Distribution of cytochrome P4501A (CYP1A) in the tissues of Baltic ringed and grey seals

Marine Environmental Research 51 (2001) 465±485 www.elsevier.com/locate/marenvrev

Distribution of cytochrome P4501A (CYP1A) in the tissues of Baltic ringed and grey seals O.M. Hyyti a, M. Nyman b,*, M.L. Willis c, H. Raunio a, O. Pelkonen a a

Department of Pharmacology and Toxicology, University of Oulu, Box 5000, FIN-90401 Finland b Finnish Game and Fisheries Research Institute, Box 6, FIN-00721 Helsinki, Finland c Environmental Conservation Division, Northwest Fisheries Science Center, NMFS/NOAA, 2725 Montlake Boulevard E, Seattle, WA 98112, USA Received 30 April 2000; received in revised form 14 August 2000; accepted 16 August 2000

Abstract Information about the expression of CYP1A in wildlife species is essential for understanding the impact of organochlorine exposure on the health status of an exposed population. Therefore, we aimed at characterising a putative CYP1A enzyme expression in both hepatic and extrahepatic tissues of ringed and grey seals from the Baltic Sea and from less polluted waters. The cellular localisation of CYP1A was identi®ed using a monoclonal antibody against scup P4501A1 (MAb 1-12-3). Immunohistochemical staining showed the highest level of CYP1A expression in liver hepatocytes, and the second highest level in the endothelial cells of capillaries and larger blood vessels in the liver and other organs. The most frequent and strongest staining was found in Baltic ringed seals. Although CYP1A-positive staining was observed in only a few tissues in the other seal populations, it was more intense in Baltic grey seals than in Canadian grey seals. The CYP1A enzyme activity, expressed as ethoxyresoru®n O-deethylation (EROD), followed a similar tissue distribution and geographical pattern as the immunohistochemistry with clearly elevated EROD activities in most tissues of both Baltic seal populations. Immunochemical characterisation by immunoblotting con®rmed the presence and elevation pattern of a putative CYP1A protein in ringed and grey seals, supporting our ®ndings using other methods. The evenly distributed elevation of CYP1A expression among most of the tissues examined indicates that Baltic seals are exposed to CYP1A inducing agents a€ecting the whole body. This may result in an increased or

* Corresponding author. Tel.: +358-205-751-274; fax: +358-205-751-201. E-mail address: madeleine.nyman@rktl.® (M. Nyman). 0141-1136/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00258-0

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decreased toxic potential of foreign substances, which may ultimately determine the biological e€ects of the contaminants. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Baltic Sea; Canada; CYP; Cytochrome P450; Grey seal; Ringed seal; Sable Island; Svalbard

1. Introduction Many species of marine mammals are vulnerable to exposure to environmental pollutants. Since they are predators at the top of the food chain, they are exposed to large amounts of contaminants resulting in an accumulation of toxic and lipophilic compounds in their large lipid reserves. Elevated contaminant levels in wild marine mammal populations have been associated with a high frequency of pathological changes and abnormalities (Aguilar and Borrell, 1994; BeÂland et al., 1993; Bergman and Olsson, 1986; DeLong et al., 1973; Martineau et al., 1994). The Baltic ringed seal (Phoca hispida baltica) and grey seal (Halichoerus grypus) populations have su€ered from a highly elevated contaminant burden since the 1970s (Jensen, Johnels, Olsson & Otterlind, 1969). Although the contaminant levels in the Baltic fauna have decreased since then, the concentrations of both polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT) are still clearly elevated. Mean levels of sum PCB and DDT levels in grey and ringed seal blubber is 30±70 mg/kg lipid weight (l.w.) and 8±40 mg/kg l.w., respectively (Koistinen, unpublished data). In these seal populations, a high frequency of pathological changes has been observed (Helle, Olsson & Jensen, 1976a, b; Bergman and Olsson; Mortensen, Bergman, Bignert, Hansen, HaÈrkoÈnen & Olsson, 1992; Olsson, Karlsson & Ahnland, 1994), although a decreasing trend has been detected more recently (Bergman, 1999). The cytochrome P450 (CYP) enzyme superfamily, consisting of hundreds of enzymes (http://drnelson.utmem.edu/nelsonhomepage.html), plays a central role in the ®rst phase of the metabolic transformation of foreign compounds. The biotransformation results in either a decrease in the toxicity of the compound followed by its excretion, or in the formation of more reactive metabolites, which leads to increased toxicity. Originally found in the mammalian liver, the CYP enzymes were later found in other animals, plants bacteria and fungi (Nelson et al., 1996). In mammals, CYP-associated activity is found in all tissues except skeletal muscle cells and erythrocytes (Guengerich, 1993a, b), but predominantly occurs in the liver in most species. In extrahepatic tissues, CYP enzymes involved in the metabolism of xenobiotics are mainly concentrated in those organs through which foreign compounds pass when entering the body, i.e. lung, skin and intestine (Raunio, Pasanen, Hakkola & MaÈenpaÈaÈ, 1995), the body's ``®rst pass'' protection. Of the xenobiotic metabolising CYP enzymes, those belonging to the CYP1A subfamily are of particular interest to environmental toxicologists because of their capability to metabolise certain environmental pollutants, especially lipophilic chlorinated organic compounds that accumulate in the food chain. The endogenous functions of CYP1A enzymes are not yet known. They have been implicated in

