Microsomal Mixed-Function Oxidation in Untreated and Polycyclic

microsomal AHH activity in control and DBA-treated little skates. (including metabolic profiles obtained from. C-benzo(a)pyrene as elucidated by high ...
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18 Microsomal Mixed-Function Oxidation in Untreated and Polycyclic Aromatic Hydrocarbon-Treated Marine Fish 1

JOHN R.BEND ,LOUISE M. BALL, TAHANI H. ELMAMLOUK, MARGARET O.JAMES ,and RICHARD M. PHILPOT Laboratory of Pharmacology, National Institute of Environmental Health Sciences/NIH, Research Triangle Park, NC 27709

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2

1

The aquatic environment is a repository for numerous foreign organic chemicals (xenobiotics) that occur as environmental pollutants. Dumping of industrial and urban wastes, leaching from landfill sites, atmospheric fallout of airborne particles, runoff from cultivated land that has been treated with insecticides or herbicides, accidental or intentional spillage during shipping, and seepage of hydrocarbons from underwater oil deposits all contribute to this pollution. As certain xenobiotics, such as the polycyclic aromatic hydrocarbon, benzo(a)pyrene, are metabolically activated to products that are toxic (mutagenic, carcinogenic, cytotoxic) to mammals, including man, the ability of aquatic species to biotransform and excrete xenobiotics is of considerable importance, especially in those species that are used for food. Based on this rationale, we have been investigating the in vitro and in vivo metabolism of xenobiotics in marine vertebrate and invertebrate species. Attention has focused upon the cytochrome P-450-dependent microsomal mixed-function oxidase (MFO) system (16), which is very important in the oxidative metabolism of most lipophilic pollutants including hydrocarbons, and those enzymes which are responsible for the further biotransformation of toxic alkene or arene oxides (4) as well as conjugation pathways for chlorinated phenoxyacetic acid herbicides (7, 8). In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinacea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPHcytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin '-' For further author affiliations see page 318. This chapter not subject to U.S. copyright. Published 1979 American Chemical Society In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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298

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

ORGANISMS

(TCDD), p o l y c h l o r i n a t e d biphenyls (Arochlor 1254), or polybrominated biphenyls (Firemaster FF1) to marine vertebrates such as l i t t l e s k a t e , winter flounder (Pseudopleuronectes amerieanus), southern flounder [Paralichthyes lethostigma), or sheepshead [Arohosargus probatocephalus) r e s u l t s i n dramatic increases (up to 3 5 - f o l d ) i n hepatic microsomal benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase [AHH]) a c t i v i t y (2, 3, 4, 6, 9, 10). This i s to be expected from s i m i l a r observations i n several mammalian species. However, i n f i s h treated with DBA or TCDD, under c o n d i t i o n s where i n d u c t i o n of AHH a c t i v i t y was observed, there was no apparent wavelength s h i f t i n the absorption maximum of the C0bound form of reduced cytochrome P-450 i n hepatic microsomes ( i . e . , no i n d i c a t i o n of cytochrome P-448 formation) nor was there a s i g n i f i c a n t increase i n microsomal cytochrome P-450 content (3). The formation o f cytochrome P-448 was a n t i c i p a t e d in l i v e r s of these DBA- or TCDD-treated f i s h because increases i n AHH a c t i v i t y r e s u l t i n g from a d m i n i s t r a t i o n of p o l y c y c l i c hydrocarbon-like inducing agents to mammals are normally associated with cytochrome P-448 (11, 12). Consequently, we have i n v e s t i g a t e d the p o l y c y c l i c hydrocarbon-induced hepatic microsomal system of a few marine f i s h i n some d e t a i l . In t h i s r e p o r t we compare several properties of hepatic microsomal AHH a c t i v i t y i n c o n t r o l and DBA-treated l i t t l e skates ( i n c l u d i n g metabolic p r o f i l e s obtained from C-benzo(a)pyrene as e l u c i d a t e d by high pressure l i q u i d chromatography [HPLC]), we d e s c r i b e the p a r t i a l p u r i f i c a t i o n of two d i f f e r e n t forms of cytochrome P-450 (cytochrome P-448 and cytochrome P-451) from hepatic microsomes of DBA-pretreated l i t t l e skates and we r e p o r t p o l y c y c l i c hydrocarbon-like induction i n large numbers of winter flounder assayed i n Maine during June, J u l y , and August, which was q u i t e d i f f e r e n t than data obtained with sheepshead examined i n F l o r i d a during the same p e r i o d . M a t e r i a l s and Methods F i s h C o l l e c t i o n , Maintenance, and Treatment. Adult f i s h were c o l l e c t e d near Mount Desert I s l a n d , Maine, or Marineland, F l o r i d a , and were acclimated i n aquaria equipped with continuously flowing seawater or i n l i v e cars immersed i n s a l t water f o r at l e a s t 24 hr before u s e . For i n d u c t i o n s t u d i e s l i t t l e skates were i n j e c t e d IP with 10 mg/kg 1,2,3,4-dibenzanthracene in corn o i l on days 1, 2, and 3 and were s a c r i f i c e d on day 10. Control f i s h were i n j e c t e d with corn o i l o n l y . Whole Homogenate and Microsome P r e p a r a t i o n , Enzyme Assays. A l l f i s h were s a c r i f i c e d between 5:00 and 9:00 a.m. Washed microsomes were prepared from l i v e r homogenates as described p r e v i o u s l y (3). When whole l i v e r homogenates were used as the enzyme s o u r c e , 10% w/v homogenates were prepared i n 0.15 M KC1, 0.02 M HEPES (N2-hydroxypiperazine-N'-2-ethane s u l f o n i c a c i d ; Sigma) buffer (pH

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

18.

