phénobarbital, Fraction C 2

A l l i n c u b a t i o n m i x t u r e s c o n t a i n e d t h e complete monoxygenase s y s t e m a n d 2 χ IQr b a c t e r i a i n a f i n a l v o l u m e o f 0 . 5 m l a s d e s c r i b e d in Table III. C y t o c h r o m e P - 4 5 0 was p u r i f i e d f r o m t h e l i v e r s o f r a t s ( 5 , 2 9 ) , r a b b i t s (6) o r m i c e (7) as p r e v i o u s l y d e s c r i b e d . The f i n a l s u b s t r a t e c o n c e n t r a t i o n w a s 75 y M . After incubation for 5 min at 37°C, t h e r e a c t i o n s were t e r m i n a t e d by t h e a d d i t i o n of 9 nmol o f m e n a d i o n e . B e n z o l a j p y r e n e h y d r o x y l a t i o n was m e a s u r e d as fluorescent phenols (28). f t


6.

LEVIN E T A L .

Activation

and Detoxification

of Benzo[a]pyrene

107

m e t a b o l i t e s by t h e p u r i f i e d and r e c o n s t i t u t e d monoxygenase system using cytochrome P-450 p u r i f i e d from animals t r e a t e d w i t h d i f f e r e n t i n d u c e r s as w e l l as from s e v e r a l d i f f e r e n t animal s p e c i e s . These r e s u l t s c l e a r l y demonstrate t h e marked d i f f e r e n c e s i n t h e m e t a b o l i s m o f BP t o f l u o r e s c e n t p h e n o l s b y v a r i o u s p u r i f i e d f o r m s of cytochrome P-450. T h e m e t a b o l i c a c t i v a t i o n o f BP t o p r o d u c t s m u t a g e n i c t o s t r a i n TA 9 8 o f S. t y p h i m u r i u m b y v a r i o u s p u r i f i e d c y t o c h r o m e P - 4 5 0 s was s i m i l a r t o t h e r a t e o f m e t a b o l i s m o f BP to fluorescent phenols (Table IV). Cytochrome P-450 i s o l a t e d from rats pretreated with e i t h e r 3-methylcholanthrene or A r o c l o r 1254 were t h e most e f f i c i e n t h e m e p r o t e i n s f o r t h e m e t a b o l i s m o f BP t o f l u o r e s c e n t p h e n o l s a n d m u t a g e n i c p r o d u c t s w h i l e t h e o t h e r p u r i f i e d c y t o c h r o m e P - 4 5 0 s w e r e much l e s s e f f e c t i v e .


1

1

E f f e c t o f Epoxide Hydrase 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 Benzol a lpyrene t o Mutagenic MetâEoTTtes. Epoxide hydrase i s an i m p o r t a n t enzyme i n t h e m e t a b o l i s m o f BP s i n c e i t c o n v e r t s t h e i n t e r m e d i a t e arene o x i d e s formed by t h e monoxygenase system to the corresponding trans dihydrodiols (15,16). In t h e absence of f u r t h e r metabolic a c t i v a t i o n , t h e d i h y d r o d i o l s produced from these arene oxides are e s s e n t i a l l y nontoxic (21,30-32). Addi t i o n o f h i g h l y p u r i f i e d e p o x i d e hydrase t o t h e monoxygenase s y s t e m d u r i n g t h e m e t a b o l i s m o f BP d e c r e a s e d t h e m u t a t i o n f r e q u e n c y b y a maximum o f 3 0 % i n S. t y p h i m u r i u m s t r a i n TA 9 8 ( F i g u r e 2 ) , i n d i c a t i n g t h a t a t l e a s t some o f t h e m u t a g e n i c m e t a b o l i t e s o f BP a r e a r e n e o x i d e s . In c o n t r a s t t o these r e s u l t s , mutations i n d u c e d b y BP 4 , 5 - o x i d e w e r e c o m p l e t e l y a b o l i s h e d b y t h e a d d i t i o n of 5 units o f epoxide hydrase (Figure 2 ) . Thus, t h e i n a b i l i t y of epoxide hydrase t o completely abolish the metabolic a c t i v a t i o n o f BP t o m u t a g e n i c p r o d u c t s s u g g e s t e d t h a t n o n - a r e n e o x i d e m e t a b o l i t e s o f BP m a y a l s o h a v e m u t a g e n i c a c t i v i t y o r t h a t some m u t a genic epoxide m e t a b o l i t e s a r e poor substrates f o r epoxide hydrase. Metabolic Activation of Benzolajpyrene Phenols. Phenols, formed e i t h e r by spontaneous i s o m e r i z a t i o n o f arene oxides (15) or d i r e c t oxygen i n s e r t i o n r e a c t i o n s ( 3 3 ) , a r e t h e p r i m a r y o x i d a t i v e p r o d u c t s when BP i s m e t a b o l i z e c T E y t h e p u r i f i e d m o n o x y genase system i n t h e absence o f epoxide hydrase ( c f Table I ) . Of t h e t w e l v e p o s s i b l e i s o m e r i c p h e n o l s o f B P , o n l y 6 - and 1 2 HOBP h a v e s i g n i f i c a n t i n t r i n s i c m u t a g e n i c a c t i v i t y i n s e v e r a l s t r a i n s o f S. t y p h i m u r i u m ( 3 2 ) . H o w e v e r , b a s e d on t h e amount o f phenols produced from B l H ï y t h e p u r i f i e d monoxygenase system (cf Table I ) , i t i s u n l i k e l y that these phenols contribute s i g n i f i c a n t l y t o t h e m u t a g e n i c i t y o b s e r v e d f r o m BP m e t a b o l i s m . S i n c e s e v e r a l s t u d i e s h a v e shown t h a t p r i m a r y o x i d a t i v e m e t a b o l i t e s o f BP, i n c l u d i n g phenols, can be f u r t h e r m e t a b o l i z e d b y t h e m o n o x y g e n a s e s y s t e m ( 2 6 , 3 4 ) , we u t i l i z e d a l l t w e l v e o f t h e phenols as s u b s t r a t e s f o r t h e p u r i f i e d monoxygenase system to determine i f f u r t h e r o x i d a t i v e metabolism would r e s u l t i n the formation o f mutagenic products. T a b l e V shows t h e m u t a g e n i c



