Concentrations, patterns and metabolites of organochlorine pesticides in relation to xenobiotic phase I and II enzyme activities in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea

Environmental Pollution 157 (2009) 2428–2434

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Concentrations, patterns and metabolites of organochlorine pesticides in relation to xenobiotic phase I and II enzyme activities in ringed seals (Phoca hispida) from Svalbard and the Baltic Sea Heli Routti a, b, *, Bert van Bavel c, Robert J. Letcher d, Augustine Arukwe e, Shaogang Chu d, Geir W. Gabrielsen a a

Norwegian Polar Institute, Polar Environmental Centre, 9296 Tromsø, Norway Centre of Excellence in Evolutionary Genetics and Physiology, Department of Biology, University of Turku, 20014 Turku, Finland ¨ rebro University, 70182 O ¨ rebro, Sweden MTM Research Centre, O d Wildlife Toxicology and Disease Program, Wildlife and Landscape Science Directorate, Science and Technology Branch, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, Ontario K1A 0H3, Canada e Department of Biology, Norwegian University of Science and Technology, 7491 Trondheim, Norway b c

Contrasting patterns of organochlorine pesticides in two ringed seal populations.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 January 2009 Received in revised form 27 February 2009 Accepted 7 March 2009

The present study investigates the concentrations and patterns of organochlorine pesticides (OCPs) and their metabolites in liver and plasma of two ringed seal populations (Phoca hispida): lower contaminated Svalbard population and more contaminated Baltic Sea population. Among OCPs, p,p0 -DDE and sumchlordanes were the highest in concentration. With increasing hepatic contaminant concentrations and activities of xenobiotic-metabolizing enzymes, the concentrations of 3-methylsulfonyl-p,p0 -DDE and the concentration ratios of pentachlorophenol/hexachlorobenzene increased, and the toxaphene pattern shifted more towards persistent Parlar-26 and -50 and less towards more biodegradable Parlar-44. Relative concentrations of the chlordane metabolites, oxychlordane and -heptachlorepoxide, to sumchlordanes were higher in the seals from Svalbard compared to the seals from the Baltic, while the trend was opposite for cis- and trans-nonachlor. The observed differences in the OCP patterns in the seals from the two populations are probably related to the catalytic activity of xenobiotic-metabolizing enzymes, and also to differences in dietary exposure. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: Organochlorine pesticide Biotransformation Ringed seal Baltic Sea Svalbard

1. Introduction High concentrations of persistent organic pollutants (POPs) have been detected in seals from the industrialized Baltic Sea compared to the remote Arctic regions (Nyman et al., 2002; Routti et al., 2008a). In the highly contaminated Baltic seals, POP levels have been associated with health effects including reproduction impairment, pathological changes (Bergman and Olsson, 1985; Helle et al., 1976) and endocrine disruption (Nyman et al., 2003; Routti et al., 2008b). However, there is still limited information concerning specific toxic mechanisms, which would link the adverse health effects to high contaminant exposures. One of

* Correspondence to: Norwegian Polar Institute, Polar Environmental Centre, 9296 Tromsø, Norway. Tel.: þ47 77750544; fax: þ47 77750501. E-mail address: [email protected] (H. Routti). 0269-7491/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2009.03.008

the major classes of POPs detected in seals from the Baltic and from the Norwegian Arctic is organachlorine pesticides (OCPs). The potential toxicity of OCPs and/or their metabolites has been demonstrated by several studies conducted in mammals in vivo and in vitro (Bondy et al., 2004; De Geus et al., 1999; Guillette et al., 2006). Biotransformation processes influence the levels and congener patterns of residual OCPs in biota (Boon et al., 1998; Wolkers et al., 2000) in addition to dietary exposure, age and gender (Gouteaux et al., 2005; Nyman et al., 2002). Therefore, knowledge of the biotransformation of OCPs, and the potentially toxic metabolites that are formed, is important for understanding the elimination capacity of OCPs and the possible mechanisms of toxic effects. Biotransformation of OCPs is a complex process involving mediation via xenobiotic-metabolizing phase I (cytochrome P450 (CYP)) and conjugating phase II enzymes, which can lead to the formation and retention of e.g. chlordane and p,p0 -dichlorodip

