Mutation Research 444 Ž1999. 427–439 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres

Biomarkers of DNA damage in marine mammals J.M. Gauthier a

a,)

´ Rassart a, W.M. Jarman b, R.S. Wells , H. Dubeau a , E.

c

Departement des Sciences Biologiques, UniÕersite´ du Quebec Canada H3C 3P8 ´ ´ a` Montreal ´ (UQAM), Montreal, ´ Quebec, ´ b UniÕersity of Utah, Department of CiÕil and EnÕironmental Engineering, Salt Lake City, UT 84108, USA c Chicago Zoological Society and Mote Marine Laboratory, Sarasota, FL 34236, USA Received 3 March 1999; received in revised form 1 June 1999; accepted 2 June 1999

Abstract Certain environmental contaminants found in marine mammals have been shown to cause DNA damage and cancer. The micronuclei ŽMN., sister chromatid exchange ŽSCE. andror chromosome aberration ŽCA. assays were used to assess baseline Žspontaneous. levels of DNA damage in blood lymphocytes of individuals of the relatively healthy and lightly contaminated Arctic beluga whale Ž Delphinapterus leucas ., Sarasota Bay, FL, bottlenose dolphin ŽTursiops truncatus . and Northwestern Atlantic grey Ž Halichoerus grypus . and harp Ž Phoca groenlandicus. seal populations. MN cell ŽMNC. frequencies ranged between 2 and 14r1000 binucleated ŽBN. cells and were statistically similar between species. In bottlenose dolphins, MNC frequency was correlated with age and was significantly higher in females than in males. No intraspecific variation in MNC frequency was found in beluga whales. Intraspecific variation was not tested in seals due to the small sample size. Frequencies of SCEs and total CAs, excluding gaps, ranged, respectively, between 1 and 15 SCEŽs.rper cell and 4–6 CAsr100 cells in beluga whales. SCE and CA frequencies did not vary with age or sex in beluga whales. The MN, SCE and CA assays were found to be practical tools for the detection of DNA damage in marine mammals and could be used in the future to compare DNA damage between relatively lightly and highly contaminated populations. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Micronuclei; Sister chromatid exchange; Chromosome aberration; Biomarker; Beluga whale; Bottlenose dolphin; Grey seal; Harp seal

1. Introduction Marine mammal populations, such as the St. Lawrence beluga whales Ž Delphinapterus leucas ., the Mediterranean striped dolphins Ž Stenella

)

Corresponding author. E-mail: [email protected]

coeruleoalba., the US Atlantic coast bottlenose dolphins ŽTursiops truncatus ., and Baltic seals, have shown high concentrations of environmental contaminants in their tissues which have been associated with diseases w1–4x. Cancer has been diagnosed as the principal cause of death in 18% of 97 necropsied St. Lawrence beluga whales, and the annual crude cancer rate was estimated to be 233r100 000; higher than in many human and domestic animal populations w5,6x ŽD. Martineau, personal communication..

1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 0 6 - 0

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J.M. Gauthier et al.r Mutation Research 444 (1999) 427–439

Chemical contamination has been suggested to be involved in the high prevalence of cancer in St. Lawrence beluga whales and California Sea lions Ž Zalophus californianus. w3,5,7x. DNA damage plays an important role in the development of cancer and can be a risk factor for teratogenesis and genetic disease w8–10x. Certain environmental contaminants found in marine mammals, such as polycyclic aromatic hydrocarbons ŽPAHs., and certain organochlorine compounds ŽOCs. and heavy metals, w3,4,11,12x have been shown to cause DNA damage w8,13–26x and cancer w27x. The mammalian cytokinesis block micronucleus ŽCBMN., sister chromatid exchange ŽSCE. and chromosome aberration ŽCA. assays are well-known cytogenetic techniques that have been used extensively to assess DNA damage at the chromosomal level in human peripheral blood lymphocytes w9,28–30x. Peripheral blood lymphocytes are considered the best non-invasive source of cells to analyze DNA damage because they circulate throughout all tissues and organs and thus provide an estimate of average whole body exposure to mutagens w10x. Micronuclei ŽMN. are chromosomal fragments or whole chromosomes that are not incorporated into daughter nuclei during mitosis because of chromosomal breakage or dysfunction of the mitotic apparatus, respectively w29x. SCEs are thought to be exchanges of chromosomal fragments between two chromatids of the same chromosome during replication of damaged DNA w31x. CAs can be analyzed in cells as structural chromatid- or chromosome-type aberrations, such as breaks and gaps within a chromosome or rearrangement within or between chromosomes w28x. These assays have been proposed for biomonitoring exposure to genotoxic compounds in the natural habitat of fish, invertebrates, terrestrial mammalian wildlife, and seals w32–36x. Studies have shown statistically higher MN or CA frequencies in fish, invertebrates, wild mammals and farm animals from environments with high levels of PAHs andror OCs andror heavy metals compared to those sampled from reference sites with low levels of these compounds w32–34,37–39x. Statistically significant correlations have been found between total mercury concentrations in blood and CAs in humans consuming fish and seafood, and between PAH pollution in air and CAs in humans w19,21,40x.

