Mineral density and biomechanical properties of bone tissue from male Arctic foxes (Vulpes lagopus) exposed to organochlorine contaminants and emaciation

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Chemosphere 71 (2008) 1214–1224

Organochlorine-induced histopathology in kidney and liver tissuefrom Arctic fox (Vulpes lagopus)

Christian Sonne a,*, Hans Wolkers b, Pall S. Leifsson c, Bjørn Munro Jenssen d, Eva Fuglei b,Øystein Ahlstrøm e, Rune Dietz a, Maja Kirkegaard a, Derek C.G. Muir f, Even Jørgensen g

a Section for Contaminants, Effects and Marine Mammals, Department of Arctic Environment, National Environmental Research Institute,

University of Aarhus, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmarkb Norwegian Polar Institute, Polar Environmental Centre, NO-9296 Tromsø, Norway

c Department of Veterinary Pathobiology, Faculty of Life Sciences, University of Copenhagen, Bulowsvej 17, DK-1870 Frederiksberg, Denmarkd Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway

e Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, N-1432 As, Norwayf National Water Research Institute, Environment Canada, Burlington, Ontario, Canada L7R 4A6

g Norwegian College of Fishery Science, University of Tromsø, N-9037 Tromsø, Norway, and Norwegian Institute for Nature Research,

Polar Environmental Centre, N-9296 Tromsø, Norway

Received 16 August 2007; received in revised form 23 November 2007; accepted 7 December 2007Available online 14 February 2008

Abstract

The effects of persistent organic pollutants on renal and liver morphology in farmed arctic fox (Vulpes lagopus) were studied underexperimental conditions. Control animals received a diet containing pork (Sus scrofa) fat with low amounts of persistent organic pollu-tants, while the diet of the exposed animals contained whale blubber, ‘naturally’ contaminated with persistent organic pollutants. Poly-chlorinated biphenyls (PCB) and organochlorine pesticide (OCP) concentrations in the whale blubber were 488 and 395 ng/g wet weight,respectively. Animals were sacrificed and sampled when they were at their fattest (winter) as well as their lowest body weight (summer).The results show that PCB and OCP exposure causes renal (and probably also liver) lesions in arctic foxes. The prevalence of glomerular,tubular and interstitial lesions was significantly highest in the exposed group (chi-square: all p < 0.05). The frequency of liver lesions (ste-atosis, intravascular granulocyte accumulations, interstitial cell infiltrations, lipid granulomas, portal fibrosis and bile duct hyperplasia)were also highest in the exposed group, although not significantly (chi-square: all p > 0.05). The prevalence of lesions was not signifi-cantly different between lean (winter) and fat (summer) foxes for any of the lesions (chi-square: all p > 0.05). We suggest that wild arcticfoxes exposed to an environmental cocktail of persistent organic pollutants, such as PCBs and OCPs, in their natural diet are at risk fordeveloping chronic kidney and liver damage. Whether such lesions may have an impact on age and health of the animals remainsuncertain.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Arctic fox; Organochlorine pesticides; Histopathology; Kidney; Liver; PCB Renal

0045-6535/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.chemosphere.2007.12.028

* Corresponding author. Tel.: +45 4630 1200 (switch board)/+45 46301954 (direct)/+45 2521 4686 (cell); fax: +45 4630 1914.

E-mail addresses: [email protected] (C. Sonne), [email protected](H. Wolkers), [email protected] (P.S. Leifsson), [email protected] (B.M. Jenssen), [email protected] (E. Fuglei), [email protected] (Ø. Ahlstrøm), [email protected] (R. Dietz), [email protected](M. Kirkegaard), [email protected] (D.C.G. Muir), [email protected] (E. Jørgensen).

URL: http://www.neri.dk (C. Sonne).

1. Introduction

The Arctic is exposed to a wide variety of persistentorganic pollutants, such as polychlorinated biphenyls(PCB) and organochlorine pesticides (OCP). These com-pounds are considered particularly harmful due to theirpersistence and bioaccumulation in the food chain. There-fore, Arctic top predators such as polar bears (Ursus

C. Sonne et al. / Chemosphere 71 (2008) 1214–1224 1215

maritimus), arctic fox (Vulpes lagopus), sledge dogs (Canis

familiaris), killer whales (Orcinus orca) and humans (Homo

sapiens) are exposed to the highest levels, and health effectsat the immune, endocrine and reproductive level are there-fore most likely to occur in these species (AMAP, 2004).

