Effects of methylmercury(II) on the viability of HeLa S3 cells

TOXICOLOGY

Effects

AND

APPLIED

PHARMACOLOGY

of Methylmercury DIETER

46,249-256

(1978)

on the Viability

W. GRUENWEDEL

of HeLa S3 Cells

AND BARRY L. FORDAN

Department of Food Science and Technology, University of California, Davis, California 95616 Received August 5,X977; accepted March 17,1978

Effects of Methylmercury(I1) on the Viability of HeLa S3 Cells. GRUENWEDEL, D. W., AND B. L. (1978). Toxicol. Appl. Pharmacol. 46,249-256. HeLa S3 cells were incubated with varying concentrations of CH,HgOH for varying periods of time and the viability of the cells was determined by using the trypan blue dye exclusion test. Cell viability was found to decrease “cooperatively” with increasing CH,Hg(II) concentration in the medium. Below a certain “threshold” value of the organomercurial, viz., at CH,Hg(II) concentrations of 1 PM and below, the cells remain alive and exhibit the viability of the control, whereas at CH,Hg(II) concentrations of 10 ,UM and above, rapid cell death occurs. The kinetics of the cell death as a function of organomercurial concentration was determined, as was the ability of the cells to recover from injury once the toxicant has been removed. The effect of methylmercury on the mitotic pattern of the cells was also evaluated. At CH,Hg(II) concentrations of 1 @I and below, the mitotic coefficient was increased by a factor of 4 over that of untreated cells after 24 hr of incubation. Treated cells became arrested preferentially during the early stages of mitosis. However, higher methylmercury concentrations, viz., 10 ,UM and above, caused coagulation necrosis in the ceils and no chromosome spreads could be obtained. The viability phenomena observed in this study suggest that the plasma membrane of the cells serves as the primary site of the organomercurial attack at lethal toxicant concentrations. FORDAN,

The well-known clinical features and gross pathological changes brought about in man and other organisms as a result of exposure to elevated levels of the environmental pollutant methylmercury(I1) (Bidstrup, 1964; Skerfving, 1972; Takeuchi, 1972: Kurland, 1973; Skerfving and Copplestone, 1976) have stimulated great interest in studying its direct action on cells and tissues in the hope that it is there, at the cellular level, that it should be possible to obtain detailed information concerning the interaction of the organomercurial with subcellular components and where, hopefully, those molecules can be identified which serve as principal targets in the organomercurial attack.

With regard to assay cultures derived from human sources, Umeda et al. (1969) used HeLa S3 cells in studying the cytotoxicity of a variety of mercury(I1) derivatives, but not methylmercury, at a few, selected concentrations while Fiskesjo (1970) employing a wider concentration range of both methylmercuric chloride and (methoxy)ethylmercuric chloride, investigated their action on human short-term leukocyte cultures. Other animal cell cultures derived from mouse cerebellum (Kim, 1971), chick cerebrum (Ammitzboll and Clausen, 1973), or mouse connective tissue (Li and Traxler, 1974) have also been used in assessing the toxic effects of mercury(I1) and its organic derivatives. 249

0041-008x/78/0461-0249$02.00/0 Copyrtght Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved. Printed in Great Britam

250

GRUENWEDEL

AND

FORDAN

The present communication describes the effects of methylmercury, CH,Hg(II), on the viability and growth of asynchronous HeLa S3 cells as a function of organomercurial concentration, as well as incubation time, and also presents some data on the mitotic coefficient of the cells. In a forthcoming paper (Gruenwedel and Cruikshank, unpublished data), the effects of CH,Hg(II) on DNA, RNA, and protein synthesis in HeLa cells will be addressed. METHODS

