Immunochemical and catalytic characterization of hepatic microsomal cytochrome P450 in the sperm whale (Physeter macrocephalus)

Aquatic Toxicology 52 (2001) 297– 309 www.elsevier.com/locate/aquatox

Immunochemical and catalytic characterization of hepatic microsomal cytochrome P450 in the sperm whale (Physeter macrocephalus) Jan P. Boon a,*, Wilma E. Lewis a, Anders Goksøyr b a b

Netherlands Institute for Sea Research (NIOZ), PO Box 59, 1790 AB den Burg, Texel, Netherlands Department of Molecular Biology, HIB, Uni6ersity of Bergen, PO Box 7800, N-5020 Bergen, Norway Accepted 7 August 2000

Abstract Liver samples from three live-stranded adult male sperm whales, that could be sampled and frozen in liquid nitrogen within 18 h post mortem, provided an opportunity to learn more about the basic properties of their cytochrome P450 (CYP) system. All samples were catalytically active and showed sharp bands of the different CYP enzymes after Western blotting, indicating that degradation of the proteins was negligible. All three sperm whales showed a similar immunochemical CYP pattern: bands of CYP1A1/2, CYP3A and CYP4A were present, but CYP2B1/2 was not detected. Significant biotransformation of the polychlorinated aromatic hydrocarbons 4, 4%dichlorobiphenyl (CB-15), 2,7-dichlorodibenzodioxin and 1,2,3,4,8-pentadibenzofuran was measured in an in vitro biotransformation assay. In contrast, 3,3%,4,4%-tetrachlorobiphenyl (CB-77) and two chlorobornanes (CHB-32 and CHB-62) occurring in the insectide toxaphene®, were not metabolised. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Physeter macrocephalus; Sperm whale; Cytochrome P450; Biotransformation; Persistent organic pollutants; PHAHs; PCBs; Chlorobornanes; Toxaphene®

1. Introduction Biotransformation plays a crucial role in the environmental fate of lipid soluble organic pollutants, since (a) it determines whether accumulation of a compound will occur from prey to predator (biomagnification), and (b) the changes * Corresponding author. Tel.: +31-222-369466; fax: + 31222-319674. E-mail address: [email protected] (J.P. Boon).

in molecular structure can have strong effects on toxicity. Cytochrome P450 (CYP) is the central enzyme system in phase I metabolism of persistent organic pollutants. In the case of lipophilic organohalogens lacking reactive groups, oxygen is usually introduced into the molecules. The CYP enzyme system is often primarily responsible for both bioactivation and detoxification mechanisms of many endogenous and exogenous compounds. Of

0166-445X/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 4 4 5 X ( 0 0 ) 0 0 1 6 5 - X

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the numerous families of CYP identified, iso-enzymes belonging to the subfamilies CYP1A, CYP2B, CYP3A, and CYP4A are particularly important in the metabolism of xenobiotics (Nebert et al., 1991; Nebert and Gonzalez, 1987). Each iso-enzyme is specialised in the transformation of a group of closely related compounds, but the iso-enzyme composition may differ between species. In marine mammals, the iso-enzymes CYP1A1 and 1A2 have been detected immunochemically in several species of seals and whales, but CYP2B is generally much more prevalent in seals (Boon et al., 1992). CYP3A and CYP4A seem to be generally present (Goksøyr, 1995). The reported CYP related data of whales concern the odontocete families delphinidae (dolphins), phocoenidae (porpoises) and monodontidae (e.g. beluga and narwhal) (Jarman et al., 1996; Muir et al., 1988, 1996; Norstrom et al., 1991; Reijnders et al., 1999; Tanabe et al., 1988; Watanabe et al., 1989) and the mysticete family balaenopteridae (Goksøyr et al., 1988, 1989). Some immunochemical evidence for the occurrence of CYP2B enzymes in whales was found in the odontocete species beluga whale (Delphinapterus leucas) and harbour porpoise (Phocoena phocoena) (White et al., 1994; Goksøyr, 1995). Thus, information is still completely lacking on the composition and properties of the CYP system of the family physeteridae, to which the sperm whale belongs. Another way to characterize the CYP system of an animal is by determination of the set of organic pollutants that can be metabolized under in vitro conditions utilizing isolated liver microsomes. In this manner, in vitro biotransformation of some PCB, PCDD and PCDF congeners (Letcher et al., 1998; White et al., 1994), and biotransformation of technical toxaphene® and some individual chlorobornanes (CHB) (Boon et al., 1998) has been demonstrated utilizing microsomes of harbour seal, whitebeaked dolphin (Lagenorhynchus albirostris) and harbour porpoise (Phocoena phocoena). Of the brominated biphenyl (PBB) and diphenylether (PBDE) congeners tested, only 4,4%-dibromobiphenyl was metabolised to a significant extent in the assay (de Boer et al., 1998a,b).

