Effects of tributyltin in vivo on hepatic cytochrome P450 forms in marine fish

Aquatic

0

Toxicology,

24 ( 1993) 2 19-240

219

1993 Elsevier Science Publishers B.V. All rights reserved 0166-445X/93/$6.00

AQTOX

0055

I

ffects of tributy~tin in vivo on hepatic 50 forms in marine fish Karl Fenta and John J. Stegemanb “Swiss Federal hstitute.for Switxrland

Wurer Resources and Water Pollution

hWoorls HOC Uceattogruphic

I~stitutio~t,

Cotztrol (EA WACIETH).

Biology Department.

Woo&

DtibemiorJ

Hole, Mas.wchusetts,

USA

(Received 10 October 1991; revision received 5 October 1992; accepted 26 October 1992)

The interaction of tributyltin chloride (TBT) in vivo with different forms of hepatic microsomal cytochrome P450 and uJluxqri~cii.ufin U-deethylase (EROD)

activity was studied in scup (Steuotottw

clrr_w~~ps). Fish were injected with single doses of 3.3, 8.1 and 16.3 mglkg TBT. Hepatic microsomes were

analyzed 24 h later for total cytochrome P450 content, and three P450 forms by immunobtotting: scup P450E. the major polyaromatic hydrocarbon-inducible form (CYPIAI

). scup P450A. the major contribu-

tor to microsomal testosterone G/3-hydroxylase activity. and scup P150B, which oxidizes testosterone at several different sites including the 15a position. Spectrally determined conversion of P450 to its degraded form cytochrome P420 occurred at all TBT doses, the conversion being considerable only at the highest dose with more P42O (65%) than P450 (35%). EROD activity tended to be decreased by TBT in all doses. with a significant reduction at 16.3 mg/kg. Microsomal pro&in degradation as revealed by SDS-polyacrylamide gel electrophoresis occurred at the highest dose. lmmunoblot analysis with ;: monoclonal antibody to PJSOE (CY PI A I ) showed a decrease of CY Pl A protein content :it all TBT doses, with a significant loss at 16.3 mg/kg. similar to the EROD activity pattern, Immunoblot imalysis with polyclonal antibodies to P45OA and P45OB showed decreases in P45OA and P45OB protein conicnt only at the highest dose. the decrease being significant for P450A only. Cytochrome bi content was unaffected. In the liver. metabolitcs ofTBT,

dibutyltin and monobutyltin, were identified at all doses, with a decreased content. however. in fish

given the highest dose, This study indicates important biochemical effects of TBT in fish liver. and suggests that exposure to TBT may alter both cytochromc P450 dcpcndent metabolism, and induction response lo other environmental po!!utsnts.

Key words: Cytochromc P450 forms: Tributyltin; In viva effects: Fish (S~crto~rrrjltrs chyop)

INTRDDUCTION

The widespread industrial and agricultural applications of organotin compounds give rise to contamination of marine and freshwater environments. Tributyltin (TBT) Corrcl.s/~atrt/~Jr?f,~~ IO: K. Fent, Swiss Fcdcral Institute for Waler Resources and Water Pollution Control

(EAWAGIETH),

CH- 8600 Diibendorf, Switzerland.

220

was detected in considerable amounts in marinas and areas with heavy shipping activity due to its application in antifouling paints. The regulation of this compound resulted in a decreased contamination, but chronicL!ly toxic concentrations are still 1989; Fent and Hunn, 1991). In addition, present in these locations (Alzieu et al., other organotin sources such as municipal and industrial wastewaters have been identified (Fent and Miiller, 199 1). In recent years, the environmental contamination and the high toxicity of TBT has been documented in some detail (Bryan et al., 1986; Alzieu et al., 1989: Martin et al.. 1989; Fent, 1992). However, in aquatic species little is known about the basis of the toxic action of organotins on the biochemical level. The sterilization of female dogwhelks. which is one of the most sensitive reactions to TBT occurring at trace levels of a few ng/litre (Bryan et al., 1986), has been assumed to be related to interferences of TBT with the steroid metabolism (Spooner et al., 1991). The hepatic cytochrome P450-dependent monooxygenase system is crucial to the metabolism of a variety of endogenous and xenobiotic compounds including hormones, carcinogens, and environmental pollutants. This enzyme system plays also an important role in the metabolism of TBT (Lee, 1986). Recently, we demonstrated that TBT can strongly interact in vitro with hepatic microsomal cytochrome P450 in fish leading to destruction of native enzyme and inhibition of enzyme activity (Fent and Stegeman, 1991). Here we investigate whether or not these effects occur in vivo as well. Emphasis in this study is placed on the identification of any specificity to different forms of cytochrome P450. Our in vitro findings have suggested that TBT might lead to a selective inactivation of the EROD catalyst, cytochrome P450E or CYPl A 1, as a complete loss of EROD activity occurred at TBT concentrations where not all P450 was destroyed (Fent and Stegeman, 1991). The questions addressed here are not only important with respect to the mode of action of organotins in aquatic species, and the biochemistry and pharmacology of P450 enzymes, but also to the problem of environmental induction. Elevated CY Pl A I protein content has been detected in liver microsomes of fish in several field studies, and associated with environmental concentrations of polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons (PAHs) as active agents in environmental induction (Elskus and Stegeman, 1989; Van Veld et al., 1990; Goksoyr et al., 199la). Recent studies have indicated that in addition to induction, a specific PCB congener may decrease P450E catalytic activity at high doses (Gooch et al., 1989), or induction of CYPl A 1 protein was not paralleled with induction of EROD activity Goks0yr et al., 1991 b; Boon et al., 1992). The catalytic efficiency of P450E, and the response to additional inducers, can also be affected by the degree of prior environmental exposure to pollutants (Monosson and Stegeman, 1991). and in the highly contaminated New Bedford Harbor, MA, USA, apparently uninduced winter flounder were found (Stegeman et al., 1987). Here we show that the environmental pollutant TBT produced a decrease of native P450E and associated catalytic activity in fish liver. The pronounced loss of cytochrome P450E (CYPlA 1) and its related catalytic

221

activity (EROD) indicates a considerable effect on this isozyme. Although cytochrome P450E tended to be decreased at all concentrations tested thus indicating a stronger or selective action on this form in vivo, the loss of P450A, and to a minor extent P450B, at high TBT doses shows that other forms are affected as well. MATERIALS

AND METHODS

Tributyltin chloride (TBTi was obtained from Fluka AG, Switzerland, with a purity of > 97%. TBT was diluted in 98% ethanol to concentrations of 10 to 50 mM, and used for injection. Doses in this study refer to the weight of tributyltin chloride, whereas residue concentrations and calculations refer to the organotin ion. _A!!other chemicals were obtained in highest purity available from commercial suppliers.