H. Routti et al. / Environmental Pollution 157 (2009) 2428–2434

henyldichloroethylene (DDE) metabolites, including oxychlordane and 3-methylsulfonyl (CH3SO2 ¼ MeSO2)-p,p0 -DDE, respectively (Jensen and Jansson, 1976; Nomeir and Hajjar, 1987). There is limited information concerning OCP biotransformation in seals. Relative comparisons of OCP patterns in diet and tissues of seals have been investigated in different seal species (Gouteaux et al., 2005; Wolkers et al., 2000). A few studies have reported residues of MeSO2-DDE in ringed, grey and harbour seals (Haraguchi et al., 1992; Jensen and Jansson, 1976; Larsson et al., 2004; Letcher et al., 1998; Troisi et al., 2001). Toxaphene metabolism has been investigated in grey and harbour seals using in vitro metabolism/inhibition assays (Boon et al., 1998; van Hezik et al., 2001). Recent findings for ringed seals (Phoca hispida) have shown that PCB concentrations are positively related to phase I and II enzyme activities, which leads to the biotransformation of certain PCB congeners to persistent hydroxy- (OH-) and MeSO2-PCB metabolites that are retained mainly in the plasma and liver, respectively (Routti et al., 2008a). Therefore, it could be assumed that the elevated enzyme activities also result to increased biotransformation of OCPs. The objective of the present study is to investigate the concentrations and patterns of OCPs in relation to xenobiotic enzyme activities in ringed seals from two geographically distinct and differentially contaminated populations: less contaminated Svalbard and more contaminated Baltic Sea. Compound-specific chlordane, toxaphene and DDE patterns and concentrations were determined and the relationships were examined with the catalytic activities of phase I and II enzymes. 2. Experimental section 2.1. Sample collection and preparation The seals from west coast of Svalbard, Norway (77470 N to 78 230 N, 17 000 E) were sampled in May and June 2007 with special permission granted to the Norwegian Polar Institute by the Governor of Svalbard and during the local hunting season under local hunting law of Svalbard. The Ministry of Forestry and Agriculture in Finland granted the Finnish Game and Fisheries permission to sample Baltic ringed seals in April 2002, 2006 and 2007 (65100 N, 24 200 E). All the seal samples were collected after the weaning period during the moulting season of the seals. Based on counting annual layers from the thin transverse sections of the canine or molar tooth, the mean age of the seals sampled from Svalbard was 11.9 years [95% confidence interval 8.6, 15.3] and from the Baltic Sea 9.4 years [7.5, 11.3], respectively. The seals were of both sexes (males/females-ratio for Svalbard 13/6 and for the Baltic 19/13). Samples for chemical analysis were stored at 20  C until analyzed. Samples for enzyme activity analysis were frozen in liquid nitrogen in the field and stored at 80  C until analyzed. 2.2. Chemical analysis The extraction and clean-up methods of OCPs and their metabolites in liver and plasma sample have been previously described (Routti et al., 2008a). Detailed information about congeners analyzed is given in Table 1. The internal standards used were 13C12-labeled PCBs for chlordanes, toxaphenes, p,p0 -DDE and hexachlorobenzene (HCB), 3-MeSO2-2-CH3-20 3 0 4 0 5 0 50 -pentachlorobiphenyl for 3-MeSO2-p,p0 -DDE, and 13C12-labeled OH-CB120 for pentrachlorophenol (PCP) and 4-OH-heptachlorostyrene (4-OH-HpCS). For liver samples, p,p0 -DDE, chlordanes and HCB were determined by gas chromatography (GC) (Agilent 6890, Agilent, Waldorff, Germany) coupled with low resolution mass spectrometry (MS) (Agilent 5973) using electron impact (EI) in the selective ion monitoring (SIM) mode. For plasma samples, p,p0 -DDE, chlordanes and HCB analyses and quantification were performed on an Autospec Ultima high-resolution GC–MS(EI) (Micromass, Manchester, UK) in the SIM mode. Toxaphenes were analyzed by the low resolution MS using electron capture negative ionization (ECNI) in the SIM mode monitoring the two most abundant ions of the molecular ion. The aryl sulfone, containing 3-MeSO2-p,p0 -DDE, fractions and methoxy-derivatized phenolic, containing PCP and 4-OH-HpCS, fractions from samples were analyzed by GC–MS (Agilent 6890 and 5973; Agilent Technologies, CA) with ECNI source in the SIM mode. The mean internal standard recoveries were 49% for p,p0 -DDE, chlordanes, toxaphenes and HCB, 92% for the PCP and 4-OH-HpCS and 127% for MeSO2-p,p0 -DDE. Laboratory blank samples did not contain any of the target compounds at levels above 10% of the levels found in the samples. The variance of individual chlordane congeners, p,p0 -DDE and HCB of the standard reference material (human adipose tissue, n ¼ 5) was less than 20%. The replicate determination (n ¼ 6) of a polar bear plasma pool (NWRC in-house plasma

