mercury-induced micronuclei in skin fibroblasts of beluga whales

2487

Environmental Toxicology and Chemistry, Vol. 17, No. 12, pp. 2487–2493, 1998q 1998 SETAC

Printed in the USA0730-7268/98 $6.00 1 .00

MERCURY-INDUCED MICRONUCLEI IN SKIN FIBROBLASTS OF BELUGA WHALES

JULIE M. GAUTHIER,* HELENE DUBEAU, and ERIC RASSARTDepartement des Sciences Biologiques, Universite du Quebec a Montreal, C. P. 8888, Succursale Centre-Ville,

Montreal, Quebec, H3C 3P8, Canada

(Received 11 November 1997; Accepted 20 April 1998)

Abstract—Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmentalpollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high incidence ofcancer observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei assay wasused to test the genotoxic potential of mercury compounds in skin fibroblasts of an Arctic beluga whale. Both mercuric chloride(Hg) and methylmercury (MeHg) induced a highly significant (p , 0.001) dose–response increase of micronucleated cells. Statis-tically significant increases in micronucleated cells were observed for 0.5, 5, and 20 mg/ml Hg and 0.05, 0.5, and 2 mg/ml MeHgwhen compared to control cultures. Concentrations of 0.5, 5, and 20 mg/ml Hg induced a two-, three- and fourfold increase ofmicronucleated cells, respectively. Treatment with MeHg was one order of magnitude more potent in inducing micronuclei and ininhibiting cell proliferation than Hg. Although results of this in vitro study do not imply that mercury compounds are involved inthe etiology of cancer in St. Lawrence beluga whales, significant increases in micronuclei frequency were found at low concentrationsof MeHg (0.05 and 0.5 mg/ml) that are believed to be comparable to concentrations present in certain whales of this population.

Keywords—Beluga whale St. Lawrence estuary Mercury Micronuclei Skin fibroblasts

INTRODUCTION

Beluga whales (Delphinapterus leucas) of the St. Lawrenceestuary form a small endangered population according to theCommittee on the Status of Endangered Wildlife in Canada.Necropsies of dead stranded whales have shown a high prev-alence of tumors, nonneoplastic lesions, and opportunistic dis-eases [1,2]. Cancer has been diagnosed as the principal causeof death in 18% of 97 examined carcasses, and the annualcrude cancer rate is estimated to be 233 per 100,000 popu-lation, which is higher than in most human and domestic an-imal populations [3] (D. Martineau, personal communication).Population modeling, field studies, and necropsy observationsof female reproductive organs have suggested a lower repro-ductive rate in this population than in Arctic populations (re-viewed by Beland et al. [4]). High concentrations of environ-mental contaminants, such as polychlorinated biphenyls, or-ganochlorine compounds, and mercury, and the presence ofbenzo[a]pyrene DNA adducts have been found in tissues ofSt. Lawrence beluga whales [2,5–7]. In contrast, Arctic belugawhales have low concentrations of environmental contami-nants [5,7], and no gross evidence of cancer has been foundin approx. 50 carcasses sampled for routine biological purposes(D.J. St. Aubin, personal communication). Factors such astumor induction, tumor promotion, and immunosuppressioninduced by environmental contaminants have been proposedto explain the elevated cancer incidence in the St. Lawrencepopulation [1,2].

DNA damage plays an important role in the developmentof cancer and can be a risk factor for teratogenesis and geneticdisease [8,9]. Mercury compounds are highly toxic, wide-spread, and long-lived bioaccumulating agents [10,11], thathave genotoxic and teratogenic activities [12,13]. Both inor-ganic and organic forms of mercury have been shown to induce

* To whom correspondence may be addressed ([email protected]).

DNA damage at the chromosomal level in cells of humans,domestic animals, and wildlife (reviewed by DeFlora et al.[13]). Methylmercury (MeHg) is a carcinogen in experimentalanimals and is considered possibly carcinogenic to humans[14].