Marine mammals are exposed to genotoxic chemicals in the marine environment, but very few studies have addressed possible genotoxic effects in wild marine mammal populations w35,41x. Increased DNA damage analyzed by flow cytometry has been demonstrated in sea otters Ž Enhydra lutris . potentially exposed to petrochemical PAHs during the Exxon Valdez oil spill in western Prince Williams Sound compared to a control group in eastern Prince Williams Sound w41x. Screening of changes in genotoxic activity inside a population or between different populations requires baseline Žspontaneous. values to be established in populations exposed to relatively low levels of putative genotoxic environmental contamination. Arctic beluga whales and Northwestern Atlantic seals are considered to be healthy populations, have relatively low concentrations of environmental contaminants, and no gross evidence of cancer has been found in 50 Arctic beluga whale carcasses sampled for routine biological purposes w3,42–44x ŽD.J. St. Aubin, personal communication.. Blood samples can easily be obtained from captive beluga whales and seals and biological parameters of these animals are usually well-known. Bottlenose dolphins residing in Sarasota Bay, FL, are considered to represent a healthy population and relatively low concentrations of environmental contaminants have been found in their tissues w45–47x. Contrary to most wild marine mammal populations, information on biological parameters is available for individual dolphins through long-term monitoring and health assessments conducted on this population since 1970 w45x. The objective of this study is to apply biomarkers of genotoxic activity in marine mammals to evaluate possible long-term effects, such as cancer and teratogenicity, induced by putative genotoxic environmental contaminants. Analysis of DNA damage and chemical compounds in blood samples of marine mammal populations exposed to low levels of environmental contamination could provide baseline data for monitoring the health status of these populations and those of populations exposed to high levels of environmental contamination. This preliminary study presents data on MN in peripheral blood lymphocytes of individual Arctic beluga whales, Sarasota Bay bottlenose dolphins, Northwestern Atlantic grey seals Ž Halichoerus grypus . and harp seals Ž Phoca

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groenlandicus. and on SCEs and CAs in Arctic beluga whales. Data on lymphocyte proliferation are also presented for each animal. Variations in MN, SCE and CA frequencies and values of proliferation indices are evaluated according to species, sex and age.

2. Methods 2.1. Culture and slide preparation Blood samples were taken between 1996 and 1998 from: Ž1. nine Arctic beluga whales held captive at the Vancouver Aquarium or the J.G. Shedd

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Aquarium; Ž2. nine Sarasota Bay, Fl, USA, resident free-ranging bottlenose dolphins during the capture–release health assessment program conducted by the Chicago Zoological Society and Mote Marine Laboratory; Ž3. two sedated wild harp seals at the Magdalan Islands, Quebec, Canada; and Ž4. three ´ grey seals held captive at the Granby Zoo, Granby, Quebec, Canada ŽTable 1.. Beluga whales and grey ´ seals had been held in captivity for at least 5 years at sampling. All samples were taken in sterile heparinized tubes. Cetacean and grey seal blood samples were shipped on ice within 3 days to l’Universite´ du Quebec a` Montreal ´ ´ ŽUQAM. and used immediately on arrival. Harp seal blood samples were kept on ice until frozen within 8 h in fetal calf serum ŽGibco.

