Cytogenetic monitoring of fishermen with environmental mercury exposure

Mutation Research, 320 (1994) 23-29 © 1994 Elsevier Science Publishers B.V. All rights reserved 0165-1218/94/$07.00

23

MUTGEN 01946

Cytogenetic monitoring of fishermen with environmental mercury exposure E. Franchi a,,, G. Loprieno b, M. Ballardin b, L. Petrozzi b and L. Migliore b a Dipartirnento di Biologia Ambientale, Viale delle Cerchia 3, Universith di Siena, 53100 Siena, Italy and b Dipartimento di Scienze dell'Ambiente e del Territorio, Universita di Pisa, Italy (Received 18 November 1992) (Revision received 15 June 1993) (Accepted 6 July 1993)

Keywords: Monitoring; Human lymphocyte micronucleus assay; Mercury

Summary Due to high mercury levels in many Mediterranean aquatic organisms, people who live in this area and consume large amounts of seafood are exposed to a toxicological hazard. A group of 51 fishermen exposed to mercury through eating contaminated seafood from the northern Tyrrhenian Sea underwent cytogenetic monitoring. This work is part of a research project consisting of the evaluation of micronuclei (MN), chromosomal aberrations (CA) and sister-chromatid exchanges (SCE) in peripheral blood lymphocytes. Here we present data on mercury levels in blood and on micronucleus frequencies in peripheral blood lymphocytes of fishermen. The range of mercury concentrations in blood was 10.08-304.11 n g / g fresh weight, the average was 88.97 + 54.09 ng/g. Micronucleus frequency was defined with at least 2000 binucleated cells scored for each person; the average was 8.74 + 2.56 expressed on 1000 binucleated cells. A statistical correlation was found between MN frequency and total mercury concentration in blood ( p = 0.00041, r = 0.674), as well as between MN frequency and age ( p =0.017). No other parameters taken into account correlated with MN frequency.

The higher mercury levels found in aquatic organisms caught in the Mediterranean compared with concentrations in the same species from the Atlantic (Baldi et al., 1979; Renzoni et al., 1979; Leonzio et al., 1981; Barghigiani et al., 1986; U N E P / F A O / W H O , 1987) seem to be due mainly to the geochemical anomaly of this basin, which is located in a 'mercury belt' (Jonasson and Boyle, 1971). It seems that anthropogenic sources

* Corresponding author. Tel. 0577-298917; Fax 0577-298806. SSDI 0 1 6 5 - 1 2 1 8 ( 9 3 ) E 0 0 7 5 - Y

do not have a great influence on the whole system, but they may cause heavy local contamination episodes. Human subjects consuming Mediterranean seafood showed elevated blood concentrations of mercury, sometimes above 200 n g / g ; at this level the earliest poisoning symptoms may appear (WHO, 1976). Despite its well-known neurotoxicity and teratogenicity (Harada, 1978; Leonard et al., 1983), the genotoxic effects of mercury on humans are not completely defined yet. Previously published data concerning humans exposed to mercury

24 through different pathways, including food consumption, are considered. In nine subjects exposed to mercury through the diet, Skerfving (1970) reported a correlation between the mercury concentration in blood and chromosome breaks in lymphocytes. In an extension of the previous work conducted on a larger number of subjects, Skerfving et al. (1974) reported significant correlations between blood cell mercury levels and frequencies of ceils with chromatid type aberrations, 'unstable' chromosome type aberrations and aneuploidy. Verschaeve et al. (1978) found an increase in hyperploids in a group of workers occupationally exposed to phenyl mercury, when compared to a group of non-exposed people. In a subsequent study, Verschaeve et al. (1979) did not find any chromosomal effects in workers of a chloralkali plant exposed to metallic mercury. This negative result was explained with the low degree of exposure. In workers exposed to metallic mercury vapors, Popescu et al. (1979) revealed an increased incidence of chromosome aberrations in exposed subjects as compared with the control group. No differences between control and exposed groups were noticed regarding the frequency of aneuploidy and polyploid cells. Wulf et al. (1986) investigated the relationship between sister-chromatid exchanges (SCE) and diet in a population of Eskimos feeding mainly on seals contaminated by heavy metals. SCE showed no linear correlation with seal diet, smoking, and mercury concentrations in blood. It has also been demonstrated that mercury exposure is related to the amount of seal or fish meat consumed (Hansen et al., 1983). Monsalve and Chiappe (1987) performed an analysis of chromosomal aberrations and SCE in 16 subjects exposed to mercury through eating fish caught in Cartagena Bay (Colombia), an area of known methylmercury contamination. A statistically significant increase in mean chromosomal aberrations was found between the control group and the exposed group only if gaps were included. SCE showed no correlation with diet or smoking habits. In a recent study, Wagida and Gabal (1991) reported an increase in mercury concentrations in the urine of workers occupationally exposed to

