Are liver and renal lesions in East Greenland polar bears (Ursus maritimus) associated with high mercury levels?

BACKGROUND: In the Arctic, polar bears (Ursus maritimus) bio-accumulate mercury as they prey on polluted ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus). Studies have shown that polar bears from East Greenland are among the most mercury polluted species in the Arctic. It is unknown whether these levels are toxic to liver and kidney tissue.
METHODS: We investigated the histopathological impact from anthropogenic long-range transported mercury on East Greenland polar bear liver (n = 59) and kidney (n = 57) tissues.
RESULTS: Liver mercury levels ranged from 1.1-35.6 microg/g wet weight and renal levels ranged from 1-50 microg/g wet weight, of which 2 liver values and 9 kidney values were above known toxic threshold level of 30 microg/g wet weight in terrestrial mammals. Evaluated from age-correcting ANCOVA analyses, liver mercury levels were significantly higher in individuals with visible Ito cells (p < 0.02) and a similar trend was found for lipid granulomas (p = 0.07). Liver mercury levels were significantly lower in individuals with portal bile duct proliferation/fibrosis (p = 0.007) and a similar trend was found for proximal convoluted tubular hyalinisation in renal tissue (p = 0.07).
CONCLUSION: Based on these relationships and the nature of the chronic inflammation we conclude that the lesions were likely a result of recurrent infections and ageing but that long-term exposure to mercury could not be excluded as a co-factor. The information is important as it is likely that tropospheric mercury depletion events will continue to increase the concentrations of this toxic heavy metal in the Sub Arctic and Arctic marine food webs.

Background

Approximately 200–300 tons mercury (Hg) is yearly transported to the Arctic from lower latitudes via the atmosphere and large scale ocean currents [1,2], hence the Arctic acts as a sink for global emitted mercury due to spring mercury depletion events [3-6]. Mercury is toxic and the organic methyl-mercury (MeHg) form mediates central nervous system toxicity pre- and postnatally mainly via the posterior cortex [7,8]. Mercury levels are high in the Greenland and Faroe populations and a special focus has been to address health effects in these regions as such effects are proposed to have a significant socioeconomic impact [9].

The inorganic form of mercury is toxic to liver and kidney tissues due to the co-enzyme inhibition via high affinity to various microsome and mitochondria SH-group enzymes [8]. Studies of Atlantic bottlenose dolphins (Tursiops truncatus) [10] and Arctic beluga whales (Delphinapterus leucas) [11] have linked mercury exposure to liver histopathological changes. Also, renal glomerular and tubular lesions have been associated with mercury toxicity in controlled laboratory studies [12-14], in humans [15,16], in wildlife [11,17] and in domestic mammals [18,19].

In the Arctic, polar bears (Ursus maritimus) bio-accumulate mercury as they prey on polluted ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) which results in a high mercury uptake [1,2,20]. Studies have shown that East Greenland polar bears are among the most mercury polluted species in the Arctic [1,2]. In target organs like liver and kidney, inorganic mercury concentrations of 2.13–13.4 and 2.87–32.0 μg/g w.w. (wet weight) have been reported [21,22]. These levels exceed threshold levels for lethal mercury toxicity [1,2]. Although mercury is a naturally occurring element in the Arctic, Dietz et al. [23] showed that up to 94% of the mercury in East Greenland polar bears is of anthropogenic origin. We therefore examined liver and kidney histology and mercury concentrations in East Greenland polars sampled during 1999 to 2002 to determine whether high mercury concentrations may cause pathological changes at the histological level in target organs of large Arctic top predators.

Methods

Sampling

Liver (n = 59) and kidney (n = 57) samples were taken by local subsistence hunters in the Scoresby Sound area in central East Greenland (69°00'N to 74°00'N) during 1999–2002. A randomly chosen single renal lobe and a tissue sample from the periphery of a right liver lobe was taken for histological examination and fixed in a phosphate buffered formaldehyde/alcohol solution (3.5% formaldehyde, 86% ETOH and 10.5% H2O) to avoid freeze damage. In addition, sub samples for mercury analyses were stored in separate Polyethylene plastic bags until arrival at the laboratory in Roskilde. All samples were taken <12 h post mortem.

The sample

The sample consisted of 32 subadults (10 females and 22 males), 15 adult females (5 ≥ 15 years) and 12 adult males (4 ≥ 15 years). Of these, 32 were caught during summer (1 June to 30 September) and 27 during winter (1 October to 31 May). The general East Greenland polar bear liver and renal histology, including micrographs, is described by Sonne et al. [24,25].

