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Does the nutrition profile of vitamins, fatty acids and microelementscounteract the negative impact from organohalogen pollutants on bone

mineral density in Greenland sledge dogs (Canis familiaris)?

Christian Sonne a,⁎, Frank F. Rigét a, Jens-Erik Beck Jensen b, Lars Hyldstrup b,Jenni Teilmann b, Rune Dietz a, Maja Kirkegaard a, Steen Andersen c,1,

Robert J. Letcher d, Jette Jakobsen e

a Section for Contaminants, Effects and Marine Mammals, Department of Arctic Environment, National Environmental Research Institute, University of Aarhus,Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark

b University Hospital of Hvidovre, Kettegaards Allé 30, DK-2650 Hvidovre, Denmarkc Vingaards Allé 51, DK-2900 Hellerup, Denmark

d National Wildlife Research Centre, Science and Technology Branch, Environment Canada, Carleton University (Raven Road), Ottawa, ON Canadae National Food Institute, Technical University of Denmark, Mørkhøj Bygade 19, DK-2860 Søborg, Denmark, Denmark

Received 5 November 2007; accepted 30 January 2008Available online 14 March 2008

Abstract

There is a great need for understanding the impact from dietary OHCs (organohalogen compounds) on bone mineral composition – andthereby osteoporosis – in especially arctic wildlife such as polar bears (Ursus maritimus) as well as humans. For that purpose, we measured BMD(bone mineral density) by DXA scanning (g/cm−2) in 15 age and weight normalized sledge dog (Canis familiaris) bitches and their 26 pupsdivided into a control group (n=26) given 50–200 g/day clean pork (Suis scrofa) fat and a treated group (n=15) given 50–200 g/day OHCpolluted minke whale (Balaenoptera acutorostrata) blubber as main lipid sources. The results showed that BMD increased significantly with age(linear regression: pb0.0001, r2=0.83, n=41) while no sex difference was found in the F-generation (two-way ANOVA: all pN0.3). Nodifferences in BMDfemur or BMDvertebrae between exposed and control individuals in the bitch generation were found (linear mixed effect model:both pN0.38). Likewise, no difference between exposed and control subadults and juveniles in the F-generation was found (two-way ANOVA: allpN0.33). Correlation analyses between BMDfemur, BMDvertebrae and groups of OHCs, respectively, did not show any statistically significantrelationships nor a clear or decreasing trend (Pearson's: p: 0.07–0.78; r: −0.2–0.59; n: 10–18). As the groups were similar regarding genetics,age and sex are the only factors that can explain this observation. Either the pollutants did not have an impact on BMD using the present timeframe and OHC concentrations (threshold levels not reached), or the difference in food composition (mainly vitamins and n3 fatty acids) concealthe potential OHC impact on BMD. Such information is important when evaluating the positive and negative health consequences from eatingpolluted marine species.© 2008 Elsevier Ltd. All rights reserved.

Keywords: Balaenoptera acutorostrata; BMD; Bone; Canis familiaris; DDTs; Greenland; Mercury; Minke whale blubber; OCs; Organochlorine pesticides; PBDEs;PCBs; Pork fat; Sledge dogs

Available online at www.sciencedirect.com

Environment International 34 (2008) 811–820www.elsevier.com/locate/envint

⁎ Corresponding author. Section for Contaminants, Effect and Marine Mammals, Department of Arctic Environment, National Environmental Research Institute,University of Aarhus, Frederiksborgvej 399; PO Box 358, DK-4000 Roskilde, Denmark. Tel.: +45 4630 1200 (switch board), +45 4630 1954 (direct), +45 2521 4686(cell); fax: +45 4630 1914.

E-mail addresses: csh@dmu.dk (C. Sonne), ffr@dmu.dk (F.F. Rigét), jebj@dadlnet.dk (J.-E. Beck Jensen), larshyldstrup@dadlnet.dk (L. Hyldstrup),jenniteilmann@hotmail.com (J. Teilmann), rdi@dmu.dk (R. Dietz), mki@dmu.dk (M. Kirkegaard), stan@dk4.dk (S. Andersen), Robert.Letcher@ec.gc.ca(R.J. Letcher), jja@food.dtu.dk (J. Jakobsen).

URL: http://www.neri.dk (C. Sonne).1 HuntersScience.com.

0160-4120/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.envint.2008.01.009

