(Canis familiaris) Dietary Exposed to - Springer Link

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C. Sonne,1 S. Fonfara,2 R. Dietz,1 M. Kirkegaard,1 R. J. Letcher,3 S. Shahmiri,3 S. Andersen,1 P. Møller1. 1 Department of Arctic Environment Section for ...
Arch. Environ. Contam. Toxicol. 53, 110–118 (2007) DOI: 10.1007/s00244-006-0135-y

Multiple Cytokine and Acute-Phase Protein Gene Transcription in West Greenland Sledge Dogs (Canis familiaris) Dietary Exposed to Organic Environmental Pollutants C. Sonne,1 S. Fonfara,2 R. Dietz,1 M. Kirkegaard,1 R. J. Letcher,3 S. Shahmiri,3 S. Andersen,1 P. Møller1 1 Department of Arctic Environment Section for Contaminant Effect and Marine Mammals, National Environmental Research Institute, University of Aarhus, Frederiksborgvej 399, P. O. Box 358, DK-4000 Roskilde, Denmark 2 FTZ-Westkste, GKSS-Forschungszentrum, Hafentoern 1, 25761 Bsum, Germany 3 National Wildlife Research Centre, Science and Technology Branch, Environment Canada, Carleton University, Raven Road, Ottawa, ON, Canada K1A OH3 4 Vingaards All 51, DK-2900 Hellerup, Denmark (HuntersScience.com)

Received: 11 July 2006 /Accepted: 5 November 2006

Abstract. Exposure levels of persistent organic pollutants, such as polychlorinated biphenyls and DDTs, are high in Arctic apex predators and Inuit peoples and are suspected to have negative impacts on their immune systems. We conducted a controlled generational study on liver tissue and ethylenediaminetetraacetic acid blood cytokine and acute-phase protein (APP) mRNA expressions using reverse transcriptionase–polymerase chain reaction in West Greenland sledge dogs (Canis familiaris); contaminated minke whale (Balaenoptera acutorostrata) blubber serves as dietary pollutant source. Two of seven blood cytokine expressions (IL-6 and IL-12) and three of five APP expressions (haptoglobin [HP], heat shock protein, and fatty acid–binding protein [FABP]) were lowest in the exposed group, whereas the remaining five blood cytokine expressions (IL-2, IL-10, IFN-c, TNF-a, and TGF-b) and two APP expressions (MT1 and MT2) were highest in the exposed group. In liver tissue, three cytokine expressions (IL-10, IFN-c, and TNF-a) and two APP expressions (MT1 and MT2) were highest in the exposed group, and the remaining cytokine and APP expressions were lowest in the exposed group. Of these, the liver tissue expression of HP and FABP was significantly lowest in the exposed group (both p < 0.05). As a consequence of our findings, we suggest that a daily intake of 50 to 200 g polluted whale blubber is associated with a genotoxic decrease in HP and FABP gene expression in the liver of sledge dog and possibly of other top mammalian marine predators and consumers in the Arctic, indicating a restricted acute-phase reaction and insufficient immune response. Finally, HP and FABP liver expression appear to be new and sensitive biomarkers of organohalogen compound exposure.

Anthropogenic organohalogen compounds (OHCs), such as polychlorinated biphenyls (PCBs) and DDTS, are highly

Correspondence to: C. Sonne; email: [email protected]