Table 1 Data on life history parameters of beluga whales, bottlenose dolphins, harp and grey seals of this study NA: non-applicable. Animal

Sex

Age a

Origin

Institution

Comments

D. leucas Aurora Immiayuk Puiji Allua Mauyak Kavna Imaq Nanuq Inuk

F F F F F F M M M

8 12 12 13 17 25 8 13 17

Churchill River Churchill River Churchill River Churchill River Churchill River Churchill River Churchill River Churchill River Churchill River

Vancouver J.G. Shedd J.G. Shedd Vancouver J.G. Shedd Vancouver Vancouver Vancouver J.G. Shedd

12.5 months gestation

T. truncatus FB109 FB003 FB079 FB111 FB144 FB128 FB142 FB010 FB046

F F F F M M M M M

2 8 16 20 2 5 5 16 20

Sarasota Bay Sarasota Bay Sarasota Bay Sarasota Bay Sarasota Bay Sarasota Bay Sarasota Bay Sarasota Bay Sarasota Bay

NA NA NA NA NA NA NA NA NA

P. groenlandicus Pg-1 Pg-2

M M

adult adult

Magdalen Islands Magdalen Islands

NA NA

H. grypus Caroline Chloe´ Chinook

F F M

6 6 7

captive born captive born New Brunswick

Granby Zoo Granby Zoo Granby Zoo

a

Age at blood sampling.

calf of FB079 lactating FB109 lactating FB144 calf of FB111

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containing 10% dimethylsulfoxide ŽDMSO. in liquid nitrogen vapour, brought back to l’UQAM by airplane, placed in liquid nitrogen and thawed 3 weeks later for use. All captive animals were in good health at the time of sampling and there was no history of major illness for any of the animals. Six of the free-ranging bottlenose dolphins were considered in good health according to body condition assessment, blood parameter analysis, ultrasound and veterinarian examination at capture. Four of the free-ranging bottlenose dolphins had health concerns. FB128 had elevated white blood cell count, mediastinal masses observed by ultrasound, and a papilloma virus infection on the prepuce. FB111 also had elevated white blood cell count. FB046 had an abscess below the throat in the gular region caused by a bacterial infection, and FB109 had a possible stingray barb embedded in its head. Harp seals appeared to be in good health at capture. Sex was determined visually for all animals. Ages of grey seals, Caroline and Chloe, ´ were determined from date of birth in captivity. Age estimations of beluga whales and grey seal Chinook were based on size, coloration and associations at time of capture. Ages of bottlenose dolphins were determined through research activities which have been carried out since 1970 w45x. Ages of FB010, FB109, FB128, FB144 and FB003 were determined by visual observations of these animals within a few days or weeks after their births through monitoring of their mothers. FB142 was estimated to be the length of a 5-year old according to age–body length curves w48x. FB111 was first observed with a calf in 1983 and thus was at least 20 years old at sampling because age at sexual maturity is about 6 years old w49x. FB079 was first caught during the capture–release health assessment program in 1985 at 218 m, which corresponds to 3–5 years of age and had her first calf in 1993 w48x. Taken together with the fact that females give birth to their first calves at 5–12 years of age w49x, FB079 was about 16 years old in 1997. FB046 was first caught in 1984 at 229 m, which corresponds to about 5–8 years of age and making him about 20 years of age at sampling w48x. Lymphocytes were isolated from whole blood using the Ficoll–Paque ŽGibco. differential centrifugation gradient method. Lymphocytes were cultured for 72 h at 3 = 10 6 cellsrml in RPMI medium

supplemented with L-glutamine, penicillin–streptomycin, 20% fetal calf serum and concavalin A ŽFlow Laboratories. mitogen to stimulate proliferation of T-lymphocytes. For the MN assay, cytochalasin B ŽSigma. Ž6 mgrml. was added for the last 28 h of culture. MN are found in cytoplasm of cells and are analyzed in cells that have divided once following animal blood sampling. Cytochalasin B prevents cytokinesis and produces binucleated ŽBN. cells which can be easily scored for MN w29x. SCE and CA assays were conducted within the same cultures. For SCErCA cultures, bromodeoxyuridine ŽBrdU. ŽICN. Ž6 mgrml. was added for the last 48 h of culture. Only first-division metaphases should be analyzed for CAs since these can be lost or appear in altered form in subsequent cell divisions w50x. For SCErCA cultures, colchicine ŽSigma. was added 1 h before harvest of cells to block dividing cells at metaphase. All cultures were harvested at 72 h by centrifugation. The cells were then swelled with hypotonic agent KCl 0.075 M for 5 min for MN cultures or 25 min for SCErCA cultures and fixed three times with methanolracetic acid Ž5:1.. Fixed cell suspensions were dropped on slides and air-dried. Slides prepared for MN assay were stained with Giemsa ŽSigma. 3% for 10 min. Slides prepared for SCErCA analysis were stained, following a modified version of the Fluorescent Photolysis Giemsa ŽFPG. technique of Perry and Wolff w51x, with 0.5 mgrml Hoescht 33258 Žbis-benzimidazole. ŽICN. 15 min, covered with 2 = SSC solution and a coverslip and exposed to 365 nm UV light at 378C 15 min, immersed in 2 = SSC at 608C 20 min, rinsed, air-dried and stained for 7 min with Giemsa 3%. 2.2. Analysis MN were scored for all cultures according to established criteria w29,52x, with previously described specifications w24x. The cytokinesis-blocked proliferation index, which measures the average number of cell cycles per cell, for the MN cultures were calculated for each culture as CBPIs w1Ž M1 . q 2Ž M2 . q 3Ž M3 q M4 .xrN, where M1 to M4 represent the number of cells with one to four nuclei and N is the total number of cells scored w53x. For each culture, at least 1000 BN cells were scored for CBPI, MN cells

