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Aquatic Toxicology 109 (2012) 185– 193

Contents lists available at SciVerse ScienceDirect

Aquatic Toxicology

jou rn al h om epa ge: www.elsev ier .com/ locate /aquatox

ercury toxicity in beluga whale lymphocytes: Limited effects of seleniumrotection

. Frouina, L.L. Losetob, G.A. Sternb, M. Haulenac, P.S. Rossa,∗

Fisheries and Oceans Canada, Institute of Ocean Sciences, 9860 West Saanich Rd, P.O. Box 6000, Sidney, BC, Canada V8L 4B2Fisheries and Oceans Canada, Freshwater Institute, 501 University Crescent, Winnipeg, MB, Canada R3T 2N6Vancouver Aquarium, 845 Avison Way, Vancouver, BC, Canada V6G 3E2

r t i c l e i n f o

rticle history:eceived 30 July 2011eceived in revised form7 September 2011ccepted 28 September 2011

eywords:ercury

eleniumymphocyteseluga whaleelphinapterus leucas

a b s t r a c t

Increasing emissions of anthropogenic mercury represents a growing concern to the health of high trophiclevel marine mammals. In its organic form, this metal bioaccumulates, and can be toxic to several physi-ological endpoints, including the immune system. In this study, we (1) evaluated the effects of inorganicmercury (mercuric chloride, HgCl2) and organic mercury (methylmercuric chloride, MeHgCl) on thein vitro function of lymphocytes isolated from the peripheral blood of beluga whales (Delphinapterusleucas); (2) characterized the potential protective effects of sodium selenite (Na2SeO3) on cell prolifer-ation of HgCl2 or MeHgCl-treated beluga whale lymphocytes; and (3) compared these dose-dependenteffects to measurements of blood Hg in samples collected from traditionally harvested beluga whales inthe western Canadian Arctic. Lymphocyte proliferative responses were reduced following exposure to1 �M of HgCl2 and 0.33 �M of MeHgCl. Decreased intracellular thiol levels were observed at 10 �M ofHgCl2 and 0.33 �M of MeHgCl. Metallothionein induction was noted at 0.33 �M of MeHgCl. Concurrentexposure of Se provided a degree of protection against the highest concentrations of inorganic Hg (3.33

and 10 �M) or organic Hg (10 �M) for T-lymphocytes. This in vitro protection of Se against Hg toxicityto lymphocytes may contribute to the in vivo protection in beluga whales exposed to high Hg concen-trations. Current Hg levels in free-ranging beluga whales from the Arctic fall into the range of exposureswhich elicited effects on lymphocytes in our study, highlighting the potential for effects on host resis-tance to disease. The implications of a changing Arctic climate on Hg fate in beluga food webs and the

lth of

consequences for the hea

. Introduction

Climate change has the potential to have a considerable impactn marine mammals (Simmons and Eliott, 2009) through directr indirect effects on ecosystems or their health (reviewed byurek et al., 2008). Direct effects such as loss of habitat, tem-erature stress, and exposure to severe weather are discussed inaidre et al. (2008). Indirect effects could result from changes inost–pathogen associations due to altered pathogen transmissionr host resistance, changes in body condition due to alterationsn predator–prey relationships or changes in exposure to contam-nants (Schiedek et al., 2007). Marine mammals including thosen the Arctic are exposed to a wide range and concentration ofnvironmental contaminants (AMAP, 2009) and are likely to be

specially vulnerable to pathogen exposure which can be affectedy climate change (Macdonald, 2007).

∗ Corresponding author. Tel.: +1 250 363 6806; fax: +1 250 363 6807.E-mail address: peter.s.ross@dfo-mpo.gc.ca (P.S. Ross).

166-445X/$ – see front matter. Crown Copyright © 2011 Published by Elsevier B.V. All rioi:10.1016/j.aquatox.2011.09.021

beluga whales remain pressing research needs.Crown Copyright © 2011 Published by Elsevier B.V. All rights reserved.

High concentrations of mercury (Hg) and selenium (Se) havebeen documented in Arctic marine biota (Wagemann et al., 1998).Phytoplankton and zooplankton are known to bioaccumulate met-als and are the principal prey for baleen whales and fish, while fishrepresent the primary prey for toothed whales and most pinnipeds.Hg and Se tend to increase with age in beluga whales (Delphi-napterus leucas) (Woshner et al., 2001) and narwhals (Monodonmonoceros) (Friberg et al., 1986).

While inorganic Hg is the major source of Hg to most aquatic sys-tems, methylmercury (MeHg) is the form of Hg that bioaccumulatesin organisms and biomagnifies up trophic levels in marine, freshwa-ter and terrestrial food webs (Gray, 2002); it is also the form knownto cause severe toxicological effects on wildlife (reviewed by Wolfeet al., 1998). Exposure to Hg impairs specific immune defences ofmany species including marine mammals (De Guise et al., 1996b;Pillet et al., 2000; Lalancette et al., 2003; Camara Pellisso et al., 2008;Das et al., 2008; Dufresne et al., 2010; Frouin et al., 2010; Schaefer

et al., 2011).

An association between both liver Hg and MeHg concentrationsand severity of disease in a sample of 57 harbour porpoises in theNorth and Baltic Seas suggested a possible effect of Hg on host

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esistance (Siebert et al., 1999). Hg liver concentrations and theg:Se molar ratio were significantly higher in harbour porpoises

rom England and Wales that died of infectious disease when com-ared to a sample of healthy animals that died from physical traumaBennett et al., 2001).

Because Se antagonizes Hg toxicosis in controlled laboratorynimal studies, the coincident increase of Se and Hg in tissues ofdontocetes seems to implicate a protective role for Se (Caurantt al., 1996; Woshner et al., 2001). Unlike Hg, which has no knownhysiological function in mammals, Se is an essential element,nd as such, its concentrations are physiologically regulated. Inddition, marine mammals, having the highest Hg burdens in thearine biota, are able to detoxify MeHg by a specific chemicalechanism involving Se (Koeman et al., 1973). This relationship has

lso been documented in Arctic marine mammals (e.g. Wagemannt al., 1998). Based on molar ratios, Hg and Se appear to accumulaten a 1:1 fashion in liver and kidney, with each element seeminglyffsetting the toxicity of the other. This may involve demethylationf organic Hg, a process that may require Se (through increasedemethylation) and result in the formation of inert complexesinorganic and/or proteinaceous) that can be stored in hepatic lyso-omes (Cuvin-Aralar and Furness, 1991; Woshner et al., 2002).

