17 Cytochrome P-450 in Fish Liver Microsomes and
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Carcinogen Activation JORMA T. AHOKAS Department of Pharmacology, University of Oulu, SF-90220 Oulu 22, Finland and Clinical Pharmacology Unit, Department of Medicine, Princess Alexandra Hospital, Ipswich Rd, Brisbane, Queensland, 4102, Australia 1
Random somatic mutation is thought to be a major event in carcinogenesis which may result from exposure to radiation, viruses, or chemicals. In recent years the role of environmental chemicals in the initiation of cancer has become more evident and the mechanisms involved have been greatly clarified (1). In mammals the cytochrome P-450 mediated monooxygenase or mixed function oxidase system involved in the elimination of lipo philic environmental contaminants and other foreign compounds, has been implicated in the carcinogen activation process. There are several distinct variants of cytochrome P-450 in mammalian tissues and there may be more than one form of this ubiquitous cytochrome also in fish. The significance of this lies in the fact that dif ferent forms of cytochrome P-450 result in different metabolite patterns, which i n turn may reflect on the carcinogenicity or toxicity of compounds being metabolized. Cytochrome P-450 mediated monooxygenase About twenty years ago a liver microsomal pigment was dis covered which in i t s reduced form was found to bind carbon mon oxide, resulting in a complex absorbing light at 450 nm (2, 3). It was established that the pigment is a hemoprotein and was named cytochrome P-450 (4, 5). It was soon realized that cytochro me P-450 plays a central role in the metabolism of a wide variety of drugs, endogenous and synthetic steroids, pesticides, polycye l i c aromatic hydrocarbons (PAH) e.g. carcinogenic benzo(α)pyrene (BP) and a large number of other compounds which come intention ally or unintentionally into contact with living organisms. Exis ting information on cytochrome P-450 and i t s functions has been extensively reviewed by a number of authors (6-13). Cytochrome P-U50 i n fish. Cytochrome P-U50 with i t s charac teristic spectral properties has since i t s discovery been detected in a wide range of organisms including several species of fish (13) Current address 1
0-8412-0489-6/79/47-099-279$05.00/0 © 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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( T a b l e I ) . The l e v e l s o f b o t h , c y t o c h r o m e P-U50 ( T a b l e I ) and i t s NADPH ( r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e p h o s p h a t e ) r e q u i r i n g r e d u c i n g component ( F i g u r e l ) ( w h i c h can be measured as NADPH d e p e n d e n t c y t o c h r o m e c r e d u c t a s e ) a r e s u b s t a n t i a l i n f i s h l i v e r m i c r o s o m e s , a l t h o u g h l o w e r t h a n i n mammals. NADPH c y t o c h r o m e c r e d u c t a s e l e v e l i n t r o u t {Salmo trutta laoustris) i s 20 nmol c y tochrome c reduced/mg m i c r o s o m a l p r o t e i n / m i n ; t h e c o r r e s p o n d i n g a c t i v i t y i n male S p r a g u e Dawley r a t l i v e r m i c r o s o m e s i s 96 nmol c y t o c h r o m e c reduced/mg m i c r o s o m a l p r o t e i n / m i n ( l U ) . Table I . Comparison o f the l i v e r microsomal l e v e l s o f cytochrome P-U50 i n v a r i o u s s p e c i e s o f f i s h w i t h t h o s e o f some mammals. Source Cytochrome P-U50 Reference concentration* Carp present (15, l 6 ) Female c a r p Ο.38 (17) Female g i b e l 0.15 (IT.) T r o u t [Salmo gairdneri) 0.22 (18) T r o u t ( 5 . gairdneri) present ( 1 9 , 20) T r o u t {S. trutta laoustvis) 0.20-0.U0 ( l U , 21) T r o u t {Salvalinus fontinalis) 0.56 (0.19 s t a r v e d ) (22) K i s s i n g gourami 1.03 (22) Bluegill 0.81 (22) Winter flounder 0.17 (23) L i t t l e skate 0.22-0.32 (23-26) Large skate Ο.36, Q.kl (21) Dogfish shark 0.23, 0.29 ( 2 3 , 26) Fundulus heteroolitus 0.009 (27)
Stenotomus
versicolor
0.35
(28)