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1. Introduction

There is a large knowledge gap in understanding the inter-action between bone mineral composition and organohalogenEDCs (endocrine disrupting contaminants) such as PCBs(polychlorinated biphenyls) and DDT (dichlorodiphenyl tri-chloroethane). For wild mammals and humans such investi-gations have only been carried out on Baltic grey seals(Halichoerus grypus) (Lind et al., 2003), East Greenland polarbears (Ursus maritimus) (Sonne et al., 2004, 2006a) andSwedish and Inuit populations (Alveblom et al., 2003; Coteet al., 2006; Glynn et al., 2000). All these studies suggest thatorganochlorines may cause endocrine and stress relateddisruption of the bone mineral composition. From variouscontrolled studies it has been suggested that such a cause–effectbetween contaminant exposure and bone mineral compositionexists (Lind et al., 1999, 2000a,b; Lundberg et al., 2006; Navasand Segner, 1998). In general, the pathogenesis of stress-inducedbone mineral changes is an activation of the hypophyseal–adrenal/thyroid axis, leading to enhanced cortisol and thyroidhormone secretion and increased osteoclastic bone resorption,while osteoblast bone formation is decreased (Colborn et al.,1993; Damstra et al., 2002; Feldman, 1995; Ganong, 1997;Selye, 1973; Tung and Iqbal, 2007 and references therein).Whether this mechanism also applies to contaminant-inducedbone mineral loss is uncertain. However, it is known that bonemineral changes also result from in uteri exposure, nutritionalcomposition of food and the age of the individual (Lundberg et al.,2006; Palacios, 2006). Of particular interest for contaminant-bonecomposition relationships is the relation to the risk of developingosteoporosis in both wildlife and humans (Alveblom et al., 2003;Beard and Jong, 2000; Cote et al., 2006; Glynn et al., 2000; Lindet al., 2003; Sonne et al., 2004, 2006a) with both conservation andsocio-economic consequences.

EDC exposure is an important issue to arctic biota (AMAP,1998, 2004). From a global perspective, arctic wildlife is amongthe most contaminated with pollutants such as PCBs, DDTs andPFAs (perfluorinated acids) (AMAP, 1998, 2004; Muir et al.,2006; Smithwick et al., 2005; Verreault et al., 2005). In general,the presence of such compounds in the arctic marine environmentis the result of long-range atmospheric transport from lowerlatitude sources in industrial areas of the world, where outputs anduse of PCBs peaked in the 1960's (AMAP, 1998, 2004). Due totheir high affinity for lipids and proteins, organohalogens andtheir metabolites persist and accumulate in the environment andassociated food webs (AMAP, 1998, 2004; Muir et al., 2006;Smithwick et al., 2005; Verreault et al., 2005). In arctic toppredators such as polar bears, arctic fox (Alopex lagopus) andsledge dogs (Canis familiaris), organochlorines are consequentlytransferred transplacentally frommother to fetus and via lactation,resulting in fetal and neonatal exposures that have the potential foradverse health effects on various organs and endocrine/immunesystems. Polar bears in East Greenland and in Svalbard have beeninvestigated with respect to their immune, endocrine and organsystems and correlative studies have indicated that OHCs mayhave an impact on steroid/peptide hormones and vitamins(Braathen et al., 2004; Haave et al., 2003; Oskam et al., 2003,

2004; Skaare et al., 2001), the immune system (Bernhoft et al.,2000; Lie et al., 2004, 2005), internal and reproductive organs(Kirkegaard et al., 2005; Sonne et al., 2005, 2006a,b, 2007a) andbone mineral density (Sonne et al., 2004, 2006a, 2007a). Incontrolled studies of sledge dogs and arctic fox, the abovefindings have been reproduced using approximately the samecocktail and concentrations as those ingested by the polar bears(Sonne et al., 2006c, 2007b,c, 2008, in press).

We presently investigated whether a known cocktail of en-vironmental pollutants (organohalogens and mercury) andvarious nutrients (vitamins, fatty acids and microelements) inarctic minke whale (Balaenoptera acutorostrata) blubber havean impact on bone mineral composition in captive sledge dogs.For that purpose, we conducted a controlled generational studyof bonemineral density in 15 captiveWest Greenland sledge dogsister bitches – including their offspring – fed either the abovementionedminke whale blubber or clean pork (Suis scrofa) fat ascontrol. The advantage of using sledge dogs in the Arctic is theirknown canine physiological parameters and the oppportunity tocontrol genetic components as well as environmental factors andclimatic oscillations.

2. Materials and methods

2.1. Experimental design

The experimental design is described in detail in e.g. Sonne et al. (2006c,2007b,c, 2008, in press). Briefly, the parent bitch generation (P) was composed ofpair-wise sisters randomly divided into an exposed and a control group includingtheir pup offspring (F) (8 control bitches and 18 control pups; 7 exposed bitchesand 8 control pups). That was done to minimize the influence from age, genderand genetics between groups. Furthermore, the pair-wise sisters and their pupswere kept at the same body weight and condition to avoid confounding. Bitcheswere fed exposed and control food, respectively, immediately after entering theproject at age two months (body OHC burden near zero as most were raisedmainly on non-marine food prior to project start) while pups were fed exposedand control food after weaning at age 6–8 weeks. To facilitate the extrapolationfrom sledge dogs to polar bears and Inuits, the exposed group was fed 50–200 g/day (equivalent of 2.9–7 g/kg and 20% of the daily total energy intake) of OHCcontaminated blubber from a West Greenland minke whale rich in polyunsatu-rated lipids and vitamins and low in mercury (Table 1). The control group wasexposed to a similar amount of pork fat/day (low in OHCs, mercury andpolyunsaturated fatty acids). The energy intake was balanced by achievingcomparable weights among siblings in the two groups. The dogs were fed equalamounts of standardized Royal Canine Energy 4300/4800 dry dog pellets tocover basic nutrients and microelements and to standardize weight (http://www.royalcanin.com/) (Table 1). All dogs were kept in the locally required chains,which also served to control food intake. The dogs were regularly exercised andhealth examined by a field veterinarian. The dogs were also immunized forcanine distemper virus, parvovirus, hepatitis virus, parainfluenza virus and rabies(Duramune 4 Vet Scanvet®). The P-generation was followed for up to 669 dayswhile the F-generation was followed for 342 days (subadults) and 49 days(juveniles). Basic biometric is given in Table 2. It is noteworthy, that the pups hadreceived OHCs transplacentally and via milk in their first 8 weeks of life. Theanimal experiments were performed on a licence granted by the Home RuleGovernment in Greenland and conducted in accordance with national andinstitutional guidelines for the protection of animal welfare.

