Expression and inducibility of members in the cytochrome P4501 (CYP1) family in ringed and grey seals from polluted and less polluted waters

Environmental Toxicology and Pharmacology 8 (2000) 217 – 225

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Expression and inducibility of members in the cytochrome P4501 (CYP1) family in ringed and grey seals from polluted and less polluted waters Madeleine Nyman a,b,*, Hannu Raunio b, Olavi Pelkonen b b

a Finnish Game and Fisheries Research Institute, P. O. Box 6, FIN-00721 Helsinki, Finland Department of Pharmacology and Toxicology, Uni6ersity of Oulu, Box 5000, FIN-90401 Oulu, Finland

Received 29 October 1999; received in revised form 11 April 2000; accepted 17 April 2000

Abstract The expression and inducibility of cytochrome P4501A (CYP1A) were investigated in hepatic microsomes of ringed seals (Phoca hispida) and grey seals (Halichoerus grypus) from polluted (Baltic Sea) and less polluted (Svalbard and Sable Island) areas. Liver CYP1A protein levels and activities were assessed by immunoblot analysis and determining catalytic activities towards ethoxyresorufin (EROD) and pentoxyresorufin (PROD). The amount of CYP1A apoprotein and its catalytic activity were increased up to 5-fold in Baltic ringed and grey seal populations in comparison with ringed seals from Svalbard and grey seals from Sable Island. EROD and PROD activities correlated in all seal groups, indicating catalysis by the same CYP form(s). Enzyme kinetic studies suggested that PROD activity is catalysed by CYP1A enzymes in both ringed and grey seals. In immunoblot analysis, a protein was revealed in liver with an antibody against human CYP1B1, indicating that a CYP1B1 like protein could be present in ringed and grey seals. In conclusion, these data strengthen the concept that CYP1A is markedly induced in seals living in polluted waters and that both EROD and PROD activities are mediated by CYP1A forms in the seal liver. In addition, this study provides the first evidence for the presence of a CYP1B like protein in seal liver. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Ringed seal; Grey seal; CYP1; Baltic Sea

1. Introduction Synthetic organic compounds, such as polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT) are problematic environmental contaminants because of their previous and present widespread use, their bioaccumulation in the food chain and resistance to biological degradation. A variety of biochemical and toxicological effects have been observed in animals exposed to these compounds (Reijnders, 1986; Brouwer et al., 1989; Safe, 1994; Ross, 1995; De Swart, 1995; Ba¨cklin, 1996). Persistent lipophilic contaminants affect especially marine mammals due to their position as top predators and accumulation of contaminants in the thick blubber layers of the animals. * Corresponding author. Tel: + 358-205-751274; fax: +358-205751201. E-mail address: [email protected] (M. Nyman).

It is commonly thought that the high prevalence of pathological changes and toxic effects in wild marine mammals are associated with elevated contaminant burdens (DeLong et al., 1973; Mortensen et al., 1992; Aguilar and Borrell, 1994; Martineau et al., 1994; Olsson et al., 1994; Jenssen et al., 1995). In the Baltic Sea, both ringed seals (Phoca hispida botanical) and grey seals (Halichoerus grypus) have suffered from various types of pathological changes (ulcers, renal failure, adrenocortical hyperplasia, skull lesions, tumours and reproductive impairments), and have displayed simultaneously high levels of contaminant burdens (Helle et al., 1976a,b; Bergman and Olsson, 1986; Bergman et al., 1992). In Baltic seals, contaminant burdens have decreased from a mean sum of PCB and DDT concentrations in blubber exceeding 100 mg/g lipid in the 1960s and early 1970s, to the current levels of roughly 60 mg/g (PCB)

