TOXICOLOGY AND APPLIED PHARMACOLOGY 68, 380-391 (1983)

Characteristics of the Hepatic Monooxygenase System of the Goldfish (Carassius auratus) and Its induction with ,&Naphthoflavone

JAY W. GCKKH AND FUMIO MATSUMURA

Pesticide Research Center, Michigan State University, East Lansing, Michigan 48824-1311

Received September 7, 1982; accepted December 30. I982

Characteristics of the Hepatic Monooxygenase System of the Goldfish (Carassius aura&s) and Its Induction with &Naphthoflavone. GOOCH, J. W., AND MATSUMURA, F. (1983). Toxicol. Appl. Pharmacol. 68, 380-391. Treatment of goldfish with fl-naphthoflavone (ip) resulted in induction of hepatic monooxygenase activity in a dose-dependent manner. Levels of ‘I-ethox- yresorufin-Odeethylase and mexacarbate NADPH-dependent oxidative metabolism as well as cytochrome(s) P-450 were increased. Other substrates which have been associated with 3-meth- ylcholanthrene (3-MC>type induction in mammals were unchanged as was b-aminoievulinic acid synthetase. Enzyme activities were inhibited by carbon monoxide and piperonyl butoxide confirming their cytochrome P-450 nature. NADH was found to stimulate metabolism in a significant manner with NADPH. pH-dependent ethyl isocyanide binding characteristics were similar in control and induced preparations while mexacarbate binding to oxidized microsomes reveakd a higher apparent affinity constant (gpp) as well as greater overall binding in induced preparations. SDS-gel electrophoresis revealed an increased band with a molecular weight of approximately 56,000.

The induction of xenobiotic metabolizing en- zymes in animals has been an intensive area of research for the past two decades. Numer- ous studies in mammalian systems have shown that there are distinct increases in mi- crosomal proteins and enzyme systems, and several scientists have made attempts to dis- cern the complex relationship between the in- duction status of the system and toxicity or carcinogenesis (for review, see Conney, 1967; Nebert and Jensen, 1979; Snyder and Rem- mer, 1979; Nebert, 1979). It has become in- creasingly apparent that one of the micro- somal detoxification enzyme systems that is clearly induced by many xenobiotics is the monooxygenase system. This cytochrome P- 450-mediated system is an intricate multien- zyme complex capable of a vast array of met- abolic transformations.

While investigations into the nature of this system in fish have received much less atten-

tion, it is now well established that fish are also capable of a wide variety of enzymatic oxidations (Buhler and Rasmusson, 1968; Chambers and Yarbrough, 1976; Bend and James, 1978; Lech and Bend, 1980). It also has been established that fish are capable of increasing microsomal monooxygenase activ- ities as a result of exposure to some of the xenobiotics that cause induction in mammals (Pederson et al., 1974; Lidman et al., 1976; Gerhart and Carlson, 1978; Vodicinik et al., 1981). As a result of these investigations, it has been proposed that fish are incapable of responding to inducers of the phenobarbital- type, while 3-methylcholanthrene (3-MC)-type compounds cause high levels of induction (Ahokas et al., 1977, 1979). These workers also point to the similarity of fish liver systems to that of the PI-450 system of mammals, al- though cytochrome P-450 spectral maxima are at approximately 449-450 nm both before

0041-008X/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.

380

HEPATIC MONOOXYGENASE SYSTEM OF THE GOLDFISH 381

and after induction. James and Bend (1980) purchased from E. Merck Company, Rahway, New Jer- have isolated a partially purified cytochrome sey. [‘%]Mexacarbate (Z&ran, 4dimethylamino-3,5-

P-450 from the sheepshead (a marine teleost) [‘4C]xylyl N-methylcarbamate) was provided by the Dow

which exhibits an absorbance maximum at Chemical Company, Midland, Michigan. 7-Ethoxyreso-

448 nm. That the system behaves in a manner rufin was purchased from Pierce Chemical Company, Rockford, Illinois. Ethyl isocyanide was synthesized in

analogous to the PI-450 system of mammals this laboratory according to the method of Casanova et

may have important implications concerning al: (1963). All biochemicals were purchased from Sigma

the carcinogenic process in these organisms Chemical Company, St. Louis, Missouri.

