Skeletal pathology and bone mineral density changes in wild muskrats (Ondatra zibethicus) and red squirrels (Tamiasciurus hudsonicus) inhabiting arsenic polluted areas of Yellowknife, Northwest Territories (Canada): A radiographic densitometry study.

The City of Yellowknife is a known hotspot of arsenic contamination and there is a growing body of evidence suggesting that local wildlife in the vicinity of the abandoned Giant Mine site may be at risk of decreased bone mineralization and various bone disorders. The purpose of this study was to preliminarily measure bone mineral density (BMD) changes and investigate the incidence, pattern, and severity of bone lesions in wild muskrats and red squirrels breeding in three (3) catchment areas at different distances from the Giant Mine Site in Yellowknife, Northwest Territories (Canada): ~2 km (location 1), ~18 km (location 2), and ~40-100 km (location 3). Full femoral bones of 15 muskrats and 15 red squirrels were collected from the three sampling locations (5 from each location) and subjected to radiographic analysis and densitometric measurements. The patterns and severities of bone lesions, including changes in bone mineral density, were evaluated and compared between groups. As levels were significantly higher in the bones of muskrats caught from location 1 and 2, relative to location 3. Further, As and Cd levels were significantly higher in the bones of squirrels caught from locations 1 and 2 relative to squirrels caught from location 3. The preliminary results from bones revealed that radiographic abnormalities such as bone rarefaction, osteopenia, and thinning of the femoral shafts with significant ossific cystic lesions and bowing were the most common skeletal pathologies found in bones of red squirrels from the three locations. Radiographic appearances of massive sclerosis and dysplasia, including severe osteocondensation and osteopathia striata-like abnormalities, were found in the bones of muskrats from all the sampling locations. Densitometric evaluation showed no significant differences between the three locations in the bone parameters measured. However, there was a statistically significant correlation between As content in the bones of muskrats and percent fat content in the femur samples, which suggests that accumulation of As could have been a causal factor for a change in percent fat in femurs of muskrats.

Comparative study of arsenic toxicosis and ocular pathology in wild muskrats (Ondatra zibethicus) and red squirrels (Tamiasciurus hudsonicus) breeding in arsenic contaminated areas of Yellowknife, Northwest Territories (Canada).

The Giant Mine is an abandoned gold mine in Yellowknife, Northwest Territories, Canada. Throughout its operation from 1948 to 2004, the Giant Mine released heavy amounts of arsenic trioxide into the environment, thus contaminating the soil and surface water within and around the vicinity of the mine site. Chronic arsenic (As) poisoning negatively impacts wildlife health and can induce multi-organ damages including neurodegeneration and visual dysfunction depending on concentration and duration of exposure. The aim of the current study was to comparatively assess retina layer changes and prevalence of ocular lesions in wild rodent populations (i.e. muskrats and red squirrels) breeding in arsenic endemic areas of Yellowknife, near the vicinity of the abandoned Giant mine site (∼2 km radius), at an intermediate location (approximately 20 km from the mine area) as well as a reference location (spanning 52-105 km from the city of Yellowknife, Canada). Eye globes were removed from euthanized muskrats and squirrels from the three sampling locations with increasing distance from the Giant mine area. Optical Coherence Tomography (OCT) was used to attempt a pan-retinal layer assessment, and histologic examination was utilized for assessment and confirmation of ocular lesions. The retinal layers were measured and statistically compared between the groups based on sampling locations to enhance the scope of histologic evaluations. The preliminary results revealed that thicknesses of ganglion cell layer (GCL), retina nerve fibre layer (NFL), and inner retina layer (IR) were statistically reduced in the muskrats from arsenic endemic area, particularly near the vicinity of the Giant mine compared to the control group. Generalized ocular pathology was histologically confirmed in all the muskrats from the arsenic endemic areas with the manifestation of moderate to severe lymphocytic plasmacytic uveitis (LPU), keratitis and subcapsular cataracts. Inner retinal degeneration was also observed in all the muskrats from the arsenic endemic areas, while muskrats from the control group were predominantly normal. Three muskrats from the control group were noted to have a mild LPU and keratitis. Significant histopathologic changes were not detected in the squirrel eyes from the three groups except for incidental mild cornea scars from all the locations. In general, these preliminary findings confirm the presence of ocular lesions and retina abnormalities in wild muskrats in the Yellowknife area and provide the first evidence of visual dysfunction and impairment in wildlife inhabiting arsenic endemic areas of Canada.

Neuropathological changes in wild muskrats (Ondatra zibethicus) and red squirrels (Tamiasciurus hudsonicus) breeding in arsenic endemic areas of Yellowknife, Northwest Territories (Canada): Arsenic and cadmium accumulation in the brain and biomarkers of oxidative stress.