mercury fulminate. In the exposed group, an increase in chromosomal aberrations and micronucleated lymphocytes was also found; no correlations were found between mercury levels in urine and CA or MN. These findings lead us to suspect that mercury exposure may cause a considerable genetic risk for human populations. Using an in vivo approach through cytogenetic assays, we try to assess the sensitivity of peripheral blood techniques in order to evaluate the degree of genetic damage in this population. Materials and methods

During two years of analysis (1990-1991), peripheral venous blood from 51 fishermen was collected in lithium heparin tubes at the local health center and then brought to the laboratory within 24 h. For each individual a complete questionnaire of about 50 questions was filled in giving information about age, seafood-based meals, and life habits like smoking and alcohol, which could be confounding factors for cytogenetic analysis according to the criteria developed by Carrano and Natarajan (1988). The total amount of blood, about 10 ml, was divided into two aliquots, one was used for micronucleus analysis, the other for mercury detection.

Cytogenetic analyses For each person two lymphocyte cultures were set up by adding 0.3 ml of heparinized whole blood to 4.7 ml of complete culture media, consisting of Ham's F10 medium (Flow Laboratories) supplemented with 10% fetal bovine serum (Flow Laboratories), with 1.5% phytohemagglutinin (PHA, Wellcome) and antibiotics (100 IU penicillin/ml and 100 ~ g streptomycin, Sigma). The cultures were grown at 37°C. Cytochalasin B (Cyt B, Sigma) was added to each culture after 44 h (final concentration 3 /zg/ml). After an incubation time of 72 h, cultures were harvested and centrifuged at 1000 rpm for 10 min. The cells were then treated twice with a buffer ( N H 4 H C O 3 0.9 mM, NH4C1 131 mM) for

25 20 m i n at 37°C, a n d c e n t r i f u g e d at 3000 r p m for 15 min. A f t e r this t r e a t m e n t they were twice r e s u s p e n d e d in cold fixative (glacial acetic acid : m e t h a n o l , 3 : 1) for 20 m i n at r o o m t e m p e r a ture a n d c e n t r i f u g e d at 1000 r p m for 10 min. A f t e r the final c e n t r i f u g a t i o n ceils were d r o p p e d o n t o wet clean slides, air-dried a n d s t a i n e d with G i e m s a (Merck, 3% in distilled water) for 10 min. T h e slides were t h e n rinsed in distilled water, air-dried a n d m o u n t e d in Eukitt. All slides were coded a n d analyzed blind. F o r each p e r s o n a total of 2000 b i n u c l e a t e ceils with preserved cytoplasm were scored at a magnification of 400 × . T o evaluate the p r e s e n c e of micronuclei, two observations of 1000 cells were p e r f o r m e d by different observers o n different slides. Criteria used for the d i s c r i m i n a t i o n of true a n d false M N were those of C o u n t r y m a n a n d H e d d l e (1976).

Mercury analysis A b o u t 1 ml of b l o o d was t r e a t e d with nitric acid in d e c o m p o s i t i o n vessels of teflon u n d e r pressure at 120°C for 6 - 8 h (Stoeppler a n d Backhaus, 1978). T h e solution o b t a i n e d was analyzed by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y (PerkinE l m e r m o d e l 2280) using the vapor stream system for mercury. Precision a n d accuracy were evaluated by calibration with certificate s t a n d a r d s (NBS, I A E A ) .

Statistical analysis All data were processed by p a r a m e t r i c statistical analysis (regression analysis), using Statgraph (Statistical G r a p h i c s C o r p o r a t i o n ) software o n a n IBM personal computer.