J.M. Gauthier et al.r Mutation Research 444 (1999) 427–439

ŽMNC. and total MN at 400 = magnification, and the presence of MN was confirmed at 1000 = magnification. MN frequencies are presented as numbers of MNCs and total MN per 1000 BN cells. Thirty second-division metaphase spreads were analyzed for SCEs for each beluga whale culture at 1000 = magnification. However, the small blood sample obtained from Mauyak, along with the low lymphocyte proliferation rate found in this animal, permitted the analysis of SCEs in only 20 second-division cells. Frequencies are shown as number of SCEs per cell. The proliferation rate index used for the SCE assay was calculated using 200 cells for each culture as PRI s w1Ž M1 . q 2Ž M2 . q 3Ž M3 .xrN, with M1 to M3 indicating, respectively, metaphases in first, second, or third cell division and N is the total number of cells scored w54x. One-hundred first-division metaphase spreads were analyzed for structural CAs in beluga whale cultures. Chromosome- and chromatid breakage-type Žbreaks accompanied or not with resulting fragment and isolated acentric fragments not evidently associated with a chromosome. and exchange-type Že.g., dicentrics, rings and tri- and quadriradial figures. aberrations were scored. Gaps were also analyzed, but were quantified separately from CAs. The mitotic index, which measures the relative proportion of cells that have divided, was calculated using 2000 cells for each culture as MI s Žnumber of cells in metaphase= 100.rnumber of cells in interphase. Frequencies of CAs excluding gaps and MI values are shown as percentages. 2.3. Statistical analysis In most cases, the data met the criteria of normality and homogeneity. In the few cases that did not meet these requirements, heterogeneity of variance and deviation from normality was small. Parametric tests were therefore used to analyse all data. Harp and grey seals were combined for interspecific analyses of MNC frequency and CBPI values due to small sample size. The small sample size precluded statistical analysis of putative intraspecific differences in seals. A one-way analysis of variance ŽANOVA. was used to test for differences in MNC frequencies and CBPI values between species andror

431

sex in beluga whales, bottlenose dolphins and seals, and in frequencies of SCEs and total CAs excluding gaps and PRI and MI values between sex in beluga whales. A one-way ANOVA was also used to test for differences between bottlenose dolphins with and without health concerns. Differences in age of individuals and proportions of females and males between species were tested using ANOVA and Chisquare test, respectively. Regression analysis was performed to test for relationships for each cytogenetic variable and age, where the y-axis represents either MNC, SCEs, or total CAs excluding gaps, or CBPI, PRI or MI values and the x-axis represents age of the animals. All statistical analyses were analyzed at the 0.05 level of significance and were conducted using Minitab ŽMinitab software release 8, State College, PA..

3. Results 3.1. MN analyses in marine mammals Typical BN cells without and with MN are shown in Fig. 1. Results for MNC and total MN frequencies and CBPI in peripheral blood lymphocytes of beluga whales, bottlenose dolphins, harp and grey seals are shown in Table 2. MNC and total MN frequencies ranged between 2 and 14. Differences between total MN and MNC frequencies indicate that some animals had certain MNCs containing more than one Žtwo or three. MN. MNC frequency was statistically similar between beluga whales and bottlenose dolphins Ž p s 0.680., beluga whales and seals Ž p s 0.190. and bottlenose dolphins and seals Ž p s 0.399..