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and 20 mg/g (DDT) lipid weight in ringed seals and 25 mg/g (PCB) and 8 mg/g (DDT) lipid weight in grey seals (Jensen, 1969; Helle et al., 1976a,b; Olsson et al., 1994; Roots and Talvari, 1997; J. Koistinen, unpublished data). The incidence of reproductive impairments has diminished simultaneously in the grey seal, but the frequency of ulcers has increased (Bergman, 1999). Uterine occlusions still occur in about 25 – 30% of the female adult ringed seals in the Baltic Sea, while the Baltic grey seals reproduce successfully (E. Helle and M. Nyman, unpublished data). The differing pathological symptoms between the two seal species could be explained by a difference in their metabolic capacities towards foreign organic compounds. In seals at the reference sites chosen for this study, the mean sum of PCB and DDT concentrations in blubber are less than 3 mg/g lipid weight in Svalbard and 0.5 – 1 mg/g lipid weight in Sable Island (Wolkers et al., 1998b; Addison et al., 1999), i.e. an about ten times lower contaminant burden than in the Baltic seal populations. The cytochrome P450 (CYP) enzymes constitute an important part of the phase I biotransformation of exogenous compounds, resulting in altered toxicity of the parent compound. The CYP enzymes are divided into a multitude of families and subfamilies according the their gene structures. The major CYP enzymes that metabolise xenobiotics belong to families CYP1, CYP2 and CYP3 (Nelson et al., 1996). CYP expression and activity profiles are well documented in humans and laboratory animals, but since substantial species differences exist (Boobis et al., 1990), it is difficult to extrapolate CYP enzyme functions to less well-known wildlife species. In true seals, the presence of CYP1 like proteins and activities have previously been found to exist in grey seals, harbour seals (Phoca 6itulina), harp seals (Phoca groenlandica) hooded seals (Cystophora cristata) and ringed seals (Engelhardt, 1982; Addison and Brodie, 1984; Addison et al., 1986; Goksøyr et al., 1992; Murk et al., 1994; White et al., 1994; Mattson et al., 1998; Wolkers et al., 1998a, 1999). Other studies have indicated elevated CYP activities in marine mammals by comparing the profiles of organochlorine derivatives in seal or whale blubber to same contaminants in their food sources (Boon et al., 1987; Tanabe et al., 1988; Duinker et al., 1989; Kannan et al., 1989; Bergek et al., 1992; Norstrom et al., 1992; Boon et al., 1994; Bruhn et al., 1995; Letcher et al., 1998). The presence of CYP1A has been reported previously in both grey and ringed seals (Engelhardt, 1982; Addison and Brodie, 1984; Mattson et al., 1998; Wolkers et al., 1998a), but more species specific information on the CYP1A forms present and their induction/inhibition pattern is needed to understand the toxic effects of the contaminant burdens found in populations from heavily polluted areas.

The expression of CYP1B1 has been reported only in human, rat and mouse tissues (Nelson et al., 1996). CYP1B1 has been studied because of its capacity to metabolise and activate polycyclic hydrocarbons, thereby often initiating carcinogenesis (Shimada et al., 1996; Baron et al., 1998; Kim et al., 1998). Overlapping substrate specificity, lack of truly specific catalytic assays and antibody cross-reactions between CYP1A1, CYP1A2 and CYP1B has made it difficult to distinguish the three members of the CYP1 family from each other (Shimada et al., 1998). Peptide based antibodies and isoform specific inhibitors for the enzyme assays have been proposed as tools to discriminate the three P450 forms (Shimada et al., 1998). In marine mammals, CYP1B1 expression has not been investigated before. The aim of this study was to characterise in detail background expression and inducibility of CYP1A in ringed and grey seals from polluted (the Baltic Sea) and less polluted (Svalbard and Sable Island) waters using enzyme assays, specific enzyme inhibitors, kinetic models and immunoblotting analysis. In addition, the expression and possible induction of CYP1B1 in these seal populations were examined.

2. Materials and methods

2.1. Sample collection and microsome preparation During the spring of 1997 and 1998, 20 ringed seals and 16 grey seals were sampled in the Bothnian Bay, Baltic Sea (Fig. 1). Reference (control) tissue samples were obtained from ten ringed seals (Phoca hispida hispida) in Svalbard in the Arctic, and from 20 grey seals on Sable Island in Canada in 1998. All samplings were conducted during the moulting season, approximately at the same phase of the annual reproductive cycle of the seal populations. The seals were aged by counting annual layers of the cementum in thin sections of canine teeth (Laws, 1952). A condition index (Table 1) was estimated for each individual, based on the thickness of the blubber measured from the sternum relative to the body length (Read, 1990; Beck and Smith, 1995). The higher condition index observed in the control ringed seals (P=0.015) could be explained by the Svalbard seals being somewhat further in the moulting phase than their Baltic relatives. The Canadian grey seals were older on average (P= 0.028), but the ranges of ages were similar in the two areas (Table 1). No effects caused by age and condition of the seal populations on EROD and PROD activities were noted in either species. Liver samples (6–8 g) were collected and freezeclamped directly in the field in liquid nitrogen. The samples were stored at − 70°C until further treatment. For the preparation of microsomes, 1 g of tissue was