108

DRUG M E T A B O L I S M

CONCEPTS

0.1 nmol CYTOCHROME

Figure I. Effect of cytochrome P-448 concentration and time of incubation on the metabolism of BP to products mutagenic to strain TA 98 of Salmonella typhimurium. Reaction mixtures were similar to those described in Table III. Each value represents the mean ± S.D. from three replicate incubation mixtures.

Figure 2. Effect of epoxide hydrase on mutations induced bu the metabolism of BP by the purified monoxygenase system (left) and on the mutagenic activity of BP 4,5-oxide (right). Incubation mixtures for the metabolic activation of BP were similar to those described in Table 111. The effect of epoxide hydrase on the mutagenic activity of BP 4,5-oxide was assayed using 0.4 nmole of BP 4,5-oxide in 0.5 ml containing 2 X 10* bacteria. All samples were incubated for 5 min at 37° before addition of the top agar.

10

Î 2 " 2 0

UNITS OF EPOXIOE HYDRASE


•i-^—k

6.

LEVIN ET A L .

Activation

and Detoxification

Table

of Benzo[a]pyrene

109

V


Metabolism o f Benzo|aIpyrene Phenols t o Mutagenic Products by a Pur i f i e T t y t o c h r o m e P-448""Dependen£ Monoxygenase System

Substrate

Cytochrome 0 Hi s

None BP 1-H0BP 2-H0BP 3-H0BP 4-HOBP 5-H0BP 6-HOBP 7-H0BP 8-HOBP 9-HOBP 10-H0BP 11-H0BP 12-H0BP

17 18 43 17 32 15 19 94 35 15 33 14 11 219

P-448 (pmol) 25 50 Revertants/Plate

-

280 109 67 117 22 19 116 43 35 93 22 21 302

17 670 140 73 158 29 24 261 49 45 180 21 21 372

I n c u b a t i o n m i x t u r e s c o n t a i n e d t h e complete monoxygenase system and t y p h i m u r i u m s t r a i n TA 98 a s d e s c r i b e d i n T a b l e I I I u s i n g f i x e d c o n c e n t r a t i o n s o f a l l components e x c e p t cytochrome P-448. The f i n a l c o n c e n t r a t i o n o f BP o r BP p h e n o l s was 25 μ Μ .