lipophilic and are relatively resistant to xenobiotic metabolism; consequently, they accumulate in adipose tissue (e.g., seal blubber) depending on the exposed organisms (Arctic Monitoring and Assessment Programme [AMAP] 2004). OHCs typically reach the Arctic by way of long-distance atmospheric transport, from midlatitude sources in North America and Eurasia, and are subsequently integrated into marine food webs. Biomagnification of OHCs in Arctic wildlife and food webs is facilitated by high lipid content (energy resource) and lower rate of metabolism and elimination relative to the uptake and accumulation rate (AMAP 2004). The levels of OHCs are particularly high in the blubber of Greenland marine mammals, and concentrations are in ranges believed to have a negative impact on wildlife health (AMAP 2004). OHCs in wildlife are known to function as xenoagonist/ antagonist endocrine disrupters (EDs) (Colborn et al. 1993; Damstra et al. 2002). For developing and young mammals, exposure to OHCs is by way of the mother. For example, female polar bears transfer a high burden of OHCs transplacentally to the developing foetus and to newborns and young by way of lactation and milk consumption (Bernhoft et al. 1997; Polischuk et al. 2002). Depending on the OHC, interference can occur with receptors in the main endocrine pathway and may result in ED of the hypothalamus–hypophysis–target organ and tissue axis. In mammals, this can result in disruption of cortisol, thyroid hormones (T3 and T4), and sex steroid hormone levels in blood plasma, increasing the potential for a negative health impact on reproductive organs and immune system (AMAP 2004; Colborn et al. 1993; Damstra et al. 2002; Lie et al. 2003, 2004; Sonne et al. 2006a). Immune system cells, such as macrophages, monocytes and also endothelial cells, produces pro- and anti-inflammatory cytokines and are responsible for the initiation and progression of immune responses (DiPrio 1997; Elenkov and Chrousos 1999). The cell subpopulations are differentiated by their contrasting and cross-regulating cytokine expressions (Mosmann & Sad 1996). Th1-cells produce proinflammatory cytokines,

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Immunotoxicologic Effects in Sledge Dogs

such as interleukin (IL)-2, IL-12, and interferon (IFN)-c, to stimulate the cellular immune response, whereas Th2-cells produce anti-inflammatory cytokines, such as IL-4, IL-5, IL10, and IL-13, which regulate the immune response (Elenkov & Chrousos 1999; Kidd 2003; Lucey et al. 1996; Mosmann & Sad 1996). Th3-cells are the source of transforming growth factor (TGF)-b, a cytokine with strong anti-inflammatory characteristics (Mills 2004; Wahl et al. 2004; Weiner 2001). During the progression of an immune reaction, proinflammatory cytokines IL-1, IL-6, and tumour necrosis factor (TNF)-a initiate the expression of acute-phase proteins (APPs). In veterinary medicine, APPs are used as inflammation and stress biomarkers (Daffada et al. 1999; Hiss et al. 2003; Murata et al. 2004; Petersen et al. 2004; Sato et al. 1994). A correlation between different pollutant (e.g., OHC) burdens and APP expressions has been previously detected in fish species, cell cultures and coke-oven users, and APP as an environmental stress monitoring tool has been discussed (Ait-Aissa et al. 2000; Mukhopadhyay et al. 2003; Wong et al. 2000; Xiao et al. 2002). We recently showed that the cellular immune system of domestic sledge dogs (Canis familiaris) was negatively impacted by a marine-based diet containing high levels of OHCs (Sonne et al. 2006a). We have also shown that a relationship between daily in utero exposure to OHCs and the presence of hypospadias is indicated in East Greenland sledge dogs (Sonne et al. [submitted]). Furthermore, polar bears from East Greenland, Svalbard. and the Western Russian Arctic are among the most OHC-polluted species and populations in the Arctic because they rely on blubber mainly from ringed (Phoca hispida) and bearded seals (Erignathus barbatus) (AMAP 2004; Ramsay & Stirling 1988). In Svalbard and East Greenland, OHCs may have an impact on internal organs (Kirkegaard et al. 2005; Sonne et al. 2005, 2006b), the skeletal system (Sonne et al. 2004), and hormones (Braathen et al. 2004; Haave et al. 2003; Oskam et al. 2003, 2004; Skaare et al. 2001) in polar bears. Furthermore, OHC levels are believed to negatively affect size of sexual organs in East Greenland bears (Sonne et al. 2006c). We conducted a controlled generational study on cytokine and APP expressions in West Greenland sledge dogs as a surrogate model for high trophic-level/top-predator Arctic wildlife and humans. The study design was based on exposures to a natural marine mammalÕs diet of local and OHC-polluted minke whale blubber. Chemical contamination, including identified OHCs, represented concentrations and composition simulating what is ingested by other top predator Arctic wildlife and Inuit peoples. We hypothesized that OHC-exposed sledge dogs and their first-generational offspring, relative to a control cohort, would have differentiated genotoxic related cytokine and APP expressions and thus effects on the humoral immune system.