SCUP(Stutzotut?llr.s ~*hr;~~~~,s) used for this study weighed between 142 to 330 g. They were caught by angling at Tarpaulin Cove, Vineyard Sound, MA, USA, and maintained for seven months in flow-through water of 18°C in the laboratory. The maintenance of the fish during this period did not lead to a complete loss of ethoxyresorufin O-deethylase (EROD) activity. The presence of P450E and its associated enzymatic activity are likely to be due to contaminants in the waters from where the animals were caught. One day prior to treatment, fish were moved to 120 1 glass aquaria and held under flow-through conditions at 18°C. Four to six fish per dose group (a total of 8 males and 1 1 females) were injected intraperitoneally with 3.3, 8.1 or 16.3 mgikg (10, 25, 50 ,crM) of TBT in 98% ethanol, and control fish received an equal volume of the solvent ethanol (injection volume in each case 1 ml/kg). Control fish and TBT-treated fish were kept in f’our different aquaria. At 16.3 mg/kg TBT one fish was found dead the next day. After 24 h, animals were killed, and gall bladders were removed to avoid contaminating the livers with bile. Atier weighing, small portions of liver were used both for l~istological investigations and organotin residue analysis. Large liver portions were homogenized in buf’fer (50 IIIM Tris-HC!, pH 7.4, containing 0. I5 M KC!), and microsomes were obtained by ultracentrif’ugation. Hepatic microsomes of individual fish were prepared in resuspension buffer (50 mM Tris- HC!, p1-I 7.4; I mM dithiothreitol; 20% v/v glycerol; 1 IIIM NamEDTA) employing the procedure previously described (Stegeman et al., 1979; Gooch et a!., 1989), and archived III liquid nitrogen until use. Microsomal protein was measured by the method of Smith et al. (1985) using bovine serum albumin as a standard.

Cytochrome P4.50(extinction coefficient 91 mM-‘cm-‘) and cytochrome P420 (extinction coefficient 111 mM-‘cm‘-‘) content of microsomal preparations were determined with a dithionite difference spectrum of CO-bound microsomes, as described by Stegeman et al. (1979). Cytochrome b5 content (extinction coefficient 185 mM-‘cm-‘) was determined with a NADH difference spectrum at 26°C. Cytochrome P450E (CYPl Al) enzymatic activity (ethoxyresorufin O-deethylase; EROD) was determined using a visible spectrophotometric assay according to the method of Klotz et al. (1984). The reaction mixture consisted of buffer (0.1 M TrisHCl, pH 8.0, 0.1 M NaCl). 2 ,uM 7-ethoxyresorufin, and 40-70 ~1 of microsomal suspension (350-800 ,ug protein). The reaction was initiated by the addition of 10 ~1 NADPH. and product formation was measured at 572 nm for 2 min (extinction coefficient 73 mM-‘cm-‘).

Western blots were used to quantitate the contents of three different forms of scup cytochrome P450: P450E, P450A. and P450B. Cytochrome P45OE is the form CYPlAl, according to the nomenclature of Nebert et al. (1991). It represents the hydrocarbon-inducible P450 form and catalyses EROD activity (Kloeppc r-Sams et al., 1987). Unlike scup cytochrome P450E. neither cytochrome P45OA nor P450B appears to be directed towards aromatic hydrocarbons. Cytochrome P450A was shown to represent the dominant catalyst of testosterone 6/?-hydroxylation, and P450B is the major contributor to testosterone 1Scr-hydroxylase activity (Klotz et al., 1986). P450B is likely a member of the CYP2B subfamily (Stegeman et al., 1990). Primary antibodies used in immunoblotting consisted of a mouse monoclonal antibody (MAb 1-12-3) against purified scup cytochrome P450E or CY Pl A 1 (KloepperSams et al., 1987) and two polyclonal antibodies developed in rabbits against purified scup cytochrome P450A (PAB 7-93) (Gray, 1988) and cytochrome P450B (PAB 7-94) (Klotz et al., 1986). The method described in Kloepper-Sams ct al. (1987) was applied with slight modi~cations for immunoblots. Microsomal samples were incubated in a steaming water bath for 10 min in 62.5 mM Tris-HCI (pH 6.8). 3% sodium dodecyl sulfate, 10% glycerol, and 5% /,?-mercaptoethanol. Microsomal proteins (30 or 45,1~g)were electrophoretically separated on gradient gels (8-l 5% acrylamide) and transferred overnight onto 0.2 pm nitrocellulose paper. The efficiency of the protein transfer was evaluated by staining of the nitrocellulose with Ponceau S (0.2% w/v. in 3% w/v trichloroacetic acid, and 3% w/v sulfosalicylic acid), and by staining of the residual proteins remaining in the SDS-polyacrylanlide gels (SDS-PAGE) with Coomassie Blue R (0.2% w/v in 40% methanol, 10% acetic acid, and 50% deionized water). Subsequent incubations of the nitrocellulose were conducted with Tris-buffered

223

saline (TBS) and 5% (w/v) dry milk solution to block nonspecific protein binding. First, the blots were incubated in 5% TBS-milk solution at 42°C for 1 h. For quantitation of P450E. the nitrocellulose was incubated with a monoclonal antibody, mouse anti-scup F450E (MA\, I-12-3) (100 /~g/,~l), for 1 h at room temperature. Following rinses (10 min) each with TBS, 0.5% Tween 20 plus TBS, and TBS again, the nitrocellulose was incubated with alkaline phosphatase conjugated goat anti-mouse IgG (Bio-Rad, Richmond, CA) (dilution of 1:200) as a secondary antibody, and rinsed as before. For quantitation of P450A and P450B, the nitrocellultrse was incubated with a polyclonal rabbit antibody (PAB 7-93) (30 pg/ml) and polyclonal rabbit antibody (PAB 7-94) (25 pg/ml), respectively, for 1 h. Following rinses with TBS-Tween, the nitrocellulose was incubated with alkaline phosphatase conjugated goat anti-rabbit IgG (Bio-Rad, Richmond, CA) (dilution of 1:200), and washed again as above. BCIP (5bromo-4-chloro-3-indolylphosphate in color buffer, 50 mglml) and NBT (50 mg/ml nitroblue tetrazolium in 70% N./V-dimethylformamide) were used for color development, and the reaction was stopped by rinsing with distilled water. The color buffer contained 0.1 M NaHCOJ and I mM MgCl,, pH 9.8. Blots were scanned and quantitated by video image analysis (Master Scan, Interpretative Densitometer NEC Multi Sync 2A, American Power Conversion). The amount of stain was expressed as picomoles of P450E protein, by comparison with staining obtained with purified scup P450E standards. P450A and P450B protein was quantitated by ccnparison with staining obtained from archived scup microsomes in which P450A and P450B content were determined with purified P450A and P450B. In vitro incubations