Cell culture. Human cervix carcinoma cells, HeLa S3, were maintained as monolayers (75~cm* plastic Falcon flasks; Becton, Dickinson & Co.) and routinely passaged in Eagle’s minimal essential medium (MEM, Joklik-modified; Grand Island Biological Co.). The medium was supplemented with 10% fetal calf serum, 50 pug/ml streptomycin, 50 units/ml penicillin, and 50 units/ml polymixin B sulfate (Grand Island Biological Co.). The cells were grown at 37°C in an atmosphere of 5% CO,. Cell densities of 2 x 10’ cells/flask were attained; cell viability was found to be 95-98% as judged from the trypan blue dye exclusion test. Chemicals. Methylmercuric hydroxide, CH,HgOH (97+%), was purchased from Alfa Products, Ventron Corporation. Stock solutions were prepared by dissolving appropriate quantities of the crystals in distilled water. Actual concentrations were determined as described elsewhere (Gruenwedel and Davidson, 1966). Stock solutions were kept in tightly sealed vials in the dark; their titer remained unchanged over a 3-month period. Methylmercury concentrations are given in pM units whereby pM = -log Phosphate-buffered saline (1 x), Ca*+ and Mg*+ free, was obtained ~=U-WH~a~w from Pacific Biologicals; trypsin (0.25%) was obtained from Grand Island Biological co. Incubation. Intoxication studies were performed as follows. Cells were removed from one of the stock cell cultures by trypsinization (2 min at 37OC), washed three times with saline, and suspended in complete medium, and both their viability and actual cell number were determined. Aliquots of IO6 cells each were then transferred into sterile plastic tubes, each tube containing new complete medium and the organomercurial at the desired concentration. After gassing with 5% CO,, the tubes were attached to a spinner wheel and rotated slowly about their long axis (3 rpm) at 37°C for the desired periods of time. Postincubation consisted of collecting the cells by centrifugation and, after suspending them in 0.5 ml of saline each, counting them anew and determining their viability again. No cell attachment to the tubes was observed within any of the given incubation periods. Hence, organomercurial treatment involved cells that can be considered as having been in suspension. Cell viability is expressed as [(total cells - dead cells)/total cells] x 100%. The trypan blue dye exclusion test was used in determining cell viability. Mitotic index. For mitotic index measurements, the methylmercury-treated cells were washed once with saline and incubated for 15 min at 37OC in hypotonic saline (saline : water = 1 :9). The cells were then fixed for 5 min in a glacial acetic acid : absolute methanol mixture (1 : 3), transferred onto a clean uncoated microscope slide, and permitted to dry in air overnight. Cells were stained with May-Griinwald stain (5 min) and then with Giemsa stain (12 min). After dehydration and clearing, the

METHYLMERCURY

AND HeLa S3 VIABILITY

251

cells were ready for mounting. The mitotic index is defined as the number of cells observed microscopically in various stages of mitosis per 1000 cells. A minimum of 500 cells was scanned per index. RESULTS

The viability of HeLa S3 cells that had been incubated for 5 hr at 37OC in the presence of varying concentrations of CH,Hg(II) is shown in Fig. 1. The curve with the

2.0

3.0

4.0

5.0

6.0

7.0

a,

PM

FIG. 1. Viability of HeLa S3 cells (percentage live cells) as a function of CH,Hg(II) concentration. pM = -log [CHrHgOHl,,,,,. (0) Complete medium: mean + SD of seven to nine measurements per data point, except extremely small SD (<2%) not shown; (0) phosphate-buffered saline: mean of triplicate measurements with variations not exceeding those given for closed-circled curve; (A) complete medium: 24.hr holding period prior to incubation with toxicant, mean of duplicate measurements with variations not exceeding those given for closed-circled curve. All data points pertain to 5 hr of incubation with CH,Hg(lI). Actual cell number is about lo6 per data point. For further details, see text.

closed circles represents Eagle’s minimal essential medium (Joklik-modified), supplemented with 10% fetal calf serum and antibiotics, while the curve with the open circles was obtained by using only phosphate-buffered saline as incubating medium. As can be seen, the shift of the saline-based curve to higher pM values, i.e., lower CH,Hg(II) concentrations, appears reasonable since in saline the attacking species will be primarily methylmercuric chloride, a small, mostly nonionic entity, that should pass a cellular membrane rather readily, thereby eliciting the cellular response at low concentrations. In the complete medium, by contrast, the organomercurial is most certainly bound to high molecular weight substances (sulfhydryl groups of proteins) and might, therefore, not be as readily available as is the chloride species. On the other hand, the