BEND ET AL.

Microsomal Mixed-Function

Oxidation

299

7.4). AHH a c t i v i t i e s were determined as p r e v i o u s l y described ~ ( 3 ) , except that a d d i t i o n a l incubation mixtures c o n t a i n i n g 10Z M and 10" M α-naphthoflavone (microsomes) or 5 χ 10" M and 10" M α-naphthoflavone (whole homogenate) were assayed simultaneously. One AHH f l u o r e s c e n c e u n i t equals the f l u o r e s c e n c e i n t e n s i t y of a 3 yg/ml quinine s u l f a t e " 2H 0 i n 0.1 Ν s u l f u r i c a c i d s o l u t i o n ( e x c i t a t i o n λ 425 nm, emission λ 555 nm). 7-Ethoxyresorufin deethylase a c t i v i t y was assayed e s s e n t i a l l y as described by Burke and Mayer (13) a t a 7-ethoxyresorufin concentration of 2 μΜ, a f i n a l pH of 7 . 8 , and an incubation temperature of 3 0 ° (14). Epoxide hydratase a c t i v i t y , with H-benzo(a)pyrene 4 , 5 - o x i d e as s u b s t r a t e , was assayed by the t h i n - l a y e r chromatographic procedure of J e r i n a et at. (15). The p r o t e i n content of microsomal and whole homogenate preparations was determined according to Lowry et al. (16), using bovine serum albumin as the standard, and m i c r o ­ somal cytochrome P-450 content was assayed by the method o f Omura and Sato (17) on an Aminco DW-2A spectrophotometer.

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Metabolic P r o f i l e of ^C-Benzo(a)pyrene i n Skate Hepatic Microsomes. Microsomal suspensions (1 mg/ml) i n 0.5 M HEPES b u f f e r (pH 7.4) were preincubated f o r 1 min with 100 nmol 7,10C-benzo(a)pyrene (4.1 mCi/mmol, radiochemical p u r i t y > 97.5%, purchased from C a l i f o r n i a Bionuclear C o r p o r a t i o n , Sun V a l l e y , CA) d i s s o l v e d i n acetone (10 y l ) . The r e a c t i o n was i n i t i a t e d by the a d d i t i o n o f NADPH and was incubated at 3 1 ° f o r up to 30 min. The r e a c t i o n was terminated by the a d d i t i o n of 2 ml ethyl a c e t a t e / a c e ­ t o n e , 2 : 1 , v / v , saturated with water, and v o r t e x i n g . After c e n t r i f u g a t i o n and removal of the upper l a y e r , the incubation mixtures were extracted twice more with 2 ml of the ethyl acetate/acetone solution. The upper phases were combined, stored overnight at - 2 0 ° to remove r e s i d u a l water, then evaporated to dryness i n the dark at room temperature under a stream of n i t r o g e n . The residues were taken up i n 40 yl o f methanol (glass d i s t i l l e d , Burdick and Jackson L a b o r a t o r i e s , Muskegon, MI) a n d , a f t e r a d d i t i o n o f a methanolic s o l u t i o n (5 y l ) o f unlabeled authentic benzo(a)pyrene metabolites ( k i n d l y supplied by the National Cancer I n s t i t u t e - N I H C a r c i n o ­ genesis Research Program) analyzed by HPLC on a 1 m 0DS Permaphase column i n a DuPont Model 830 instrument, e s s e n t i a l l y as described by Holder et al. (18). R a d i o a c t i v i t y was quantitated by l i q u i d s c i n t i l l a t i o n counting (Beckman LS 9000 instrument) using 10 ml Instagel (Packard Instrument C o . , I n c . , Downers Grove, IL) and a p p r o p r i a t e l y quenched standards. S o l u b i l i z a t i o n and P a r t i a l P u r i f i c a t i o n of Cytochrome P-450 from Hepatic Microsomes of Male, DBA-Pretreated L i t t l e Skates" Washed hepatic microsomes (3) from the l i v e r s of 10 skates were suspended i n 0.25 M sucrose and f r o z e n under nitrogen (-5 to - 1 0 ° ) at the Maine l a b o r a t o r y . They were then packed i n dry i c e and transported to NIEHS, Research T r i a n g l e Park, NC, w i t h i n 14 days of preparation and were stored a t - 6 2 ° C u n t i l u s e . Microsomes

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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300