The mammalian cytokinesis–block micronuclei (MN) assayis a well-known cytogenetic technique used to assess DNAdamage induced by environmental contaminants [15]. Micro-nuclei are chromosomal fragments or whole chromosomes thatare not incorporated into daughter nuclei during mitosis be-cause of chromosomal breakage or whole chromosome loss(i.e., aneuploidy), respectively [15]. Cytochalasin B preventscytokinesis and produces binucleated (BN) cells, which canbe easily scored for MN [16]. The MN assay is an ideal testfor the detection of mercury-induced genotoxicity becausemercury-compounds can induce both chromosomal breakageand aneuploidy [13].

Very few studies exist on the genotoxic effects of environ-mental contaminants in marine mammals. Recently, inductionof single-strand breakage by MeHg has been demonstrated invitro in lymphocytes of bottlenose dolphins (Tursiops trun-catus) [17]. In this study, primary skin fibroblasts of an Arcticbeluga whale were used to test the sensitivity of these cells tothe genotoxic effects of mercuric chloride (Hg) and MeHg atconcentrations relevant to those found in St. Lawrence belugawhales.

METHODS

Cell culture

Primary beluga whale (D. leucas) fibroblastoid cells wereisolated from a skin biopsy sample taken by S. DeGuise froma subadult or adult (3.9 m) male Arctic beluga whale collectedby subsistence Inuvialuit hunting at Arviat, west Hudson Bay,Canada. The biopsy sample was placed in fetal calf serum

2488 Environ. Toxicol. Chem. 17, 1998 J.M. Gauthier et al.

(Gibco, Burlington, Ontario, Canada) containing 10% dimeth-yl sulfoxide (DMSO) in a cryopreservation vial and stored inliquid nitrogen until use. The skin sample was thawed, placedin a 60-mm plastic petri dish, rinsed several times in phos-phate-buffered saline (PBS) without Ca21 and Mg21 (Gibco),sliced into small pieces with a scalpel, and rinsed again. Thesample was then transferred to an Erlenmeyert flask and dis-persed twice for 20 min with a magnetic stirrer at 378C in PBScontaining 0.125% trypsin and 1.33 mM ethylenediaminetetra-acetic acid (Gibco). After each treatment with trypsin, the cellsuspension was pipetted into a centrifuge tube and fetal calfserum was added to arrest treatment. The pooled cell suspen-sions were centrifuged at 1,900 rpm for 20 min, and the cellpellet was resuspended in modified Eagle’s medium (Gibco)supplemented with penicillin-streptomycin and 15% fetal calfserum in a 60-mm petri dish and incubated at 378C in a hu-midified atmosphere with 5% CO2. The medium was replen-ished after 24 h, and cells were maintained thereafter by chang-ing the medium every 1 to 2 d. The same serum batch wasused during the whole course of the study.

Treatment and slide preparation

Experiments were performed using pooled, exponentiallygrowing cells at passages 7 to 10. Confluent cultures weretrypsinized, and cells were seeded at 5 3 105 cells/ml onsterilized microscopic slides placed individually in 100-mmpetri dishes and incubated for 48 h. Mitomycin C (MMC)(Sigma Chemical, Oakville, ON, Canada) and Hg (generouslydonated by F. Denizeau) were dissolved in sterile distilledwater (Gibco), and MeHg (Ultra Scientific, distributed byVWR, Mississauga, ON, Canada) was dissolved in DMSO.Distilled water or DMSO was used as the negative control,and MMC was used as the positive control. Final distilledwater and DMSO concentrations were 1% for all cultures. Cellswere treated (three replicates per culture) with MMC for 3 hand Hg or MeHg for 24 h at concentrations shown in Tables1 and 2. Ranges of experimental doses were chosen accordingto those expected to be found in the St. Lawrence belugawhales and according to preliminary cell-killing dose–re-sponse experiments. The medium was then aspirated and re-placed by fresh medium containing 6 mg/ml cytochalasin B(Sigma), and cells were incubated another 48 h. Cells werefixed in situ once with half PBS and half methanol:acetic acid(4:1, v/v) and twice with methanol:acetic acid (4:1, v/v), air-dried, and stained with 10% Giemsa (Sigma) for 30 min.