The arctic fox has a circumpolar distribution and thehabitat is the Arctic tundra and pack ice (Macpherson,1969; Wrigley and Hatch, 1976). Based on feeding ecology,they can be divided into two different ecotypes, such as‘‘inland” (mainly living on lemmings) and ‘‘coastal” (feed-ing on birds and marine mammals) foxes (Braestrup, 1941).Such differences in habitat and feeding ecology seems toresult in differences in tissue concentrations of PCBs/OCPswhere coastal arctic foxes in Svalbard have higher levelscompared to the inland arctic foxes from Canada andAlaska (Hoekstra et al., 2003; Fuglei et al., 2007). Arcticfoxes living in Svalbard have a more marine diet and feedhigher in the food chain compared to foxes in Canadaand Alaska (Hoekstra et al., 2003; Fuglei et al., 2007).For coastal foxes at Svalbard, the food availabilitybecomes limited during autumn and winter when they relyon carcasses of reindeer (Rangifer tarandus platyrhynchus),seals, Svalbard rock ptarmigan (Lagopus muta hyperbo-

rean) and food stored during spring and summer (Frafjord,1993; Prestrud, 1992). To some extent, the arctic foxes fol-low the polar bears to feed on killed seal remains (Hirukiand Stirling, 1989). Finally the arctic fox are capable ofkilling and eating seal spp. (Phocidae) pups (Roth, 2002).

Studies of tissue concentrations of PCBs (polychlori-nated biphenyls) in arctic foxes at Svalbard have shownconsistently high levels (Norheim, 1978; Wang-Andersenet al., 1993; Severinsen and Skaare, 1997; AMAP, 2004;Fuglei et al., 2007). PBDEs (polybrominated diphenylethers) are also detected in arctic foxes from Svalbard,but in significantly lower levels and with a somewhat differ-ent metabolism and excretion of BDE congeners comparedto other top predators (Fuglei et al., 2007; Wolkers et al.,2004; Muir et al., 2006). The PCB levels in arctic foxesfrom Svalbard are up to 40% higher than those found inmale polar bears from Svalbard (AMAP, 2004; Bernhoftet al., 1997; Fuglei et al., 2007; Norheim, 1978; Severinsenand Skaare, 1997; Wang-Andersen et al., 1993). The PCBcongener pattern found in arctic foxes is similar to thatfound in polar bears, suggesting a similar metabolismand possible effects on vital functions such as reproduction,immunity (disease resistance) and endocrine homeostasis(AMAP, 2004; Fuglei et al., 2007; Norheim, 1978; Wang-Andersen et al., 1993). Besides the endocrine and immunesystems, also internal organs might be directly affected.In rats (Rattus norwegicus) and mink (Mustela vision),PCBs exposure has been associated with renal lesions andhepatotoxicity (e.g. Bergman et al., 1992; Bruckner et al.,1974; Chu et al., 1994; Jonsson et al., 1981; Kelly, 1993;Kimbrough et al., 1971; McCormack et al., 1978; Wadeet al., 2002). In wildlife species such as polar bears (Sonneet al., 2005, 2006a, 2008) and ringed seals (Phoca hispida)(Bergman et al., 2001) as well as sledge dogs (Sonne

et al., 2007a, in press-b, 2008) PCB/OCPs exposure hasbeen linked to liver and renal lesions.

Adaptations to Arctic conditions make animals particu-larly sensitive to the effects of PCB/OCPs. For example, tocope with high variations in food availability, animalsbuild up large fat reserves when food is available, and uti-lize these reserves when food intake is limited. Hence, Arc-tic animals display marked seasonal cycles of ‘‘fattening”

and emaciation. The deposition of lipophilic contaminantsoccurs mainly in these adipose tissues. However, when lip-ids are mobilized to meet energy demands, accumulatedPCB/OCPs become bioavailable and may reach sensitivetissues like liver and kidney. Conclusions on previouslyconducted studies of negative health effects from PCB/OCPs in Arctic top predators have typically been basedon correlations between the effect parameters and individ-ual PCB/OCP levels (e.g. Braathen et al., 2004; Haaveet al., 2003; Skaare et al., 2001; Sonne et al., 2004,2006b). Cause-effect relationships are difficult to establishin such wildlife studies and long-term contamination exper-iments under controlled conditions in the lab has thereforebeen warranted. Hence, the present study was undertaken,in which the impact from PCB/OCPs on liver and kidneymorphology were investigated in farmed arctic fox fed foralmost 2 years a diet containing ‘‘naturally” contaminatedminke whale (Balaenoptera acutorostrata) blubber.

2. Materials and methods

2.1. Animals and housing

The experimental animals were farmed arctic foxes ofthe blue colour type. Blue fox farming in Norway startedabout 90 years ago with wild arctic foxes caught in Green-land, Alaska, Iceland and Svalbard. The farmed foxes havesimilar yearly cycles in body fatness as the wild foxes andare therefore a good model for feral arctic foxes. Duringcaptivity, they have been bred for larger body weight toincrease skin size. The experimental animals from whichhistopathology is reported in the present investigationcomprised 31 blue fox male cubs (16 exposed and 15 con-trols), which were siblings from 21 litters born in the periodMay 23 to June 11 2003. However, the present projectstarted out with 42 siblings that were distributed amongthe two treatment groups (exposed vs. controls) in such away that each pair of siblings was allocated both groups.This was to obtain information about weights and contam-inant concentrations in the initial period of the experiment.Information about this is given in Table 1. The entire exper-imental period was from August 13 2003 to June 19 2005(Table 1). The animals were kept in semi-outdoor housesin individual cages (1.5 m � 1.2 m � 1.0 m) equippedwith a resting platform and a wooden box for shelter.The animals were regularly examined by the daily staffand by a veterinarian when requested. The animals wereeuthanized by electrocution carried out with AuthanatosType 3 (Lima A/S, Sandnes, Norway). The apparatus

