Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells

Chemico-Biological Interactions 141 (2002) 259 /274 www.elsevier.com/locate/chembiont Methyl mercury uptake and associations with the induction of c...

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Chemico-Biological Interactions 141 (2002) 259 /274 www.elsevier.com/locate/chembiont

Methyl mercury uptake and associations with the induction of chromosomal aberrations in Chinese hamster ovary (CHO) cells C. Ehrenstein a, P. Shu b, E.B. Wickenheiser c, A.V. Hirner b, M. Dolfen d, H. Emons d, G. Obe a,* a

b

Department of Genetics, University of Essen, FB 9, 45117 Essen, Germany Institute of Environmental Analytical Chemistry, University of Essen, Essen, Germany c Department of Chemistry, Northern Michigan University, Marquette, MI, USA d Institute of Phytospheric Research, Research Center Ju¨lich, Ju¨lich, Germany

Received 25 March 2002; received in revised form 15 July 2002; accepted 23 July 2002

Abstract In order to evaluate possible health effects of environmental exposure of humans towards methyl mercury species, relevant exposure experiments using methyl mercury chloride in aqueous solution and Chinese hamster ovary (CHO) cells were performed. The solution was monitored for the presence of monomethyl, dimethyl and elemental mercury by several analytical techniques including chromatographic as well as atomic absorption and mass spectrometric methods. Methyl mercury induces structural chromosomal aberrations (CA) and sister chromatid exchanges (SCE) in CHO cells. At a concentration of methyl mercury in the culture medium of 1.0/10 6 M where the frequencies of CA and SCE are significantly elevated, the intracellular concentration was 1.99 /10 16 mol/cell. Possible biochemical processes leading to the cytogenetic effects are discussed together with toxicological consequences, when humans (e.g. workers at waste deposits) are exposed to environmental concentrations of methyl mercury. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Methyl mercury; Chinese hamster ovary (CHO) cells; Chromosomal aberrations

* Corresponding author. Tel.: /49-201-183-3388; fax: /49-201-183-4397 E-mail address: [email protected] (G. Obe). 0009-2797/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 0 2 ) 0 0 0 7 9 - 0

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1. Introduction Methyl mercury has been classified as a hazardous environmental pollutant since the Minamata episode in Japan [1]. A major source of exposure to methyl mercury is consumption of fish that accumulated this compound from water polluted with mercury or its derivatives by industrial activities or by leaching of inorganic mercury from earth crust and microbial transformation to methyl mercury [2,3]. Besides exhibiting significant neurotoxicological effects [4,5], organomercurials induce different types of genetic damage in vitro and in vivo such as gene mutations, chromosomal aberrations (CA), micronuclei, dominant lethals, sister chromatid exchanges (SCE) and aneuploidy [6 /13]. As a lipophilic compound, methyl mercury passes through cell membranes relatively easily. In cells mercury binds to sulfhydryl groups in neurofilaments or microtubules but also to glutathione. Binding to sulfhydryl groups in glutathione may lead to disturbances of cellular elimination of free radicals [11]. Binding of mercury to sulfhydryl groups in microtubules disrupts them and suppresses tubulin polymerization which leads to aneuploidy [7,11,14 /16]. The aim of the present study was to determine the portion of methyl mercury in solution entering Chinese hamster ovary (CHO) cells and to correlate the intracellular concentration with the frequencies of CA and SCE. We found that at the intracellular concentration of methyl mercury of 1.99 /10 16 mol/cell the frequencies of CA and SCE are significantly elevated. Besides a quantitative determination of monomethyl mercury, the sample solutions were qualitatively screened for the presence of elemental and dimethyl mercury to test for possible species transformation/degradation processes. Finally, possible health effects of environmental concentrations of methyl mercury will be discussed.