Sperm whales normally occur only in deep-sea environments. In 1995 and 1997, a few massstrandings on the beaches of the North Sea have occurred of live sperm whales which entered the North Sea apparently by accident (Kompanje and Reumer, 1995; Smeenk, 1997). Post mortem investigations have shown that these animals had apparently been unable to feed in the shallow North Sea, since their intestinal sytem was empty (Jauniaux et al., 1998). All stranded animals were adult males of about 20 yr of age, that were returning from their summer feeding grounds in the NorthEast Atlantic ocean and the Norwegian sea, to mate in the area near the Azores. These animals usually migrate along the continental slope and pass to the west of Ireland. Three of the stranded animals could be sampled within about 18 h after death, and therefore provided a rare opportunity to learn more about the properties of the parts of their hepatic CYP system expected to interact with organic pollutants. Therefore, the presence of enzymes of the CYP1-4 families was investigated by Western blotting. The metabolic capacity of the sperm whale microsomes was investigated towards a selection of planar polychlorinated aromatic hydrocarbons compounds and two chlorobornanes occurring in the insecticide toxaphene®.

2. Materials and methods Animals and sample preparation: The liver microsomes were prepared from animals that stranded alive on the North Sea coast of the Netherlands or, in one case, Denmark. All three sperm whales (Physeter macrocephalus) were young adult males of about 20 yr old that entered the North Sea apparently by accident on their return from their feeding grounds in the Norwegian Sea to the Azores. The first animal was designated c95PMb. This animal stranded near the Hague (NL) on 12th January 1995. It was first sighted alive at 9 A.M. Upon arrival at the beach at 19:00 h, the body cavity had already been opened by Dr. R. Kastelein of the Marine Mammal Centre in Harderwijk (The Netherlands). Liver samples of 0.5 –1

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kg were taken without any special preference for a particular lobe or area. They were cut in fine slices of a few grams which were homogenised on the beach with an Ultra turrax TP 18/10 after the addition of 1 volume of glycerol to two volumes of liver (electrical current was available from a mobile generator). The samples were frozen in liquid nitrogen (−196°C) at 20:15 h. Upon arrival in the laboratory, the samples were stored at − 80°C until the preparation of the microsomes. The second sperm whale, designated c 97PMa, also stranded near the Hague, on 28th of November 1997. It stranded during the night and died in the morning. The body cavity was opened at about 17:00 h under the responsibility of Dr. C. Smeenk of the museum of natural history ‘Naturalis’ in Leiden (NL) by Dr. M. Garcia Hartmann (Duisburg Zoo, Germany). A liver sample was obtained at approximately 19:00 h. Preparation and homogenisation occurred at the beach as described above. All samples were frozen in liquid nitrogen at 21:30 h. Length of animal: 12.5 m. The third sperm whale was designated c 97PMc. This animal stranded on a sandbank near the Danish Wadden Sea island of Rømø and died in the night of the 4th of December 1997. The salvage operation of the animals was carried out under the responsibility of Dr. S. Tougaard of the museum of natural history in Esbjerg (DK). The body cavity was opened by our own team in the morning on the 5th December around 08:30 h.

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A part of about 1 kg was cut from the liver at approximately 10:00 h. Since electrical current was unavailable this time, the samples were cut in fine slices, which were flushed with a 0.15 M KCl solution, dried on a tissue, and individually packed in aluminium foil. All samples were frozen in liquid nitrogen by 12:00 h. Length of animal: 15 m. The basic data of the three sperm whales are summarised in Table 1, together with the basic data of a whitebeaked dolphin, a harbour porpoise, and three harbour seals that were analysed on the same gels for comparison of the CYP system of other marine mammal species with the sperm whales, and to validate the methods used. A more extensive description of the history of these animals is given in Boon et al. (1998), and van Hezik et al., (2001).

2.1. Ethoxyresorufin-O-deethylase assay Total microsomal protein content was measured with the Biorad assay based on the method developed by Bradford (1976). The ethoxyresorufin-O-deethylase (EROD) activity is specific for CYP1A activity, but it was also used as a pre-screen for the viability of the microsomes. EROD was determined by the method of Eggens and Galgani (1992). The activity of a pool of harbour seal microsomes was determined on every plate as a positive control to check the general performance of the assay. The activity of this pool

Table 1 Basic data of the animals Species

Id. no.