The brain is one of the critical organs particularly susceptible to the damaging effects of chronic arsenic poisoning and there is a growing body of evidence that suggest that oxidative stress plays a key role in the pathogenesis of neurodegenerative disorders. The aim of this present work was to comparatively assess biomarkers of oxidative stress and status of antioxidant enzyme activities in the brains of muskrats and squirrels breeding in arsenic endemic areas, specifically near the vicinity of the abandoned Giant mine site (~2 km radius), and an intermediate location approximately 20 km from the mine area and in reference locations spanning 52-105 km from the city of Yellowknife, Northwest Territories (Canada). Analysis included measurement of total arsenic and cadmium concentration in the nails, brain, and stomach content of muskrats and squirrels, in addition to biochemical evaluation of lipid peroxidation levels and antioxidant enzymes defense: catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the brain tissues. The results revealed that arsenic concentration in the nails of muskrats collected closest to the vicinity of the mine area was in the range of 11 to 35.1 times higher than those from the reference site. The maximum concentration of arsenic in the nails of muskrats from the intermediate location was 47.6 times higher than the maximum concentration observed in the reference muskrats. Cadmium was generally undetected in the nails of muskrats and squirrels from the three sampling locations. Arsenic in the gut contents of muskrats from the arsenic affected area was 4.5 to 49.1 times higher than those from the reference site. Cadmium levels in the guts of muskrats from the mine area almost doubled those from the reference site. Arsenic accumulated in the nails of squirrels from the areas closest to the mine but was undetected in the squirrel nails from the reference location. The maximum arsenic levels in the stomach content of squirrels from the mine area was ~40 times higher than those from the reference site. Arsenic did not accumulate in the brains of muskrats, but cadmium was detected in a few brains of muskrats. Brains of squirrels from the mine area and intermediate locations accumulated both arsenic and cadmium. The brains of squirrels and muskrats from the arsenic affected area showed no evidence of increased lipid peroxidation compared to the animals from the reference site. However, SOD, CAT and GPx activities in the brains of animals from the arsenic endemic areas tended to be higher compared to the control sites. This is the first study documenting evidence of oxidative stress and altered antioxidant enzyme activities in brains of wild rodent population in arsenic endemic areas of Canada.

Altered neurotransmission and neuroimaging biomarkers of chronic arsenic poisoning in wild muskrats (Ondatra zibethicus) and red squirrels (Tamiasciurus hudsonicus) breeding near the City of Yellowknife, Northwest Territories (Canada).

Chronic arsenic poisoning has been shown to be a risk factor for the development of intellectual disability. Numerous human and animal studies have also confirmed that low-level arsenic exposure has deleterious effects on neurotransmission and brain structures which have been further linked to neurobehavioral disorders. The aim of this present work was to comparatively assess structural brain volume changes and alteration of two (2) neurotransmitters, specifically dopamine (DA) and serotonin (5-HT) in the brains of wild muskrats and squirrels breeding in arsenic endemic areas, near the vicinity of the abandoned Giant mine site in Yellowknife and in reference locations between 52 and 105 km from the city of Yellowknife. The levels of DA and 5-HT were measured in the brain tissues, and Magnetic Resonance Imaging (MRI) was used to attempt brain volume measurements. The results revealed that the concentrations of DA and 5-HT were slightly increased in the brains of squirrels from the arsenic endemic areas compared to the reference site. Further, DA and 5-HT were slightly reduced in the brains of muskrats from the arsenic endemic areas compared to the reference location. In general, no statistically significant neurotransmission changes and differences were observed in the brain tissues of muskrats and squirrels from both arsenic endemic areas and non-endemic sites. Although MRI results showed that the brain volumes of squirrels and muskrats were not statistically different between sites after multiple comparison correction; it was noted that core brain regions were substantially affected in muskrats, in particular the hippocampal memory circuit, striatum and thalamus. Squirrel brains showed more extensive neuroanatomical changes, likely due to their relatively smaller body mass, with extensive shrinkage of the core brain structures, and the cortex, even after accounting for differences in overall brain size. The results of this present study constitute the first observation of neuroanatomical changes in wild small mammal species breeding in arsenic endemic areas of Canada.

Chronic arsenicosis and cadmium exposure in wild snowshoe hares (Lepus americanus) breeding near Yellowknife, Northwest Territories (Canada), part 2: Manifestation of bone abnormalities and osteoporosis.

Various bone abnormalities, including osteoporosis, have been associated with chronic arsenic and cadmium exposure in experimental animal models, but information regarding the bone pathology of wild population of small mammals breeding in contaminated environment is limited. This present study was conducted to comparatively assess the prevalence and pattern of skeletal abnormalities in free ranging snowshoe hares inhabiting an area heavily contaminated by arsenic and other trace metals, near the vicinity of the abandoned Giant mine, and in a reference location approximately 20km from the city of Yellowknife, Northwest Territories, Canada. The femur and vertebrae of snowshoe hares from the mine area and reference location were subjected to bone densitometry examination and biomechanical testing using dual energy X-ray absorptiometry (DXA) and 3-point bending test. t-test results indicated that femoral densitometry parameters such as bone mineral density (BMD) (p=0.5), bone mineral content (BMC) (p=0.675), bone area (BA) (p=0.978) and tissue area (TA) (p=0.549) were not significantly different between locations. All densitometry parameters of the vertebrae (BMD, BA and TA) differed between locations (p<0.05), except for BMC (p=0.951) which showed no significant difference between the two locations. Vertebrae from the mine area also showed relatively lower BA and TA compared to the reference location. A constellation of skeletal abnormalities were also observed along the axial and appendicular bones respectively. Specifically, growth defects, osteoporosis, cortical fractures, sclerosis, and cyst like changes were commonly observed in the femurs and vertebrae of hares from both locations. With respect to biomechanical properties, only bone stiffness and peak load tended to be relatively reduced in specimens from the mine area, whereas work to failure was notably increased in specimens from the reference site compared to those from the mine area. Taken together, the results of this preliminary study suggest that chronic concomitant exposure to arsenic and cadmium may be involved in the etiology of various bone abnormalities, including osteoporosis in wild population of snowshoe hares from the Yellowknife area. The result presented in this study represent the first evaluation of osteological effects in free-ranging furbearers (snowshoe hares) diagnosed with arsenicosis, and concomitantly exposed to environmental levels of cadmium.