Incubation of microsomes with TBT in vitro was accomplished as previously described (Fent and Stegeman, 1991). Tributyltin chloride was added in ethanol to EDTA-free buffer, which was then incubated with microsomal protein (0.026 mg) in a total volume of 10 ~1, to yield TBT at final concentrations of 0.1, 0.2 or 0.5 mM, similar to concentrations used in our previous in vitro study (Fent and Stegeman, 1991). The TBT concentrations represent approximately 0.29, 0.58 and 1.45 pug of TBT (as ion) per incubation. Assuming that 70-90% of the TBT added is adsorbed to the microsomal vesicles, then approximately 8-10, 16-20, and 40-50 ,ug TBT (ion) would be associated with 1 mg of microsomal protein at the low, medium and high concentrations, respectively. Buffer alone was added to control incubations. After incubation for 20 min and 120 min, the protein was prepared further for SDS-gel electrophoresis and immunoblotting as described above. Organ0 tin residue mtff Iysis

Organotin residue analysis of liver samples was performed at EAWAG, Switzerland, by high-performance capillary gas chromatography with flame photometric

detection similar to the procedure of Fent and E-funn (I991 ). Liver samples (each 0.1-0.5 g wet weight) of one fish per dose group were homogenized, and internal standards tripropyltin, mono-, di- and tripentyltin were spiked to the samples. After acidification and extraction with deethylether/hexane (3:2 v/v) containing 0.25% tropolonc the extract was ethylated” After a cleanup on silica gel the samples were analyzed for TBT, dibutyltin (DBT) and monobutyltin (MBT) on a 30 m DB-5 capillary column. Analysis was performed in duplicates and means are given. The butyltin residues data are corrected for recovery (40-70% depending on the compound), and given as the respective ion.

Data were analyzed using the analysis of variance portion of the Statview 512TM program on an Apple Macintosh SE computer. When signi~cant ANOVA F-values were obtained, the data were analyzed further using the Fisher PLSD test for multiple comparisons to determine which animal groups were significantly different at the 0.05% level.

TBT induced the formation of P420 in scup hepatic microsomes 24 h after single injections at all doses of TBT. Fig. 1 shows typical spectrog~ms from a ~ontro1 and a TBT-treated fish with both cytochrome P450 and P42Osimultaneously present. The formation of cytochrome P420 appears to be reIated to dose, as minor P420 contents could be observed in two of the four fish at 3.3 m&kg and 8.1 mglkg (Table 1). At the highest TBT dose, more cytochrome P420 (65%) was present than cytochrome P450 (35%). It should be noted that a certain interindivjdual variability in the conversion of P450 to P420 occurred. Between fish. the spectrally measured P420 content ranged from O-15% and 9-91% of the total content of P450 (P420 plus P450) at 8.1 mg/kg and 16.3 mg/kg TBT, respectively. These data indicate that TBT led to an in vivo denaturation of cyto~hrome P450, but compared to control values, total cytochro~ne P45O (P420 plus P450) content was not decreased after treatment with TBT. The occurrence of P420 indicates that P450 forms are denatured, but not yet degraded by proteases. The TBT-induced destruction of P450 observed in vivo is consistent with effects observed in vitro, where a dose-dependent destruction of P450 was measured (Fent and Stegeman, 1991).

Fig. 2 shows that TBT tended to inhibit EROD activity at all doses, The activity fell

225 0.02

0.02

-0.02 400

500

Wavelength

(nm)

Fig. 1. Formation of cytochrome P420 after TBT treatment in vivo. The difference spectra of hepatic microsomes of a control fish (left), and a fish injected with 16.3 mg/kg TBT (right) are shown. Note that more P420 was present than P450 in the TBT-treated fish.

TABLE 1 Effect of TBT in vivo on total cytochromc separate determinations _+SEM.

P450 and cytochromc

b,. All values represent averages of

TBT dose

Animals

(m&kg)

01)

Total cytochrome P450 (pm01 mg-’ protein)”

Cytochrome P420 (pm01 mg-’ protein)“

Cytochrome b, (pm01 mg-’ protein)”

0 (I LTO ethanol) 3.3 8.1 16.3

5 4 4 5

83.4 112.3 91.9 61.9

0 5.1 f 5.9 3.5 t 3.4 113.5 !I 38.1

38.3 49.8 48.9 44.2

t 10.5 &-11.6 + 11.6 + 11.6 -~~

“Average of 2 to 3 determinations

per animal.

+ 5.0 & 7.8 + 7.6 f 6.0

EROD activity

Control

3.3

8.1

16.3

TBT Dose (mglkg)

EROD turnover 80 60

Control

3.3 8.1 TBT Dose (mglkg)

Fig. 2. Inhibition of ethoxyresorufin O-deethylase (EROD)

16.3

activity after treatment with three doses of TBT

as compared to controls. The values t SEM represent the average of all fish 01 = 4 or 5) in a dose group. *, statistical significance at the 0.05 level.

to 56-67% of control values at 3.3 and 8.1 mglkg TBT, respectively, and to 33% at the highest dose. The loss of EROD activity was statistically significant only at the highest dose. The inhibition of the catalytic activity by TBT in vivo is consistent with the effect observed in vitro, where incubation of microsomes with TBT produced a dosedependent inhibition (Fent and Stegeman, 1991). The turnover number for EROD activity, or the catalytic efficiency of P450E (activity/pmol P450E enzyme) as quantified by immunoblotting, showed no statistically significant difference between controls and TBT-treated fish (Fig. 2). Therefore, the majority of loss of EROD activity is not due to an altered catalytic efficiency.

At the highest TBT dose, both the residual proteins

on the polyacrylamide

gel and

227

S

0

3.3

8.1

16.3

mg / kg Tl3T

Fig. 3. Ponceau S stained microsomal proteins on nitrocellulose from controls and scup treated with TBT. The hands represent proteins that were ele~trophoreti~all~ separated on a S~S-polya~~Iamide transferred to nitrocellulose prior to immunoblotting.

gel and

For each group tuo fish are shown. and the same

amounts of microsomal protein (4.5 pug)were used. The protein pattern is identical to that on the polyacrylamide gel which contains remaining proteins. The respective immunoblot is shuan in Fig. 4. P4SOE standard (SI.

the transferred proteins on Ponceau~stained nitrocellulose showed a different pattern than in the controls and in the low and medium dose groups. The proteins on the shown Ponceau-stained nitrocellulose (Fig. 3) demonstrate a substantial increase in low molecular weight bands ~degraded proteins) that did not appear in controls or at lower TBT doses, and some higher molecular weight bands were not identifiable. This indicates that at the highest dose, a number of different proteins were degraded after TBT treatment. Histopathologi~~~ obser~tions of hemato~ylin-eosins stained hver sections did not reveal any significant TET-related alterations or necrosis in the liver of these fish at all doses (R.M. Smolowitz, personal communication). Thus. degradation of hepatic m~crosomal proteins at the highest dose are not related to visible histopathologica~ changes.