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oxidative stress response, cell cycle control, and apoptosis, but ®rm evidence is still lacking (Nebert, Roe, Dieter, Solis, Yang & Dalton, 2000). However, their xenobiotic metabolising function has been well conserved during evolution since similar detoxifying mechanisms are present in a broad range of animal species (Stegeman, 1998). Members of the CYP1A subfamily are induced by planar aromatic hydrocarbons, including some PCBs and dioxins. The induction of CYP1A enzymes is used as a bio-indicator of contaminant exposure in ®eld studies of marine mammals (Fossi et al., 1997; Marsili et al., 1998; Troisi & Mason, 1997; White, Hahn, Lockhart & Stegeman, 1994; Wolkers, Witkamp et al., 1998; Wolkers, Burkow, Monshouwer, Lyderson, Dahle & Witkamp, 1999). The induction of CYP1Amediated metabolism may also produce compounds with increased toxicity, leading to harmful biological e€ects. Knowledge of CYP1A and its induction patterns is, therefore, of importance when studying exposure to and e€ects of environmental contaminants in wild animals. Although the cellular distribution of CYP1A in various tissues has been extensively studied in human and experimental animals (Baron, Redick & Guenerich, 1981; Foster et al., 1986; Gonzales & Gelboin, 1994), little is known about organ speci®c CYP1A expression in marine mammals. CYP1A-associated catalytic activities have been found in the liver, kidneys, adrenal glands, intestines, lungs and spleen of a few species of pinnipeds and cetaceans (review in Boon et al., 1992, Goksùyr, 1995; Murk, Morse, Boon & Brouwer, 1994; Wolkers, Witkamp et al., 1998; Wolkers et al., 1999). In addition, putative CYP1A proteins have been demonstrated in some marine mammal species using CYP1A speci®c antibodies (review in Boon et al., 1992; Goksùyr, 1995; White et al., 1994). In our previous studies, a putative CYP1A enzyme was demonstrated in the liver of both ringed and grey seals (Mattson, Raunio, Pelkonen & Helle, 1998; Nyman, Raunio, Pelkonen & Helle, 2000). Furthermore, a clear hepatic induction of CYP1A, in terms of both enzyme activity and protein level, was observed in the Baltic seal populations when compared to seal populations from less polluted areas. The primary objective of this study was to localise and quantify a putative CYP1A expression in several tissues of ringed and grey seals as a means to study the distribution of the organochlorine exposure in the body of seals living in a heavily polluted environment. Further, we compared the applicability of three methods in detection of CYP1A expression in contaminated marine mammal populations namely, immunohistochemistry, enzyme activity assays and immunoblot analysis. Samples collected from waters around Svalbard and Sable Island, Canada, were used as reference material. In both reference populations, the mean sum PCB and DDT concentrations have been reported to be less than 3 mg/kg l.w. (Wolkers, Burkow, Lydersen, Dahle, Wittkamp & Monshouwer, 1998). The contaminant load of the animals used in this study showed mean sum PCB levels of 0.4 and 8 mg/kg l.w. in the reference ringed and grey seals, respectively (Koistinen, unpublished data). The corresponding levels for mean sum DDT was 0.4 and 2 mg/kg l.w., respectively. As there probably is no marine mammal population in the world free from contamination and the contaminant levels measured in the Svalbard and Canadian seals are well below threshold levels proposed for development of