2. Materials and methods 2.1. Chemicals Water was purified by a Seradest SD200 purification system (Allhauser). Unless otherwise stated, reagents were of analytical grade. One molar hydrochloric acid was prepared by dilution of concentrated acid (suprapur from Merck). Five percent sodium borotetrahydride (m/m) was prepared from 99% sodium borotetrahydride (Aldrich) in a beaker and then purged with helium for 60 min (in order to decrease the mercury blank) before it was used. All glassware was soaked 10 min in a potassium hydroxide bath, then in an 8% phosphoric acid bath overnight and rinsed with distilled water. Between runs the hydride reaction flask was rinsed first with 1 M hydrochloric acid and then with distilled water. All plasticware used was rinsed with 1 M HCl and distilled water and finally dried in an oven. Stock solutions of 0.01 mol/l methyl mercury chloride (Strem Chemicals) and 1000 mg Hg/ml as dimethyl mercury (TRI Chemical) were prepared in methanol. Both stock solutions were stored in a refrigerator. A stock solution of 1000 mg/l Hg2 in

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the form of mercury chloride (Lancaster) in 0.5 mol/l nitric acid was used for the addition of mercuric ions to cells, and this solution was stored at room temperature. Working standards were prepared weekly by successive dilution of stock solutions, and standards for calibration were made daily. 2.2. Cell cultures CHO9 cells a strain derived from CHO cells (from A.T. Natarajan, Leiden) were cultured in McCoy’s 5A medium (C. C. pro), supplemented with 10% fetal calf serum (FCS; Invitrogen) and antibiotics (penicillin, streptomycin; Biochrom) in a humidified atmosphere with 5% carbon dioxide at 37 8C. 2.3. Chromosomal aberrations Cells were grown in 60 mm plastic Petri dishes. Before treatment, medium was removed, cell monolayers were washed twice with McCoy’s 5A medium and exposed to methyl mercury chloride for 2 h. Respective concentrations of methyl mercury chloride were prepared by dilution of the stock solution with methanol so that the desired treatment concentrations were reached when 30 ml were contained in 3 ml medium, i.e. cell monolayers were exposed to methyl mercury chloride in a total of 3 ml medium resulting in final concentrations of 0.75 /10 /106 M. Controls were exposed to 30 ml methanol in 3 ml medium. After treatment cell monolayers were washed three times with prewarmed McCoy’s 5A medium and recovered in the presence of 5-bromodeoxyuridine (BrdUrd, final concentration 2/105 M) for 18 h in 3 ml medium including exposure to Colcemid (0.08 mg/ml) for 2 h. The cells were recovered in the presence of BrdUrd to make sure that exclusively first post-treatment metaphases are analyzed which are uniformly stained by the FPG method [17]. Colcemid was added to accumulate mitotic divisions in a metaphase like stage (C-metaphases). The test compound was added to the cells in the absence of BrdUrd, which was only present during recovery. BrdUrd is incorporated in the chromosomal DNA during the S-phase of the cell cycle, which in the first post-treatment cells to be analyzed starts a couple of hours after the addition of the test compound. It is not to be expected that this has an influence on the induction of CA by methyl mercury chloride. 2.4. Sister chromatid exchanges For the analyses of SCE, cells were prelabeled with BrdUrd for 18 h (concentration see above), exposed to methyl mercury chloride and recovered as described for CA but in the absence of BrdUrd for 18 h including exposure to Colcemid (0.08 mg/ ml) for 2 h. Controls were exposed to methanol as described for the CA experiments. SCE are generally visualized in cells whose chromosomal DNA is differentially substituted with BrdUrd. A contribution of incorporated BrdUrd to the observed SCE frequencies cannot be determined.