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GRUENWEDEL

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FORDAN

90 80 70 60 50 40 30 20 IO 4

8

12

16

20

24

28

32

36

TIME (hr) FIG. 2. Viability of HeLa S3 cells (percentage live cells) as a function of incubation time in complete medium and at the indicated concentration levels of CH,Hg(II). pM = -log[CH,HgOHl,,,,,. Unless indicated otherwise, all data points are mean + SD of five to seven individual measurements. (0) Control (PM co), pM 7, and pM 6; SD not shown since ~2%; (0) pM 5; (0) pM 4.4: the 24-hr data point represents a single measurement; (0) pM 3; the data points pertaining to 6-hr and longer periods of incubation represent single measurements. Actual cell number is about 10’ cells per data point. For further details, see text.

shift by about 0.5-l pM units may very well be fortuitous, since by taking the somewhat reduced viability of the control (PM co) in saline into account, one can almost superimpose the various dose-response data points. In an attempt to see whether trypsinization has any effect on cell viability, experiments were also performed in which the cells, after trypsinization, were kept for 24 hr in suspension (spinner wheel) prior to their incubation with CH,Hg(II). The triangular data points displayed in Fig. 1 show the corresponding cell response. It is apparent that after correcting for the somewhat decreased viability of the control (PM co) both triangles and closed circles will overlap and that the effect of trypsinization can be neglected as far as the methylmercury-dependent decrease in cell viability is concerned. (D b ii ;i 3 8 IJ zj

5.6 5.2 4.0 4.4 4.0 3.6 3.2 2.8 2.4

k 2.0 B 1.6 g 1.2 3 0.0 5 0.4 IO

20

30

40

50

60

70

80

90

100

TIME (hr) FIG. 3. Plot of number of live HeLa S3 cells against time of incubation in complete, methylmercury-free medium after the cells had been exposed to CH,Hg(II) for 5 hr at the indicated concentrations. (A) Control (PM co), PM 7, and pM 6; (n) pM 5; (0) pM 3: each data point is the average of duplicate measurements with variations amounting to about + 7%.

METHYLMERCURY

AND

HeLR

s3

253

VIABILITY

In Fig. 2, the time course of HeLa cell viability gathered at a few selected methylmercury concentrations is shown. The incubation time is the time period the cells were in contact with CH,Hg(II). It is readily seen that cell viability decreases rapidly between pM 4.4 and 3.0 but that at pM 5.0 the number of dead cells exceeds those found in the control only after an incubation period beyond 8-10 hr. In fact, at still lower methylmercury concentrations, viz., pM 7-6, the viability response of the cells does not differ from the one exhibited by the control, at least not at incubation periods up to 36 hr. One other way to assessCH,Hg(II) toxicity is to monitor the growth behavior of cells after they have been removed from the intoxicating medium. As shown in Fig. 3, HeLa cells that had been incubated for 5 hr at the CH,Hg(II) concentrations shown were removed from the medium, washed once with saline, then resuspended in new, TABLE MITOTIC

INDEX

1

HeLa S3 CELLS AS A FUNCTION OF INCUBATION ANDABSENCE (PM CO;CONTROL)OFMETHYLMERCURY(II)

OF ASYNCHRONOUS

THEPRESENCE (pM6)

Mitotic

-CH,HgW) PM’ 00 03 co 03

6 6 6 6 u pM = -log

Incubation time (W

5 12

24 36

5 12 24 36

index

__-

TIME

IN

.- ~~~

Metaphase Prophase

2 6 12 12 2 2 40 33

Early

2 14 3 4 0 12 20 10

Late 2 12 1

10 6 30 40 40

Anaphase

0 0 1 2 0 0 0 0

Telophase

4 4 5 4 0 0 0 0

Total

IO 36 28 32 8 44 100 83

[CH,HgOHI,,,,,

complete medium now void of the organomercurial, and incubated for the time spans indicated. While cells that had been exposed to CH,Hg(II) concentrations of pM 7 and 6 grew in a fashion indistinguishable from that of the control, cells kept at pM 5 displayed a more or less “stationary” behavior, although there were indications pointing to a certain degree of recovery. Cells kept at pM 3 did not recover: The number of live cells had virtually decreased to zero within 36 hr of incubation in the methyhnercury-free medium. The effect of organomercurial treatment on the mitotic index of asynchronously growing HeLa cells is illustrated in Table 1. Attempts were made not only to give the total index as a function of incubation time at the organomercurial concentrations selected, but also to provide a distribution of the index across the various stages of mitosis. The mitotic stages prophase, early metaphase, late metaphase, anaphase, and telophase were selected. If the number of cells found in a particular mitotic stage was below 0.1% of the total, this number was arbitrarily set to zero in the table. In general, pM 7- and pM 6treated cells produced mitotic patterns which were indistinguishable from those of the