Micronuclei analysis

Micronuclei were scored according to established criteria[18] with the following specifications: (1) cells were scoredonly if there was intact cytoplasm, a distinct nuclear mem-brane, and little or no debris and overlap with adjacent cells;(2) no more than four MN within a BN cell was scored; and(3) MN were scored only if their texture and focal plane weresimilar to those of main nuclei and if there was absence of abridge between the MN and main nuclei. However, MN thatoverlapped the boundaries of the main nuclei were scored [15]if the complete membranes of both objects could be readilydistinguished by transparency. The cytokinesis-blocked pro-liferation index (CBPI), which measures the average numberof cell cycles per cell, was calculated for each treatment asCBPI 5 [1(M1) 1 2(M2) 1 3(M3 1 M4)]/N, where M1 toM4 represent the number of cells with 1 to 4 nuclei and N isthe total number of cells scored [19]. For each treatment, at

least 1,000 BN cells were scored for MN at 4003 magnifi-cation, and the presence of MN was confirmed at 1,0003magnification.

Statistical analysis

A one-way analysis of variance (ANOVA) was used tocompare CBPI and frequency of micronucleated BN cells foreach treatment with the respective control. Regression analysiswas performed to test for dose–response relationships for eachtest compound, where the y axis represents either the CBPI orfrequency of micronucleated BN cells and the x axis representsthe concentration of the compound. All statistical analyseswere conducted using Minitab software, version 8 (Minitab,State College, PA, USA).

RESULTS

Typical BN cells with and without a MN are shown inFigure 1. Results for MN induction and CBPI in beluga whaleskin fibroblasts treated in vitro with MMC, Hg, and MeHg aresummarized in Tables 1 and 2. The CBPI and micronucleatedcell frequency in distilled water or DMSO control cultures ofbeluga whale skin fibroblasts were similar and ranged from1.48 to 1.59‰ and 5 to 11‰ BN cells, respectively (Tables1 and 2).

A significant increase in micronucleated cell frequency wasfound for all tested concentrations of positive control MMC(Table 2). Treatment with 0.1 mg/ml MMC induced a twofoldmean increase in micronucleated cells, whereas both the 1-and 2- mg/ml concentrations induced a similar mean increasein micronucleated cells of about sixfolds when compared tothe distilled water control. Treatment with MMC caused ahighly significant dose–response increase in micronucleatedcells (y 5 14.7 1 19.0x, R2 [adjusted] 5 68.7%, p , 0.001),which was accompanied by a significant decrease in CBPI (y5 1.49 2 0.138x, R2 [adjusted] 5 59.9%, p 5 0.002) (Fig.2).

Beluga skin fibroblasts treated with Hg and MeHg showedhighly significant dose–response increases in micronucleatedcells (Hg: y 5 10.4 1 1.0x, R2 [adjusted] 5 64.9%, p , 0.001;MeHg: y 5 12.2 1 7.59x, R2 [adjusted] 5 68.5%, p , 0.001)with concomitant highly significant decreases in cell prolif-eration (Hg: y 5 1.56 2 0.0114x, R2 [adjusted] 5 78.4%, p, 0.001; MeHg: y 5 1.62 2 0.203x, R2 [adjusted] 5 84.3%,p , 0.001) (Figs. 3 and 4). However, MMC was generallymore potent in inducing these effects, as the regression co-efficient (slope) for micronucleated cell frequency was 2.5-and 19-fold greater for MMC than for MeHg and Hg, respec-tively, and regression coefficients for CBPI were 10.1 and 17.8times greater for MMC and MeHg, respectively, than for Hg.In contrast to MMC cultures, frequency of micronucleatedcells in mercury-treated cultures did not reach a threshold butcontinued to increase up to the last tested concentration thatdid not induce total mortality. According to concentrationstested in this study, the CBPI statistically decreased startingat 5 mg/ml Hg and 2 mg/ml MeHg (Table 1). This correspondsto a mean decrease in BN cell frequency of 12 and 44% at 5and 20 mg/ml Hg, respectively, and to a 64% mean decreasein BN cell frequency at 2 mg/ml MeHg compared to controlcultures (Table 1). Total mortality (absence of cells on slide)was observed at concentrations of 50 mg/ml Hg and 5 mg/mlMeHg (Table 1). However, treatment with 20 mg/ml Hg re-sulted in notable cytotoxicity, because approx. 40% of cellsthat had not detached from the slide had a rounded appearance

