American Journal of Pathology, Vol. 137, No. 5, November 1990 Copyright © American Association of Pathologists
Flow Cytometric Analysis of the Mechanism of Methylmercury Cytotoxicity Robert M. Zucker,t Kenneth H. Elstein,t Robert E. Easterling,* and Edward J. Massaro* From the Developmental Toxicology Division (MD-67),* Health Effects Research Laboratory, United States Environmental Protection Agency, and NSI Technology Services Corp., Environmental Services Division,
Research Triangle Park, North Carolina
Flow cytometric analysis of murine erythroleukemic cells (MELC) exposed in vitro to 2.5 to 7.5 ,umol/l (micromolar) methylmercury (MeHg) reveals a dose-dependent decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle), manifested as the accumulation of most of the cells in the S phase, and a modest accumulation of cells in the G2/M phase of the cycle. Light microscopy reveals a progressive increase in chromosomal damage (condensation, pulverization). At or above 10 ,umol/l MeHg, progression through all the phases of the cell cycle is blocked and mitotic cells are arrested irreversibly in anaphase, with most exhibiting arrangement of chromosomes in a wreathlike ring formation. Also the cells exhibit both nuclear propidium iodide (PI) fluorescence (indicative of loss of viability) and concurrent cytoplasmic carboxyfluorescein (CF) fluorescence (viable cells exhibit CF fluorescence and exclude PI). In addition, there is a dose-dependent increase in cellular refractive index (900 light scatter), an apparent decrease in cell volume (axial light loss), and progressive resistance to detergent (NP-40)-mediated cytolysis. Resistance to detergent-mediated cytolysis is indicative of fixation (protein denaturation, cross-linking, and so on) of the plasma membrane/cytoplasm complex. Our findings indicate that DNA synthesis is the primary target of MeHg cytotoxicity and that apparent targets and degree of cytotoxicity are a complexfunction of dose. (Am JPathol 1990, 13 7:1187-1198)
Methylmercury (MeHg) interferes with the functioning of a broad spectrum of cellular systems and is a potent environmental neurotoxicant1 and teratogen.24 It has been reported to inhibit DNA, RNA, and protein synthesis78;
perturb
microtubule assembly919 and cell-cycle enhance lipoperoxidation8; alter membrane properties and function22-25; and impair immune cell function26-28 and neural signal transduction.9 Methylmercury inhibits mitosis and/or decreases rate of progress through cell cycle.221 Methylmercury-induced mitotic arrest (the accumulation of cells in the G2/M phase of the cycle) apparently results from inhibition of microtubule assembly,9-19 while decreased rate of progression through the cycle has been attributed to lengthening of the duration of the G1 phase as a consequence of inhibition of protein synthesis.' Whether the duration of other premitotic phases of the cell cycle is altered similarly is not clear. However Costa et al21 reported that exposure to mercuric chloride induces an S-phase-specific block in Chinese hamster ovary cells in vitro. We used flow cytometry (FCM) to investigate MeHginduced perturbation of the cell-cycle kinetics of the murine erythroleukemic cell (MELC). We observed that exposure to relatively low levels of MeHg (2.5 to 7.5 ,umol/l [micromolar]) predominately inhibits progression through the S phase of the cell cycle (in a dose-dependent manner). Accumulation of cells in the G2/M phase of the cycle also occurs, but to a considerably lesser extent. Light microscopic analysis of cytologic preparations reveals a dosedependent increase in incidence of chromosomal aberrations (condensation, pulverization). These observations indicate that DNA synthesis is the primary target of MeHg cytotoxicity. Exposure to 10 to 50 ,umol/l MeHg results in the formation of wreathlike chromosomal structures, apparently involving the entire chromosomal complement, and progressive perturbation of the plasma membrane/cytoplasm complex as a function of dose. The latter is manifested as increased 900 light scatter (refractive index3a), de-
kinetics'2-;
The research described in this article has been reviewed by the Health Effects Research Laboratory of the United States Environmental Protection Agency and was approved for publication. Approval of this article by the United States Environmental Protection Agency does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Address reprint requests to Edward J. Massaro, Developmental Toxicology Division (MD-67), Health Effects Research Laboratory, U. S. Environmental Protection Agency, Research Triangle Park, NC 27711. Accepted for publication June 25, 1990.
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creased axial light loss (apparent cell volume31), simultaneous propidum iodide (PI) and carboxyfluorescein (CF) fluorescence, and resistance to detergent (NP-40)-mediated cytolysis.32-33 These observations indicate that apparent targets and degree of MeHg cytotoxicity are complex functions of dose. Previously we reported perturbation of the plasma membrane/cytoplasm complex of MELC exposed to trialkyltin compounds.34 These observations, in conjunction with those reported herein, suggest that fixation (denaturation, cross-linking, and so on) of the proteins of the plasma membrane/cytoplasm complex may be a common toxic endpoint of exposure to relatively high levels of organometals.
density was determined and nuclei were prepared by nonionic detergent-mediated solubilization of the plasma membrane/cytoplasm complex by incubating approximately 5 X 105 cells in 1 ml of 0.2% Nonidet P-40 (NP40: Sigma N6507) in PBS containing 0.5 mg/ml RNase A (Sigma R4875) for 30 minutes at room temperature followed by cooling on ice. Fluorescein isothiocyanate (FITC; Sigma F7250), 1.5 ,g/ml, was used to stain protein and 50,ug/ml Pi was used to stain DNA.'"3'37 The dyes were added to the lysates at 00C and incubated at 00C for 10 minutes before analysis. To minimize variability of the staining reactions, the number of NP-40-treated cells per sample was kept constant.