Moreover, Se has been well documented to influence theistribution, kinetics and toxic effects of Hg (Cuvin-Aralar andurness, 1991). As a component of the amino acid selenocys-eine, Se is a constituent of numerous proteins, including theelenium-dependent glutathione peroxidases and the thioredoxineductases. The glutathione peroxidase system quenches free rad-cals and reactive intermediates (reactive oxygen species) and, inurn, affects demethylation of Hg. However, mercury toxicity canffect the proteins of the mammalian thioredoxin system (Carvalhot al., 2008). Finally, metallothionein (MT) is important for detoxi-cation of Hg in terrestrial mammals, but appears to be much lesso for marine mammals (Caurant et al., 1996).

The present study focuses on the immunotoxic effect ofnorganic (HgCl2) and organic (MeHgCl) Hg on beluga whale lym-hocytes. This study (1) evaluated the toxic effects of both mercuricompounds on lymphocyte proliferation, intracellular thiol levelsnd metallothionein induction; (2) characterized the degree of sele-ium protection against Hg toxicity in lymphoblastic proliferation;nd (3) compared these dose-dependent effects to measurementsf blood Hg in samples collected from 10 traditionally harvestedeluga whales in the western Canadian Arctic.

. Material and methods

.1. Animals and sample collection

Blood samples were obtained regularly from four captive bel-gas (one male and three females) housed at the Vancouverquarium, BC, Canada. Three of them had been captured earliers immature animals in the Churchill River in Manitoba, while theoungest was born in captivity at the Vancouver Aquarium. Bloodamples were drawn from the tail fluke into heparinised tubesVacutainer, Becton Dickinson, Franklin Lakes, USA), with approx-mately 10–30 mL collected from each animal. The blood was keptt 4 ◦C until the time of analysis, which took place 4–10 h afterampling.

In addition, blood, serum and plasma were collected from 10ild beluga whales (10 adult males) harvested by Inuit hunters for

ood consumption in July 2010 at the Hendrickson Island whale

amp, close to the community of Tuktoyaktuk, Northwest Territo-ies, Canada. Once whales were harvested and brought to shoreor sampling the jugular vein was cut immediately, and 5–7 mLf blood was collected in sterile glass bottles. The whole blood

ogy 109 (2012) 185– 193

samples were frozen in cryoshippers (−154 ◦C). Ages of animalswere determined from a thin section of a tooth by counting growthlayer groups in the dentine (Stewart et al., 2006).

2.2. Isolation of mononuclear cells

Beluga blood samples were diluted in an equal volume of phos-phate buffer saline (PBS; Sigma-Aldrich, Oakville, Canada), withthis the cellular suspension then carefully laid on a LymphoprepTM

density solution (1.077 g/mL; Axis-Shield, Oslo, Norway). Theleukocytes were then isolated by centrifugation for 20 min at800 × g. The peripheral blood leukocytes (PBLs) layer were washedthree times with PBS at 800 × g and resuspended in RPMI-1640(Sigma-Aldrich) supplemented with 10% heat inactivated foetalbovine serum (FBS), 1% Pen-Strep (Sigma-Aldrich) and 10 mMHepes (Sigma-Aldrich) (complete medium). The ratio of live/deadcells was assessed using trypan blue (Sigma-Aldrich) dye exclusionand visual examination under a microscope with a hemacytometer.

2.3. Chemical preparation

Stock solution of HgCl2 and Na2SeO3 (Sigma-Aldrich) were pre-pared in distilled water at a concentration of 10−2 M. MeHgCl wereobtained from Sigma-Aldrich and was first dissolved in alcohol anddiluted in dissolved water to achieve a stock solution of 10−3 M.Subsequent dilutions were performed with RPMI 1640.

2.4. In vitro exposures

For lymphocyte proliferation, metallothionein induction, intra-cellular thiol levels and associated viability, cells were adjusted to2 × 106 cells/mL. Incubations took place at 37 ◦C in a humidified5% CO2 atmosphere for up to 48 h. Exposures comprised HgCl2 orMeHgCl at concentrations of 0, 0.03, 0.1, 0.33, 1, 3.3 or 10 �M, aswell as co-exposures to several ratios of Hg:Se (1:0; 1:0.5; 1:1; 1:2;1:3 and 1:4) in complete medium.

2.5. Viability

Viability of lymphocytes after exposure was evaluated by flowcytometry using propidium iodide (PI) (100 �g/mL) as described byBrousseau et al. (1999). PI enters in all cells but is actively excretedonly by living cells. Samples were analyzed with a FACScaliburflow cytometer (Becton Dickinson, Mississauga, Canada) and thefluorescent of 5000 events was read in FL3.

2.6. Determination of T-lymphocyte proliferation

PBLs (100 �L) were plated with 100 �L mitogen, 10 �L of Hg orvehicle and 10 �L of Se or vehicle for 48 h at 37 ◦C with 5% CO2 in96-well round-bottom plates. The optimal concentration used tostimulate T lymphocytes was 1 �g/mL concanavalin A Con A (DeGuise et al., 1996a). Samples were tested in triplicate.

Lymphocyte proliferation was evaluated as the incorporationof 5-bromo-2′-deoxyuridine (BrdU), a thymidine analogue, andfurther detected with a monoclonal antibody and a colorimetricenzymatic reaction (Cell Proliferation ELISA BrdU [colorimetric],Roche Diagnostics, Laval, Canada) according to the instructionsof the manufacturer. Briefly, after mitogen stimulation for 48 h,20 �L of the BrdU labelling solution were added to each well. After18 h of incubation at 37 ◦C with 5% CO2, plates were centrifuged(300 × g, 10 min), cells were dried, fixed, and the DNA denatured.

The Peroxidase-conjugated anti-BrdU monoclonal antibody wasadded to the cells and incubated for 90 min at room temperature.After three washing steps (washing solution) the bound peroxi-dase was detected by substrate reaction. The reaction was stopped

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y adding 1 M H2SO4 and quantified by measuring the optical den-ity of the yellow product at 450 nm and a reference wavelengthf 690 nm using a Dynatech MRX enzyme-linked immunosorbentssay (ELISA) reader.

.7. Determination of intracellular thiol levels

The intracellular thiol concentration was measured by CMFDAInvitrogen, Burlington, Canada) staining in flow cytometry accord-ng a method modified from Brousseau et al. (1999). After exposureuring 48 h (as described above), 0.5 × 106 cells were collected andesuspended in 500 �L sterile PBS-G (1 g glucose/L) and cell sam-les were stained with CMFDA at a final concentration of 50 �M for5 min at 37 ◦C. The cells were analyzed immediately after by flowytometry. Green fluorescence from CMFDA was measured in theL1 channel. The level of intracellular thiols was indicated by meanuorescence intensity of stained probes.