Atlantic stingray 0Λ3 (26) Bluntnose stingray 0.32 (26) Sheephead 0.29 (26) Mangrove s n a p p e r 0.25 (26) B l a c k drum O.lh (26) L a r g e mouth b a s s 0.0215** (29) Mammals Rat 0.72 (lU) Mouse 0.99 (30) Rabbit 1.55 (*+, 5) *nanomoles o f c y t o c h r o m e Ρ-ί+50/mg m i c r o s o m a l p r o t e i n . **Absorbance/mg m i c r o s o m a l p r o t e i n ; c o m p a r a t i v e v a l u e s f o r r a t and mouse a r e 0.0860 and 0.09T0, r e s p e c t i v e l y ( 2 9 ) ; t h e s e a u t h o r s j u s t i f i a b l y a v o i d c a l c u l a t i n g m o l a r q u a n t i t i e s as t h e e x t i n c t i o n c o n s t a n t a v a i l a b l e ( 9 1 mM~ cm" ) i s f o r mammalian c y t o c h r o m e P^50 (k 5 ) . 1
1
9
S p e c t r a l p r o p e r t i e s o f c y t o c h r o m e P-U50. The s p e c t r a o f r e d u c e d c y t o c h r o m e P-U50.CO complex i n f i s h l i v e r m i c r o s o m e s d i s p l a y c o n s i s t e n t l y a peak a t U20 nm when t h e Omura and S a t o method ih 5) i s u s e d (lh 19, 2 2 , 2 7 ) . I t w o u l d a p p e a r t h a t t h e f i s h 9
9
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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AHOKAS
Cytochrome P-450
281
P-450 (Fe .Q ) ++
2
Figure 1. Simplified scheme for the electron transfer in the Cytochrome P-450 mediated monooxygenase activity. In the liver, the flavoprotein is Cystochrome c reductase. R is the compound being metabolized. NAD and Cytochrome b have not been included (36). 5
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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l i v e r m i c r o s o m a l c y t o c h r o m e P-l+50 i s more s e n s i t i v e and i s c o n v e r t e d i n t o i n a c t i v e f o r m o f c y t o c h r o m e P-l+50 upon n o r m a l h a n d l i n g . I f t h i s i s t h e c a s e many o f t h e e s t i m a t e s o f c y t o c h r o m e P1+50 i n f i s h l i v e r may be e r r o n e o u s l y l o w . However, a c c o r d i n g t o S t a n t o n a n d Khan ( 2 2 ) t h e 1+20 nm peak i s n o t due t o c y t o c h r o m e P1+20, a s i t i n c r e a s e s w i t h t i m e w i t h o u t c o n c o m i t a n t d e c r e a s e i n t h e c y t o c h r o m e P-l+50 l e v e l . A l s o , i t h a s b e e n n o t e d b y u s t h a t i f t h e method o f G r e i m et al. ( 3 l ) i s u s e d t h e 1+20 nm peak does n o t a p p e a r ( 2 1 ) . Fukami et al. ( l 6 ) r e p o r t e d an a b s o r b a n c e peak a t 1+30 nm f o r r e d u c e d c a r p m i c r o s o m a l p r e p a r a t i o n + CO, however i t must be n o t e d t h a t t h e y b u b b l e d t h e p r e p a r a t i o n s u n u s u a l l y l o n g (10 min) w i t h carbon monoxide. R e c e n t l y Bend et al. have s u c c e e d e d t o s o l u b i l i z e a n d p a r t i a l l y p u r i f y l i t t l e s k a t e l i v e r m i c r o s o m a l c y t o c h r o m e P-l+50 a n d h a v e thus been a b l e t o r e c o r d t h e a b s o l u t e s p e c t r a o f f i s h cytochrome P-l+50 (25.). The s p e c t r a a r e v e r y s i m i l a r t o t h o s e o f r a t l i v e r m i c r o s o m a l c y t o c h r o m e P-l+50 a n d p u r i f i e d r a t l i v e r c y t o c h r o m e P1+50 a n d P-l+1+8 ( T a b l e I I ) . Table I I . Comparison o f t h e s p e c t r a l p r o p e r t i e s o f p a r t i a l l y p u r i f i e d l i t t l e s k a t e l i v e r c y t o c h r o m e P-l+50 w i t h p a r t i a l l y p u r i f i e d r a t l i v e r c y t o c h r o m e P-l+50 a n d h i g h l y p u r i f i e d r a t l i v e r c y t o c h r o me P-l+50 a n d P-UU8, L i t t l e Skate Rat l i v e r cytochrome P-1+5C S p e c i f i c content (nmol/mg p r o t e i n ) Molecular weight A b s o r p t i o n maxima (nm) : o x i d i zed 1+20, 1+82, 571 reduced 1+21, r e d u c e d + CO 1+50, 553
P-l+50**
P-1+50***
P-l+1+8***
~3
-17
-20
1+8,000
53,000
360, I+18, I+18, 5 3 5 , 1+17, 535, 568 568 537, 568 1+1*+, 5^6 1+11, 5^5 U l 8 , 5^5 552 1+1+7, 552 1+23, 1+50, 5I+8 * P a r t i a l l y p u r i f i e d L i t t l e S k a t e l i v e r c y t o c h r o m e P-1+50. The d a t a i s f r o m ( 2 5 ) a n d i s a s a c c u r a t e a s c a n be r e a d f r o m p u b l i s h e d s p e c t r a . - * * P a r t i a l l y p u r i f i e d r a t l i v e r c y t o c h r o m e P-l+50; t h e d a t a i s f r o m (6).***Highly p u r i f i e d c y t o c h r o m e P-l+50 a n d P-l+1+8 f r o m r a t l i v e r ; t h e data i s from ( 3 2 ) .