Sex

Length (m)

Weight (kg)

Age(yr)

Stranded at

Date

Sperm whale

95PMb 97Pma 97PMc

m m m

14.5 12.5 15

30 × 103 – –

20 20 20

The Hague (NL) The Hague (NL) Rømø (DK)

12 Jan 1995 28 Nov 1997 4 Dec 1997

Harbour porpoise Whitebeaked dolphin

91Ppa 95LAa

f f (preg.)

2.5

– –

Adult Adult

Texel (NL) Texel (NL)

19 Dec 1991 24 Jan 1995

Harbour seal

92PVa 94PVa 96PVa

m f f

– 1.1 1.35

46 31 49.5

Adult Juvenile 4

Texel (NL) Texel (NL) Camperduin (NL)

4 Jan 1992 5 Jan 1994 16 Oct 1996

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was measured six times over a period of 1.5 yr and was determined to be 575 9 86 pmol mg − 1 protein min − 1 (mean 9 S.D.; CV = 15%). Western blotting: Western blotting combines the separation power of SDS-polyacrylamide gels with the specificity of immunochemical detection. The methods used have been described by Nilsen et al. (1998). The following combinations of primary and secondary antibodies were utilised to detect the different CYP enzymes: CYP1A1/2: the primary antibody was a monoclonal mouse anti-rat CYP 1A1/1A2 antibody (Oxford Biomedical Research, Oxford, Michigan, USA, cat. no. PM 16), dilution 1:1000. The secondary antibody was horseradish peroxidase (HRP) conjugated anti-mouse IgG (BioRad Lab, Hercules, CA, USA, cat. no. 170-6516), dilution 1:3000. CYP1A1: the primary antibody was a polyclonal rabbit anti-rat CYP1A1 (ECL kit RPN 256 of Amersham International plc, Little Chalfont, UK), dilution 1:200. The secondary antibody was a donkey anti-rabbit Ig-biotinylated antibody from the same ECL kit, dilution 1:2000. Development was performed with streptavidin-horseradish peroxidase conjugate and ECL reagents (from ECL-kit). CYP2B1/2: the primary antibody was a monoclonal mouse anti-rat 2B1/2B2 antibody (Oxford Biomedical Research, cat. no. PM 20), dilution 1:100. The secondary antibody was a goat anti-mouse IgG (Bio-Rad Lab, cat. no. 170-6516), dilution 1:3000. CYP3A: the primary antibody was a polyclonal rabbit anti-rat CYP3A antibody; (ECL-kit RPN 259 of Amersham International plc), dilution 1:200. The secondary antibody was a donkey anti-rabbit Ig-biotinylated antibody from the same ECL kit, dilution 1:2000. Development was performed with streptavidinhorseradish peroxidase conjugate and ECL reagents (from kit). CYP4A: the primary antibody was a polyclonal sheep anti-rat CYP4A (ECL kit RPN 260 of Amersham International plc.), dilution 1:200. The secondary antibody was an anti-goat IgG

(DAKO Patts, Glostrup, Denmark, code E466), dilution 1:500. The bands containing the complexes of the different P450 isoforms with the primary and secondary antibodies were visualised with enhanced chemoluminescence (ECL) substrates (Amersham International plc). The molecular weights (MWs) were estimated from a comparison of the migration of the bands of the different CYP cross-reacting proteins with the added molecular weight markers of the ECLkits (visible on the films), or pre-stained SDSPAGE standards (Bio-Rad Lab, cat. no. 161-0305; visible on the nitrocellulose membranes).

2.2. Polyhalogenated aromatic hydrocarbon mixture All standards and organic solvents (picograde® quality) were obtained from Promochem (Wesel, Germany). A mixture containing 4,4%-dichlorobiphenyl (CB-15), 3,3%,4,4%-tetrachlorobiphenyl (CB-77), 2,2%,4,4%,5,5%-hexachlorobiphenyl (CB153), 2,7-dichloro-p-dibenzodioxin (2,7-DiCDD), and 1,2,3,4,8-pentachloro dibenzofuran (1,2,3,4,8PnCDF), 4–11 ng/ml acetone was used. Acetone was used as the solvent because it readily mixes with water, which is a necessary prerequisite for the substrates to reach the reactive site of the CYP enzymes. CB-153 is known to be highly resistant towards environmental degradation and is not metabolised in the assay. Therefore it could be used as an internal standard.