Table 1Daily mean consumption of food (g/d), metabolizable energy (MJ/d),pork fat (g/d) and whale blubber (g/d). BW: Mean body weight (kg)development in the entire experimental period (672 d). n = number ofsamples in each group

Date Control Exposed

Food(g/d–MJ/d)

Porkfat(g/d)

BW(kg)

Food(g/d–MJ/d)

Whaleblubber(g/d)

BW(kg)

Period 1 August 11 2003 – High energy diet 143 d, n = 21

August 11 2003 3.38 3.36September12 6.00 5.70October17 8.11 7.64December 1 10.36 9.55December 31 11.12 10.31Mean 668–4.98 51.4 645–4.47 49.7

Period 2 January 5 2004 – Low energy diet 213 d, n = 16

January 28 2004 10.63 10.04February 27 9.18 8.56March 25 8.22 7.61April 30 6.77 6.44May 21 6.66 6.05July 5 5.78 5.29July 28 5.75 5.49Mean 325–1.64 6.5 349–1.78 7.0

Period 3 August 9 2004 – High energy diet 111 d, n = 16

September 3 6.78 6.58October 7 8.01 7.46November 9 9.11 8.81November 29 9.52 9.28Mean 458–3.42 36.6 496–3.44 39.7

Period 4 November 29 2004 – Low energy diet 205 d, n = 8

December 28 9.10 8.67January 31 2005 8.68 8.38March 1 8.28 7.93March 29 7.62 7.20May 12 6.44 6.11June 1 6.15 5.83June 19 5.54 5.39Mean 314–1.58 6.3 359–1.83 7.1

Table 2Feed composition and metabolizable energy (ME) data

Ingredients (%) August 13 2003 toJanuary 4 2004August 8 2004 toNovember 28 2004

January 5 2004 toAugust 7 2004November 29 2004to June 16 2005

Control Exposed Control Exposed

Whale blubber – 7.7 – 2.0Pork fat 7.7 – 2.0 –Cod scraps 30.7 30.7 40.0 40.0Poultry by-products 11.5 11.5 4.0 4.0Fishmeal 2.5 2.5 2.0 2.0Meat-and-bone meal 2.5 2.5 3.0 3.0Slaughterhouse by-products 9.6 9.6 8.0 8.0Precooked carbohydrates 11.5 11.5 12.0 12.0Vitamin mineral mixturea 0.1 0.1 0.2 0.2Water 23.9 23.9 28.8 28.8Sum 100.0 100.0 100.0 100.0

Chemical content (%)Dry matter 35.5 34.7 30.3 30.0Ash 3.8 3.8 4.4 3.9Crude protein 13.6 14.0 12.1 12.4Crude fat 10.8 9.0 4.5 4.5Carbohydrates (by difference) 7.3 7.9 9.3 9.2ME content, MJ/kg feedb 7.46 6.93 5.04 5.09

1216 C. Sonne et al. / Chemosphere 71 (2008) 1214–1224

produced 110 V/0.3 A and is made and approved for elec-trocution of farmed foxes only. The electrocution time wasminimum 4 s. The animal experiment was performed on alicence granted by the Norwegian National AnimalResearch Authority and experimental procedures followedNorwegian protocols for ethical standards for the use oflive animals.

ME content, MJ/kg dry matter 21.01 19.97 16.63 16.97Energy distributionc 31-56-13 34-51-15 40-35-25 41-34-25

a Norsk Mineraln�ring, Hønefoss, Norway. Ingredients per kg: VitaminA; 2000000 IU, vitamin D3; 200000 IU, vitamin E; 50000 mg, vitamin B1,15000 mg, vitamin B2; 3000mg, vitamin B6; 3000 mg, vitamin B12; 20 mg;pantothenic acid; 3000 mg, niacin; 5000 mg, biotin; 30 mg, folic acid;300 mg, Fe (amino acid-chelated); 20000 mg, Zn oxide; 7500 mg, Mnoxide; 15000 mg and Cu sulphate; 1250 mg.

b For calculating content of ME, digestibility values found in blue foxesand these factors were applied (kJ/g digestible nutrient): crude protein;18.8, fat; 39.7, carbohydrates; 17.6.

c Percentage of ME from protein, fat and carbohydrates.

2.2. Diet composition and feeding

One group received a diet based on pork (Sus scrofa)fat (control group) and one group received a diet basedon naturally contaminated whale blubber (exposed group)(Table 1). The whale blubber was from minke whalescaught in Norwegian waters and a homogenized batchwas analysed for organochlorine concentrations. Theproduct was packed by Ellingsen A/S, Skrova, Norway.