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homogenised in four volumes of ice cold 0.1 M K –Na– phosphate buffer (pH 7.4). The homogenate was centrifuged for 30 min at 10 000×g, after which the supernatant was centrifuged for 1 h at 100 000 × g. The microsomal fractions were resuspended and homogenised in the same buffer and stored at −70°C until analysis. For comparative purposes in enzyme kinetic and immunoblot analyses, microsomes were also prepared from male Wistar rats. One group of rats was treated with the potent CYP1A inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a single 100 mg/kg intraperitoneal injection) and another group received the CYP2B inducer phenobarbital (PB, 500 mg/l in drinking water for 1 week). Control rats were untreated. Liver microsomes were prepared as described above.

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2.2. Reagents Reagents and solutions used in the enzyme assays, gel electrophoresis and immunoblotting have been described previously by Raunio et al. (1988, 1990). aNaphthoflavone was purchased from Sigma (St. Louis, MO). Specific antibodies against human CYP1A1 and CYP1A2 as well as human and mouse CYP1B1 were produced by immunising rabbits with pentapeptides corresponding to areas in the C-termini of these CYP proteins. The preparation, validation and specificity of these antibodies have been described in detail previously (Edwards et al., 1998). These antibodies were generously donated by Alan Boobis (Imperial College School of Medicine, London, UK). A monoclonal antibody against rat CYP1A1 (MAb 1-7-1) (Gelboin and

Fig. 1. Map of sampling areas for ringed and grey seals. The specific areas are marked (1) The Bothnian Bay, Baltic Sea, (2) Kongsfjorden, Svalbard and (3) Sable Island, Canada. Baltic Sea (BS), Gulf of Bothnia (GF), Gulf of Finland (GF). Table 1 Age and body condition index of ringed and grey seals sampled from the Baltic Sea, Svalbard and Canada Sample size n

Age mean 9 S.D. (range)

Condition index (%) mean 9 S.D. (range)

Ringed seal Baltic Sea Svalbard

20 10

10.69 6.4 (1–25) 8.1 9 4.9 (2–19)

2.6 90.8 (1.6–4.4) 3.490.6 (2.7–4.1)

Grey seal Baltic Sea Canada

16 20

13.19 8.0 (5–33) 18.69 7.1 (7–35)

2.190.5 (1.4–3.3) 1.7 90.6 (0.8–2.6)

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Friedman, 1985) was kindly provided by Harry Gelboin (National Cancer Institute, Bethesda, MD, USA).

2.3. Protein and enzyme assays Protein concentrations were analysed by the method of Bradford (1976). Bovine serum albumin was used as an external standard. The fluorometric end point assays of Burke et al. (1977) for 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) catalytic activities were used in this study. For the individual activity analyses of EROD and PROD, the substrate levels were 1 and 2 mM, respectively. Specific enzyme activities are expressed as pmol/mg microsomal protein/min.

2.4. Kinetic and inhibition studies Three to five individual liver samples per species and sampling location were used in EROD and PROD inhibition assays. The substrate affinity constants (Km) and the maximal velocities (Vmax) of the reactions were determined using standard Michaelis – Menten enzyme kinetic analysis. To determine the inhibition profiles of these enzyme activities, a-naphthoflavone (ANF), a specific chemical inhibitor of CYP1A enzymes, was used at concentrations ranging from 0.001 to 0.01 mM for the EROD and from 0.01 to 100 mM for the PROD assays. Inhibition constants (Ki) were determined graphically from Dixon plots.

