Molecular and Neurochemical Biomarkers in Arctic Beluga Whales

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Molecular and neurochemical biomarkers in Arctic beluga whales (Delphinapterus leucas) were correlated to brain mercury and selenium concentrations Sonja Ostertag, Alyssa C. Shaw, Niladri Basu, and Hing-Man Chan Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es501369b • Publication Date (Web): 29 Aug 2014 Downloaded from http://pubs.acs.org on September 2, 2014

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Environmental Science & Technology

Molecular and neurochemical biomarkers in Arctic beluga whales (Delphinapterus leucas) were correlated to brain mercury and selenium concentrations. Names and Affiliations of Authors:

Sonja K. Ostertag1, Alyssa C. Shaw2t, Niladri Basu3, Hing Man Chan,2,4*

1. Natural Resources and Environmental Studies, University of Northern British Columbia, Prince George, British Columbia, Canada, V2N 4Z9 2. Community Health Sciences, University of Northern British Columbia, Prince George, British Columbia, Canada, V2N 4Z9 3. Faculty of Agricultural and Environmental Sciences, Macdonald Campus, McGill University Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9 4. Center for Advanced Research in Environmental Genomics, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5

t

Alyssa Shaw passed away on August 29th, 2011

*

Corresponding author: [email protected]; Tel: (613) 562-5800 x7116; Fax: (613) 562-5385

1

ACS Paragon Plus Environment


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Abstract

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leucas) since the industrial revolution. Methylmercruy (MeHg) is a known neurotoxicant, yet

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little is known about the risk of exposure for beluga whales. Selenium (Se) has been linked to

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demethylation of MeHg in cetaceans, but its role in attenuating Hg toxicity in beluga whales is

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poorly understood. The objective of this study is to explore relationships between Hg and Se

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concentrations and neurochemical biomarkers in different brain regions of beluga whales in

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order to assess potential neurotoxicological risk of Hg exposure in this population. Brain tissue

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was sampled from hunter-harvested beluga whales from the western Canadian Arctic in 2008

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and 2010. Neurochemical and molecular biomarkers were measured with radioligand binding

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assays and quantitative PCR, respectively. Total Hg (HgT) concentration ranged from 2.6 – 113

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mg kg-1 dw in temporal cortex. Gamma-amminobutyric acid type A receptor (GABAA-R)

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binding in the cerebellum was negatively associated with HgT, MeHg and total Se (SeT)

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concentrations (p ≤ 0.05). The expression of mRNA for GABAA-R subunit α2 was negatively

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associated with HgT and labile inorganic Hg (iHglabile; p ≤ 0.05). Furthermore, GABAA-R

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binding was positively correlated to mRNA expression for GABAA-R α2 subunit, and negatively

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correlated to the expression of mRNA for GABAA-R α4 subunit (p ≤ 0.05). The expression of N-

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methyl-D-aspartate receptor (NMDA-R) subunit 2b mRNA expression was negatively associated

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with iHglabile concentration in the cerebellum (p ≤ 0.05). Variation of molecular and/or

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biochemical components of the GABAergic and glutamatergic signaling pathways were

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associated with MeHg exposure in beluga whales. Our results show that MeHg exposure is

Mercury (Hg) concentrations have increased in western Arctic beluga whales (Delphinapterus

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associated with neurochemical variation in the cerebellum of beluga whales and Se may partially

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protect MeHg neurotoxicity.

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Introduction

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Mercury is transported to the Arctic from southern latitudes at a rate of 200 to 300 tonnes per

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year, from both anthropogenic and natural sources.1 Anthropogenic activities are responsible for

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the release of the majority of Hg into the environment.2 In Arctic biota, mercury levels are an

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order of magnitude higher today than in the pre-industrial period, and approximately 74 to 94%

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of Hg in biota is estimated to originate from anthropogenic Hg emissions.1 Modern beluga

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whales have Hg concentrations that are 4- to 17- times higher than pre-industrial levels,

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according to the analysis of Hg in beluga whale teeth collected from archeological sites and

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harvest camps.3, 4 Furthermore, an increase in age-adjusted Hg concentrations was observed in

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beluga whales from the Beaufort Sea in the 1990s.5 Mercury concentrations continue to be

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significantly higher than concentrations observed in the 1980s in this population of whales.6

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There are concerns about the potential risk of elevated Hg exposure for people and animals in the

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Arctic.7

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Methylmercury (MeHg) can pass the blood brain barrier and placenta to exert toxic effects on the

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central nervous system of adults and fetuses.8, 9 Overall, the toxicity of MeHg has been linked to

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its reactivity with sulfhydryl groups.10 Mercury neurotoxicity varies with its speciation (i.e.