The whale product consisted of subcutaneous fat (4–6 cm) and corresponding skin. The skin was removedfrom the blubber before use. The pork fat was deliveredfrom Gilde, A/S, Rudshøgda, Norway. All other ingredi-ents were supplied from the commercial market for furanimal feed in Norway. The wet ingredients were fro-zen-stored and partly thawed and ground before mixing.The diet was produced two times weekly and kept in arefrigerator until feeding. The diet composition andmetabolizable energy (ME) data is shown in Table 2.The food was given once daily on a feeding board.Remaining food was recorded and food consumptionwas calculated on a group basis. To support natural bodyfat deposition and mobilization for the two periods, thefoxes were given a high energy diet during summer andautumn, and a low energy diet during winter (Tables 1and 2). The control diet and the exposed diet wereplanned to be isoenergetic, but analyses of the pork fatand whale blubber revealed that the whale blubber con-tained significantly less fat and more protein than thepork fat. Both fat sources were difficult to homogenizebefore analyses and parallel analyses of the same sample

Table 3Polychlorinated biphenyl (PCB) and chlorinated pesticide (OCP) concen-trations (ng/g wet weight) in the minke whale blubber and pork fat feeddiet given to exposed and control foxes

Class Minke whale blubber feed(n = 1)

Difference Pork fat feed(n = 1)

PPCBa 488 >*** 18

POCPb 395 >*** 9

Liver weight 187 > 162Kidney weight 15.5 > 14.9

Furthermore, the average total weight (g) of liver and kidney organswithin the minke whale blubber (exposed) and pork fat (control) group isgiven.***: Significantly highest concentration in the minke whale blubber atp < 0.0001 based on a one-way ANOVA.

a Sum of 104 congeners: 1, 3, 4–10, 7–9, 6, 8–5, 19, 30, 18, 15–17, 24–27,16–32, 54–29, 26, 25, 31–28, 50, 33–20, 53, 51, 22, 45, 46, 52, 43, 49, 47–48,44, 59, 42, 71–41–64, 40, 100, 63, 74, 70–76–98, 66, 95, 91, 55, 56–60, 92,84, 101, 99, 119, 83, 97, 81–87, 85, 136, 110, 82, 151, 135–144, 147, 107,149, 118, 133, 114, 134–131, 146, 153, 132, 105, 141, 179, 137, 176, 130,163–138, 158, 129, 178, 175, 182–187, 183, 128, 167, 185, 174, 177, 202–171, 156, 173, 157–200, 172, 197, 180, 193, 191, 199, 170–190, 198, 201,203–196, 189, 208–195, 207, 194, 205, 206, 209.

b Sum of 1,3-DCB, 1,4-DCB, 1,2-DCB, 1,3,5-TCB, 1,2,4-TCB, 1,2,3-TCB, Hexachlorobutadiene, 1,2,3,4-TTCB, PECB, a-HCH, HCB,Pentachloroanisole, b-HCH, g-HCH(Lindane), Heptchlor, Aldrin,Octachlorostyrene, Heptachlor epoxide, Oxychlordane, g-Chlordane,a-Endosulfan, o,p-DDE, a-Chlordane, trans-Nonachlor, Dieldrin,p,p-DDE, o,p-DDD, Endrin, b-Endosulfan, cis-Nonachlor, p,p-DDD,o,p-DDT, p,p-DDT, Methoxychlor, Mirex.

C. Sonne et al. / Chemosphere 71 (2008) 1214–1224 1217

gave variable results (pork fat: 77.0–85.0% fat, 2.6–5.3%crude protein; whale blubber: 46.7–63.6% fat, 10.3–17.8% crude protein). Thus, during the first part of theexperiment the energy content in the feed was higher forthe control group than in the exposed group. Thisresulted in slightly higher body weights for the controlafter the first part of the experiment (Table 1). Later,the feed allowance was harmonized to attain comparableweights among individuals in the exposed and controlgroup (Table 1).

Time periods in Table 1 show the mean food and energyintake for the two groups with coherent body weight fluc-tuations. In the period of low energy intake, the extensivebody fat was utilized as energy source. The food rationsprovided in this period of the year therefore containedinsufficient energy amounts, but adequate amount of pro-tein to cover maintenance requirements.

On November 29 2004, seven control foxes (one animaldied on August 12 of unknown reasons) and eight exposedfoxes were sacrificed for histopathological examination. Atthe time they were sacrificed the animals had peak body fatdeposits due to several months of excessive energy intake,approximately 250% of daily ME maintenance requirement(375 kJ/kg0.75/d) as determined by Tauson et al. (2002).The remaining 16 foxes were subjected to a period ofrestricted rations (approximately 80 % of ME maintenancerequirement) from December 2004 to June 2005. Therestrictions took place in a period in which the farmed bluefox experienced reduced energetic food intake and subse-quently mobilized body lipid reserves. During winter thefoxes lost 30–50% of their body weight and utilized almostall body fat. On June 16 2005, the remaining 16 foxes (eightexposed and eight controls) were sacrificed for the sameanalyses as described above.