2.2. Osteodensitometry

Bone material was taken less than 30 min after euthanasia from eachindividual and frozen at −20 °C. The right femur and two lumbar vertebrateswere taken from each individual. However, due to preparation techniques (bone

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sutures were not fully closed) it was not possible to scan vertebrates from thejuvenile F-generation. X-ray osteodensitometry was applied to detect osteopenia(osteoporosis) in the bone samples by use of a Norland XR 26 X-ray bonedensitometer (Norland Corporation, Wisconsin, USA), which determined thebone mineral density (calcium-phosphate; hydroxyapatite) during a dual X-rayabsorptiometry (DXA). Both entire femur and vertebrates were scanned in“Small subject” mode (Resolution: 1.0×1.0 mm; Scan width: 6.0 cm; ScanSpeed: 40 mm/s) and analysed in XR software revision 2.4®, which generated apicture of the bone segment and calculated the bone mineral density ofhydroxyapatite (BMDfemur and BMDvertebrae; g cm−2) (Fig. 1). The DXA-scanner was daily calibrated using a phantom with known mineral densityshowing reproducibility N99% and accuracy of ±2SD. Based on an 11-foldrescanning of BMDfemur CV was 1.3%.

2.3. Chemical analyses of dog diet and plasma

Organohalogen contaminant analyses of dog diet and blood plasma samples(Tables 1 and 3) were conducted in the labs of R.J. Letcher at the NWRC(Environment Canada, Carleton University, Ottawa, Canada) according methodsdescribed and validated elsewhere (Dietz et al., 2004; Muir et al., 2006;Verreault et al., 2005). For contaminant analysis the blood was sampled from thecava vein using a Vacutainer™ blood collection system with an 18 gauge needleand sterile Lithium-Heparin Greiner 9 mLVacuette and centrifuged at 3600 rpmfor 10 min. Subsequently 4 mL blood plasma from each dog was sampled in

10 mL sterile Nunc 348224 cryo tubes and kept at −20 °C prior to the OHCanalyses. All analyses for metal and vitamins were performed accreditedaccording to ISO 17025 (ISO, 2000). Metal analyses of food were performed atNERI-DAE Laboratory (Roskilde, Denmark; www.neri.dk) by Flow InjectionAnalyse System for mercury and selenium, and for zinc and iron by AtomicAbsorption Spectrometry. In food, analyses for total vitamin C (ascorbic acidand dehydroascorbic acid) and E (α-tocopherol) were performed at EurofinsLab (Kolding, Denmark, www.eurofins.dk) and for vitamin D (vitamin D3 and25-hydroxyvitamin D3) at National Food Institute (DTU, Søborg, Denmark,www.food.dk) by use of HPLC. Fatty acid and cholesterol analyses of food wereconducted at Biochemistry and Nutrition Group (DTU, Lyngby, Denmark;www.dtu.dk). Fatty acids were analysed using gas-chromatography and flameionization (GC-FID) while cholesterol was measured by high performance thinlayer chromatography and scanning densitometry (Sonne et al., 2006a, 2007b,c).

2.4. Statistical analyses

The statistical analyses were performed with the free software R version2.1.0 and a significance level of p=0.05 was used. To evaluate the relationshipbetween BMDfemur and BMDvertebrae, the linear metric Pearson's correlationanalysis was used while a linear regression was used to assess the relationshipbetween age and BMDfemur. The further data handling was, due to agedependency and generational differences, conducted separately on the P- and F-generations (the F-generation was further divided into a group of juveniles andsubadults). Furthermore, that avoided confounding from age and body weight/condition (please also see above). In the P-generation, it was not necessary toanalyse the effect from age on BMD in the statistical analyses as the pair-wisesisters of same age were split into the control and exposed group, respectively(Fig. 3). A linear mixed effect model with BMDfemur and BMDvertebrae,respectively, as dependent variable, bitch as a random or grouping factor andtreatment as a fixed factor was conducted to evaluate the influence fromtreatment (control vs. exposed) on BMD. From the model we estimated thevariation between pair-wise sister pairs to be 0.0242 for BMDfemur and 0.013

2 forBMDvertebrae which was several folds higher than the treatment (control vs.exposed) variation for BMDfemur (0.05

2) and BMDvertebrae (0.0752).