TABLE 1 DATA CONCERNING AGE, BLOOD MERCURY LEVELS (Hg), MICRONUCLEUS FREQUENCY (MN %c), SEAFOOD CONSUMPTION, AND SMOKING HABITS OF THE SAMPLES COLLECTED IN 1990 Code Age Hg MN%~ Seafood Smoking (individ- (years) (ng/gf.w.) meals/ habit uals) week A B C D E G H L M N O P Q R S T U V AA AB AC AD AE AF AG AH AI AR AS AT AU AV AZ BA BB

25 26 59 45 59 52 74 27 29 39 65 69 65 24 51 45 40 45 42 54 45 44 36 35 50 33 39 50 52 35 28 49 28 45 22

44.58 135.08 93.78 52.69 153.59 52.02 89.97 31.51 28.66 43.61 54.78 49.77 29.86 68.73 89.72 100.24 50.13 126.72 90.34 100.27 27.49 137.51 252.25 74.08 119.36 91.12 133.69 44.18 70.23 96.39 29.32 83.55 43.61 119.89 10.08

5.5 7.5 12.0 5.0 12.0 7.5 7.0 9.0 8.0 9.0 7.0 8.5 6.5 7.0 6.5 8.5 10.0 8.0 9.5 11.0 11.5 10.5 9.5 13.5 11.5 10.5 10.0 10.0 10.0 9.0 4.5 7.0 9.0 11.5 1.5

5 2 7 5 10 10 4 6 8 6 5 2 2 4 7 7 7 13 6 14 2 10 11 12 7 12 7 1 7 10 7 7 12 5 2

S NS NS NS NS S NS S S S S S NS NS NS S S NS NS S S S NS S S S S S NS S NS NS NS NS NS

Results

S, smoker; NS, nonsmoker.

A total of 35 f i s h e r m e n were e x a m i n e d d u r i n g the first year of analysis; in T a b l e 1 we report all p a r a m e t e r s considered: age, blood m e r c u r y levels, M N frequencies, smoking, a n d seafood consumption. T h e age r a n g e was 2 2 - 7 4 years, a n d the average n u m b e r of seafood b a s e d m e a l s / week was 6.97 + 3.49. Blood m e r c u r y levels r a n g e d from 10.08 n g / g to 252.25 n g / g with a m e a n of 81.97 + 49.96. T h e average f r e q u e n c y of

m i c r o n u c l e a t e d lymphocytes was 8.7%0 with a s t a n d a r d deviation of 2.47. W h e n data were analyzed with linear regression analysis, a significant c o r r e l a t i o n was f o u n d b e t w e e n m i c r o n u c l e u s freq u e n c y a n d b l o o d m e r c u r y levels ( p = 0.0118, r = 0.786) (Fig. 1). W h e n we c o n s i d e r e d the n u m b e r of seafood b a s e d m e a l s / w e e k vs. total blood m e r c u r y level, we also f o u n d a weakly significant

26 correlation. O t h e r p a r a m e t e r s were t a k e n into account, b u t did n o t supply any significant correlation. I n 1991, 28 blood samples were collected a n d T a b l e 2 s u m m a r i z e s all the data. A g e r a n g e was 1 6 - 7 4 years, a n d the m e a n n u m b e r of seafood m e a l s / w e e k was 7.93 + 3.04. T h e average mercury c o n c e n t r a t i o n in b l o o d was 97.72 n g / g (SD = 58.57 n g / g ) , a n d r a n g e d from 27.25 n g / g to 304.11 n g / g . T h e r e was a significant correlation b e t w e e n M N f r e q u e n c y a n d blood m e r c u r y conc e n t r a t i o n ( p = 0.017, r = 0.706) (Fig. 2). N u m bers of seafood meals did n o t affect blood mercury levels, while age showed a significant correlation with m i c r o n u c l e u s frequency. I n o r d e r to assess w h e t h e r the m i c r o n u c l e u s frequency in the exposed group was d u e to the genotoxic effects of mercury, we also correlated all the data o b t a i n e d d u r i n g the two years of analysis. W h e n c o n s i d e r i n g the total n u m b e r of samples (63), o b t a i n e d from 51 f i s h e r m e n 12 of w h o m were analyzed again in the second year, M N frequency showed a significant correlation with m e r c u r y c o n c e n t r a t i o n in blood (Fig. 3). I n linear regression analysis, the frequency of micronuclei also showed a significant correlation with age of each subject; n o correlation was f o u n d with seafood meals. Since the correlations b e t w e e n M N frequency a n d age of subjects a n d also b e t w e e n Hg levels

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Code Age Hg MN %0 Seafood Smoking (individ- (years) (ng/gf.w.) meals/ habit uals) week E F G H I S T U AA AB AD AE AH AL AM AN AO AQ BA BC BD BE BF BG BH BI BL BM

60 51 53 75 58 52 46 41 43 54 44 36 33 57 43 60 36 50 45 51 53 57 55 55 22 26 20 16