2.3. PCB/OCP concentrations in pork fat and whale blubber

The whale blubber diet contained 488 ng RPCB/g wetweight and 395 ng ROCP/g wet weight (Table 3). ThePCB/OCP concentration of the exposed group over theca. 2-year-study period reached a contaminant body bur-den within the range found in wild arctic foxes at Svalbard(i.e. 10–60 lg RPCB/g fat) (AMAP, 2004; Fuglei et al.,2007). The RPCB ratio in pork fat diet vs. whale blubberdiet was 1:27 and for ROCP 1:44 (Table 3). PCB/OCPanalyses were conducted using methods described inJohansen et al. (2004). In brief samples were homogenizedand Soxhlet extracted with dichloromethane. PCB/OCPswere isolated from lipid co-extractives by gel permeationchromatography followed by fractionation on a silica gelcolumn. Extracts were analysed for 104 PCB congenersand 35 OCPs and chlorinated byproducts (see list in Table3) using gas chromatography with electron capture detec-tion. Certified reference materials from the National Insti-tute of Standards and Testing (NIST 1774b mussel, NIST1588a cod liver oil), and laboratory blanks consisting ofall reagents, were also analysed with each batch of samples.

2.4. Sampling and histopathology

A representative sample of liver and renal tissue fromeach individual was taken within 30 min after euthanasiaand preserved in phosphate buffered formaldehyde. Thetissue was trimmed, processed conventionally, embeddedin paraffin, sectioned at about 4 lm and stained with Hae-matoxylin (Al-Haematein)-Eosin (HE) and periodic acid-Schiff (PAS) for routine diagnostics, Van Gieson and Mas-son Trichrome to detect fibrous tissue (collagen), Best’scarmine to demonstrate glycogen storage, Sudan III todetect lipid (frozen tissue) and Perls’ Prussian blue reactionand Smorl for detecting haemosiderin and lipofuscin pig-ments, respectively (Bancroft and Stevens, 1996; Lyonet al., 1991). The presence of lesions was evaluated fromlow and high power Leica microscope fields (5–40� magni-fication) and grouped as ‘‘present” or ‘‘not present”because it was not possible to grade into severity (e.g. mild,moderate and severe).

2.5. Statistical analyses

Pearson’s chi-squared test with Yates’ continuity correc-tion (2 � 2 Tables) was applied to test for differences in theprevalence of renal lesions between exposed and controlgroups (SAS statistical software package V8 and enterpriseguide V3.0, SAS Institute Inc., Cary, NC, USA). The dif-ference in contaminant concentrations and organ weightswas analysed within a one-way ANOVA incorporating

1218 C. Sonne et al. / Chemosphere 71 (2008) 1214–1224

concentration and weight as dependent variables andgroup as class variable. The level of statistical significancewas set to p 6 0.05 while 0.05 < p < 0.1 was designated asa trend.

3. Results

It should be noted that we – due to field logistics –did not have the opportunity to measure clinical-chemi-cal blood and urine parameters. However, we measuredliver and renal weights and the average of these is givenin Table 3. The weights were all highest in the exposedgroup – most pronounced in the liver – although not sta-tistically significantly (both p > 0.05). Furthermore, levelsof PCBs and chlorinated pesticides was significantlyhighest in the minke whale blubber given the exposedgroup (both p < 0.0001).

Fig. 1. (A) Normal glomeruli from a male arctic fox fed to pork fat (control groand thickening of the glomerular capillary basement membrane (arrow head)group) for 15 months (PAS � 40). C: Glomerular sclerosis (GS), tubular degen(MC) in a male arctic fox exposed to polluted minke whale blubber (exposed

3.1. Renal lesions

Three types of glomerular lesions were found (Fig. 1).Over all, the most prominent changes were PAS positiveglomerular mesangial deposits (GMD) (74%) – accompa-nied by dense Masson Trichrome and PAS-positive colla-geneous fibrosis (glomerular sclerosis) in the most severecases – and glomerular basement membrane thickening(GBMT) (45%). Furthermore, dilatation of glomerularcapillaries (DGC) was found in six individuals (19%).The prevalence of all three lesions was significantly highestin the exposed group (chi-square: all p < 0.05) (Fig. 2),while no significant difference was found between fat andlean animals (chi-square: all p > 0.05).

In general, two types of tubular lesions were found:degeneration of proximal tubules (61%) and medullartubular casts (81%) (Fig. 1). The most prominent feature

up) for 15 months (PAS � 40). (B) Glomerular capillary dilatation (arrow)in male arctic fox fed PCB/OCP polluted minke whale blubber (exposed

eration (TD), tubular protein casts (TC) and mononuclear cell infiltrationsgroup) for 22 months (PAS � 10). Bar = 50 lm.

0

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30

40

50

60

70

80

90

100

TCHC GMD HTBM ILI GBMT DGC IFRenallesion

Prev

alen

ce (%

)

ExposedControls

**

* **

* *

Fig. 2. Prevalence of renal lesions in arctic fox fed PCB/OCP polluted minke whale blubber (exposed group) and pork fat (controls) for 15–22 months.DGC: dilated glomerular capillaries; GMD: glomerular mesangial deposits; GBMT: glomerular basement membrane thickening; HTBM: hyalinization oftubular basement membrane; TCHC: tubular cylindrical hyaline casts; ILI: interstitial lymphohistiocytic infiltration; IF: Interstitial fibrosis. *:Significantly higher prevalence in the exposed group at p < 0.05 level (chi-square).