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2.5. Preparation of metaphases and scoring Mitotic preparations were made following a routine protocol. In short, mitotic cells were collected by shake off, hypotonically treated with sodium citrate (1%) for 8 min at room temperature and fixed two times with acetic acid/methanol 1:3 (v/v). Cell suspensions were dropped on slides and dried at room temperature. Metaphases were differentially stained by using the FPG method [17]. For CA, 100 well spread uniformly stained first post-treatment metaphases and for SCE, 50 differentially stained first post-treatment metaphases were analyzed per dose point per experiment. All experiments were repeated three times. 2.6. Chemical analysis Cell monolayers were exposed to methyl mercury chloride as described for CA but without BrdUrd and only for 0.5 or 1 h. After exposure, cells were washed three times with medium, cell monolayers were trypsinized, cells were counted and exposed to distilled water for 15 min to destroy cell membranes. Intracellular concentrations of mercury species were determined with three different analytical methods (M I, M II, M III). 2.7. M I 0.5 ml of the sample were taken and diluted to 20 ml with triply distilled water. An aliquot of this solution was administered into a reaction vessel containing the reducing agent, a mixture of SnCl2/H2SO4. There the species Hg2 was converted quantitatively into elemental mercury. The elemental mercury (Hg0) was then separated from the solution by a defined stream (3 l/min) of purified, pressurized air. The air was purified by passing through traps filled with sodium hydroxide twice (20 and 10%, respectively) and once through deionized water. Finally, it was dried using silicagel. The mercury was preconcentrated on a gold wool trap as amalgam. To release the mercury from the gold wool, the quartz tube (5 mm i.d. and 6 mm o.d.) containing the gold wool was heated to 600 8C. The mercury was transported to the quartz cuvette by means of a separate air stream (0.1 l/min) and the absorption signal of a mercury vapor lamp was recorded in transmission. In another aliquot of the diluted sample solution, dimethyl mercury was removed by purging the solution with nitrogen. The methyl mercury in solution was then determined indirectly after its destruction by UV radiation of a 15 W Hg vapor lamp for 0.5 /2 h. The further procedure is the same as described above for mercury. 2.8. M II A sample aliquot was distilled with a mixture of H2SO4/NaCl/H2O (1:1:8). The mixture was heated to about 150 8C and a constant stream of air was bubbled through the solution. Two milliliters of concentrated hydrochloric acid were added to the distillate and the acidified distillate was passed through an ion exchange

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column where the ionic mercury was retained [18]. The eluted methyl mercury was converted to Hg2 by UV radiation and determined as described for M I. 2.9. M III After distillation as described in M II the mercury species were separated and quantified by an on-line coupling of HPLC /UV /PCO /CV /AAS (high performance liquid chromatography/ultraviolet /post-column oxidation /cold vapor atomic absorption spectrometry) [19]. In brief the distillate was adjusted to pH 6 and a sodium pyrrolidinedithiocarbamate (SPDC) solution (0.5%) was added to form the corresponding complexes of the mercury species. The resulting solution was pumped through a preconcentration column (Hypersil ODS, 3 mm). After switching of valves the eluent (acetonitrile/water, 60/40 v/v, containing 0.5 mmol/l SPDC) was pumped through the preconcentration column. The complexed mercury species were separated on a reversed phase-C18-column and afterwards destroyed on-line by UV radiation. The on-line reduction of the resulting ionic mercury was performed in a PTFE reaction coil of 2 m length after adding sodium borohydride solution. The resulting elemental mercury was separated with a gas /liquid separator and measured after passing a drying tube (filled with calcium chloride) by a cold vapor-atomic absorption spectrometer [19]. Quality assurance of the quantitative determinations was performed by parallel analysis of a certified reference material (NIST SRM 2974 Mussel Tissue) and the obtained data were within the certified ranges for both methyl and total mercury. All three methods detect the separated mercury species as elemental mercury. Therefore, the intracellular amount of the test compound had to be converted into methyl mercury chloride by using the atomic masses. In a further set of experiments, cell monolayers were exposed to methyl mercury chloride for 10, 30, 60, 120 or 240 min (conditions of treatment see above). Monolayers were washed three times with medium and trypsinized. The cells were counted and exposed to deionized water for 15 min. Monomethyl, dimethyl, and elemental mercury as well as their diffusion into cells was monitored (after purging) using a GC/ICP-MS method [20]. The volatile species dimethyl and elementary mercury were directly measured by this technique, monomethylmercury was mainly derivatized by 0.8 ml 5% sodium borotetrahydride in 0.1 ml 1 M hydrochloric acid to yield the volatile methyl mercury hydride, but part of it was also transformed into elemental mercury [21,22]. The chemical analyses were performed with cells which were not cultured in the presence of BrdUrd as done in the cytogenetic studies. It is not very probable that this could have influenced the cellular uptake of methyl mercury chloride.