As TBT led to formation of cytochrome P420 the question arises whether or not this is due to similar effects on al1 P450 forms. or due to a more specific deg~dation of certain P450 forms. To address this question IK measured the effect on three

S

0

3.3

8.1

16.3

mg /kg TBT

Fig. 4. Immunoblot of liver microsomes from controls and scup treated with TBT and probed with antiscup P450E (MAb I-12-3). The immunoreacting band has a relative molecular mass of 54-55 k&i. For each group two fish are shown, and the same amounts of microsomal protein (45 ,Ug) were used. WSOE standard (S) was 0.5 pmol. Cytochrome P450E is estimated to represent about 12% of the total cytochrome P4.50 in controls.

P450 forms identified and quantitated by immunoblotting with specific and polyclonal antibodies. The content of P450E (CUP1 Al) was considerably reduced at all TBT doses. Fig. 4 shows an immunoblot of liver microsomes of two fish in each group. A single band was recognized at about 54-55 kDa, which was present in controls, but was reduced in intensity at 3.3 and 8.1 mg/kg TBT. This band was virtually absent at 16.3 mg/kg. As shown in Fig. 5, the immunoquantitated content of cytochrome P450E of all fish was lower after TBT treatment, the decrease being statistically significant at 16.3 mg/kg TBT. At 3.3, 8.1 and 16.3 mg/kg, the P450E content fell to 53%, 62% and 25% of control values, respectively. There is a striking similarity between the decreases in cytochrome P450E content and EROD activity (Fig. 2) in all doses, consistent with the identity of scup cytochrome P450E 9s the EROD catalyst. As shown in Fig. 2 the catalytic efficiency of P450E (activity/ pmol P450E enzyme) did not change after TBT treatment, Hence, the loss of EROD activity can be ascribed primarily to the loss of native P450E. Proteolytic fragments resulting from protease digestion of microsomes can immunoreact with P450E antibody (Gokscayr et al., 1991~). However, no breakdown products of P450E could be detected; there were no low molecular peptides found immunoreacting with the antibodies (Fig. 4). This suggests that P450E apoprotein could have been completely degraded. Like scup cytochrome P450E, P450A also showed a strongly decreased content at the highest TBT dose. Immunoblotting of liver microsome= of the same fish (Fig. 6)

different

monoclonal

229 ~mmunodetected P450E

Control

3.3 8.1 16.3 TESTDose (mglkg)

lmmunodetected P4fiOA @-I

1

-

3a

20

IO

0

Control

3.3 8.1 T3T Dose ~rn~g)

16.3

lmmunodetected P45OB

Control

3.3 8.1 16.3 TBT Dose (mg/kg)

Fig. 5. ~mmuno~uantitated data of scup cytochrome P450E fCYP1 Al), P45OAand P45OBin controls and TBT-treated fish. Values are derived from immunoblots. and represent averages + SEM of 4 to 5 fish in each group. Cytochrome P45OE content is depressed at alt doses, P45OA and P45OBonly at the highest dose. *, statistical significance at the 0.05 level.

230

S

0

3.3

8.1

16.3

mg /kg TBT

Fig. 6. lmmunoblot of liver microsomes from controls and scup treated with TBT and probed with antiscup P450A. The major immunoreacting band has a relative molecular mass of 52-53 kDa. and represents cytochrome PJSOA. The appearance of additional, less stained, bands is due to the polyclonal nature of this antibody. as it cross-reacted with proteins of different molecular weight (Gray. 1988). For each dose, the same two fish as in Figs. 3 and 4 are shown, and same amounts of microsomal protein (30 kg) were used for each fish. P450A standard (S) was 2 pmoi. Cytochrome P450A is estimated to represent about 30% of the total cytochrome P450 in controls.

showed recognition of a major band, about 52-53 kDa, representing scup cytochrome P450A (Gray, 1988). Cytochrome P450A was strongly present in controls, and at 3.3 and 8.1 mg./kg TBT, but could virtually not be detected in fish given the highest dose. As with P450E, no degradation products of P450A were detected in the blots. Fig. 7 shows an immunoblot for cytochrome P450B of the same fish of each dose group, with the recognition of a band at about 45-46 kDa. Only at the highest TBT dose was the staining reduced as compared to controls. The immunoquantitation data of all fish showed that at the highest TBT dose, P450A was depressed by 85% and P450B by 40%, compared to control values (Fig. 5). It is important to note that at the highest dose, proteins other than cytochrome P450 forms were degraded, as was indicated by the low molecular weight protein residues on the nitrocellulose (Fig. 3). However, comparison of the results for P450E, P45OA, and P450B (Figs. 4-7) illustrates the possibility of a stronger effect on cytochrome P450E: P450E tended to be depressed even at the lowest TBT dose. These data also imply that TBT has a different activity against different cytochrome P450 forms. Cytochrome P450A, which represents the dominant catalyst of testosterone 6p-hydroxylation. and P450B, which is the major contributor to testosterone 15a-hydroxylase activity, show a decrease only at the highest dose. As indicated by the more pronounced loss of cytochrome P450A, this form appears to be more vulnerable to high TBT doses than cytochrome P450B.

231

In vitro incubations The possibility that a direct interaction of TBT with microsomes might reduce the immunostaining signal of P450E was tested by incubating scup liver microsomes directly with TBT and then analyzing the proteins by immunoblotting. The results show that 0.1,0.2 or 0.5 mM TBT had little or no effect on MAb l-12-3 detection of P45OE content over a short (20 min) period of incubation (Fig. 8), indicating that TBT did not bind to and mask the epitope. There was also little effect of 0.1 mM TBT on P450E content measured after 2 h, However, 0.2 mM TBT did decrease the content of P450E after 2 h and 0.5 mM TBT abolished the staining signal (Fig. 8). The loss of P450E staining could result from TBT binding to the P450E protein and masking the MAb l-l 2-3 epitope. However, the loss of immunodetected P450A and to a lesser extent P450B, neither of which is recognized by MAb l-12-3, at the highest in vivo doses indicates that the effect on P450E is not an epitope specific effect. The mechanism of this in vitro effect is not clear, but the time dependence of the effect indicates that it is not the result of a rapid binding alone. Cytochrome b, Cytochrome bg, another component of the hepatic microsomal electron transport system, was not adversely affected by TBT. Table 1 shows that the content of cytochrome b5 was not altered in the TBT-treated fish, even at the highest dose. A ; .llllar

S

0

3.3

8.1

16.3

mg / kg Tf3T

Fig. 7. Immunoblot of liver microsomes from controls and scup treated with TBT and probed with antiscup P450B. The major immunoreacting band has a relative molecular mass of 45 -46 l&a. The appearance of additional, less stained, bands is due to the polyclonal nature of this antibody, as it cross-reacted with other proteins. For each dose group, the same two fish as in Figs. 3,4 and 6 are shown. and same amounts of microsomal protein (3Opg) were used. P450B standard (S) was I .5 pmol. Cytochrome P450B is estimated to represent about 40% of the total cytochrome P4SO in controls.