C r i t e r i a f o r monooxygenase a c t i v i t y . Metabolism o f f o r e i g n compounds b y microsomes was f i r s t d e s c r i b e d b y M u e l l e r a n d M i l l e r (33, 3*0. They n o t e d t h a t NADP, NAD ( n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ) a n d m o l e c u l a r o x y g e n a r e r e q u i r e d i n t h e o x i d a t i v e N-dem e t h y l a t i o n o f aminoazo d y e s . I n 1955 B r o d i e et al. ( 3 5 ) showed t h a t l i v e r m i c r o s o m e s m e t a b o l i z e d many d r u g s and f o r f u l l a c t i v i t y a NADPH g e n e r a t i n g s y s t e m , c o n s i s t i n g o f g l u c o s e - 6 - p h o s p h a t e (G-6P) a n d G-6-P d e h y d r o g e n a s e o r NADPH i t s e l f i s r e q u i r e d . The e l e c t r o n t r a n s f e r s y s t e m i n v o l v e d i n t h e m e t a b o l i s m o f f o r e i g n com-
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
17.
AHOKAS
Cytochrome
P-450
283
pounds has b e e n e x t e n s i v e l y s t u d i e d and s e v e r a l schemes have b e e n s u g g e s t e d . F i g u r e 1 shows a s i m p l i f i e d scheme f o r t h e e l e c t r o n t r a n s f e r ( m o d i f i e d a f t e r Coon et al. ( 3 6 ) ) s h o w i n g a l s o t h e c h a r a c t e r i s t i c l i g h t r e v e r s i b l e i n h i b i t i o n o f t h e c y t o c h r o m e P-U50 m e d i a t e d s y s t e m by c a r b o n m o n o x i d e . The e l e c t r o n t r a n s f e r s y s t e m has n o t b e e n s t u d i e d i n d e t a i l i n f i s h , b u t t h e m e t a b o l i s m o f compounds s u c h as b i p h e n y l ( 3 7 ) b e n z o ( a ) p y r e n e (21) and 2 , 5 - d i p h e n y l o x a z o l e (38) by f i s h l i v e r m i c r o s o m e s has b e e n shown t o r e q u i r e o x y g e n and NADPH g e n e r a t i n g s y s t e m . The m e t a b o l i s m o f BP ( 2 1 ) , 2 , 5 - d i p h e n y l o x a z o l e ( A h o k a s , u n p u b l i s h e d o b s e r v a t i o n ) and a l d r i n (27) by f i s h l i v e r m i c r o s o m a l enzyme s y s t e m i s i n h i b i t e d s t r o n g l y b y c a r b o n m o n o x i d e . T h i s i n f o r m a t i o n and t h e f a c t t h a t c y t o c h r o m e P-U50, as w e l l as NADPH cytochrome c r e d u c t a s e system are p r e s e n t i n f i s h , suggest s t r o n g l y t h a t f i s h have a c y t o c h r o m e P-U50 m e d i a t e d monooxygenase s y s t e m w h i c h i s v e r y s i m i l a r t o t h a t d e s c r i b e d i n mammals.
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5
M u l t i p l e forms o f c y t o c h r o m e P-U50. I t i s now c l e a r t h a t t h e r e a r e more t h a n one f o r m o f c y t o c h r o m e P-U50. Thomas et al. have r e c e n t l y shown by immunochemical means t h a t t h e r e a r e a t l e a s t s i x f o r m s o f mammalian c y t o c h r o m e P-U50 ( 3 9 ) . I n I 9 6 0 s i t was n o t e d t h a t t h e r e a r e a t l e a s t two c a t a l y t i c a l l y and s p e c t r a l l y d i s t i n c t c y t o c h r o m e P - U 5 0 s , viz. c y t o c h r o m e P-U50 and c y t o c h r o m e P-Ul+8 o r P^i+50 ( U o ^ J a ) . Cytochrome P-Ul+8 i s i n d u c i b l e b y PAH s s u c h as 3 - m e t h y l c h o l a n t h r e n e (MC) and BP. I t m e t a b o l i z e s p r e f e r e n t i a l l y P A H s ( s u c h as t h e above c a r c i n o g e n i c i n d u c e r s ) . C y t o chrome P-UU8 d e r i v e s i t s name f r o m t h e f a c t t h a t when r e d u c e d and c o m p l e x e d w i t h c a r b o n m o n o x i d e i t has an a b s o r b a n c e maximum a t hkQ nm. Cytochrome P-U50 i n d u c e d b y compounds s u c h as phénobarbital (PB) a p p e a r s s i m i l a r t o t h e c o n t r o l c y t o c h r o m e P-U50 b o t h s p e c t r a l l y and c a t a l y t i c a l l y . I n a d d i t i o n t o t h e 2 nm s h i f t i n t h e a b s o r p t i o n maximum, t h e two c y t o c h r o m e s c a n be d i s t i n g u i s h e d b y t h e u s e o f e t h y l i s o c y a n i de i n t e r a c t i o n s p e c t r a ( 6 , 7) and v a r i o u s i n h i b i t o r s o f t h e monoo x y g e n a s e a c t i v i t y ( F i g u r e 2 and T a b l e I I I ) . The r e l a t i v e m a g n i t u d e o f t h e e t h y l i s o c y a n i d e - c y t o c h r o m e P-U50 i n t e r a c t i o n s p e c t r a l p e a k s a t Λ30 and ~^55 nm i s pH dependent (6_) and i f t h e a b s o r b a n c e d i f f e r e n c e s a r e p l o t t e d as f u n c t i o n s o f pH, t h e r e i s a c r o s s - o v e r p o i n t a t a c e r t a i n pH w h i c h i s c h a r a c t e r i s t i c f o r a p a r t i c u l a r f o r m o f c y t o c h r o m e P-U5O; pH -6.9 f o r c y t o c h r o m e P-UU8 and pH -7.5-7.6 f o r PB i n d u c e d o r c o n t r o l c y t o c h r o m e P-U50 ( 6 , 21). The c y t o c h r o m e P-^50 o f a p p a r e n t l y u n i n d u c e d t r o u t s p e c i e s (Salmo trutta laoustris) has b e e n shown by us t o have t h e pH c r o s s - o v e r p o i n t f o r e t h y l i s o c y a n i d e i n t e r a c t i o n s p e c t r u m a t pH 7.8 (21) and t h e a b s o r p t i o n maximum o f t h e r e d u c e d t r o u t l i v e r c y t o c h r o m e P-U50 .CO complex i s U50 nm, n e v e r t h e l e s s i t s c a t a l y t i c and i n h i b i t o r y p r o p e r t i e s ( 2 1 ) ( T a b l e I I I ) a r e s i m i l a r t o t h o s e o f c y t o c h r o m e P1
!