(Ahokas et al., 1977, 1979; Nebert and Jen- Goldfish (Carassius auratus, Common Comet variety,

20 to 75 g) were initially obtained from a local pet store sen, 1979) or may provide insight into the whereas in later studies they were obtained from the Blue nature of the mammalian PI-450 enzvme. Ridge Fish Hatchery, Inc., Kernersville, North Carolina.

We have begun our studies with the gold- Based on levels of cytochromes P-450, levels of 7&h-

fish for several reasons. First, the vast majority oxyresorufin induction, and mexacarbate binding behav-

of work done on fish has been with coldwater ior, no obvious differences between groups were noted. Fish were kept at 2 1 “C f 1 “C for at least one week prior

species while there is a lack of data on warm- to use. Fish were initially fed Goldfish Food Flakes (War-

water species. The goldfish is particularly suited in this regard since a much greater amount of physiological literature has been generated that can support toxicologic stud- ies. Second, it has been shown that some warmwater fish have relatively high metabolic capabilities. Indeed it has been generally rec- ognized that warmwater fish are less sensitive to chemical pollution (Johnson, 1968). For example, work from this laboratory (Hinz and Matsumura, 1977) has demonstrated a greater capability of goldfish to degrade 2,5,2’-tri- chlorobiphenyl over that of rainbow trout or bullhead catfish. Third, goldfish are readily available in many locations even during the winter. In this study we have made an attempt to characterize the general nature of the gold- fish monooxygenase system by using P-naph- thoflavone (a known 3-MC-type inducer) as a model inducer. Special attention has been paid to compare similarities and differences of the goldfish system to other fish systems and to mammalian systems.

METHODS

Materials. &Naphthoflavone (5.6~benzoflavone), ami- nopyrine, and pnitroanisole were purchased from Ald- rich Chemical Company, Milwaukee, Wisconsin. 3- Methylmonomethyl aminoazobenzene (3-MMAB) was kindly supplied by Dr. J. A. Miller, McArdle Laborato- ries, University of Wisconsin, Madison, Wisconsin. Benzphetamine was a gift from Dr. O’Connor, Upjohn Company, Kalamazoo, Michigan. Ethylmorphine was

dley Products Company, Inc., Secaucus, N.J.) but were later fed Biodiet pelIets (Bioproducts Inc., Warrenton, Ore.) once daily. The last feeding was approximately 24 hr prior to termination.

Preparation of microsomes. Fish were killed by cervical dislocation, livers pooled (5 to 10 fish), and microsomes prepared as described by Elcombe and Lech (1979). Mi- crosomes thus obtained were either used fresh, or quick frozen as a pellet in 150 mM phosphate buffer containing 20% glycerol, 50 mM sucrose, and 1 mM dithiothreitol, and stored in liquid N, , Under these conditions enzyme activity was stable for several weeks.

Enzyme assays. All enzyme assays, with the exception of 7-ethoxyresorufmOthylase, were carried out in 25- ml Erlenmeyer flasks in a shaking water bath at 29°C. Mixtures contained enzyme, substrate, 150 mM phos- phate buffer (pH 7.4), and a standard NADPH generating system consisting of 2 pmol of NADP, 10 pmol glucose 6-phosphate, and four units glucose-6-phosphate dehy- drogenase. 7-Ethoxyresorufin-O-deethylase was measured by a direct fluorometric procedure in a Varian Model 634s UV/VIS spectrophotometer equipped with a fluo- rescence attachment. Excitation was at 560 nm while broad band emission below 575 nm was followed. Under these conditions approximately 20 pmol of resorufin could be detected. pNitroanisole-O-demethylase was determined by measuring the amount of pnitrophenol formed, with a modified method of Bell and Ecobichon (1975) as de- scribed by Madhukar and Matsumura (1979). Ndemeth- ylation of 3-methylmonomethyl aminoazobenzene (3- MMAB) was measured as described by Madhukar and Matsumura (1979). Benzphetamine-N-demethylase, ethylmorphine-Ndemethylase, and aminopyrine-Nde- methylase were determined by measuring the amount of formaldehyde formed with a modified Nash reagent (Nash, 1953). Oxidative degradation of mexacarbate was assayed by the method of Esaac and Matsumura (1979) with anal- ysis of “‘C metabolic products by the TLC method of Benezet and Matsumura ( 1974).