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reproductive impairments in seals (Helle et al., 1976a, b), we consider the chosen reference seal populations appropriate as reference material for this study. 2. Materials and methods 2.1. Tissue sampling Our study was carried out in a total of 32 seals. Tissue samples were taken from the liver, lungs, kidneys, adrenal glands, heart, spleen, uterus, placenta, testes and epididymis of ringed and grey seals in the Bay of Bothnia, in the Baltic Sea, during 1997 and 1998 (eight animals per species). The uterus and epididymis samples were only used in the immunohistochemical study, and the placenta samples were only used to determine enzyme activity and protein content. All other samples were used in all the studies. The seals were primarily sampled to monitor the health status of the populations, as agreed internationally for conservation purposes. Reference samples from corresponding tissues were taken from areas of low contamination: eight ringed seals around Svalbard in the Arctic, and eight grey seals on Sable Island, Canada, in 1998. The seals were sampled approximately at the same phase of the annual reproduction cycle, during the moulting period, and aged by counting the annual layers of the cementum from thin transverse sections of a canine tooth (Laws, 1952). A condition index was determined for each individual by dividing the total body length by the thickness of the blubber layer, measured from the sternum. The condition index was modi®ed from methods used by Beck and Smith (1995), and Read (1990). No di€erence in age or condition was observed between the Baltic seals and the reference seals for either species. No correlation was found between age or condition and the CYP1A-associated parameters determined. 2.2. Reagents The following chemicals were purchased from Sigma (St. Louis, MO): Bovine serum albumin; normal goat serum; and 3,30 -diaminobenzidine tetrahydrochloride (DAB). A Super Sensitive Concentrated Detection Kit, including biotinylated goat anti mouse IgG and the peroxidase conjugated Streptavidin, was purchased from Bio Genex (San Ramon, CA). A monoclonal antibody (MAb) 1-12-3 against scup liver cytochrome P450E (CYP1A1) was generously provided by John J. Stegeman (Woods Hole Oceanographic Institution) and Harry V. Gelboin (National Cancer Institute, Bethesda, MD, USA; NCI). The preparation of MAb 1-12-3 is described in Park, Miller, Klotz, Klopper-Sams, Stegeman and Gelboin (1986). This antibody has shown cross-reactivity with several vertebrate species, speci®cally recognising CYP1A1 in mammals (Stegeman & Hahn, 1994). A monoclonal antibody against rat CYP1A1 (MAb 1-7-1) was generously provided by Dr. Gelboin (NCI). Puri®ed mouse non-speci®c myeloma protein UPC-10, IgG, was purchased from Organon Teknika (Costmesa, CA). Reagents and solutions used in the enzyme assays and

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immunoblot analysis have been described previously by Raunio et al. (1988) and Raunio, Liira, Elovaara, RiihimaÈki and Pelkonen (1990). 2.3. Sample preparation and analysis 2.3.1. Immunohistochemical analysis Tissue samples were collected in the ®eld and placed in cassettes in 10% neutral bu€ered formalin for ®xation overnight, after which they were transferred into 70% alcohol until further processing. The samples were processed and embedded in paran using standard methods (Husùy, Myers, Willis, Collier, Celander & Goksùyr, 1994). An indirect peroxidase labelling method was used for immunohistochemical staining (Myers et al., 1994). In short, paran sections, cut at 5 mm, were mounted on Biotech Probe On Plus charged slides, deparanated in xylene substitute and hydrated in 0.1% BSA/phosphate-bu€ered saline in a graded ethanol series. During this process, sections were incubated in 3% hydrogen peroxide in deionised water for 5 min to block endogenous peroxidase activity (Van Noorden & Polak, 1983). After hydration, sections were placed in a Shandon immunostaining centre and incubated in normal goat serum for 20 min to block non-speci®c binding of the secondary antibody (biotinylated goat anti mouse IgG; Van Noorden & Polak, 1983). The sections were incubated for 2 h at room temperature in 1.5 mg protein/ml dilution of MAb 1-12-3, followed by incubations with the secondary antibody (20 min) and with streptavidin (20 min; Myers et al., 1994). Matching serial sections were incubated simultaneously with puri®ed mouse non-speci®c myeloma protein UPC-10 (1.5 mg protein/ml), to provide negative controls for the method. To develop colour, the sections were incubated in DAB chromogen with NiCl, after which they were washed, dehydrated and cover slipped with xylene substitute mountant. Parallel serial sections of each specimen were stained with Harris' hematoxylin and eosin according to Luna's method (1968). Relative staining occurrence and intensity were determined subjectively by light microscopy. Staining occurrence was scored as 0=no staining (or equal to UPC staining), 1=very rare, 2=rare, 3=multifocal and 4=di€use. Staining intensity was scored as 0=none (or equal to UPC staining), 1=mild, 2=moderate, 3=strong and 4=very strong. A scaled product of the staining occurrence and intensity (staining index) was used to quantify the expression in each cell type (Woodin, Smolowitz & Stegeman, 1997). 2.3.2. Enzyme and immunoblot assays Tissue samples were collected, immediately freeze-clamped in liquid nitrogen and stored at 70 C until further treatment. Microsomes were extracted from 1±4 g of tissue as described in Mattson et al. (1998). Protein content was determined by spectrophotometry, as described in Bradford (1976). The ¯uorometrical end point assay of Burke, Prough and Mayer (1977) was used for measuring ethoxyresoru®n O-deethylase activity (EROD). Enzyme activities are expressed as pmol/mg protein/ minute. Immunoblot assays were performed on microsome samples (1 mg/ml) from all the seals using the same methods as in Mattson et al. (1998). The CYP1A protein

