Pesticide and Xenobiotic Metabolism in Aquatic Organisms - American

17 Cytochrome P-450 in Fish Liver Microsomes and

Downloaded by GEORGE MASON UNIV on February 25, 2016 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch017

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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PESTICIDE AND XENOBIOTIC M E T A B O L I S M IN AQUATIC ORGANISMS


( 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.


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



282


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-


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.


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


PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

284

RAT

TROUT


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).



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


T

286


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


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


AHOKAS

17.

Cytochrome

P-450

287


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



288

PESTICIDE AND XENOBIOTIC M E T A B O L I S M I N AQUATIC

ORGANISMS

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).


17.

AHOKAS

Cytochrome


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.



290

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


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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Literature Cited 1 Heidelberger, C.: Chemical carcinogenesis. Ann. Rev. Biochem. (1975) 44, 79-121. 2 Klingenberg, M.: Pigments of rat liver microsomes. Arch. Biochem. Biophys. (1958) 75, 376-386. 3 Garfinkel, D.: Studies on pig liver microsomes. I. Arch. Biochem. Biophys. (1958) 77, 493-509. 4 Omura, T. and Sato, R.: The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. (1964) 239, 2370-2378. 5 Omura, T. and Sato, R.: The carbon monoxide-binding pigment of liver microsomes. II. Solubilization, purification, and properties. J. Biol. Chem. (1964) 239, 2379-2385. 6 Mannering, G.J.: Microsomal enzyme systems which catalyze drug metabolism. In La Du, B.N., Mandel, H.G. and Way, E.L. (Eds.): Fundamentals of Drug Metabolism and Disposition. Baltimore, Williams. (1971). 7 Mannering, G.J.: Properties of cytochrome P-450 as affected by environmental factors: Qualitative changes due to administration of polycyclic hydrocarbons. Metabolism (1971) 20, 228-245. 8 Gillette, J.R., Davis, D.C. and Sasame H.A.: Cytochrome P450 and its role in drug metabolism. Ann. Rev. Pharmacol (1972) 12, 57-84. 9 Cooper, D.Y.: Discovery of the function of the heme protein P-450: A systematic approach to scientific research. Life Sci. (1973) 13, 1151-1161. 10 Orrenius, S. and Ernster, L.: Microsomal cytochrome P-450linked monooxygenase systems in mammalian tissues. In Hayaishi, O. (Ed.): Molecular Mechanisms of Oxygen Activation (1974). Academic Press, New York. pp 215-244. 11 Gunsalus, I.C., Pederson, T.C. and Sligar S.G.: Oxygenasecatalyzed biological hydroxylations. Ann. Rev. Biochem. (1975) 44, 377-407. 12 Orrenius, S.: Reaction mechanisms of cytochrome P-450. Proceedings Sixth International Congress of Pharmacology, Helsinki, Finland (1975) 6, 39-52. 13 Wickramasinghe, R.H.: Biological aspects of cytochrome P450 and associated hydroxylation reactions. Enzyme (1975) 19, 348-376. 14 Ahokas, J.T., Pelkonen, O. and Karki, N.T.: Cytochrome P-450 and drug-induced spectral interactions in the hepatic microsomes of trout, Salmo trutta lacustris. Acta Pharmacol. et Toxicol. (1976) 38, 440-449. 15 Garfinkel, D: A comparative study of electron transport in microsomes. Comp. Biochem. Physiol. (1963) 8, 367-379. 16 Fukami, J-I., Shishido, T., Fukunaga, K. and Casida, J.E.: Oxidative metabolism of rotenone in mammals, fish and insects and its relation to selective toxicity. J. Agr. Food. Chem.