133.81 86.64 106.57 117.37 66.43 119.23 131.79 68.48 66.04 78.08 118.20 101.19 63.28 156.16 88.01 112.10 77.51 34.22 179.35 27.25 61.41 60.37 190.75 304.11 35.08 48.33 32.30 76.86

9.0 12.0 8.5 7.5 11.0 9.0 7.0 10.5 11.5 8.0 8.5 7.5 8.0 9.0 7.0 12.5 8.5 14.5 13.5 6.5 6.5 5.0 10.0 13.0 5.0 6.5 3.5 7.0

10 7 10 3 7 7 10 7 4 14 10 2 10 14 7 12 12 7 5 5 10 5 10 7 1 7 6 7

NS S S NS NS NS S S NS S S NS S NS S NS NS NS NS NS NS NS NS NS NS S NS NS

250 200

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SUMMARY OF INDIVIDUALS, AGE, BLOOD MERCURY LEVELS (Hg), MICRONUCLEUS FREQUENCY (MN %0), SEAFOOD MEALS/WEEK, AND SMOKING HABITS IN SAMPLES COLLECTED IN 1991

S, smoker; NS, nonsmoker.

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TABLE 2

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a n d age were positive ( p < 0.001 a n d p < 0.005, respectively), to evaluate the effective c o r r e l a t i o n b e t w e e n M N f r e q u e n c y a n d m e r c u r y exposure we correlated M N frequencies adjusted for age a n d Hg levels adjusted for age. Also in this case, a positive linear regression b e t w e e n M N f r e q u e n cies a n d Hg c o n c e n t r a t i o n s in blood was f o u n d ( F = 16.79; p < 0.0005). All the data were logtransformed.

15

Micronucleated l y m p h o c y t e s Fig. 1. Regression analysis between MN frequencies and mercury concentrations in blood (ng/g fresh weight) of samples collected in 1990.

Discussion O u r findings show a n increase in cytogenetic d a m a g e in p e r i p h e r a l blood lymphocytes of fish-

27 .,.,. 4 0 0

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Micronucleated lymphocytes Fig. 2. Regression analysis between micronucleated lymphocytes and blood mercury levels (ng/g fresh weight) of samples collected in 1991.

ermen exposed to mercury through seafood consumption. When the data were processed by regression analysis a positive correlation was found between total mercury concentration in blood and micronucleus frequency ( p <0.001) evaluated in peripheral lymphocytes. Humans studies have shown a good correlation between mercury intake with food and blood mercury concentrations (Sherloch et al., 1984; Grandjean et al., 1992). In a study performed by Renzoni (1987) on the same population, it was found that seafood is responsible for the high levels of mercury in the

400 01

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300

> 200

E

100

"o o o

0

3

6

9

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Micronucleated lymphocytes Fig. 3. Linear regression analysis of all samples collected. MN frequencies versus mercury concentrations in blood (ng/g fresh weight).

hair of fishermen; a good correlation has been found between mercury levels in hair and blood with a ratio 250 : 1 (WHO, 1976). A positive correlation was found between MN frequency and age; this finding is in agreement with other studies (Fenech and Morley, 1986; Sorsa et al., 1988; Migliore et al., 1991) and confirms the importance of this factor in human lymphocyte MN assays. The increase of MN frequency with age may be due to spindle disturbances. However, the correlation between the MN frequency and mercury concentrations in the blood found in both years of cytogenetic monitoring confirms the persistence of exposure to mercury through diet, so that the contaminant can be considered the main cause of the increase in micronucleated lymphocytes. Moreover, since the half-life of methylmercury (which accounts for more than 95% of the total mercury content) is about 70 days, our study was likely able to detect both acute and chronic exposure. MN frequency in a group of workers exposed to mercury fulminate was reported by Wagida and Gabal (1991); they found an increase in micronucleated lymphocytes in the exposed group, but they could not establish a statistically significant correlation between mercury levels in urine and MN frequency. Failure to establish a correlation between chromosomal aberration or MN frequency and the amount of mercury in the urine could mean that this biological material is not suitable for monitoring the level of exposure. Subjects excreting more mercury may have not a higher exposure but more efficient excretion. Previous studies (Verschaeve et al., 1979; Mabille et al., 1984) considering mercury levels in urine have failed to find a correlation with cytogenetic damage. The in vitro genotoxic effects of this heavy metal seem to be related to the capability of mercury to interfere with spindle activity during cell division (Verschave et al., 1985; Betti et al., 1992). Mercury is also know to interact with the sulfhydryl groups of cell spindle proteins. The positive results found with the micronucleus assay in our study could be indirect confirmation of this molecular mechanism of action, since micronuclei may be derived from acentric fragments or whole chromosomes. On the basis

28

of our findings, we suppose that a large percentage of micronuclei found in our samples could include entire chromosomes. Another aim of our project was to perform a cytogenetic surveillance of the monitored population, in order to assess the sensitivity of this technique at environmental levels of exposure. The data obtained so far indicate that the MN assay satisfactorily detects cytogenetic damage induced by mercury.