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inorganic or MeHg).8, 11 Given that iHg cannot easily cross the blood-brain barrier, the presence

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of iHg in the brain is likely due to in-situ demethylation of MeHg.8 The brain is expected to be

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more sensitive to MeHg than iHg, based on brain pathology and symptoms associated with

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MeHg poisoning.12 Selenium (Se) may play a role in MeHg detoxification in the central nervous

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system of certain species of cetaceans.13 Marine mammals and perhaps also some bird species

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are able to demethylate MeHg and immobilize it in the liver as HgSe.14 Co-exposure to Se and

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Hg may reduce Hg toxicity by reducing oxidative stress associated with Hg exposure and by

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forming inert compounds with Hg.15

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Neurochemical changes are potential indicators of neurological harm because they may precede

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functional or structural damage of the nervous system.16 Neurochemical changes may represent

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early and reversible indicators of neurological harm because they occur prior to the onset of

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overt functional or structural damage.16, 17 A neurochemical biomarker approach may provide

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valuable information about potential neurotoxicity in wildlife exposed to MeHg and other

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neurotoxins; however, challenges exist in relating biological endpoints to contaminant exposure

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in a wildlife setting, given the opportunistic nature of these sampling programs. Messenger RNA

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transcription levels may provide additional insight into toxic effects on organisms, prior to

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effects on the whole organism.18 Gene transcription may be downregulated or upregulated in

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association with stress; however, the abundance of mRNA is not a proxy for the abundance,

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function and activity of proteins in yeast or animal cell lines, yet protein function is a key

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component of how an organism responds to stress.18

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The interaction of MeHg or iHg with cysteine residues may inhibit, stimulate or damage

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components of the dopaminergic, cholinergic, γ-aminobutyric acid (GABA), and glutamatergic

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signaling pathways. 19, 20, 21, 22, 23 The N-methyl-D-aspartate receptor (NMDA-R) is a class of

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glutamate-gated ion channels; glutamate is thought to be one of the major excitatory transmitters

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in the brain and may be used by up to 40% of synapses.24 In contrast, the GABAA receptors form

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GABA-gated chloride ion channels that are responsible for inhibitory synaptic transmission.25

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NMDA-Rs are involved in the formation and maintenance of synapses and are also required for

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learning and memory processes.26 GABAA receptors have been linked to sedation, sleep, anxiety,

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seizures and amnesia.27 Previous studies suggest that the toxicity of MeHg and iHg exposure

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may be modulated by the interaction of this toxicant with NMDA and GABAA receptors. 21, 28, 29

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The activation of NMDA receptors was found to play a key role in glutamate-associated

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excitotoxicity of astrocyte cultures and inhibition of the GABAA receptor could potentially lead

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to an excitatory effect as well. 23, 30 Therefore, these receptors may respond to MeHg and iHg

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exposure to protect neurons from excitotoxicity. 31

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The objective of this study was to characterize the relationship between the concentrations of

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different chemical forms of Hg (total Hg (HgT) MeHg and labile iHg (iHglabile)), total Se (SeT),

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and the molar ratio of Hg to Se, and various neurochemical and molecular biomarkers in

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different brain regions of beluga whales. The overall hypothesis was that neurochemical and

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molecular variation in components of the GABAergic and glutamatergic signaling pathways

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would be associated with environmentally relevant Hg concentrations currently found in brain

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tissue of Arctic beluga whales.

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Methods

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Sample collection

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Harvested beluga whales were sampled on Hendrickson Island, NT, Canada in 2008 (n = 20) and

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2010 (n = 15) as described previously.13 Prior to each sampling season, a research permit and

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scientific license to fish were obtained from the Aurora Research Institute and Department of

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Fisheries and Oceans (DFO), respectively. The sampling program was approved and supported

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by the Tuktoyaktuk and Inuvik Hunters and Trappers Committees for all sampling years.