292

17 18


19 20 21 22 23 24

25

26 27 28

PESTICIDE

AND

XENOBIOTIC

METABOLISM

IN

AQUATIC

ORGANISMS

(1969) 17, 1217-1226. Ichikawa, Y. and Yamano, T.: Electron spin resonance of microsomal cytochromes. Arch. Biochem. Biophys. (1967) 121, 742-749. Lidman, U., Förlin, L., Molander, O. and Axelson, G.: Induction of the drug metabolizing system in rainbow trout (Salmo gairdnerii) liver by polychlorinated biphenyls (PCBs). Acta Pharmacol. et Toxicol. (1976) 39, 262-272. Chan, T.M., Gillett, J.W. and Terriere, L.C.: Interaction between microsomal electron transport systems of trout and male rat in cyclodiene epoxidation. Comp. Biochem. Physiol. (1967), 20, 731-742. Pedersen, M.G. and Hershberger, W.K.: Metabolism of 3,4benzpyrene in rainbow trout (Salmo Gairdneri). Bull. Environ. Contam. Toxicol. (1974) 12, 481-486. Ahokas, J.T., Pelkonen, O. and Kärki, N.T.: Characterization of benzo(a)pyrene hydroxylase of trout liver. Cancer Res. (1977) 37, 3737-3743. Stanton, R.H. and Khan, M.A.Q.: Components of the mixed-function oxidase of hepatic microsomes of fresh water fishes. Gen. Pharmac. (1975) 6, 289-294. Pohl, R.J., Bend, J.R., Guarino, A.M. and Fouts, J.R.: Hepatic microsomal mixed-function oxidase activity of several marine species from coastal Maine. Drug Metab. Dispos. (1974) 2, 545-555. Bend, J.R., Pohl, R.J., Davidson, N.P. and Fouts, J.R.: Response of hepatic and renal microsomal mixed-function oxidases in the little skate, Raja erinacea, to pretreatment with 3-methyl-cholanthrene or TCDD (2,3,7,8tetrachlorodibenzo-p-dioxin). Bull. Mt. Desert Is. Biol. Lab. (1974) 14, 7-11. Bend, J.R., Pohl, R.J., Arinc, E. and Philpot, R.M.: Hepatic microsomal and solubilized mixed-function oxidase systems from the little skate, Raja erinacea, a marine elasmobranch. In Ullrich, V., Hildebrandt, Α., Roots, I., Eastabrook, R.W. (Eds.): Microsomes and Drug Oxidations (1976). Pergamon Press, Oxford, pp 160-169. Bend, J.R., James, M.O. and Dansette, P.M.: In Vitro metabolism of xenobiotics in some marine animals. Ann. N.Y. Acad. Sci. (1977) in press. Burns, K.A.: Microsomal mixed function oxidases in an estuarine fish, fundulus heteroclitus, and their induction as a result of environmental contamination. Comp. Biochem. Physiol. (1976) 53B, 443-446. Chevion, M., Stegeman, J.J., Peisach, J. and Blumberg, W.E.: Electron paramagnetic resonance studies on hepatic micro somal cytochrome P-450 from a marine teleost fish. Life Sci. (1977) 20, 895-900.


17.

29 30


31 32 33

34 35 36

37 38 39 40

AHOKAS

Cytochrome

P-450

293

Kulkarni, A.P., Smith, E. and Hodgson, E.: Occurrence and characterization of microsomal cytochrome P-450 in several vertebrate and insect species. Comp. Biochem. Physiol. (1976) 54B, 509-513. Wada, F., Higashi, T., Ichikawa, Y., Tada, K. and Sakamoto, Y.: The relationship between electron spin resonance signals and haemoproteins of liver microsomes. Biochim. Biophys. Acta (1964) 88, 654-655. Greim, H., Schenkman, M., Klotzbucher, M. and Remmer, H.: The influence of phenobarbital on the turnover of hepatic microsomal cytochrome b, and cytochrome P-450 in the rat. Biochim. Biophys. Acta (1970) 201, 20-25. Ryan, D., Lu, A.Y.H., Kawalek, J., West, S.Β. and Levin, W.: Highly purified cytochrom P-448 and P-450 from rat liver microsomes. Biochem. Biophys. Res. Commun. (1974) 64, 1134-1141. Mueller, G.C. and Miller, J.A.: The reductive cleavage of 4-dimethylaminoazobenzene by rat liver: the intracellular distribution of the enzyme system and its requirement for triphosphopyridine nucleotide. J. Biol. Chem. (1949) 180, 1125-1136. Mueller, G.C. and Miller, J.A.: The metabolism of methyl ated aminoazo dyes. II. Oxidative demethylation by rat liver homogenates. J. Biol. Chem. (1953) 202, 579-587. Brodie, B.B., Axelrod, J., Cooper, J.R., Gaudette, L., La Du, B.N., Mitoma, C., and Udenfriend, S.: Detoxication of drugs and other foreign compounds by liver microsomes. Science (1955) 121, 603-604. Coon, M.J., Ballou, D.P., Haugen, D.A., Krezoski, S.O., Nordblom, G.D. and White, R.E.: Purification of membranebound oxygenases: isolation of two electrophoretically homogeneous forms of liver microsomal cytochrome P-450. In Ullrich, V., Hildebrandt, Α., Roots, I., Eastabrook, R.W. (Eds.): Microsomes and Drug Oxidations (1976). Pergamon Press, Oxford, pp 82-94. Creaven, P.J., Parke, D.V. and Williams, R.T.: A fluorimetric study of the hydroxylation of biphenyl in vitro by liver preparations of various species. Biochem. J. (1965) 96, 879-885. Ahokas, J.T.: Metabolism of 2,5-diphenyloxazole (PPO) by trout liver microsomal mixed function monooxygenase. Res. Commun. Chem. Pathol. Pharmacol. (1976) 13, 439-447. Thomas, P.E., Lu, Α.Y.H., Ryan, D., West, S.B., Kawalek, J. and Levin, W.: Immunochemical evidence for six forms of rat liver cytochrome P450 and p448. Mol. Pharmacol. (1976) 12, 746-758. Sladek, N.E. and Mannering, C.J.: Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene treated rats. Biochem. Biophys. Res. Commun. (1966) 24, 668-674.