PESTICIDE

A N D XENOBIOTIC

METABOLISM

IN AQUATIC

ORGANISMS

from male and female skates (both control and DBA-pretreated) were pooled s e p a r a t e l y . Thawed microsomal preparations (500-700 mg protein) from l i t t l e skates were digested with sodium cholate (1 mg/mg protein) i n 10 mM potassium phosphate buffer (pH 7.7) to make a f i n a l con­ c e n t r a t i o n of 10 mg p r o t e i n / m l , i n the presence of 0.1 mM EDTA, 0.1 mM d i t h i o t h r e i t o l , and 20% g l y c e r o l . Digestion was c a r r i e d out at room temperature f o r 20 min with constant s t i r r i n g . The digested microsomal preparation was c e n t r i f u g e d at 176,000x0 f o r 60 min. The p r e c i p i t a t e was discarded and the c l e a r supernatant was a p p l i e d to a DEAE-cellulose column (2.5 χ 38 cm) p r e v i o u s l y e q u i l i b r a t e d with 10 mM potassium phosphate b u f f e r , pH 7 . 7 , c o n t a i n i n g 0.1 mM EDTA, 0.1 mM d i t h i o t h r e i t o l , 0.1% sodium c h o l a t e , and 20% g l y c e r o l (Buffer I). The column was eluted with 1000 ml b u f f e r I c o n t a i n i n g 0.5% Emulgen 913 (obtained from KaoA t l a s , Tokyo, Japan; Buffer II). The column was subsequently eluted with B u f f e r II c o n t a i n i n g a l i n e a r KC1 gradient (0-0.5 M) at a flow r a t e of 60 ml/hr as described by P h i l p o t et al. (19). F r a c t i o n s (250 d r o p s , 8-15 ml) obtained from the column were monitored at 418 nm, and f o r t h e i r cytochrome P-450 content, NADPH-cytochrome c reductase a c t i v i t y (determined according to Williams and Kamin [ 2 0 ] ) , and epoxide hydratase a c t i v i t y . Cytochrome P-450 f r a c t i o n s were pooled and the f r e e Emulgen 913 removed from the enzyme preparation by s t i r r i n g with Amberlite XAD-2 beads followed by f i l t r a t i o n . The f i l t r a t e was concentrated i n an Ami con u l t r a f i l t r a t i o n c e l l using a YM 10 D i a f l o membrane. D i a l y s i s was c a r r i e d out i n 2 l i t e r s of Buffer I f o r 24 hr when required. The f r a c t i o n s c o n t a i n i n g cytochrome P-450 were stored under nitrogen i n 0.5 ml a l i q u o t s at - 6 2 ° . R e c o n s t i t u t i o n o f Benzo(a)pyrene Hydroxylase A c t i v i t y i n Systems Containing Cytochrome P-448 Obtained from Hepatic M i c r o ­ somes of DBA-Treated L i t t l e Skates. Rabbit hepatic microsomal phospholipids (125 y g , only where required) and sodium cholate (125 yg) i n acetoneimethanol, 3 : 1 , v / v , were mixed and evaporated to dryness. The cytochrome P-450 preparation (0.02 nmol) and NADPH-cytochrome ο reductase (115 u n i t s , or as s p e c i f i e d i n 2-10 yl of 10 mM phosphate b u f f e r , pH 7.7; p u r i f i e d from r a b b i t l i v e r and generously supplied by C. S e r a b j i t - S i n g h , Laboratory of Pharma­ c o l o g y , NIEHS) were added and preincubated f o r 40 min at 3 1 ° p r i o r to i n i t i a t i n g the r e a c t i o n . HEPES b u f f e r (0.5 M, pH 7.4, 1.0 ml) was added as well as benzo(a)pyrene (100 nmol in 10 yl acetone). NADPH (0.6 mg i n 50 yl HEPES b u f f e r ) was added to s t a r t the r e a c ­ t i o n , which was incubated at 31° f o r up to 30 min. The r e a c t i o n was terminated and analyzed f o r AHH a c t i v i t y by fluorescence according to Nebert and Gelboin (21), using 3-hydroxybenzo(a)pyrene as the reference standard. For incubation mixtures con­ t a i n i n g 1 , 1 , 1 - t r i c h l o r o p r o p e n e oxide or α-naphthoflavone (both from A l d r i c h Chemical C o . , Milwaukee, WI), the a d d i t i o n was made j u s t a f t e r the substrate and 1 min before s t a r t i n g the r e a c t i o n with NADPH.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

18.

BEND

ET AL.

Microsomal

Mixed-Function

Oxidation

301

,tf

When C-benzo(a)pyrene (100 nmol) was incubated with the r e c o n s t i t u t e d MFO system, the r e a c t i o n components were increased 1 0 - f o l d (maintaining the o r i g i n a l incubation volume, and substrate and NADPH c o n c e n t r a t i o n s ) . Metabolites were extracted and analyzed by HPLC as described f o r the microsomal i n c u b a t i o n s .

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Results and Discussion Several p r o p e r t i e s of hepatic microsomal AHH a c t i v i t y were compared in c o n t r o l and DBA-pretreated male l i t t l e skates as shown i n Table I. Following treatment there was an approximately 35f o l d increase i n s p e c i f i c enzyme a c t i v i t y , as quanti tated by f l u o r e s c e n c e of the phenolic metabolites formed (3, 21). The pH optimum, which was f a i r l y broad, and the concentration of benzo(a)pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme a c t i v i t y were the same f o r both control and induced skate hepatic microsomes. The shorter periods observed f o r l i n e a r i t y of product formation with microsomes from the i n duced skates i s thought to be r e l a t e d to the much higher AHH a c t i v i t y p r e s e n t , and may be due to substrate d e p l e t i o n or the formation o f products which are i n h i b i t o r y ( i . e . , compete with the MFO system as they are substrates themselves). A s i m i l a r explanat i o n may be relevant f o r the l o s s of l i n e a r product formation at lower microsomal p r o t e i n concentrations in the induced animals. The apparent k i n e t i c constants were obtained from Lineweaver-Burk p l o t s o f AHH a c t i v i t i e s recorded in the presence of i n c r e a s i n g concentrations of benzo(a)pyrene (0.001-1.0 mM). The p l o t s were l i n e a r f o r both untreated and DBA-induced animals. The apparent V was 20- to 3 0 - f o l d higher i n hepatic microsomes from the inâuced skates whereas the apparent Κ values were of the same magnitude i n c o n t r o l and t r e a t e d f i s h . An obvious d i f f e r e n c e was a l s o noted between control and induced skate hepatic^microsomal AHH a c t i v i t y i n the presence of α - n a p h t h o f l a v o n e (10" M). This compound, when added in vitro at t h i s or higher c o n c e n t r a t i o n s , caused s i g n i f i c a n t s t i m u l a t i o n of AHH a c t i v i t y i n c o n t r o l animals (about 3 - f o l d ) but i n h i b i t i o n (80%) was found i n DBA-pretreated s k a t e s . S i m i l a r r e s u l t s were e a r l i e r reported f o r control and 3-methylcholanthrene-treated r a t s (23), where i t appears that the response i s due to d i f f e r e n t i a l effects o f α - n a p h t h o f l a v o n e on hepatic microsomal cytochrome P-450 (stimu­ l a t e d ) and cytochrome P-448 ( i n h i b i t e d ) (24). Our data suggests that there may be a novel form of cytochrome P-450 synthesized i n skate l i v e r i n response to p o l y c y c l i c hydrocarbon a d m i n i s t r a t i o n , even though there was no hypsochromic s h i f t i n the carbon monoxide d i f f e r e n c e spectrum of d i t h i o n i t e reduced hepatic microsomes from DBA-treated skates ( r e l a t i v e to hepatic microsomes from control fish). Benzo(a)pyrene i s converted by the microsomal MFO system of mammals (18) and t r o u t (25) to a number of o x i d i z e d products.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. 25°) 25°)