2.3. Chlorobornane mixture A solution consisting of 5 ng ml − 1 of each of the individual chlorobornanes, i.e. CHB-26, -32, -50 and -62 in 2,2,4-trimethylpentane (TMP) was purchased from Promochem (Wesel, Germany). The different structures are shown in Fig. 1. The original solvent (TMP) was evaporated and replaced by acetone. The final concentration of each congener was 7 ng ml − 1 acetone. An external standard solution (ESTD) was prepared by diluting 3 ml of this mixture to a final volume of 0.5 ml with TMP.

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Fig. 1. Chemical structures of the chlorobornanes (CHBs) used in the in vitro studies with sperm whale microsomes. Either Cl- or H-atoms can be bound to the carbon atoms numbered 2,3,4,5,6,8, 9 and 10 in the general structure. Only the positions of the Cl-substituents are indicated in the structures of the individual congeners CHB-26, -32, -50, and -62. (*) indicates a likely site for metabolic attack by the CYP 450 system (see also Boon et al., 1998).

2.4. In 6itro biotransformation assay This assay was performed according to the protocol of Boon et al. (1998). All treatment groups consisted of four replicate samples. The experiments were carried out in a water bath at 37°C. The incubation mixture consisted of 1 ml of a 80 mM NaH2PO4 buffer (J.T. Baker Chemicals, Deventer, The Netherlands) and 0.1 ml of standardised 10 mg/ml microsomal suspension. The pH was adjusted to 7.6. Subsequently, 3 ml of the contaminant mixture (CHB or PHAH mixtures) was added to the incubation mixture and pre-incubated for 3 min at 37°C. Since the reference assays consisted of only these constituents, biotransformation reactions could not occur because of the lack of an electron donor. To the four assay samples, 0.1 ml of an 11 mM NADPH (Boehringer Mannheim, Almere, The Netherlands) solution was added to initialise the biotransformation reactions, followed by additions of

0.1 ml every 10 min up to the 90 min mark. At this time, the reaction was terminated by transfer of the entire incubation mixture to 1 ml of icecold methanol.

2.5. Sample clean-up and GC-ECD analysis PHAHs: the isolation and gas chromatography with electron capture detection (GC-ECD) determination of the unmetabolised PHAHs followed the methodology for CHBs described by Boon et al. (1998). Briefly, the residual PHAHs were extracted by liquid –liquid partitioning with n-hexane, followed by H2SO4 (98%) treatment to remove co-extracted lipids. After removal of the acid, the residual acid was neutralised with portions of ca. 5 ml of a 25 mM NaHCO3 buffer solution until the pH reached a value of 6.0. After addition of TMP, the volume was reduced by heat-assisted evaporation to about 250 ml, which was transferred to a GC-vial. The reaction tube

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was washed twice with TMP, and the final volume in the GC vial was gravimetrically adjusted to 500 ml (TMP, l = 0.69 g/ml). The PHAHs were analysed by GC-ECD. The sample was injected by splittless injection of 1 ml solution. The injector temperature was 250°C. The GC column was a CP-Sil 8 CB (50 m × 0.25 mm i.d. × 0.25 mm film thickness; Chrompack, Middelburg, the Netherlands, cat. no. 486998). The carrier gas was hydrogen, with a column pressure of 100 kPa, and a linear gas velocity of 107 cm s − 1. The oven temperature programme started at 90°C (2 min isothermal), followed by sequential temperature increases of 10°C min − 1 to 215°C (followed by an isothermal phase of 15 min), 8°C min − 1 to 270°C (1 min isothermal), and 8°C min − 1 to 275°C (9 min isothermal). The detector temperature was 340°C. The peak heights of the congeners in the chromatograms of the reference group were compared to those in the ESTD mixture. The results were accepted only if the recovery was at least 75% for each of the congeners. Chlorobornanes: The clean-up was done as described previously (Boon et al., 1998). The extracts were prepared for analysis in a final volume of 0.5 ml TMP in GC-vials for automated injection. Aliquots of 1 ml were injected in the splitless mode on a Carlo Erba 5160 GC-ECD. The injector temperature was 250°C. The column was a CP-Sil 8CB (25 m × 0.32 mm × 0.12 mm; Chrompack, Middelburg, The Netherlands, cat. no. 908952). The carrier gas was hydrogen, with a column pressure of 100 kPa, and a linear gas velocity of 107 cm s − 1. The oven temperature programme started at 90°C (isothermal phase of 3 min) followed by sequential temperature increases of 10°C min − 1 to 215°C (followed by an isothermal phase of 5 min), and 20°C min − 1 to 300°C (16 min isothermal). The peak heights of the four CHB congeners in the chromatograms of the reference group were compared to those in the ESTD. The results were accepted only if the recovery was more than 75% for all four congeners relative to the ESTD. Calculations: For assays that fulfilled the minimum 75% recovery condition, calculations were made to determine the differences between different levels of inhibitor. Since CB-153 and CHB-50 were