110


CONCEPTS

a c t i v i t y o f t h e t w e l v e i s o m e r i c p h e n o l s o f BP i n s t r a i n T A 9 8 b e f o r e and a f t e r m e t a b o l i c a c t i v a t i o n by v a r i o u s amounts o f c y t o chrome P-448. The number o f r e v e r t a n t c o l o n i e s i n t h e a b s e n c e of the cytochrome i s a measure o f the i n t r i n s i c mutagenic a c t i v i t y of the phenols s i n c e the r e c o n s t i t u t e d system i s i n a c t i v e in the absence of the cytochrome. Of t h e t w e l v e p o s s i b l e p h e n o l s o f B P , o n l y 1 - , 2 - , 3 - , 6 - , 9 - and 12-HOBP w e r e m e t a b o l i c a l l y a c t i v a t e d to mutagenic products. However, s i n c e none o f t h e p h e n o l s was as e f f e c t i v e l y c o n v e r t e d t o m u t a g e n i c p r o d u c t s as B P , i t a p p e a r s t h a t f u r t h e r m e t a b o l i s m o f BP p h e n o l s t o m u t a g e n i c products is not responsible f o r the mutagenic a c t i v i t y of metab o l i c a l l y a c t i v a t e d BP. This data, together with the lack of i n h e r e n t m u t a g e n i c a c t i v i t y o f s i x p o s s i b l e BP q u i n o n e s (32), s t r o n g l y s u g g e s t s t h a t n o n e p o x i d e d e r i v a t i v e s o f BP a r e n o t r e s p o n s i b l e f o r t h e m u t a t i o n s o b s e r v e d w h e n BP i s m e t a b o l i z e d t o mutagenic p r o d u c t s i n t h e p r e s e n c e of t h e p u r i f i e d monoxygenase s y s t e m and e p o x i d e h y d r a s e . Metabolic A c t i v a t i o n of Benzolajpyrene Dihydrodiols. Since a d d i t i o n of e p o x i d e hydrase t o t h e p u r i f i e d monoxygenase system was u n a b l e t o b l o c k t h e f o r m a t i o n o f m u t a g e n i c m e t a b o l i t e s o f B P , i t w a s p o s s i b l e t h a t a n a r e n e o x i d e o f BP w a s c o n v e r t e d t o a d i h y d r o d i o l b y a d d i t i o n o f e p o x i d e h y d r a s e w h i c h was t h e n f u r t h e r m e t a b o l i c a l l y a c t i v a t e d t o a potent mutagen. In t h i s r e g a r d , B o r g e n e t a l . (35) have shown t h a t m e t a b o l i c a c t i v a t i o n o f BP 7 , 8 - d i h y d r o d i o l " T 5 y l i v e r m i c r o s o m e s r e s u l t s i n a m u c h g r e a t e r b i n d i n g t o DNA t h a n d o e s s u c h m e t a b o l i c a c t i v a t i o n o f B P , BP 4 , 5 - d i h y d r o d i o l o r BP 9 , 1 0 - d i h y d r o d i o l . Sims e t a l . ( 3 6 ) have p r e s e n t e d e v i d e n c e t h a t a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x " u i e ~ T s Tiïe b i o a c t i v a t e d m e t a b o l i t e o f BP 7 , 8 - d i h y d r o d i o l w h i c h b i n d s t o DNA. I t h a s s i n c e b e e n e s t a b l i s h e d t h a t t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epoxides a r e among t h e m o s t p o t e n t m u t a g e n s y e t described (21,31,37-39). We h a v e u t i l i z e d t h e p u r i f i e d m o n o x y g e n a s e s y s t e m i n an a t t e m p t t o m e t a b o l i c a l l y a c t i v a t e t h e f o u r BP d i h y d r o d i o l s w h i c h h a v e b e e n s y n t h e s i z e d i n o u r l a b o r a t o r i e s (21). A s s h o w n i n F i g u r e 3 , BP 7 , 8 - d i h y d r o d i o l w a s a c t i v a t e d t o a t l e a s t a 4 - f o l d g r e a t e r e x t e n t t h a n was B P . In c o n t r a s t t o t h e s e r e s u l t s , t h e BP 4 , 5 - , 9 , 1 0 - a n d 1 1 , 1 2 - d i h y d r o d i o l s were not m e t a b o l i c a l l y a c t i v a t e d to mutagenic m e t a b o l i t e s . The h i g h m u t a g e n i c a c t i v i t y o f a m e t a b o l i t e ( s ) o f BP 7 , 8 - d i h y d r o d i o l t o w a r d s S. t y p h i m u r i u m s t r a i n TA 98 p r o m p t e d f u r t h e r s t u d i e s o 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 BP 7 , 8 - d i h y d r o d i o l b y t h e p u r i f i e d monoxygenase system. Microsomal epoxide hydrase converts the a r e n e o x i d e s f o r m e d by t h e c y t o c h r o m e P-450 monoxygenase system into trans, v i c i n a l dihydrodiols (15). T a b l e VI shows t h a t ç i s BP 7 , 8 - d i h y d r o d i o l c a n a l s o b e m e t a i ï o l i c a l l y a c t i v a t e d t o a p o t e n t mutagen(s) by t h e monoxygenase s y s t e m , a l t h o u g h i t i s s l i g h t l y less a c t i v e than the trans isomer. These r e s u l t s i n d i c a t e that the r e l a t i v e p o s i t i o n of the hydroxyl groups i s not a c r i t i c a l f a c t o r in determining the mutagenic a c t i v i t y of the