m a x

(FU/min/mg p r o t e i n )

(mM)

4

D a t a from r e f e r e n c e

(+10"

22.

Range of 2 experiments.

d

0.73

e

e

d

1.16

e

e

5.23

4.2

6.9

d

0.008 - 0.010

2, and 3 and s a c r i f i c e d on day 10.

- 0.21

0.006^

0.06

1i near to 1.2

d

mg/ml

D a t a from a s i n g l e experiment. The experiment was repeated twice and s i m i l a r data obtained each time, f α - N a p h t h o f l a v o n e was added to the r e a c t i o n mixture in vitro.

e

Mean ± SD (N).

c

f

0.23

0.20

0.005 -

0.06

mg/ml

f o r 15 min

to 1.6

(4)

linear

± 4.32

linear

8.08

f o r 20 min

c

linear

(4)

optimum, pH 7 . 4 - 8 . 0

± 0.06

DBA-PRETREATED SKATES

IN CONTROL AND

optimum, pH 7 . 4 - 8 . 0

0.23

CONTROL SKATES

with DBA (10 mg/kg) on days 1,

M α-naphthoflavone)

^Skates were t r e a t e d (IP)

a

AHH A c t i v i t y

AHH A c t i v i t y (no α - n a p h t h o f l a v o n e )

Apparent V

Apparent 1^ (mM)

Enzyme-Saturating Concentration

Substrate (BP) C o n e . - A c t i v i t y R e l a t i o n s h i p s (at 2 5 ° )

Protein C o n e . - A c t i v i t y R e l a t i o n s h i p (at

T e m p . - A c t i v i t y R e l a t i o n s h i p (at

25°)

(FU/min/mg p r o t e i n )

p H - A c t i v i t y R e l a t i o n s h i p (at

AHH A c t i v i t y

PARAMETER

SOME PROPERTIES OF HEPATIC MICROSOMAL BENZO(a)PYRENE HYDROXYLASE (AHH) ACTIVITY DBA-PRETREATED MALE LITTLE SKATES {Raja erinaceaf

TABLE I

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Downloaded by UNIV OF PITTSBURGH on May 3, 2015 | http://pubs.acs.org Publication Date: May 24, 1979 | doi: 10.1021/bk-1979-0099.ch018

18.

BEND E T A L .

Microsomal

Mixed-Function

Oxidation

303

Since the f l u o r e s c e n c e assay only quantitates phenolic metabolites ( p r i m a r i l y 3- and 9-hydroxybenzo(a)pyrene i n rats (26) and d o e s , not a c c u r a t e l y measure t o t a l metabolism, we have a l s o compared Cbenzo(a)pyrene metabolism i n hepatic microsomes from control and DBA-treated male skates using HPLC a n a l y s i s . The metabolism of benzo(a)pyrene (per mg microsomal protein) was a l s o much f a s t e r (about 1 5 - f o l d a f t e r incubation f o r 5, 15, or 30 min) i n microsomes from DBA-pretreated f i s h than i n those from untreated,skates ( F i g . 1). As shown i n F i g . 2, the metabolites formed from C-benzo(a)pyrene by hepatic microsomes from untreated and DBA-pretreated male skates are q u a l i t a t i v e l y very s i m i l a r , i f not i d e n t i c a l , and the combined r a d i o a c t i v i t y e l u t i n g with the 9-hydroxybenzo(a)pyrene (9-OH) and 3-hydroxybenzo(a)pyrene (3-OH) standards ( i . e . , the phenolic metabolites) accounted f o r greater than 50% of the t o t a l biotransformation products in each c a s e . The major q u a n t i ­ t a t i v e d i f f e r e n c e i n these metabolic p r o f i l e s was the greater amount of r a d i o a c t i v i t y chromatographinq with the standards i n the Q + Ε region of the chromatogram ( b e n z o ( a ) p y r e n e - l , 6 - , - 3 , 6 - , and -6,12 quinones and benzo(a)pyrene 4 , 5 - o x i d e ) . It i s l i k e l y that t h i s i s in part due to the accumulation of benzo(a)pyrene 4 , 5 - o x i d e i n the incubation mixtures containing microsomes from DBA-pretreated skates s i n c e epoxide hydratase a c t i v i t i e s i n l i t t l e skate hepatic microsomes, with benzo(a)pyrene 4 , 5 - o x i d e as sub­ s t r a t e , are low (0.19 ± 0.05 nmol/min/mg microsomal p r o t e i n , mean ± SD, Ν = 3) and are unaffected by pretreatment with DBA (4). It was a l s o i n t e r e s t i n g that s i g n i f i c a n t amounts of benzo(a)pyrene 7 , 8 - d i h y d r o d i o l were formed by hepatic microsomes of both control and DBA-pretreated s k a t e s . This d i h y d r o d i o l i s the metabolic precursor f o r the isomeric 9 , 1 0 - e p o x i d e - 7 , 8 - d i h y d r o - 7 , 8 dihydroxybenzo(a)pyrenes, which are at l e a s t one ultimate c a r ­ cinogenic and mutagenic form of benzo(a)pyrene (27, 28). Should the formation of benzo(a)pyrene 7 , 8 - d i h y d r o d i o l be an important metabolic pathway f o r benzo(a)pyrene i n aquatic s p e c i e s , there i s a d i s t i n c t p o s s i b i l i t y that p o t e n t i a l l y dangerous l e v e l s of t h i s compound might b u i l d up i n l i v e r and muscle of f i s h used f o r human food. Lee et al. (29) p r e v i o u s l y suggested that benzo(a)pyrene 7 , 8 - d i h y d r o d i o l was the predominant benzo(a)pyrene metabolite found i n l i v e r , g u t , g i l l , f l e s h , and heart of mudsucker (Gillichthys mirabilis), s c u l p i n (oligooottus maculosus) and sand dabs (cithariohthys stigmaeus) although the metabolites were not rigorously i d e n t i f i e d . The e l u t i o n p r o f i l e of cytochrome P-448 (absorption at 418 nm) and epoxide hydratase a c t i v i t y from a sodium c h o l a t e - s o l u b i l i z e d hepatic microsomal preparation (from DBA-treated male skates) a p p l i e d to a DEAE-cellulose column and eluted with Buffer II i s shown i n F i g . 3. The void volume of the column contained s i g n i f i ­ cant amounts of epoxide hydratase a c t i v i t y . Fractions 40-70 ( F i g . 3) were combined, and concentrated. The carbon monoxide d i f f e r e n c e spectrum, which had an absorption maximum at 448 nm in the induced s t a t e , i s shown i n F i g . 4. This form of the cytochrome ( i . e . ,