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content in the extrahepatic tissues was too low for detection using this method. Two monoclonal antibodies (a) against scup P450E (MAb 1-12-3) and (b) against rat CYP1A1 (MAb 1-7-1) were used to detect protein binding to cytochrome P4501A in the two seal species. Liver microsomes (3 mg) from Wistar rats, which had been treated with a single intraperitoneal injection of 100 mg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin, served as a positive control for the anti-rat antibody. Samples from b-naphtho¯avone induced rainbow trout were used as a positive control for the anti-scup antibody. The protein bands were detected using an enhanced chemiluminescence kit and visualised on autoradiography ®lms. 2.4. Statistics The data were divided into four groups for statistical analyses: Baltic ringed seals; Baltic grey seals; reference ringed seals (Svalbard); and reference grey seals (Canada). Di€erences between animal groups were analysed using the Kruskall-Wallis analysis of variance, with P<0.05 considered signi®cant and n=4 as the minimum sample size. The sample sizes of the reproductive organs in each seal population were too small for a geographical comparison. The Spearman rank correlation test was used to compare CYP1A content and activity between the various tissues and between the di€erent methods used. The data were grouped according to species for the correlation analyses. Results are presented as meansS.D.

3. Results 3.1. Immunohistochemical analysis A comparison of the CYP1A-associated expression between tissues revealed a clearly stronger CYP1A expression in the liver compared to any other tissue. The CYP1A expression was observed primarily in the hepatocytes in the liver, and in the endothelial cells of capillaries and larger blood vessels in the liver and the other organs. The staining indices of all tissues examined are presented in Table 1. In general, CYP1A-speci®c staining was found most frequently in the Baltic ringed seals, positive staining being found in only a few tissue types in the other seal populations. In the liver, 90% of seals of both species from the Baltic Sea showed strong CYP1A-associated expression distributed di€usely in the hepatocyte cytoplasm (Fig. 1a and c), whereas expression in hepatocytes in seals from the reference areas was only mild to moderate with a scattered distribution (Fig. 1b and d). In general, the staining was weaker in periportal areas. Moderate to strong expression was seen in the endothelial cells of the hepatic vasculature in 40% of the Baltic ringed seal samples. In contrast, the other seal populations showed only very mild or no expression in the hepatic vasculature. No staining was seen in the sinusoidal lining cells in any of the specimens examined.

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Table 1 Staining index (meanS.D.) of CYP1A-associated expression in various tissues of ringed and grey seals from di€erent geographical areas Ringed seals

Grey seals

Baltic Sea

Svalbard

Baltic Sea

Canada

Liver Hepatocytes Vascular endothelium

12.84.7** 3.15.5

4.02.1 <1

12.43.5* <1

6.44.9 <1

Lung Vascular endothelium Mucous glands

14.33.6*** 8.45.9***

<1 <1

4.60.1* <1

1.01.4 <1

Kidney Vascular endothelium

7.83.4***

0

1.81.8*

Adrenal Vascular endothelium

2.02.8***

<1

<1

<1

Heart Vascular endothelium

5.85.7***

<1

<1

<1

Spleen Vascular endothelium

7.12.8***

0

0

0

Uterus Vascular endothelium

6.54.8

<1

<1

<1

Testis Vascular endothelium

4.85.5

0

0

Epididymis Vascular endothelium

12.55.0

0

2.71.5

<1

1.00.0 <1

*P<0.05. **P<0.01. ***P<0.001.