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Table 1 Concentrations of the main organochlorine pesticides in ringed seals from the Svalbard and the Baltic (geometric mean and 95% confidence intervals ng/g wet weight). Svalbard

Baltic

n

Mean

95% CI

n

Mean

95% CI

Liver Lipid % p,p0 -DDE MeSO2-DDEa P -CHLb P6 c 8-TOX HCB P -OCPd

18 18 21 18 18 18 18

3.2 14 10 2.8 0.25 28

2.9, 3.6 9.2, 21 <0.3–2.5 7.2, 15 2.3, 3.4 0.19, 0.31 19, 40

31 31 27 31 31 31 31

3.7 113 5.4 55 5.2 0.41 184

3.4, 4.0 85, 152 3.1, 9.4 40, 75 3.6, 7.7 0.34, 0.50 137, 247

Plasma Lipid % p,p0 -DDE P -CHLb P6 c 8-TOX HCB PCP 4-OH-HpCS P -OCPd

18 18 18 18 18 19 19 18

0.69 2.1 0.76 0.44 0.05 0.42 0.06 4.2

0.61, 0.77 1.2, 3.6 0.45, 1.3 0.34, 0.56 0.04, 0.06 0.28, 0.63 0.04, 0.09 2.6, 6.8

30 30 30 29 30 30 30 30

0.65 8.0 3.9 1.6 0.05 0.96 0.52 16

0.59, 0.73 6.0, 11 2.6, 5.8 1.2, 2.0 0.04, 0.07 0.80, 1.14 0.42, 0.66 13, 21

a Only range is given for the Svalbard population because 3-MeSO2-p,p0 -DDE was detected in 19% of the samples from Svalbard. b P 6-CHL: cis-heptachlorepoxide, cis-chlordane, oxychlordane, trans-nonachlor/ MC6 and cis-nonachlor. c P 8-TOX: Tox 2, Parlar-26, -38, -40/-41, -44, -50, -62. P P d P -OCP: p,p0 -DDE, 6-CHL, 8-TOX, HCB.

