Inhibition of hepatic microsomal monooxygenase system by organotins in vitro in freshwater fish

ELSEVIER

AOIIATIC TOXICOLOGY Aquatic Toxicology 28 (1994) 107-126

Inhibition of hepatic microsomal monooxygenase system by organotins in vitro in freshwater fish Karl Fent*, Thomas D. Bucheli Swiss FederalInstitute for Environmental Science and Technology ( EA WAG), Oberlandstrasse133, CH-8600 Diibendorf Switzerland (Received 25 March 1993; revision received 7 September 1993; accepted 27 September 1993)

Abstract

The interaction in vitro of organotins tributyltin (TBT) and triphenyltin (TPT) with the hepatic microsomal monooxygenase systems in the freshwater fish, rainbow trout (Oncorhynchus mykiss), European eel (Anguilla anguilla), and bullhead (Cottus gobio) was studied. Hepatic microsomes were incubated in vitro with TBT and TPT and various components analyzed. Ethoxyresorufin O-deethylase (EROD) activity was strongly inhibited by TBT and TPT in a concentration-dependent manner in all fish. Rainbow trout microsomes were more sensitive than were eel or bullhead microsomes. Total inhibition of EROD activity occurred both at 0.5 mM TBT and TPT in rainbow trout, whereas I mM decreased EROD activity to 15% (TBT) or 3% (TPT) in eel, and 35% (TBT) or 18% (TPT) in bullhead, respectively. As this effect may be caused by inhibition of different components of the microsomal electron transport system, different enzymes were studied separately. In all fish, both organotins led to a time- and concentration-dependent decrease in spectral total microsomal P450 content, and formation of cytochrome P420. TPT led to a greater inactivation of P450 enzyme than TBT, and induced a 50% loss in all fish at 0.08 mM TPT, whereas in case of TBT a 50% loss occurred at 0.18 mM in rainbow trout, 0.30 mM in bullhead, and 0.83 mM in eel. Cytochrome b5 content was not affected, but both organotins led to an almost selective inhibition of either N A D H or N A D P H cytochrome c reductase activity in trout and eel, or of both in bullhead. Whereas TBT inhibited N A D H cytochrome c reductase, TPT acted strongly on N A D P H cytochrome c reductase. The study demonstrates species-related significant and selective effects of TBT and TPT on different components of the microsomal monooxygenase system in freshwater fish. This leads to inactivation of native enzymes and inhibition of enzyme activities.

Key words: Freshwater fish; Tributyltin; Triphenyltin; Microsomal Monooxygenase System; Cytochrome P450; N A D P H cytochrome P450 reductase; N A D H cytochrome b 5 reductase

* Corresponding author. Phone: +41-41-482 149. Fax: +41-41-482 168 0166-445X/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0166-445X(93)E0047-F

108

K. Fent, 72D. Bucheli/Aquatic Toxicology 28 (1994) 10~126

1. Introduction

Tributyltin (TBT) and triphenyltin (TPT) are of environmental concern owing to their high toxicities and direct introduction into the aquatic environment mainly by leaching from antifouling paints and employment in agriculture. Even after regulation of these compounds in antifouling paints in many countries, contamination persists at chronic levels (Alzieu et al., 1989; Fent and Hunn, 1991). Other sources including municipal and industrial wastewaters, agricultural applications, and employment in materials protection may further cause pollution (Fent and Mfiller, 1991). The high toxicities of TBT and TPT have been demonstrated in a number of aquatic animals including fish (for review, Hall and Pinkney, 1985; Jarvinen et al., 1988; Fent, 1992; Fent and Meier, 1992, 1993), but only a few studies have focused on biochemical effects in these biota (Spooner et al., 1991; Fent and Stegeman, 1993). The hepatic microsomal monooxygenase or cytochrome P450 systems play a vital role in the metabolism of both endogenous and xenobiotic compounds. Thus, interference of xenobiotics with these crucial enzyme systems are of importance. Most studies on the interaction of environmental pollutants with hepatic microsomal cytochrome P450 have been focused on the induction of this enzyme system (for review, Stegeman and Kloepper-Sams, 1987). Recently, we provided evidence that in fish, environmental pollutants may also inhibit these enzymes. TBT was shown to have a strong inhibitory effect in vitro and in vivo on microsomal cytochrome P450 in a marine fish (Fent and Stegeman, 1991; Fent and Stegeman, 1993). lmmunoblot analysis of three different isoforms in Western blots with specific antibodies showed that TBT led to loss of P450 forms and associated enzyme activity in vivo. Cytochrome P450 1A1 (CYP1A1) was demonstrated to be more selectively affected than other hepatic P450 forms. Here, the investigation into the interactions of organotins with hepatic monooxygenase systems is extended to freshwater fish, in a study not only of TBT, but also of TPT. The latter is a fungicide used in agriculture, and employed in antifouling paints as well. The in vitro interaction of these organotins was studied in fish species with different ecological niches. Whereas rainbow trout (Oncorhynchus mykiss) is a predator living in the open water, both European eel (Anguilla anguilla) and bullhead (Cottus gobio) are sediment-dwelling predators in the benthic food chain. Bullheads are thought to be susceptible to anthropogenic influences including environmental chemicals, whereas eels are much more pollutant tolerant. The aim of this study was to investigate and compare effects of TBT and TPT on the hepatic monooxygenase systems in these representative freshwater fish. The data provide evidence that both organotins, TBT and TPT, have a strong and selective effect upon different components of this enzyme system. Moreover, they show marked species differences in inhibitory effects. These findings have importance not only for the acute toxicity of organotins, but also for the use of P450 and associated enzyme activity as biomarkers for exposure and toxicity.