Flow Cytometry
Materials and Methods Cells Friend murine erythroleukemic cells (MELC: T3CL2, obtained from Dr. Clyde Hutchison, University of North Carolina, Chapel Hill, NC) were grown in suspension culture in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 25 mmol/l (millimolar) HEPES (Sigma H3375, St. Louis, MO). Cell density was monitored by Coulter Counter (Model ZBI: Coulter Electronics, Inc., Hialeah, FL) and the cells were passed every 2 to 3 days to maintain logarithmic growth.
Viability Assay Viability was determined by the carboxyfluorescein diacetate (CFDA)/PI assay.3' Before use, a stock solution containing 2 mg/ml CFDA (Molecular Probes, Eugene, OR) in acetone was diluted 1:50 with phosphate buffered saline (PBS: GIBCO, pH 7.4) and added to approximately 2 X 1 05 MELC (in logarithmic growth) per milliliter growth medium to a final concentration of 3.6 ,ug/ml. The cells were incubated for 10 minutes at 37°C, chilled on ice for 10 minutes, and PI (Sigma P5264, from a stock solution of 50 jig/ml in PBS) was added to a final concentration of 4.5 ,ug/ml. The cells were analyzed by FCM at 0°C. Viable cells exhibit green CF fluorescence (resulting from intracellular esterase-catalyzed hydrolysis of CFDA) and exclude Pl.' Nonviable cells exhibit the red fluorescence of PI bound to nucleic acids.'-'
Preparation of Nuclei for Cell-cycle Analysis Cells were harvested and washed twice in PBS by centrifugation (200g for 5 minutes) at room temperature. Cell
Flow cytometric analyses were accomplished with an Ortho Flow Cytometer (Model 5OH; Becton-Dickinson, Westwood, MA) equipped with an analytic flow cell (30H). A 5-W argon (blue) laser (Innova 90-5; Coherent, Palo Alto, CA) emitting 488-nm light at 200 mW was used to quantify the green fluorescence of CF or FITC, the red fluorescence of PI, and 900 light scatter (a measure of internal structure/refractive index and protein content38). Two-color suppression circuitry prevented green (CF or FITC) fluorescence from entering the red (PI) channel and vice versa. A helium-neon (HeNe) laser emitting 633-nm (red) light at 0.8 mW was used to obtain the axial light loss signal, a measure of apparent cell volume.3' A 645nm Schott long-pass filter was used to eliminate red 900 light (HeNe laser) scatter from entering the PI fluorescence channel.'3 The flow rate was maintained at less than 200 cells per second and the data were collected, stored, and analyzed in an Ortho 2150 computer system.
Light/Fluorescence Microscopic Analysis Cellular and chromosomal morphology and cellular fluorescence properties were investigated with a Leitz Orthoplan fluorescence/interference microscope.
MeHg Exposure Protocol Methylmercury (11) chloride (Alfa 37123, Danvers, MA) was dissolved in methanol before addition to logarithmically growing MELC. The methanol concentration of the culture medium was 0.1%, which had no effect on cell viability (measured by the CFDA/PI assay) or growth rate. The MeHg concentrations investigated were 0.1, 0.25, 0.5, 1.0,2.5,5.0, 7.5,10, 25, or 50 Amol/l. Duration of exposure was 1, 2, 4, or 6 hours. To investigate recoverability from the effects of MeHg exposure, cells were exposed to
Flow Cytometric Analysis of MeHg Cytotoxicity 1189 AJP November 1990, Vol. 13 7, No. 5
MeHg for 6 hours, washed in prewarmed FBS-supplemented medium, and reincubated for 18 hours.
Progression Assay Colcemid treatment, which inhibits mitosis by blocking microtubule assembly, was used to investigate effects of MeHg on cell-cycle progression by monitoring the rate of accumulation of cells in the G2/M phase. Following 4 hours of exposure to MeHg, each cell culture was divided into two aliquots. One aliquot received 0.2 ,ug/ml Colcemid (Demecolcine, Sigma D7385) from a stock solution of 0.1 mg/ml 95% ethanol; the other (control) received an equivalent amount of 95% ethanol. After 2-hour incubation (6 hours total), the cells were harvested and nuclei prepared for cell-cycle (DNA distribution) analysis. Quantification of the DNA distribution in the Go/Gl, S, and G2/M compartments of the cell cycle, as a function of time, allows estimation of the rate of progression of cells through the cycle (progression assay). In our experiments, exposure of control cells to Colcemid for 2 hours insured accumulation of cells in the G2/M phase of the cycle without total depletion of the GO/G1 population, thereby permitting quantification of the phase distribution of cells by Multicycle, a cell-cycle analysis personal computer software package (Phoenix Flow Systems, San Diego, CA).