Table 1Pollutants and nutrients in minke whale blubber, pork fat and Royal Canine (RC)feed to exposed and control bitches and pups

Minke whaleblubber (n=3 a)

Pork fat(n=3 a)

RC 4300(n=3 a)

RC 4800(n=3 a)

Lipid-% 72 97 21 30

Pollutants (ng/g lipid weight) b

∑PCBc 1716 b0.01 1.0 0.5Chlordanes 296 b0.01 b0.01 b0.01∑DDT 595 b0.01 b0.01 2.2Dieldrin 94 b0.01 b0.01 b0.01∑HCH 20 b0.01 b0.01 b0.01HCB 28 b0.01 b0.01 b0.01∑PBDEd 63 b0.01 0.9 0.4Total mercury 5 1 1 2

Lipids (mg/g wet weight)Total lipid (mg/g) 571 954 196 263n−3 fatty acids (mg/g) 64.30 8.56 4.53 4.08% n−3 fatty acids of total

fatty acids17.5 1.3 3.4 2.5

Vitamins and microelements (mg/kg wet weight)Vitamin E 75 15 650 650Vitamin D3 0.028 0.005 b0.001 b0.00125OHD3 b0.001 0.002 . .Vitamin C b10 b10 320 320Vitamin A e 6–24 0.09 . .Iron b10 b10 250 250Zinc 0.6 0.1 190 190Selenium 0.15 b0.01 0.25 0.25a The number of replicate analyses.b For BDE-99, concentrations detected in the method blanks were subtracted

from the concentrations in the food samples.c In the Energy 4300 and 4800, theΣPCB concentrations consisted entirely of

CB-153.d ΣPBDE concentration in the pork fat was comprised of 51% BDE-99, 28%

BDE-153 and 21% BDE-183. ΣPBDE concentration in the Energy 4300 and4800 was comprised of 54% BDE-47 and 46% BDE-99.e Based on literature studies (Andersen, 2005; www.foodcomp.dk).

Table 2Information about age (days), body weight (kg), length of femur (mm) and bonemineral density (BMD; g/cm

−2 ) in 41 Greenland sledge dogs exposed to minkewhale blubber (exposed) and pork fat (controls), respectively, and divided intoadults (P-generation), juvenile and subadult pups (F-generation)

Cohortgrouping

Exposedmean±SD (n)

Difference Controlsmean±SD (n)

F-generation subadultsAge 342 (3) = 342 (7)Body weight 22.6±2 (3) Nn.s. 21.3±3 (7)Lengthfemur 194.5±7.8 (3) bn.s. 195±6.7 (7)BMDfemur 0.69±0.04 (3) Nn.s. 0.57±0.17 (7)BMDvertebrae 0.69±0.07 (3) Nn.s. 0.68±0.07 (5)

F-generation juvenilesAge 25.6±21.4 (5) Nn.s. 22.8±8.5 (11)Body weight 1.8±1.4 (5) Nn.s. 1.4±0.7 (11)Lengthfemur 50.4±16 (5) Nn.s. 48.5±22 (11)BMDfemur 0.163±0.027 (5) bn.s. 0.173±0.081 (11)

P-generationAge 524±102 (7) bn.s. 542±107 (8)Body weight 24.6±2.8 (7) Nn.s. 24.4±3.1 (8)Lengthfemur 184±6.9 (7) Nn.s. 181±7.7 (8)BMDfemur 0.691±0.07 (7) bn.s. 0.716±0.03 (8)BMDvertebrae 0.719±0.08 (7) Nn.s. 0.699±0.07 (8)

n.s.: Not significant (pN0.05); F-generation BMD: according to the two-wayANOVAs; P-generation BMD: according to the linear mixed effect model.Other: according to one-way ANOVAs.

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In the subadult F-generation, a two-way ANOVA with BMDfemur andBMDvertebrae, respectively, as dependent variable and treatment and sex asexplanatory variables as well as their inaction links, was conducted to evaluatethe effect from treatment (control vs. exposed). As none of the interaction linkswere significant the relationships were evaluated from the treatment factor(exposed vs. control) and age. A similar model was conducted for BMDfemur inthe juvenile generation. Furthermore, two subadults (lowest observations) andone juvenile (highest observation) pup were evaluated as outliers and thereforeexcluded from the statistical analyses (Fig. 2). Regarding the juvenile andsubadult pups in the F-generation, the statistical analyses were conducted in thetwo groups, separately, and therefore age was not taken into account (Fig. 2).

3. Results

3.1. Diet composition

The contaminant concentrations were several folds higher in theminke whale blubber compared to both pork fat and the Royal Caninepellets (Table 1). N−3 fatty acids, vitamin E, D3 and A, zinc and

selenium were higher in minke whale blubber compared to pork fat,whereas 25-hydroxyvitamin D3, vitamin C and iron were present incomparable amounts. N−3 fatty acids and vitamin D3 were lower inthe Royal Canine dog feed whereas vitamin E, vitamin C, iron and zincwere significantly higher in the Royal Canine pellets compared to theminke whale blubber and pork fat. Total lipids were highest in pork fat,followed by minke whale blubber, Royal Canine 4800 and RoyalCanine 4300. Even though the Royal Canine pellets to some extentcompensated for the differences between the whale blubber and thepork fat, the differences could have the potential of influencing thebone mineral status, being investigated in the present study and asdiscussed later.