K. Fent, T.D. BuchelilAquatic Toxicology28 (1994) 107-126

109

2. Materials and Methods

Materials Tributyltin chloride (TBT) and triphenyltin chloride (TPT) both with a purity of > 97% were purchased from Fluka AG, Buchs, Switzerland. Dilutions of TBT and TPT were made either in 98% ethanol or in dimethylsulfoxide (DMSO > 99.5%), and used in the incubation experiments. Dithionite was purchased from Aldrich, Milwaukee, WI, USA. Horse heart cytochrome c, NADPH and NADH were obtained from Sigma, St. Louis, MO, USA. Ethoxyresorufin was from Molecular Probes, Eugene, OR, USA. All chemicals were of highest purity available. Fish Rainbow trout (Oncorhynchus mykiss) weighing 350 and 475 g were from our laboratory breeding stock held in outdoor tanks in running Lake Lucerne water. One female was taken on 23 April 1992 just prior spawning, and one male on 22 July. Twenty-one bullheads (Cottus gobio) of both sexes weighing between 6.8 and 23.2 g, and three European eels (Anguilla anguilla) of both sexes weighing 163, 218 and 681 g were both caught by electrofishing in the River Rhine, Switzerland, on 23 April and 26 May 1992, respectively, and transported live to the laboratory. Bullheads were from the Alpine Rhine at Werdenberg, Canton St. Gallen, and eels from the High Rhine at Eglisau-Rheinsfelden, Canton Ziirich. Bullheads were caught at the spawning period, and five fish contained eggs. One eel had an estimated age of 9+2 years, and two were 6 + 2 years old, one was gonadally immature. In the laboratory, bullheads were maintained in aquaria in flow-through Lake Lucerne water at 8°C for 1 week, and eels for 1 day prior to preparation of microsomes. Microsome preparation Animals were killed after having been anaesthetizised briefly with 2-phenoxyethanol (99%, 20 ml in 40 L water), and livers were homogenized in ice-cold buffer (50 mM Tris-HC1, pH 7.4, containing 0.15 M KCI). Hepatic microsomes of rainbow trout, and eel were prepared individually. Bullheads of both sexes were randomly divided in two groups of either ten or eleven individuals, and livers were pooled. Microsomes were prepared in resuspension buffer (50 mM Tris-HC1, 1 mM NaEDTA, pH 7.4, 1 mM dithiothreitol, 20% v/v glycerol) by differential centrifugation. After eliminating cell debris and mitochondria, microsomes were obtained by centrifugation of the 10 000 ReM supernatant with 20 000 R1,Mfor 90 min in a Centrikon H-401 B centrifuge (Kontron Instruments, Ziirich, Switzerland). The obtained pellet was resuspended, washed in resuspension buffer, and spun down at 20 000 gem for 120 min. Investigations by Stegeman (1987) showed that fish microsomes obtained by this procedure are very similar to those obtained by ultracentrifugation. Microsomes resuspended in EDTA-free resuspension buffer were archived in liquid nitrogen until used. Microsomal protein was measured by the method of Bradford (1976) using BSA as standard.

110

K. Fent, TD. Bucheli/Aquatic Toxicology 28 (1994) 10~126

Organotin incubations Microsomes were incubated either in glass tubes in a water bath, or in glass cuvettes within the spectrophotometer (Uvikon 810, Kontron Instruments, Zfirich, Switzerland) at 30°C for up to 20 min. Different concentrations of TBT or T P T in carrier solvent ethanol or DMSO, or equal amounts of solvent alone were used. Maximal solvent concentrations were 5% for ethanol, and 2% for DMSO. Various components of the microsomal electron transport system including ethoxyresorufin O-deethylase activity (EROD), contents of cytochrome P450, cytochrome P420, and cytochrome bs, and N A D ( P ) H cytochrome c reductase activity were determined after different incubation times. EROD assays In vitro inhibition of ethoxyresorufin O-deethylase (EROD) activity, catalyzed by cytochrome P450 1A (CYP1A), was first determined by the method of Klotz et al. (1986) in rainbow trout and eel to optimize assay conditions. Activities were higher at 30 than at 15°C, and they were higher at 5 than at 15 min incubation. Moreover, the influence of carrier solvent was tested. Ethanol or DMSO had no influence on spectral total P450, whereas ethanol (2%), but not DMSO (2%), led to inhibition of E R O D activity. Consequently, DMSO was used as a solvent for TBT and T P T in E R O D assays. The time point of optimal and linear E R O D activity differed between fish. Whereas in rainbow trout and bullheads activity was highest at the beginning, activity in eel occurred only after a time-delay, and was highest 5 rain after the start of the reaction. Based on these results the following assay was employed. Inhibition of E R O D activity was determined after incubation of microsomes in glass vials with different concentrations of TBT or T P T in D M S O (2%), or 2% DMSO alone at 30°C for 5 min. TBT in DMSO was added to a 50 pl micro somal suspension (a total of 0.31-1.07 mg protein). The reaction mixture (440 pl) with buffer (0.1 M Tris-HCl, pH 8.0, 0.1 M NaC1) and 2 p M 7-ethoxyresorufin was preincubated separately at 30°C for 5 min, and the reaction was initiated by the addition of 10pl N A D P H (4.3 mg ml-l). Product formation was measured at 572 nm for 6.5 min in 2-min intervals (e = 73 mM -~ cm-~). Cytochrome P450 and cytochrome b5 determinations Cytochrome P450 (e = 91 m M -1 cm -1) and cytochrome P420 (e -- 111 mM -L cm 1) content of microsomal preparations were determined by CO difference spectra of dithionite reduced microsomes. A total volume of 700/.tl resuspension buffer containing 20 pl microsomal suspension (a total of 0.31 2.70 mg protein) and 5/.tl N A D H (5 mg m1-1) was bubbled for 5 s with CO in a glass vial and then distributed into two cuvettes for P450 and P420 determination. Difference spectra were run at 0, 2, 5, 10, 15, and 20 min at 30°C. Influences of ethanol and DMSO were indistinguishable, and thus both solvents were used. In general, freshwater fish microsomal P450 was observed to be susceptible to degradation by CO. Thus, bubbling for only a short time (5 s), and addition of N A D H prior to determination of P450 was crucial for obtaining P450 spectra without P420 contamination. Cytochrome b5 content (e = 185 m M -~ cm -1) was spectrally determined in glass