reference material) showed 32 and 29% variation in the PCP and 4-OH-HpCS concentrations, respectively. The minimum level of quantification (MLOQ) for chlordanes, p,p0 -DDE, toxaphenes and HCB was defined as a signal-to-noise ratio of 3:1, and for 3-MeSO2-p,p0 -DDE, PCP and 4-OH-HpCS as signal-to-noise ratio of 10:1, respectively. 2.3. Enzyme assays Methods for analysis of hepatic activities of phase I (microsomal ethoxyresorufin-O-deethylase (EROD), benzyloxyresorufin-O-dealkylase (BROD), methoxyresorufin-O-demethylase (MROD) and pentoxyresorufin-O-dealkylase (PROD)) and II (microsomal uridine-diphosphate glucuronosyltransferase (UDPGT), cytosolic glutathione S-transferase (GST)), and total amount of protein were based on previously described methods (Routti et al., 2008a). 2.4. Data analysis Congeners/metabolites detected in 60% or more of the samples for each seal P population were included in sum ( ) concentrations of OCPs of a given class (Table 1). For these congeners/metabolites, the samples with concentrations below the minimum level of quantification (MLOQ) were replaced by randomly generated normally distributed data, assuming one half of the MLOQ as the mean with 40% variation. 28% of the 3-MeSO2-p,p0 -DDE and 0.7% of the remaining OCP data were below MLOQ. Statistical analyses were carried using R version 2.8.1 (R Development Core Team, 2008). Relationships between ln-transformed OCPs, geographical areas and time trends were investigated using linear models. Parameter estimates (b) with 95% confidence intervals are given in the text and in Table 2. Diagnostic plots of residuals were used to verify that the model assumptions were met (most importantly constant variance between residuals). Hepatic lipid concentrations were more elevated in seals from the Baltic compared to the seals from Svalbard (Routti et al., 2008a) (Table 1). In order to avoid the confounding effect of lipid concentration with area, hepatic OCP concentrations were lipid normalized for the linear models. One P P -CHL and -TOX in individual had extremely high concentration of p,p0 -DDE, P plasma. This individual was removed from the data for plasma p,p0 -DDE, -CHL and P -TOX analysis, which did not result in substantial changes on relationships between the given OCP in plasma and geographical area. Principal component analyses (PCAs) were used to investigate OCP patterns (Rao, 1964). Because proportions summing up to one were used, the PCA was derived from the covariance matrix of centred log-ratio of proportions (ln(OCPx/ P -OCP)) (Aitchison and Greenacre, 2002). Congeners showing high uncertainty were not included in the PCA (Tox 2 and Parlar-38). In consequence, no data included in the PCAs were below MLOQ. Correlations are shown as Pearson correlation coefficients with 95% confidence intervals. All the absolute concentrations of POPs P and enzyme activities were ln-transformed for correlation analysis. -POP P concentration used for PCA and correlations refers to the sum of hepatic -PCBs P (Routti et al., 2008a) and -OCPs.

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Table 2 Geographical differences for ln-transformed hepatic (ng/g lipid weight) and plasma (ng/g wet weight) organochlorine pesticides in ringed seals from the Baltic and Svalbard indicated as parameter estimates from linear models (b and 95% confidence intervals (CI)), F-statistics and p-values from analysis of variance. df: degrees of freedom.

b

95% CI

df

F

p

Liver p,p0 -DDE P -CHL P6 8-TOX HCB

1.40 1.10 0.35 0.27

1.07, 1.73 0.77, 1.43 0.03, 0.72 0.06, 0.49

1,47 1,47 1,47 1,47

71.0 44.5 3.51 6.63

<0.001 <0.001 0.067 0.013

Plasma p,p0 -DDE P -CHL P6 8-TOX HCB PCP 4-OH-HpCS

1.11 1.31 0.97 0.04 0.56 1.59

0.78, 1.43 0.89, 1.73 0.70, 1.24 0.25, 0.33 0.29, 0.83 1.28, 1.89

1,45 1,45 1,44 1,46 1,46 1,47

46.2 40.6 52.6 0.08 17.2 108

<0.001 <0.001 <0.001 0.783 <0.001 <0.001

3. Results and discussion 3.1. OCP levels and enzyme activities The sum concentrations of OCP classes or individual OCP concentrations in both liver and plasma were significantly higher in the ringed seals from the Baltic Sea compared to the seals from Svalbard (Tables 1 and 2). For seals from both populations OCPs were comprised mainly of p,p0 -DDE and chlordanes (Table 1). Hepatic p,p0 -DDE concentrations (geometric mean ng/g lipid weight) in ringed seals from the present study were 13% of the levels detected in the ringed seals sampled in the Baltic in 1997–98 (b ¼ 2.02 [2.46, 1.59]), which indicates that DDE levels have decreased over this time period in the Baltic seals. A significant decreasing trend has also been observed for PCB concentrations in these same seals (Routti et al., 2008a). Parameter estimates for linear models (Table 2) were used to estimate geographical variation between the different OCP classes and between liver and plasma. The geographical differences of OCP P -CHLs concentrations were greater for hepatic p,p0 -DDE and P compared to hepatic -TOXs and HCB, which is in accordance with the long-range transport potential of these compounds (Beyer et al., P 2000). Spatial variation for -CHLs and p,p0 -DDE was similar in both liver and plasma. In contrast, the geographical difference for P P plasma -TOXs was greater compared to hepatic -TOXs. This suggest that toxaphenes may be less bioaccumulative in ringed seal P liver compared to -CHLs and p,p0 -DDE. This hypothesis is supported by the results of Wolkers et al. (2000), who reported bioaccumulation pattern of pesticides from polar cod to harp seal blubber. Data on the hepatic phase I and II enzyme activities in the present ringed seals have been reported elsewhere (Routti et al., 2008a). Briefly, EROD, PROD, BROD, MROD and GST activities were higher in the Baltic seals compared to the seals from Svalbard. UDPGT activity was not found to differ between the populations. Phase I enzyme and P GST activities were positively correlated to hepatic -OCP concentrations (r ¼ 0.48 [0.23, 0.67], 0.63 [0.42–0.77]).