Fig. 1. Typical BN lymphocyte without ŽA. and with MN ŽB. from a beluga whale. The MN is indicated by an arrow.

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Table 2 Frequencies of MNCs and total MN per 1000 BNCs and CBPI values in blood lymphocytes of beluga whales, bottlenose dolphins, harp and grey seals Animal

Sex

CBPI

MNCsr 1000 BNCs

Total MNr 1000 BNCs

F F F F F F M M M

1.40 1.42 1.45 1.42 1.41 1.35 1.42 1.38 1.43 1.41 " 0.03 1.41 " 0.03 1.41 " 0.03

3 5 8 4 13 7 8 6 4 6.7 " 3.6 6.0 " 2.0 6.4 " 3.0

3 5 12 4 14 7 8 6 4 7.5 " 4.5 6.0 " 2.0 7.0 " 3.8

F F F F M M M M M

1.37 1.54 1.41 1.48 1.55 1.55 1.55 1.51 1.49 1.45 " 0.08 1.53 " 0.03 1.49 " 0.07

3 11 7 12 2 4 7 7 11 8.3 " 4.1 6.2 " 3.4 7.1 " 3.7

4 12 7 12 2 4 7 7 11 8.8 " 4.0 6.2 " 3.4 7.3 " 3.7

M M

1.21 1.35

7 6

7 6

F F M

1.49 1.44 1.51 1.48 " 0.04

7 13 9 10.3 " 3.1

9 13 11 11.0 " 2.0

Seals All

1.41 " 0.12

8.8 " 3.0

9.2 " 2.9

Marine mammals All

1.44 " 0.08

7.1 " 3.2

7.6 " 3.5

D. leucas Aurora Immiayuk Puiji Allua Mauyak Kavna Imaq Nanuq Inuk Females Males All T. truncatus FB109 FB003 FB079 FB111 FB144 FB128 FB142 FB010 FB046 Females Males All P. groenlandicus Pg-1 Pg-2 H. grypus Caroline Chloe´ Chinook All

Similar results were obtained when only grey seal samples were used to compare interspecific differences with beluga whales Ž p s 0.085. and bottlenose dolphins Ž p s 0.202.. Age of individuals and proportions of females and males were statistically similar

between belugas, dolphins and grey seals Ž p s 0.218 and x 2 s 1.270, p s 0.736, respectively., indicating that these are not factors influencing interspecific variations in MNC frequency. No significant differences were found between MNC frequencies in fe-

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Table 3 Mean frequencies of SCEs per cell for a total of 30 cells a and PRI values in blood lymphocytes of beluga whales

Fig. 2. Effect of age on MNC frequency in lymphocytes of beluga whales Ž`. and bottlenose dolphins Žv ..

Beluga

Sex

PRI

SCEsrcell

Range of SCEsrcell

Aurora Immiayuk Puiji Allua Mauyak a Kavna Imaq Nanuq Inuk Females Males All

F F F F F F M M M

1.27 1.06 1.08 1.26 1.08 1.09 1.18 1.29 1.35 1.14"0.10 1.27"0.09 1.18"0.11

5.8"2.1 6.3"2.1 5.3"1.9 5.5"1.8 5.7"1.8 4.9"1.5 5.5"2.3 4.2"1.7 4.5"1.5 5.6"0.5 4.7"0.7 5.3"0.7

3–15 1–10 2–9 2–9 3–9 2–8 1–8 2–7 1–11 1–15 1–11 1–15

a

male and male beluga whales Ž p s 0.779., but the highest value was observed in the 12.5-month pregnant female. Contrary to beluga whales, female bottlenose dolphins had significantly greater frequencies of MNCs than males Ž p s 0.044.. MNC frequencies in lactating females appeared to be similar to those of other dolphins. A significant positive relationship was found between MNC frequency and age in bottlenose dolphins Ž y s 2.98 q 0.344 x, R 2 adjusted s 45.6%, p s 0.027. ŽFig. 2., but this relationship was not observed for beluga whales Ž p s 0.511.. Lymphocytes of young bottlenose dolphins Ž2–4 years old. showed the lowest MNC frequencies. No significant differences were found for MNC frequen-

Only 20 second-division metaphases were analyzed in this animal.

Fig. 3. Example of a second-division metaphase spread for a beluga whale lymphocyte. The SCEs are indicated by arrows.