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

II

M)

c

125

450

500

275

b

Assayed by f l u o r e s c e n c e a f t e r incubation with benzo(a)pyrene (100 nmol) and NADPH (0.16 mg) i n 0.5 M HEPES b u f f e r , pH 7.6 (1 ml) at 31° f o r 20 min. Product formation was l i n e a r f o r at l e a s t 20 min.

S e e reference 30 f o r more d e t a i l s .

b

c

hepatic

15

57

63

35

80

28

0

100

Activity

% Maximum

Cytochrome P-448 (0.02 nmol), NADPH-cytochrome a reductase (115 u n i t s ; p u r i f i e d from r a b b i t microsomes) and sodium cholate (125 μg) preincubated at 31° f o r 30 min.

Plus α - n a p h t h o f l a v o n e (5 χ 10" M)

4

7

oxide (50 μΜ)

Plus α - n a p h t h o f l a v o n e (5 χ 10"

Plus 1 , 1 , 1 - t r i c h l o r o p r o p e n e

Plus r a b b i t hepatic microsomal phospholipids (125 u g )

625

Minus sodium c h o l a t e

0

775

225

3

formed/min/nmol cytochrome P-448

pmoles 3-hydroxybenzo(a)pyrene

Minus NADPH-cytochrome a reductase

Minus NADPH

Complete system

Incubation Conditions

BENZO(a)PYRENE HYDROXYLASE (AHH) ACTIVITY OF A RECONSTITUTED MIXED-FUNCTION OXIDASE SYSTEM CONTAINING HEPATIC CYTOCHROME P-448 FROM DBA-TREATED MALE SKATES

TABLE

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BEND E T A L .

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20p

Microsomal

Mixed-Function

Oxidation

305

• untreated skates ο DBA-induced skates

ç φ

2 Q. σ»

Ε \

9'

S

2 Φ Ε ρ

ô Ε c

10

20 Time (min)

30

Figure 1. Production of total BP metabolites by hepatic microsomes from control and DBA-pretreated male little skates. Each point is the result of a single incuba­ tion and HPLC determination: (O O ) , control skates; — DBA-pre­ treated skates. The DBA-dosing schedule and incubation conditions are described

in Materials and Methods.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

306

PESTICIDE

A N D XENOBIOTIC

METABOLISM

I N AQUATIC

ORGANISMS

50

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untreated skates

25h

o

ϊ oi Β

ELn.

n.

DBA-induced skates

"S as 25

n

9,10-D 4,5-D

7,8-D

Q+E

9-OH 3-OH

Average of 2 HPLC determinations after 15 min. incubation Figure 2. Profile of radioactive metabolites obtained upon incubation of C-BP with hepatic microsomes from control or DBA-pretreated male little skates. 14

The hepatic microsomes were aliquots from pools of 10 control or pretreated fish that were also used for the partial purification of various forms of Cytochrome P-450: 9,10-D, BP-9,10-dihydrodiol; 4,5-D, BP-4,5-dihydrodiol; 7,8-D, BP-7,8-dihydrodiol; Q + E, BP1,6-, -3,6-, and -6,12-quinones and BP-4,5-oxide; 9-OH, 9-hydroxy-BP; 3-OH, 3-hydroxyBP. The DBA-dosing schedule and incubation conditions are described in Materials and

Methods.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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18.

BEND

ET AL.

Microsomal Mixed-Function

Oxidation

307

Figure 3. Elution profile of partially purified Cytochrome P-448 and epoxide hydratase activity of solubilized hepatic microsomes from DBA-treated male skates from a DEAE-cellulose column with Buffer II. Epoxide hydratase activity was determined with BP-4,5-oxide as the substrate (see Materials and Methods).