3.2. DDE compounds The compound 3-MeSO2-p,p0 -DDE has been shown to be a metabolite of p,p0 -DDE formed in for example Baltic grey seals and Canadian polar bears (Larsson et al., 2004; Letcher et al., 1998). In the present ringed seals, 3-MeSO2-p,p0 -DDE was detected in 93% of the Baltic ringed seals and the geometric mean concentration was

18 times higher than MLOQ. In contrast, in the seals from Svalbard 3-MeSO2-p,p0 -DDE was detected only in 19% of the liver samples. Although p,p0 -DDE shows strong bioaccumulation potential compared to its parent compound p,p0 -DDT in both lower and more contaminated seal populations (Routti et al., 2005; Wolkers et al., 2000), our results suggest that ringed seals are able to metabolize p,p0 -DDE and form 3-MeSO2-p,p0 -DDE to some extent. The 3MeSO2-p,p0 -DDE concentration showed a strong correlation to P -POPs (r ¼ 0.67 [0.42, 0.82]), which indicates that the capacity of ringed seals to metabolize p,p0 -DDE and to 3-MeSO2-p,p0 -DDE increases with contaminant exposure. Dietary bioaccumulation of 3-MeSO2-p,p0 -DDE is suggested to be minor. Letcher and coworkers (1998) showed that 3-MeSO2-p,p0 DDE was not detectable in polar cod, but was present in ringed seal blubber for individuals collected from the Canadian Arctic. In invertebrates and fish from Canadian Arctic and from Great Lakes, MeSO2-metabolites of PCB have been detected only in sculpin (Bright et al., 1995; Stapleton et al., 2001), which is a minor diet species of the ringed seal from the Baltic Sea (Tormosov and Rezvov, 1978). Biotransformation of p,p0 -DDE to 3-MeSO2-p,p0 -DDE has been suggested to involve the activity of CYP2B-like enzymes, GST and the mercapturic acid pathway (Letcher, 1996) as is similarly the case for PCB biotransformation to MeSO2-PCBs (Letcher et al., 2000). Therefore, elevated activity of GST and CYP2B-like enzymes in the Baltic ringed seals compared to the Svalbard seals (Routti et al., 2008a) may enhance the formation of 3-MeSO2-p,p0 -DDE from p,p0 -DDE in the Baltic seals. The ratio of hepatic 3-MeSO2-p,p0 DDE/p,p0 -DDE in the Baltic ringed seals from the present study was similar to previous reports for Baltic grey seals (Larsson et al., 2004). A comparison of hepatic 3-MeSO2-p,p0 -DDE/p,p0 -DDE ratios with other species indicates that ringed seals have better capacity to biotransform p,p0 -DDE to 3-MeSO2-p,p0 -DDE than highly contaminated harbour porpoises from the North Sea (Chu et al., 2003), but lower than polar bears (Gebbink et al., 2008). 3.3. Toxaphenes P In the ringed seals from Svalbard and the Baltic, -TOX consisted mainly of Parlar-44, Parlar-26 and Parlar-62 (Fig. 1A). PCA showed P that the relative concentrations of Parlar-26 and -50 to -TOX were higher in the Baltic seals compared to the Svalbard seals while Parlar-44 showed the opposite trend (Fig. 2A and B). Observed differences in the hepatic toxaphene patterns between the Baltic and Svalbard seals may be related to biotransformation and/or to dietary intake. Biotransformation may occur via oxidation, dechlorination or dehydrochlorination (De Geus et al., 1999) and it is mainly catalyzed by CYP enzymes in rats (De Geus et al., 1999). In vitro studies on marine mammals show that biotransformation depends on the chlorine (Cl) substitution pattern of the congeners and that hydroxylated metabolites are formed (Boon et al., 1998). In the present study, relative concentrations of Parlar-26 and -50 to P P -TOX increased and Parlar-44 decreased with increasing -POP concentration (Fig. 2A). This finding indicates the persistence of Parlar-26 and -50 and metabolism of Parlar-44. Parlar-26 and -50 possess Cl-substituents at each carbon atom positioned at the lateral ring making them unsuitable for enzymatic attack in seal liver microsomes (Boon et al., 1998). Parlar-44 possesses Clunsubstituted sites at the positions C-3 and C-6 of the lateral carbon ring. These sites may react with xenobiotic-metabolizing enzymes (Boon et al., 1998). Relative concentration of Parlar-44 was negatively correlated to phase I enzyme activities measured as PROD (r ¼ 0.43 [0.64, 0.16] and BROD (r ¼ 0.42 [0.63, 0.15]) P (Fig. 2A). Relative concentration of Parlar-44 to -TOX was also negatively correlated to the activities of EROD (0.57 [0.74, 0.34] and MROD (0.47 [0.66, 0.21]). PROD has been used as a model