3. Results Methyl mercury chloride induced CA and SCE in a linear concentration dependent manner in the concentration range of 0.75 /10 /106 M (at higher

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Chromosomal aberrations MeHgCl concentration /10 6 M Number of Number of aberrations cells analyzed Gaps Chromatid and isochromatid/ chromosome breaks 0 0.75 1 2.5 5 7.5 10

300 300 300 300 300 300 300

1 2 4 4 7 2 18

0 1 2 15 36 87 93

SCE

Chromatid exchanges

Dicentric and ring chromosomes

Percent aberrant cells9/ Number of SEM cells analyzed With Without gaps gaps

0 3 2 11 67 124 112

0 2 0 4 1 0 0

0.39/0.3 2.79/0.9 2.79/0.9 7.09/2.3 17.39/4.4 29.79/0.9 35.39/0.3

0.09/0.0 2.09/0.6 1.39/0.3 5.79/2.2 15.09/3.6 25.39/4.2 34.39/0.3

150 150 150 150 150 150 150

SCE per cell 9/SEM

6.69/0.2 7.59/0.5 7.69/0.1 8.79/0.4 11.09/0.5 13.19/0.7 13.79/0.7

Pooled data from three independent experiments and the standard errors of the means (9/SEM) are given. Linear regression for percent aberrant cells: y/ 6.2024x/11.238, R2 /0.8953. Linear regression for SCE per cell: y/1.424x/1.3273, R2 /0.9429. The frequencies of CA and SCE are significant at the 5% level for all concentrations tested (CA: x2-test; SCE: t -test).

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Table 1 CA and SCE induced by methyl mercury chloride (MeHgCl) in CHO cells

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concentrations no mitoses were found) (Table 1). Mainly chromatid type aberrations (chromatid exchanges and breaks) were induced. Statistical analyses performed with the pooled data from three independent experiments using the t-test for SCE and the x2-test for CA revealed that the frequencies of both end-points are significantly elevated at the 5% level at all concentrations tested. The qualitative pattern of mercury species in the treatment solution and inside cells is shown in Figs. 1 /3. When the treatment solution was spiked with methyl mercury chloride (Fig. 1), three separate peaks appeared in the chromatogram. During analysis, a major portion of methyl mercury is transformed to methyl mercury hydride due to the addition of sodium borotetrahydride [23]. Some of the methyl mercury is reduced by sodium borotetrahydride to elemental mercury [24,25]. Divalent ionic mercury in solution will also be reduced to elemental mercury. Dimethyl mercury may have been formed by a transmethylation involving methyl mercury (2MeHg 0/Hg2/ Me2Hg), or by some other methylation processes. Compared to methyl mercury chloride, the concentration of the mercury species inside the cells was negligible when spiking the solutions with dimethyl or divalent ionic mercury (Fig. 2). The low levels of intracellular mercury following treatment with inorganic and dimethyl mercury can be attributed to the inability of elemental and divalent ionic mercury to pass the cell membrane, and to the escape of volatile dimethyl mercury from the treatment solution. The absence of intracellular mercury following treatment with dimethyl mercury implies that the relatively high dimethyl mercury peak of the inner cell solution following treatment with methyl mercury chloride (Fig. 1) cannot be caused by diffusion of dimethyl mercury into the cells following its extracellular production. It seems more probable that the transmethylation process 2MeHg  0/Hg2/Me2Hg will happen. For the Hg2 generated suitable coordination partners such as sulfide ions or sulfhydryl groups may be present which are abundant inside the cells. The cellular content of methyl mercury is shown in Table 2. The results indicate that cellular uptake of methyl mercury is nearly completed after 30 min (compare also Fig. 3), when exposure to 10 7 or 106 M led to an intracellular concentration of 2.9 /1017 and 1.9 /1016 mol/cell, respectively. The fast uptake of methyl mercury is the reason why in these experiments we did not use an exposure time of 2 h as we did in the cytogenetic tests.