Quantification of the Mitotic Fraction of G2/M Nuclei The percentage of G2/M nuclei was determined flow cytometrically by computerized cell-cycle analysis of highresolution DNA histograms obtained from nuclei prepared by detergent-mediated cytolysis in PBS buffer, as described above. To quantify the percentage of cells in the M phase of the cell cycle, nuclei were prepared and analyzed in Pollack's nuclear isolation buffer,39 which allows for flow cytometric discrimination of the M subpopulation.3 Approximately 1 X 1 06 cells/sample were washed twice in ice-cold PBS and suspended in 0.3 ml of Pollack's nuclear isolation buffer (0.5% v/v NP-40, 0.05 mol/l TRIS, 0.05 mol/l NaCI, 1 mmol/l EDTA: pH 7.4) to which 0.3 ml of NaCI-NaHCO3 buffer (0.03 mol/l NaCI, 0.03 mol/l NaHCO3: pH 8.1), 0.3 ml of 70 jig/mI PI in NaCI-NaHCO3 buffer, and 0.06 ml of 3 ,ug/ml FITC were added. Samples were incubated for 30 minutes on ice and then analyzed
atO°C.
PBS by centrifugation (5 minutes, 1 20g). The pellets were resuspended in 10 ml hypotonic (75 mmol/l) KCI solution and fixed in methanol-acetic acid. Chromosome spreads were obtained by centrifuging an aliquot of the fixed cell suspension (-20,000 cells) onto glass slides at 5OOg for 10 minutes at room temperature in Leif cytobuckets (Coulter kit 322, Coulter Electronics, Inc., Hialeah, FL), followed by drying on a slide warmer, staining with 6% Giemsa (Fisher SG-28-100, Raleigh, NC), and mounting with mounting medium (Baxter Scientific M7635-1, McGaw Park, IL). Chromosome morphology was analyzed microscopically and the percentage of cells (nuclei) with normal chromosomes, condensed chromosomes, pulverized chromosomes, and ring formations was obtained from 200 cells. The mitotic index was obtained microscopically from 500 cells. The data represent the mean ± standard deviation of three experiments.
Data For each cytometric parameter investigated (Pi or FITC fluorescence, 900 light scatter, axial light loss), the distribution or mean of 104 events (cells or nuclei) per condition (dose, duration of exposure) or combination of conditions was determined. Quantification of the distribution of cells (nuclei) across the cycle was obtained with a cell-cycle analysis personal computer software package (Multicycle, Phoenix Flow Systems, San Diego, CA). Following establishment of precise dose (x duration of exposure)/response (viability) data, effects of MeHg on growth rate, the cellular and nuclear biophysical properties obtained by FCM and cytogenetic parameters were established through extensive preliminary investigation. The results
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Figure 1. MELC viability as afunction of MeHg concentration and duration of exposure. Viability was investigated by the P1 exclusion assay. At concentrations s 5 Amol/l, MeHg bad no significant effect on viabilityfor exposures up to 6 bours. At 10 Is mol/l MeHg, decrease in viability was afunction of duration of exposure.
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we report were obtained from two complete repetitions of the entire array of experiments we describe in this communication. The data were consistent both between the repetitions and with those established in preliminary studies. Data derived from cells exposed to MeHg concentrations less than 2.5 Amol/l did not differ from the control condition and are not included.
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Exposure of MELC to 5 ,umol/l MeHg for up to 6 hours has no significant effect on viability (Figure 1), 900 light scatter (Figure 2), or axial light loss (Figure 2) but does inhibit rate of growth (Figure 3) following MeHg washout and reincubation for 18 hours in fresh MeHg-free medium. Following exposure to 10 ,umol/l MeHg, cells lose viability and become permeable to PI (indicating damage to the plasma membrane) in a time-dependent manner (Figure 1). Microscopically (data not shown) these cells exhibit simultaneous PI (red) and CF (green) fluorescence, suggesting persistence of the capacity to convert CFDA to CF, despite plasma membrane damage (PI uptake). Flow cytometry analysis (Figure 2) reveals decreased apparent cell volume (axial light loss) and increased refractive index (90° light scatter), perturbations indicative of cellular
damage.33 The 90° light scatter (refractive index) and FITC fluorescence (protein content) of nuclei obtained (by detergent-mediated cytolysis) from MELC exposed to 5 to 50
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(GLM)
Figure 3. Effect ofMeHg on MELC growth rate. Following 6hours of exposure to 2.5-50,umol/l MeHg, the cells were washed and reincubatedfor 18 hours in MeHg-free medium. MELC doubling time following exposure to 2.5 zmol/l MeHg was essentially equal to that of control cells.
,umol/l MeHg for 6 hours increase as a function of MeHg concentration (Figure 4) independent of DNA distribution across the cell cycle (Figure 5). Fluorescence/interference microscopy (data not shown) reveals that these increases correlate with increasing amounts of proteinaceous (FITC fluorescent) material (cytoplasmic tags) adherent to the nuclei, indicating increased resistance of the plasma membrane/cytoplasm complex to detergent-mediated dissolution.32 Exposure to MeHg alters the cell-cycle distribution of the MELC (Figures 5 and 6). To evaluate the effect of MeHg on the ability of cells to traverse the cycle, MeHgtreated cells were exposed to Colcemid for 2 hours. In the control (untreated) condition, Colcemid blocks microtubule assembly, preventing M-phase cells from dividing
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10. Dose-dependent effects of MeHg on the cell cycle. Figure Nuclei were preparedfrom MELC exposed either to 5.0 or 7.5 itmol/l MeHg for 6 hours, as described in the text. 5 ,umol/l MeHg blocked the cells in mid to late Sphase, whereas exposure
to 7.55mol/l MeHg blocked the cells in the early S phase.