2.5. Immunoblotting Immunoblot analysis was done in microsomal samples pooled from four individual grey or ringed seals as described previously (Mattson et al., 1998). Briefly, the microsomes were separated in a 9 – 12% polyacrylamide gel by electrophoresis, and transferred to nitrocellulose filters. The filters were blocked in Tris-buffered saline (TBS) containing 5% fat-free milk for 3 h, after which they were incubated overnight at +4°C with the primary antibodies. After extensive washing, the filters were incubated for 1 h with the secondary antibodies. All antibodies were suspended in fresh TBS-1% milk solution. For the monoclonal antibody (MAb 1-7-1), a biotinylated anti-mouse antibody conjugated with horseradish peroxidase was used as the secondary antibody. Anti-rabbit antibody, conjugated with horseradish peroxidase, was used as the secondary antibody for the anti-human peptides (CYP1A1, 1A2 and 1B1) and for the polyclonal anti-mouse CYP1B1 antibody. As positive controls, 3 mg of liver microsomes of TCDD-treated rats (induced CYP1A1), 10 mg of liver microsomes of PB-treated rats (induced CYP2B) and 1 mg of recombinant human CYP1B1 (74 pmol/mg) expressed in lymphocyte microsomes (Gentest, Woburn,

MA, USA) were used. The protein bands were detected by enhanced chemiluminescence (ECL+ ) kit and visualised by autoradiography films (both from Amersham Pharmacia). The intensities of the bands were quantitated with ImageMaster VDS gel analysis system and software (Amersham Pharmacia).

2.6. Statistical analysis Although males showed higher EROD (P=0.033) and PROD (P= 0.000) activities than females in the Canadian sample, the sex difference did not influence the geographical difference observed for these two parameters. For further statistical comparisons, the data were divided into four groups according to species and geographical location (Table 1). The parameters examined were tested for normal distribution, and LOG or SQR transformed when needed. For the statistical analyses, ANOVA, regression and Pearson%s correlation analyses were used.

3. Results

3.1. Geographical and species 6ariation in enzyme acti6ities EROD and PROD enzyme activities were clearly higher in Baltic seals than in control groups for both species (Fig. 2). The difference in EROD activity was 5-fold between the two grey seal populations (P= 0.000), while a 3-fold increase was observed in the exposed ringed seals (P= 0.000, Fig. 2, upper panel). PROD activity differed with a roughly 2-fold increase in the elevated groups (ringed seal P= 0.003, grey seal P= 0.000, Fig. 2, lower panel). EROD and PROD activities showed a strong correlation in both seal species (0.92B rB0.96, P=0.000 for both species). A negative correlation of both enzyme activities with age was found for grey seals ( −0.45B rB − 0.42, P=0.01 for both enzymes). In ringed seals, the condition index showed a weak negative correlation with the enzyme activities, only PROD being significant (r= −0.48, P= 0.01). A species difference in enzyme activity was observed between the reference groups, where the grey seals showed significantly lower EROD (P= 0.016) and PROD (P=0.004) activities compared to the ringed seals in Svalbard. For the Baltic group, a corresponding difference was observed only in PROD activities (P= 0.040).

3.2. CYP1A enzyme kinetics and inhibition The main kinetic parameters of EROD and PROD are summarised in Table 2. The Km values of EROD were of the same magnitude in all four seal groups, the

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were markedly higher in the Baltic seals than in the respective reference populations. In the Baltic ringed seals, the EROD Vmax values exceeded the Vmax of a phenobarbital treated rat and approached the very high level of TCDD treated rat liver microsomes. The inhibition pattern of EROD with ANF was similar in both species from all sampling areas, with inhibition profiles resembling that of the TCDD-induced rat (Table 2). Due to the much lower activity of PROD, only approximate IC50 values could be determined, and the ANF inhibition profile of PROD appeared to resemble that of EROD. ANF also efficiently inhibited PROD activity in TCDD-induced rats, with characteristics closely resembling those in the seals.

3.3. Immunoblot analysis Using anti-peptide antibodies against human CYP1A1 and CYP1A2 proteins, no specific CYP1A1/ 2 like protein bands were observed in any of the seal microsome samples (data not shown). In contrast, monoclonal antibody MAb 1-7-1, raised against rat CYP1A1/2, revealed a clear band in the contaminant exposed groups of both species (Fig. 3, upper panel). This band comigrated with the 56-kDa CYP1A1 protein in TCDD-induced rat liver microsomes. Low amounts of the 56-kDa protein were observed in the Svalbard ringed seals, but none were observed in the Canadian grey seal samples. A relative quantitation of the protein bands revealed an about 3-fold increase of this CYP1A-like protein in highly exposed ringed seals compared to the reference population. The CYP1A like protein levels in the Baltic grey seals did not differ between the sexes or from the Baltic ringed seals.