Strong CYP1A-associated expression was observed in the lungs of 90% of the Baltic ringed seals, distributed di€usely in the endothelia of the capillaries within the alveolar septa as well as in the larger pulmonary blood vessels (Fig. 2a). A more moderate expression in the endothelium of the pulmonary vasculature was observed in all the Baltic grey seals (Fig. 2c), but only 50% of the reference seals showed any staining in the lungs, and this was very rare and mild (Fig. 2b and d). A moderate to very strong positive CYP1A-associated staining was seen in the mucous glands surrounding the bronchi and smaller bronchioles, in the Baltic ringed seal population. The staining of the mucous glands was very rare and always mild in the other seal groups (Table 1). Occasionally, there was scattered expression in the basal region of the bronchial epithelium, with Baltic ringed seals showing the highest staining index. The strongest CYP1A-associated expression in the kidney was found in the endothelium of the capillaries and larger blood vessels within the interstitium of the

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Fig. 1. Immunohistochemical CYP1A-associated staining in hepatic tissue of ringed and grey seals from the Baltic Sea and reference areas. The CYP1A-associated staining in the hepatocyte cytoplasm is indicated with an arrow. Magni®cation 127. (a) Very strong staining in cytoplasm of hepatocytes in Baltic ringed seals. (b) CYP1A staining is absent in Svalbard ringed seals. Small dark spots represent lipofuscin and hemosiderin pigments normally seen in the liver of older mammals. (c) Strong CYP1A-associated staining in Baltic grey seal hepatocyte cytoplasm. (d) No CYP1A staining is seen in hepatic tissue from grey seals on Sable Island.

cortical region, and in the upper region of the medulla (Fig. 3). Tubular epithelium generally exhibited mild and scattered expression, although occasionally groups of tubules with very strong staining were observed. For both types of tissue in the kidney, the staining indices were highest in the Baltic ringed seals, followed by lower values in Baltic grey seals (Table 1). In the reference seals, no expression was observed in the endothelium of the vasculature in the kidneys. In 90% of the Baltic ringed seals, sections of the adrenal gland showed mild to moderate, but multifocal, staining in the endothelia of the blood vessels in the cortex (Fig. 4a). The strongest expression was found in the blood vessels in the zona glomerulosa, with less expression in the zona fasciculata and reticulata, and very rare expression in the medulla. Occasionally, a very mild expression in the endothelium of the adrenal gland vasculature was seen in the other seal populations (Fig. 4b±d). A fairly strong CYP1A-speci®c expression was found in the endothelia of capillaries and larger blood vessels within the myocardium of the heart in 25% of the Baltic ringed seals, but only rarely and with a very mild staining in the other seal populations (Table 1). Some scattered staining was occasionally seen in the endocardial lining.

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Fig. 2. Immunohistochemical localisation of CYP1A-like proteins in the lungs of ringed and grey seals. Magni®cation 127. (a) Very strong staining in endothelium of arteries (arrow) and alveolar capillaries of Baltic ringed seals. (c) Similar staining pattern but with moderate intensity in Baltic grey seals. (b) and (d) No CYP1A-associated staining in lung tissue from seals in the reference areas.

In the spleens of all the Baltic ringed seals, there was a moderate to strong expression in the endothelium of the smaller blood vessels in the red pulp and moderate, but rare expression in the lymphoid follicles (white pulp; Table 1). No CYP1A-associated staining was found in the spleens of seals in the other groups. Mostly mild and rare CYP1A-associated staining was found in the vasculature of the reproductive organs. In the uterus, expression was con®ned to the capillaries and smaller blood vessels of the myometrium and endometrium. The Baltic ringed seals showed mild and rare staining in both the myometrium and the endometrium, apart from one individual which showed moderate staining in the myometrium (Table 1). Occasionally, very mild and rare expression was observed in the other females. Sections of ovaries showed mild and rare CYP1A-speci®c expression in capillaries and blood vessels of the stroma. In the testis, rare and mild to moderate CYP1A-associated expression was present in the endothelium of the interstitial blood vessels around the seminiferous tubules (Table 1). CYP1A expression in the vasculature within the connective tissue of the epididymis was moderate to strong in Baltic ringed seals, but mild in Baltic grey seals (Table 1). When quantifying CYP1A-associated immunohistochemical expression, an overall trend was con®rmed: Baltic ringed seals showed stronger staining in all tissues than the other seal populations (Table 1). In ringed seals, a signi®cantly elevated staining index was revealed for all tissues in the Baltic population, except the hepatic

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Fig. 3. CYP1A-associated staining in kidney tissue of ringed and grey seals. Magni®cation 127. (a) Strong CYP1A staining in the vascular endothelium of glomerular capillaries (arrow) and of the proximal tubule in Baltic ringed seals. (b) CYP1A-associated staining is absent in kidney tissue from Svalbard ringed seals. (c) Moderate staining of CYP1A in the vascular endothelium of the renal cortex in Baltic grey seals. (d) No CYP1A-associated staining in kidney tissue from grey seals on Sable Island.