294

PESTICIDE

AND

XENOBIOTIC

METABOLISM

IN

AQUATIC

ORGANISMS

41 Alvares, A.P., Schilling, G., Levin, W. and Kuntzman, R.: Studies on the induction of CO-binding pigments in liver microsomes by phenobarbital and 3-methylcholanthrene. Biochem. Biophys. Res. Commun. (1967) 29, 521-526. 42 Ahokas, J.T., Pelkonen, O. and Kärki, N.T.: Metabolism of polycyclic hydrocarbons by a highly active aryl hydrocarbon hydroxylase system in the liver of a trout species. Biochem. Biophys. Res. Commun. (1975) 63, 635-641. 43 Atlas, S.A., Boobis, A.R., Felton, J.S., Thorgeirsson, S.S. and Nebert, D.W.: Ontogenetic expression of polycyclic aromatic compound-inducible monooxygenase activities and forms of cytochrome P-450 in rabbit. J. Biol. Chem. (1977) 252, 4712-4721. 44 Pedersen, M.G., Hershberger, W.K., Zachariah, P.K. and Juchau, M.R.: Hepatic biotransformation of environmental xenobiotics in six strains of rainbow trout(Salmogairdneri) J. Fish Res. Board Can. (1976) 33, 666-675. 45 Goujon, F.M., Nebert, D.W. and Gielen, J.E. : Genetic expression of aryl hydrocarbon hydroxylase induction. IV. Interaction of various compounds with different forms of cytochrome P-450 and the effect on benzo(a)pyrene metabolism in vitro. Mol. Pharmacol. (1972) 8, 667-680. 46 Nebert, D.W., Kumaki, K., Sato, M. and Kon, H.: Association on Type I, Type II, and reverse Type I difference spectra with absolute spin state of cytochrome P-450 iron. In Ullrich, V., Hildebrandt, Α., Roots, I., Eastabrook, R.W. (Eds.): Microsomes and Drug Oxidations (1976). Pergamon Press, Oxford, pp224-231. 47 Ahokas, J.T., Pelkonen, O. and Kärki, N.T.: Cytochrome P450 and drug-induced spectral interactions in the hepatic microsomes of a trout sp. Second Congress Hungarian Pharmacological Society, Budapest (1974) pp 11-14. 48 Ashley, L.M.: Animal model of human disease. Am. J. Path. (1973) 72, 345-348. 49 Harsbarger, J.C.: Activities Report, Registry of Tumors in Lower Animals 1965-73. Smithsonian Institution, Washington, D.C. (1973). 50 Harsbarger, J.C.: Activities Report, Registry of Tumors in Lower Animals 1974. Smithsonian Institution, Washington, D.C. (1974). 51 Harsbarger, J.C.: Activities Report, Registry of Tumors in Lower Animals, 1975. Smithsonian Institution, Washington, D.C. (1975). 52 Wood, E.M. and Larson, C.P.: Hepatic carcinoma in rainbow trout. Arch. Pathol. (1961) 71, 471-479. 53 Halver, J.E.: Crystalline aflatoxin and other vectors for trout hepatoma. Res. Rep. US Fish. Wildl. Serv. (1967) 70:78-102. 54 Kraybill, H.F. and Shimkin, M.B.: Carcinogenesis related to foods contaminated by processing and fungal metabolites.


17.