Fig. 2. EROD and PROD activities in grey and ringed seals from the Baltic Sea, Svalbard and Sable Island are presented as individual points () and means 9 SD (horizontal bar 9vertical bars). n is the number of individuals studied. Statistically significant differences between the two areas within each species are indicated with asterisks (*** P B 0.001, ** P B0.01).

Baltic Sea ringed seals exhibiting a somewhat higher value (0.47 mM) than the other groups (0.12–0.18 mM). As expected, the calculated EROD Vmax values Table 2 Enzyme kinetics and inhibition parametersa Sample

EROD

PROD

Km (mM substrate)

Vmax (pmol/mg protein/min)

Ringed seal Baltic Sea Svalbard

0.47 0.18

3790 253

0.21 0.05

100 100

0.06 0.02

1.18 n.m

94 n.m.

Grey seal Baltic Sea Canada

0.12 0.16

1191 95

0.28 0.04

100 100

0.32 0.34

0.78 0.08

92 75

Wistar rat TCDD PB Control

1.47 0.57 0.69

7440 1920 28

0.10 12 50

100 85 60

0.05 5.80 64

0.70 50 –

80 80 20

a

IC50 (mM ANF)

maximal inhibition (%)

Ki (mM ANF)

IC50 (mM ANF)

Maximal inhibition (%)

IC50 (concentration of inhibitor needed for a 50% reduction in activity); Ki (inhibition constant); Km (affinity constant); Vmax (maximal velocity); n.m., not measurable due to very low basic activity levels; TCDD rat, Wistar rat treated with TCDD; PB rat, Wistar rat treated with phenobarbital.

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Fig. 3. Immunoblots of grey and ringed seal hepatic microsomes using monoclonal antibody MAb 1-7-1, raised against rat CYP1A1/2 (upper panel) and anti-peptide antibody against human CYP1B1 (lower panel). The following samples were loaded on the lanes. Upper panel: 1, Baltic female ringed seal; 2, Baltic male ringed seal; 3, control female ringed seal; 4, control male ringed seal; 5, TCDD rat (CYP1A = 56 kDa); 6, blank; 7, Baltic female grey seal; 8, Baltic male grey seal; 9, control female grey seal; 10, control male grey seal. Lower panel: 1, recombinant human CYP1B1 (50 kDa); 2, blank; 3, Baltic female ringed seal; 4, Baltic male ringed seal; 5, control female ringed seal; 6, control male ringed seal; 7, Baltic female grey seal; 8, Baltic male grey seal; 9, control female grey seal; 10, control male grey seal.

In immunoblots with the anti-peptide antibody against human CYP1B1, a protein comigrating with the human 50-kDa CYP1B1 protein was revealed in both seal species (Fig. 3, lower panel). Quantitation of the protein bands showed no clear differences between the seal groups examined neither between area, sexes nor species.

4. Discussion

4.1. Geographical and species 6ariation This study extends our previous observations on the induction of CYP1 family members in seals in highly contaminated waters. As in the previous study on ringed seal CYP1 induction (Mattson et al., 1998), the induced seal populations in the Baltic Sea both showed EROD and PROD levels approaching those of a TCDD-induced rat. Thus, the degree of induction in seals in polluted waters is quite remarkable. Both EROD and PROD activity levels in the Svalbard ringed seal population are in agreement with a previous report