3.2. Bone mineral density

In the F-generation, no difference in BMD values was foundbetween males and females or in comparison with the juvenile group(two-way ANOVA: p=0.3) or the subadult group (two-way ANOVAs:

Table 3Blood plasma levels of OHC classes (ng/g lipid weight) in Greenland sledge dogs exposed to minke whale blubber (exposed) and pork fat (controls), respectively

OHCs Exposed Difference Controls BMDfemur BMDvertebrae

Mean±SD (n) Mean±SD (n) (r, p, n) (r, p, n)

∑PCB 11.9±4.25 (10) N⁎⁎⁎ 1.38±0.86 (8) 0.07, 0.78, 18 0.22, 0.38, 18∑PBDE 0.13±0.24 (8) b 0.54±0.45 (4) −0.05, 0.87, 12 −0.21, 0.52, 12∑Chlordane 4.62±1.50 (10) N⁎⁎⁎ 0.13±0.12 (4) 0.25, 0.39, 14 0.40, 0.15, 14∑DDT 0.39±0.32 (8) N 0.11±0.10 (3) 0.19, 0.58, 11 0.44, 0.17, 11Dieldrin 2.70±1.08 (10) N 0 0.59, 0.07, 10 0.32, 0.37, 10∑HCB 0.56±0.21 (9) N 0.08 (1) 0.45, 0.18, 10 0.21, 0.57, 10

Furthermore, the p, r and n values from Pearson's correlation analyses between OHC classes and BMDfemur and BMDvertebrae, respectively, are given.⁎⁎⁎: Significantly different in plasma mean values of OHCs between exposed and control dogs at pb0.0001 (one-way ANOVA).∑PCB: CB-18, −17, −16/32, −31, −28, −33/20, −22, −52, −49, −47/48, −44, −42, −64/41, −74, −70/76, −95, −66, −56/60, −92, −101/90, −99, −97, −87, −85,−110, −151, −149, −118, −114, −146, −153, −105, −179, −141, −130, −176, −137, −138, −158, −178, −187, −183, −128, −167, −174, −177, −202, −171,−156, −200, −157, −172, −180, −170/190, −189, −199, −196/203, −208, −195, −207, −194, −206, −201).∑PBDE: BDE-25, −28, −75, −47, −66, −100, −119,−99, −116, −85, −155, −154, −153, −138, −183, −191, −181, −190, −203, −206.∑Chlordane: heptachlor epoxide, oxychlorodane, trans- and cis-chlorodane, andtrans- and cis-nonachlor. ∑DDT: p,p′-DDE, p,p′-DDD, p,p′-DDT.∑HCB: tetrachloroBz, pentrachloroBz.

Fig. 1. DXA scanning image of femur (left) and two vertebrae (right) from a 342 day old female sledge dog fed pork fat (control). Note the light high density areas (H)of cortical bone tissue (light) and the lower dark density areas (L) of trabecular bone tissue (dark).

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both pN0.53). Furthermore, the BMD variation seemed low (Table 2,Fig. 2). BMD in the femur (mix of cortical and trabecular bone tissue)increased significantly up to ca. 300 days of age (puberty) (linearregression: pb0.0001, r2=0.83, n=41), while that could not be

investigated for BMDvertebrae (mainly trabecular bone) (Figs. 2 and 3).However, the BMDfemur and BMDvertebrae were highly correlated(Pearson's: pb0.0001, r=0.89, n=24) which justified the use ofBMDfemur (and BMDvertebrae) as measurement sites for the mineral

Fig. 3. BMDvertebrae (top) and BMDfemur (bottom) (g/cm−2) vs. age (days) divided on exposed (whale blubber=Δ; broken line) and controlled (pork fat=■; solid line)P-generational sledge dog bitches. Lines do not indicate growth patterns.

Fig. 2. BMDfemur (g/cm−2) vs. age (days) in exposed (whale blubber=Δ) and controlled (pork fat=■) F-generational subadult (age: 342 days) and juvenile (age:

10–49 days) sledge dog pups.

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status of the skeletal system in sledge dogs. Regarding treatment effectin the subadults of the F-generation no clear trend was found. BothBMDfemur and BMDvertebrae was highest in the exposed group althoughnot statistically significant (two-way ANOVA: both pN0.5) whileBMDfemur in the juveniles of the control group was non-significantlyhighest (two-way ANOVA: p=0.34) (Table 2).