K. Fent, T.D. Bucheli/Aquatic Toxicology28 (1994) 107-126

111

cuvettes from NADH difference spectra after incubation of a 700 gl resuspension buffer containing a 20/A microsomal suspension (a total of 0.31-1.17 mg protein) with TBT or TPT in 2% DMSO, or 2% DMSO alone at 30° C. N A D ( P) H cytochrome c reductase activity NAD(P)H cytochrome c reductase activity was assayed with a reaction mixture containing 460/zl horse heart cytochrome c (1.1 mg m1-1 in 0.2 M K-phosphate buffer, pH 7.5, 1 mM KCN), and 10/.tl microsomal suspension (containing 0.0230.053 mg protein in different samples). After incubation of this mixture with 10/zl TBT or TPT of different concentrations in DMSO (2%) or 2% DMSO alone for 2 min at 30°C, the reaction was started by addition of 20/.tl NADH (7.1 mg m1-1) or NADPH (4.3 mg ml-1), and the reduction of cytochrome c was measured at 550 nm for 2 min (e = 21.1 mM -1 cm-l). Substrate-induced spectral determinations Rainbow trout hepatic microsomes (70 gl microsomal suspension) were prepared in 700/11 resuspension buffer (protein content 2.34 mg m1-1) and equally divided between two cuvettes. The baseline was corrected for equal absorbance prior to the addition of substrates, TBT and TPT, which were dissolved in ethanol. Increasing concentrations of TBT or TPT were added to the sample cuvette, while an equal volume of ethanol alone was added to the reference cuvette at 30°C, and the spectra recorded after each addition. Data processing TBT and TPT concentrations refer to the weight of the respective chloride. Every measurement was performed at least three times, and data are given as means with standard error of means (SEM). Statistical evaluation of the data was performed employing t-tests, and ANOVA with Dunnett's t-test in the case of pooled bullhead microsomes. Statistical significance is related to the level of P -< 0.05.

3. Results 3.1. P450 spectral studies

Table 1 gives data on the monooxygenase systems in the fish species studied. Total spectral P450 content was higher in the male than female rainbow trout, and higher than in eel or bullhead. Generally, highest P450 difference spectra were found 2 min after reduction with dithionite. Cytochrome b5 content and activities of the NAD(P)H cytochrome c reductases in all fish were in the same order of magnitude. EROD activities in eel were low, but with rainbow trout microsomes, relatively high values occurred as compared to activities reported typical for unexposed fish (Goksoyr et al., 1987). Eels were caught at the same location, where a sample of 19 fish was analyzed for residues of polychlorinated biphenyls (PCB) two years earlier (Vecsei-

K. Fent, 72D. Bucheli/Aquatic Toxicology 28 (1994) 107-126

112

EROD activity

12(~ eq

TBT

1o* ,.,

....

-~.

© 8(

.

",

6(

r..)

"~--.

4(

...... D..... Rainbow trout o Eel - - - a - - . Bullhead

20 •

0.0

,

0.2

.

,

,

•

0.4 0.6 TBT [raM]

q

0.8

.

,

1.0

EROD activity TPT ca

L)

...... D..... o ---a--. 0.0

0.2

0.4 0.6 TPT [mM]

0.8

Rainbow trout Eel Bullhead

1.0

Fig. 1. Concentration dependence of the inhibition of ethoxyresorufin O-deethylase (EROD) activity after 5 rain incubation at 30°C in presence of TBT (top) and TPT (below) in DMSO in rainbow trout, eel, and bullhead. Averages + SEM of at least three separate determinations.

TABLE 1 Content and activities of different components of the microsomal electron transport system in freshwater fish Component

Rainbow trout

Eel

Bullhead

Cytochrome P450 (nmol mg -1) EROD (pmol rain -1 mg -l) Cytochrome b 5 (pmol mg ~) N A D H cytochrome c reductase (nmol rain -l mg -~) N A D P H cytochrome c reductase (nmol min -1 mg 1)

0.295 _+ 0.105

0.179 + 0.002

0.141 + 0.029

367 + 8

12 + 0

18 _+4

37 + 2

47 _+ 12

132 _+ 0

99 + 4

72 _+ 17

40 + 2

20 _+ 1

38 + 3

Averages + SEM of at least three determinations in at least one fish

186 + 9

113

K. Fent, TD. Buchelil Aquatic Toxicology 28 (1994) 107-126

l

0.01

O

0.00

<

-0.01 i

400

450

500

400

450

500

400

450

500

Fig. 2. Decrease of absorbance at 450 nm (cytochrome P450) and increase of absorbance at 420 nm (formation of cytochromeP420) in rainbow trout microsomes. The difference spectra were recorded after incubation of microsomal suspension with 2% DMSO (left), 0.2 mM TPT (middle), and 0.5 mM TPT (right) in DMSOfor 5 min at 30°C. With increasingconcentrations of TPT, suspensions becameturbid and the baseline shifted.

Hohl et al., 1992). Fish of similar size had average total PCB residues in muscle tissue between 1.68 and 2.85 mg/kg (wet weight). Both organotin compounds significantly inhibited hepatic microsomal enzyme activity in all fish species. E R O D activity, catalyzed by cytochrome P450 1A1 (CYP 1A1) in rainbow trout, was strongly inhibited by TBT and TPT in a concentrationdependent manner (Fig. 1). Significant inhibitions occurred at 0.1 m M TBT and TPT, and effects of both compounds were similar. Moreover, species-related differences occurred at concentrations higher than 0.1 mM. Rainbow trout microsomes were more sensitive to both organotins than were eel or bullhead microsomes. For both compounds a 50% loss in E R O D activity occurred at 0.15-0.17 m M in rainbow trout and eel, and 0.48 m M TPT or 0.73 m M TBT in bullhead, respectively. In rainbow trout, E R O D activity was completely lost at 0.5 m M TBT or TPT. Incubation of microsomes in the presence of TBT and TPT led to a time- and concentration-dependent decrease in total spectrally-determined microsomal P450 content. As illustrated in Fig. 2, the organotin-induced decrease in absorbance at 450 nm was accompanied by an increase of absorbance at 420 nm. At 0.5 m M TPT and more, microsomal suspensions became turbid probably due to denaturation of proteins, and so the spectral baseline shifted. At 0.75 m M TPT, no spectral P450 was detectable. In rainbow trout a significant concentration-dependent change of absorbance took

114

K. Fent, T.D. Bucheli/Aquatic Toxicology 28 (1994) 10~126 C y t o c h r o m e P450 100

Rainbow trout

8°iI

~-

Control EtOH (2%) ,t 0.05 mM s 0.10 mM ---O-- 0.20 mM -0.50 mM ---'&--- 0.75 mM 1.00 mM