&Aminolevulinic acid synthetase (L-ALAS) was mea-

382 GOOCH AND

sured in whole liver homogenates by the method of Marver et al. (1966). This method involves homogenizing the liver in 3 vol of 0.9% NaCl containing 0.5 mM EDTA and 10 mM Tris-HCl (pH 7.4). The incubation mixture contained 0.5 ml of homogenate, 200 Mmol glycine, 20 pmol EDTA, and 150 pmol Tris-HCI in a final volume of 2.0 ml. The reaction was for 60 min at 29°C in a shaking water bath. The reaction was stopped with 0.5 ml of 25% TCA, and the incubation mixture was centri- fuged. The supernatant fluid was poured off, and 0.7 ml of 1.0 M sodium acetate (pH 4.6) plus 50 ~1 of acetyl- acetone was added. The preparation was then heated in a boiling water bath for 15 min. L-ALA was estimated by the methylene chloride extraction method of Poland and Glover (1973).

Binding studies. Ethyl isocyanide (EtNC) binding at various pH was accomplished by diluting a concentrated microsomal suspension in the standard buffer (150 mM phosphate, 50 mM sucrose) with 0.25 M buffer of the desired pH. pH above 7.4 were achieved with a Tris-HCl buffer. All pH measured were final (i.e., after addition of 0.25 M buffer, sodium dithionite, and EtNC [EtNC = 3.0 mM]). Spectra were recorded within 1 min after the ad- dition of EtNC as it was noted that the peak ratio changed steadily with time.

Microsomes used for mexacarbate binding were sus- pended in 50 mM Tris-HCl buffer, pH 7.5. Substrate was dissolved in absolute ethanol and added to oxidized mi- crosomes in the sample cuvette at various concentrations. Equivalent amounts of ethanol were added to the refer- ence cuvette. Microsomal protein concentration was ap- proximately 3.0 m&ml. Data were plotted according to the method of Ebel et al. (1978).

Cytochrome P-450 was measured by the method of Estabrook et al. (1972) with an extinction coefficient of 100 mrK’ cm-‘. This method measures the reduced car-

bon monoxide ligated cytochromes P-450 minus the ox- idized CO ligated cytochrome thus reducing interferences due to hemoglobin. Cytochrome bS was measured by the method of Omura and Sato ( 1964). NADPH-cytochrome c reductase was determined at room temperature by the method of Williams and Kamin (1962) as modified by Masters et al. (1965). Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin as standard.

Sodium dodecyl sulfate-polyacrylamide gel electro- phoresis (SDS-PAGE) was performed in a 1.5-mm slab gel apparatus (Bit-Rad, Model 221). The upper stacking gel (6 to 8 cm) contained 3% acrylamide while the lower resolving gel (26 to 28 cm) contained 7.5% acrylamide. A discontinuous buffer system similar to that described by Dent ef al. (1978) was used with a constant current of 10 mA. Staining for protein was by Coomassie Blue R-250 in isopropanol:acetic acid:water (25:10:65 v/v). Destaining of the background was achieved by repeated bathing in isopropanokacetic acid:water (IO: 10180, v/v). Staining for peroxidase activity was by the 3,3’,5,5’-tetra- methylbenzidine method of Thomas et al. (1976).

MATSUMURA

RESULTS

Studies on the Patterns of Induction of p-Na- phthoflavone

The time course of induction of 7-eth- oxyresorufin-Odeethylation by a single 150 mgfkg dose of P-naphthoflavone was studied. As shown in Fig. 1, a maximal level of in- ducJ&n was achieved between 48 and 96 hr. Although enzyme activities were highly vari- able, induction was already significant (p -c 0.01) at 24 hr.