.8. Determination of metallothionein induction

The relative intracellular MT protein levels were measured byow cytometry according a method described by Yurkow andakhijani (1998) and Pillet et al. (2002). After exposure during

8 h (as described above), 1 × 106 cells were washed with PBS, cen-rifuged at 300 × g for 10 min, fixed with 0.3% paraformaldehydeSigma-Aldrich), and stored at 4 ◦C for 4 days. Cells were permeabi-ized with PBS containing 3% FBS, 0.1% sodium azide (Sigma), and.1% saponin (Sigma-Aldrich). Cells were then incubated for 30 minith a monoclonal mouse anti-MT antibody 1 �g/106 cells (Dako,arkham, Canada). Cells were then washed with staining buffer

3% FBS and 0.1% sodium azide in PBS) and incubated with a fluores-ein isothiocyanate (FITC)-conjugated goat anti-mouse secondaryntibody (Sigma-Aldrich). Cells probed with an irrelevant (isotypic)ontrol primary antibody (mouse IgG1; BD Biosciences) in con-unction with the FITC-conjugated secondary antibody served asegative controls. The analysis was performed using a FACScal-

bur flow cytometer. Cells were also exposed to cadmium chlorideCdCl2; Sigma-Aldrich) at 10−5 and 10−6 M for 48 h as a positiveontrol.

.9. Chemical analysis

Beluga whole blood samples were subsampled for THg, MeHgnd Se analysis. All samples were weighed to approximately 1 gor THg analysis. Samples were digested with a hydrochloric/nitriccid mixture (Aqua Regia) heated to 90 ◦C. The digested samplesere analyzed for THg by cold vapour atomic absorption spec-

roscopy (CVAAS) (Armstrong and Uthe, 1971). The detection limitas 0.2 ng/g wet weight of tissue.

For MeHg analysis, beluga whole blood samples were extractedccording to the procedure of Uthe et al. (1972). Wet tissue samplesapproximately 0.5 mL) were homogenized in an acidic bromidend copper sulphate solution. This was extracted with toluenend partitioned into a thiosulphate solution where the addition ofotassium iodide allowed back-extraction into toluene. The extractas analyzed on a gas chromatograph with an electron captureetector. The absolute detection limit for MeHg by GC-ECD was

pg of Hg in a 1 �L injection.Selenium concentrations were determined using an atomic fluo-

escence spectrophotometer following a modified method of Vijannd Wood (1976). Whole blood samples (approximately 0.8 mL)ere digested with a mixture of nitric, sulphuric and perchloric

cids. Hydrochloric acid was added to digested samples, whichere then heated to ensure that all selenium was in the Se4+ oxi-ation state. Selenium was then reduced to Se2+ by the additionf sodium borotetrahydride. The generated selenium hybride gas

ogy 109 (2012) 185– 193 187

(SeH2) is then atomised using a chemically generated hydrogen dif-fusion fame. Atomic fluorescence is measured after excitation usinga selenium boosted discharge hollow cathode lamp. The detectionlimit was 0.05 �g/g wet weight of tissue.

Certified standard reference materials (TORT-2 and DOLT-3 forTHg; DOLT-3 and DORM-3 for MeHg; TORT-2, DORM-3 and CRM2976 for Se) were analyzed in duplicate in every run. Recoverywithin 10% of the certified values was used as a batch validationfor samples.

2.10. Statistical analysis

All data are presented as the mean ± standard error. The resultswere analyzed using Statistica for windows (version 7.0, StatSoftInc., 1995). Normality (Kolmogorov–Smirnov test) and homogene-ity of variance (Levene–Median test) were first checked and aparametric one-way analysis of variance (ANOVA) was subse-quently performed on the data. The error ̨ was fixed at p ≤ 0.01or p ≤ 0.05 for all tests.

For the proliferation assay, triplicates were averaged, and themean and standard error were determined. An ANOVA was usedto compare the different experimental groups to the control, usingp ≤ 0.01 and p ≤ 0.05 for statistical significant.

Non-normally distributed variables included: THg and MeHg.Logarithmic transformation was used for both.

Dose values for inhibition of 50% of proliferation response (ID50)by Hg were obtained statistically by linear regression.

3. Results

3.1. Immunotoxicology

Cell viability was always greater than 90%.The sensitivity of lymphocytes to different concentrations of

HgCl2 or MeHgCl after 48 h of exposure is shown in Fig. 1. Viabilitywas significantly decreased (p < 0.01) after 48 h of incubation with10 �M of HgCl2 and with 1 �M up to 10 �M of MeHgCl.

Both organic and inorganic Hg affected the proliferation of Tlymphocytes (Fig. 1). The proliferation was significantly reduced by1 �M (p < 0.05) up to 10 �M (p < 0.01) of HgCl2. MeHgCl significantlyreduced (p < 0.01) the proliferation at 0.33 �M.

Dose values for IC50 indicated a difference in proliferativeresponses of lymphocytes exposed to inorganic or organic Hg.Beluga T-lymphocytes were 10 times more sensitive to organic(0.24 �M) than inorganic (2.62 �M) Hg.

There was a reduction in intracellular thiol levels in lymphocytestreated in vitro with 10 �M HgCl2 (p < 0.01) or 0.33 �M MeHgCl(p = 0.02; Fig. 2).

The MT levels in lymphocytes were not affected by HgCl2 buta significant induction was observed after exposure to 0.33 �M ofMeHgCl (Fig. 3). Cadmium chloride as a positive control increasedthe MT levels in beluga lymphocytes (p < 0.01).

Thiol and MT levels varied in proliferating lymphocytes thatwere exposed to inorganic and organic Hg (Fig. 4). A line of “bestfit” was applied to the data (quadratic), depicting this relation-ship whereby the highest inorganic and organic Hg concentrationsinduced a simultaneous decrease in lymphocyte proliferation andthiol levels. On the contrary, the highest concentrations of organicHg, but not inorganic Hg, induced a simultaneous decrease oflymphocyte proliferation associated with an induction of metal-

A selenium-dependent protection was evidenced by an increasein T-lymphocyte proliferation only in cells exposed to the highestHgCl2 concentrations (3.33 and 10 �M) and to the highest MeHgClconcentration (10 �M) at Hg:Se ratios of 1:2 to 1:4 (Fig. 5).

188 H. Frouin et al. / Aquatic Toxicology 109 (2012) 185– 193

Fig. 1. The proliferation of T lymphocytes (bars) and their viability (lines) following different exposures to inorganic mercury (A) and organic mercury (B). Results aree ercury# l2 or 1t

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xpressed as a (%) of the control response (unexposed cells) (n = 3 for inorganic mp ≤ 0.01. Viability was significantly decreased after an 48 h-exposure to 10 �M HgCo 1 �M of HgCl2 and 0.33 �M of MeHgCl.