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100
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become arrested in a premitotic phase in which their nuclei exhibit the same biophysical properties as those of Mphase cells. It is conceivable, also, that exposure to MeHg 2 5.0 ,gmol/l results in increased nuclear fragility and loss of chromosomes during preparation. But fixation of the plasma membrane/cytoplasm complex is observed at MeHg concentrations of more than 5 ,mol/l (Figures 2 and 4), which would argue against increased fragility.
%Viability
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%Normal 92± 6 84 ± 8 74 ± 22 3±4 90 4
Figure 11. Contour cytograms of nuclear 900 scattervs PIfluorescence. Nuclei were preparedfrom cells exposedfor 0, 2.5, 5.0, or 7.5 mol/l MeHg before treatment with Pollack's nuclear isolation buffer.39Mitotic nuclei appear as a distinct subpopulation exhibiting decreased 900 light scatter and PIfluorescence. Following MeHg exposure, the relative percentage of this phase increases with dose.
Exposure to concentrations of MeHg 2 5 ,umol/l had little effect on viability or nuclear FCM parameters (Figures 1, 3, and 4). However morphologic analysis of chromosome preparations reveals a dose-dependent increase in both the mitotic index (although considerably less than that caused by Colcemid) and the percentage of condensed and pulverized chromosomes (Table 1, Figure 12). At a 10-Mmol/l MeHg dose, chromosome spreading was inhibited and the chromosomes of more than one
Chromosomal Aberrations %Condensed %Pulverized
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Table 1. Cytogenetic effects of MeHg. The mitotic index and percentage of chromosomal aberrations were obtainedfrom MELC exposedfor 6 hours to 0 to 50 mol/l MeHg or 0.2 ytg/ml Colcemid. The mitotic index is based on the evaluation of 500 cells. The percentage of chromosomal aberrations is based on the evaluation of 200 mitotic cells. The data represent the combined results of three experiments.
Flow Cytometric Analysis of MeHg Cytotoxicity 1195 AJP November 1990, Vol. 13 7, No. 5
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Figure 12. Effiects ofMeHg on MELC cbromosome morpbology. Pbotomicrograpbs (63OX) of representative cbromosome preparations from cells exposed to MeHg for 6 bours. A: Control; B: 10 /lmoo/ MeHg; C: 25 ,umoo/ MeHg. The percentage of cells exhibiting condensed and pulverized cbromosomes (B) increases as a function of dose. At 10 Almol/ MeHg, the cbromosomes of more than one hagf of the cells in wbicb they are delineated appear in the form of dense ring structures. At bigber doses, the cbromosomes are found exclusively in tbe form of ring structures (C).
half of the mitotic cells appeared in the form of condensed, wreathlike ring structures. Following exposure to 25 or 50 iimol/l MeHg for as little as 1 hour (the shortest time period investigated), all spreads appeared as ring structures.
Discussion The mechanism through which MeHg exerts its toxicity has been postulated to involve binding to sulfhydryl groups and disruption of disulfide bonds. Indeed inhibition of microtubule assembly, disruption of assembled microtubules, and inhibition of mitosis have been attributed to the binding of MeHg to sulfhydryl groups of tubulin.919,40 It is expected that sulfhydryl binding also would affect other cellular functions, including the synthesis, repair and structure of DNA, RNA and protein,8 and chromosome structure.41
In our study, dose-dependent effects of MeHg on logarithmically growing MELC were quantified by FCM. The parameters investigated included cell-cycle kinetics, viability (by the PI exclusion method), apparent cell volume (axial light loss) and refractive index (900 light scatter), and nuclear 900 scatter and FITC fluorescence. Chromosome morphology (of centrifugal cytologic preparations) also was investigated. Two distinct levels of MeHg cytotoxicity, differing in target specificity, are observed. Following exposure to relatively low levels of MeHg (2.5 to 7.5 ,umol/I), most cells exclude PI (ie, are viable), exhibit levels of axial light loss and 900 scatter within the normal (control) range, and
maintain growth, albeit at a reduced rate (Figures 1 to 3). Cell-cycle analysis reveals a dose-dependent increase in the percentage of cells in the S compartment, little change in the percentage of cells in the G2/M compartment, compared to the control condition, and depletion of the Go/ G1 compartment (Figures 5 and 10). Depletion of the Go/ G, compartment indicates retardation/inhibition of mitosis. Ordinarily retardation/inhibition of mitosis results in an increase in the size of the G2/M compartment (a colchicinelike effect). That this is not observed indicates retardation of S phase transit, which is supported by the increase in the size of the S compartment (Figures 5 to 8). However these cells exhibit partial, if not complete, recovery from these perturbations following reincubation in MeHg-free medium (Figure 9). To obtain more precise information on cell-cycle effects, we investigated the time course of interaction of MeHg (5 ,mol/l) with logarithmically growing MELC (Figures 7 and 8). The DNA histogram suggests reduction of the rate of DNA synthesis, resulting in retardation of the rates of both S-phase traverse and efflux (Figures 7 and 10). Computerized mathematical analysis42 of the histograms indicates that the percentage of cells in S phase increases with time, reaching a maximum after 2 hours of exposure and remaining constant thereafter (Figures 7 and 8). During the same time period, the percentage of cells in GO/Gl decreases and continues to decrease as a function of exposure up to 4 hours, indicating reduction of the rate of influx of cells into this compartment. The percentage of cells in G2/M changes modestly during the course of the experiment, suggesting retardation of influx