H. Routti et al. / Environmental Pollution 157 (2009) 2428–2434

A

2431

Baltic Sea (Fromberg et al., 2000; Koistinen et al., 2008; McHugh et al., 2004). The only study, which has investigated the same toxaphene congeners in polar cod as the present study, has been conducted in the east side of Svalbard (Wolkers et al., 2006). However, the toxaphene pattern may vary considerably between the east and west coast of Svalbard, because toxaphene levels are several times higher in the east coast compared to the west coast of Svalbard (Wolkers et al., 2000), where the present seal sampling was conducted. 3.4. Chlordanes

B

Fig. 1. Concentration ratios (mean and 95% confidence intervals) for the main hepatic P P (A) toxaphenes (Parlarx/ -TOX) and (B) chlordanes (CHLx/ -CHL) in ringed seals from Svalbard (open circles) and from the Baltic Sea (solid circles). CHC: cis-heptachlorepoxide; CCL: cis-chlordane; OCL: oxychlordane; TNCL: trans-nonachlor; CNCL: cisnonachlor.

substrate for rat CYP2B (Burke et al., 1985), while in dogs, BROD is catalyzed by CYP2B11 (Klekotka and Halpert, 1995). EROD has been used as model substrates for CYP1A1/2 in dogs (Jayyosi et al., 1996) and MROD for CYP1A2 in rats (Nerurkar et al., 1993). This suggests that phase I enzymes may be involved in the metabolism of Parlar44 in ringed seals. However, it is unlikely that CYP1A would be involved in the toxaphene biotransformation, since planar compounds are preferred by CYP1A enzymes (Lewis et al., 1998). Parlar-62, which has a similar structure to Parlar-44, has been suggested to be partly biotransformed in seals (Boon et al., 1998; Gouteaux et al., 2005; van Hezik et al., 2001). In the present study, relative concentrations of Parlar-62 did not differ between seals from different areas (Fig. 1A) and it was not correlated to phase I enzyme activities (Fig. 2A). There is no clear explanation for the absence of this phenomenon. Toxaphene biotransformation may be influenced by several other factors in addition to the phase I enzymes measured in the present study. CYP3A-like enzyme has been suggested to be involved in Parlar-62 metabolism in harbour and grey seal microsomes (van Hezik et al., 2001). Previously, CYP3A activity has been reported to be higher in the Svalbard ringed seals compared to the Baltic ringed seals (Nyman et al., 2001). Geographical differences in dietary accumulation of individual toxaphene congeners in the present ringed seals are difficult to assess. To our knowledge, there is not enough published information on toxaphenes in seal prey items from the same locations investigating the same congeners as in the present study. Only three toxaphene congeners (Parlar-26, -50, -62) have been reported in ringed seal prey items (Labansen et al., 2007; Tormosov and Rezvov, 1978) from Svalbard (Wolkers et al., 2000) and from the