Age estimation

The age determination was carried out by counting the cementum Growth Layer Groups (GLGs) of the lower I3 tooth after decalcification, thin sectioning (14 μm) and staining (toluidine blue) using the method described by Dietz et al. [26]. Adult males were categorised as ≥ 6 years, adult females as ≥ 5 years, old as ≥ 15 years and the remaining as subadults [27,28]. Furthermore, seasonal differences were investigated as summer: 1 June to 30 September vs. winter: 1 October to 31 May [27].

Liver histology

The liver tissue was trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 μm and stained with Haematoxylin (Al-Haematein)-Eosin (HE) and periodic acid-Schiff (PAS) for routine diagnostics, Van Gieson and Masson Trichrome to detect fibrous tissue (collagen), Best's carmine to demonstrate glycogen storage, Sudan III to detect lipid (frozen tissue) and Perls' Prussian blue reaction and Smorl for detecting haemosiderin and lipofuscin pigments, respectively [29,30].

Six histological features were evaluated: 1. Portal mononuclear cell infiltrations (absent, unifocally, multifocally or diffuse). 2. Random mononuclear cell infiltrations (average no. in 5 fields at 10× magnification). 3. Lipid granulomas (average no. in 5 fields at 10× magnification). 4. Hepatocytic steatosis (intracellular lipid; absent, foamy, multifocally macrovesiculary or diffuse macrovesiculary). 5. Visible Ito cells (average no. in 5 fields at 20× magnification). 6. Mild multifocally bile duct proliferation accompanied by portal fibrosis (absent or present). Each histological change was grouped semi-quantitatively as: 1. Portal mononuclear cell infiltrations: absent = 0; mild = unifocally; moderate = multifocally and severe = diffuse. 2. Random cell infiltrations: absent = 0; mild = Index ]0;1]; moderate = Index ]1;3] and severe = Index ]3;8]. 3. Lipid granulomas: absent = 0; mild = Index ]0;1 [; moderate = Index [1;2[and severe = Index [2;5]. 4. Hepatocytic steatosis: mild = foamy; moderate = multifocally macrovesiculary and severe = diffuse macrovesiculary. 5. Ito cells: absent = 0; mild = Index ]0;10]; moderate = Index ]10;50] and severe = Index ]50;200]. 6. Bile duct proliferation; see above.

Renal histology

Kidney tissue was trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 μm and Periodic acid-Schiff (PAS) and periodic acid silver methenamine (PAS-M) were used to demonstrate glomerular (capillary and mesangial) and proximal convoluted tubular changes; Van Gieson and Masson Trichrome to detect fibrous tissue (collagen) in the glomeruli (glomerulofibrosis) and in the interstitium (interstitial fibrosis) [30]. Seven histological features were evaluated: 1. Glomerular capillary wall thickening (10 randomly selected 5–40× fields). 2. Glomerular mesangial deposits (10 randomly selected 5–40× fields). 3. Tubular epithelial cell hyperplasia (10 randomly selected 5–40× fields). 4. Proximal convoluted tubular hyalinization/atrophy/dilations/necrosis (10 randomly selected 5–40× fields). 5. Tubular medullar hyaline casts (10 randomly selected 5–40× fields). 6. Interstitial fibrosis (10 randomly selected 5–40× fields). 7. Mononuclear cell infiltrations (10 randomly selected 5–40× fields). Each histological change was grouped semi-quantitatively as: 1. Glomerular capillary wall thickening: absent, mild and moderate. 2. Glomerular mesangial deposits: absent, mild and moderate. 3. Tubular epithelial cell hyperplasia: mild: focally; moderate: multi focally. 4. Proximal convoluted tubular hyalinization/atrophy/dilations/necrosis: mild: focally; moderate: multi focally. 5. Tubular medullar hyaline casts: mild: focally; moderate: multi focally. 6. Interstitial fibrosis: mild: focally; moderate: multi focally. 7. Mononuclear cell infiltrations: mild: focally; moderate: multi focally.

Analyses of mercury

Liver (n = 59) and kidney (n = 57) samples were analysed for mercury levels (μg/g w.w.) according to Dietz et al. [23]. The principle was atomic absorption spectrometry (AAS; hydride generation and the flow injection analyses) have previously been described by Asmund et al. [31]. The detection limit was 0.005 mg/kg of dry weight. Analytical quality was ensured by repeated analyses and by frequent analysis of various certified reference materials [TORT-2 (lobster hepatopancreas), DORM-2, and Dolt-3] supplied by the National Research Council of Canada (Marine Analytical Chemistry Standards Program).

Statistical analyses

The statistical analyses were performed with the SAS statistical software package (SAS V8 and enterprise guide V1) and the level of significance was set to p ≤ 0.05, while significance at 0.05