Acknowledgements We are very grateful to Professors R. Barale, N. Loprieno and A. Renzoni for comments, helpful discussions and reading the manuscript.

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Leonard, A., P. Jacquet and R.R. Lauwerys (1983) Mutagenicity and teratogenicity of mercury compounds, Mutation Res., 114, 1-18. Leonzio, C., E. Bacci, S. Focardi and A. Renzoni (1981) Heavy metals in organisms from the Northern Tyrrhenian Sea, Sci. Total Environ., 20, 131-146. Mabille, V., H. Roels, P. Jacquet, A. Leonard and R. Lauwerys (1984) Cytogenetic examination of leukocytes of workers exposed to mercury vapor, Int. Arch. Occup. Environ. Health, 53, 257-260. Migliore, L., P. Guidotti, C. Favre, M. Nardi, M.R. Sessa and E. Brunori (1991) Micronuclei in lymphocytes of young patients under antileukemic therapy, Mutation Res., 263, 243-248. Monsalve, M.V., and C. Chiappe (1987) Genetic effects of methyl mercury in human chromosomes: I. A Cytogenetic study of people exposed through eating contaminated fish, Environ. Mol. Mutagen., 10, 367-376. Popescu, H.I., L. Negru and I. Lancranjan (1979) Chromosome aberrations induced by occupational exposure to mercury, Arch. Environ. Health, 34, 461-463. Renzoni, A. (1987) Mercury levels in human hair and their relevance to health, 6th International Conference on Heavy Metals in the Environment, pp. 80-83. Renzoni, A., M. Bernhard, R. Sar~ and M. Stoeppler (1979) Comparison between the mercury concentration of Thunnus thynnus from the Mediterranean and Atlantic, J. Etud. Pollut. CIESM, 4, 255-260. Sherloch, J., J. Hislop, D. Newton, G. Topping and K. Whittle (1984) Elevation of mercury in human blood from controlled chronic ingestion of methylmercury in fish, Hum. Toxicol., 3, 117-131. Skerfving, S. (1970) Chromosome breakage in humans exposed to methyl mercury through fish consumption, Arch. Environ. Health, 21, 133-139. Skerfving, S., K. Hansson, C. Mangs, J. Lindsten and N. Ryman (1974) Methylmercury-induced chromosome damage in man, Environ. Res., 7, 83-98. Sorsa, M., L. Pyy, S. Salomaa, L. Nylund and J.V. Yager (1988) Biological and environmental monitoring of occupational exposure to cyclophoshamide in industry and hospital, Mutation Res., 204, 456-479. Stoeppler, M., and F. Backhaus (1978) Pretreatment studies with biological and environmental materials. I. System for pressurized multisample decomposition, Fresenius Z. Anal. Chem., 29, 116-120. U N E P / F A O / W H O (1987) Assessment of the state of pollution of the Mediterranean Sea by mercury and mercury compounds, MAP Tech. Rep. Set., 18, UNEP, Athens. Verschaeve, L., M. Kirsch-Volders, L. Hens and C. Susanne (1978) Chromosome distribution studies in phenyl mercury acetate exposed subjects and in age-related controls, Mutation Res., 57, 335-347. Verschaeve, L., J.P. Tassignon, M. Lefevre, P. De Stoop and C. Susanne (1979) Cytogenetic investigation on leukocytes of workers exposed to metallic mercury, Environ. Mutagen., 1, 259-268.

29 Wagida, A., and S. Gabal (1990) Cytogenetic study in workers occupationally exposed to mercury fulminate, Mutagenesis, 6, 189-192. WHO (1976) Environmental Health Criteria 1: Mercury, WHO, Geneva. Wulf, H.C., N. Kromann, N. Kousgaard, J.C. Hansen, E.

Niebuhr and K. Alboge (1986) Sister chromatid exchanges (SCE) in Greenlandic Eskimos. Dose-response relationship between SCE and seal diet, smoking, and blood cadmium and mercury concentrations, Sci. Total Environ., 48, 81-94.