: i 60" ~ ' ~ 40" 20" 0

5

0

10 Time [min]

Cytochrome

15

20

P420 Rainbow trout

~ 10G ~ 80 o ~, ~ 60

~ ~

~ -----O--& " -----O----""@""--0"--

~ 40 r~'~ 20 0

5

I0 Time [min]

i

i

15

20

Control EtOH(2%) 0.05 mM 0.10 mM 0.20 mM 0.50 mM 0.75 mM 1.00mM

Fig. 3. In vitro time-dependent change of absorbance in the presence of different concentrations of tributyltin chloride (TBT); loss of cytochrome P450 (top), and formation of cytochrome P420 (below). Microsomes were prepared from the liver of a female rainbow trout. The microsomal suspensions in a total volume of 700/11 were incubated at 30°C in presence of TBT in ethanol. The values are given as percentage of maximal total CO-binding cytochrome occurring at 2 min without solvent, and represent the average+sEM of at least three separate determinations. After 20 min incubation with 1 mM TBT P420 could not be measured due to baseline shift.

place rapidly after addition of T B T to the microsomal suspension, with subsequent minor losses of P450 over time (Fig. 3). A statistically significant decrease was observed at 0.2 m M T B T after 2 min, and 0.1 m M T B T after 5 min incubation. Further decreases occurred within 20 min incubation, but these m a y have involved instability of the microsomal enzymes, due to factors other than organotins, as the control incubations showed similar changes over this period (Fig. 3). With ethanol as the solvent (2%), the decrease in P450 was not significant. The increases in P420 content followed the same time course as loss of P450. After 15-20 min incubation with 1 m M TBT, as much as 100% of CO-binding cytochrome was present as cytochrome P420. T P T acted even stronger on the shift of absorbance from 450 to 420 nm in rainbow trout than TBT. The concentration-dependence of the conversion of the cytochrome P450 into P420 is shown in Fig. 4. After 5 min incubation with 0.1 m M T P T cyto-

K. Fent, T.D. BuchelilAquatic Toxicology 28 (1994) 10~126 Cytochrome P450

TPT Rainbow trout

100' ~=

80: •

~

40

,t

~)

20-

--

5 10 Time [mini

Control DMSO (2%) 0.10 mM 0.20 mM 0.50 mM 0.75 mM

15

Cytochrome P420

TPT

100"

~" ~

115

Rainbow trout

60 ;

Control

40 o (~ ~" 20

,I. 0.10 mM --'O'-- 0.20 mM -0.50 mM •

0

i

5 10 Time [min]

,

i

15

Fig. 4. In vitro time-dependent change of absorbance in the presence of different concentrations of triphenyltin chloride (TPT); loss o f cytochrome P450 (top), and formation of cytochrome P420 (below). Microsomes were prepared from the liver of a male rainbow trout. The microsomal suspensions in a total volume of 700 pl were incubated at 30°C in the presence of T P T in DMSO. The values are given as percentage of maximal total CO-binding cytochrome in controls and represent averages +SEM of at least three separate determinations. At 0.75 m M T P T P420 could not be quantified•

chrome P450 was significantly lowered from its maximal content. The solvent DMSO did not have a significant influence. No P450 was detectable at 0.75 m M TPT. As with rainbow trout, TBT and T P T led to conversion of P450 to P420 in eel and bullhead microsomes. Consequently, only the loss of P450 is demonstrated in Figs. 5 and 6. Both organotins led to a concentration-dependent decrease of total P450, and T P T had a stronger effect than TBT. A significant loss of P450 was found in eel and bullhead at 0.2 m M TBT and 0.1 m M TPT, respectively. Total conversion of total P450 to P420 occurred after 2 min with 0.75 mM T P T in eel, and 0.5 m M T P T in bullhead. Fig. 7 shows the inactivation of total spectral P450 for the three species after 5 min incubation. With TBT, species-dependent differences occurred at higher concentrations, for which bullhead microsomes were more susceptible than were rainbow trout and eel. In eel, the remaining P450 content was 60% at 0.5 m M TBT, and 47% at 1

K. Fent, T.D. Bucheli/Aquatic Toxicology 28 (1994) 107 126

116

C y t o c h r o m e P450 TBT Eel

,Al"-----e----~,l~.____._-

100 O

~

80

~-"~ 60" "~

• + ........o ....... ----d23---

40"

20" i

5

1

0

i

10 Time [min]

i

i

15

20

C y t o c h r o m e P450 TPT 0 ~

Eel

~~ E',q 60 [ \\\xlKE E I ~\~---~

.=o

40 1 \it

~",'~"'

I

I ~" [- - - 0 - -

at

. . . ."

\~,,

201

~-...,....g

Control DMSO(2%)

O.lmM

1-""0 ...... 0.2mM

\

0.,mM

0.75 mM

0-f----~-

0

Control EtOH(2%) 0.2 mM 0.5 mM 1.0 mM

.

5

•

10

-

~

15

Time [rain]

Fig. 5. Time-dependent decrease in absorbance at 450 nm (loss of cytochrome P450) in presence of different concentrations of TBT in ethanol (top), or TPT in DMSO (below) in eel (Anguilla anguilla). Microsomes were incubated at 30°C. Values are given as percentage of maximal total CO-binding cytochrome in controls at 2 min incubation and represent averages + SEN of at least three separate determinations.

mM. In all fish, TPT led to a stronger inactivation of P450 than TBT (Fig. 7). TPT induced a 50% loss of P450 in all fish at 0.08 m M TPT, whereas in the case o f TBT a 50% loss occurred at 0.18 m M in rainbow trout, 0.30 m M in bullhead, and 0.83 m M in eel. Complete loss o f P450 occurred at 0.5 m M TPT in bullhead, and at 0.75 m M TPT in rainbow trout and eel. Contrary to TBT, TPT did not cause significant species-related differences m the loss o f total spectral P450. Comparisons o f E R O D inhibition and inactivation o f total spectral P450 reveal that in rainbow trout E R O D activity was completely lost at 0.5 m M TBT or TPT (Fig. 1), at concentrations where not all spectral P450 was inactivated (Fig. 7). In eel E R O D activity also tended to be more decreased than total spectral P450.

3.2. Electron transport system studies The hemeprotein cytochrome b5 transfers electrons from N A D H cytochrome c reductase to P450. In all fish, the content o f cytochrome bs, measured spectrophoto-

K. Fent, T.D. Buchelil Aquatic Toxicology 28 (1994) 107-126

117

Cytochrome P450 TBT Bullhead

100" ~r~80 ~) "e-,

~'~

60

~

40

r..)