In ringed seals from Svalbard and the Baltic, the major chlordane compounds were trans-nonachlor/MC6 and oxychlordane (Fig. 1B). The results of PCA of hepatic chlordanes indicate that relative concentrations of oxychlordane, cis-heptachlorepoxide and cisP chlordane to -CHL were higher in the seals from Svalbard compared to the seals from the Baltic, while the trend was opposite for trans-nonachlor/MC6 and cis-nonachlor (Fig. 2C and D). Both biotransformation and dietary exposure may have and influence on the chlordane composition observed in the present ringed seals. Biotransformation of chlordanes includes several steps and pathways (Nomeir and Hajjar, 1987). Briefly, trans- and cis-nonachlor are dechlorinated to trans- and cis-chlordane, respectively. The oxidation of these compounds through several reaction steps by phase I and II enzymes favors the formation of bioaccumulative oxychlordane and to a lesser amount to heptachlorepoxide and other compounds. In the present study, chlordane pattern was shifted more towards trans-nonachlor/MC6 and cis-nonachlor, and less towards cis-chlordane, oxychlordane, and cis-heptachlorepP oxide with increasing hepatic -POP concentration and phase I enzyme activities (Fig. 2C and D). In an experimental study with cohorts of differentially exposed and captive Greenland sledge P dogs, -CHL consisted mainly of oxychlordane in the control group fed on pork fat, while in the exposed group fed on minke whale blubber oxychlordane was a minor compound (Verreault et al., 2009). The authors suggested that in the exposed dogs, induction of catalytic activity and substrate turnover yield of xenobioticmetabolizing enzymes may be saturated by elevated POP concentrations, which would lead to lower formation of oxychlordane in the more contaminated animals. Similarly, in the higher contaminated Baltic seals elevated levels of PCBs (Routti et al., 2008a) and p,p0 -DDE (Table 1) may result in reduced formation of oxychlordane compared to the lower contaminated Svalbard seals. Another possibility is that CYP3A may play a role in chlordane metabolism. In rats, trans-nonachlor induces both CYP3A and CYP2B, which possibly leads to formation of oxychlordane (Bondy et al., 2004). Elevated CYP3A activity in Svalbard ringed seals compared to Baltic ringed seals (Nyman et al., 2001) could result in increased formation of oxychlordane in the seals from Svalbard compared to the Baltic seals. Geographical differences of the chlordane composition in seals may also be related to the contaminant exposure. However, bioaccumulation cannot be assessed based on literature, because previous studies indicate variable ratios of the main chlordane components, oxychlordane and trans-nonachlor, in arctic cod from Svalbard/Barents Sea (Borgå et al., 2007; Wolkers et al., 2000, 2006) and in herring from the Baltic (Strandberg et al., 1998). 3.5. PCP and HpCS Hydroxylated compounds, PCP and 4-OH-HpCS, were both higher in the Baltic seals compared to seals from Svalbard (Tables 1 and 2). PCP has been used as a pesticide, but it is also the main metabolite of two other pesticides, namely, HCB (van Ommen et al., 1985) and pentachloroanisole (Ikeda and Sapienza, 1995). PCP and

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H. Routti et al. / Environmental Pollution 157 (2009) 2428–2434

A

B

P62 MROD PROD BROD

P40.41

POPs

EROD

S

P50

P26

B

P44

C

B=Baltic S=Svalbard

D

CHC POPs UDPGT MROD

EROD

CNCL PROD

CCL OCL

S

B

GST

TNCL.MC6 BROD

B=Baltic S=Svalbard Fig. 2. Ordination plots from PCA based on covariance matrix of log-ratio toxaphene (A–B) and chlordane (C–D) compounds in ringed seals livers from Svalbard and the Baltic Sea. P Sample scores are grouped by geographical area. Enzyme activities and -POPs are shown as supplementary variables. The 1st axis explains 73% of the variation of chlordanes and the 2nd axis 17%, respectively. The variation of toxaphenes is explained 76% by the 1st axis and 18% by the 2nd axis, respectively. See Fig. 1 for abbreviations.