Table 1. Data on the sister-wise paired 15 Greenland sledge dog bitches in the P generation and their 8 pups (F generation) at time of samplinga Weight (kg) Sex (generation)

Age (days)

Exposed

Control

Bitches (P) Bitches (P) Bitches (P) Bitches (P) Bitches (P) Bitches (P) Bitches (P) Bitch (P) Pup (F) Pup (F) Pup (F) Pup (F) Pup (F) Pup (F) Pup (F) Pup (F)

371 399 497 553 553 630 630 658 336 336 336 357 357 357 357 357

22.3 24.1 23.0 27.5 28.6 25.7 21.5

21.9 24.1 23.3 28.0 28.2 27.5 21.3 20.7

22.0 24.9 21.1 24.1 21.9 18.9 17.4 24.2

a

Bitches were followed-up to 82 weeks and pups for 48 and 51 weeks respectively.

were performed with a licence granted by the Home Rule Government in Greenland. The experimental design was conducted as a randomized blind intervention study in Aasiaat, West Greenland, using reallife exposure and taking into account seasonal fasting from yearly climatic oscillations and environmental factors, such as infectious agents. The parent bitch group (P generation) was composed of pairwise sisters randomly divided into an exposed group and a control group (eight controls and seven exposed because one bitch died before sampling) and their eight pups (four in each group, all siblings; F generation) (Table 1) to minimize influence from age, sex, and genetics. Furthermore, all bitches were mated with the same 5-yearold male sledge dog. The exposed group was fed 50 to 200 g/d (equivalent of 2.9 to 7.0 g/kg) OHC- and mercury-contaminated West Greenland minke whale blubber rich in polyunsaturated lipids. The control group was likewise fed 50 to 200 g pork fat/d (low in OHCs, mercury, and polyunsaturated fatty acids) to balance energy intake by attaining a comparable weight among siblings in the two groups (Table 2). Bitches were fed exposed and control food, respectively, immediately after entering the project at age 2 months, whereas pups were fed exposed and control food after weaning at age 6 to 8 weeks. All dogs were kept in the locally required chains, which also served to control food intake. The dogs were exercised regularly and examined regularly by a field veterinarian. The dogs were also immunized for canine distemper virus, parvovirus, hepatitis virus, parainfluenza virus, and rabies (Duramune 4 Vet Scanvet). They were fed equal amounts of standardized Royal Canin Energy 4300/4800 dry dog pellets (50 to 200 g/d) to cover basic nutrients and microelements (see http://www.royalcanin.com/). The P and F generations were followed for up to 82 and 51 weeks, respectively. It is noteworthy that the pups had received OHCs transplacentally and by way of milk during their first 8 weeks of life (Polischuk et al. 2002).

Materials and Methods Experimental Design

Analyses of Cytokines and APPs

The present field study is part of a larger study of persistent organic pollutants in West Greenland sledge dogs. The animal experiments

All dogs were fasted for 24 hours before they were killed and sampling begun. A volume of 10 mL ethylenediaminetetraacetic acid

C. Sonne et al.

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Table 2. Composition of minke whale blubber, pork fat, and Royal Canin (RC) feed to exposed and control Greenland sledge dog bitches and their pups. Compound Pollutants (ng/g)b P PCBc Chlordanes P DDT Dieldrin P HCH HCB P PBDEd Total mercury Lipids (mg/g) Total lipid n-3 fatty acids n-3 Fatty acids of total fatty acids (%) Vitamins and microelements (mg/kg) Vitamin E Vitamin C Iron Zinc Selenium

Minke whale blubber (n = 3)a

Pork fat (n = 3)a

RC Energy 4300 (n = 3)a

RC Energy 4800 (n = 3)a

1716 296 595 94 20 28 63 0.005

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