232

result has been observed in vitro (Fent and Stegeman, 1991). These results show that not all components of the microsomal electron transport system are affected by TBT. TBT residues and metabolism

T is indicated to be metabolized in fish liver by cytochrome P450 via successive dealkylation to dibutyltin (DBT), monobutyltin (MBT) and tin (IV) (Lee, 1986) the question arises of whether or not metabolism ofTBT took place in these fish. Fig. 9 shows a typical gas chromatogram of a fish liver extract, and Table 2 gives the butyltin residues. Besides TBT, residues of the metabolites DBT and MBT were present. Of the total TBT injected, between l-5% was recovered in the fish livers as MBT, DBT and TBT. Residues of DBT occurred in significant concentrations in the liver of low and medium dose groups. The concentrations were in the range of about 20% for DBT, and l-2% for MBT of the total butyltins measured. At the highest dose, however, DBT and MBT residues were less than 9% and 0.4%, respectively. These data indicate that at low and medium doses metabolism of TBT occurred, but was reduced at the highest dose. DISCUSSION

This study demonstrates a significant interaction in vivo of TBT with cytochrome

140

A

160

B

120

60

20 0

0

0.1

0.2

0.5

0

Tribdyltin

0.1

0.2

0.5

(rnM)

Fig. 8. Effect of tributyirin in vitro on P450E content of scup liver microsomes. A, incubation for 20 min: B, incubation for 2 h. The incubations were carried out as described in the Materials and Methods Results of immunoblotting with MAb I-12-3 were analyzed by dcnsitometry. Note that 100% values were different for the two time points: at 2 h the control incubations had lost greater than 50% of the signal. The TBT effects are thus superimposed

on this time-dependent

loss.

233 Sl

(TPrT)

S2 (DPeT) 53 (TPeT)

TBT

DBT

Fig. 9. Capillary GC-FPD chromatogram of a liver extract of a fish dosed with 8.1 me/kg TBT for 24 h. Besides TBT, melabolites dibutyltin (DBT) and monobutyltin (MBT) are present. Internal standards, tripropyltin (Sl, TPrT), dipentyltin (S2, DPeT) and tripentyltin (S3. TPeT) used for quantitation were spiked into the homogenate prior to extraction. Possible other metabolites such as hydroxylated dibutyltins may also be present (peak between S2 and S3).

TABLE 2 Residues of organotins (as ions) in scup liver (Aglg wet weight: percentage) of one fish per dose group (mean of duplicate samples), and relative percentage of TBT injected. TBT dose (mg/kg) 3.3 8.1 16.3

DBT

MBT

Wg/g)

Wglg)

WE/g)

Relative to injection

8.0 (80%) 14.7 (74%) 202 (91%)

1.9 (19%) 4.7 (24%) 19.7 (9%)

0.1 (1%) 0.3 (2%) 0.8 (0.4%)

1% 1% 5%

TBT

234

~450 content and activity, with a destruction and loss of several P450 forms, but no measurable effects on cytochrome b5 content. The degradation of native cytochrome ~450, indicated by the occurrence of cytochrome P420, implies that the catalytic activity would also be reduced. Cytochrome P420 is produced by denaturation of ~450, and unlike P450, P420 cannot catalyze biological oxidations. The 10~s of EROD activity at 3.3 @kg TBT and higher confirms that the catalytic function was lost. A pronounced decrease in ER0D activity occurred at a TBT dose that yielded only low concentrations of spectrally measured P420. This may indicate that the inhibition of catalytic activity could represent a selective inactivation of the EROD catalyst, P45OE or CYPl A 1. A selective inactivation of P450E is consistent with our in vitro tmdings (Fent and Stegeman, 199 1), in which a complete loss of EROD activity occurred at TBT concentrations where not all cytochrome P450 was destroved. Fl!rthermore, it was shown that the NADPH-cytochrome P450 reductase _ activity was not inhibited by TBT. In the present in vivo study, the catalytic efficiency of P450E, or the turnover number for EROD activity, was unaffected by TBT. Thus, the loss of EROD activity is due to the destruction of native P450E, and not to an alteration of catalytic activity. Immunoblot analysis of hepatic microsomes showed considerable losses in P450E, but not in cytochrome P450A or P450B at 3.3 and 8.1 mg/kg TBT, indicating an apparent selective action on P450E. The loss of P450A and P450B at he highest dose shows that these forms are also destroyed, although they are less sensitive to the action of TBT. To our knowledge, the question of specificity of cytochrome P450 isozymc destruction of TBT has not been addressed thus far. As cytochrome P450A represents the dominant catalyst of testosterone 6P-hydroxylation, and P450B is the major contributor to testosterone 15a-hydroxylase activity (Klotz et al., 1986) and likely a member of the CYP2B subfamily (Stegeman et al., 1990) effects on steroid metabolism are expected to occur at high TBT doses. It is possible that TBT might also affect other species of cytochrome P450 including others responsible for steroid metabolism such as aromatase, or other microsomal proteins in addition to the major inducible P450E. The measured concentrations of 8 to 14 pug TBT/g liver (Table 2) could represent 0.6 to 1.2 yg TBWmg microsomal protein, assuming that the TBT in the liver was entirely in the endoplasmic reticulum (microsomes) and given a microsomal protein yield of 12-l 3 mg per g liver, typical for scup (Stegeman et al., 1979). The higher value (1.2 ,q$ng) is 6 to 16-fold less than the estimated values of 8 to 20 pug TBT/mg (see In vitro incubations) associated with a slight effect on P450E in direct incubation. The 200 fig TM’ per g liver in high-dose fish could result in 20 lug TBT/mg microsomal protein, half the estimate of 40-50 pg/mg associated with “loss” of P450E in vitro. However, TBT in homogenized liver is certain to be distributed among the subcellular fractions, meaning that less than the total amounts measured (Table 2) would occur in the microsomes. Moreover-, even the high concentrations in vitro exerted effects slowly enough to show that loss of immunostaining is not an immediate result (Fig.8).