20

~Control -"--13--- EtOH (2%) it 0.05 mM 0.10 mM -0.20 mM 0.50 mM i

0

5

10 Time [min]

i

i

15

20

Cytochrome P450 TPT A

100

A

Bullhead

6o

"~

•

40 20

0

5 10 Time [minl

Control DMSO (2%) 0.10 mM 0.50 mM

15

Fig. 6. Time-dependent decrease in absorbance at 450 n m (loss ofcytochrome P450) in presence of different concentrations of TBT in ethanol (top), or T P T in D M S O (below) in bullhead (Cottus gobio). Microsomes were incubated at 30°C. Values are given as percentage of maximal total CO-binding cytochrome in controls at 2 min incubation and represent averages_+SEM of at least three separate determinations.

metrically, was not altered after addition of TBT or TPT to the incubation cuvette, and it remained constant for up to 15 min (data not shown). No significant decrease occurred at 0.2 TBT and TPT, respectively, and 0.5 mM TBT. However, baselines of the spectra shifted considerably, partly due to turbidity in the microsomal suspensions at higher organotin concentrations. The baseline-shift was more pronounced with TPT than TBT. TBT and TPT led to inhibition of NADH and NADPH cytochrome c reductase activity, but the organotins differed in their specificity. In rainbow trout, TBT inhibited NADH more strongly than NADPH cytochrome c reductase activity, whereas TPT acted strongly on NADPH cytochrome c reductase, and had only minor effects on the other reductase (Fig. 8). Inhibitions were significant at 0.2 mM TBT for the NADH, and 0.2 mM TPT for the NADPH cytochrome c reductase. Similar, but weaker effects occurred in eel microsomes (Fig. 8). Hence, TBT selectively inhibited NADH cytochrome c reductase, or NADH cytochrome b5 activity, whereas TPT selectively inhibited the NADPH cytochrome c, or NADPH cytochrome P450 reduc-

K. Pent, 72D. Bucheli/Aquatic Toxicology 28 (1994) 107-126

118

Cytochrome

P450

100 8O 60

~E

40

r..)

20

..... 12---

trout

Eel - - --/x--i

0.0

Rainbow

~

0.2

•

J

0.4 0.6 TBT [mM]

•

i

0.8

.

Bullhead

i

1.0

Cytochrome P450 lO0

~ ca.,

,~ 80 ,..,

i=2 60 "=~ 40 . . . . . 13----

~)

20 - - ~

0 0.0

Rainbow

trout

Eel --

Bullhead

i

0.2

0.4 0.6 TPT [mM1

0.8

1.0

Fig. 7. Concentration dependence of the shift in absorbance from 450 to 420 nm (conversion of the cytochrome P450 into P420) after 5 min incubation at 30°C. Averages +SEM of at least three separate determinations with TBT (top), and TPT (below) are given for rainbow trout, eel and bullhead.

tase in r a i n b o w t r o u t a n d eel. H o w e v e r , with b u l l h e a d m i c r o s o m e s , i n h i b i t i o n o f N A D H a n d N A D P H c y t o c h r o m e c r e d u c t a s e s were f o u n d with b o t h o r g a n o t i n s (Fig. 8), w h i c h indicates species-related differences. S i m i l a r l y to the o t h e r fish species, T P T a c t e d m o r e s t r o n g l y to the N A D P H c y t o c h r o m e P450 r e d u c t a s e t h a n TBT. Therefore, o r g a n o t i n s i n h i b i t o t h e r c o m p o n e n t s o f the m i c r o s o m a l m o n o o x y g e n a s e system besides P450.

3.3. Binding spectra To f u r t h e r investigate the i n t e r a c t i o n s o f o r g a n o t i n s with fish m i c r o s o m a l P450, s u b s t r a t e - b i n d i n g s p e c t r a were d e t e r m i n e d . I n c u b a t i o n o f r a i n b o w t r o u t liver m i c r o somes in the presence o f either T B T o r T P T led to different spectra (Fig. 9). W i t h T B T a t y p e I s u b s t r a t e - i n d u c e d difference s p e c t r u m was p r o d u c e d with an a b s o r b a n c e p e a k at 387 n m a n d a t r o u g h at 427 nm. A n a d d i t i o n a l difference s p e c t r u m was s u p e r i m p o s e d o n the s u b s t r a t e - i n d u c e d s p e c t r u m . This resulted in a s e c o n d p e a k at 406 nm. This s p e c t r u m m i g h t o r i g i n a t e f r o m r e d u c e d P450 1A1 t h a t has its a b s o r p -

K. Fent, T.D. BuchelilAquatic Toxicology 28 (1994) 107-126

119

NAD(P)H cytochrome c reductase activity R a i n b o w

\

4°

~ot

~ ....

~ ........

ol . . . . . . . o.o o.o

t r o u t

~= ¢

NADH ('rBT) NADH (TPT)

..... O'-"-. . . . O----

NADPH fYBT) NADPH OPT)

,.o

TBT or TPT [mM] NAD(P)H cytochrome e reduetase activity

Eel 100:

8o

8

.~

60

<

40

-~-

NADH (TBT) NADH OPT)

2O ..... £3...... NADPH (TBT) ...... O .... NADPH OPT) 0.0

0.2

0.4

0.6

0.8

1.0

TBT or Tiff ImM] NAD(P)H cytoehrome c reductase activity

Bullhead 100

~ 8

so

~ "r.

60

I~

4O --" 20-

.

0.0

,

0.2

•

,

0.4

•

,

0.6

•

,

0.8

•

NADH (TBT)

•

NADHO'PT)

....-12..... .... "O......

NADPH (TBT) N ADPH (TPT)

,

1.0

TBT or TPT [mM]

Fig. 8. Concentration dependence of the inhibition of N A D H and N A D P H cytochrome c reductase activity in presence of TBT and T P T in D M S O in rainbow trout (top), eel (middle), and bullhead (below). Activities were determined after 2 min incubation at 30°C. Averages +_ sEu of at least three separate determinations.