vasculature. In grey seals, a corresponding geographical di€erence was observed in the hepatocytes and lung and kidney vasculature. A di€erence between the species was observed between the two Baltic seal populations. The ringed seals showed stronger staining than grey seals in all tissues except for the liver (P<0.003 for all tissues). No species di€erence was seen between the reference groups. A strong positive correlation of the staining indices between most tissue types was observed for the ringed seal (Table 2). In the grey seals, a corresponding relationship was observed only between the mucous glands of the lungs and the adrenal vasculature (r=0.64, P<0.01). The species di€erence is explained by the fact that only a few tissues showed positive CYP1A-associated staining in grey seals (Table 1). 3.2. Enzyme activity Hepatic EROD activities showed three and eight times higher activities in the Baltic ringed and grey seal populations, respectively, when compared to their reference groups (Fig. 5). A similar geographical pattern was observed for all extrahepatic tissues, except the reproductive organs (P<0.03, Fig. 6). Extrahepatic EROD activity followed the same pattern in all four seal populations: EROD activity was clearly highest in the lungs, followed by the adrenal glands, kidneys,

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Fig. 4. CYP1A-associated staining in adrenal tissues of ringed and grey seals. Magni®cation 127. (A) Very strong CYP1A-associated staining in the vascular endothelium (arrow) in the adrenal cortex of Baltic ringed seals. CYP1A-associated staining is absent from adrenal tissue in all other seal populations.

heart, spleen and the reproductive organs. When the tissue distribution of EROD activity was examined, a strong positive correlation was found between most tissues in both seal species (Table 3). A species di€erence was observed for the hepatic EROD levels, where ringed seals showed clearly higher activities in both areas (P<0.05). In the Baltic Sea, a similar trend was observed for the enzyme activity in the adrenal glands, kidney and heart, although only signi®cantly in the kidney (P=0.007), while the EROD activity in the lungs was clearly higher in the grey seals (P=0.01). In the reference areas, ringed seals showed higher activities only in the adrenal glands (P=0.03). 3.3. Immunoblot analysis Immunoblot analysis was performed to: (1) verify the speci®city of the antibodies used; and (2) ascertain that the observed elevation also occurs at the speci®c apoprotein level. Both monoclonal antibodies displayed a similar highly speci®c binding to CYP1A protein in the seal samples examined (Fig. 7). MAb 1-12-3 di€ered from MAb 1-7-1 in that it cross-reacted with rainbow trout CYP1A as well as with rat and seal CYP1A. This result veri®es the speci®city of the immunohistochemical staining. A clear elevation of CYP1A apoprotein levels was also observed in both Baltic Sea seal species.

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Table 2 Correlation (r presented as r100) between various tissues measured as staining index in ringed sealsa Lung

Liver Hepatocytes Vascular endothelium Lung Vascular endothelium (1) Mucous glands (2)

Kidney

Adrenal

Heart

Uterus

Spleen

1

2

76.8 ±

70.1 ±

66.2 62.4

80.9 ±

67.4 ±

± ±

± ±

63.2 64.7

75.8

91.3 78.7

84.6 76.3

82.6 79.1

± 91.1

87.3 ±

87.0 83.7

88.1

89.6

±

86.8

92.5

86.3

±

±

87.3

±

91.9

95.3

Kidney Vascular Endothelium Adrenal Vascular Endothelium Heart Vascular Endothelium Uterus Vascular Endothelium a

Testis

89.9

Signi®cance P<0.01.

3.4. Comparison of methods used A comparison of the CYP1A content, quanti®ed by immunohistochemical staining, and the CYP1A-mediated EROD activity, revealed a strong positive correlation in most ringed seal tissues (Table 4). In grey seals, a corresponding strong positive correlation was found in the liver (r=0.71, P<0.01), although the magnitude of the CYP1A elevation di€ers between the two methods. 4. Discussion 4.1. Cellular localisation and tissue distribution The cellular distribution of CYP1A in various seal tissues showed a pattern common to both seal species in all areas. The CYP1A expression was several times stronger in the liver compared to the other tissues. This is explained by the central role of the liver in CYP1A-mediated xenobiotic metabolism. In the liver, the expression was strongest in hepatocytes, followed by the vascular endothelium. The most common site for CYP1A expression in extrahepatic tissues was in the endothelial cells of capillaries and other blood vessels in seals from both polluted and less

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Fig. 5. Hepatic ethoxyresoru®n O-deethylase activity (pmol/mg protein/min) in ringed and grey seals from the Baltic Sea, Svalbard and Sable Island, Canada. Results are presented as meansS.D. Signi®cance of geographical di€erence is marked with *P<0.05, **P<0.01, ***P<0.001.