1
1
hkQ. C u r i o u s l y t h i s p a r a l l e l s t h e d i s c o v e r y o f MC i n d u c i b l e c y t o chrome P-U50 i n f e t a l and n e o n a t a l r a b b i t s (U3) w h i c h , a) f a i l s t o
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC
284
RAT
TROUT
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ORGANISMS
Cytochrome P-450:Cytochrome P-450 EtNC interaction spectra : — EtNC:
pH
curves
BP hydroxylation:
+++
-inhibition by SKF 525 A
+
a-naphthoflavone
+++
Covalent binding of BP to D N A
+++
++ +
+ +
n.d.
+ + ++ ++ + +
Figure 2. Spectral properties of Cytochrome P-450s from various sources related to the BP hydroxylase activity, the inhibition of BP hydroxylase, and the extent of covalent binding of BP to DNA. Cytochrome P-450 spectra (reduced + CO) show the 2-nm shift to the blue as a result of 3-MC induction. No such shift is observed in the trout, control rat, and PB-induced rat liver microsomes. Cytochrome P-450 EtNC spectra were recorded at pH 7.4, 2 min after the samples were reduced with dithionite. The absorption peaks are at 430 and 455 nm. For pFL curves the Δ Absorbance represents ΔΑ (430-490 nm) and ΔΑ (455490 nm). The number of (Λ-) signifies only the relative activity or inhibition; with respect to BP hydroxylation and covalent binding of BP to DNA (+++) signifies 2-4 times and (++++) signifies 10-36 times the control rat microsomal activity; (n.d.), not determined. Figure 2 is based on data from (6, 21, 69, 70).
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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17.
AHOKAS
Cytochrome
P-450
285
T a b l e I I I . I n h i b i t i o n o f monooxygenase ( a r y l h y d r o c a r b o n h y d r o x y l a s e ) a c t i v i t y i n f i s h and mammalian h e p a t i c m i c r o s o m e s ( b a s e d on G o u j o n et al. (1+5) and A h o k a s et al. ( 2 1 ) ) . Inhibitor Rat Trout c y t o Cytochrome Cytochrome chrome P-l+50 P-l+50 P-l+1+8 ++ SKF 525A ++ Metyrapone + + ++ DDT ++ Pyridine ++ n-Octylamine ++ ++ α-Naphtho f l a v o n e ++ ++ Lindane ++ ++ Testosterone ++, s t r o n g i n h i b i t i o n ; ±, i n t e r m e d i a t e i n h i b i t i o n ; -, p o o r i n h i b i t i o n . A p p l i e s t o mouse; no i n h i b i t o r y d i f f e r e n t i a t i o n b e t w e e n r a t c y t o c h r o m e s P-l+1+8 and P-l+50 was o b s e r v e d (21.) . 1
1
show a s p e c t r a l b l u e s h i f t i n t h e S o r e t maximum o f t h e r e d u c e d c y t o c h r o m e P-l+50.CO c o m p l e x , b) i s r e a d i l y i n h i b i t e d b y a - n a p h t h o f l a v o n e (no e t h y l i s o c y a n i d e i n t e r a c t i o n s p e c t r a l d a t a was p r e s e n ted) . Induction of a r y l hydrocarbon hydroxylase a c t i v i t y i n t e l e o s t f i s h ( 2 8 , kk) and e l a s m o b r a n c h (25) has a l s o b e e n o b s e r v e d w i t h o u t a h y p s o c h r o m i c s h i f t i n t h e s p e c t r u m o f t h e c y t o c h r o m e P1+50.CO c o m p l e x . E l e c t r o n p a r a m a g n e t i c r e s o n a n c e (EPR) e x a m i n a t i o n o f h e p a t i c microsomes f r o m d i f f e r e n t l y p r e t r e a t e d a n i m a l s has l e a d t o t h e c o n c l u s i o n t h a t MC p r e t r e a t m e n t l e a d s t o t h e f o r m a t i o n o f c y t o chrome P-l+50 (P-l+1+8) w i t h h i g h s p i n i r o n (6, 1+6) . C h e v i o n et al. (28) f a i l e d t o d e m o n s t r a t e t h e p r e s e n c e o f h i g h s p i n c y t o c h r o m e P-l+50 i n a t e l e o s t f i s h e v e n a f t e r i n d u c t i o n w i t h MC. The work o f C h e v i o n et al. (28) i n d i c a t e s f u r t h e r t h a t t h e f i s h c y t o c h r o m e P1+50, w h i c h i s i n d u c i b l e w i t h P A H s and m e t a b o l i z e s r e a d i l y P A H s , i s n o t i d e n t i c a l w i t h t h e mammalian c y t o c h r o m e P-l+1+8. C e r t a i n compounds ( o t h e r t h a n e t h y l i s o c