The dose-response relationship of &na- phthoflavone-induced-Odeethylation changes was then studied. The results shown in Fig. 2 indicate that there was a general increase in the response with increased dosage. From these studies, a single 150 mg/kg dose fol- lowed by termination of fish at 96 hr was cho- sen as a standard induction treatment for fur- ther investigations on the nature of the gold- fish monooxygenase system. The 150 mg/kg dose was chosen due to the difficulty in de- livering the suspension required for a 200 mg/ kg treatment.

Characterization of Induced Microsomal Monooxygenase Systems

The effect of /3-naphthoflavone pretreat- ment on various electron transfer compo-

Time, hr

FIG. 1. Time course of induction of 7ethoxyresorutin- O-deethylase activity following a single ip dose of j3- naphthotlavone (100 mg/kg).

HEPATIC MONOOXYGENASE SYSTEM OF THE GOLDFISH 383

50 loo En 200 Dose, mg/kg

FIG. 2. Dose-response curve for 7-&oxyresoruf1n-O- deethylation induction. Fish received a single ip injection of &naphthoflavone and were killed 72 h later.

nents in the liver microsomes of the goldfish was investigated, and the results are shown in Table 1. Under the standard test condi- tion the cytochrome P-450 level increased nearly twofold, while levels of cytochrome b5 and NADPH cytochrome c reductase were unaffected.

A variety of substrates which had previ- ously been shown to indicate the character- istics of induced monooxygenase activities in higher animals were tested on the &naphtho- flavone-induced liver microsomes of the gold- fish. The results shown in Table 2 indicate that 7-ethoxyresorufin-O-deethylase activity, known as an indicator of enzyme activity for 3-MC-type inducers (Burke and Mayer, 1974), was induced nearly eightfold. This level is very similar to that seen by Melancon et al. ( 198 1)

in the closely related species, the carp. Oxi- dative metabolism of mexacarbate, a carba- mate insecticide, was also significantly in- creased. Metabolism of all other substrates was either unaffected or slightly depressed. It was also noted preliminarily that Mirex, a phe- nobarbital-type inducer in mammalian sys- tems, at a 100 mg/kg dose was unable to stimulate 7-ethoxyresorufin, benzphetamine, mexacarbate, or ethylmorphine metabolism, a result also seen by Vodicinik et al. (198 1).

Also shown in Table 2 is the effect of p- naphthoflavone on d-aminolevulinic acid synthetase (A-ALAS), the rate-limiting en- zyme in heme biosynthesis (Urata and Gran- ick, 1963). Mammalian induction studies of- ten have shown increases in this enzyme ac- tivity apparently reflecting synthesis of new monooxygenase enzymes (Greim et al. 1970). In the case of the goldfish system, &ALAS activity was unchanged by Bnaphthoflavone treatment.

The absence of induction of 3-MMAB N- demethylase activity is surprising since this system has been useful in recognizing 3-MC- type induction in higher animals (Madhukar and Matsumura, 1979).

To further examine the nature of the monooxygenase system of the goldfish, the ef- fect of in vitro addition of inhibitors and co- factors on 7-ethoxyresorufin-O-deethylation and benzphetamine-N-demethylation was studied (Table 3). The O-deethylase activity with 7-ethoxyresorufin was slightly less sen- sitive to carbon monoxide inhibition than was

TABLE 1

THE EFFECT OF @-NAPHTHOFLAVONE PRETREATMENT ON VARIOUS PARAMETERS OF THE HEPATK MON~~XYGENASE SYSTEM OF THE GOLDFISH (Carassius auratus)

cyt P-45@ W b,” NADPH-Cyt C reductase“

Control 0.23 k 0.09 0.070 + 0.02 51.01 -+ 0.53 Treated 0.43 + 0.08* 0.074 + 0.06 49.86 f 3.55

r? Fish received a single 150 me/kg dose in corn oil and were killed after 96 hr. ’ nmole &I50/mg protein. ’ nmol b,/mg protein. d nmole cyt c reduced/min/mg protein. * Significantly greater than control (p -z 0.05).