polluted waters. The perivenous localisation of the CYP1A expression is in agreement with previous studies on the zonation of cytochrome P450 enzymes in rat liver (Oinonen & Lindros, 1998). In the Arctic and Canadian seal populations, hepatic EROD levels were comparable to previous studies on the same species (Mattson et al., 1998, Nyman et al., 2000, Wolkers, Witkamp et al., 1998) and to activities reported for untreated rat and human liver microsomes (Raza, John, Lakhani, Ahmed & Montague, 1998). The extrahepatic EROD activity pattern in these seal populations is in the same range as in non-exposed rats showing 1.9 and 4.8 pmol/mg protein/min in lung and kidney, respectively (Jewell & O'Brien, 1999). Goksùyr (1995) reported a 500-fold higher CYP1A activity in liver compared to the extrahepatic tissues in minke whales (Balaenoptera acutorostrata). The hepatic CYP1A activity was 14±900 times higher in than in lung and testis, respectively, in all seal populations. 4.2. Activity pattern The induction of both hepatic and extrahepatic xenobiotic metabolising CYP enzymes determines the e€ects of xenobiotic exposure. The production of metabolites and the induction of CYP activity itself may both result in increased oxidative stress and other toxic e€ects in the target organ (review in Stegeman & Hahn, 1994). PHAHs induce oxidative stress that causes increased production of reactive oxygen species, lipid peroxidation and DNA damage both in vitro and in vivo in laboratory animals. As the liver is the main xenobiotically metabolising organ, it is also the main target for possible toxic e€ects. However, induction of CYP1A expression may also be of importance in extrahepatic tissues that are sensitive to oxidative stress. The geographical comparison of CYP1A protein levels and activity revealed that the CYP1A elevation pattern was consistent in the tissues studied. A 2- to 9-fold

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Fig. 6. Ethoxyresoru®n O-Deethylase activity (pmol/mg prot./min) in extrahepatic tissues of (a) ringed and (b) grey seals from di€erent geographical areas. Results are presented as meansS.D.

elevation of EROD activity in both the liver and extrahepatic tissues of both Baltic seal species suggests that the whole body is a€ected by CYP1A-inducing compounds. In comparison to the liver, the localisation of elevated CYP1A expression in extrahepatic organs was generally limited to the endothelial cells of the vasculature, not to organ speci®c cell types. This indicates a distribution of CYP1A inducing agents via the blood. A corresponding induction pattern has been observed in rats, mice, rabbits and ®sh after treatment with typical CYP1A1 inducers (Anderson et al., 1987; Dees, Masters, Muller-Eberhard & Johnson, 1982; Stegeman, Miller & Hinton, 1989; Thirman et al., 1994). An induced CYP1A metabolism and oxidative stress in the vascular endothelia may have a considerable impact on endogenous functions in various parts of the body, and may ultimately lead to the initiation of

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Table 3 Correlation (r presented as r100) between various tissues measured as ethoxyresoru®n O-deethylase activity in A) ringed seals and B) grey sealsa

A) Ringed seals Liver Lung Kidney Adrenal Heart B) Grey seals Liver Lung Kidney Adrenal Heart a

Lung

Kidney

Adrenal

Heart

76.0

84.0 69.7

85.7 82.4 83.1

86.5 75.4 86.4 81.5

93.5

73.2 71.3

92.1 91.2 67.3

82.6 83.2 81.3

Spleen

77.6 73.0 82.2 82.6 66.5 85.8 77.8

Signi®cance P<0.01.

Fig. 7. Immunoblots of ringed and grey seal hepatic microsomes using monoclonal antibodies (a) MAb 17-1 against rat CYP1A1, and (b) MAb 1-12-3 against scup liver cytochrome P450E (CYP1A1). Hepatic microsomes (40 mg) were used for the seal samples: Canada grey seals (lanes 1 and 2); Svalbard ringed seal (lane 3); Baltic grey seals (lanes 4 and 5); and Baltic ringed seal (lanes 6 and 7). Microsomes from bnaphtho¯avone-induced rainbow trout (1.5 mg, lane 8) and from 2,3,7,8-tetrachlorodibenzo-p-dioxininduced rat (3 mg, lane 9) were used as positive controls.