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into and efflux out of this compartment (the size of the G2/M compartment is limited by the relative rates of Sphase efflux and mitosis). Increasing the MeHg dose to 10 ,Amol/I does not increase the size of the G2/M compartment (Figure 6). If the primary target of MeHg were microtubule assembly/ disassembly, it would be expected that increasing dose, below cytotoxic levels, would progressively inhibit mitosis and increase the mitotic index and overall size of the G2/ M compartment. However this is not the case (Figures 5 and 6, Table 1). Indeed cells accumulate in the S compartment. Quantification of the relative contribution of M-phase cells to the G2/M compartment (Figure 11) reveals that, although the overall percentage of cells in G2/M increases modestly as a function of dose (Figure 6), the M subpopulation appears to increase substantially following exposure to MeHg concentrations of more than 2.5 gmol/I. Thus it would appear that, at concentrations of MeHg above which influx into G2 is retarded (more than 2.5 ,umol/ I), efflux out of M (ie, mitosis) is retarded to a greater extent. However morphologic analysis reveals that the mitotic index changes little as a function of MeHg dose (Table 1). This suggests that the apparent increase in the percentage of M-phase nuclei observed using the preparative method of Pollack et al3 results from the cells leaving G2 and becoming arrested in a premitotic phase in which their nuclei exhibit biophysical properties similar to those of M-phase nuclei. This argues against the hypothesis that the mitotic spindle (ie, the microtubule) is the primary target of MeHg,919 as does our observation of only limited accumulation of cells in the G2/M phase of the cell cycle (far less than those seen after treatment with Colcemid, an agent that specifically blocks microtubule assembly). Following exposure to concentrations of MeHg .10 ,umol/l, viability decreases (Figure 1), growth is completely inhibited (Figure 3), and traverse through all phases of the cell cycle is blocked (Figures 5 and 6). Cytotoxicity is manifested flow cytometrically as 1) increased cellular refractive index (90° scatter) and decreased apparent cell volume (axial light loss; Figure 2), 2) simultaneous cellular PI and CF fluorescence, and 3) increased nuclear FITC fluorescence and 900 scatter resulting from inhibition of detergent-mediated cytolysis (Figure 4). These perturbations are an apparent manifestation of irreversible alteration of the plasma membrane/cytoplasm complex resulting from fixation (protein denaturation, cross-linking, and so on'3233Fixation may play a role in MeHg-induced mitotic arrest (eg, see references 5, 6, 15). Cytologic examination reveals that MeHg induces chromosome aberrations (condensation, pulverization) and that the incidence and severity of such effects increase with dose (Table 1, Figure 12). Perturbation of chromosome structure is observed following exposure (6
hours) to MeHg concentrations as low as 2.5 ,gmol/l (Table 1). At low MeHg concentrations, condensation is the predominant chromosomal aberration observed. Exposure at or above 10 umol/I MeHg results in the induction of wreathlike chromosomal ring structures that appear to be formed by chromosomal fusion. In human leukocytes, Fiskejo28 observed chromosomes clustered into dense ring structures after in vitro exposure for 1 hour to 0.2 to 2 mmol/l MeHg or methoxyethyl mercury (chloride) in balanced salt solution (free of potentially available MeHg-reactive extraneous SH groups). In addition, the cells exhibited increased resistance to squashing compared to control cells, which is indicative of fixation. Rozynkowa and Raczkiewicz17 also described formation of chromosomal ring configurations resulting from 'sticky' chromatin bridges in spreads prepared from PHA-stimulated human lymphocytes exposed in vitro to 40 mg/ml MeHg for 10 minutes at 37°C in Eagle's minimal essential medium supplemented with serum. Although the mechanism of ring structure formation is unknown, direct interaction of MeHg with chromatin and/ or perturbation of the plasma membrane/cytoplasm complex resulting in alteration of the intracellular environment may be involved. Indeed in vitro exposure to MeHg has been shown to affect the membranes of many cell lines. For example, in cultured mouse neuroblastoma cells, Koerker13 reported that exposure to 1 MAmol/l MeHg for 24 to 72 hours at 370C in Ham's F-12 medium supplemented with serum resulted in perturbation of the function of the plasma membrane, lysosomes, mitochondria, and endoplasmic reticulum. Our findings indicate two thresholds (at least) of cytotoxicity following MeHg exposure: at relatively low doses, MeHg specifically perturbs cell-cycle progression into, through, and out of the S and G2 phases (Figures 5 to 1 1). Exposure to higher concentrations, however, results in a fixationlike alteration of the plasma/cytoplasm complex (Figures 2 and 4). The fixation phenomenon is observed flow cytometrically as increased 90° light scatter, decreased axial light loss, simultaneous CF and PI fluorescence, and resistance to detergent-mediated cytolysis (seen as increased nuclear FITC fluorescence and 900 scatter). It is interesting to note that fixation also is observed following exposure of MELC to short-chain trialkyltins (trimethyltin, triethyltin, and tributyltin) at doses specific for each compound.34 This suggests that such severe and nonspecific toxicity may be a common endpoint of exposure to high levels of organometals.