4-OH-HpCS detected in marine mammals have been suggested to result from biotransformation of HCB or pentachloroanisole, and of octachlorostyrene, respectively (Hoekstra et al., 2003; Sandau et al., 2000). In the present study, circulating PCP/HCB ratio was posiP tively related to -POPs (r ¼ 0.38 [0.11, 0.61]) and to EROD activity (r ¼ 0.51 [0.26, 0.70]). In addition, concentrations of PCP and 4-OHHpCS were positively correlated to EROD activity (r ¼ 0.49 [0.24, 0.68] and (r ¼ 0.74 [0.58, 0.85], respectively). Similar significant trends, although less pronounced, were obtained between PCP and 4-OH-HpCS and other phase I enzyme activities. These results strengthen the hypothesis that PCP and 4-OH-HpCS are products of phase I biotransformation in marine mammals. Comparisons with other studies indicated that PCP and 4-OHHpCS concentrations in the present Svalbard seals were at a similar range to those in the ringed seals from Canadian Arctic (Sandau et al., 2000). Interestingly, in the present ringed seals PCP concentrations were lower compared to low contaminated bowhead whales (Hoekstra et al., 2003) but similar or slightly higher in comparison to polar bears (Gebbink et al., 2008; Sandala et al., 2004; Sandau et al., 2000). 3.6. Implications of OCP biotransformation OCPs and their metabolic products have been related to several toxicological effects in experimental animals including changes in liver and in endocrine system (Bondy et al., 2004; De Geus et al.,1999; Guillette et al., 2006). In rodents, 3-MeSO2-p,p0 -DDE accumulates in adrenal cortex leading to histological changes and inhibition of steroid secretion (Lindhe et al., 2001; Lund et al., 1988). In rats, oxychlordane shows a several times greater toxicity towards thyroid, liver and thymus compared to trans- and cis-nonachlor (Bondy et al., 2003). PCP and 4-OH-HpCS show a higher affinity compared to its natural ligands to mammalian transthyretin (Sandau et al., 2000; van

den Berg, 1990), which is one of the main thyroid hormone transport proteins (McNabb, 1992). In ringed seals, high levels of contaminants have been related to adrenal hyperplasia (Bergman and Olsson, 1985), altered liver function (Nyman et al., 2003) and changes in thyroid hormone levels (Routti et al., 2008b). 4. Conclusions The results of the present investigation show significant relationships between contaminant exposure, catalytic activities of xenobiotic-metabolizing enzymes, changes in OCP patterns and formation of metabolites. Based on these results we suggest that contaminant exposure induces phase I and II enzyme activities leading to partial biotransformation of p,p0 -DDE, toxaphenes and HCB and formation of MeSO2-p,p0 -DDE, PCP and 4-OH-HpCS. In contrast, the geographical difference in chlordane pattern suggests that metabolism of chlordanes and nonachlors to oxychlordane decreases with increasing contaminant exposure. Another possibility is that dietary exposure of chlordane compounds differs geographically. Previous studies report that MeSO2-p,p0 -DDE, PCP and 4-OH-HpCS show high toxic potential towards endocrine system e.g. by binding to carrier proteins and by inhibiting hormone secretion. Elevated levels of these toxic metabolites in addition to OH- and MeSO2-PCBs raise concern about the possible role of OH- and MeSO2-substituted metabolites in endocrine disruption observed in the Baltic ringed seals. Therefore, further studies are warranted to investigate the role of OH- and MeSO2metabolites in endocrine disruption in ringed seals. Acknowledgements We acknowledge Eero Helle, Mervi Kunnasranta, Madeleine Nyman and Finnish Game and Fisheries Research Institute for the

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sampling in the Baltic Sea and Jukka Ikonen, Øystein Overrein, Tommy Sandal and Hans Wolkers for their help in sampling in Svalbard. We thank Hele´n Bjo¨rnhoft for the assistance in OCP analysis and Christina Lockyer for aging the seals from Svalbard. We acknowledge Åke Bergman at Stockholm University for supplying 3-MeSO2-p,p’-DDE standard. This study was financed by Nordic Council of Ministers, Kone Foundation, Research Council of Norway, Norwegian Polar Institute and the Biological Interactions Graduate School.

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