Mercury-induced micronuclei in skin fibroblasts Environ. Toxicol. Chem. 17, 1998 2489

Table 1. Distribution of cells with different numbers of nuclei, cytokinesis-blocked proliferation index, and analysis of variance results for belugawhale skin fibroblasts treated in vitro with mitomycin C, Hg, and MeHg

Chemicalandconcn.(mg/ml) Replicate

Cells with indicated no. ofnuclei (per 1,000 cells)

1 2 3 4 CBPI Mean 6 SD pa

H2O1% 1

23

457423437

533563554

812

7

222

1.551.591.57 1.57 6 0.02

DMSO1% 1

23

451530456

530456531

118

10

863

1.571.481.57 1.54 6 0.05 0.404b

MMC0.1

1

2

123123123

578518537780790770739692724

418477456213203227259303273

456673233

001100020

1.431.491.481.231.221.231.261.311.28

1.47 6 0.03

1.23 6 0.01

1.28 6 0.03

0.009

,0.001

,0.001

Hg0.05 1 446 542 8 4 1.63

0.5

5

20

23123123123

473455398455504504509496678637736

516528588534479479477499317355261

101511

6131311

3584

12354432000

1.571.541.561.621.561.511.501.511.331.371.27

1.58 6 0.05

1.58 6 0.03

1.51 6 0.01

1.32 6 0.05

0.746

0.687

0.006

0.00350 1–3 All cultures failed

MeHg0.05

0.5

2

123123123

314455389416434413834795823

663535603575555581164203173

1886994223

522022001

1.711.551.621.591.581.591.181.211.18

1.63 6 0.08

1.59 6 0.01

1.19 6 0.02

0.191

0.197

,0.0015 1–3 All cultures failed

CBPI 5 cytokinesis-blocked proliferation index; DMSO 5 dimethyl sulfoxide; MMC 5 mitomycin C.a Based on a one-way analysis of variance (ANOVA) comparing the CBPI per treatment with the respective control.b Based on a one-way ANOVA comparing DMSO and the distilled water control.

with different degrees of cell deterioration grossly consistingof vacuolized cytoplasm and reduction of nuclei and cell size.Statistically significant increases in micronucleated cell fre-quency were observed for both compounds at all concentra-tions when compared to control cultures, with the exceptionof 0.05 mg/ml Hg (Table 2). A highly significant increase inmicronucleated cells was observed at 2 mg/ml MeHg. Con-centrations of 0.5, 5, and 20 mg/ml Hg induced 1.7-, 2.8-, and3.6-fold mean increases in micronucleated cells, respectively,compared to controls. Treatment with MeHg was more potentin inducing MN than Hg, because the regression coefficientwas 7.6 times higher, and similar micronucleated cell fre-

quencies were observed for 0.05, mg/ml MeHg and 0.5 mg/mlHg, and for 2 mg/ml MeHg and 20 mg/ml Hg (Table 2).

Cells with two MN were observed in both control andtreated cultures, but frequencies were consistently greateracross replicates at higher concentrations, such as 5 and 20mg/ml Hg and 2 mg/ml MeHg (Table 2). Multimicronucleatedcells (3–4 MN per cell) were never observed in control culturesand rarely found in mercury-treated cultures. However, mul-timicronucleated cells were mainly found at concentrationsthat significantly increased micronucleated cell frequency.Similar results for cells with two or more MN were obtainedwith MMC.


Fig. 1. Typical binucleated beluga whale fibroblasts without (A) andwith (B) a micronucleus (indicated by arrow).