known to be persistent in earlier assays (Boon et al., 1998), they were used as internal standards (ISTD) to decrease the analytical variation in the respective assays. To compare the results of the different treatments, the ratios of the peak heights (H) of the potentially metabolisable PHAHs and CHBs to their respective ISTDs were calculated in each sample according to: HCompound-X Ratio(Compound-X) = HISTD The statistical significance of the difference of these Ratio(Compound-X) values for the different compounds in the group of assay samples (+NADPH) and the group of reference samples (− NADPH) was tested with a student’s T-test after a log transformation of the Ratio(Compound-X) values to increase homogeneity of variance. Both groups contained four replicate samples. In order to plot the data, single samples incubated with NADPH (assay group) and samples incubated without NADPH (reference group) were paired randomly. For each assay/reference pair, the fractions remaining (Fr) after treatment with NADPH, was calculated from the Ratio(Compound-X) values according to: Ratio(Compound-X)(+ NADPH) Fr(Compound-X)= Ratio(Compound-X)(− NADPH) Subsequently, means and standard deviations were calculated from the values of the four sample pairs. 3. Results

3.1. Immunochemical analyses The patterns of the enzymes CYP1A, CYP2B, CYP3A and CYP4A are given in Figs. 2–5. Patterns in the three individual sperm whales were virtually identical to each other. Bands were visible for all CYP enzymes except the CYP2B1/2. All samples seem to have been in a good condition, since the bands in the Western blots appeared sharp. A measurable degradation of the proteins would have resulted in an increased background smear after separation of the compounds by SDSPAGE.

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In order to validate the results obtained for the three sperm whales, samples of a whitebeaked dolphin, a harbour porpoise, some harbour seals and pre-induced rat were also investigated on the same gels. In the case of the rat, the inducing agent depended on the choice of antibodies against a particular CYP enzyme. The MWs obtained with the PM16 monoclonal antibody were 55 kDa for the CYP1A1/2 band in all sperm whales and harbour seal samples, and 52 kDa in the whitebeaked dolphin and the harbour porpoise samples. The MW of the CYP1A1 band treated with the polyclonal antibody of the enhanced chemoluminescence kit of Amersham, was 57 kDa. The cross-reactions with this antibody showed multiple bands. The migration of the band with the lowest molecular weight corresponded to the positive control from the rat and gave the strongest signal with

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the sperm whale and harbour seal samples. Weak bands at this MW were observed in the whitebeaked dolphin and the harbour porpoise. The other bands are attributed to unspecific binding of the antibody, but they could also be due to protein aggregation. The whitebeaked dolphin and the harbour porpoise showed only a weak band after crossreaction with the CYP1A1 antibody. In contrast, the band after treatment with the CYP 1A1/1A2 antibody was relatively strong, but it had slightly lower molecular weight than the other animals. This implicates that these small odontocete species may have relatively high values of CYP1A2 compared to the sperm whales and the harbour seals. The monoclonal antibody PM20 cross-reacted with a CYP2B1/2B2-like protein with a MW of 53 kDa only in the harbour seal samples.

Fig. 2. Western blots of liver samples of odontocete species and harbour seal probed with: upper lane: a monoclonal mouse antibody against rat CYP 1A1/1A2 (Oxford Biomedical Research, cat. no. PM 16), dilution 1:1000. The secondary antibody was a horseradish peroxidase (HRP) conjugated anti-mouse IgG (Bio-Rad Lab, cat. no. 170-6516), dilution 1:3000. Detection by enhanced chemoluminescence; incubation time 1 min. Lower lane: a polyclonal rabbit antibody against rat CYP1A1 (ECL kit RPN 256 of Amersham International plc), dilution 1:200. The secondary antibody was a donkey anti-rabbit Ig-biotinylated antibody from the same ECL kit, dilution 1:2000. Development was performed with streptavidin-horseradish peroxidase conjugate and ECL reagents (from kit). Detection by enhanced chemoluminescence; incubation time 10 min (odontocetes), or 3 min (seals).