In the P-generation, the variation in BMDfemur and BMDvertebrae wasrelatively low and that the pair-wise sisters carried similar BMD values(Fig. 3). Regarding treatment effect in the P-generation, BMDfemur waslowest in the exposed group while BMDvertebrae was lowest in thecontrol group although none of these comparisons were significant(both pN0.05) (Table 2, Fig. 3). Regarding the scatter plots in Fig. 3 noobvious trend for treatment (control vs. exposed) was found and thiswas also supported by the statistical analyses (linear mixed effectmodel: both pN0.38).

3.3. Bone mineral density and OHC concentrations

Although all ANOVAs suggested that there was no effect fromtreatment (control vs. exposed) on BMD, we tested the correlationbetween BMDfemur and BMDvertebrae and individual plasma levels ofPCBs, PBDEs, Chlordane, DDTs, dieldrin and HCB, respectively(Table 3). None of the analyses showed any statistically significantrelationships (all p: 0.07–0.78)while the correlation coefficients varied toa large degree (r: −0.2 to 0.59; n: 10–18). The same result was foundwhen the analyses were employed separately on the P-generation,F-generation subadult and F-generation juveniles (results not shown here).

4. Discussion

BMD seemed to increase up to ca. 400 days of agecorresponding to puberty and onset of first mating attempt/breeding. This is in accordance with the results of earlierinvestigations on arctic wildlife. Sonne et al. (2004, 2006a) andSonne-Hansen et al. (2002) found that BMD increased up to theonset of puberty of breeding consistent with an asymptoticGompertz growth curve. Furthermore, the correlations showedthat both femur and vertebrae osteoid tissues are possiblemeasurement sites of BMD, and thereby osteoporosis, in caninespecies which is in accordance with Sonne et al. (2004) andSonne-Hansen et al. (2002), where it was shown that this was thecase for femur, vertebrae and skulls of polar bears and ringed seals(Phoca hispida). As the adult dogs were all females we did nothave the opportunity to follow the male dogs until they were fullymatured in the present study and therefore we could not determinethe potential BMD sex difference that exists in mammals (e.g.Van Langendonck et al., 2000; Sonne et al., 2004).

Female mammals mobilize and transfer large amounts ofcalcium and phosphate during the gestation and postpartum(suckling) period (Maghbooli et al., 2007 and references therein)and one would therefore expect to see a BMD decrease in thepostpartum bitches. However, we did not observe such a decreaseas the dogswere not examined prior to themating event, due to therelative short study period and the lack of BMD data from oldersledge dog bitches. In the F-generation we did not detect an effectfrom sex onBMD,whichwe explain by the young age of the pupsincluded in the study. According to BMD studies of other Arcticmammals such a sex difference exists in e.g. polar bears but not inringed seals (Sonne-Hansen et al., 2002, Sonne et al., 2004).

However, if a difference exists it will not be obvious until after theonset of puberty (Sonne et al., 2004). A similar pattern exists inhumans where the distinguishing BMD difference between menand women is partly a result of highermuscle mass and strength inmales, leading to higher biomechanical loading of the bone andthereby an increased bone formation through the stimulation of theosteocytic mechanotransduction system (e.g. Van Langendoncket al., 2000).

4.1. BMD and contaminant exposure

Few BMD studies in relation to contaminant exposure havebeen carried out on arctic wildlife species. To our knowledge thishas been done in polar bears (Sonne et al., 2004, 2006a) and ringedseals (P. hispida) (Sonne-Hansen et al., 2002). At southernlatitudes, BMD has beenmeasured in Baltic male grey seals (Lindet al., 2003) and inAmerican alligators (Alligatormississippiensis)in Lake Apopka (Lind et al., 2004). In the case of polar bears andthe grey seals, a negative relationship was found between periodsof PCB/DDT pollution and BMD in skull and radius, respectively.Furthermore, a negative correlation was found between individuallevels of BMD and various OHC groups in polar bears. The OHCgroups found in wildlife were the same groups as those found intheminke whale blubber of the present study. In fact, the exposureof East Greenland polar bears being approximately 2-fold highercompared to the present sledge dogs were similarly based on bodyweight and concentrations of OHCs inEast Greenland ringed sealsand West Greenland minke whale (blubber) (Sonne, 2004,unpublished data). However, Lind et al. (2004) reported aninteresting relationship between tibia BMD and exposure to DDTin juvenile female American alligators. When compared to a non-polluted reference alligator subpopulation, the tibia trabecularBMD in alligators from the polluted Lake Apopka was increasedand it was suggested that environmental estrogenic compounds(DDTs and its metabolites) disrupted the normal bone remodellingprocess (inhibition of osteoclast activity), which had resulted in theobserved BMD increase. In humans, Alveblom et al. (2003)investigated the incidence of osteoporotic fractures in fishermenand their wives from the Baltic Sea (high pollution) and comparedthese to fishermen from the west coast of Sweden (low pollution)as controls. For vertebral fractures there was a significantly higherIRR (incidence rate ratio) for east coast (Baltic) women comparedto west coast women and a similar but non-significant trend wasfound for men. The PCB concentration (10 congeners) in serumwas 2000 ng/g lipid weight which was significantly highercompared to the west coast population and way above the presentsledge dogs (Table 3).