235

Thus we conclude that a direct effect of TBT on P450 during or after microsome preparation contributes little to the observed effects of in vivo TBT. Further analysis of the distribution and concentrations of TBT in various fractions of the liver, and the time course of deposition in those fractions, is necessary to fully evaluate this possibility. Similar to the results presented, George (1989) showed for cadmium that injections in fish strongly reduced EROD activity. Preliminary immunological studies suggested that this was due to a decrease in enzyme protein rather than direct inhibition of activity by cadmium. Also, acrylamide led to significant decreases in EROD activity and microsomal CYPI A protein in rainbow trout (Petersen and Lech, 1987). Rosenberg et al. (1985) suggested that apoproteins of various cytochrome P450 forms were affected by tricyclohexyltin, and their results indicated differential effects on specific P450 forms, similar to our study. In conclusion, our immunoblot data indicate that TBT acts differently on different P450 forms, with a stronger effect on cytochromc P450E. The loss of cytochrome P450E with no measurable effect on other isozymes at the low and medium dose may also explain why the spectrally measured content of total P450 did not decrease after TBT treatment (Table 1). Mode of action on P4.50 The mechanisms responsible for the loss and inactivation of cytochrome P450 forms in vivo are uncertain. The following processes could account for the loss of immunodetectable cytochrome P450: (1) binding of TBT to P450, dirzc: destruction of the native enzyme and formation of P420 with subsequent breakdown, (2) inhihition of P450 synthesis in addition to the P450 degradation, (3) accelerated turnover of P450 due to induction of the heme degrading enzyme heme oxygenase. Our results favor the hypothesis that, although all processes may be relevant, the direct destrurtion of P450 and a subsequent, rapid degradation of the apoprotein are more important than the inhibition of P450 synthesis and the induction of heme oxygenase. This is based on .he following. Direct de~~ir~ctiov~ of P450 Earlier in vitro data showed that binding of TBT resulted in the destruction of native P450 and loss of EROD activity (Fent and Stegeman, 1991). Those in vitro results showed that TBT produced a denaturation of P450 directly (i.e. formation of P420), and not indirectly through heme oxygenase (Fent and Stegeman, 1991). The rapid loss of P450 integrity was found in the absence of added NADPH, indicating that this effect is not the result of mechanism-based inactivation, or suicide processing by P450. in which metabolites of TBT would be responsible. The present study shows also that the loss of EROD activity in vivo is associated with a decrease in immunodetectable P450E, and not with an alteration of the catalytic efficiency of this enzyme. Thus, the in vitro and in vivo data lead to the hypothesis that binding of TBT to

236

P4SOled to the denaturation

of the apoprotein and formation of F42U with following breakdown by proteases. Protease digests of microsomes can in principle immunoreact with P45OE antibody (Gokssyr et al., 1991~). However, no breakdown products of P450E or P45OA were detected (Figs. 4,6) indicating that these apoproteins were completefy degraded. Similarly, there was a lack of i~unorea~tio~ with degraded P450 in microsomes from rat hepatocyte cultures treated with Arodor 1254 and macrolite antibiotics (Watkins et al., 1986). The failure to detect immunoreactive fragments in scup microsomes might indicate that the cytochromes are removed from the endoplasmic reticulum prior to, or shortly after the initiation of proteolysis.

An inhibitory effect of TBT on the synthesis of P450 could contribute to the loss of immunoreactive P45OE, or EROD activity, or P45OA. However, P450E apoprotein was shown to have a half-life of about 40 h (Kloepper-Sams and Stegeman, unpublished results), and the microsomes here were prepared only 24 h after treatment with TBT. Hence, if the decline here was solely due to an inhibition of protein synthesis, immuncrdetectable P450 should always ha.ve been present. We conclude that the inhibition of P45Osynthesis is nut primarily responsible for the loss of P450E and P45OA at the highest TBT dose, but might be a secondary efGectin addition to the degradation of these cytochromes, and important during longer term exposure. Inductiun of heme oxygenase

Metals including tin can induce heme oxygenase, with a decline in pools of the heme prosthetic group and consequently in the amount of P450 holoenzyme (Maines and Kappas, 1977). These mammalian studies showed a direct correiation between the elevation of heme oxygenase and the decrease in microsomal content of P450. Additional studies showed that the heme moiety of intact P450 was in fact not degraded by heme oxygenase, rather it was the heme of ~yto~hrome P420, the denatured form of P450, that was the substrate for the enzyme (Maines, 1976; Maines and Kappas, 1977; Kutty et al., 1988). In rats, organotins produced substantia1 alterations in the regulation of cellular heme metabolism. Elevation of hepatic heme oxygenase activity and depression of cytochrome P450 content were found after S.C.injections of tri~y~lohexyltin, bis(tri-~-butyltin) oxide (TBTU) and diethyltin (Rosenberg et al., 19&l), and other organotin compounds (Rosenberg et al., 1980). Cytochrome P450 dependent mixed function oxidase activity in rat liver was also impaired in a dosedependent manner, and mitochondrial ALA-synthetase activity underwent initial inhibition followed by rebound induction with TBTO (Rosenberg et al., 1981). These results suggest that in fish other enzymes critical to heme metabolism such as heme oxygenase and ALA-synthetase may be affected in addition to cytochrome P450. Based on the in vitro effects (Fent and Stegeman, 1991) it appears that the degradation of P450E involves formation of P420, a direct effect on the enzyme. A possible induction of heme oxygenase may further lower the cytochrome P45Q content, since

237

cytochrome P420 was present at all TBT doses. Besides the direct denaturation of P450, the occurrence of P420 may be due to the fact that the heme oxygenase (and/or protease) was not yet activated to such an extent that all P420 could be metabolized. Due to the short-term nature of our ex~rjment (24 h), however, we suggest that the induction of heme oxygenase was not mainly responsible for the TBT effects observed in scup. However, the heme oxygenase may come into play after a longer time period. The question whether or not TBT leads to a further P450 degradation by induction of heme oxygenase and thus to an acceleration of heme turnover should be investigated in forthcoming experiments.