384 GOOCH AND MATSUMURA

TABLE 2

THE E~;ECT OF B-NAPHTHOFLAVONE TREATMENT ON SELECTED HEPATIC MICRO~~MAL MON~~XYGENASE ENZYMES AND ON b-ALA-SYNTHETASE

Enzyme Control

7-Ethoxyresorufin-Odeethylase (nmol resorufin/min/mg protein)

pNitroanisole-Odemethylase (nmol pnitrophenol/min/mg protein)

3-MMAB-Ndemethylase” (nmol HCHO/min/mg protein)

Benzphetamine-Ndemethylase (nmol HCHO/min/mg protein)

Ethylmorphine-Ndemethylase (nmol HCHO/min/mg protein)

Aminopyrine-Ndemethylase (nmol HCHO/min/mg protein)

Mexacarbatec &ALA-synthetase

(nmol S-ALA/g liver/hr)

0.015 +- 0.006’

0.313 + 0.004

0.787 5 0.266

0.846 + 0.445

0.214 +- 0.104

0.853 + 0.265 4.84 t- 2.29

12.72 + 2.80

Treated

0.224 + 0.092*

0.358 -+ 0.054

0.748 k 0.109

0.605 + 0.135

0.257 -t 0.126

0.831 k 0.211 11.20 * 1.77**

9.98 2 2.36

’ All data are expressed as X rt SE. b 3-Methyl-4-monomethylaminoazobenzene. ’ Radioassay: percentage of metabolites detected via TLC analysis (Benezet and Matsumura, 1974). The Nde-

methylated product represented approximately 1.8 1 and 3.26% in the control and treated preparations, respectively. * Significantly greater than control (p < 0.02). ** Significantly greater than control (p < 0.005).

the benzphetamine-N-demethylase activity. benzphetamine-N-demethylase reaction ves- 7-Ethoxyresorufin activity was completely in- sels contained considerably more protein (and hibited by 0.5 mM piperonyl butoxide. On the hence P-450) than the 7-ethoxyresorufin-O- other hand, benzphetamine was less sensitive deethylase reaction vessels, and thus may re- to the same level of inhibitor. Caution should quire more piperonyl butoxide for complete be used in interpretation of these data since inhibition. Addition of 1.67 mM NADH in

TABLE 3

THE EFFECT OF ADDED COFACTORS AND INHIBITORS in Vitro ON MON~~XYGENASE ACTIVITY FROM CONTROL AND B-NAPHTHOFLAVONE-TREATED GOLDFISH”

Treatment

7-Ethoxyresorufin-O- deethylase

Control Treated

Benzphetamine-N- demethylase

Control Treated

Standard mixture lOO.Ob 100.0 100.0 loo.0 Carbon monoxide 20.0 18.5 0.0 0.0 Piperonyl butoxide (0.5 mM) 0.0 0.0 66.8 72.9 l/2 NADPH + 1.67 mivr NADH 162.1 144.1 223.2 217.0 NADH alone (1.67 mM) 0.0 0.0 96.1 99.5

u All data are expressed as the percentage of activity compared to the standard incubation mixture containing an NADPH-generating system (described under Methods). Injected fish received the standard treatment of &NF (150 mg/kg) followed by termination at 96 hr.

b For approximate specific activities refer to Table 2.

HEPATIC MONOOXYGENASE SYSTEM OF THE GOLDFISH 385

the presence of one-half of the normal NADPH-generating system resulted in a sig- nificant enhancement of enzyme activities with both substrates. NADH (1.67 mM) alone was unable to support 7&hyoxyresorufin-O- deethylase, while benzphetamine-N-demeth- ylase was maintained at near normal levels.

Spectroscopic Examination of Induced Cyto- chrome P-450

The spectrum of ethyl isocyanide (EtNC) binding to reduced goldfish cytochrome P-450 as a function of pH is shown in Fig. 3. In accordance with observations made in pre- vious studies with higher animals (Dahl and Hodgson, 1978), change in pH brought a pro- found change in the peak ratio. On the other hand, no clear-cut difference in the pH-in- duced spectral changes was observed between

.a0 -

2 g.050 -

3 I .

&40 -

i- c 8 p.030 -

4 3 4.020 -

4 g n a .OIO - a

I ’ c I 650 1.00 7.56 6.00 8.50 8.00

PH

FIG. 3. Ethyl isocyanide binding to sodium dithionite- reduced goldfish microsomes as a function of pH from control (A) and Snaphthoflavone (.)-induced prepara- tions. Fish received the standard 150 m&kg ip dose fol- Iowed by termination at 96 hr. Other aspects of the assay are described under Methods. Data are expressed ~2 + SE. The variability for the treated preparation at pH 8.75 was +0.003.