120

I~ Fent, T.D. Bucheli/Aquatic Toxicology 28 (1994) 107-126

TBT

TPT

0.045

Y~ 2

-0.045 t_ 350

~ 400

450

500 350

J 400

I 450

500

Wavelength [nm] Fig. 9. Substrate-induced difference spectra induced by incubation of rainbow trout liver microsomes (2.34 mg ml-~) in the presence of either 100,uM TBT or TPT. TBT produced a type I substrate-induced spectrum with a peak at 387 nm and a through at 427 nm. An additional difference spectrum superimposed on the substrate-induced spectrum leads to a second peak at 406 nm. Addition of TBT also led to a baseline shift. TPT, however, did not show a substrate-induced difference spectrum.

tion m a x i m u m at 409 n m (Keck-Oertle, 1986). The o r g a n o t i n - i n d u c e d spectral c h a n g e was first observed at 10 ~tM TBT, a n d the m a g n i t u d e increased with c o n c e n tration. N e i t h e r type I n o r type II s u b s t r a t e - i n d u c e d difference spectra could be observed with T P T in c o n c e n t r a t i o n s o f 1 0 / t M to 2000/.tM (Fig. 9). Therefore, only T B T p r o d u c e d a s u b s t r a t e - i n d u c e d b i n d i n g spectrum, which indicates a significant difference between the organotins.

4. Discussion I n fish, T B T a n d T P T can strongly interact with m i c r o s o m a l m o n o o x y g e n a s e systems, which leads to i n a c t i v a t i o n of the m a j o r c o m p o n e n t , P450 enzyme, i n h i b i t i o n o f P450 enzyme activity, i n h i b i t i o n o f N A D ( P ) H c y t o c h r o m e c reductase activity, a n d

K. Fent, T.D. Buchelil Aquatic Toxicology 28 (1994) 107-126

121

thus the loss of an enzyme system responsible for the detoxification of environmental pollutants and the metabolism of endogenous substances. These in vitro findings are relevant for effects in vivo as well. In the marine scup, similar effects occurred with TBT in vitro (Fent and Stegeman, 1991) and in vivo (Fent and Stegeman, 1993), and effects of TPT in vivo are now under investigation. However, the implications of these findings for chronic toxicity have to be shown. The inhibition of the hepatic mono-oxygenase system is in concert with additional biochemical effects of organotins. TBT was shown to inhibit oxidative phosphorylation (Aldridge, 1976), and to act as a mitochondrial uncoupler (Connerton and Griffiths, 1989). In addition, TBT and TPT inhibit glutathione S-aryltransferase (Mannervik et al., 1985), and organotins interfere with heme metabolism (Rosenberg et al., 1981). Hemolytic action of TBT has been demonstrated in human erythrocytes, so this compound may disrupt membranes and act as a membrane toxicant (Gray et al., 1987). Moreover, organotins may negatively affect additional detoxication enzymes in fish, as TPT is a potent inhibitor of glutathione S-transferase (George and Buchanan, 1990). Among the most susceptible organism affected by TBT are stenoglossan gastropod molluscs. Females produce reproduction anomalies (imposex) at trace quantities of a few ng/l TBT in seawater Bryan et al., 1986). In female dogwhelk, an increase in penis length and increased levels of testosterone were found after exposure to 40 ng/1 TBT, and testosterone injections in snails resulted in imposex (Spooner et al., 1991). Inhibition or stimulation of P450 systems may result in disturbances of hormone production. Hence, it is possible that the inhibition of the P450 system responsible for the conversion of testosterone to estradiol-17fl by TBT and/or TPT may result in the observed disturbances in steroid patterns in snails. The findings of this study may have implications for the use of P450 as a biomarker in environmental monitoring. Inhibitory effects of these organotins on cytochrome P450 catalytic activity (EROD activity) may modulate the induction response by organic environmental pollutants, an effect hitherto widely neglected in the evaluation of this biochemical response as a biomarker. 4.1. E R O D activity and total P450

The hepatic microsomal monooxygenase system of rainbow trout has been characterized by a number of laboratories (e.g. Williams and Buhler, 1982; Goksoyr et al., 1987). P450 contents in eel were reported recently (Lemaire-Gony and Lemaire, 1992), but the bullhead system has not yet been described. Marked species-related differences in the interactions of freshwater fish microsomal monooxygenase systems with TBT and TPT were observed. This might be interpreted as species-related differences in these hepatic systems. The TBT results were similar to those in the marine scup (Fent and Stegeman, 1991). However, freshwater fish microsomal P450 was found to be maximal in content only after 2 min incubation. It was generally unstable during incubations longer than 5-10 min even without organotins. Further, differences between scup and freshwater fish occurred in the activity of the reductases. This cannot, however, be interpreted in terms of different importance of either reductases as electron donors for P450 (Kloepper-Sams et al., 1987).

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K. Fent, 7~D. Bucheli/Aquatic Toxicology 28 (1994) 10~126

In rainbow trout, E R O D activity was completely lost at organotin concentrations where not all total spectral P450 was inactivated (Figs. 1 and 7). In eel, E R O D activity also tended to be more decreased than total P450, but this was not the case in bullhead. As E R O D inhibition and reduction of spectral P450 did not occur in parallel, the inhibition of catalytic activity could represent a selective inhibition of CYP1A. Similarly, in the marine scup, E R O D activity was abolished at TBT concentrations where not all the total spectral P450 was inactivated (Fent and Stegeman, 1991). Subsequent immunoblot analysis showed that TBT in vivo led to a decrease of CYP1A protein content at all TBT doses, but decreases of two additional P450 forms at the highest dose only (Fent and Stegeman, 1993). The question of specificity towards different P450 forms, especially in case of T P T are investigated in forthcoming experiments using different substrates and selective immunological methods. 4.2. In vitro mechanism o f T B T and T P T action

