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.

Influence of 3-aminobenzamide, an inhibitor of poly(ADP-ribose)polymerase, in the evaluation of the genotoxicity of doxorubicin, cyclophosphamide and zidovudine in female mice.

Testing new chemical entities for genotoxicity is an integral part of the preclinical drug-development process. Lowering the detection limit and enhancing the sensitivity of genotoxicity assays is required, as the standard test-battery fails to detect some carcinogens (non-genotoxic) and weak genotoxins. One of the mechanisms that affect the detection of weak genotoxins is related with the DNA-repair efficiency of the cell system used. In the present study, 3-aminobenzamide (3-AB, 30 mg/kg body-weight), a poly(ADP-ribose)polymerase inhibitor, was used to evaluate the DNA-damaging potential of zidovudine (AZT, 400 mg/kg bw), doxorubicin (DOX, 5 mg/kg bw) and cyclophosphamide (CP, 50 mg/kg bw, as a positive control) and sucrose (SUC, 3 g/kg bw, as a negative control) in Swiss female mice. The endpoints considered included micronucleus formation, DNA breakage (in peripheral blood lymphocytes, bone marrow and liver; comet assay) and chromosome aberrations, as well as immunohistochemistry of PARP-1 and phosphorylated histone H2AX (γ-H2AX). The results clearly indicate that the genotoxicity of zidovudine (AZT), doxorubicin (DOX) and cyclophosphamide (CP) was significantly increased in the combination treatments (3-AB+AZT, 3-AB+DOX, 3-AB+CP) as compared with the respective controls (treatment with AZT, DOX and CP alone). There was no increase in the genotoxicity per se after treatment with SUC, 3-AB or 3-AB+SUC, compared with the control (saline). Correlation analysis suggests that all genotoxicity parameters are well correlated with each other. The results clearly show that the genotoxicity of weak genotoxins can be enhanced and detected in the presence of 3-AB in mice. Thus, this approach can be used in the pre-clinical genotoxicity screening of weak genotoxins.

Pretreatment with valproic acid, a histone deacetylase inhibitor, enhances the sensitivity of the peripheral blood micronucleus assay in rodents.

Micronucleus (MN) assay is widely used for the determination of the genotoxic potential of new chemical entities. Improvement in the sensitivity of MN assay will be advantageous for the successful detection of marginally active genotoxins. In the past, several improvements have been made in the automated scoring of micronuclei, while very few attempts have been taken to improve the sensitivity of manual micronuclei detection. The present study aims to validate the effect of valproic acid (VPA) pretreatment on the sensitivity of peripheral blood micronucleus (PBMN) assay using cyclophosphamide (CP, 50mg/kg), methotrexate (MTX, 20mg/kg) and zidovudine (AZT, 400mg/kg) in rodents. However, to find out the optimum VPA pretreatment time as well as to detect the effect of species and age difference, separate experiments were conducted on young Swiss albino mice (24-28 days) and Sprague-Dawley rats (21-24 days), in which significant increase in MN induction was observed with 3-day VPA pretreatment in both the species. Based on these results, studies on adult mice were conducted with 3-day VPA pretreatment along with CP or MTX or AZT. The results of the present study clearly demonstrate that the 3-day VPA pretreatment significantly enhances the sensitivity of PBMN assay in peripheral blood (PB) in adult mice. After validation with other standard genotoxins as well as other HDAC (histone deacetylase) inhibitors, this model may be useful for the detection of marginally active DNA damaging agents.