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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308

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

420

440 460 Wavelength (nm)

480

500

Figure 4. Carbon monoxide difference spectrum of partially purified hepatic microsomal Cytochrome P-448 from DBA-treated little skates. The cuvettes contained dithionite-reduced cytochrome (0.10 mg protein/mL) in lOmM phosphate buffer, pH 7.7, containing 20% glycerol, O.lmM EDTA and O.lmM dithiothreitol.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

18.

BEND E T A L .

Microsomal

Mixed-Function

309

Oxidation

cytochrome P-448) was p u r i f i e d 4- to 5 - f o l d during e l u t i o n from the D E A E - c e l l u l o s e column, was f r e e of cytochrome b and contained n e g l i g i b l e epoxide hydratase a c t i v i t y . Consequently, i t has been f u r t h e r used to study the metabolism of C-benzo(a)pyrene i n a r e c o n s t i t u t e d mixed-function oxidase system. When a l l of the material absorbing at 418 nm (associated with the cytochrome P-448 f r a c t i o n s ) was eluted from the DEAE-cellulose column (which i n some experiments required more than 1 l i t e r of B u f f e r II), e l u t i o n was continued with a l i n e a r KC1 gradient (00.5 M) in Buffer II, as shown i n F i g . 5. A d i f f e r e n t form(s) of cytochrome P-450 ( f r a c t i o n s 130-155), having maximal absorption near 451 nm i n the carbon monoxide l i g a t e d and reduced form ( F i g . 6 ) , was obtained although only 2- to 3 - f o l d p u r i f i c a t i o n , r e l a t i v e to microsomes, was achieved. This form of cytochrome P-450 was e x t e n s i v e l y contaminated with epoxide hydratase a c t i v i t y . However, by combining f r a c t i o n s 130-150 ( F i g . 5 ) , i t was p o s s i b l e to obtain cytochrome P-451 e s s e n t i a l l y f r e e of cytochrome b . The r e l a t i v e content of cytochrome P-448 and cytochrome P-451 Tn the DEAEcolumn eluates ranged from 1:1.1 to 1:1.6 in several d i f f e r e n t experiments. The absorbance at 418 nm of f r a c t i o n s 155-168 ( F i g . 5) was p r i m a r i l y a s s o c i a t e d with cytochrome b whereas that in f r a c t i o n s 170-180 was p r i m a r i l y due to NADPH-cyt8chrome ο reductase. Very s i m i l a r r e s u l t s to those described i n F i g . 3-6 were obtained when sodium cholate s o l u b i l i z e d hepatic microsomes from DBA-treated female l i t t l e skates were subjected to chromatography on D E A E - c e l l u l o s e as described above (data not shown). Also not shown are the r e s u l t s obtained with hepatic microsomes from un­ treated male and female l i t t l e skates. With untreated a n i m a l s , 80-90% of the cytochrome P-450 eluted from the DEAE-cellulose column only at higher i o n i c strength ( i . e . , with the KC1 g r a d i e n t ) . However, i n a l l preparations s t u d i e d , an appreciable amount of cytochrome P-450 (10-20%), having i t s absorption maximum i n the carbon monoxide-1igated and reduced s t a t e at 450 nm, was eluted from the column with buffer II, as was observed with cytochrome P448 of hepatic microsomes from DBA-treated s k a t e s . The f u r t h e r p u r i f i c a t i o n of the various forms of cytochrome P-450 from control and DBA-pretreated l i t t l e skate l i v e r s i s c u r r e n t l y i n progress in our laboratory. C o l l e c t i v e l y , these experiments have demonstrated that sub­ s t a n t i a l amounts of cytochrome P-448 are indeed present i n l i v e r of DBA-pretreated male and female skates but not i n untreated skates. S u f f i c i e n t cytochrome P-448 i s present to account f o r the increased AHH a c t i v i t i e s observed i n hepatic microsomes of DBA-treated s k a t e s . A f t e r removal of f r e e Emulgen 913 from p a r t i a l l y p u r i f i e d hepatic cytochrome P-448 of DBA-treated male skates an a c t i v e mixed-function oxidase system was r e c o n s t i t u t e d by preincubating the cytochrome with p u r i f i e d r a b b i t hepatic NADPH-cytochrome ο

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5

5

5

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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310

PESTICIDE AND XENOBIOTIC METABOLISM IN AQUATIC ORGANISMS

Fraction no. Figure 5. Elution profile of partially purified Cytochrome P-451 and epoxide hydratase activity of solubilized hepatic microsomes from DBA-treated male skates from a DEAE-cellulose column with a linear KCl (0-0.5M) gradient in Buffer II. The KCl gradient was initiated only after all of the Cytochrome P-448 (Figure 3) had been eluted from the DEAE-cellulose column with Buffer II (see

Materials and Methods).

_ i

420

.

ι

.

ι

440 460 Wavelength (nm)

.

1

-L-

480

1

500

Figure 6. Carbon monoxide difference spectrum of partially purified hepatic microsomal Cytochrome P-451 from DBA-treated male little skates. The cuvettes contained dithionite-reduced cytochrome (0.15 mg protein/mL) in lOmM phos­ phate buffer, pH 7.7, containing 20% glycerol, O.lmM EDTA and O.lmM dithio­ threitol.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

18.

BEND E T A L .