Chronic arsenicosis and cadmium exposure in wild snowshoe hares (Lepus americanus) breeding near Yellowknife, Northwest Territories (Canada), part 1: Evaluation of oxidative stress, antioxidant activities and hepatic damage.

Previous gold mining activities and arsenopyrite ore roasting activities at the Giant mine site (1948 to 2004) resulted in the release of high amounts of arsenic and trace metals into the terrestrial and aquatic ecosystems of Yellowknife, Northwest Territories, Canada. While elevated levels of arsenic has been consistently reported in surface soils and vegetation near the vicinity of the Giant mine area and in surrounding locations, systematic studies evaluating the overall health status of terrestrial small mammals endemic to the area are lacking. The purpose of this present study was to evaluate and comparatively assess the biochemical responses and histopathological effects induced by chronic arsenic and cadmium exposure in wild snowshoe hares breeding near the city of Yellowknife, specifically around the vicinity of the abandoned Giant mine site and in reference locations. Analysis included measurement of total arsenic and cadmium concentration in nails, livers, kidneys, bones, stomach content of hares, in addition to histopathological evaluation of hepatic and ocular lesions. Biochemical responses were determined through measurement of lipid peroxidation levels and antioxidant enzymes activities (catalase, superoxide dismutase, glutathione peroxidase, and glutathione disulfide). The results revealed that arsenic concentration was 17.8 to 48.9 times higher in the stomach content, and in the range of 4 to 23 times elevated in the nails of hares from the mine area compared to the reference location. Arsenic and cadmium levels were also noted to be increased in the bones, renal and hepatic tissues of hares captured near the mine area compared to the reference site. Specifically, hares from the mine area showed nail cadmium levels that was 2.3 to 17.6 times higher than those from the reference site. Histopathological examination of the eyes revealed no specific ocular lesions, such as lens opacity (cataracts) or conjunctivitis; however, hares from both locations exhibited hepatic steatosis (fatty liver change). Lipid peroxidation levels were relatively increased and accompanied with reduced antioxidant enzyme activities in hares from the mine area compared to the hares from the reference site. The results of this preliminary study suggest that the snowshoe hares breeding near the vicinity of Yellowknife, including near the Giant mine area have been chronically exposed to elevated levels of arsenic and cadmium, which consequently led to the increased levels of oxidative stress and perturbation of antioxidant defense system in exposed animals. The results of this present study constitute the first observation of chronic arsenicosis in wild small mammal species in Canada.

Heavy metal bioaccumulation and histopathological alterations in wild Arctic hares (Lepus arcticus) inhabiting a former lead-zinc mine in the Canadian high Arctic: A preliminary study.

A preliminary study was undertaken to determine post-mining baseline accumulation of selected trace metals, and histopathological alterations in free-living arctic hares (Lepus arcticus) inhabiting the vicinity of a former lead-zinc mine located on North Baffin Island in the Canadian High Arctic. Trace metal analysis included measurement of As, Cd, Fe, Pb and Zn in tissues, and histopathological assessment comprised of evaluation and scoring the severity of metal-induced hepatic and renal lesions. Metal contents in hepatic and renal tissues from hares from the mine area compared with the reference locations did not differ significantly suggesting that the animals are not uniformly exposed to background levels of metals in the environment. However, relatively higher accumulation pattern of Pb and Cd were noted in liver tissues of hare from the mine area compared to the background area, but did not induce increased lesions. Surface soils near the mine area contained relatively higher levels of trace metals (Zn>Mn>Pb>Cd>As) compared to reference soils, and with soil levels of Cd showing strong correlation with Cd accumulation in kidney tissues. Generally, both case and reference animals showed similar but varying severities of hepatic and renal lesions at the sublethal level, notably vascular congestion, occasional large hepatocyte nuclei, binucleate hepatocytes, yellow-brown pigmentation in the cytoplasm of hepatocytes and clustering of lymphocytes. Only hares with relatively higher accumulation of Pb from the mine area showed evidence of renal edema and hemorrhage of the capsular surface. This study constitutes the first assessment of metal induced histopathological alterations in arctic hares exposed to a historical mining area in the high arctic.