The similar effects of TBT in freshwater and marine fish (Fent and Stegeman, 1993) indicate a similar mode of action towards P450. Moreover, T P T inactivated total spectral P450, but acted differently on the reductases than TBT. These organotin compounds may affect either the heme moiety of cytochrome P450 or bind to amino acids such as cysteine at the active site, or on other sites resulting in binding to the globin portion of the enzyme which leads to inactivation of catalytic activity. TBT and T P T are lipophilic, as indicated by their octanol-water partition coefficients of 3.54 (Laughlin et al., 1986) and 2.11 (Tsuda et al., 1986), respectively. Therefore, they likely penetrate the hydrophobic membrane environment in which the cytochrome P450 is embedded, and thereby gain access to this enzyme. However, the specific mechanism of action of TBT and T P T on P450 is apparently different. Visible spectrophotometry has been used to determine the binding characteristics of a variety of compounds in proximity to oxidized P450 (Fe3+). Incubation of rainbow trout microsomes with TBT resulted in the formation of a type I binding spectrum (Fig. 9), similar to that observed in rats (Rosenberg and Drummond, 1983). These binding spectra are typical of a lipophilic hydrocarbon interaction with P450 (Schenkman et al., 1967), and have been correlated with a low to high spin transition of the heme iron. Based on studies of the interaction of triethyltin with cat hemoglobin (Taketa et al., 1980), and on the formation of a type 1 substrate-binding spectrum, TBT may not affect the heme moiety itself in P450. Rather, this compound may bind to amino acids such as cysteine and react with thiol groups. This would result in perturbations at the substrate binding center and inactivation of catalytic activity. This mechanism has also been proposed in rats by Rosenberg and Drummond (1983), where no decrease in specific content of microsomal heme was found after treatment with bis(tributyltin)oxide. TPT, however, did not show any substrate binding spectrum, indicating that this compound did not bind to the P450 molecule in a way similar to TBT. Hence, TBT and T P T bind differently on the P450 molecule, and this may be based on the different molecular structure of both compounds. The difference between T P T and TBT thus may be related to steric effects of the phenyl rings, hence not allowing oxidation to

K. Fent, T.D. BuchelilAquatic Toxicology 28 (1994) 107-126

123

Microsomal cytochromeP450 dependentmonooxygenasesystem

NADPH

V///////////////A Y/ NADPH ~1 e- >~cytochrome P450~A reductase ~] V///////////////A

e- )

~

Substrate + O2 Substrate-OH + H20

e-l NADH

e- ~ c y t o c h r o m e bsx~-I reductase k \ " ~ \ ", \ X \ \ X \ \ X \ \ \ \ 1

~ i Cytochrome b5

I

~ 7 " ~ : Affected by TPT ~x~:

Affected by TBT

Fig. 10. Effects of TBT and TPT in vitro on components of hepatic microsomal monooxygenase system in freshwater fish. Enzymes affected by TBT, cytochrome P450 and NADH cytochrome b5 reductase, as well as by TPT, cytochrome P450 and NADPH cytochrome P450 reductase, are marked.

occur. This might provide one explanation as to why TPT did not get hydroxylated after incubation in vitro with rat liver microsomes as was the case with TBT (Fish et al., 1976; Kimmel et al., 1977). Differences of TBT and TPT in the mode of action on the microsomal monooxygenase system are further underlined by their different action on the reductases. Further studies must show, however, how these organotins act on the P450 enzymes. The specific action of TBT and TPT on P450 is reinforced by the lack of any strong effect on cytochrome bs, another hemeprotein in the microsomal electron transport system. Furthermore, TBT and TPT acted on flavoproteins NADH cytochrome b5 reductase and NADPH cytochrome P450 reductase, respectively. Interestingly, there was a selectivity towards the different reductases in rainbow trout and eel, but to a lesser extent in bullhead. Thus, the mechanism of action of TBT and TPT on these flavoproteins differ, probably by binding to different sites. Alternatively, the structure of the reductases, or their embedment within the microsomal membrane may be different. Further studies are required, however, to elucidate the mechanisms by which TBT and TPT interact with cytochrome P450 and NAD(P)H cytochrome c reductase. This study demonstrates significant in vitro interactions of TBT and TPT with fish hepatic microsomal monooxygenase systems. Initial experiments revealed a significant organotin-induced inhibition of EROD activity in all fish, indicating the catalytic function of P450 1A was lost after incubation with organotins. The loss of catalytic activity may either be based on adverse effects upon the major component, cytochrome P450 1A, or on the inhibition of NAD(P)H reductases thus preventing oxidizing equivalents (electrons) from being transferred to P450. Alternatively, organotins

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K. Fent, 7~D. Bucheli/Aquatic Toxicology 28 (1994) 10~126

m a y affect c y t o c h r o m e bs, or they m a y act towards different c o m p o n e n t s in concert, thus inhibiting the catalytic activity. This question has been tackled in subsequent experiments by studying different c o m p o n e n t s o f the electron transport system separately. The findings show that organotins exerted effects on different components. T B T and T P T inactivated P450 enzyme, and N A D ( P ) H c y t o c h r o m e c reductase activity, but had no measurable effects on spectrally-determined c y t o c h r o m e b5 content. Fig. 10 summarizes the effects on the hepatic microsomal m o n o o x y g e n a s e system in fish. The rapid inactivation o f spectral P450 that occurred in the absence o f added N A D P H indicates a direct effect o f the organotins, and not a result o f mechanismbased inactivation, or suicide processing o f T B T or T P T by P450. The inactivation o f P450 protein measured spectrophotometrically implies that other catalytic activities besides E R O D would also be affected. Furthermore, the organotins acted selectively on reductase activities in trout and eel. Whereas T B T inhibited c y t o c h r o m e b5 reductase, T P T inhibited c y t o c h r o m e P450 reductase. In summary, organotins act on different c o m p o n e n t s o f the microsomal electron transport system, the hemeprotein P450 and the flavoproteins.

Acknowledgements We t h a n k J. H u n n for valuable help and assistance, W. D6nni, A. Peter and M. Zeh for providing fish, M. Keck-Oertle, University o f Ziirich, for helpful discussions and valuable suggestions on the manuscript, and C. M o n t a g u e for reading the m a n u script.

References Aldridge, W.N. (1976) The influence of organotin compounds on mitochondrial functions. In: New Chemistry and Applications, edited by J.J. Zuckerman. Adv. Chem. Ser. 157, American Chemical Society, Washington, DC, pp. 186-196. Alzieu, C., J. Sanjuan, P. Michel, M. Borel and J.P. Dreno (1989) Monitoring and assessment of butyltins in Atlantic coastal waters. Mar. Pollut. Bull. 20, 22-26. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 254. Bryan, G.W., P.E. Gibbs, L.G. Hummerstone and G.R. Burt (1986) The decline of the gastropod Nucella lapillus around south-west England: evidence for the effects of tributyltin from antifouling paints. J. Mar. Biol. Ass. UK 66, 611~640. Connerton, I. and D.E. Griffiths (1989) Organotin compounds as energy-potentiated uncouplers of rat liver mitochondria. Appl. Organomet. Chem. 3, 545 551. Fent, K. (1992) Embryotoxic effects of tributyltin on the minnow Phoxinus phoxinus. Environ. Pollut. 76,

187-194. Fent, K. and J. Hunn (1991) Phenyltins in water, sediment, and biota of freshwater marinas. Environ. Sci. Technol. 25, 956-963. Fent, K. and M.D. Miiller (1991) Occurrence of organotins in municipal wastewater and sewage sludge and behavior in a treatment plant. Environ. Sci. Technol. 25, 489493. Fent, K. and J.J. Stegeman (1991) Effects of tributyltin chloride on the hepatic microsomal monooxygenase system in the fish Stenotomus chrysops. Aquat. Toxicol. 20, 159 168.