References 1. Atchison WD: Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury. J Pharm Exp Ther 1987, 241:131-139
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2. Olson FC, Massaro EJ: Effects of methylmercury on murine fetal amino acid uptake, protein synthesis and palate closure. Teratology 1977, 16:187-194 3. Slotkin TA, Bartolome J: Biochemical mechanisms of developmental neurotoxicity of methylmercury. Neurotoxicology 1987, 8:65-84 4. Slotkin TA, Kavlock RJ, Cowdry T, Orband L, Bartolome M, Whitmore WL, Bartolome J: Effects of neonatal methylmercury exposure on adrenergic receptor binding sites in peripheral tissues of the developing rat. Toxicology 1986, 41: 95-10 5. Howard JD, Mottet NK: Effects of methylmercury on the morphogenesis of the rat cerebellum. Teratology 1986, 34: 89-95 6. Rodier PM, Aschner M, Sager PR: Mitotic arrest in the developing CNS after prenatal exposure to methylmercury. Neurobehav Tox and Terat 1984, 6:379-385 7. Gruenwedel DW, Glaser JF, Cruikshank MK: Binding of methylmercury (II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis. Chem Biol Int 1981, 36:259-274 8. Verity AM, Cheung M, Sarafian T: Biochemical mechanisms of methyl mercury neurotoxicity. Abstracts of the symposium Methyl Mercury Exposure: Effects on Human Neurotoxicity. National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, July 6-7, 1989 9. Sager PR, Doherty RA, Olmstead JB: Interaction of methylmercury with microtubules in cultured cells and in vitro. Exp Cell Res 1983,146:127-137 10. Sager PR, Syversen TLM: Differential responses to methylmercury exposure and recovery in neuroblastoma and glioma cells and fibroblasts. Exp Neurol 1984, 85:371-382 11. Sager PR, Syverson TLM: Disruption of microtubules by methylmercury. In Clarkson TW, Sager PR, Syversen TLM, eds. The Cytoskeleton: A Target for Toxic Agents. New York, Plenum, 1986, pp 97-116 12. Vogel DG, Margolis RL, Mottet NK: The effects of methylmercury binding to microtubules. Toxicol Appl Pharmacol 1985, 80:473-486 13. Koerker RL: The cytotoxicity of methylmercuric hydroxide and colchicine in cultured mouse neuroblastoma cells. Toxicol Appl Pharmacol 1980, 53:458-469 14. Ramel C: Methylmercury as a mitosis disturbing agent. J Japan Med Assoc 1969, 61:1072-1081 15. Miura K, Inokawa M, Imura N: Effects of methylmercury and some metal ions on microtubule networks in mouse glioma cells and in vitro tubulin polymerization. Toxicol Appl Pharmacol 1984, 73:218-231 16. Miura K, Suzuki K, Imura N: Effects of methylmercury on mitotic mouse glioma cells. Environ Res 1978, 17:453-471 17. Rozynkowa D, Raczkiewicz B: Destructive effect of methylmercury chloride on human mitoses in living cells in vitro. Mutat Res 1977, 56:185-191 18. Abe T, Haga T, Kurokawa M: Blockage of axoplasmic transport and depolymerization of reassembled microtubules by methyl mercury. Brain Res 1975, 86:504-508 19. Sager PR: Selectivity of methylmercury effects on cytoskeleton and mitotic progression in cultured cells. Toxicol App Pharmacol 1988, 94:473-486
20. Vogel DG, Rabinovitch PS, Mottet NK: Methylmercury effects on cell cycle kinetics. Cell Tissue Kinet 1986, 19:227-242 21. Costa M, Cantoni 0, Demars M, Swartzendruber DE: Toxic metals produce an S-phase specific cell cycle block. Res Comm Chem Pathol Pharmacol 1982, 38:405 22. Bienvenue E, Boudou A, Desmazes JP, Gavach C, Georgescauld D, Sandeaux J, Sandeaux R, Seta P: Transport of mercury compounds across biomolecular lipid membranes: Effect of lipid composition, pH and chloride concentration. Chem Biol Interactions 1984, 48:91-101 23. LeBlanc RM, July LP, Paiement J. pH-dependent interaction between methyl mercury chloride and some membrane phospholipids. Chem Biol Interactions 1984, 48:237-241 24. Goodman DR, Fant ME, Harbison KD: Perturbation of aminoisobutyric acid transport in human placental membranes: Direct effects by HgCI2, CH3HgCI, and CdCI2. Teratogen Carcinogen Mutagen 1983, 3:89-100 25. Peckham NH, Choi BH: Surface charge alterations in mouse fetal astrocytes due to methyl mercury: An ultrastructural study with cationized ferntin. Exp Mol Pathol 1986, 44:230-234 26. Verschaeve L, Kirsch-Volders M, Hens L, Susanne C: Comparative in vitro cytogenetic studies in mercury-exposed human lymphocytes. Mutat Res 1985, 157:221-226 27. Verschaeve L, Kirsch-Volders M, Susanne C, Groetenbriel C, Haustermans R, LeComte A, Roossels D: Genetic damage induced by occupationally low mercury exposure. Environ Res 1976, 12:306-316 28. Fiskejo G: The effect of two organic mercury compounds on human leukocytes in vitro. Hereditas 1970, 64:142-146 29. Traxinger DL, Atchison WD: Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release. J Pharm Exp Ther 1987, 240:451-459 30. Shapiro H: Practical Flow Cytometry, Second Edition. New York, Alan R. Liss, 1988 31. Cambier JC, Monroe JG: Flow cytometry as an analytical