The observed 10s~of P450E at low and medium TBT doses is relevant in terms of subacute and acute toxicity, but inactivation of P45OE and associated enzyme activities may occur at lower TBT concentrations as well. At the lowest dose the TBT residue in the liver was 8 ,&g which is in the same range of residues measured in an acute toxicity test in the liver of rainbow trout (Martin et al., 1989), and in mi~now’s early life stages fJ+nt, 19911.Additional experiments should show, if and how lower concentrations of TBT affect cytoch, ome P453 forms in fish after long-term chronic exposure. TBT is shown to be metabolized in mammals (Fish et al., 1936; Kimmel et al., 1977), fish and crab (Lee, 1986) via hydroxylated inte~ediates to d~butyltin (DBT) and monobutyltin (MBT). The cytochrome P450 dependent monooxygenase system appears to be responsible for the oxidation of TBT, but the isozymes involved are not known. Residues of the metabolite DBT were present in significant amounts (Table 2) indicating that metabolism of TBT has taken place during the 24 h exposure period in fish given the low and medium dose. However, at the highest dose, low quantities of metabolites were found. Even though DBT and MBT may partly originate from impurities in the TBT solution injected, metabolism was apparently decreased (or even inhibited) in fish given the high TBT dose. This suggests that P450 responsible for TBT metabolism may be inactivated as well. ~imiIarly, virtually no TBT metabolism was detected in fish early life stages at whole body residue concentrations (as TBT ion) of 1.48 to 4.5 ,ug/g TBT (Fent, 1991). Therefore, the reduction of TBT metabolism at high TBT residue concentrations may represent an inhibition of cytochrome P450 forms responsible for TBT metabolism. The highest TBT dose led to the appeamn~e of low molecular weight bands in the polyacrylamide gels and nitrocelluloses representing degraded proteins (Fig. 3). This points to a rapid degradation of other microsomal proteins in addition to cytochromes P450. Proteins ~ontai~j~~ sulfhydryl groups are likely candidates far being inhibjted and subsequently degraded. Studies in mammals have shown that TBT inhibits oxidative phospho~lation (Aldridge, 13177).and acts as an uncoupler (Connerton and Griffiths, 1989) in mitochondria. The glutathione-Saryhransferase was

2%

shown to be inhibited in liver of mammals (Henry and Byington, 1976). and fish (George and Buchanan, 1990). Also, TBT exerted a hemolytic action on human erythrocytes. indicating that this compound can disrupt membranes and act as a membrane toxicant (Gray et al.. 1987). From these studies, it can be concluded that additional proteins are also affected by TBT in fish. Interference with en vironrnen tal induction oj‘ P4.50

Elevated content of hydrocarbon-inducible P450 and high rates of EROD activity and aryl hydrocarbon hydroxylase activity (both catalyzed by CYPl A) have been observed in fish from many sites (Stegeman et al., 1987; Elskus et al., 1989; Van Veld et al., 1990). Thus far, only 1it;le attention has been paid to inhibitory action of environmental chemicals on this cytochrome. Gooch et al. (1989) found a decrease in catalytic activity in fish given high doses of a nonortho-PCB-congener that is known to be a potent inducer of CYPl A at low doses. Recently, a study further indicated that the induction response may be affected by the degree of prior environmental exposure to pollutants (Monosson and Stegeman, 1991). The strong action of TBT on C’r’PlA content and catalytic activity indicates that exposure to this environmental pollutant may result in a reduced induction response to other pollutants such as PAHs, PCBs, dibenzodioxins, and dibenzofurans. This may be relevant for the use of CYPl A as a biomarker. Given the widespread contamination of aquatic environments by organotins, the possibility and the potential of organotins to modify the function and induction of CYPIA in fish should be evaluated in future experiments. ACKNOWLEDGEMENTS

We thank B.R. Woodin, WHOI, for valuable help and assistance, R.M. Smolowitz, WHOI, for histopathological investigation. and M.E. Hahn, WHOI, for reading the manuscript. Support for this study came from the EAWAG and PHS grant ES 04220. Contribution no. 7850 from the Woods Hole Oceanographic Institution. REFERENCES Aldridge, W.N..

1977. The influence of organotin compounds on mitochondrial functions. In: Organotin

compounds: new chemistry and applications, edited by J.J. Zuckerman. American Chemical Society, Washington, D.C. Adv. Chem. Ser. 157, 186 196. Alzieu. C., J. Sanjuan, P. Michel, M. Bore1 and J.P. Dreno, 1989. Monitoring and assessment of butyltins in Atlantic coastal waters. Mar. Pollut. Bull. 20. 22 -26. Boon. J.P.. J.M. Everaarts, M.T.J. Hillebrand, M.L. Eggens. J. Pijnenburgand A. Gokssyr. 1992. Changes in levels of hepatic biotransformation

enzymes and haemoglobin levels in female plaice (PlmrtmJctc~s

pIuks.s~~)after oral administration of a technical polychlorinatcd biphcnyl mixture (Clophen A40). Sci. Total Environ. I 14, I 13.- 133. Connerton. I.F. and D.E. Griffiths, 1989. Organotin compounds as energy-potentiated uncouplers of rat liver mitochond:ia. Appl. Organomct. Chcm. 3. 545 551.

239 Elskus, A.A. and J.J. Stegeman, 1989. Induced cytochrome P450 in Fut~~l~l~~.s lie~L~t-oc&u.~ associated with environmental contaminalion by polychlorinated biphenyls and polynuclear aromatic hydrocarbons. Mar. Environ. Res. 27. 31-50. Fent, K., 1991. Bioconcentration and elimination of tributyltin chloride by embryos and larvae of minnows Phxinus pfm~ims. Aquat. Toxicol. 20. i47- 158. Fent, K., 1992. Embryotoxic effects of tributyltin on the minnow Pizusinusphosit~us. Environ Pollut. 76, 187-194. Fent. K. and J. Hunn. 1991. Phenyltins in water, sediment and biota of freshwater marinas. Environ. Sci. Technol. 25,956963. 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 in vitro on the hepatic microsomal monooxygenase system in the fish Src~atanius chr_wops. Aquat. Toxicol. 20. 15%168. Fish, R.H., EC. Kimmel and J.E. Casida. 1976. Bioorganotin chemistry: reactions of tributyltin derivatives with a cytochrome P-450 dependent monooxygenase enzyme system. J. Organomet. Chem. 118,4l-54. George, S.C., 1989. Cadmium eff,ects on plaice liver xenobiotic and metal detoxication systems: doseresponse. Aquat. Toxicol. 15,303-3 10. George, S.G. and G. Buchanan, 1990. Isolation, properties and induction of plaice liver cytosolic glutathione-S-transferases. Fish Physiol. Biochem. l&437-449. Goksoyr. A., T.S. Solberg and B. Serigstad, 199Ia. lmmunochemical detection of cytochrome P4SOIAl induction in cod larvae and juveniles exposed to a water soluble fraction of Nort!i Se;i crude oil. Mar. Pollut. Bull. 22, 122-127. Goksayr. A., A. Hussy, H.E. Larsen, J. Klungseyr, S. Wilhelmsen, A. Maage, E.M. Brevik. T. Andersson, M. Celander, M. Pesonen and L. Flirlin. I991 b. Environmental contaminants and biochemical responses in flatlish from the Hvaler Archipelago in Norway. Arch. Environ. Contam. Toxicol. 21,486496. Goksoyr, A.. T. Andersson, D.R. Buhler, J.J. Stegeman, D.E. Williams and L. Forlin. 1991~. Immucochemical cross-reactivity of/%naphthoflavone-inducible cytochrome P450 (P4501A) in liver microsomes from different fish species and rat. Fish Physiol. Biochem. 9, l-13. Gooch, J.W., A.A. Elskus, P.S. Kloepper-Sams. M.E. Hahn and J.J. Stegeman. 1989. Effects of ortho- and non-ortho-substituted polychlorinated biphenyl congeners on the hepatic monooxygenase system in scup (Srrtrorottru.~ dzrysops). Toxic01 Appl. Pharmacol. 98.422.~433. Gray, B.H.. M. Porvaznik. C. Flemming and L.H. Lee. 1987. Tri-n-butyltin: a membrane toxicant. Toxicology 47. 3554. Gray, ES.. 19%. Sexual patterns of monooxygenase function in the liver of marine teleosts and the regulation of activity by estradiol. Ph.D. thesis, MITIWHOI, WHOI-88-34. 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. 1.59.-209. Henry. R.A. and K.H. Byington, 1976. Inhibition of gluthatione-S-aryltransferase from rat liver by organoge~anium, lead and tin compounds. Biochem. Pharmacol. 2X2291-2295. Kimmel, EC.. 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. 2.5. l-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 P450 of the marine fish scup. Arch. Biochem. Biophys. 253.268-278. Klotz. A.V.. J.J. Stegeman and C. Walsh. 1984. An alternative 7-ethoxyresorufin 0-deethylase activity assay: a continuous visible spectrophotometric method for measurements ofcytochrome P450 monooxygenasc activity. Anal. Biochem. 140. 138-145. Klotz. A.V.. J.J. Stegeman. B.R. Woodin, E.A. Snowberger, P.E. Thomas and C. Walsh. 1986. Cytcchrome P4.50 isozymes from the marine telcost S~crrolor~rrrschr:l~o~~.s:their roks in steroid hydrovlation and the iiuluence ofcytochrome b,. Arch. Biochem. Biophys. 249.326-338.