DISCUSSION

This study is part of a long-term project on possible mech-anisms of action of environmental contaminants in the patho-genesis of disease in the St. Lawrence beluga whale popula-tion. Approximately 37% of tumors reported worldwide incetaceans have been observed in this highly contaminated pop-ulation [2]. Chromosome breakage and aneuploidy is an in-dication of exposure to genotoxic compounds that may in-crease the risk of cancer, teratogenesis, and genetic disease[8,9]. Tumor induction by environmental contaminants maybe partly responsible for the high cancer rate observed in theSt. Lawrence beluga whale population [1,2]. Although envi-ronmental contaminants exist in mixtures, it is important tofirst characterize the genotoxic hazard of individual com-pounds in beluga whale cells. In the present study, the MNassay was successfully used to test the in vitro genotoxic ef-fects of mercury compounds in beluga whale skin fibroblasts.

Most cancers are of epithelial origin, including those ob-served in St. Lawrence beluga whales [3,20]. However, epi-thelial cells of target organs are difficult or impossible to obtainin a noninvasive manner, especially in wild cetaceans. Pe-ripheral blood lymphocytes are considered the best noninva-sive source of cells to analyze DNA damage because theycirculate throughout all organs and tissues and thus providean estimate of average whole-body exposure to mutagens [9].However, blood lymphocytes were not available in sufficient

amounts from beluga whales for in vitro MN studies. Fibro-blasts are the major cell type of conjunctive tissue of organsand body tissues and are thus omnipresent in the organism[21]. Fibroblasts have been widely used in genotoxicity testingstudies using the MN assay and other cytogenetic endpoints.Mice injected intraperitoneally with MeHg in vivo have shownincreases in chromosome aberrations in skin fibroblasts [22].Beluga whale fibroblast cell cultures were easily establishedfrom a small skin biopsy sample, and cells proliferated vig-orously in culture. Therefore, these cells were found to bepractical for the analysis of genotoxic effects of environmentalcontaminants in this species.

Spontaneous micronucleated cell frequency in beluga skinfibroblasts (5–11‰) were in the lower range reported for hu-man skin fibroblasts (3–22%) taken from healthy, karyotypi-cally normal individuals and cultured for three to 14 passages[23,24]. Mitomycin C is a direct alkylating agent commonlyused as a positive control in the MN assay because it is wellrecognized as a clastogen and may also act as an aneugen [25].Induction of MN by MMC in beluga fibroblasts was similarto or lower than that reported for Chinese hamster fibroblasts(fourfold increase at 0.1 mg/ml) and human skin fibroblasts(six- to eightfold increase at 1 mg/ml and ninefold increase at2 mg/ml) [25–27].

Although MMC was a more potent inducer of micronu-cleated cells, highly significant dose–response increases wereobtained with both mercury compounds, indicating the stronggenotoxicity capacity of these compounds in beluga whalefibroblasts in vitro. Mercury compounds may induce breakageof single-stranded DNA and, more importantly (particularlyfor MeHg), c-mitosis–induced aneuploidy through binding tosulfhydryl groups of tubulin proteins of the mitotic apparatus[13]. Reduction of cell proliferation and cytotoxicity inducedby mercury compounds may be due to inhibition of proteinsynthesis and interactions with microtubules, membranes, andorganelles [10,28]. It is difficult to compare sensitivity of fi-broblasts of only a single beluga whale to genotoxic and cy-totoxic effects of mercury compounds with cells of other mam-mals because methodologies and endpoints vary between stud-ies. Impairment of cell division and survival have been re-ported for similar concentrations of Hg and MeHg in differentmammalian cell types [28–30]. DNA strand breakage has beenreported in dolphin lymphocytes exposed to 1 and 2 mg/mlMeHg [17]. Sensitivity of beluga whale fibroblasts to geno-toxic activity of Hg and MeHg appears to be greater than thatof Chinese hamsters fibroblasts and human lymphocytes cul-tured using a protocol similar to the one used in this studyand treated with mercury compounds for 20 to 24 h, becauseno increase in MN and chromosome aberration frequency wasfound when these cells were exposed to concentrations rangingfrom 0.27 to 3 mg/ml Hg and from 0.3 to 1.25 mg/ml MeHg,and two- to threefold increases were induced at concentrationsranging from 2.7 to 15 mg/ml Hg and from 1.5 to 4.5 mg/mlMeHg [29–31]. These comparisons must be interpreted withcaution, because only a limited number of studies that usedsimilar methodologies were found. No threshold in MN in-duction was observed for concentrations of Hg and MeHgtested in this study. This is similar to results from other studiesthat have tested similar concentrations of MeHg in humanlymphocytes [29,30]. Binucleated beluga fibroblasts contain-ing two to four MN were mainly observed for the highesttested mercury concentrations (5 and 20 mg/ml Hg and 2 mg/ml MeHg). An increased number of chromosome aberrations