4. Discussion Our measurements of cellular uptake and intracellular content of methyl mercury gave similar results as obtained by using a different method of measuring [26]. These authors [26] used HeLa S3 cells and 203Hg labeled methyl mercury chloride and found 2/1016 mol/cell following incubation with 1/106 M methyl mercury in medium. At all concentrations of methyl mercury tested we found significant elevations of CA and SCE frequencies. At concentrations of 10 6 and 10 7 M in the medium we found intracellular concentrations of 1.99 /10 16 and 2.70 /1017

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Fig. 1. Chromatogram of inner cell solution and treatment solution (treat solution) after methyl mercury chloride spike (exposure time 15 min) measured by GC/ICP-MS (mass 202).

mol/cell, respectively, following an incubation time of 1 h and quite similar values were already found after 30 min. The concentration of 10 6 M led to a significant elevation of CA and SCE frequencies and so did 0.75 /106 M, 10 7 M was not used in the cytogenetic tests.

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Fig. 2. Chromatograms of inner cell solution and treatment solutions (treat solution) after dimethyl mercury (a) and (b) Hg (II) spiking (exposure time 60 min) measured by GC/ICP-MS (mass 202).

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Incubation time (h)

Concentration of CH3HgCl in treatment solution (M)

Method I: CH3Hg as Hg after expulsion of (CH3)2Hg (ng Hg/ml)

Method II: CH3Hg as Hg after distillation (ng Hg/ml)

Method III: CH3Hg as Hg after HPLC separation (ng Hg/ml)

Mean value of CH3Hg  from M I, M II and M III (ng Hg/ml)

Number of cells analyzed

Amount of CH3HgCl per cell (ng)

Amount of CH3HgCl per cell (mol)

0.5 1 0.5 1

1/10 7 1/10 7 1/10 6 1/10 6

11.00 8.80 85.10 101.70

7.10 8.40 78.20 73.10

9.00 12.00 82.70 119.60

9.03 9.73 82.00 98.13

7.70/106 8.75/106 1.06/107 1.22/107

7.3 /10 6 6.9 /10 6 4.84 /10 5 5.0 /10 5

2.90/10 17 2.70/10 17 1.90/10 16 1.99/10 16

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Table 2 Analytical determination of methyl mercury by different approaches (see text)

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Fig. 3. Chromatograms of derivatized mercury species in inner cell solutions dependent on different times of exposure (15, 30 and 60 min (a); 1, 2 and 4 h (b)) measured by GC/ICP-MS (mass 202).

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Fig. 4. Transformation of methyl mercury inside cells (hypothetical scheme).

We conclude that the critical intracellular concentration of methyl mercury leading to CA and SCE is in the range of 1016 mol/cell. Cytotoxicity tests were not done but test concentrations higher than the ones given in Table 1, namely, ]/2.5 /105 M did not reveal enough divisions to score 100 metaphases. Our data confirm that cells in an aquatic environment take up mercury dissolved in the water body and therefore, preconcentration of the analyte by bacteria has been proposed as a method to analyze mercury in water samples [27]. However, the interaction between mercury and biota is dependent on the chemical form (i.e. species) of the element. In particular, unlike many other metal ions, mercury as free (hydrated) cation is not a potential candidate for the penetration of plasma membranes. The same applies to elemental mercury, but not to methyl mercury (see Figs. 1 and 2). Besides organic mercury compounds, other species like small neutral and hydrophobic complexes of mercury may freely diffuse across cell membranes [28]. Once inside the cell, methyl mercury is not regarded to be stable, but will eventually be degraded/transformed by enzymatic/chemical processes. In Fig. 4, a respective hypothetical scheme is given in accordance to what is known about mercuric ion reductase/organomercurial lyase in microorganisms [29]. However, still no information exists about a possible homology between microbial reductase/lyase and similar enzymes in eukaryotic cells (V. Aposhian, personal communication). Attempts to isolate an enzyme converting monomethyl mercury to Hg2 in liver and