.2. Chemical analysis

The mean length of wild beluga whales sampled in 2010 was.06 m (SD = 0.42; range: 3.56–5.13; n = 10) and the mean age was4.3 (SD = 4.6; range: 16–28; n = 10). Concentrations of THg, MeHgnd Se in whole blood are reported in Table 1 and are based on wetfresh) weight. The log10THg correlated with log10MeHg (r = 0.99;

< 0.001; n = 10). However, mean log10THg concentrations wereigher than mean log10MeHg (p = 0.04; t = −2.39; n = 10; paired t-est), with MeHg (�g/g) accounting for 95.4 ± 5.9% (range: 87–102;

= 10) of the THg (�g/g) in whole blood samples.Se concentration (�g/g) was correlated with beluga length

r = −0.64; p < 0.05; n = 10). No correlation was observed betweenog10THg (r = 0.15; p = 0.69) or log10MeHg (r = 0.12; p = 0.74) andength. No correlation was observed between Se (�g/g; r = −0.03;

= 0.93), log10THg (r = 0.55; p = 0.10) or log10MeHg (r = 0.54; = 0.11) and age.

There was no apparent association between log10THg and Se

oncentrations (nMol/g wet wt; r = 0.07; p = 0.86), length and theHg:Se molar ratio (r = 0.37; p = 0.29), or between age and theHg:Se molar ratio (r = −0.01; p = 0.98); the THg:Se molar ratiosarying between 0.10 and 0.33.

ig. 2. The intracellular thiol levels in lymphocytes following different exposures to inorgaesponse (unexposed cells) (n = 4 per dose group; mean ± standard error). *p ≤ 0.05, #p ≤0 �M HgCl2 or 0.33 �M MeHgCl.

and n = 4 for organic mercury per dose group; mean ± standard error). *p ≤ 0.05, �M MeHgCl. T-lymphocyte proliferation was inhibited following an 48 h-exposure

4. Discussion

The overall health of an individual animal is a complexinteraction between its immune status, body condition, toxi-cant load, pathogen characteristics, and environment. ElevatedHg levels represent a significant risk to the health of belugawhale. Possible mechanisms of action include contaminant-relatedimmunosuppression which could represent a factor that facil-itates the emergence of infectious disease induces by climatechange.

4.1. Immunotoxic effects of Hg

We observed a dose-dependent effect of in vitro HgCl2 andMeHgCl on beluga whale lymphocyte proliferation, intracellularthiol levels and metallothionein induction. Exposure to 10 �M ofHgCl2 and 1 �M of MeHgCl decreased lymphocyte viability, indicat-ing acute toxicity. Similarly, in vitro MeHg exposure induced DNA

single-strand breaks and cytotoxicity in bottlenose dolphin (Tur-siops truncatus) lymphocytes at concentrations equal to or greaterthan 0.5 �g/mL (approximately 2.3 �M; Betti and Nigro, 1996).In our experiment, the reduced proliferative response occurred

nic mercury (A) and organic mercury (B). Results are expressed as a (%) of the control 0.01. Intracellular thiol level was significantly decreased after an 48 h-exposure to

H. Frouin et al. / Aquatic Toxicology 109 (2012) 185– 193 189

Table 1Concentrations (wet weight) of total mercury (THg), methylmercury (MeHg) and selenium (Se) in whole blood of beluga whales from Hendrickson Island (Beaufort Sea,Arctic, 2010).

THg blood (�g/g) THg blood (�M) MeHg blood (�g/g) MeHg blood (�M) Se blood (�g/g) Se blood (�M)

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t 1 �M of HgCl2 and 0.33 �M of MeHgCl, suggesting that themmunotoxic effect observed was not linked to the cytotoxic effect.gCl2 inhibited the proliferation of bottlenose dolphin lympho-ytes at 1 ppm (corresponding to ∼3.7 �M; Camara Pellisso et al.,008), and grey (Halichoerus grypus) and harbour seal (Phoca vit-lina) lymphocytes at 0.1 mM (Dufresne et al., 2010). Moreover,-lymphocyte proliferation was reduced by MeHgCl at 10 �M inarbour seals (Das et al., 2008; Dufresne et al., 2010) and grey sealsLalancette et al., 2003). Our results suggest that T-lymphocytesrom cetaceans are more sensitive to the immunotoxic effects ofg than T-lymphocytes from seals.

The 10-fold higher relative immunotoxicity of the organic Hgompared to the inorganic Hg suggests that the chemical structuref the Hg form is important in shaping its potency. Similar resultsere found in a study of beluga whale splenocytes, where cells

xposed in vitro to MeHgCl showed a 10-fold lower proliferation

han cells exposed to HgCl2 (De Guise et al., 1996b). Moreover, inn vitro cultured beluga skin fibroblasts, the methylated form of Hg

as one order of magnitude more potent in inducing micronucleind inhibiting cell proliferation than HgCl2 (Gauthier et al., 1998).

ig. 3. The metallothionein induction in lymphocytes following different exposures to caC). Results are expressed as a (%) of the control response (unexposed cells) (n = 4 per doseffected by inorganic mercury but a significant induction was observed after exposure to

10 10 101.20 ± 0.45 527.8 ± 105.6 7.50 ± 1.550.83–2.07 312.8–695.9 4.30–9.80

Thiols such as glutathione (GSH) (Messina and Lawrence, 1989)and cysteine (Angeline et al., 2002) have been suggested to play animportant role in T cell proliferation. Depletion of GSH in humanT cells was shown to impair IL-2 production, which is known tostimulate T cell proliferation (Yim et al., 1994). The reduction ofintracellular thiol concentrations of beluga whale lymphocytes byexposure to both inorganic and organic Hg supports a role for thiolsin altered T lymphocyte proliferation. Schurz et al. (2000) observedthat intracellular GSH concentrations increased in murine embry-onic fibroblast cells NIH 3T3 cells co-incubated with 0.5 and 1 �MHg2+, but they decreased at higher concentrations.

Metallothioneins are a family of low molecular weight, cysteine-rich metal binding proteins that have been detected in marinemammals (Das et al., 2000). MTs play a central role in the home-ostatic regulation of essential metals, and the detoxification ofnon-essential metals, such as Hg (Lee et al., 1977). However,

there are relatively few studies on the interaction between MeHgand MT. In the present study, 0.33 �M MeHgCl affected MTconcentrations in beluga whale peripheral blood lymphocytes.Similarly, Fortier et al. (2008) reported an increase in MT in human

dmium chloride (positive control; A), inorganic mercury (B) and organic mercury group; mean ± standard error). *p ≤ 0.05, #p ≤ 0.01. Metallothionein level was not

0.33 �M MeHgCl.