Table 2. Distribution of micronuclei and total number of micronucleated cells per 1,000 binucleated beluga whale skin fibroblasts treated in vitrowith mitomycin C, Hg, and MeHg

Chemicalandconcn.(mg/ml) Replicate

No. of MNCs/BNC

0 1 2 3 4Total

MNCs Mean 6 SD pa

H2O1% 1

23

988994993

937

230

000

000

1167 8.0 6 2.7

DMSO1% 1

23

991992995

883

102

000

000

985 7.3 6 2.1 0.733b

MMC0.1

1

2

123123123

985984984948946951953954957

151213464743394239

022665742

011011100

001000002

151517525449484643

15.6 6 1.0

49.1 6 6.1

45.1 6 2.1

0.010

,0.001

,0.001

Hg0.05 1 995 5 0 0 0 5

0.5

5

20

23123123123

995994984988987981980972973972964

45

161211151624242431

10000444332

01002000103

00000000000

56

161213192028282836

5.2 6 0.7

13.5 6 2.1

22.2 6 4.8

28.6 6 6.7

0.163

0.049

0.011


MeHg0.05

0.5

2

123123123

986984982983983978973970977

121417141517212818

221202424

000011201

000102000

141618171622273023

15.8 6 1.9

18.6 6 3.2

26.7 6 3.2

0.006

0.007


BNC 5 binucleated cell; DMSO 5 dimethyl sulfoxide; MMC 5 mitomycin C; MNCs 5 micronucleated cells.a Based on a one-way analysis of variance (ANOVA) comparing the total number of MNCs observed per 1,000 BNCs for each treatment withthe respective control.

b Based on a one-way ANOVA comparing the total number of MNCs observed per 1,000 BNCs for DMSO and the distilled water control.

per cell has also been reported for similar concentrations ofMeHg in human lymphocytes [29].

Low concentrations of Hg (0.5 mg/ml) and MeHg (0.05 and0.5 mg/ml) induced significant increases in MN frequency withno accompanying decrease of cell proliferation in beluga whalefibroblasts. Mercury has not been analyzed in blood or skinof beluga whales. Mean total mercury concentrations on a wet-weight basis in liver of St. Lawrence beluga whales is 34 mg/g (range 5 0.4–202 mg/g), compared to 6.6 mg/g (range 50.02–25 mg/g) in western Hudson Bay Arctic beluga whales[7]. According to total mercury concentrations (wet-weightbasis) in liver (63 mg/g, range 5 0.2–218 mg/g), blood (0.4

mg/g, range 5 0.008–1.5 mg/g), and skin (1.9 mg/g, range 50.002–6.1 mg/g) of spotted dolphins (Stenella attenuata) ofthe Pacific Ocean [32], concentrations (parts-per-million basis)in blood and skin of certain St. Lawrence beluga whales arewithin the range of concentrations (parts-per-million basis) thatresulted in significant DNA damage in fibroblasts. Althoughin vitro effects of mercury compounds are expected to begreater than in vivo effects [13], significant correlations havebeen found between MN or chromosome aberration frequencyand blood mercury concentrations ranging from 0.01 to 1.1mg/g wet weight in humans [33,34].

In vitro induction of MN by mercury compounds in beluga


Fig. 2. Effect of 0.1, 1, and 2 mg/ml mitomycin C (MMC) on mi-cronuclei induction and cell proliferation in beluga whale fibroblasts.MNCs 5 micronucleated cells; l 5 MNC frequency per 1,000 bi-nucleated cells; m 5 mean cytokinesis-blocked proliferation index(CBPI).