the treated and control cytochromes. It should be noted that with the methods employed here (described in Methods) a marked instability in the ratio of the two peaks was observed as time progressed. It was also noted that pH above 7.5 caused a shift in the position of the 430-nm peak to the extent that at a pH of 8.0, the peak was shifted to approximately 436 nm. This phenomenon is demonstrated graphically in Fig. 4. To maintain consis- tency, the absorbance at 430 nm was adopted throughout in plotting the data for Fig. 3.

Since no discernable differences were ob- served in binding to the heme iron moiety (as exemplified by EtNC), a suitable type I sub- strate was chosen to examine the effect of /% naphthoflavone induction on binding to the protein moiety of cytochromes P-450. Prelim- inary tests established that mexacarbate was a suitable monooxygenase substrate for the goldfish system. This substrate binds in a type I manner in the rat, rabbit, mouse, and sheep liver microsomes (Kulkami et al., 1975).

Figure 5 demonstrates a typical type I spec- trum obtained upon addition of mexacarbate to oxidized goldfish liver microsomes. This binding spectrum may be characterized by a fairly narrow absorption minimum at 420 nm followed by a broad maximum around 387 nm. A double reciprocal plot of the magni- tude of the absorbance change as the micro- somes were titrated with increasing amounts of mexacarbate is shown in Fig. 6. An ap- proximately 2.4-fold increase in the apparent affinity constant (ePp) over the control is ev- ident. It should also be noted that the maxi- mal (i.e., the point at which further addition of substrate did not increase the absorbance change) spectrum from the control was ap- proximately one-half that of the treated mi- crosomes.

Gel Electrophoresis ’

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a valuable tool for examining the constitutive proteins

386 GOOCH AND MATSUMLJRA

-

0.01 A

-

pH=7.25

pH=6.60

PH =7.45

/“I b pH=6.00

1lllllllllllll,,,,,II 500 450 400

WAVELENGTH,nm

FIG. 4. Ethyl isocyanide binding spectra of reduced goldfish microsomes showing a gradual shift in the ab- sorbance of the 430-nm peak with increasing pH.

from complex mixtures. Figure 7 shows the electrophoretic pattern of &naphthoflavone- induced and noninduced goldfish liver mi- crosomes. The appearance of an increased tration was 2.8 m&ml.

FIG. 5. Computer-assisted representation of mexacar- bate binding to oxidized goldfish liver microsomes from control (-) and &naphthotlavone-induced (- - -) fish. [Mexacarbate] = 0.11 mM. Microsomal protein concen-

protein band at apparent molecular weight of 56,000 daltons is noted in the treated prep- aration. This band is the general region where hemoprotein(s) P-450 migrate and is also ap- proximately the same molecular weight as the enhanced protein found in rainbow trout af- ter fl-naphthoflavone induction (Elcombe and Lech, 1979). An effort to demonstrate the presence of heme in this band region by per- oxidase staining failed. Simultaneous electro- phoresis of microsomes from rat liver, how- ever, did reveal peroxidase activity indicating a marked species difference in this regard. Ap- parently all of the heme from the goldfish preparations had dissociated from the micro- somes quite easily since peroxidase activity was noted at the migration front of the gel.

DISCUSSION

The results of this study indicate that gold- fish possess a /I-naphthoflavone-inducible monooxygenase system as has been described in other marine and freshwater species (El- combe and Lech, 1979; James and Bend, 1980). Dose response studies showed a gen- eral linear relationship within the dose range

” 0

<--\ ,’ ’

1; *

\ : \ : m ‘-’

0 0

‘490 / / / /

470 450 430 410 390 370 WAVECENGTHLNMI

HEPATIC MONOOXYGENASE SYSTEM OF THE GOLDFISH 387

A-control

.-Treated

l/mM Mexacarbate

FIG. 6. Double reciprocal plot of mexacarbate binding to goldfish microsomes. Binding was done in 50 mM Tris buffer (pH 7.5) at a protein concentration of 2.9 mg/ml. The apparent affinity constants (cm) were 6.05 X 10e5 M for control and 2.6 X lo-’ M for induced preparations.

tested, while time course experiments dem- onstrated maximal induction 4 to 5 days post- treatment.