254

GRUENWEDEL

AND

FORDAN

control only in that they appeared with higher frequency. On the other hand, pM 5 treated cells did not yield microscopically discernible phases; instead, the cells displayed coagulation necrosis and thus could not be evaluated. DISCUSSION

The most striking feature of the results presented in Fig. 1 is that control cells (PM co) as well as cells that had been exposed to CH,Hg(II) concentrations ranging from 0.1 (PM 7) to 10 PM (PM 5) maintained a high degree of viability (> 95%) under the experimental conditions given, while cells that had been in contact with CH,Hg(II) concentrations exceeding 10 PM (PM (5) in the medium decreased in viability in a dramatic fashion. Cells die, so to speak, “cooperatively” between pM 5 and 4 after 5 hr of incubation; in fact, the transition curve assumes the appearance of a step function curve at incubation periods exceeding 12 hr (Fig. 2). From the experimental evidence on hand, it is concluded that there exists a “threshold” value of methylmercury concentration, lying between pM 6 ad 5, below which HeLa S3 cells remain alive but above which they become injured to such an extent that cell death is brought about in a very short period of time (Fig. 2). Sublethal CH,Hg(II) concentrations do affect the cells: They interfere with the functioning of the mitotic apparatus and arrest cells preferentially in the early stages of mitosis (Table 1). This, however, does not lead to an increase in cell mortality, at least not within an incubation period of up to 36 hr. By contrast, methylmercury concentrations exceeding the “threshold” value produce immediate necrosis in the cells and it becomes impossible to prepare chromosome spreads. We would like to point out that “cell viability,” as defined here, is nothing but the ability of live HeLa cells to actively prevent trypan blue dye from entering the cell interior. Dead cells, by contrast, have lost this ability and permit the stain to enter freely. The dye exclusion test, then, may be taken as a measure of the intactness of the outer cellular membrane. The fact that lethal concentration levels of CH,Hg(II) increase the number of permeable cells (Fig. 1) leads to the conclusion that the plasma membrane is among the first sites of organomercurial attack and that it ceases to function in a -‘normal” fashion if and when the methylmercury concentration exceeds the “threshold” value. In view of the differences that exist in regard to the cell type employed and the mercury derivatives used, there is little merit in comparing the results of this study, particularly at the quantitative level, with the results obtained by others (Fiskesjii, 1970; Kim, 1971; Thrasher and Adams, 1972; Ammitzboll and Clausen, 1973; Li and Traxler, 1974). The study with which our own can, perhaps, be most readily compared is the one of Umeda et al. (1969). They found that the organomercurials phenyl-, ethyl-, and butylmercuric chloride are lethal to HeLa S3 cells at concentrations exceeding 3.2 pg/ml and after 6 days of incubation. This translates roughly into pM 4.9, using our notation, and would indicate that methylmercury, particularly on a time basis, is considerably more toxic than are the higher alkyl or aryl derivatives of Hg(II). This seems reasonable since methylmercury is generally believed to represent the most toxic form of all organic mercury derivatives. The high cytotoxicity of CH,Hg(II) might also bc responsible for the fact that we found a 10% increase in the mitotic index after 24 hr of