190 H. Frouin et al. / Aquatic Toxicology 109 (2012) 185– 193

Fig. 4. Relationship between proliferative response relative to control (%) and the thiol levels relative to control (%) when cells from male (square) and females (circle) areexposed to inorganic mercury (A) and organic mercury (B). Relationship between proliferative response relative to control (%) and the metallothionein induction relativeto control (%) when cells from male (square) and females (circle) are exposed to inorganic mercury (C) and organic mercury (D). Highest inorganic and organic mercuryconcentrations induced simultaneous a decrease of lymphocyte proliferation associated with a decrease of thiol levels, suggesting that intracellular thiols play an importantr ons ofl ing thT

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ole in the regulation of T-cell functions, i.e. cell proliferation. Highest concentratiymphocyte proliferation associated with an induction of metallothionein suggest-lymphocyte proliferation.

ymphocytes following in vitro exposure to 33 �g/L MeHg (approx-mately 0.15 �M). In our experiment, 10 �M HgCl2 failed to induceny increase in MT, whereas Yamada and Koizumi (1991) showedn increase of thioneins (apoproteins of MTs) in human lympho-ytes exposed to 1 �M HgCl2. Schurz et al. (2000) reported anncrease in intracellular MT concentrations in NIH 3T3 cells exposedo concentration of Hg2+ as low as 0.5 �M. Exposure to 10 �M CdCl2nduced MT in human lymphocytes to higher levels than grey sealymphocytes, suggesting that marine mammal lymphocytes mighte less sensitive to the effects of this metal (Pillet et al. (2002).

MT is thought to be an intermediary in stress-inducedmmunomodulation. For example, MT interacts with thelasma membrane of immune cells, including lymphocytesnd macrophages (Youn et al., 1995; Borghesi et al., 1996).lthough one consequence of the interaction of MT with lym-hocytes is increased cell proliferation (Borghesi et al., 1996), ourbserved inverted quadratic relationship between MT inductiony MeHg-associated T-lymphocyte proliferation (Fig. 5) did notupport this finding. Lynes et al. (1990) found that MT induction

y some metals (such as Hg and copper) are inhibitory in Con-induced proliferation assays when added alone to lymphocyteultures. At least in these circumstances, MT does not appear tolay a role as a protective agent against metal toxicity.

organic mercury, but not inorganic mercury, induced simultaneous a decrease ofat, in beluga whale peripheral blood lymphocytes, MT induced by MeHg inhibited

4.2. Se protective effect

Demethylation of MeHg and protection by Se have been sug-gested as possible Hg detoxification mechanisms in cetaceans(Thompson, 1990). It has been suggested that Hg is detoxified bySe via the formation of an equimolar Hg–Se complex in liver ofmarine mammals (Koeman et al., 1973). Indeed, crystalline mer-curic selenide (tiemannite; HgSe) has been observed in tissues,including white blood cells, of some marine mammals (Martoja andBerry, 1980; Nigro, 1994; Rawson et al., 1995; Nigro and Leonzio,1996). Selenium also reduces the toxicity of Hg through mecha-nisms other than the formation of crystalline HgSe. Palmisano et al.(1995) found in liver of striped dolphins (Stenella coeruleoalba)that, besides HgSe, there might also exist non-labile Hg attachedto Se compounds (presumably selenoproteins) and forming aHg–selenoprotein complex whose molar ratio of Se and Hg washigher than one.

In marine mammals, a molar ratio of Hg:Se approaching unitysuggests that Se is acting to reduce the toxicity of Hg through ulti-

mate binding to high molecular weight substances (Ikemoto et al.,2004). In the present study, concurrent exposure to Na2SeO3 andHgCl2 or MeHgCl decreased the toxicity of Hg in beluga whalelymphocytes. When the Hg:Se molar ratio was similar or higher

H. Frouin et al. / Aquatic Toxicology 109 (2012) 185– 193 191

Fig. 5. Selenium proved to exert a protective effect against inorganic mercury (A)and organic mercury (B) toxicity in lymphocyte proliferation assays (concurrentexposures to HgCl2 or MeHgCl and Na2SeO3). Results are expressed as a (%) relativeto ratio Hg 1:Se 0 (HgCl2 or MeHgCl exposed cells) (n = 3 for inorganic mercurya#

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Fig. 6. Hg concentrations (mean ± SD) reported in blood of cetaceans and lowestconcentrations of MeHgCl or HgCl2 inducing a decrease of proliferative response ofT-lymphocytes (stimulated with Con A).

bottlenose dolphin lymphocytes was caused by MeHg in a dose-

nd n = 4 for organic mercury per dose group; mean ± standard error). *p ≤ 0.05,p ≤ 0.01.

han 1:2, Se provided protection following exposure to highestoncentrations of HgCl2 (3.33 and 10 �M) or MeHgCl (10 �M).his is consistent with reports in Atlantic spotted dolphin (Stenellalagiodon) renal cells (Sp1K cells), where concurrent exposure to0 �M Na2SeO3 provided full protection against the decrease inell proliferation induced by 20 �M HgCl2 (Wang et al., 2001).nterestingly, the maximum protection was seen at a ratio ofgCl2:Na2SeO3 = 1:1 in human whole-blood cultures (Morimotot al., 1982). Therefore, although cetacean cells appear to be moreesistant to Hg toxicity in a selenium-independent manner thanuman and rat cells (Betti and Nigro, 1996), they may also requireelatively more selenite for protection.

.3. Hg and Se blood levels

As observed in our Arctic beluga samples, previous studies haveoncluded that MeHg and THg in blood of cetaceans (Itano et al.,984; Woshner et al., 2008) were equivalent, such that all Hg inhe blood may be inferred to be in the form of MeHg. This is unlike

ome tissues in odontocetes, like liver and kidney, where MeHgonstitutes a minor (<10%) proportion of the total Hg, especially inlder animals (Wagemann et al., 1998; Woshner et al., 2001).

Data were cited from a: Itano et al. (1984); b: Nielsen et al. (2000); c: Woshner et al.(2008); d: André et al. (1990); e: this study and f: Camara Pellisso et al. (2008).

In the present study, blood Se concentrations but not THg andMeHg concentrations correlated with length of the beluga whales.Moreover, our observations suggest that age has no effect on Se orHg concentrations in whole blood, perhaps due to rapid turnoverand recent exposures via feeding. Similarly, in the pantropical spot-ted dolphin (Stenella attenuata), no relationship was establishedbetween blood Hg concentrations and age of individuals (Andréet al., 1990). However, Woshner et al. (2008) previously demon-strated that THg and Se concentrations in the blood of bottlenosedolphins were correlated to age of dolphins. A possible explanationis that our sample numbers is small (10 individuals) to adequatelyexplore this (possible) relationship.