AHOKAS

Cytochrome

P-450

295

Adv. Cancer Res. (1964) 8, 191-248. 55 Lee, D.J., Wales, J.H., Ayres, J.L. and Sinnhuber, R.O.: Synergism between cyclopropenoid fatty acids and chemical carcinogens in rainbow trout (Salmo gairdneri). Cancer Res. (1968) 28, 2312-2318. 56 Sato, S., Matsushima, T., Tanaka, N., Sugimura T. and Takashima, F.: Hepatic tumors in the guppy (Lebistes reticulatus) induced by aflatoxin B dimethylnitrosamine, and 2-acetylaminofluorene. J. Natl. Cancer Inst. (1973) 50, 765-778. 57 Pliss, G.B. and Khudoley, V.V.: Tumor induction by carcino genic agents in aquarium fish. J. Natl. Cancer Inst. (1975) 55, 129-136. 58 Matsushima, T. and Sugimura, T.: Experimental carcinogenesis in small aquarium fishes. Prog. Exp. Tumor Res. (1976) 20, 367-379. 59 Dawe, C.J., Stanton, M.F. and Schwartz, F.J.: Hepatic neo plasms in native bottom-feeding fish of Deep Creek Lake, Maryland. Cancer Res. (1964) 24, 1194-1201. 60 Kimura, I., Miyake, T., Kubota, S., Kamata, Α., Morikawa, S. and Ito, Y.: Adenomatous polyps in the stomachs of hatchery -grown salmonids and other types of fishes. Prog. Exp. Tumor Res. (1976) 20, 181-194. 61 Kraybill, H.F.: Distribution of chemical carcinogens in aquatic environments. Prog. Exp. Tumor Res. (1976) 20, 3-34. 62 Jerina, D.M. and Daly, J.W.: Arene oxides: A new aspect of drug metabolism. Science (1974) 185, 573-582. 63 Grover, R.L. and Sims, P.: Enzyme-catalyzed reactions of polycyclic hydrocarbons with deoxyribonucleic acid and prot ein in vitro. Biochem. J. (1968) 110, 159-160. 64 Gelboin, H.V.: A microsome-dependent binding of benzo(a) pyrene to DNA. Cancer Res. (1969) 29, 1272-1276. 65 Lotlikar, P.D., Miller, E.C., Miller, J.A. and Halver, J.E.: Metabolism of the carcinogen 2-acetylaminofluorene by rain bow trout. Proc. Soc. Exp. Med. (1967) 124, 160-163. 66 Diamond, L. and Clark, H.F.: Comparative studies on the interaction of benzo(a)pyrene with cells derived from poikilothermic and homeothermic vertebrates. I. Metabolism of benzo(a)pyrene. J. Natl. Cancer Inst. (1970) 45, 10051011. 67 Clark, H.F. and Diamond, L.: Comparative studies on the interaction of benzpyrene with cells derived from poikilo thermic and homeothermic vertebrates. II. Effect of temperature on benzpyrene metabolism and cell multiplication. J. Cell. Physiol. (1971) 77, 385-392. 68 Pedersen, M.G., Hershberger, W.K. and Juchau, M.R.: Metabolism of 3,4-benzpyrene in rainbow trout(Salmogaird neri). Bull, Environ. Contam. Toxicol. (1974) 12, 481-486.


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PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

69 Ahokas, J.T., Pelkonen, O. and Kärki, N.T.: The possible role of trout liver aryl hydrocarbon hydroxylase in activating aromatic polycyclic carcinogens. In Jollow, D.J. Kocsis, J.J., Snyder, R. and Vainio, H. (Eds.): Biological Reactive Intermediates. Formation, Toxicity, and inactivation (1977). Plenum Press, New York, pp 162-166. 70 Ahokas, J.T., Saarni, H., Nebert, D.W. and Pelkonen, O.: The in vitro metabolism and covalent binding of benzo(a) pyrene to DNA catalysed by trout liver microsomes. Manuscript in preparation. 71 Ames, B.N., McCann, J. and Yamasaki, E.: Methods for detecting carcinogens and mutagens with Salmonella/mammalianmicrosome mutagenicity test. Mutat. Res. (1975) 31, 347. 72 Ahokas, J., Pääkkönen, R., Rönnholm, K., Raunio, V., Kärki, N. and Pelkonen, O: Oxidative metabolism of carcinogens by trout liver resulting in protein binding and mutagenicity. In Ullrich, V., Roots, I., Hildebrandt, Α., Eastabrook R.W. and Conney, A.H. (Eds.): Microsomes and Drug Oxidations (1977). Pergamon Press, Oxford pp 435-441. 73 Payne, J.F., Martins, I. and Rahimtula, Α.: Crankcase oils: Are they a major mutagenic burden in the aquatic environment? Science (1978) 200, 329-330. 74 Stott, W.T. and Sinnhuber, R.O.: Trout hepatic enzyme activ ation of aflatoxin-B1 in a mutagen assay system and inhibit ory effects of PCBs. Bull. Environ. Contam. Toxicol. (1978) 19, 35. RECEIVED January 2, 1979.


Pesticide and Xenobiotic Metabolism in Aquatic Organisms - American

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