METHYLMERCURY

AND

HeLa

S3 VIABILITY

255

incubation and at pM 6, while they found that ethylmercuric chloride increases the index to almost 30% after the same period of incubation and at pM 5.2. This concentration level is close to that which produces cell necrosis in our case. On the other hand, the higher mitotic activity observed by Umeda et al. (1969) in the case of ethylmercuric and phenylmercuric chloride, viz., 30 and 20%, respectively, as compared to the 10% observed by us, might be independent of cytotoxicity and might indicate a true decrease in the sequence aryl z higher alkyl > methyl, since they found HgCl, to be completely ineffective in altering the mitotic coefficient of HeLa S3 cells. We are unaware of studies in which the mitotic coefficient of mercury-treated cells is expressed in terms of the various stages of mitosis and, thus, cannot compare our results with those possibly obtained by others. Our finding that control cells exhibit a mitotic coefficient of 2-3% is in complete agreement with what is known from the literature for HeLa cells (Terasima and Tolmach, 1963). While we are not certain what importance should be attributed to the individual numerical data listed in Table 1, we find it of interest to note that cells in anaphase and telophase are conspicuously absent once they have become exposed to methylmercury and that the organomercurial, at sublethal concentration levels, arrests the cells preferentially during the first stages of mitosis. Lastly, the CH,Hg(II) concentrations listed here represent total mercury in the medium and not organomercurial taken up by the cells. Consequently, we are at present unable to correlate our dose-response data, say, the pM value pertaining to the 50% viability point (Fig. l), with toxic in viva doses as they are known from LD50 data involving whole animals. Attempts to measure the internal methylmercury concentration of HeLa S3 cells are in progress. ACKNOWLEDGMENTS The HeLa S3 cells used in this work were a gift generously provided by Dr. L. Levintow, Department of Microbiology, University of California, San Francisco. The authors would also like to thank the Cytokinetic Laboratory of the Radiobiology Laboratory of the University of California, Davis, in particular Mrs. Nancy Taylor and her assistants, for the help and advice received in determining the mitotic coefficients. This work was supported by U.S. Public Health Service Grant No. 1 RO 1 ES0 1115. REFERENCES AMMITZBOLL. T., AND CLAUSEN, J. (1973). The toxic effect of methylmercury chloride on brain cell cultures from chick embryo. Environ. Physiol. Biochem.. 3, 248-254. BIDSTRUP, P. L. (1964). Toxicity ofMercury and Its Compounds. Elsevier, Amsterdam. FISKESJ~, G. (1970). The effect of two organic mercury compounds on human leukocytes in vitro. Hereditas 64, 142-146. GRUENWEDEL, D. W., AND DAVIDSON, N. (1966). Complexing and denaturation of DNA by methylmercuric hydroxide. I. Spectrophotometric studies. J. Mol. Biol. 21, 129-144. KIM, S. U. (197 1). Neurotoxic effects of alkyl mercury compound on myelinating cultures of mouse cerebellum. Exp. Neural. 32, 237-246. KURLAND, L. T. (1973). An appraisal of the epidemiology and toxicology of alkylmercury compounds. In Mercury, Mercurials and Mercaptans (M. W. Miller and T. W. Clarkson, eds.), pp. 23-55. Thomas, Springfield, Ill. Lr, M. F., AND TRAXLER, G. S. (1974). Effect of mercuric chloride on cellular morphology and acid phosphatase of tissue culture cells cultivated in suspension. Environ. Physiol. Biochem. 4,263-269.

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S. (1972). Organic mercury compounds. In Mercury in the Environment (L. Friberg andJ. Vostal, eds.)pp. 141-168. CRC Press,Cleveland,Ohio. SKERFVING, S., AND COPPLESTONE, J. F. (1976). Poisoningcausedby the consumptionof organomercury-dressed seedin Iraq. Bull. WHO 54, 101-l 12. TAKEUCHI, T. (1972). Biological reactions and pathological changesin human beingsand animals caused by organic mercury contamination. In Environmental Mercury Contamination (R. Hartung and B. D. Dinman, eds.), pp. 247-289. Ann Arbor Science,Ann Arbor, Mich. TERASIMA, T., AND TOLMACH, L. J. (1963).Growth and nucleicacid synthesis in synchronously dividing populationsof HeLa cells.Expt. Cell Res. 30, 344-362. THRASHER, J. D., AND ADAMS, J. F. (1972). The effects of four mercury compoundson the generationtime and cell division in Tetrahymena pyriformis, WH 14. Environ. Res. 5, 443SKERFVING,

450. UMEDA, M., SAITO, K., HIROSE, K., AND SAITO, M. (1969). Cytotoxic effect of inorganic, phenyl, andalkyl mercuriccompoundson HeLa cells.Japan. J. Exp. Med. 39,47-X