C. Sonne et al. / Chemosphere 71 (2008) 1214–1224 1219

was mild to moderate PAS-positive hyalinization of thetubular basement membrane (HTBM) and in severe casesaccompanied by tubular dilatation, atrophy and necrosisas well as interstitial fibrosis and glomerular sclerosis.The tubular cylindrical hyaline casts (protein) (TCHC) inthe medulla indicated tubular or glomerular protein loss.

Fig. 3. (A) Mild bile duct hyperplasia accompanied by portal fibrosis in arc(HE � 20). (B) Interstitial lymphohistiocytic infiltration (LI) similar to granulpolluted minke whale blubber for 15 months (HE � 20). (C) Intravascular leupolluted minke whale blubber for 22 months (HE � 40). (D) Focal liver necromonths (HE � 5). Bar = 50 lm.

The prevalence of both lesions was significantly highest inthe exposed group (chi-square: all p < 0.05) (Fig. 2), whileno significant difference was found between fat and leananimals (chi-square: all p > 0.05). Beside this, lipofuscin/haemosiderin (Smorl and Perls’) negative intracellular yel-low–brown pigments were found in the entire nephron of

tic fox male fed PCB/OCP polluted minke whale blubber for 22 monthsomas, steatosis (St) and Ito cell (IC) in an arctic fox male fed PCB/OCPkocyte accumulation in a hepatic vein from an arctic fox male exposed tosis in an arctic fox male exposed to polluted minke whale blubber for 22

1220 C. Sonne et al. / Chemosphere 71 (2008) 1214–1224

all animals and therefore probably represent haemoglobin,melanin or metabolite by-products from digested plantmaterial. Furthermore, hyaline droplet degeneration wasfound mainly in proximal tubules.

Interstitial lymphohistiocytic infiltrations (ILI) andinterstitial fibrosis (IF) was found in 58% and 19% of theindividuals, respectively (Figs. 1 and 2). In three cases ofintense cell infiltrations these were accompanied by tubulardilatation, atrophy and necrosis, but rarely fibrosis ortubular hyaline casts. In the most severe cases of interstitialfibrosis, total PAS-positive glomerular obliteration (fibro-sis) was found (see above). The prevalence of both intersti-tial lymphohistiocytic infiltrations and interstitial fibrosiswas significantly highest in the exposed group. Again, nosignificant difference was found between fat and lean ani-mals (chi-square: all p > 0.05).

3.2. Liver lesions

Six types of liver lesions were found in decreasing order(1) Steatosis (St); Intravascular granulocyte accumulations(IGA); Lymphohistiocytic infiltrations (LI); Lipid granulo-mas (LG); Portal fibrosis (PF); Bile duct hyperplasia (BH)(Fig. 3). For all lesions, the prevalence was highest, butnot significantly, in the exposed group (chi-square: allp > 0.05). A non-significant trend was found for LI andPF (chi-square: both p < 0.08) (Fig. 4). Steatosis in the per-iacinar zone was found in 81% of the animals. Further-more, non-parenchymal lipid vacuoles of diverging sizeand numbers were found in vitamin A storing Ito-cells,mainly in the periacinar zone. Intravascular granulocyteaccumulations were found in 68% of the animals and wereconsidered a reaction towards infection (bacterial or viral).Portal fibrosis accompanied by mild bile duct hyperplasiawas found in three animals from the exposed group. Inter-stitial lymphohistiocytic infiltrations were found in 29% of

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100

St IGA LILive

Prev

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ce (%

)

n.s.

n.s.

n.t.

Fig. 4. Prevalence of liver lesions in arctic fox exposed to minke whale blubbeSt: steatosis; IGA: intravascular granulocyte accumulations; LI: lymphohistiochyperplasia. n.t.: non-significantly trend of a higher prevalence in the exposeexposed and control group (chi-square: p > 0.1).

the animals, while lipid granulomas were found in 26% ofthe animals. These changes were categorized/diagnosed aschronic multifocal hepatitis. Furthermore, multifocalnecrosis was found in two individuals fed minke whaleblubber. The frequencies were not significantly differentbetween fat and lean animals for any of these lesions(chi-square: all p > 0.05).

4. Discussion

Since the foxes were of very similar genetics (breed andbrother-pairs), had the same age and body condition,received feed of the same composition, and had the sameenergy intake, the present study design allowed us to elim-inate important confounding factors which may otherwisehave impacted the histological parameters. However, thelipid classes in the feed differed between the control andthe exposed group, as did some of the metal and vitaminconcentrations. We can therefore not eliminate lipid andvitamin/element signals as a course of treatment differencesin histological parameters (Sonne et al., 2006c). On theother hand, the aim of the study was to evaluate the effectfrom an environmental ‘‘cocktail” of high lipid levels andPCB/OCPs vs. a diet high in lipids but low in pollutants,which facilitate the extrapolation to correlative wildlifestudies. The current study is one of the very few studieswhere a wildlife species (i.e. arctic fox) has been exposedto an ecologically relevant concentration and compositionof PCB/OCPs over a longer period. Furthermore, concen-trations of brominated flame retardants and perflurinatedacids (PFAs) were not analysed for and therefore we can-not exclude these groups of organic pollutants as co-factorsin the present study.