BP 7 , 8 -

2±1

( ± ) - t r a n s BP 7 , 8 dihydroxy-7,8,9, 10-H BP

76+10

130±8

9±2

2

34±3

6±4

320+18

10±3

60±5

20

9±2

120±10

(pmol)

ΉΓ

Revertants7P1ate

522±45

His

+

Cytochrome P-448 5 80

Products

29±4

800±45

t o Mutagenic

I n c u b a t i o n m i x t u r e s c o n t a i n e d t h e c o m p l e t e monoxygenase system as d e s c r i b e d i n T a b l e I I I . The f i n a l s u b s t r a t e c o n c e n t r a t i o n was 25 y M . Background mutation f r e q u e n c i e s have been s u b t r a c t e d . V a l u e s r e p r e s e n t t h e mean + S . E . f o r t h r e e determinations.

4

1±0.5

6±1

1±0.6

( t ) - c i s BP 7 , 8 dihydrodiol

dihydrodiol

(±)-trans

BP

VI

A c t i v a t i o n o f Benzol aipyrene 7 , 8 - D i h y d r o d i o l

Substrate

Metabolic

Table


112

DRUG M E T A B O L I S M CONCEPTS


bioactivated metabolite(s). However, 7,8-dihydroxy-7,8,9,10t e t r a h y d r o B P , a c o m p o u n d r e l a t e d t o BP 7 , 8 - d i h y d r o d i o l b u t w i t h t h e d o u b l e bond removed f r o m t h e 9 , 1 0 - p o s i t i o n o f t h e m o l e c u l e , c a n n o t be m e t a b o l i c a l l y a c t i v a t e d t o mutagenic m e t a b o l i t e s by t h e monoxygenase system. These r e s u l t s suggest t h a t the mutag e n i c p r o d u c t f o r m e d o n m e t a b o l i c c o n v e r s i o n o f BP 7 , 8 - d i h y d r o d i o l i s a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e s i n c e t h e d o u b l e b o n d i n t h e 9,10-position of the molecule is a requirement f o r the formation o f an e p o x i d e a t t h a t p o s i t i o n . Metabolic A c t i v a t i o n of Benzolajpyrene 7,8-Oxide. Since BP 7 , 8 - d i h y d r o d i o l w a s r e a d i l y m e t a b o l i z e d t o a m u t a g e n i c p r o d u e t t s ) b y t h e c y t o c h r o m e P - 4 4 8 m o n o x y g e n a s e s y s t e m , we e x a m i n e d t h e e f f e c t o f t h e monoxygenase s y s t e m on t h e m u t a g e n i c a c t i v i t y o f i t s a r e n e o x i d e p r e c u r s o r , BP 7 , 8 - o x i d e ( T a b l e V I I ) . In t h e a b s e n c e o f a d d e d e p o x i d e h y d r a s e , t h e weak i n t r i n s i c m u t a g e n i c a c t i v i t y o f BP 7 , 8 - o x i d e ( 4 0 ) w a s u n a f f e c t e d b y a d d i t i o n o f t h e p u r i f i e d monoxygenase system. Addition of epoxide hydrase to t h e monoxygenase system r e s u l t e d i n a marked i n c r e a s e i n the m u t a t i o n f r e q u e n c y i n S. t y p h i m u r i u m s t r a i n TA 9 8 . These r e s u l t s d e m o n s t r a t e t h a t BP 7 , ï ï - o x i d e i s h y d r a t e d t o t h e c o r r e s p o n d i n g d i h y d r o d i o l w h i c h i n t u r n i s o x i d a t i v e l y m e t a b o l i z e d t o an a c t i v e m u t a g e n ( s ) , p r e s u m a b l y t h e s t e r e o i s o m e r i c BP 7,8-diol-9,10-epoxides. Metabolism of Benzolajpyrene 7,8-Dihydrodiol b^ the P u r i f i e d Monoxygenase System. Based on t h e s t u d i e s o f Borgen e t a l . ( 3 5 ) , Sims e t a l . ( 3 6 ) p r o p o s e d a BP 7 , 8 - d i o l - 9 , 1 0 - e p o x i d e a s t h e DTOa c t i v a t e ï ï " m e T â b o l i t e o f B P 7 , 8 - d i h y d r o d i o l w h i c h b