on the same population (Wolkers et al., 1998a). In addition, elevated EROD activities have been associated with individual contaminant burdens in a number of marine mammal species, suggesting that CYP1A induction might serve as an exposure biomarker for environmental contamination (Watanabe et al., 1989; Fossi et al., 1992; Goksøyr et al., 1992; White et al., 1994; Fossi et al., 1997; Troisi and Mason, 1997; Marsili et al., 1998). Consistent with earlier studies on seals (Addison and Brodie, 1984; Wolkers et al., 1999; Goksøyr et al., 1992; Wolkers et al., 1998a), EROD activities in most seal groups studied here did not differ significantly between the sexes. PROD activities have not been reported for grey seal liver microsomes before. The extent of EROD induction was somewhat more pronounced in grey seals than in ringed seals. This can be explained by the lower constitutive levels of EROD activity in control grey seals, resulting in a higher degree of induction than in the ringed seals with a higher constitutive EROD activity. The higher EROD induction seen in grey seals, reaching similar activity levels as in ringed seals although the contaminant burden is lower in grey seals, indicate that grey seals are more sensitive to respond to contaminant burdens. Contaminant analyses of individual seals are needed to speculate further on this issue. The lack of a corresponding species difference in PROD induction pattern could be explained by the very low enzyme levels, being close to the detection limit in all seal populations studied.

4.2. Enzyme kinetics and inhibition The close correlation with EROD and PROD activities and their kinetic as well as inhibition characteristics indicate that PROD is mainly catalysed by CYP1A enzymes in both ringed and grey seals, contrary to rats in which PROD activity is mainly catalysed by CYP2B (Burke et al., 1985). These results confirm our previous findings and are consistent with previous results from other wildlife species (White et al., 1994; Van der Oost et al., 1996; Letcher et al., 1998; Mattson et al., 1998; Wolkers et al., 1998a). The induction and inhibition patterns of both EROD and PROD in the PB-induced rat, in which the induction is known to go through CYP2B forms, differ clearly from the patterns in the seal. The distinct ANF inhibition profiles of PROD activity in PB- and TCDD-induced rats indicate that TCDD-induced PROD activity is mainly mediated by CYP1A also in the rat. Three lines of evidence suggest that CYP1 enzymes catalyse both the constitutive and induced EROD activities in ringed and grey seals: (1) The EROD Km values were practically the same in non-exposed and exposed seals. (2) Maximal inhibition of EROD activities were obtained with ANF, a specific CYP1A inhibitor, in all

M. Nyman et al. / En6ironmental Toxicology and Pharmacology 8 (2000) 217–225

seal groups. (3) The Ki values for ANF were in the same order of magnitude in the less exposed and higher exposed seals. This is in clear contrast with the rat, in which ANF inhibited EROD with a very high affinity (Ki =0.05 mM) in liver microsomes from the TCDD treated rat, and with a very low affinity in the control rat (Ki = 64 mM). This implies that several CYP forms participate in the constitutive EROD activity of the rat. Similar inhibition patterns of CYP1A-like activity using ANF have been observed also previously in marine mammals (White et al., 1994; Wolkers et al., 1998a, 1999).

4.3. Expression of CYP1 proteins in seals This study confirms that a protein closely resembling CYP1A is present in the livers of ringed seals and grey seals, in analogy with previous findings (Goksøyr, 1995; Mattson et al., 1998; Wolkers et al., 1998a). Relative quantitation of the CYP1A-like protein in ringed seal immunoblots showed a similar 3-fold induction pattern as EROD activities. We attempted to specify whether this CYP1A-like protein is CYP1A1 or CYP1A2, using specific anti-peptide antibodies raised against human CYP1A1 and CYP1A2 proteins in immunoblot analysis. Unfortunately, both of the antibodies failed to reveal any proteins in seal liver microsomes, probably due to the highly specific nature of these antibodies. Wolkers et al. (1998a) found a strong cross reactivity with human probes for CYP1A1 in ringed seals using mRNA, and one band in immunoblot analysis with a monoclonal anti-rat CYP1A antibody in both ringed and harp seals (Wolkers et al., 1998a, 1999). This is contrary to another study on harp and hooded seals where two bands were detected using anti-cod CYP1A1 antibodies (Goksøyr et al., 1992), indicating that these seal species may have two CYP1A forms. Thus, this issue remains unsettled, and the exact answer will probably come only from characterisation of the respective genes in seals. A protein was also revealed in seal liver microsomes with an antibody against CYP1B1. The intensity of this band was about the same in all seal groups studied, suggesting that it is not inducible by the same pollutants that strongly elevate CYP1A levels. This is the first indication that a CYP1B1 protein could be present in seal liver, but further studies are needed to verify this finding.