There was no relationship between Hg and Se in the blood ofbeluga whales or between the Hg:Se molar ratio and length or age.This is not surprising considering the lack of inorganic Hg in blood,which is involved in Hg–Se interaction reported in odontocete liveras well as the important role that the liver plays in detoxificationprocesses (Martoja and Berry, 1980). Previous studies have demon-strated that molar relationships between Se and Hg occurred inthe blood of bottlenose dolphins (Woshner et al., 2008) and pilotwhales (Globicephala melas) (Nielsen et al., 2000) but not in theblood of sperm whales (Physeter catodon) (Nielsen et al., 2000), indi-cating also the potential existence of species-specific differences.A relationship between the Hg:Se molar ratio in liver and age inharbour porpoises (Phocoena phocoena) (Bennett et al., 2001) sug-gested a reduced protective action of Se against Hg toxicity in olderanimals.

With the exception of pilot whales from the Faroe Islands, con-centrations of Hg in whole blood from our Arctic beluga whaleswere lower than those reported in other cetacean species fromother locations (Fig. 6). As Hg concentrations in blood reflects recentexposure via food, the factors influencing the species differencesinclude feeding and habitat, as well as age (Loseto et al., 2008).

Toothed cetaceans are typically high trophic level feeders andrepresent the apex species for MeHg biomagnification in food webs(Itano et al., 1984; Woshner et al., 2008). Hg concentrations incetacean blood from the literature were higher than the lowestMeHg concentration which induced an immunotoxic effect on bel-uga whale lymphocytes (0.33 �M; Fig. 6). DNA fragmentation in

related manner (Betti and Nigro, 1996). However, these dolphinleukocytes were significantly less susceptible to both genotoxicand cytotoxic effects of MeHg, when compared with human and

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at cells (Betti and Nigro, 1996). Taddei et al. (2001) establishedhat repair enzymes are able to rejoin most of the DNA repair sys-ems induced by MeHg in bottlenose dolphin leukocytes, and thathe efficiency of DNA repair systems in dolphin leukocytes is higherhan in human cells. A more efficient repair activity could accountor the higher resistance of dolphins to the toxic effects of organicg previously observed by Betti and Nigro (1996). In light of theseonsiderations, the elevated ability of cetaceans to repair the DNAamage induced by MeHg, the immobilization of Hg as mineralranules and the selenium-associated protection could be inter-reted as defence strategies developed to cope with elevated Hg

n marine food webs. In fish, Ralston et al. (2008) suggested thatlood Hg:Se molar ratios were a better predictor of risk comparedo blood Hg or MeHg concentrations.

The lack of integrated long-term data on health, diseases, andoxic effects in Arctic beluga whale, as well as the variable changesn climate, limit our ability to predict the effects of Arctic climatehange on its health. Because climate change have the potential toncrease pathogen development and survival rates, disease trans-

ission, and host susceptibility (Harvell et al., 2002), beluga whales a top predator, would be at greatest risk to an epidemic due toheir relatively high levels of Hg (Ross, 2002).

. Conclusions

Some caution is needed as we interpret our findings, as ourtudy had a limited sample size, and relied on extrapolations ofur in vitro results to beluga whales. However, beluga lymphocytesere affected by exposure to MeHg at concentrations that are cur-

ently observed in free-ranging populations. A lingering questionemains the degree to which cetaceans are able to cope with theseffects as they deal with elevated Hg levels in their prey. Our obser-ations suggest that Hg levels may already be sufficient to elicitn adverse effect on immune function in free-ranging populations.s climate change alters beluga habitat in the Arctic and changesost–pathogen associations due to altered pathogen transmissionr host resistance, any alterations to the transport and fate of Hgay have consequences for the health of Arctic beluga populations.

cknowledgements

This study was supported by Northern Contaminant ProgramNCP) of Aboriginal Affairs Canada, and by the Ecosystem Researchnitiative of Fisheries and Oceans Canada. We are grateful for theartnerships with the Tuktoyaktuk Hunters and Trappers Commit-ee who supported the program and the hunters who providedhales for sampling at Hendrickson Island. The authors wish to

hank Stephen Raverty, Marie Noel and summer students for sam-ling of Arctic beluga whales, Gail Boila and Joanne DeLaronde forercury and selenium analysis, Neil Dangerfield for laboratory sup-

ort, and the Vancouver Aquarium team for captive beluga whalelood sampling.

eferences

MAP, 2009. Arctic Pollution 2009. Arctic Monitoring and Assessment Programme(AMAP), Oslo, Norway.

ndré, J.M., Ribeyre, F., Boudou, A., 1990. Mercury contamination levels and distri-bution in tissues and organs of delphinids (Stenella attenuata) from the Easterntropical Pacific, in relation to biological and ecological factors. Mar. Environ. Res.30, 43–72.

ngeline, G., Gardella, S., Ardy, M., Ciriolo, R., Filomeni, G., Di Trapani, G., Clarke,

F., Sitia, R., Rubartelli, A., 2002. Antigen-presenting dendritic cells provide thereducing extracellular microenvironment required for T lymphocyte activation.Proc. Natl. Acad. Sci. U.S.A. 99, 1491–1496.

rmstrong, J.A.J., Uthe, J.F., 1971. Semi-automated determination of mercury in ani-mal tissue. At. Absorpt. Newslett. 10, 101–103.

ogy 109 (2012) 185– 193

Bennett, P., Jepson, P., Law, R., Jones, B., Kuiken, T., Baker, J., Rogan, E., Kirkwood,J., 2001. Exposure to heavy metals and infectious disease mortality in harbourporpoises from England and Wales. Environ. Pollut. 112, 33–40.

Betti, C., Nigro, M., 1996. The comet assay for the evaluation of the genetic hazard ofpollutants in cetaceans: preliminary results on the genotoxic effects of methyl-mercury on the bottle-nosed dolphin (Tursiops truncatus) lymphocytes in vitro.Mar. Pollut. Bull. 32, 545–548.

Borghesi, L.A., Youn, J., Olson, E.A., Lynes, M.A., 1996. Interactions of metalloth-ionein with murine lymphocytes: plasma membrane binding and proliferation.Toxicology 108, 129–140.

Brousseau, P., Payette, Y., Tryphonas, H., Blakley, B., Boermans, H., Flipo, D., Fournier,M., 1999. Manual of Immunological Methods. CRC Press, Boston.

Burek, K.A., Gulland, F.M.D., O’Hara, T.M., 2008. Effects of climate change on Arcticmarine mammal health. Ecol. Appl. 18, S126–S134.

Camara Pellisso, S., Munoz, M.J., Carballo, M., Sanchez-Vizcaino, J.M., 2008. Determi-nation of the immunotoxic potential of heavy metals on the functional activityof bottlenose dolphin leukocytes in vitro. Vet. Immunol. Immunopathol. 121,189–198.

Carvalho, C.M.L., Chew, E.H., Hashemy, S.I., Holmgren, A., 2008. Inhibition of thehuman thioredoxin system. J. Biol. Chem. 283, 11913–11923.