Cytochrome P-450 levels of uninduced goldfish were similar to those reported for rainbow trout (Elcombe and Lech, 1979) sheepshead (James and Bend, 1980), lake trout (Ahokas et al., 1977), and the common carp (Guiney et al., 1980). Significant induction, while variable, was readily achieved. The lev- els of control and &naphthoflavone-induced preparations were very similar to that ob- tained in a closely related species, the carp (Melancon et al., 198 1). Cytochrome b5 levels were generally greater than those reported for rainbow trout or brook trout (Stegeman and Chevion, 1980). Levels of NADPH cyto- chrome c reductase were also not induced and compared favorably with other species of fish tested (James and Bend, 1980; Stanton and Khan, 1975; James et al., 1979). This enzyme, however, appeared to be less active than in

most mammalian systems (reviewed by Kato, 1979).

The substrates chosen for examination of inductive effects covered a variety of the types of cytochrome(s) P-450 found in mammalian systems (e.g., Madhukar and Matsumura, 198 1). Some interesting differences were noted between goldfish and mammals, particularly with respect to 3-MMAB-Ndemethylase and &ALA-synthetase. Both of these activities are inducible by 3-MC-type inducers in mam- malian systems (Madhukar and Matsumura, 198 I), but they are not induced in goldfish. Indeed, 3-MMAB-N-demethylase induction appears not to be specific for 3-MC-type in- ducers since Madhukar and Matsumura (1979) showed nearly a twofold induction of 3-MMAB-N-demethylase activity following phenobarbital treatment of rats. Curiously, mexacarbate oxidative metabolism, which proceeds primarily through a Ndemethyla- tion in our system (see footnote, Table 2), is

388 GOOCH AND MATSUMURA

FIG. 7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hepatic microsomes from control (b, c) and p-naphthoflavone-treated (d, e) goldfish. Well (a) contained standard proteins with molecular weights of 14,400, 21,500, 31,000, 45,000 (shown), 66,200 (shown), and 92,500. Wells contained 75 pg protein and the resolving gel was 7.5% acrylamide.

apparently stimulated by P-naphthoflavone induction. This observation is supported by the increase in the apparent affinity constant (e**) based on mexacarbate binding to the microsomal preparation. Mexacarbate may prove to be a useful substrate when examining the induction of monooxygenase activity in fish.

&ALA-synthetase did not increase after fl- naphthoflavone induction even though SDS- PAGE indicated formation of an increased protein band in the hemoprotein region. There are at least two possible explanations for this: (1) There was a high enough titer of the por- phyrin-heme pool to supply the new protein synthesized in the goldfish liver, or (2) the increased band in the electrophoresis gel was not of a hemoprotein nature since we were unable to detect peroxidase activity in this or any other region of the gel. Based on the in- creases in enzyme activity, binding of sub- strate, and the work of others (Elcombe and Lech, 1979; Vodicinik et al., 198 l), the former explanation appears to be more plausible.

The results in Table 3 apparently represent observations noted only recently in fish liver monooxygenase systems (Schwen and Man- nering, 1982). The synergism seen when NADH is added with NADPH for both of the substrates tested is quite similar to the phe- nomenon seen in rabbits (Cohen and Esta- brook, 197 1). It is also interesting to note that benzphetamine-N-demethylation was sup- ported by NADH nearly as well as with NADPH. Schwen and Mannering (1982) noted a similar phenomenon when investi- gating pnitrophenetole metabolism in brown trout. Whether or not these effects are me- diated through cytochrome b5 in fish as they are in mammals (Staudt et al., 1974; Ka- mataki and Kitagawa, 1977) remains to be seen. However, based on other qualitative similarities between the two systems, cyto- chrome b5 involvement would appear to be likely.