In contrast to correlative field studies, where the designusually does not allow conclusions to be made regardingcause-effect relationships, this study has shown that farmed

LG PF BHr lesion

ExposedControls

n.s.

n.s.n.t.

r as PCB/OCP source (exposed) and pork fat (controls) for 15–22 months.ytic infiltrations; LG: lipid granulomas; PF: portal fibrosis; BH: bile duct

d group (chi-square: p < 0.1). n.s.: no significantly difference between the

C. Sonne et al. / Chemosphere 71 (2008) 1214–1224 1221

arctic foxes subjected to a contaminated diet exhibit mor-phological abnormalities in kidney – and to some degreealso in liver – when compared to control animals. Althoughthere were some differences in fatty acid composition, vita-min and mineral content in the feeds given to exposed andcontrol groups, it is unlikely that these were the cause ofthe observed differences. This because the pattern of tissuelesions observed corresponds well with the findings in othercontaminant studies (Bergman et al., 2001; Koponen et al.,2001; Sonne et al., 2006a).

4.1. Renal lesions

The main finding of the current study was the presenceof a significantly higher incidence of renal lesions in theexposed animals. The types of glomerular, tubular andinterstitial lesions found in the farmed foxes have previ-ously been ascribed to age and infectious diseases in vari-ous domestic and wild mammals (Confer and Panciera,1995; Bergman et al., 2001; Maxie, 1993; Woshner et al.,2002) as well as in humans (Churg et al., 1995; Cotranet al., 1999). This type of glomerular pathogenesis isthought to be due to exposure to immune complexes initi-ated by chronic infections which subsequently induces mes-angial phagocyte and podocyte proliferative changes(Maxie, 1993; Confer and Panciera, 1995). However, theimmune complex deposition on the epithelial side of theglomerular capillary basement membrane has also beenassociated with various chemicals and toxic agents (Maxie,1993; Churg et al., 1995; Confer and Panciera, 1995;Cotran et al., 1999). In wildlife, renal lesions have beenassociated with exposure to PCB/OCPs and heavy metalsin European coastal waters (Bergman et al., 2001) and inthe Arctic (Sonne et al., 2006a). In Baltic seal species, glo-merular lesions similar to those found in the farmed foxeswere associated with individual levels of PCB/OCP whilethe nature of tubular lesions also pointed towards PCB/OCP exposure as a possible co-factor (Bergman et al.,2001). In fish, similar dilatations of the glomerular capillar-ies and mesangial deposits as those found in the foxes havebeen found in environmentally contaminated bream (Abr-

amis brama) and asp (Aspius aspius) (Koponen et al., 2001).Also polar bears from East Greenland showed similar

renal lesions that could be linked to PCB/OCPs derivedmainly from ringed seal blubber (Ramsay and Stirling,1988; AMAP, 2004; Sonne et al., 2006a). The renal lesionsreported by Sonne et al. (2006a) could be correlated withvarious sum classes of PCBs, OCPs and polybrominateddiphenyl ethers. Similar morphological renal changes/lesions were also found by Sonne et al. (2007a) in a studyon renal morphology in PCB/OCP-contaminated Green-land sledge dog bitches and their pups. Furthermore, theprevalence of interstitial cell infiltrations were most pro-nounced in the PCB/OCP exposed farmed foxes.

The above mentioned indirect pathogenesis could bemediated via PCB/OCP-induced immunosuppression assuggested for polar bears (Lie et al., 2004, 2005) and sledge

dogs (Sonne et al., 2006c, 2007b), which increases the ani-mal’s susceptibility toward infections from micro patho-gens. For some of the other renal lesions a more directrenal cellular toxic mechanism could be suspected (Maxie,1993; Confer and Panciera, 1995). Although the age of theanimals were a maximum of 2 years, age and recurrentinfections cannot be excluded as co-factors in the develop-ment of interstitial fibrosis, as previously reported in e.g.wild ringed seal and grey seal (Halichoerus grypus) in WestGreenland and the Baltic (Bergman et al., 2001; Sonne-Hansen et al., 2002).

In winter the arctic fox rely mainly on scavenges (Pres-trud, 1992; Frafjord, 1993) and the yearly cycling in foodavailability subsequently induce an oscillation in theamount of lipid reserves (Prestrud and Nilssen, 1992). Ina study of tissue samples from wild arctic foxes from Sval-bard, Fuglei et al. (2007) found lower levels of contaminantconcentrations in fat arctic foxes compared to lean foxes.In the present study, foxes were held on a restricted feedration during winter to mimic the fat mobilization, andconcurrent contaminant mobilization in wild foxes (Polis-chuk et al., 1995; Prestrud and Nilssen, 1992; Wolkerset al., 1998). There was, however, no difference in the prev-alence of renal lesions between fat and lean farmed foxes,indicating that these lesions have most likely developedover the entire study period (2003–2005).