Microsomal

Mixed-Function

311

Oxidation

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50i

I

25\

w



=

9,10-D 4,5-D

=_ 7,&D

Q+E

9-OH 3-OH

Figure 7. Profile of radioactive metabolites obtained upon incubation of C-BP with a reconstituted system containing partially purified Cytochrome P-448 from DBA-pretreated male little skates. 14

The reconstituted system consisted of Cytochrome P-488 (0.2 nmol), NADPH-Cytochrome c reductase (1500 units) and sodium cholate (1.25 mg). It was preincubated for 30 min at 31°. The final reaction mixture (which was incubated at 31° for 20 min) contained the preincubated system described above, excess NADPH and C-BP (100 nmol; 4.1 mCi/mmol) in a final volume of 1 mL 0.5M HEPES buffer, pH 7.6. Rate of BP metabolism was 665 pmol/min/nmol Cytochrome P-488. Abbreviations used for metabolites are described in legend to Figure 2. 14

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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312

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

ORGANISMS

t i e s i n hepatic microsomes from f i s h captured there may a c t u a l l y be lower than i n f i s h captured from r e l a t i v e l y unpolluted waters (32). F i n a l l y , there i s the p o t e n t i a l problem of migration of marine f i s h from p o l l u t e d to l e s s p o l l u t e d a r e a s , and v i c e v e r s a . In s p i t e of these l i m i t a t i o n s there seems l i t t l e doubt that i n d u c t i o n of the hepatic MFO system i n c e r t a i n f i s h can i n d i c a t e the presence of s e l e c t e d t o x i c chemicals (to f i s h and humans) i n both freshwater and marine environments. For t h i s reason we have been studying various aspects of t h i s question f o r the l a s t few years i n f r e s h l y captured f i s h (winter flounder and l i t t l e skate i n Maine and the sheepshead i n F l o r i d a ) and in f i s h induced by p o l y c y c l i c hydrocarbon a d m i n i s t r a t i o n . One of our more i n t e r e s t i n g observations i s i l l u s t r a t e d i n Table III. The a d m i n i s t r a t i o n of DBA to winter flounder increased hepatic microsomal AHH and 7 - e t h o x y r e s o r u f i n deethylase a c t i v i t i e s as expected, and AHH a c t i v i t y was s t r o n g l y i n h i b i t e d i n the DBAtreated flounder by 10" M α-naphthoflavone as we have p r e v i o u s l y reported f o r both l i t t l e skate (4) and sheepshead (9). However, the presence of high AHH and 7-ethoxyresorufin deethylase a c t i v i ­ t i e s i n one c o n t r o l f l o u n d e r , and the i n h i b i t i o n o f AHH a c t i v i t y by α-naphthoflavone i n t h i s animal suggested that the hepatic microsomal MFO system of t h i s f i s h was already induced. Subsequently, we assayed hepatic microsomal AHH a c t i v i t y ( i n the presence and absence of 10" and 10" M α-naphthoflavone) and 7 - e t h o x y r e s o r u f i n deethylase a c t i v i t y i n t h i r t e e n winter flounder during June-August, 1977 (33). There was a marked v a r i a t i o n i n microsomal AHH a c t i v i t y (over 6 0 - f o l d ) and eleven of the f i s h had AHH a c t i v i t y that was i n h i b i t e d in vitro by α-naphthoflavone as well as higher 7 - e t h o x y r e s o r u f i n deethylase a c t i v i t i e s . This again suggested i n d u c t i o n of the MFO system i n many of the f i s h s i m i l a r i n nature to that which occurs f o l l o w i n g treatment with p o l y c y c l i c h y d r o c a r b o n - l i k e inducing agents. During June-August, 1978 we assayed these same three para­ meters ( i . e . , AHH a c t i v i t y i n the presence and absence of a naphthoflavone and 7-ethoxyresorufin deethylase a c t i v i t y ) i n l i v e r homogenates from more than two hundred winter flounder i n Maine, a f t e r f i r s t demonstrating t h a t the α-naphthoflavone e f f e c t s ( i . e . , s t i m u l a t i o n or i n h i b i t i o n ) were i d e n t i c a l i n microsomes and whole homogenates of the same f i s h (Foureman, G. L . , B e n - Z v i , Z . , D o s t a l , L . , and Bend, J . R., unpublished r e s u l t s ) . The data obtained with female winter flounder are shown i n Table IV. Once again there was a very l a r g e range (over 6 0 - f o l d ) between the lowest AHH a c t i v i t y (0.11 FU/min/mg protein) and the highest AHH a c t i v i t y (7.32) observed. Moreover, there was a good c o r r e l a t i o n between increases i n AHH a c t i v i t y i n the various a r b i t r a r y groups of flounder and 7 - e t h o x y r e s o r u f i n deethylase a c t i v i t y (which at l e a s t i n r a t s i s a s s o c i a t e d with the induction of cytochrome P-448 (13). C o l l e c t i v e l y , the three parameters tested i n d i c a t e that p o l y c y c l i c hydrocarbon-type i n d u c t i o n of the hepatic microsomal MFO system of winter flounder i s common i n the waters near Mt. Desert I s l a n d ,

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979. III

' i n j e c t e d I.P.

a

fish

DBA-Treated

Control

23 146 157 187 261

0.19 0.83 0.63 1.01 0.78

0.40 1.76 1.11 1.67 1.84

2, and 3 and s a c r i f i c e d on day 10.

0

0.19

(ΙοΛί)

7-ERF A c t i v i t y (pmol/min/mg protein)

0.05

with 10 mg/kg DBA i n corn o i l on days 1,

Fish

ameriaanus)

AHH A c t i v i t y (FU/min/mg protein) Without α-Naphthoflavone With α-Naphtboflavone

{Pseudopleuroneates

EFFECT OF 1,2,3,4-DIBENZANTHRACENE (DBA)-PRETREATMENT ON HEPATIC MICROSOMAL 3ENZ0(a)PYRENE HYDROXYLASE (AHH) AND 7-ETH0XYRES0RUFIN DEETHYLASE (7-ERF) ACTIVITIES IN WINTER FLOUNDER

TABLE

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In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3

i n h i b i t e d AHH a c t i v i t y

a-Naphthoflavone

in a l l

in a l l

of these f i s h .

in others.

of these f i s h .