K. Fent, T.D. BuchelilAquatic Toxicology 28 (1994) 107-126

125

Fent, K. and W. Meier (1992) Tributyltin-induced effects on early life stages of minnows Phoxinus phoxinus. Arch. Environ. Contam. Toxicol. 22, 428438. Fent, K. and J.J. Stegeman (1993) Effects of tributyltin in vivo on hepatic cytochrome P450 forms in marine fish. Aquat. Toxicol. 24, 219-240. Fent, K. and W. Meier (1993) Effects of triphenyltin on fish early life stages. Arch. Environ. Contam. Toxicol. (submitted). Fish, R.H., E.C. Kimmel and J.E. Casida (1976) Bioorganotin chemistry: Reactions of tributyltin derivatives with a cytochome P-450 dependent monooxygenase enzyme system. J. Organomet. Chem. 118, 41 54. George, S.G. and G. Buchanan (1990) Isolation, properties and induction of plaice liver cytosolic glutathione-S-transferases. Fish Physiol. Biochem. 8,437~49. Goksoyr, A., T. Andersson, T. Hansson, J. Klungsoyr, Y. Zhang and L. F6rlin (1987) Species characteristics of the hepatic xenobiotic and steroid biotransformation systems of two teleost fish, Atlantic cod (Gadus morhua) and rainbow trout (Salmo gairdneri). Toxicol. Appl. Pharmacol. 89, 347-360. Gray, B.H., M. Porvaznik, C. Flemming and L.H. Lee (1987) Tri-n-butyltin: a membrane toxicant. Toxicology 47, 35-54. Hall, L.W., Jr. and A.E. Pinkney (1985) Acute and sublethal effects of organotin compounds on aquatic biota: an interpretative literature evaluation. CRC Crit. Rev. Toxicol. 14, 159-209. Jarvinen, A.W., D.K. Tanner, E.R. Kline and M.L. Knuth (1988) Acute and chronic toxicity of triphenyltin hydroxide to fathead minnows (Pimephalespromelas) following brief or continuous exposure. Environ. Pollut. 52, 289-301. Keck-Oertle, M. (1986) Heterogeneity of cytochrome P-450: biochemical and biophysical studies. Ph.D. thesis, Swiss Federal Institute of Technology, ETH, Ziirich. Kimmel, E.C., R.H. Fish and J.E. Casida (1977) Bioorganotin chemistry. Metabolism of organotin compounds in microsomal monooxygenase systems and in mammals. J. Agric. Food Chem. 25, 1-9. Kloepper-Sams, P.J., S.S. Park, H.V. Gelboin and J.J. Stegeman, 1987. Specificity and cross-reactivity of monoclonal and polyclonal antibodies against cytochrome P450E of the marine fish scup. Arch. Biochem. Biophys. 253, 268-278. Klotz, A.V., J.J. Stegeman and C. Walsh (1986) An alternative 7-ethoxyresorufin O-deethylase activity assay: A continuous visible spectrophotometric method for measurements of cytochrome P450 monooxygenase activity. Anal. Biochem. 140, 138-145. Laughlin, R.B., Jr., H.E. Guard and W.M. Coleman III (1986) Tributyltin in seawater: Speciation and octanol-water partition coefficient. Environ. Sci. Technol. 20, 201-204. Lemaire-Gony, S. and P. Lemaire (1992) Interactive effects of cadmium and benzo(a)pyrene on cellular structure and biotransformation enzymes of the liver of the European eel Anguilla anguilla. Aquat. Toxicol. 22, 145-160. Mannervik, B., P. Alin, C. Guthenberg, H. Jennson, M.K. Tahir, M. Warholm and H. Johrnvall (1985) Identification of three classes of cytosolic glutathione transferase common to several mammalian species: Correlation between structural data and enzymatic properties. Proc. Nat. Acad. Sci. USA 82, 7202-7206. Rosenberg, D.W., G.S. Drummond and A. Kappas (1981) The influence of organometals on heme metabolism. In vivo and in vitro studies with organotins. Mol. Pharmacol. 21,150 158. Rosenberg, D.W. and G.S. Drummond (1983) Direct in vitro effects of bis(tri-n-butyltin)oxide on hepatic cytochrome P-450. Biochem. Pharmacol. 32, 3823-3829. Schenkman, J.B., H. Remmer and W. Estabrook (1967) Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol. 3, 113-123. Spooner, N., P.E. Gibbs, G.W. Bryan and L.J. Goad (1991) The effect of tributyltin upon steroid titres in the female dogwhelk, Nucella lapillus, and the development of imposex. Mar. Environ. Res. 32, 3749. Stegeman, J.J. (1987) Monooxygenase systems in marine fish. In: Pollutant Studies in Marine Animals, edited by C.S. Giam and L.E. Ray. CRC Press, Boca Raton, FL, pp. 65-92. Stegeman, J.J. and P.J. Kloepper-Sams (1987) Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals. Environ. Health Perspect. 71, 87-95. Taketa, F., K. Siebenlist, J. Kasten-Jolly and N. Palosaari (1980) Interaction of triethyltin with cat hemo-

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globin: identification of binding sites and effects on hemoglobin function. Arch. Biochem. Biophys. 203, 466472. Tsuda, T., H. Nakanishi, S. Aoki and J. Takebayashi (1986) Bioconcentration of butyltin compounds by round crucian carp. Toxicol. Environ. Chem. 12, 137 143. Vecsei-Hohl, R., L. Gourec, M. Bruna, M. Zeh and K. Fent (1992) Chlorinated hydrocarbons in eels (Anguilla anguilla L.) from the River Rhine. Naturwissenschaften 79, 371 374. Williams, D.E. and D.R. Buhler (1982) Purification of cytochromes P-448 from fl-naphthoflavone-treated rainbow trout. Biochim. Biophys. Acta 717, 398-404.