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Fig. 3. Western blots of liver samples of odontocete species and harbour seal probed with a monoclonal mouse antibody against rat 2B1/2B2 (Oxford Biomedical Research, cat. no. PM 20), dilution 1:100. The secondary antibody was a goat anti-mouse IgG (Bio-Rad Lab, cat. no. 170-6516), dilution 1:3000. Detection by enhanced chemoluminescence; incubation time 3.5 min.

Cross-reacting proteins of approximately 57 kDa were found with the polyclonal CYP3A antibody, whereas cross-reacting proteins of approximately 55 kDa were found with a CYP4A antibody. Both enzymes were detected in the hepatic microsomes of all animals. It has to be borne in mind that differences in MWs of up to 2 – 3 kDa can be attributed to differences in the migration of the proteins on the gels of about 1 mm. In summary, the samples of both small odontocetes showed bands for the same CYP enzymes as the three sperm whales (i.e. CYP1A1/2, CYP3A, and CYP4A), whereas the samples of the harbour seals showed an additional band for CYP2B1/2.

3.2. Catalytic acti6ities EROD: The EROD activities of the animals in this study are given in Table 2. The values span a range of more than two orders of magnitude with the sperm whales (15 – 28 pmol mg − 1 microsomal protein min − 1) and the whitebeaked dolphin (49 pmol mg − 1 microsomal protein min − 1) at the lower end, and the harbour porpoise (417 pmol mg − 1 microsomal protein min − 1) and the three harbour seals (404 – 2460(!) pmol mg − 1 microsomal protein min − 1) at the higher end of the range. PHAHs: CB-15, 2,7-diCDD and 1,2,3,4,8-pentaCDF were all metabolised to a significant extent by the microsomes of sperm whale cPM97a

(Student’s T-test on the Ratio(Compound-X) values of the samples belonging to the assay and the reference groups (n = 4), P B 0.05). The fractions of the parent compounds remaining after biotransformation (Fr values) are plotted in Fig. 6. The Fr values of CB-15, 2,7-diCDD, and 1,2,3,4,8-pentaCDF were, respectively, 0.22, 0.03 and 0.52. CB-77 still showed an Fr value of 1.0, indicating the absence of any measurable degree of biotransformation. Chlorobornanes: In these assays only CB-15, which was added as a positive control to the pollutant mixture, was metabolised to a significant extent in the assay samples (Student’s T-test, n = 4, P B 0.05). Fig. 7 shows that the mean Fr values for CB-15 were 0.06 for sperm whale c 97PMa and 0.42 for sperm whale c 97PMc. In contrast, the Fr values of all CHBs were \ 0.95 for all congeners in both animals. Thus, microsomal chlorobornane metabolism could not be demonstrated in these two sperm whales.

4. Discussion The enzymes in the microsomal preparations obtained from stranded marine mammals within a relatively short-period post mortem, were still catalytically active. This means that the results obtained in this study add to the knowledge about the fate of toxic compounds which have reached

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the marine environment in general, and in the case of sperm whales, even deep-ocean waters. The CYP enzyme patterns of the three male sperm whales investigated were very similar among themselves, as well as compared to those of the other cetacean families. Especially the relatively strong intensity of the bands of CYP1A1/2 is surprising in view of relatively low EROD activities compared to most other animals investigated, and the habitat of these animals, i.e. the deep-ocean environment. This may reflect differences in affinity for the primary antibodies used. Nevertheless, the detection of bands of CYP1A in the three sperm whales indicates that the levels of Ah-responsive pollutants in deep-sea food-chains might already be high enough to induce the enzymes of this subfamily to some degree. In the present study, bands of CYP2B1/2 were only observed in samples of seals, but enzymes cross-reacting with CYP2B antibodies have been reported in odontocete whales. In a beluga whale (Delphinapterus leucas), a band was recognised by rabbit CYP2B4 antibodies, whereas no cross-reaction occurred with rat CYP2B1/2 antibodies (White et al., 1994). In seals, CYP2B1/2 bands seem to be much more common (Goksøyr, 1995). CYP3A and CYP4A were detected in all cetacean and seal samples tested earlier (Goksøyr, 1995),