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brain of mouse was not successful (R.A. Zakharyan, personal communication) but, a free radical mechanism may be an alternative explanation for this conversion. Possible chemical species resulting from the demethylation process are also indicated in Fig. 4. If the demethylation of organic mercury does not lead to methane (by protonation of methyl (like bacterial demethylation), anionic, cationic, or radical methyl species could be formed similar to the methylation process [30,31]. These methyl species may bind to a variety of sites on DNA, which can be explained by the concepts of electrophilicity (‘acids’) and nulceophilicity (‘bases’) [32]. According to the scheme of Fig. 4 enzymatic demethylation of organic mercury entering cells and its subsequent fixation may lead to long-term accumulation of this element inside cells. Thus when humans are accidentally exposed to organic mercury, mercury will irreversibly accumulate on a long-term scale in brain in accordance to microscopic observations, which showed neuronal necrosis and extensive gliosis in the Wetterhahn case [33]. In this respect it is interesting to note that ingested methyl mercury will not leave the human body in this chemical form [34], but may be transformed to elemental mercury or stored inside the brain as just described. When discussing Fig. 4 it must be assumed that the system considered outside the cell membrane is open to the atmosphere. In closed systems, however, dimethyl mercury would be prevented from escape and will diffuse into the cytoplasm. Therefore, it must be mentioned that the behavior of dimethyl mercury inside of organisms cannot be simulated with the experimental set-up used in this study. In addition, organomercury compounds may facilitate the intracellular accumulation of reactive oxygen species (ROS) by reacting with cellular antioxidants such as glutathione [5,11]. It was shown that exposure of human lymphocytes in culture to methyl mercury chloride leads to a significant elevation of CA at concentrations ]/ 2 /106 M and of 8-hydroxydeoxyguanosine (8-OHdG) in cellular DNA resulting from oxidative damage of guanine at a concentration of 5/106 M [11]. In human lymphocytes, mercury chloride induced a significant elevation of CA at concentrations ]/10 /106 M and of 8-OHdG at concentrations ]/2 /10 6 M [11]. These data show that methyl mercury chloride is more effective to induce CA and 8-OHdG than mercury chloride. In CHO9 cells we found nearly no effect of mercury chloride on CA and SCE at concentrations from 106 to 10 4 M (data not shown). CA induced by methyl mercury chloride is mainly of chromatid type ([11] and data in present study). This together with the induction of SCE indicates that the compound does not directly induce DNA strand-breaks which would give rise to chromosometype aberrations [35 /37]. This is in line with what has been discussed above, namely that methyl mercury chloride leads to methyl radicals, CH 3 moieties and ROS. By nucleophilic attack these reactants will lead to chemical changes in chromosomal DNA such as 8-OHdG which, during S-phase, can be transformed to chromatidtype aberrations and to SCE. Methylated metal and metalloid compounds occur in the air above waste disposal sites at doses exceeding 10 mg/m3 for As, Sb, Sn, even higher concentrations could be detected for organic mercury in soil and waste gases [38,39]. It is interesting to note that in peripheral blood lymphocytes of employees from waste disposal sites significant elevations of aberration frequencies were found [40 /42]. Hundreds of

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different compounds are known to occur in gaseous releases from rubbish dump areas [40] and a decision which of these are responsible for the induction of CA in lymphocytes of workers is not yet possible but, it can be expected, that methylated metal compounds may take part in the mutagenic activities observed. In aquatic ecosystems metal(loid) organic compounds including methyl mercury have been found up to the high mg/l range paralleled by respective concentrations in fish in the order of mg/kg [43,44], associated toxicological effects have been reported as will be expected from the results of this study.

Acknowledgements We would like to thank the German Research Association (DFG: GRK 153) for financial support. These data are part of one of the authors’ PhD degree work (CE).

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