Ethyl isocyanide binding to reduced mi- crosomes has been used in the past (Sladek and Mannering, 1966; Imai and Siekevitz, 197 1) to demonstrate differences in the bind-

HEPATIC MONOOXYGENASE SYSTEM OF THE GOLDFISH 389

ing behavior of microsomal preparations be- fore and after induction. Generally an in- crease in the ratio of the absorbance at 455 and 430 nm has been observed after treat- ment with 3-MC-type inducers. This effect has not been observed in rainbow trout (Elcombe and Lech, 1979). In a similar manner, in- duced rat liver microsomes show a shift in the pH-dependent crossover point (the point at which the absorbance of the 455- and 430- nm peaks is equal) when compared to unin- duced preparations. In contrast, we were unable to demonstrate any significant differ- ences in the nature of the pH-dependent bind- ing between control and induced preparations from goldfish. This observation is consistent with other observations in goldfish liver sys- tems, i.e., the characteristics of control and induced preparations are qualitatively very similar.

The gradual shift in the absorbance of the 430~nm peak to 436 nm at higher pH is an observation we have not seen described by any other authors. This shift could be due to a conversion of a portion of the cytochrome(s) P-450 to P-420 which exhibits a single ab- sorbance at 433 nm when complexed with ethyl isocyanide (Imai and Sato, 1967). How- ever, the fact that the shift appeared to be gradual as the pH was changed would seem to indicate that other phenomena were in- volved. Also, Imai and Sato (1967) reported that the absorbance of the P-420-ethyl iso- cyanide complex was insensitive to changes in pH.

Substrate-induced spectral interactions with oxidized microsomes have been seen in vir- tually every system investigated (reviewed by Schenkman et al., 1981). Those substrates that elicit a type I binding spectrum are believed to bind to the apoprotein moiety of the cy- tochrome P-450 enzyme. In our system the qualitative nature of the mexacarbate-in- duced type I difference spectrum was similar with both control and treated preparations. The fact that titration with substrate revealed differences in apparent affinity and also in the maximum inducible spectrum could be due

to the synthesis of a different form of cyto- chrome P-450 capable of catalyzing monoox- ygenation at lower substrate concentrations. Alternatively, it could be postulated that a change had occurred in the protein environ- ment of a preexisting cytochrome P-450 en- zyme which would affect the nature of the spin equilibrium surrounding that particular cytochrome P-450 enabling the low to high spin transition to occur more readily (Cinti et al., 1979). Schenkman et al. (1981) sug- gested that this process could lead to an en- hanced rate of cytochrome P-450 reduction which in turn would lead to increased rates of metabolism. Both of these possibilities could manifest themselves as the observed phenom- enon.

Although SDS-PAGE showed an increased protein band of approximately 56,000 MW in microsomes from /3-naphthoflavone-treated fish, this alone was not sufficient to distin- guish between the two possibilities.

Other investigations (Vodicinik et al., 198 I; Bend et al., 1973; Addison et al., 1977) have demonstrated that fish are apparently refrac- tive to inducers other than the 3-MC type. Apparently, goldfish are also incapable of re- sponding to Mirex (a barbituate-type inducer) at 100 mg/kg. Preliminary data from this lab- oratory showed no changes in metabolism of ethylmorphine, benzphetamine, 7-ethoxyres- orufin, or mexacarbate 96 hr after a single injection. Also, no significant changes in lev- els of cytochrome P-450 were noted.

In summary, the pattern of induction in goldfish liver is much simpler than that found in mammalian livers. In the former, induc- tion does not extend to &ALAS, NADPH, cytochrome c reductase, cytochrome b5, and monooxygenases to metabolize 3-MMAB, p- nitroanisole, ethylmorphine, and aminopy- rene. The induced monooxygenase system is qualitatively similar to the noninduced con- trol system. This information makes the gold- fish system particularly suitable for further study of inductive mechanisms without com- plex interactions from other factors related to phenobarbital-type induction.

390 GOOCH AND MATSUMURA

ACKNOWLEDGMENTS

Supported by the Michigan Agricultural Experiment Station (Journal Article No. 10565), Michigan State Uni- versity, and by Research Grant ES01963 from the Na- tional Institute of Environmental Health Sciences.

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