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and thus these enzymes seem to be common in odontocete whales as well as seals. The molecular weights of the CYP cross-reacting forms were all in the 52–57 kDa range, which is similar to earlier results with marine mammals (Goksøyr, 1995). All major classes of the ‘classical’ organochlorines were detected in blubber of the sperm whales that stranded around the North Sea in the 1994/ 1995 winter (Law et al., 1996; Wells et al., 1997). The highest concentrations were those of the DDTs (especially 4,4%-DDE and 4,4%-DDT), followed by the PCBs, the chlordanes, dieldrin and hexachlorobenzene. Trace values of the hexachlorocyclohexanes were present. Besides these organochlorines, some polybrominated diphenylether (PBDE) congeners were also detected in these animals (de Boer et al., 1998a). These compounds are being applied as flame retardants in plastics of electronic equipment, furniture upholstery, carpets, textiles, foam, building material, and in the interiors of vehicles and aircraft. PBDEs were quite common in estuarine and coastal sediments, and some tetra- and pentabrominated congeners were also detected in marine invertebrates and fish from areas around the UK (Allchin et al., 1999). In former in vitro assays, the major congeners used in technical mix-

Fig. 4. Western blots of liver samples of odontocete species and harbour seal probed with a polyclonal rabbit antibody against rat CYP 3A (ECL-kit RPN 259 of Amersham International plc), dilution 1:200. The secondary antibody was a donkey anti-rabbit Ig-biotinylated antibody from the same ECL kit, dilution 1:2000. Development was performed with streptavidin-horseradish peroxidase conjugate and ECL reagents (from kit). Detection by enhanced chemoluminescence; incubation time 1 min (sperm whales) or 0.5 min (all the other animals).

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Fig. 5. Western blots of liver samples of odontocete species and harbour seal probed with a polyclonal sheep antibody against rat CYP4A (ECL kit RPN 260 of Amersham International plc), dilution 1:200. The secondary antibody was an anti-goat IgG (DAKO Patts, Glostrup, Denmark, code E466), dilution 1:500. Detection by enhanced chemoluminescence; incubation time 15 s.

tures, 2,2%,4,4%-tetra-BDE (PBDE-47), 2,2%,4,4%,5penta-BDE (BDE-99) and the fully brominated decabromodiphenyl ether (BDE-209), all behaved persistently (de Boer et al., 1998b) in the in vitro assays with hepatic microsomes of sperm whale c95PMb, whitebeaked dolphin c 95LAa, and harbour seal c 94PVa. The inducing capacity of BDE-47 and BDE-99 in rats was strongest for CYP2B (PROD) and weaker for CYP1A1 (EROD) and 1A2 (MROD), whereas BDE-209 did not induce any CYP-form (Hallgren and Darnerud, 1998). These results show that other compounds than those usually analysed may also affect the induction state of the CYP system in marine mammals. An important question in this field of research is, whether the set compounds that can be metabolised by an animal can be derived from the CYP enzymes present in its hepatic microsomes. In this respect, it is important that 3,3%,4,4%-tetrachlorobiphenyl (CB-77) was not metabolised in the in vitro assay with sperm whale microsomes, whereas it was biotransformed to its 4-, 5- and 6-hydroxy metabolites by the hepatic microsomes of the harbour seal c 92PVa and harbour porpoise c91PPa (Murk et al., 1994). In seal microsomes the metabolism of CB-15 and CB-77 was already inhibited in the presence of 1 mM of the selective CYP1A1/2 inhibitor ellipticine (Letcher et al., 1998), indicating that the enzymes CYP1A1 and/or 1A2 play a major role in the metabolism

of these planar CB congeners that both lack ortho-Cl substituents and possess vicinal hydrogen atoms in the ortho- and meta- positions. Thus, although CYP1A1/2 was immunochemically present in the sperm whales, their microsomal preparations metabolised only CB-15 rapidly, but not CB-77. The CHBs tested were also persistent to enzymatic attack in the in vitro assays with the microsomes of sperm whale PM97a and PM97c. The same result was obtained earlier for animal c 95PMb (Boon et al., 1998). In contrast, CHB32 was rapidly metabolised by hepatic microsomes of the whitebeaked dolphin c 95LAa and the harbour porpoise c 91PPa. Harbour seal microsomes even metabolised CHB-32 and CHB-62 (Boon et al., 1998). Since the metabolism of both Table 2 EROD activities of the hepatic microsomal preparationsa Species

Identification no.

EROD

Sperm whale

95PMa 97PMa 97PMc

17 28 15

Whitebeaked dolphin Harbour porpoise

95LAa 91PPa

9 417

Harbour seal

92Pva 94PVa 96PVd

2460 537 404

a The values are expressed in pmol mg−1 microsomal protein min−1.