Fig. 4. Example of a normal first-division metaphase spread Ž2 ns 44. of a beluga whale lymphocyte.

cies between bottlenose dolphins with and without health concerns Ž p s 0.796.. The CBPI values in marine mammal lymphocytes ranged between 1.21 and 1.55, indicating a 2.7-fold difference in frequencies of BN cells between individuals ŽTable 2.. However, when harp seal frozen samples were omitted from the comparison, differences were only 1.4-fold. Thus, harp seal samples were excluded from further statistical analysis on CBPI values. CBPI values were significantly lower

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Fig. 5. Examples of chromatid breaks in otherwise normal first-division beluga whale lymphocytes.

in beluga whales than in bottlenose dolphins Ž p s 0.003. and grey seals Ž p s 0.002., but similar in bottlenose dolphins and grey seals Ž p s 0.729.. CBPI values were similar in both sexes of beluga whales Ž p s 0.763. and bottlenose dolphins Ž p s 0.062.. No relationship between CBPI values and age was found for beluga whales Ž p s 0.490. and bottlenose dolphins Ž p s 0.833.. No significant differences were found for CBPI values between bottlenose dolphins with and without health concerns Ž p s 0.405.. 3.2. SCE and CA analyses in beluga whales An example of a second-division metaphase with SCEs of a beluga whale peripheral blood lymphocyte is shown in Fig. 3. Frequencies of SCEs per cell varied between 1 and 15 and overall mean frequency was 5.3 ŽTable 3.. An example of a normal first-divi-

sion beluga whale lymphocyte with a 2 n s 44 karyotype is illustrated in Fig. 4. Examples of CAs observed in beluga whale lymphocytes are presented in Fig. 5. Structural CAs were found to be chromatid-type aberrations, except for acentric fragments which were either observed as chromatid- or chromosome-type aberrations. No exchange-type aberrations were observed. Two-thirds of observed acentric fragments were considered chromatid-type lesions. Breaks were found accompanied by the resulting acentric fragment in 22% of cases ŽTable 4.. Notwithstanding gaps, all metaphases contained only one CA event, with the exception of one cell from Nanuq which contained two different CAs. Mean frequencies of SCEs were 0.9 SCErcell higher in females than in males, but these differences were not statistically significant Ž p s 0.065.. Frequencies of total CAs excluding gaps were similar in both sexes Ž p s 0.170. ŽTable 5.. No significant relationships were found between age and frequencies of SCEs Ž p s 0.863. and CAs excluding gaps with age Ž p s 0.280.. The PRI values ranged between 1.06 and 1.35, indicating a 5-fold difference in frequencies of second-division metaphases between individuals ŽTable 3.. MI values varied between 0.55 and 1.19, showing a 2-fold variability in this division index ŽTable 4.. PRI and MI values were similar in female and males Ž p s 0.086 and p s 0.868, respectively.. No signifi-

Table 4 Mean frequencies of individual categories of CAs and total CAs, excluding gaps per 100 cells, in blood lymphocytes of beluga whales Frequencies of gaps and MI values are also presented. All structural CAs were found to be chromatid-type aberrations, except for acentric fragments which were either observed as chromatid- or chromosome-type aberrations. Beluga

Aurora Immiayuk Puiji Allua Mauyak Kavna Imaq Nanuq Inuk a c

Sex

F F F F F F M M M

MI

1.02 0.55 0.69 1.19 0.40 0.97 1.01 0.85 0.44

Gaps

4 4 4 5 4 6 4 2 8

Breaks 2a 5 4 1a 2 2 2a 5a 4b

Chromatid

Acentric fragment Chromosome

Total CAs excluding gaps c

3 0 0 4 1 4 2 2 4

0 1 0 0 1 0 2 0 0

4 6 4 4 4 6 5 6 6

and b : One a or two b of the breaks were found with resulting acentric fragment, respectively. Breaks with resulting fragment were counted as one clastogenic event when compiling total number of CAs.

J.M. Gauthier et al.r Mutation Research 444 (1999) 427–439 Table 5 Mean frequencies of total CAs excluding gaps per 100 cells, frequencies of gaps and MI values in blood lymphocytes of female and male beluga whales Sex

MI

Gaps

Total CAs without gaps

Females Males All

0.80"0.31 0.77"0.29 0.79"0.28

4.5"0.84 4.7"3.06 4.56"2.13

4.67"1.03 5.67"0.58 5.00"1.00

cant relationship was found between PRI or MI and age Ž p s 0.118 and p s 0.554, respectively..