Kutty, R-K., R.F. Daniel, D.E. Ryan, W. Levin and M.D. Maine& I?%. Rat liver cytochrome P450b, P42Ob, and ~420~ are degraded to biliverdin by heme oxygenase. Arch. Bio:hem. Biophys. 260,638~644. Lee. R.F., 1986. Metabolism ofbis(tributyltin)oxide by estuarine animals. In: Proceedings of the organotin symposium Oceans ‘86, Washington, D.C. IEEE and Marine Technology Society, New York. 1986, pp. 1182-l 188. Maines, M.D.. 1976. Evidence for the catabolism of polychlorinated biphenyi-induced by microsomal heme oxygenase and the inhibition of &aminolevulinate dehydratase

cytochrome P-448 by polychlorinated

biphenyls. J. Exp. Med. 144, 1509-1519. Maines, M.D. and A. Kappas, 1977. Metals as regulators of heme metabolism. Science 198, 1215-1221. Martin, R.C., D.G. Dixon, R.3. Maguire, P.V. Hodson and R.J. Tkacz, 1989. Acute toxicity, uptake, depuration and tissue distribution of tri-n-butyltin in rainbow trout, Suhnoguirdneri. Aquat. Toxicol. 15, 37-52. Monosson. E. and J.J. Stegeman, 1991. Cytochrome P450E (P4501A) induction and inhibition in winter flounder by 3,3’,4,4’-tetrachlorobiphenyl: comparison of response in fish from Georges Bank and Narragansett Bay. Environ. Toxicol. Chem. IO. 765-774. Nebert. D.W. and D. Nelson et al., 1991. The P450 superfamily: update on new sequences. gene mapping, and recommended nomenclature. DNA Cell Biol. IO, I-14. Petersen. D.W. and J.J. Lech, 1987. Hepatic effects of acrylamide m rainbow trout. ioxicoi. Appi. Piiarmacot. 89,249-255. Rosenberg, D.W.. G.S. Drummond, H.C. Cornish and A. Kappas, 1980. Prolonged induction of hepatic haem oxygenase and decreases in cytochrome P450 content by organotin compounds. Biochem. J. 190. 465468. Rosenberg. D. W.. G.S. Drummond and A. Kappas. 198 I. The influence of organometals on heme metabolism. In vivo and in vitro studies with organotins. Molec. Pharmacol. 21, 150-158. Rosenberg, D.W., M.K. Sardana and A. Kappas, 1985. Altered induction response of hepatic cytochrome P-450 to phenobarbital, 3-methylcholanthrene. and/?-naphthofiavone in organotin-treated animals. Biothem. Pharmacol. 34,997-1005. Smith, P.K., R.I. Krohn. G.T. Hermanson. A.K. Mallia, i:.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goek;, B.J. Olson and D.C. Kienk, !985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150,7&85. Spooner, N.. P.E. Gibbs, G.W. Bryan and L.J. Goad, 1991. The effect of tributyltin upon steroid titres in the female dogwhelk, Ntrcrllu lupilius. and the development of imposex. Mar. Environ, Res. 32, 37-49. Stegeman. J.J.. R.L. Binder and A. Orren, 1979. Hepa:ic and extrahepatic microsomal electron transport components and mixed-function oxygenases in the marine fish Stenotonm setxicolor. Biochem. Pharmacol. 28,343 l-3439. Stegeman, J.J. and P.J. KIoepper-Sams. 1987. Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals. Environ. Health Perspect. 7 I. 87-95. Stegeman. J.J.. F.Y. Teng and E.A. Snowberger, 1987. Induced cytochrome P450 in winter flounder (PsL~uri~pl~urone~t~.swnericcmrs) from coastal Massachusetts evaluated by catalytic assay and monoclonai antibody probes. Can. J. Fish. Aquat. Sci. 44, 1270- 1277. Stegeman, J.J.. B.R. Woodin and R.M. Smolowitz. 1990. Structure. function and regulation of cytochrome P450 forms in fish. Biochem. Sot. Trans. 18, 19~.21. Van Wd. P.A.. D.J. Westbrook. B.R. Woodin, R.C. Hale, C.L. Smith, R.J. Huggett and J.J. Stegeman, 1990. Induced cytochrome P459 in intestine aud liver of spot (Lri.stottm,r.\-~tttt/~tfri(.s)from a polycyclic aromatic hydrocarbon contaminated environment. Aquat. Toxicol. 17, I I7 -132. Watkins. P.B., S.A. Wrighton, E.G. Schuetz. P, Maurel and P.S. Guzelian. 1986. Macrolide antibioticc selectively inhibit the degradation of the glucocorticoid-responsive cytochrome P-450~ in rdt hepatocytcs in viv0 and in primary monolayer culture. J. Biol. Chem. 261, 6264-6271.