Bone density expresses the bone mineral compositiondetermined by the activity of osteoblastic bone formation andosteoclastic bone resorption, which is regulated by androgensand estrogens through cytokines forming the calcium-helix-structures of the collagen matrix (DeLillis, 1989; Manalagas andJilka, 1995; Manalagas et al., 1995). Environmental xenoestro-gens – such as certain PCBs and DDTs – may behave asendocrine disrupters and thereby indirectly influence the bonemetabolism and composition. For example, in vitro and in vivostudies have shown PCBs and DDT to be weak agonists/

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antagonists to estrogen receptor-mediated activity and thatOHC-mediated induction of CYP450 activity can impactcirculating sex hormone levels (e.g. estrogens) (Navas andSegner, 1998). Relationships between 4,4′-DDE concentrationsand BMD in humans have been reported (Beard and Jong, 2000;Glynn et al., 2000). Glynn et al. (2000) found significant nega-tive correlations between 4,4′-DDE and BMD in 68 sedentarywomen (where concentrations are lower compared to the presentpolar bears), and concluded that 4,4′-DDE may also have anegative effect on BMD in men (with contaminant levelscomparable to those found in East Greenland polar bears). Theopposite relationship was found for DDT and BMD in LakeApopka alligators as described in the above section. In anotherstudy by Lundberg et al. (2007) of BMD in European commonmale frogs (Rana temporaria) exposed to p,p′-DDE showed thatfemoral cortical bone tissue was negatively affected. Althoughthis is on a lower vertebral species it shows again the complexinteractions between BMD and contaminants.

In the present study we investigated the impact fromenvironmental pollutants on femoral and vertebrae BMD.However, the wildlife studies referenced in the above sectionshows that the relationships may be much more complex thanhypothesized, and that both increases and decreases are possible.Furthermore, it must be noted that Lind et al. used pQCT(peripheral quantitative computed tomography) in their studies,which made it possible to distinguish between cortical andtrabecular bone while the DXA scanning used in the presentstudy gave the average of trabecular and cortical bone density(vertebrates reflected a larger portion of cortical bone tissuewhile femur reflected a larger portion of trabecular bone tissue;Fig. 1). However, it must be noted that the DXA BMD mea-surement is sufficient to detect BMD decreases relevant forclinical osteoporosis research. When comparing the sledge dogsin this study to East Greenland polar bears, the range in ΣPCBand ΣDDT levels were higher in the wild polar bears due tothe shorter exposure time of the sledge dogs and the relatively ca.2-fold higher exposure of the polar bears (Dietz et al., 2004;Sandala et al., 2004; Sonne, 2004, Table 1). Studies on Svalbardpolar bears, which carry levels similar to those from EastGreenland, have shown that PCB may negatively influenceplasma testosterone levels (Oskam et al., 2003) and plasma retinoland thyroid hormone levels (Skaare et al., 2001). These studiesindicate that organochlorines in Svalbard polar bears potentiallyaffect the endocrine homeostasis, which again may lead to bonemineral loss (osteoporosis). Another polar bear study fromSvalbard associated high levels of organochlorines with lowlevels of IgG, suggesting possible immunotoxic effects (Bernhoftet al., 2000; Lie et al., 2004, 2005). This potential effect maylower the immune response and enhance stress with increasedcortisol levels, which have the potential of affecting the bonemineral compositionwhich inworst casemay lead to osteoporosis(e.g. Tung and Iqbal, 2007 and references therein).

4.2. Diet composition and nutrition

Lundberg et al. (2006) showed that perinatal (in uteri) PCBexposure of female goat (Capra aegagrus hircus) offspring

altered the bone mineral composition. In fact, the trabecularbone mineral density measured by pQCT was increased in thegroup exposed to PCB-153 when compared to the control. Inthe F-generation of the present study we did not find such agenerational effect and the frog study by Lundberg et al. showedthat the BMD vs. contaminant relationship is much morecomplex than formerly assumed. As mentioned in the abovesection, sledge dog exposure to OHCs was approximately 0.5that of East Greenland polar bears while the sledge dog BMDvalues was approximately 0.6 the polar bears' reported bySonne et al. (2004). Then, why could we not detect a relationshipbetween OHCs and BMD in the sledge dogsmeasured as a BMDdifference between groups? For the F-generation, the low andunequal sample size of 3 vs. 5, 3 vs. 7 and 5 vs. 11, respectively,could be one of the reasons (Table 2). However, taking thecontrolled age, sex, weight and genetic factors into account, it isnot likely that a larger sample size would have any impact on theresults as the variance and distribution were more or lessthe same between the F-generation groups (Table 2, Figs. 2and 3). We would of course like to have increased the numbersof the F-generation but that was simply not possible due tofield logistics (the same is the case for the P-generationas viewed in Table 2 and Figs. 2 and 3). In fact, clinicallycontrolled case–control laboratory studies are conductedwith groups that include only 7 individuals (e.g. Olsen et al.,2002).