Acknowledgements We would like to thank Merja Luukonen, Marcus Wikman and Kalle Ja¨rvinen for their assistance in the sample collection and the laboratory work. We are grateful for Christian Lydersen and the staff at the

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Norwegian Polar Institute assisting in the sample collection in Ny A, lesund, Svalbard. Many thanks to Richard Addison, Wayne Stobo and Chris HarveyClark for arranging and assisting in the sample collection on Sable Island. The study was supported by the Research Council for Environmental and Natural Resources of the Finnish Academy of Science. References Addison, R.F., Brodie, P.F., 1984. Characterisation of ethoxyresorufin-O-deethylase in grey seal Halichoerus grypus. Comp. Biohem. Physiol. 79C, 261 – 263. Addison, R.F., Brodie, P.F., Edwards, A., Sadler, M.C., 1986. Mixed function oxidase activity in the harbour seal (Phoca 6itulina) from Sable Is. N.S. Comp. Biochem. Physiol. 85C, 121 – 124. Addison, R., Ikonomou, M.G., Stobo, W., 1999. Polychlorinated dibenzo-p-dioxins and furans and non-ortho- and mono-orthochlorine substituted plychlorinated biphenyls in grey seals (Halichoerus grypus) from Sable Island, Nova Scotia, in 1995. Mar. Environ. Res. 47, 225 – 240. Aguilar, A., Borrell, A., 1994. Abnormally high polychlorinated biphenyl levels in striped dolphins (Stenella coeruleoalba) affected by the 1990 – 1992 Mediterranean epizootic. Sci. Total Environ. 154, 237 – 247. Ba¨cklin, B.-M., 1996. Studies on reproduction in female mink (Mustela 6ison) exposed to polychlorinated biphenyls. Swedish University of Agricultural Sciences, Stockholm, Sweden Thesis. Baron, J.M., ZwadloKlarwasser, G., Jugert, F., Hamann, W., Rubben, A., Mukhtar, H., Merk, H.F., 1998. Cytochrome P450 1B1: A major P450 isoenzyme in human blood monocytes and macrophage subsets. Biochem. Pharmacol. 56, 1105 – 1110. Beck, G.G., Smith, T.G., 1995. Distribution of blubber in the Northwest Atlantic harp seal, Phoca groenlandica. Can. J. Zool. 73, 1991 – 1998. Bergek, S., Bergqvist, P.-A., Hjelt, M., Olsson, M., Rappe, C., Roos, A., Zook, D., 1992. Concentrations of PCDDs and PCDFs in seals from Swedish waters. Ambio 21, 553 – 556. Bergman, A., Olsson, M., 1986. Pathology of Baltic grey seal and ringed seal females with special reference to adrenocortical hyperplasia: is environmental pollution the cause of a widely distributed disease syndrome? Finnish Game Res. 44, 47 – 62. Bergman, A., Olsson, M., Reiland, S., 1992. Skull-bone lesions in the Baltic grey seal (Halichoerus grypus). Ambio 21, 517 – 519. Bergman, A., 1999. Health condition of the Baltic grey seal (Halichoerus grypus) during two decades — gynaecological health improvement but increased prevalence of colonic ulcers. APMIS 107, 270 – 282. Boobis, A.R., Sesardic, D., Murray, B.P., Edwards, R.J., Singelton, A.M., Rich, K.J., Murray, S., De La Torres, R., Segura, J., Pelkonen, O., Pasanen, M., Kobayashi, S., Zhi-Guang, T., Davies, D.S., 1990. Species variation in the response of the cytochrome-P450-dependent monooxygenase system inducers and inhibitors. Xenobiotica 20, 1139 – 1161. Boon, J.P., Reijnders, P.J.H., Dols, J., Wensvoort, P., Hillebrand, M.T.J., 1987. The kinetics of individual polychlorinated biphenyl congeners in female harbour seals (Phoca 6itulina), with evidence for structure-related metabolism. Aquat. Toxicol. 10, 307–324. Boon, J.P., Oostingh, I., van der Meer, J., Hillebrand, M.T.J., 1994. A model for the bioaccumulation of chlorobiphenyl congeners in marine mammals. Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. 270, 237 – 251. 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. 71, 248 – 254.

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