y a n i d e ) a r e known t o c a u s e s p e c t r a l changes when added t o m i c r o s o m a l s u s p e n s i o n s (6, 1 0 ) . The s p e c t r a l c h a n g e s , i n t e r a c t i o n s p e c t r a , have b e e n c l a s s i f i e d as f o l l o w s : Type I (an a b s o r p t i o n peak a t a b o u t 390 nm and a t r o u g h a t a b o u t 1+20 nm) , Type I I ( a t r o u g h n e a r 390 nm and a peak b e t w e e n 1+25 and 1+35 nm) and m o d i f i e d Type I I o r r e v e r s e Type I s p e c t r a l change ( a t r o u g h n e a r 390 nm and a peak n e a r 1+20 nm). A l l t h e s e s p e c t r a l changes h a v e b e e n o b s e r v e d t o o c c u r w i t h f i s h l i v e r m i c r o s o m a l c y t o c h r o m e P-l+50 ( Ik, 2 1 , 2 2 , 1+7). S t a n t o n and Khan ob s e r v e d Type I s p e c t r a l changes i n t r o u t l i v e r m i c r o s o m a l p r e p a r a t i o n s ( w i t h i s o d r i n , a l d r i n and h e x o b a r b i t a l as l i g a n d s ) and i n two o t h e r s p e c i e s o f f i s h b o t h Type I and Type I I s p e c t r a l changes w e r e o b s e r v e d ( 2 2 ) . On t h e o t h e r h a n d , Ahokas et al. ( l U , 2 1 , 1+7) c o u l d n o t d e m o n s t r a t e Type I s p e c t r a l change i n m i c r o s o m a l p r e p a r a t i o n s o f a n o t h e r s p e c i e s o f t r o u t u s i n g h e x o b a r b i t a l , SKF 525A 1
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
T
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o r l T o t - h y d r o x y p r o g e s t e r o n e as l i g a n d s . I t was p o s s i b l e t o demons t r a t e Type I s p e c t r a l change i n t h e s e p r e p a r a t i o n s by u s i n g BP (21) and t e t r a c h l o r o e t h e n e ( P e l k o n e n and A h o k a s , u n p u b l i s h e d obs e r v a t i o n ) as l i g a n d s . These r e s u l t s s u g g e s t t h a t t h e r e a r e d i f f e r e n c e s i n t h e c y t o c h r o m e P-l+50 s o f e v e n two r e l a t i v e l y c l o s e l y related fish. T
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A c t i v e m e t a b o l i t e s and
carcinogenicity
S a l m o n i d f i s h have become a c l a s s i c a l example o f o r g a n i s m s h i g h l y s u s c e p t i b l e t o c h e m i c a l c a r c i n o g e n s (1+8). The i n c i d e n c e o f t u m o r s i n s a l m o n i d and o t h e r s p e c i e s o f f i s h h a s b e e n e x t e n s i v e l y documented (1+8, 1+9, 50, 51) w i t h Wood and L a r s o n r e p o r t i n g t h e most a l a r m i n g 50% o c c u r r e n c e o f g r o s s t u m o r s among 250,000 a d u l t r a i n b o w t r o u t (5.2). A w i d e s p r e a d o u t b r e a k o f hepatoma i n c u l t i v a t e d r a i n b o w t r o u t i n i 9 6 0 marked t h e b e g i n n i n g o f e x t e n s i v e s e a r c h f o r a caus a t i v e a g e n t . T h i s was d e t e r m i n e d t o be a l m o s t c e r t a i n l y a f l a t o x i n ( 5 3 ) . The c a r c i n o g e n i c i t y o f s e v e r a l o t h e r compounds has b e e n i n v e s t i g a t e d i n f i s h and a t l e a s t t h e f o l l o w i n g have i n c r e a s e d t h e i n c i d e n c e o f t r o u t hepatoma b y more t h a n 20% compared w i t h c o n t r o l s : d i m e t h y l n i t r o s a m i n e , a m i n o a z o t o l u e n e , DDT and 2 - a c e t y l a m i n o f l u o r e n e (2-AAF) ( 5 ^ 0 . The c a r c i n o g e n i c i t y o f a f l a t o x i n B i ( A T B i ) and 2-AAF t o t r o u t has b e e n f o u n d t o be p r o m o t e d b y c y c l o p r o p e n o i d f a t t y a c i d s (55.). H e p a t i c t u m o u r s have b e e n i n d u c e d a l s o i n t h e guppy (Lebistes reticulatus) by A T B i , d i m e t h y l n i t r o s a m i n e and 2AAF ( 5 6 ) . The e f f e c t s o f f o u r c l a s s e s o f c a r c i n o g e n s on a q u a r i u m f i s h were i n v e s t i g a t e d b y P l i s s and K h u d o l e y (57) who s t u d i e d