tool for studies of neuroendocrine function. In Conn PM, ed. Methods in Enzymology, Vol. 103. New York, Academic Press, 1983, pp 227-245 32. Zucker RM, Elstein KH, Easterling RE, Ting-Beall HP, Allis JW, Massaro, EJ: Effects of tributyltin on biomembranes: Alteration of flow cytometric parameters and inhibition of Na+, K+-ATPase two-dimensional crystallization. Toxicol Appl Pharmacol 1989, 96:393-403 33. Zucker RM, Elstein KH, Easterling RE, Massaro EJ: Flow cytometric analysis of the cellular toxicity of tributyltin. Toxicol Lett 1988, 43:201-218 34. Zucker RM, Elstein KH, Easterling RE, Massaro EJ: Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell. Toxicology 1989, 58:107-119 35. Rotman B, Papermaster BW: Membrane properties of living cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 1966, 55:134-141 36. Crissman HA, Steinkamp JA: Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol 1973, 59:766771 37. Crissman HA, Darzynkiewicz Z, Tobey RA, Steinkamp JA: Correlated measurements of DNA, RNA and protein in in-
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dividual cells by flow cytometry. Science 1986, 228:13211324 38. Zucker RM, Elstein KH, Easterling RE, Massaro EJ: Flow cytometric discrimination of mitotic nuclei by right-angle light scatter. Cytometry 1988, 9:226-231 39. Pollack A, Moulis H, Block NL, Irvin GL Ill: Quantitation of cell kinetic responses using flow cytometric measurements of correlated nuclear DNA and protein. Cytometry 1984, 5:
473-481
40. Brown DL, Reuhl KR, Bormann S, Little JE: Effects of methyl mercury on the microtubule system of mouse lymphocytes. Toxicol Appl Pharmacol 1988, 94:66-75 41. Miura K, Imura N: Mechanism of methylmercury cytotoxicity. CRC Critical Reviews in Toxicology 1987, 18:161-188 42. Dean PN, Jett JH: Mathematical analysis of DNA distributions derived from flow cytometry. J Cell Biol 1974, 60: 523-527
Flow Cytometric Analysis of the Mechanism of Methylmercury Cytotoxicity Robert M. Zucker,t Kenneth H. Elstein,t Robert E. Easterling,* and Edward J. Massaro* From the Developmental Toxicology Division (MD-67),* Health Effects Research Laboratory, United States Environmental Protection Agency, and NSI Technology Services Corp., Environmental Services Division,
Research Triangle Park, North Carolina
Flow cytometric analysis of murine erythroleukemic cells (MELC) exposed in vitro to 2.5 to 7.5 ,umol/l (micromolar) methylmercury (MeHg) reveals a dose-dependent decrease in the rate of DNA synthesis (rate of passage through the S phase of the cell cycle), manifested as the accumulation of most of the cells in the S phase, and a modest accumulation of cells in the G2/M phase of the cycle. Light microscopy reveals a progressive increase in chromosomal damage (condensation, pulverization). At or above 10 ,umol/l MeHg, progression through all the phases of the cell cycle is blocked and mitotic cells are arrested irreversibly in anaphase, with most exhibiting arrangement of chromosomes in a wreathlike ring formation. Also the cells exhibit both nuclear propidium iodide (PI) fluorescence (indicative of loss of viability) and concurrent cytoplasmic carboxyfluorescein (CF) fluorescence (viable cells exhibit CF fluorescence and exclude PI). In addition, there is a dose-dependent increase in cellular refractive index (900 light scatter), an apparent decrease in cell volume (axial light loss), and progressive resistance to detergent (NP-40)-mediated cytolysis. Resistance to detergent-mediated cytolysis is indicative of fixation (protein denaturation, cross-linking, and so on) of the plasma membrane/cytoplasm complex. Our findings indicate that DNA synthesis is the primary target of MeHg cytotoxicity and that apparent targets and degree of cytotoxicity are a complexfunction of dose. (Am JPathol 1990, 13 7:1187-1198)
Methylmercury (MeHg) interferes with the functioning of a broad spectrum of cellular systems and is a potent environmental neurotoxicant1 and teratogen.24 It has been reported to inhibit DNA, RNA, and protein synthesis78;
perturb
microtubule assembly919 and cell-cycle enhance lipoperoxidation8; alter membrane properties and function22-25; and impair immune cell function26-28 and neural signal transduction.9 Methylmercury inhibits mitosis and/or decreases rate of progress through cell cycle.221 Methylmercury-induced mitotic arrest (the accumulation of cells in the G2/M phase of the cycle) apparently results from inhibition of microtubule assembly,9-19 while decreased rate of progression through the cycle has been attributed to lengthening of the duration of the G1 phase as a consequence of inhibition of protein synthesis.' Whether the duration of other premitotic phases of the cell cycle is altered similarly is not clear. However Costa et al21 reported that exposure to mercuric chloride induces an S-phase-specific block in Chinese hamster ovary