4. Discussion This study is part of a long-term monitoring program on the toxicological health of St. Lawrence beluga whales and Sarasota Bay bottlenose dolphins w3,55x. Since increases in chromosome damage in peripheral blood lymphocytes may reflect risk of cancer, teratogenicity and other diseases w9,28,30, 56,57x, the MN, SCE and CA assays could be used to assess the long-term health of marine mammal populations. Because these assays analyze different types of DNA damage which can have dissimilar sensitivities to environmental chemicals, the three assays should be used complementary w10,28,29x. The MN, SCE and CA assays were successfully used as biomarkers of genotoxic damage in lymphocytes of beluga whales, bottlenose dolphins, grey and harp seals. Estimates of lymphocyte proliferation rates were also obtained on the same slides that are used for analysis of chromosome damage. Comparisons of values obtained from marine mammal lymphocytes of this study with lymphocytes of healthy terrestrial mammals must be interpreted with caution since culture conditions and methodologies often vary between studies. MNC and total MN frequencies in marine mammals of this study were within the lower range reported for cows Žmean s 6.8–24.6 depending on the study, range s 4–42. and humans Žmean s; 10, range s 1–80. w57–59x. SCE frequencies in beluga whales were in the lower range than that reported in cows Žmean s 4.8–6.4, range s 0–22., and lower than in goats Žmean s 6.6, range s 0–18., sheep Žmean s 6.5 and

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9.5., humans Žmean s 8.3, range s 1–21. and river buffalo Žmean s 8.8. w50,60–65x. Total CA frequencies, excluding gaps in beluga whales, were higher than or in the higher range of those reported in other mammals Žhumans: mean s 0.08–4.5, range s 0–15; deer: mean s 2.3; pigs: mean s 3.6; cows: mean s 1.9–3.7. w37–39,50,66,67x. Mean frequencies of gaps in beluga whales were in the mid-range of those reported in humans Žmean s 0.6–8.1. w50,67,68x. CBPI values in marine mammals of this study were similar to those reported in cows Žmean s 1.46. w58x. PRI values in beluga whales were lower than in cows Žmean s 1.82. and sheep Žmean s 1.40–1.58. and MI values were lower than in humans Žmean s 4.66, range s 1.05–9.87. w60,63,65,66x. Factors such as species, sex, age, reproductive status, general health status, genetic factors and environmental contamination can be important variables affecting levels of chromosomal damage and cell proliferation in human and animal cells w28,69x. Health concerns were found in four dolphins, but CBPI values and MN frequencies were similar to those of healthy dolphins. This may indicate that these health concerns did not play a role on MN induction and lymphocyte proliferation for these animals. Higher MNC frequencies observed in female compared to male bottlenose dolphins have been reported in the majority of studies on human lymphocytes and have been explained by the preferential involvement of X chromosomes, and particularly the inactive X chromosome, in somatic aneuploidy in females w10,59,69,70x. An age-related increase in MNC was found in bottlenose dolphins. Several studies have reported increased chromosomal damage as a function of age in mammals, which may be due to accumulation of DNA damage, including loss of X chromosomes in females, and decrease of DNA repair competence w10,60,65,67,69–72x. However, some studies have shown absence of sex-related differences in chromosome damage w50,73x. A greater sample size is needed to further test these hypotheses in beluga whales. Similarly to beluga whales, most studies have found that the baseline frequency of SCEs is slightly, but not statistically, higher Žmean of 0.5 SCErcell. in females than in males w28,64x. Higher frequencies of MNC, but not SCEs and CA, were observed in 12.5-month pregnant beluga whale, Mauyak. Lymphocytes of women and muntjacs in