Caurant, F., Navarro, M., Amiard, J.-C., 1996. Mercury in pilot whales: possible limitsto the detoxification process. Sci. Total Environ. 186, 95–104.

Cuvin-Aralar, M.L.A., Furness, R.W., 1991. Mercury–selenium interaction: a review.Ecotoxicol. Environ. Saf. 21, 348–364.

Das, K., Debacker, V., Bouquegneau, J., 2000. Metallothioneins in marine mammals.Cell Mol. Biol. 46, 283–294.

Das, K., Siebert, U., Gillet, A., Dupont, A., Di-Poï, C., Fonfara, S., Mazzucchelli, G., DePauw, E., De Pauw-Gillet, M.-C., 2008. Mercury immune toxicity in harbour seals:links to in vitro toxicity. Environ. Health 7, 52–68.

De Guise, S., Bernier, J., Dufresne, M.M., Martineau, D., Béland, P., Fournier, M.,1996a. Immune functions in beluga whales (Delphinapterus leucas): evaluation ofmitogen-induced blastic transformation of lymphocytes from peripheral blood,spleen and thymus. Vet. Immunol. Immunopathol. 50, 117–126.

De Guise, S., Bernier, J., Martineau, D., Béland, P., Fournier, M., 1996b. Effects of invitro exposure of beluga whale splenocytes and thymocytes to heavy metals.Environ. Toxicol. Chem. 15, 1357–1364.

Dufresne, M., Frouin, H., Pillet, S., Hammill, M., Lesage, V., De Guise, S., Fournier,M., 2010. Comparative sensitivity of harbour and grey seals to severalenvironmental contaminants using in vitro exposure. Mar. Pollut. Bull. 60,344–349.

Fortier, M., Omara, F., Bernier, J., Brousseau, P., Fournier, M., 2008. Effects of physio-logical concentrations of heavy metals both individually and in mixtures on theviability and function of peripheral blood human leukocytes in vitro. J. Toxicol.Environ. Health A 71, 1327–1337.

Friberg, L., Nordberg, G.F., Vouk, V.F. (Eds.), 1986. Handbook on the Toxicology ofMetals. Volume II, 2nd edition. Specific Metals. Elsevier Applied Science Pub-lishers, New York.

Frouin, H., Ménard, L., Measures, L., Brousseau, P., Fournier, M., 2010. T lymphocyte-proliferative responses of a grey seal (Halichoerus grypus) exposed to heavymetals and PCBs in vitro. Aquat. Mamm. 36, 365–371.

Gauthier, J.M., Dubeau, H., Rassart, É., 1998. Mercury-induced micronuclei in skinfibroblasts of beluga whales. Environ. Toxicol. Chem. 17, 2487–2493.

Gray, J.S., 2002. Biomagnification in marine systems: the perspective of an ecologist.Mar. Pollut. Bull. 45, 46–52.

Harvell, C.D., Mitchell, C.E., Ward, J.R., Altizer, S., Dobson, A.P., Ostfeld, R.S., Samuel,M.D., 2002. Climate warming and disease risks for terrestrial and marine biota.Science 296, 2158–2162.

Ikemoto, T., Kunito, T., Anan, Y., Tanaka, H., Baba, N., Miyazaki, N., Tanabe, S.,2004. Association of heavy metals with metallothionein and other proteins inhepatic cytosol of marine mammals and seabirds. Environ. Toxicol. Chem. 23,2008–2016.

Itano, K., Kawai, S.i., Miyazaki, N., Tatsukawa, R., Fujiyama, T., 1984. Mercury andselenium levels in striped dolphins caught off the pacific coast of Japan. Agric.Biol. Chem. 48, 1109–1116.

Koeman, J., Peeters, W.H.M., Koudstaal-Hoi, C.H.M., Tijioe, P., de Goeij, J., 1973.Mercury–selenium correlations in marine mammals. Nature 245, 385–386.

Laidre, K.L., Stirling, I., Lowry, L.F., Wiig, Ø., Heide-Jorgensen, M.P., Ferguson, S.H.,2008. Quantifying the sensitivity of Arctic marine mammals to climate-inducedhabitat change. Ecol. Appl. 18, S97–S125.

Lalancette, A., Morin, Y., Measures, L., Fournier, M., 2003. Contrasting changes ofsensitivity by lymphocytes and neutrophils to mercury in developing grey seals.Dev. Comp. Immunol. 27, 735–747.

Lee, S.S., Mate, B.R., von der Trench, K.T., Rimerman, R.A., Buhler, D.R., 1977.Metallothionein and subcellular localization of mercury and cadmium in theCalifornian sea lion. Comp. Biochem. Physiol. 57, 45–53.

Loseto, L., Stern, G.A., Deibel, D., Connelly, T.L., Prokopowicz, A., Lean, D.R.S., Fortier,L., Ferguson, S.H., 2008. Linking mercury exposure to habitat and feedingbehaviour in Beaufort Sea beluga whales. J. Mar. Syst. 74, 1012–1024.

Lynes, M.A., Garvey, J.S., Lawrence, D.A., 1990. Extracellular metallothionein effectson lymphocyte activities. Mol. Ecol. 27, 211–219.

Macdonald, R.W., 2007. Contaminants, global change and cold regions. In: Ørbaek,

J.B., Kallenborn, R., Tombre, I., Hegseth, E.N., Falk-Petersen, S., Hoel, A.H. (Eds.),Arctic Alpine Ecosystems and People in a Changing Environment. Springer, NewYork.

Martoja, R., Berry, J.P., 1980. Identification of tiemannite as a probable product ofdemethylation of mercury by selenium in cetaceans. Vie Milieu 30, 7–10.

oxicol

M

M

N

N

N

P

P

P

R

R

R

S

S

S

S

H. Frouin et al. / Aquatic T

essina, J.P., Lawrence, D.A., 1989. Cell cycle progression of glutathione-depletedhuman peripheral blood mononuclear cells is inhibited at S phase. J. Immunol.143, 1974–1981.

orimoto, K., Iijima, S., Koizumi, A., 1982. Selenite prevents the induction of sister-chromatid exchanges by methyl mercury and mercuric chloride in humanwhole-blood cultures. Mutat. Res. 102, 183–192.

ielsen, J.B., Nielsen, F., Jørgensen, P.-J., Grandjean, P., 2000. Toxic metals andselenium in blood from Pilot whales (Globicephala melas) and Sperm whales(Physeter catodon). Mar. Pollut. Bull. 40, 348–351.

igro, M., 1994. Mercury and selenium localization in macrophages of the stripeddolphin, Stenella coeruleoalba. J. Mar. Biol. Assoc. U.K. 74, 975–978.