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Following the beluga harvest, the animal head was removed from the body by severing the joint

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between the skull and atlas with a knife according to the instructions given by Noel Raymond, an

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Inuvialuit beluga harvester (personal communication, July 1, 2006). The brain was removed

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from the skull using an autopsy saw (MOPEC®, Elmira, Michigan) and subsamples were

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collected and frozen within 3 h of animal death. Subsamples of cerebellum and temporal cortex

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were collected from harvested beluga whales in 2008 and 2010 to measure metal concentrations

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and speciation,, neurochemical biomarkers and mRNA expression. Samples (~ 0.5 – 3 g) for

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metals and speciation analyses were frozen at ~ -20 oC, samples for neurochemical analyses (~

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0.5-1 g) were frozen in a cryoshipper (~ -170 oC ) and samples (~ 0.07 g) for mRNA expression

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assays were frozen in a cryoshipper in 2008. In 2010, samples were placed in RNALaterTM at ~ 4

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o

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subsample availability for the separate analyses. Age was estimated by counting individual

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growth layer groups (GLG) in the dentine from a thin section of tooth at the Freshwater Institute,

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Winnipeg, MB. 32 Age estimates were based on one GLG deposited per annum.33 The

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concentrations of HgT and SeT were determined previously for the cerebellar cortex and temporal

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cortex;13 however, iHglabile and MeHg analyses were only published previously for beluga whales

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harvested in 2008.13 Chemical forms of Hg were analyzed for whales harvested in 2010

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following the method developed by Krey et al. as described previously. 34,13

C for ~ 24 h prior to freezing at -20 oC. Sample numbers varied for the different assays due to

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Membrane preparation

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For receptor binding assays, membrane preparations were prepared by homogenizing frozen

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brain tissue (~ 2 g wet weight or ww) in 50 mM sodium phosphate buffer (5 mM KCl, 120 mM

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NaCl, 50 mM HaH2PO4; pH 7.4; pH 7.4 (10 mL/g tissue) with a tissue tearer for 30 s using a

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previously described method.35 Endogenous ligands were removed by centrifugation three times

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with sodium phosphate buffer and membranes were re-suspended in buffer, aliquoted, flash

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frozen in liquid nitrogen and stored at -80 oC. Protein concentration was determined with the

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Bradford assay and bovine serum albumin was used as the standard.

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Receptor binding assays

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Receptor binding assays were adapted from previous studies by Basu et al. 36-38 In brief, thirty µg

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of membrane preparation was re-suspended in buffer and added to a microplate containing a 1.0

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mM GF/B glass filter (Millipore, Boston, MA, USA). Membrane protein was suspended in 100

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µL Tris buffer (pH 7.4; NMDA: buffer contained 100 µM glycine and glutamate) and incubated

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for 30 min on ice with [3H]-flunitrazepam ([3H]-FNP; 0.5 nM) or 120 min at room temp with

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[3H]-MK-801 (16 nM), respectively for GABA and NMDA. All incubation steps were carried

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out with gentle shaking and binding reactions were ended by vacuum filtration. The filters were

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rinsed three times with buffer and were then soaked for 72 h in 25 µL OptiPhase Supermix

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Cocktail (PerkinElmer). Radioactivity retained by the filters was quantified in a microplate

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detector (Wallac Microbeta, PerkinElmer) and counting efficiency was approx 40%. Specific

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binding was calculated as the difference between radioligand binding in the presence and

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absence of inhibitor: 20 µM clonazepam for GABAA-R, and 100 µM MK-801 for NMDA-R.

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Receptor binding is reported as fmol of radioisotope bound per mg of membrane protein (fmol

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mg-1).

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Expression of mRNA

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Total RNA was extracted from approx. 80 mg samples using 1 mL Trizol using the Qiagen

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RNeasyTM Lipid Tissue kit. Total RNA was treated with AmbionTM DNase | buffer (6 µL),

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rDNAse | (1 µL) and DNase Inactivation reagent (6 µL), prior to quantification of RNA-40 using

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a spectrophotometer (Nanodrop, ND-1000). Taqman reverse transcription reagents (Applied

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Biosystems) were used for the generation of complementary DNA (cDNA) for all samples. A

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master mix made up of 10 µL 10 X Taqman Reverse Transcriptase buffer, 22 µL MgCl2, 20 µL

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random hexamers (50 µM), 2 µL RNase inhibitors, 2.5 µL reverse transcriptase was made that

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was sufficient for all samples for one brain region (each sample: 1 µg RNA, total volume of 100

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µL). Concurrently, the identical reaction was performed without reverse transcriptase, to ensure

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the absence of genomic DNA (No Reverse Transcriptase (NRT) control). The following

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thermocycler parameters were used for the generation of cDNA archive: 25 ºC for 10 min, 37 ºC

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for 60 min, and 95 ºC for 5 min (DYADTM, DNA Engine).