t h e c a r c i n o g e n i c i t y o f PAH (MC and 7 , 1 2 - d i m e t h y l b e n z ( a ) a n t h r a c e n e ) , a r o m a t i c amino compounds ( b e n z i d i n e and 2-AAF), azo compounds ( o a m i n o a z o t o l u e n e and l+-dimethylaminoazobenzene) and n i t r o s o compounds ( d i e t h y l n i t r o s a m i n e , d i m e t h y l n i t r o s a m i n e and n i t r o s o m o r p h o l i n e ) . A l l e x c e p t b e n z i d i n e and t h e two PAH compounds c a u s e d h e p a t i c t u m o r s . However, M a t s u s h i m a and S u g i m u r a (58.) d e m o n s t r a t e d t h e p r o d u c t i o n o f e p i t h e l i o m a s i n a q u a r i u m f i s h by P A H s (MC and B P ) . There have a l s o been r e p o r t s o f neoplasms i n n a t i v e bottomf e e d i n g f i s h w i t h a s u g g e s t i o n t h a t c a r c i n o g e n i c hydrocarbons from m o t o r b o a t e x h a u s t s , r o t e n o n e and i n s e c t i c i d e s s u c h as DDT may be i n v o l v e d as c a u s a t i v e a g e n t s (59.). S i m i l a r l y (an) u n i d e n t i f i e d c a r c i n o g e n ( s ) a r e s u s p e c t e d i n t h e c a s e o f adematous p o l y p s o f g a s t r i c mucosa o f f i s h , r e p o r t e d r e c e n t l y ( 6 0 ) . The d i e t o f t h e s e f i s h was f r e e o f a f l a t o x i n s . I t has b e e n p r o p o s e d t h a t i n as many as 70-80% o f t h e c a s e s o f human c a n c e r e n v i r o n m e n t a l c h e m i c a l s a r e t h e c a u s a t i v e f a c t o r s (l). T h e r e i s no r e a s o n why s i m i l a r e s t i m a t e s w o u l d n o t be v a l i d f o r animals. I t i s noteworthy t h a t the occurrence of chemical c a r cinogens i s widespread i n the a q u a t i c environment. For example, o f t h e PAH's, BP i s f o u n d i n t h e c o n c e n t r a t i o n o f 50 t o 100 yg/m i n what i s c o n s i d e r e d m o d e r a t e l y p o l l u t e d s u r f a c e w a t e r ; i n w a s t e w a t e r as much as 100,000 yg/m has b e e n m e a s u r e d ( 6 l ) . S e v e r a l 1
3
3
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
AHOKAS
17.
Cytochrome
P-450
287
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o t h e r proven c a r c i n o g e n s ( b e n z i d i n e , v i n y l c h l o r i d e ) and suspected c a r c i n o g e n s ( p o l y c h l o r i n a t e d b i p h e n y l s , DDT, c h l o r d a n e , l i n d a n e ) are c a u s i n g c o n c e r n as w i d e s p r e a d water p o l l u t a n t s o r p o t e n t i a l l y as s u c h ( 6 l ) . C y t o c h r o m e Ρ-Λ50 i n c a r c i n o g e n m e t a b o l i s m . In spite of the b e w i l d e r i n g number o f c a r c i n o g e n s i n v o l v e d t h e i m p o r t a n t a n d u n i f y i n g f a c t i s , t h a t most o f t h e o r g a n i c c a r c i n o g e n s a r e n o t c a r c i n o g e n i c per se, b u t 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 in situ b y c y t o chrome Ρ-Π50 m e d i a t e d a r y l h y d r o c a r b o n h y d r o x y l a s e (AHH, a l s o known a s BP h y d r o x y l a s e (EC l . l H . l U . 2 ) ) . A r y l hydrocarbon hydroxylase has a t t r a c t e d c o n s i d e r a b l e i n t e r e s t i n t h e r e c e n t y e a r s a s i t was n o t e d t h a t t h e h y d r o x y l a t i o n o f a r o m a t i c r i n g s t r u c t u r e s o c c u r s v i a an e p o x i d e i n t e r m e d i a t e ( 6 2 ) . These a r e n e o x i d e i n t e r m e d i a t e s h a v e s i n c e b e e n i n c r i m i n a t e d as a c t i v e i n t e r m e d i a t e s r e s p o n s i b l e f o r t h e c o v a l e n t b i n d i n g o f P A H s t o t i s s u e m a c r o m o l e c u l e s , a s i t was known f r o m t h e work o f G r o v e r a n d Sims (63.) a n d G e l b o i n (6k), t h a t s u c h b i n d i n g i s depen d e n t on m e t a b o l i c a c t i v a t i o n . I t i s w e l l known t h a t h i g h AHH a c t i v i t y i s a s s o c i a