igro, M., Leonzio, C., 1996. Intracellular storage of mercury and selenium in differ-ent marine vertebrates. Mar. Ecol. Prog. Ser. 135, 137–143.

almisano, F., Cardellicchio, N., Zambonin, P.G., 1995. Speciation of mercury in dol-phin liver: a two-stage mechanism for the demethylation accumulation processand role of selenium. Mar. Environ. Res. 40, 109–121.

illet, S., Fournier, M., Measures, L.N., Bouquegneau, J.-M., Cyr, D.G., 2002. Presenceand regulation of metallothioneins in peripheral blood leukocytes of grey seals.Toxicol. Appl. Pharmacol. 185, 207–217.

illet, S., Lesage, V., Hammill, M., Cyr, D.G., Bouquegneau, J.M., Fournier, M., 2000.In vitro exposure of seal peripheral blood leukocytes to different metals reveala sex-dependent effect of zinc on phagocytic activity. Mar. Pollut. Bull. 40,921–927.

alston, N.V.C., Ralston, C.R., Blackwell III, J.L., Raymond, L.J., 2008. Dietary and tissueselenium in relation to methylmercury toxicity. Neurotoxicology 29, 802–811.

awson, A.J., Bradley, P.J., Teetsov, A., Rice, S.B., Haller, E.M., Patton, G.W., 1995. Arole for airborne articulates in high mercury levels of some cetaceans. Ecotoxicol.Environ. Saf. 30, 309–314.

oss, P.S., 2002. The role of immunotoxic environmental contaminants in facilitat-ing the emergence of infectious diseases in marine mammals. Hum. Ecol. RiskAssess. 8.

chaefer, A.M., Stavros, H.-C.W., Bossart, G.D., Fair, P.A., Goldstein, J.D., Reif, J.S., 2011.Associations between mercury and hepatic, renal, endocrine, and hematologi-cal parameters in Atlantic bottlenose dolphins (Tursiops truncatus) along theeastern coast of Florida and South Carolina. Arch. Environ. Contam. Toxicol.,doi:10.1007/s00244-011-9651-5.

chiedek, D., Sundelin, B., Readman, J.W., Macdonald, R.W., 2007. Interactionsbetween climate change and contaminants. Mar. Pollut. Bull. 54, 1845–1856.

churz, F., Sabater-Vilar, M., Fink-Gremmels, J., 2000. Mutagenicity of mercury chlo-ride and mechanisms of cellular defence: the role of metal-binding proteins.

Mutagenesis 15, 525–530.

iebert, U., Joiris, C., Holsbeek, L., Benke, H., Failing, K., Frese, K., Petzinger, E., 1999.Potential relation between mercury concentrations and necropsy findings incetaceans from German waters of the North and Baltic Seas. Mar. Pollut. Bull.38, 285–295.

ogy 109 (2012) 185– 193 193

Simmons, S., Eliott, W.J., 2009. Climate change and cetaceans: concerns and recentdevelopments. J. Mar. Biol. Assoc. U.K. 89, 203–210.

Stewart, R.E.A., Campana, S.E., Jones, C.M., Stewart, B.E., 2006. Bomb radiocarbondating calibrates beluga (Delphinapterus leucas) age estimates. Can. J. Zool. 84,1840–1852.

Taddei, F., Scarcelli, V., Frenzilli, G., Nigro, M., 2001. Genotoxic hazard of pollutants incetaceans: DNA damage and repair evaluated in the bottlenose dolphin (Tursiopstruncatus) by the comet assay. Mar. Pollut. Bull. 42, 324–328.

Thompson, D.R., 1990. Metals levels in marine vertebrates. In: Furness, R.W., Rain-bow, P.S. (Eds.), Heavy Metals in the Marine Environment. CRC Press Inc, BocaRaton, pp. 143–182.

Uthe, J.F., Solomon, J., Grift, N., 1972. Rapid semi-micro method for the determinationof methyl mercury in fish tissue. J. Assoc. Off. Anal. Chem. 55, 583–589.

Vijan, P.N., Wood, G.R., 1976. An automated submicrogram determination ofselenium in vegetation by quartz-tube furnace atomic-absorption spectropho-tometry. Talanta 23, 89–94.

Wagemann, R., Trebacz, E., Boila, G., Lockhart, W.L., 1998. Methylmercury and totalmercury in tissues of arctic marine mammals. Sci. Total Environ. 21, 19–31.

Wang, A., Barber, D., Pfeiffer, C.J., 2001. Protective effects of selenium against mer-cury toxicity in cultured Atlantic spotted dolphin (Stenella plagiodon) renal cells.Arch. Environ. Contam. Toxicol. 41, 403–409.

Wolfe, M.F., Schwarzbach, S., Sulaiman, R.A., 1998. Effects of mercury on wildlife: acomprehensive review. Environ. Toxicol. Chem. 17, 146–160.

Woshner, V.M., Knott, K., Wells, R., Willetto, C., Swor, R., O’Hara, T., 2008. Mercuryand selenium in blood and epidermis of bottlenose dolphins (Tursiops truncatus)from Sarasota Bay, FL: interaction and relevance to life history and hematologicparameters. EcoHealth 5, 360–370.

Woshner, V.M., O’Hara, T.M., Bratton, G.R., Suydam, R.S., Beasley, V.R., 2001. Con-centrations and interactions of selected essential and non-essential elements inbowhead and beluga whales of Arctic Alaska. J. Wildl. Dis. 37, 693–710.

Woshner, V.M., O’Hara, T.M., Eurell, J.A., Wallig, M.A., Bratton, G.R., Suydam, R.S.,Beasley, V.R., 2002. Distribution of inorganic mercury in liver and kidney ofbeluga and bowhead whales through autometallographic development of lightmicroscopic tissue sections. Toxicol. Pathol. 30, 209–215.

Yamada, H., Koizumi, S., 1991. Metallothionein induction in human peripheral bloodlymphocytes by heavy metals. Chem. Biol. Interact. 78, 347–354.

Yim, C.Y., Hibbs Jr., J.B., McGregor, J.R., Galinsky, R.E., Samlowski, W.E., 1994. Useof N-acetyl cysteine to increase intracellular glutathione during induction ofantitumor responses by IL-2. J. Immunol. 152, 5796–5805.

Youn, J., Borghesi, L.A., Olson, E.A., Lynes, M.A., 1995. Immunomodulatory activities

of extracellular metallothionein: II. Effects on macrophage functions. J. Toxicol.Environ. Health 45, 397–413.

Yurkow, E.J., Makhijani, P.R., 1998. Flow cytometric determination of metalloth-ionein levels in human peripheral blood lymphocytes: utility in environmentalexposure assessment. J. Toxicol. Environ. Health 54, 445–457.