i n some of these f i s h and i n h i b i t e d i t

M) stimulated AHH a c t i v i t y

d

3

stimulated AHH a c t i v i t y

and 5 χ 10"

a-Naphthoflavone

(10~

C

Mean ± SD.

a-Naphthoflavone

b

a

358.5 ± 197

1.10

5.71

11

d

4.00 - 7 . 3 6

2.00

±

190.9 ± 85.7

2.85 ± 0.47

31

d

- 4.00

2.01

111.5 ± 57.6

± 0.27

1.33

29

C

-

1.01

71.0 ± 34.0

0.78 ± 0.15

1.00

-

0.51

27

12.7 ± 18.3

c

b

7-ERF A c t i v i t y (pmol/min/mg p r o t e i n )

0.28 ±

- 0.50

0.11

0.10

AHH A c t i v i t y (FU/min/mg p r o t e i n )

19

No. of Fish i n Group

a

AHH A c t i v i t y (FU/min/mg p r o t e i n )

BENZO(a)PYRENE HYDROXYLASE (AHH) AND 7-ETHOXYRESORUFIN DEETHYLASE (7-ERF) ACTIVITIES IN. LIVER HOMOGENATE OF FEMALE WINTER FLOUNDER SEPARATED INTO ARBITRARY GROUPS BASED ON AHH ACTIVITY

TABLE IV

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18.

BEND ET A L .

Microsomal

Mixed-Function

Oxidation

315

Maine, during the summer months. A very s i m i l a r response was noted i n the male flounder s t u d i e d . There was a l s o no apparent change i n the r e l a t i v e number of induced flounder over the three month period monitored. Since these studies were not conducted during the spawning season i t seems u n l i k e l y that spawning i s a factor. Further studies are in progress to e l u c i d a t e whether or not t h i s i n d u c t i o n i s due to exposure to environmental p o l l u t a n t s . However, a f u r t h e r complicating f a c t o r i s that l i t t l e skates captured from the same waters during June-August do not have induced hepatic MFO systems, even though they are s u s c e p t i b l e to i n d u c t i o n by p o l y c y c l i c hydrocarbon-like compounds (Table I). S i m i l a r s t u d i e s conducted at our F l o r i d a laboratory with the sheepshead have produced q u a n t i t a t i v e l y d i f f e r e n t r e s u l t s (James, M. 0 . , unpublished d a t a ) . Of twenty-nine sheepshead assayed i n June-August over two y e a r s , only four have had p a r t i a l l y induced hepatic MFO systems. Obviously we must obtain c o n s i d e r a b l y more information c o n ­ cerning the c a u s a t i v e nature of t h i s i n t e r e s t i n g enzyme response before any of the species we are c u r r e n t l y i n v e s t i g a t i n g can be r o u t i n e l y used to monitor the marine environment f o r p o l l u t a n t s . Acknow!edgments We are g r a t e f u l to C. Ballow, Z. B e n - Z v i , L. D o s t a l , and G. Foureman f o r t h e i r e x c e l l e n t a s s i s t a n c e i n t h i s study. This research was p a r t i a l l y supported by NSF Grant No. PCM 77-26790 and NIH Biomedical Research Support Grant No. S07 RR 05764 to the Mount Desert Island B i o l o g i c a l Laboratory. We a l s o appreciate the support of the United States Environmental P r o t e c t i o n Agency under an agreement r e l a t i n g to the Federal Interagency E n e r g y / E n v i r o n ­ ment R and D Program.

Literature Cited 1. Bend, J. R., Pohl, R. J., and Fouts, J. R.: Some properties of the microsomal drug-metabolizing enzyme system in the little skate, Raja erinacea. Bull. Mt. Desert Island Biol. Lab. (1972) 12: 9-12. 2. 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. 3. 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 "Microsomes and Drug Oxidations." Ullrich, V., Roots, I., Hildebrandt, Α., Estabrook, R. W., and Conney, A. H. (eds.), pp. 160-169, Pergamon, Oxford, 1977.

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METABOLISM

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AQUATIC

ORGANISMS

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In "Origins of Human Cancer." Hiatt, H., Watson, J. D., and Winsten, I. (eds.), pp. 639-658. Cold Spring Harbor Laboratory, New York, 1977. Lee, R. F., Sauerheber, R., and Dobbs, G. H.: Uptake, metabolism and discharge of polycyclic aromatic hydrocarbons by marine fish. Mar. Biol. (1972)17:201-208. Wolf, C. R., Szutowski, M. M., Ball, L. M., and Philpot, R. M.: The rabbit pulmonary monoxygenase system: Characteristics and activities of two forms of pulmonary cytochrome P-450. Chem.-Biol. Interactions (1978) In press. Payne, J. F.: Field evaluation of benzpyrene hydroxylase induction as a monitor for marine petroleum pollution. Science (1976) 191: 945-946. Ahokas, J. T., Kärki, N. T., Oikari, Α., and Soivio, Α.: Mixed-function monooxygenase of fish as an indicator of pol­ lution of aquatic environment by industrial effluent. Bull. Environ. Cont. Toxicol. (1976) 16: 270-274. Bend, J. R., Foureman, G. L., and James, M. O.: Partially induced hepatic mixed-function oxidase systems in individual members of certain marine species from coastal Maine and Florida. In "Aquatic Pollutants: Transformation and Biologi­ cal Effects." Hutzinger, O., Van Lelyveld, L. H., and Zoeteman, B. C. J. (eds.), pp. 483-486, Pergamon, Oxford, 1978.

1

Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672 C. V. Whitney Marine Laboratory, University of Florida, Marine­ land, FL 32084

2

RECEIVED

January 2, 1979.

In Pesticide and Xenobiotic Metabolism in Aquatic Organisms; Khan, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.