t e d w i t h c y t o c h r o m e P-UU8. However, h i g h AHH a c t i v i t y i n i t s e l f may n o t r e s u l t i n c o v a l e n t b i n d i n g , m u t a g e n i c i t y o r c a r c i n o g e n i c i t y . The p r e d o m i n a n c e o f c e r t a i n m e t a b o l i t e s a n d s a t u r a t i o n o f competitive e l i m i n a t i o n processes c o n t r i b u t e towards e v e n t s l e a d i n g t o c a r c i n o g e n i c i t y ( F i g u r e s 3 a n d k). I t i s known t h a t c y t o c h r o m e P-^50 a n d P-Ui+8 m e t a b o l i z e c e r t a i n compounds q u i t e d i f f e r e n t l y ( F i g u r e 3 ) . Most o f t h e i n f o r m a t i o n h a s b e e n o b t a i n e d u s i n g r a t a n d o t h e r common l a b o r a t o r y mammals a n d r e l a t i v e l y l i t t l e i s known how v a r i o u s c a r c i n o g e n s a n d o t h e r a r o m a t i c compounds a r e m e t a b o l i z e d b y f i s h l i v e r m i c r o s o m e s . I n 1966 L o t l i k a r et al. (65.) r e p o r t e d t h e m e t a b o l i s m o f a p o t e n t c a r c i n o g e n 2-AAF w h i c h i n r a i n b o w t r o u t c a u s e d a l o w i n c i d e n c e o f l i v e r t u m o r s . They f o u n d t h a t 2-AAF was m e t a b o l i z e d b y t r o u t l i v e r p r e p a r a t i o n s , b u t i t was n o t N - h y d r o x y l a t e d t o an i n t e r m e d i a t e known t o b e h i g h l y c a r c i n o g e n i c . T h i s w o u l d s u g g e s t t h a t t h e n a t u r e o f t h e c y t o c h r o m e P-U50 i n v o l v e d r e s e m b l e s e n z y m a t i c a l l y c o n t r o l c y t o c h r o m e P-U50 ( c f . F i g u r e 3 ) . S i m i l a r l y , b i p h e n y l m e t a b o l i s m ( n o n - c a r c i n o g e n ) was c a t a l y z e d b y t r o u t l i v e r m i c r o s o m e s a s i f t h e c y t o c h r o m e i n v o l v e d was c o n t r o l r a t l i v e r c y t o c h r o m e P-U5O; t h e m e t a b o l i t e f o r m e d was khydroxybiphenyl (37). I t has been noted t h a t c e l l c u l t u r e s d e r i v e d from t r o u t ( 6 6 , 67) a n d t r o u t l i v e r m i c r o s o m e s (Î2, Î7» 68) may have r e l a t i v e l y h i g h AHH a c t i v i t y (BP h y d r o x y l a s e ) w h i c h sometimes e x c e e d s t h e a c t i v i t y o b s e r v e d i n c o n t r o l r a t l i v e r m i c r o s o m e s . The m e t a b o l i t e p a t t e r n o b t a i n e d u s i n g t r o u t l i v e r microsomes resembled t h a t p r o d u c e d b y MC t r e a t e d r a t l i v e r m i c r o s o m e s (P-UU8)(6£ a n d A h o k a s , S a a r n i , Nebert and Pelkonen, manuscript i n p r e p a r a t i o n ; Table I V ) . A s s o c i a t e d w i t h t h i s p a t t e r n o f BP m e t a b o l i t e s was c o v a l e n t b i n d i n g t o DNA w h i c h was t h r e e t i m e s a s h i g h as o b t a i n e d b y u s i n g r a t l i v e r m i c r o s o m e s . A n o t h e r s p e c i e s o f f i s h ( r o a c h ) , on t h e o t h e r h a n d was f o u n d t o be a l m o s t i n a c t i v e i n c a t a l y z i n g in vitro bind1
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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CH>"
OH Figure 3. Preferential routes of metabolism of 2-AAF, biphenyl, and BP (from top to bottom) by different forms of rat liver microsomal Cytochrome P-450. Trout has been reported to metabolize 2-AAF (65) and biphenyl (37) like rat liver Cytochrome P-450 and BP like rat liver Cytochrome P-448 (69,70).
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
17.
AHOKAS
Cytochrome
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Procarcinogen (PAH)
Dihydrodiol
P-450s
*
6SH conjugates
289
P-450
^ Phenol
C
» Conjugates
Epoxide
Covalent binding to DNA RNA protein
Carcinogenesis Mutation Terratogenesis Tissue necrosis Autoimmune response Figure 4.
Proposed scheme for Cytochrome P-450 mediated metabolism (inactivation and activation) of procarcinogens (e.g. PAHs).