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Table 4 Correlation (r presented as r100) between CYP1A-associated expression, measured using immunohistochemistry (staining index) and enzyme activity (ethoxyresoru®n O-deethylase) in ringed sealsa Tissue

Correlation

Liver Hepatocytes Vascular endothelium

72.8

Lung Vascular endothelium Mucous glands

67.5 71.0

Kidney Vascular endothelium

88.5

Adrenal Vascular endothelium

69.5

Heart Vascular endothelium

91.3

Spleen Vascular endothelium

73.1

a

Signi®cance P<0.01.

carcinogenesis or vascular diseases (Hennig, Slim, Toborek & Robertson, 1999; Stegeman et al., 1992). 4.3. Species di€erence The surprisingly high contaminant loads reported in marine mammals have been explained by a lower capacity to metabolise some groups of PHAHs in these animals as compared to polar bears or terrestrial mammals (Boon et al., 1992; Tanabe, Watanabe, Kan & Tatsukawa, 1988). A similar contaminant pro®le and CYP1A expression would be expected in both Baltic seal species as they feed at approximately the same trophic level of the Baltic food web (SoÈderberg, 1974; Tormosov & Rezvov, 1978). The PHAH composition is very similar in gill breathing animals on lower trophic levels as it is controlled rather by the contaminant concentrations in the ambient water than in their diet, while the main route of intake of the lipophilic PHAHs is through the diet in seals (Tanabe, 1988). Despite this, the PHAH levels are clearly lower in the grey seals. Judging by the EROD activities in the two seal species, the elevation of CYP1A activity in grey seals was stronger in liver and lung although the geographical di€erence in PHAH levels was smaller in this species. This could indicate that grey seals have a better capacity to react to PHAH exposure than ringed seals, or that the Svalbard ringed seals are exposed to other CYP1A inducing agents not included in this study. The higher EROD activity level in reference ringed seals compared to reference grey seals suggests a higher constitutive CYP1A expression in ringed seals, or induced CYP1A levels in the Svalbard population.

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481

The elevation pattern of EROD in the lungs, kidneys, adrenal glands and spleen of both seal species is similar to that reported for experimental animals and humans (Black, Wang, Henry & Shaw, 1998; Germolec et al., 1996; Raunio et al., 1995), showing a clear induction when exposed to CYP1A inducing compounds. In contrast, Schultz, Nueben, Davies & Edwards (1996) showed a similar induction of EROD by dioxin in the liver and lungs of marmoset monkeys as in the seals, but no induction was observed in the kidneys, adrenal glands or small intestines. The variation in extrahepatic CYP1A expression and induction between species indicates that caution must be used when making cross-species extrapolations. Immunochemical characterisation by immunoblot analysis con®rmed the presence and elevation of a putative CYP1A protein in ringed and grey seals. Immunohistochemistry and enzyme activity are compatible methods for analysing CYP1A-associated expression in the seal species studied. When quantifying the CYP1A enzyme expression in these seal species with the methods available, the combined use of EROD together with immunoblotting is recommended. A very high exposure to PHAHs may inhibit CYP1A activity while the protein level is still clearly elevated (review in Stegeman & Hahn, 1994). Additional information about the cellular localisation of CYP1A can be obtained using immunohistochemistry, although our subjective quanti®cation of this method provides only a crude indication of the relative CYP1A protein levels in various tissues. 5. Conclusion The results of this study indicate that a CYP1A protein is present in both hepatic and extrahepatic tissues of ringed and grey seals. The clear elevation of CYP1A expression in all tissues of both Baltic seal populations suggests that the Baltic seals are exposed to compounds that a€ect the whole body. An elevation of CYP1A expression may disturb the metabolism of endogenous compounds or in¯uence the toxic potential of foreign substances, which ultimately determines the biological e€ects of contaminant exposure. Further research, using various methods to connect the individual contaminant load with the observed CYP1A expression, will give more information about the usefulness of CYP1A induction, as a biomarker for contaminant exposure and e€ects in ringed and grey seals. Acknowledgements We are very grateful to Mark Myers and Tracy Collier of the Environmental Conservation Division, Northwest Fisheries Science Center, NMFS/NOAA, for their guidance and assistance in the immunohistochemical work. Monoclonal antibody (MAb) 1-12-3, against scup CYP1A1, was generously provided by John J. Stegeman (WHOI, MA) and Harry V. Gelboin (NCI). We thank Bruce Woodin (WHOI, MA) for providing useful information about the use of the 1-12-3 antibody. We are grateful to Eero Helle, Marcus Wikman and Bjùrn Kraft for collecting the

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ringed seal samples, and for the assistance of Christian Lydersen and the sta€ at the Norwegian Polar Institute in Ny AÊlesund, Svalbard. We thank Eero Helle, Richard Addison, Wayne Stobo and Chris Harvey-Clark for their help in the grey seal sampling on Sable Island, Canada; Kalle JaÈrvinen for ®eld assistance in the Bay of Bothnia; Merja Luukkonen, Ritva Tauriainen and PaÈivi Tyni for their help in the laboratory, and Hannu PoÈysaÈ for statistical assistance. This study was funded by the Finnish Academy of Science.

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