i n d s t o DNA. T h e o x i d a t i v e m e t a b o l i s m o f BP 7 , 8 - d i h y d r o d i o l a t t h e 9 , 1 0 - p o s i t i o n o f t h e m o l e c u l e c o u l d r e s u l t i n t h e f o r m a t i o n o f two p o s s i b l e stereoisomers of the d i o l epoxide (Figure 4). Recently o u r l a b o r a t o r i e s (41) s y n t h e s i z e d and u n e q u i v o c a l l y assigned t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e t w o s t e r e o i s o m e r s o f BP 7 , 8 d i o l - 9 , 1 0 - e p o x i d e ( d i o l e p o x i d e s 1 and 2 ) , b o t h o f w h i c h a r e p o t e n t mutagens ( 3 1 , 3 7 - 3 9 ) . A d e t a i l e d study of the metabolism of BP-7,8-dihydrodiol is thus necessary in order to determine w h e t h e r one o r b o t h o f t h e s t e r e o i s o m e r s a r e r e s p o n s i b l e f o r t h e m u t a g e n i c a c t i v i t y o f m e t a b o l i c a l l y a c t i v a t e d BP 7 , 8 - d i h y d r o diol. R e c e n t l y , h i g h p r e s s u r e l i q u i d chromatography has been u s e d t o s e p a r a t e m e t a b o l i t e s o f BP 7 , 8 - d i h y d r o d i o l (42«43). Although a d i r e c t demonstration of the formation of the d i o l e p o x i d e s has n o t been p o s s i b l e due t o t h e i r e x t r e m e i n s t a b i l i t y i n aqueous s o l u t i o n s , t e t r a o l s have been i d e n t i f i e d as m e t a b o l i t e s f r o m BP 7 , 8 - d i h y d r o d i o l ( F i g u r e 4 ) . D i o l e p o x i d e s 1 and 2 u n d e r go c i s and t r a n s a d d i t i o n o f w a t e r a t t h e 1 0 - p o s i t i o n o f t h e oxirane (42,43) to form stereoisomeric p a i r s of (±)-7,8,9,10tetrahydroxy-/,8,9,10-tetrahydrο BP d e r i v a t i v e s i n w h i c h t h e r e l a t i v e s t e r e o c h e m i s t r y o f t h e 7 - and 8 - h y d r o x y l group i s a l w a y s t r a n s and t h e r e l a t i v e s t e r e o c h e m i s t r y b e t w e e n t h e 8 - and 9 -


6.

Activation

LEVIN E T A L .

and Detoxification

Table


Metabolic

BP

7,8-oxide

(25 μΜ)

Benzo[z]pyrene

113

VII

A c t i v a t i o n o f BP 7 , 8 - o x i d e

Substrate

of

t o Mutagenic

Metabolites

Monoxygenase System

Epoxide Hydrase

-

-

91±4

+

-

132±10

+

+

514±22

+

His

Revertants/Plate

The c o m p l e t e monoxygenase s y s t e m c o n s i s t e d o f 0 . 0 5 nmol o f c y t o chrome P-448, 150 u n i t s o f NADPH-cytochrome c r e d u c t a s e , 50 ug o f p h o s p h a t i d y l c h o l i n e , 0 . 1 p m o l o f NADPH a n d 2 χ 1 0 b a c t e r i a i n a f i n a l volume o f 0.5 m l . Four u n i t s o f p u r i f i e d epoxide h y d r a s e was added t o t h e a p p r o p r i a t e r e a c t i o n m i x t u r e s ( s e e Table I f o r d e f i n i t i o n of u n i t s of epoxide hydrase activity). The m u t a t i o n s o b s e r v e d i n t h e absence o f t h e monoxygenase system and e p o x i d e h y d r a s e a r e a r e s u l t o f t h e i n h e r e n t m u t a g e n i c i t y o f BP 7 , 8 - o x i d e . Background m u t a t i o n f r e q u e n c i e s have been s u b tracted. V a l u e s r e p e s e n t t h e mean + S . E . f o r t h r e e d e t e r m i n a t i o n s . 8



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