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Fig. 6. In vitro biotransformation of four planar polyhalogenated aromatic hydrocarbons (PHAHs) by hepatic microsomes of sperm whale c97PMa in a set of samples that received NADPH as electron donor (n = 4) compared to a set of reference samples that did not receive NAPDH (n = 4). Biotransformation rates are calculated by the decrease of the parent compound within the incubation time of the assay (90 min). A fraction remaining (Fr) of 0 denotes complete biotransformation of the parent compound in the presence of NADPH, whereas an Fr value of 1 denotes the absence of any biotransformation. (*) indicates a significant (i.e. P B 0.05) biotransformation in the assay group ( + NADPH) as established with a student’s T-test for two groups of samples (n = 4 for both groups) on the log-transformed values of Ratio (Compound-X) of both groups.

CHBs could be fully (CHB-32) or largely (CHB62) be inhibited in a dose-dependent manner by the inhibitor ketoconazole, an important role was attributed to CYP3A in the biotransformation of these CHBs in harbour and grey seal (van Hezik et al., 2001). Thus, the information obtained from the in vitro assays with microsomes from different marine mammal species shows that despite the fact that the enzymes that catalysing the metabolism of CB-77, CHB-32 and CHB-62 in other species marine mammals were also immunochemically detected in the sperm whales, they could not metabolise these substrates. Apparently the representatives of different marine mammal species cannot always metabolise the same substrates, even when the composition of their sets of immunochemically detected CYP forms is identical. Differences in the shape of the docking area of the substrates may play an important role here.

5. Conclusions The liver samples taken from three livestranded adult male sperm whales and frozen in

307

liquid nitrogen within hours after death, provided a good opportunity to learn more about the basic properties of their CYP system. The samples were catalytically active and showed sharp bands of the different CYP cross-reacting proteins after Western blotting, indicating that degradation of the proteins was negligible at the time of sample collection. All the three sperm whales showed a similar immunochemical CYP pattern: bands of CYP1A1/2, CYP3A and CYP4A-like patterns were present, but CYP2B1/2 cross-reaction was not detected. These results are similar to those generally found for other odontocetes. Significant biotransformation of 4,4%-chlorobiphenyl (CB-15), 2,7-dichlorodibenzodioxin and 1,2,3,4,8-pentadibenzofuran was measured in the in vitro biotransformation assays with sperm whale microsomes. In contrast, 3,3%,4,4%-tetrachloro-

Fig. 7. In vitro biotransformation of chlorobornanes (CHBs) by hepatic microsomes of two sperm whale in a set of samples that received NADPH as electron donor (n = 4) compared to a set of reference samples from the same animal that did not receive NAPDH (n = 4). Biotransformation rates are calculated by the decrease of the parent compound within the incubation time of the assay (90 min). A fraction remaining (Fr) of 0 denotes complete biotransformation of the parent compound in the presence of NADPH, whereas an Fr value of 1 denotes the absence of any biotransformation. (*) indicates a significant (i.e. P B 0.05) biotransformation in the assay group ( + NADPH) as established with a student’s T-test for two groups of samples (n = 4 for both groups) on the logtransformed values of Ratio (Compound-X) of both groups.

308

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biphenyl (CB-77) and two CHBs (CHB-32 and CHB-62) were persistent to enzymatic attack, although metabolism of these compounds has been observed for other marine mammal species. Thus, the metabolic capacity of the sperm whale microsomes appears to be relatively restricted, despite the immunochemical detection of the same CYP1, CYP3, and CYP4 enzymes as usually present in other odontocetes. This indicates that the set of organic pollutants that can be metabolised by an animal, cannot be derived from the assortment of immunochemically detected CYP enzymes.

Acknowledgements The authors are very grateful for the assistance of Ms. Kjersti Helgesen when carrying out the Western blots. Without the help of a great number of people and instances, it would not have been possible to obtain the liver samples of the animals at such an early stage post mortem. Special thanks to Dr. C. Smeenk and Ms. M. Addink (Museum of Natural History ‘Naturalis’, Leiden, The Netherlands), Dr. M. Garcia Hartmann (Duisburg Zoo, Germany), Dr. R. Kastelein (Marine Mammal Centre, Harderwijk, The Netherlands), Dr. S. Tougaard (Museum of Natural History, Esbjerg, Denmark), Mr. K. Camphuysen and Ms. M.J. Greve (Netherlands Institute for Sea Research (NIOZ), Texel, The Netherlands). The samples of the small cetaceans and the harbour seals were obtained through the help of Mr. H. Brugge, Mr. R. van der Zwaag and Mr. W. Sietsma of ECOMARE, the Educational Centre for North Sea and Wadden Sea (Texel, The Netherlands). This is NIOZ publication no. 3386.

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