Therefore, another answer could be the food composition. Inour study, we did not have the possibility of measuring vitaminA (retinol and esters) in the pork fat and whale blubber.Furthermore, we could not find any literature about vitamin Ain minke whale blubber. However, based on earlier publisheddata from other Greenland marine mammals like walrus(Odobenus rosmarus), narwhale (Monodon monoceros) andringed seal by Andersen (2005), we estimated the vitamin Aconcentration in the minke whale blubber to be in the range of6–24 mg/kg. Regarding the pork fat, the vitamin A concentra-tion is 0.09 mg/kg measured in Danish pigs used in the presentstudy (www.foodcomp.dk). The concentration of fatty acids,vitamins and microelements was 5–15-folds higher in theexposed minke whale group depending on the compound inquestion. As for the n3−FA difference, studies of women in thepostmenopausal transition have showed that especially theintake of n3−FA was highly associated with femoral BMDmeasured by DXA scanning (Macdonald et al., 2004). Thewomen were followed over a 5-year-period and BMD seemedto decrease with increasing levels of n3−FA, retinol andvitamin E, while BMD increased with increasing levels ofcalcium, magnesium, potassium and vitamin C. However, theeffects found in a male rat (Rattus rattus) study was different(Shen et al., 2006). Here, n3−FA increased BMD and ingeneral was beneficial for local factors, such as enzymeactivities and growth factors, as well as the systemiccalcitrophic hormones such as PTH (parathyroid hormone)and vitamin D. A similar study by Watkins et al. (2005) showedthat a dietary intake of n3−FA similar to that of the presentstudy have a beneficial effect on BMD in ovariectomy femalerats. In the review article by Palacios (2006) a protective effect

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from calcium, protein, magnesium, phosphorus, vitamin D,potassium and fluoride on BMD is documented while theeffects from n3−FA, retinol, vitamin B and vitamin C is moreuncertain. However, oral vitamin C administration seems tohave a positive effect on femur bone formation in Wistar ratstreated with Aroclor 1254 in which bone formation increased,bone resorption decreased and antioxidant enzymes increased(Ramajayam et al., 2007). Regarding vitamin A some studiessuggest that an excess intake of vitamin A (at least 2-foldshigher food concentrations than the present sledge dogs) doesnot have an impact on rat bone composition measured by BMD(Lind et al., 2006), while other studies suggest that a subclinicalhypervitaminosis A increased the risk of bone fractures in rats(Johansson et al., 2002). In conclusion, it is therefore verylikely that the difference in fat source composition on BMD inthe present sledge dog groups could be the explanation to whywe did not see an OHC impact on BMD. At least, suchrelationships may benefit arctic wildlife, such as polar bears, asit at least to some degree could decrease the risk for OHCinduced osteoporosis. The other scenario is that neither the fatcomposition nor the OHC loads have any impact on CanineBMD. However, that is unlikely as discussed in the sectionsabove. Furthermore, minke whale blubber and pork fat were theonly achievable contaminant/adipose sources to use whenaiming at investigating the impact from the OHC “cocktail” in areal-life arctic simulation, as cleaning up the blubber wasexpensive out of all proportions and for some OHCcomponents chemically impossible as well as extractionswould have altered other properties of the fat and vitamincompositions.

In a study by Stern et al. (2005), female Sprague–Dawleyrats were treated with fractions of contaminated herring oil richin n3−FA, vitamin A and D vs. a non-polluted soy oil fractiongiven to a control group. Their BMD findings were consistentwith the present observation in exposed vs. non-exposed sledgedogs. Stern et al. (2005) measured femoral BMD using pQCT(peripheral quantitative computed tomography) and found nosignificant BMD differences between the groups and suggestedthat the various nutritional compounds had synergistic and/orantagonistic effects on BMD and that the potential impact frompollutants and food composition could not be separated. This isthe dogma when using real-life exposures in controlled studies,as discussed by e.g. Sonne et al. (2006c).

5. Conclusions

No obvious BMD difference was found between control andexposed sledge dogs. As most of the biological factors in thestudy were controlled we postulate that the higher levels ofvitamins, n3−FA and microelements in the minke whaleblubber could have counteracted the expected effects. However,in addition it cannot be excluded that the present combination oftime frame and OHC concentrations was not sufficient to reachtoxic threshold levels. Clearly more research is warranted. Suchinformation is important when evaluating the positive andnegative impacts on health and osteoporosis from eatingpolluted marine species.

Acknowledgement

We thank the Danish Cooperation for Environment in theArctic, Natural Science and Engineering Research Council(NSERC) Canada (to R.J. Letcher) and Lundbeck Foundationfor funding, Royal Canine provided reduced prices for pellets andKruuse donated clinical equipment (centrifuge). Thomas DauRasmussen, Anne Silverbau, Mikael and Jane Rasmussen, JørnBreinholt and Jens Kjeldsen are acknowledged for their help andskilled daily dog handling. We also thank Wouter Gebbink andSoheila Shahmiri (NWRC/CWS, Environment Canada) in Dr.Letcher's research group for assisting with the OHC analysis, PerMøller for conducting lipid analyses in the various food items andSigga E. Joensen and Lene Bruun for conducting metal analyses.A conflict of interest was not reported.

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