Renal lesions similar to those described above have beenassociated with renal failure in domestic animals (Maxie,1993; Confer and Panciera, 1995). However, in such casesall lesions were diffuse and more severe compared to thepresent study where the lesions were multifocal. It cantherefore not be concluded if the lesions caused any renalfailure in the farmed foxes in the most severe cases(Fig. 1C), although an impact on the most susceptible indi-viduals must be expected.

4.2. Liver lesions

Several studies have shown that OHCs – such as PCBs –induce hepatomegaly (enlarged liver) probably due to thehigher metabolic requirements (Parkinson, 1996). Wefound evidence of hepatomegaly in the present studyalthough the ca. 13% higher liver weight in the exposedfoxes relative to the control group was not significantlyhigher. No significant difference in the prevalence of liverlesions existed between the exposed and the control groupalthough a trend toward higher frequencies of lymphohist-iocytic infiltrations and portal fibrosis was found in theexposed foxes. All incidences of lesions were, however,higher in exposed than in control foxes. Although mostof these differences were small and not significant, the over-all picture reveals a consistent trend. Such a trend is sup-ported by the findings in a similar controlled study onsledge dogs (Sonne et al., in press-b, 2008). In the sledgedog study, the prevalence of portal fibrosis, mild bile ducthyperplasia and vascular leukocyte infiltrations was signi-ficantly highest in the exposed group. Furthermore, field

1222 C. Sonne et al. / Chemosphere 71 (2008) 1214–1224

studies on polar bears from East Greenland showed thatchanges in hepatic morphology were associated withPCB/OCP exposure (Sonne et al., 2005, in press-a). Thelesions observed in the polar bear liver tissue were similarto those from controlled laboratory studies on variousmammals exposed to chlorinated industrial compoundssuch as PCBs and pesticides (e.g. Bergman et al., 1992;Chu et al., 1994; Jonsson et al., 1981; Kelly, 1993; Kimb-rough et al., 1971). In the polar bears, steatosis increasedsignificantly with concentrations of hexacyclohexanes(HCHs) in adult females, while lipid granulomas increasedsignificantly with hexachlorobenzene (HCB) in adult males.

Hepatic lesions similar to those observed in the presentfox study have also been found in controlled laboratorystudies. Birnbaum et al. (1990) showed that TCDD (dioxin)induced bile duct hyperplasia in mice and Hori et al. (1982)showed that non-coplanar PCBs induced bile duct hyper-plasia and portal fibrosis in female Cynomolgus monkeys(Macaca fascicularis). Furthermore, lymphohistiocytic cellinfiltrates were associated with subacute PCB exposure inmink and Cynomolgus monkeys (Hori et al., 1982; Berg-man et al., 1992). However, cell infiltrations are non-spe-cific inflammatory reactions against local pathogens andare caused by even minor tissue damage (Kelly, 1993; Mac-Lachlan and Cullen, 1995). Since cell infiltrations and por-tal fibrosis were highest in the exposed foxes, we concludethat PCB/OCPs may be important co-factors in the devel-opment of such chronic inflammation.

Steatosis occurs during high lipid ingestion, starvation,abnormal hepatocytic function, excessive dietary intakeof carbohydrates and decreased syntheses of apoproteins(lipoproteins) (Kelly, 1993; MacLachlan and Cullen,1995; Parkinson, 1996). The large content of hepatic intra-cellular lipids in both exposed and control foxes were prob-ably a function of the high lipid ingestion. However, anadditive effect from PCB/OCPs in the exposed group can-not be excluded as the PCB/OCP signal may be hiddenby the high lipid ingestion. In case of vascular leukocyteaccumulations, the prevalence was almost the same inexposed and control animals and no conclusion or sugges-tions can be made, as the aetiology of such accumulationsare unknown. It should however be noted that Sonne et al.(2008) speculated if the exposed sledge dogs could be moresusceptible to infections from microorganisms based onprevious studies of the immune response in PCB/OCP con-taminated sledge dogs (Sonne et al., 2006c, 2007b).

5. Conclusions

Foxes fed contaminated whale blubber exhibited a sig-nificantly larger prevalence of renal lesions than foxes fedpork fat diet and similar indications were found for liverlesions. The current experiment were conducted in an eco-logically realistic manner, both with regard to contaminantduration and dose, and the results are in agreement withearlier reports on wild polar bears, seal species and sledgedogs chronically exposed to environmental cocktail of sim-

ilar PCB/OCP levels. The most severe lesions may have animpact on the general health status of the most susceptibleindividuals in populations and should therefore be includedin future biomonitoring studies of wildlife top predatorsand humans from regions contaminated by persistentorganic pollutants. We therefore suggest that wild arcticfoxes exposed to persistent organic pollutants in their nat-ural diet are at risk for developing kidney and liver lesions.

6. Conflict of interest – full disclosure

No conflicts of interest were reported.

Acknowledgements

We thank the staff at As for proper care of the foxes andKaroline Sivertsen and Ingeborg G. Hallanger for labora-tory assistance. The Norwegian Research Council (ProjectNo. 153484/S30) and the Lundbeck Foundation areacknowledged for funding of the study.

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