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Primer pairs were designed within conserved nucleotide regions of the genes of interest based on

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the alignment of genes from multiple species (human, cow, goat, pig, primate (chimpanzee and

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macaque), dog, cat and sheep) using the National Centre for Biotechnology Information (NCBI)

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website. These primers were used in polymerase chain reaction (PCR) reactions on cDNA from

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beluga cerebellum (n = 2) to develop species-specific primers and probes. The PCR products

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were direct-sequenced at UBC Okanagan (Fragment Analysis and DNA Sequencing Services,

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Kelowna, CA), which were then used to design species-specific primers. Chromatograms were

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edited and the retrieved sequences were run through the NCBI website (Standard Nucleotide

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BLAST) to ensure they matched the conserved sequences of genes. The sequence obtained for

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NMDA-2c did not match the conserved sequence; therefore, the data for this target gene are not

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presented in this study. Species-specific primers and fluorogenic probes (IDT, Coralville, Iowa)

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were designed using Genscript RT-PCR primer design (see supporting information, Table S1).

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All PCR runs were performed under identical conditions using the 7300 Real-Time PCR System

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(Applied Biosystems). Real time assays were performed in 96-well optical plates in duplicate.

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Simplex assays were run with iTaq™ Supermix with ROX kit (Biorad). PCR mastermix was

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prepared and each 25 µL reaction contained 12.5 µL iTaq mix, 500 nM of appropriate forward

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and reverse primers, 250 nM probes and 2.5 µL template for cerebellar cortex and 5.0 µL

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template for temporal cortex samples. The thermocycle program included an enzyme activation

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step at 95 oC (10 min) and 40 cycles of 95 oC (15 sec) and 60 oC (1 min).

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Standard curves were generated for all genes from serial dilutions of cDNA to calculate

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efficiencies for target and reference genes. The mean CT value for the three lowest Hg-exposed

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animals was used as the control. Pooled sample was included in triplicate for each plate to

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monitor inter-plate variability of s9 expression (reference gene). No reverse transcriptase (NRT))

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samples were included in each plate to ensure the absence of genomic DNA. The difference in

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thermal cycles for the control and sample were calculated for each target gene and reference (s9).

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Fold changes for each sample and target gene were calculated relative to the reference gene s9 39.

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RNA purity and quality was evaluated using a NanoDrop (ND-1000, NanoDrop Technologies,

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USA). The optical density (OD) ratio of 260 nm/280 nm wavelengths was used as an indicator of

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RNA quality, with ratios greater than 1.8 indicating good RNA quality (Fleige and Pfaffl 2006).

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RNA integrity was evaluated using Experion (Bio-Rad Laboratories, USA). The ratio of 28S:18S

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and RNA Quality Indicator (RQI) value were used to evaluate degradation, with a 28S:18S ratio

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of 2.0 40 or RQI of 10 indicating perfect integrity 41.

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Data Analysis

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Mercury concentrations were log-transformed to meet assumptions of normality and

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homogeneity of variance. Fold-change ratios and receptor densities were log-transformed when

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necessary to meet assumptions of normal distribution and homogeneity of variance. Non-

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parametric tests were used when necessary if transformations were unable to improve the fit of

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data. The relationships between neurochemistry and the concentration of HgT, Hg species, Se,

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Hg to Se molar ratio, and age were explored using Pearson correlation coefficients followed by

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multiple linear regression analysis. A backwards stepwise approach was used to evaluate the

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statistical significance of age, Hg (HgT, iHglabile, MeHg, SeT, and Hg:Se) and sampling year on

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neurochemical or molecular biomarkers. We removed influential outliers after examination of

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studentized residuals, leverage and Cook’s D influence. Pearson correlation coefficients were

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calculated for the expression of target genes and corresponding receptor binding levels.

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Correlations (Pearson and Spearman), F-values, Wilcoxon rank-sum test, and t-tests were

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considered to be statistically significant if p ≤ 0.05. The sample size for females was

Molecular and Neurochemical Biomarkers in Arctic Beluga Whales

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