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late pregnancies have shown higher frequencies of SCEs and X-ray-induced CAs and this has been explained by altered hormonal and physiological status during pregnancy w74x. Contrary to observations in other mammalian species w60,66,71,72x, no age-related decrease in mitogen-induced lymphocyte proliferation was observed in beluga whales or bottlenose dolphins. A wider age span is needed to further test this relationship in these cetaceans. Studies have shown statistically higher MN and CA frequencies in human, ungulate, wild rodent, fish and invertebrate populations exposed to high concentrations of PAHs, OCs andror heavy metals compared to those exposed to low levels of these compounds w19,21,32–34,37–40x. Lack of interspecific differences in MN frequencies between beluga whales, bottlenose dolphins and seals in this study may be due to comparable exposure to putative genotoxic environmental contamination andror similar age span and female and male proportions observed between species. West Hudson Bay Arctic beluga whales, Northwestern Atlantic seals and Sarasota Bay bottlenose dolphins are considered to be healthy populations and are exposed to relatively low concentrations of environmental contaminants w42,43,45–47x ŽD.J. St. Aubin, personal communication.. Although certain free-ranging grey seals have relatively high concentrations of certain environmental compounds in their tissues w43x, low concentrations are expected to be found in animals that were born in captivity or have been in captivity for long periods of time. Analyses of contaminants in blood of these animals are needed to verify the pertinence of this hypothesis. Differences in lymphocyte CBPI values between beluga whales and bottlenose dolphins and grey seals may be explained by interspecific differences in cell cycle kinetics, as reported for other mammalian species w38,75x. Frequencies of SCEs in beluga whales were similar to those reported in lymphocytes of Baltic grey seal pups and adult ring seals Ž P. hispida. ŽSCEs: mean s 4.5, range s 0–14. w35x. Total CA frequencies, excluding gaps in beluga whales, were within the same range or lower than those found in Baltic grey seal pups Žmean s 3.3–7.7 depending on year of sampling, range s 0–21. and higher than in Baltic adult ring seals Žmean s 2.2, range s 0–3.. Because Baltic seals are more highly exposed to environmen-

tal contamination than arctic beluga whales w4,42x, lower frequencies of SCEs and CAs were expected in Arctic beluga whales. However, contrary to Arctic beluga whales, metaphases with multiple CAs, multiple fragmentations of the chromosomes and rarer aberrations such as quadriradials were found in Baltic seals w35x. Rearrangements such as quadriradials are considered to be very rare and are more usually observed in humans with cancer andror exposed to genotoxic compounds than in healthy human subjects w56,76x. However, in addition to differences in culture conditions and methodologies, there may be interspecific differences in genetic factors, sex proportions and age span contributing to differences in SCE and CA endpoints between the studies w28x. In the future, analysis of MN, SCEs and CAs as biomarkers of genotoxicity could be used to compare DNA damage between relatively unexposed and highly exposed populations of marine mammals of the same species. A greater number of samples, especially from individuals in their natural habitat, are needed to conclude a more reliable frequency of spontaneous chromosome damage in each marine mammal species. Future possibilities to obtain additional bottlenose dolphin samples for DNA damage assessment through well-tested and safe capture–release operations in Sarasota Bay represent ideal opportunities to increase knowledge of DNA damage in a large sample size from a well-documented wild marine mammal population. Analysis of OC compounds and heavy metals could be used as a general contamination index and as a basis for analysis of a putative association between chemical contamination and genotoxic potential in bottlenose dolphins and other marine mammals. Genotoxicity biomarkers could act, together with measurement of other health parameters and analysis of environmental contamination, as a basis for a multiple response assessment in a non-destructive approach to predict the impact of pollution in marine mammals.

Acknowledgements We would especially like to thank Clint Wright and the marine mammal staff at the Vancouver Aquarium, Jeff Boehm, Ben Housten and Ken Ramirez at the J.G. Shedd Aquarium, Clement Lan´

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thier of the Societe ´ ´ Zoologique de Granby, and Marc Dufresne for the collection of beluga and seal blood samples. We would like to extend special thanks to Michael Scott, Howard Rhinehart, Jocelyn Vedder, Blair Irvine, Jay Sweeny, Forrest Townsend, Sue Hofmann, Kim Urian, and all the other members of the 1997 dolphin capture–release crew. Funding for the Sarasota Bay bottlenose dolphin capture–release project was provided by Dolphin Quest, through the efforts of Jay Sweeney and Rae Stone. Sampling of Sarasota Bay bottlenose dolphins was conducted under the US National Marine Fisheries Service ŽNMFS. Scientific Research Permit No. 945. We thank Nicole Lemieux for helpful advice and suggestions on the manuscript. This work was funded by the Fonds pour la Formation de Chercheuses et Chercheurs et l’Aide a` la Recherche ŽFCAR., Quebec, the US Environmental Protection Agency ´ ŽEPA., the Programme d’aide a` la Recherche de l’UQAM and the Energy and Geoscience Institute at the University of Utah. Graduate support for J.M. Gauthier was supplied by a FCAR PhD scholarship and the EPA.

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