Fig. 4. Effect of 0.05, 0.5, and 2 mg/ml MeHg on cell proliferationand induction of micronuclei in beluga whale fibroblasts. MNCs 5micronucleated cells; l 5 MNC frequency per 1,000 binucleatedcells; m 5 mean cytokinesis-blocked proliferation index (CBPI).

Fig. 3. Effect of 0.05, 0.5, 5, and 20 mg/ml Hg on micronuclei in-duction and cell proliferation in beluga whale fibroblasts. MNCs 5micronucleated cells; l 5 MNC frequency per 1,000 binucleatedcells; m 5 mean cytokinesis-blocked proliferation index (CBPI).

whale fibroblasts does not signify that these compounds areinvolved in the pathogenesis of cancer in St. Lawrence belugawhales. However, because MeHg is considered a carcinogenin experimental animals and possibly carcinogenic to humans[14], it cannot be excluded as a possible participant in thecarcinogenesis process in St. Lawrence beluga whales exposedto high concentrations of this compound. It can be argued thatMeHg has yet to be established as a potent carcinogen in fish-eating human populations, but its carcinogenic potential oncetaceans is unknown, and there may be differential interspe-cific sensitivity to the carcinogenic potential of MeHg. More-over, there may be additive or synergistic effects of mercuryand other environmental pollutants in these highly contami-nated whales.

Sources of mercury in the habitat of St. Lawrence belugawhales are principally anthropogenic, mainly attributed to pastdischarges from a chloroalkali plant [35]. Fish constitute themajor source of contaminants for beluga whales in the St.Lawrence estuary [36,37]. Methylmercury is the predominantform of mercury found in fish [11]. Although a fraction ofMeHg is converted to Hg in blood [14], almost all mercuryin the blood of mammals, including striped dolphins, existsas MeHg [38,39]. Although beluga whales are predominatelyexposed to MeHg, both inorganic and organic mercury are

genotoxic [13]. However, induction of MN by Hg was oneorder of magnitude less than that by MeHg. This is consistentwith the greater genotoxic potential of MeHg compared to Hgshown in several other in vitro studies [13].

In conclusion, significant increases in MN frequency werefound at low concentrations of MeHg that are believed to becomparable to concentrations present in blood and skin ofcertain St. Lawrence beluga whales. However, the ecotoxi-cological relevance of these findings is unknown because ofthe higher sensitivity of in vitro systems compared to in vivosystems and the lack of an established association betweengenotoxicity and carcinogenicity of mercury compounds. Dataare needed on exposure and tissue distribution of both inor-ganic and methylated forms of mercury in Arctic and St. Law-rence beluga whales to enable further interpretation of thesedata. Further studies are in progress to analyze the genotoxicpotential of other suspected or known genotoxic environmentalcompounds and mixtures in beluga whale fibroblasts and lym-phocytes using different genotoxic endpoints. This will enableinvestigations of possible synergistic, additive, and/or antag-onistic genotoxic effects of environmental mixtures found inSt. Lawrence beluga whales. Future MN studies will includetechniques using an antikinetochore antibody that discrimi-nates between MN containing chromosome fragments andwhole chromosomes [23,34] to elucidate the mechanisms ofMN formation in beluga fibroblasts.

Acknowledgement—We thank the Arviat Inuvialuit hunters and Syl-vain DeGuise for collection of the beluga whale skin biopsy sampleand Darrell J. Tomkins, Emilien Pelletier, and Nicole Lemieux foradvice and suggestions on the manuscript. This work was funded bythe Fonds pour la Formation de Chercheuses et Chercheurs et l’Aidea la Recherche Quebec (FCAR), the U.S. Environmental ProtectionAgency, and the Programme d’aide a la recherche de l’Universite duQuebec a Montreal. Graduate support for J.M. Gauthier was suppliedby an FCAR PhD scholarship and the U.S. Environmental ProtectionAgency.

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mercury-induced micronuclei in skin fibroblasts of beluga whales

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