Conversion of epoxides (arene oxides) into phenoh is spontaneous. The conversion of epoxides into dihydrodiols is catalyzed by EH (EC 4.2.1.63). Hydroxyl containing PAHs can act as substrates for conjugases (C) (UDP glucuronsyl transferase (EC 2.4.1.17) and phenol sulphotransferase (EC 2.8.2.1 )). This pathway usually leads to inactive excretable products. Epoxides are scavenged by GSH and the reaction is catalyzed by GSHt (EC 2.5.1.18). When GSH is depleted and/or the other pathways are saturated, epoxides of dihydrodiols (particularly 7,8-diol-9,10-epoxides in the case of BP) and phenol metabolites react with cellular macromolecules such as DNA, RNA, and protein. If repair mechanisms are exceeded the detrimental effects of PAH may result.
In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M. A. Q., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.
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T a b l e I V . C o m p a r i s o n o f t h e r e l a t i v e amounts o f BP m e t a b o l i t e s f o r m e d by t r o u t , c o n t r o l r a t and MC t r e a t e d r a t l i v e r m i c r o s o m e s . Metabolite MC-rat Control rat Trout % % % Total 100* 137
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62k
20 9,10-dihydrodiol 22 19 16 Τ,8-dihydrodiol 28 10 h h,5-dihydrodiol 1 6 3-phenol 33 9-phenol k 2 1 quinones 11+ 20 13 * T o t a l amount o f m e t a b o l i t e s f o r m e d by c o n t r o l r a t l i v e r m i c r o s o m a l f r a c t i o n i s a r b i t a r i l y t a k e n as t h e r e f e r e n c e f o r t h e o t h e r two t o t a l s ( d a t a f r o m (7Ό) ). ing
o f BP t o DNA. The f a c t t h a t t r o u t l i v e r m i c r o s o m a l p r e p a r a t i o n can p r o duce s i g n i f i c a n t amounts o f n u c l e o s i d e a d d u c t s o f BP 7,8-diol-9,10 e p o x i d e and a c t i v a t e d BP p h e n o l s (70) i n d i c a t e s t h a t f i s h h a v e enzymatic c a p a b i l i t y to a c t i v a t e carcinogens to r e a c t i v e interme d i a t e s . The in vivo s i g n i f i c a n c e o f t h e s e f i n d i n g s c a n n o t be p r e d i c t e d a t t h e moment. A l t h o u g h t h e c r i t i c a l c e l l u l a r t a r g e t s o f c h e m i c a l mutagens and c a r c i n o g e n s a r e n o t f u l l y known, DNA i s a p r i m e s u s p e c t . Whether o r n o t t h e r e i s any c o r r e l a t i o n b e t w e e n in vitro a c t i v a t i o n and c o v a l e n t b i n d i n g o f a c a r c i n o g e n and i t s in vivo e f f e c t s , r e m a i n s t o be s e e n . However, a t l e a s t i n one i n s t a n c e w i t h r e s p e c t t o m u t a g e n e s i s i n Salmonella typhimurium m u t a g e n e s i s a s s a y ( 7 1 ) , we have shown t h a t t h e r e a c t i v e i n t e r m e d i a t e s f o r m e d by t r o u t l i v e r m i c r o s o m e s c a n p r o d u c e b i o l o g i c a l e f f e c t s ( 7 2 ) . T h i s f i n d i n g has b e e n r e c e n t l y c o n f i r m e d ( 7 3 , 7*0 · Conclusions I t appears t h a t v a r i o u s f i s h possess d i f f e r e n t types o f c y t o c h r o m e P-l+50, w i t h r e s u l t a n t 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 c a r c i n o g e n s and r e l a t e d compounds. There a r e marked s p e c i e s d i f f e r e n c e s i n t h e p r o d u c t i o n o f DNA b i n d i n g m e t a b o l i t e s o f BP. Several reports indicate that f i s h l i v e r microsomes can a c t i v a t e promutagens i n t o mutagenic i n t e r m e d i a t e s . However, t h e r e i s a l a c k o f u n i f i e d p i c t u r e , m a i n l y b e c a u s e t h e c h a r a c t e r i z a t i o n o f c y t o c h r o m e P-l+50, c y t o c h r o m e P-l+50 m e d i a t e d c a r c i n o g e n m e t a b o l i s m and i n d u c t i o n o f c a n c e r h a v e b e e n p e r f o r m e d i n most c a s e s b y d i f f e r e n t l a b o r a t o r i e s , u s i n g d i f f e r e n t s o u r c e s o f f i s h and o f t e n d i f f e r e n t s p e c i e s o f f i s h . Not u n t i l a c o n s o r t e d e f f o r t i s made t o c h a r a c t e r i z e c y t o c h r o m e P-l+50 m e d i a t e d AHH and c a r c i n o g e n e s i s i n one p o p u l a t i o n o f f i s h , can we e x p e c t c o n c l u s i v e r e s u l t s on i n d u e i b i l i t y , c a r c i n o g e n a c t i v a t i o n and r e s u l t i n g s o m a t i c m u t a t i o n and c a r c i n o g e n e s i s .
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