cells in vitro. We used flow cytometry (FCM) to investigate MeHginduced perturbation of the cell-cycle kinetics of the murine erythroleukemic cell (MELC). We observed that exposure to relatively low levels of MeHg (2.5 to 7.5 ,umol/l [micromolar]) predominately inhibits progression through the S phase of the cell cycle (in a dose-dependent manner). Accumulation of cells in the G2/M phase of the cycle also occurs, but to a considerably lesser extent. Light microscopic analysis of cytologic preparations reveals a dosedependent increase in incidence of chromosomal aberrations (condensation, pulverization). These observations indicate that DNA synthesis is the primary target of MeHg cytotoxicity. Exposure to 10 to 50 ,umol/l MeHg results in the formation of wreathlike chromosomal structures, apparently involving the entire chromosomal complement, and progressive perturbation of the plasma membrane/cytoplasm complex as a function of dose. The latter is manifested as increased 900 light scatter (refractive index3a), de-
kinetics'2-;
The research described in this article has been reviewed by the Health Effects Research Laboratory of the United States Environmental Protection Agency and was approved for publication. Approval of this article by the United States Environmental Protection Agency does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use. Address reprint requests to Edward J. Massaro, Developmental Toxicology Division (MD-67), Health Effects Research Laboratory, U. S. Environmental Protection Agency, Research Triangle Park, NC 27711. Accepted for publication June 25, 1990.
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creased axial light loss (apparent cell volume31), simultaneous propidum iodide (PI) and carboxyfluorescein (CF) fluorescence, and resistance to detergent (NP-40)-mediated cytolysis.32-33 These observations indicate that apparent targets and degree of MeHg cytotoxicity are complex functions of dose. Previously we reported perturbation of the plasma membrane/cytoplasm complex of MELC exposed to trialkyltin compounds.34 These observations, in conjunction with those reported herein, suggest that fixation (denaturation, cross-linking, and so on) of the proteins of the plasma membrane/cytoplasm complex may be a common toxic endpoint of exposure to relatively high levels of organometals.
density was determined and nuclei were prepared by nonionic detergent-mediated solubilization of the plasma membrane/cytoplasm complex by incubating approximately 5 X 105 cells in 1 ml of 0.2% Nonidet P-40 (NP40: Sigma N6507) in PBS containing 0.5 mg/ml RNase A (Sigma R4875) for 30 minutes at room temperature followed by cooling on ice. Fluorescein isothiocyanate (FITC; Sigma F7250), 1.5 ,g/ml, was used to stain protein and 50,ug/ml Pi was used to stain DNA.'"3'37 The dyes were added to the lysates at 00C and incubated at 00C for 10 minutes before analysis. To minimize variability of the staining reactions, the number of NP-40-treated cells per sample was kept constant.
Flow Cytometry
Materials and Methods Cells Friend murine erythroleukemic cells (MELC: T3CL2, obtained from Dr. Clyde Hutchison, University of North Carolina, Chapel Hill, NC) were grown in suspension culture in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) and 25 mmol/l (millimolar) HEPES (Sigma H3375, St. Louis, MO). Cell density was monitored by Coulter Counter (Model ZBI: Coulter Electronics, Inc., Hialeah, FL) and the cells were passed every 2 to 3 days to maintain logarithmic growth.
Viability Assay Viability was determined by the carboxyfluorescein diacetate (CFDA)/PI assay.3' Before use, a stock solution containing 2 mg/ml CFDA (Molecular Probes, Eugene, OR) in acetone was diluted 1:50 with phosphate buffered saline (PBS: GIBCO, pH 7.4) and added to approximately 2 X 1 05 MELC (in logarithmic growth) per milliliter growth medium to a final concentration of 3.6 ,ug/ml. The cells were incubated for 10 minutes at 37°C, chilled on ice for 10 minutes, and PI (Sigma P5264, from a stock solution of 50 jig/ml in PBS) was added to a final concentration of 4.5 ,ug/ml. The cells were analyzed by FCM at 0°C. Viable cells exhibit green CF fluorescence (resulting from intracellular esterase-catalyzed hydrolysis of CFDA) and exclude Pl.' Nonviable cells exhibit the red fluorescence of PI bound to nucleic acids.'-'
Preparation of Nuclei for Cell-cycle Analysis Cells were harvested and washed twice in PBS by centrifugation (200g for 5 minutes) at room temperature. Cell
Flow cytometric analyses were accomplished with an Ortho Flow Cytometer (Model 5OH; Becton-Dickinson, Westwood, MA) equipped with an analytic flow cell (30H). A 5-W argon (blue) laser (Innova 90-5; Coherent, Palo Alto, CA) emitting 488-nm light at 200 mW was used to quantify the green fluorescence of CF or FITC, the red fluorescence of PI, and 900 light scatter (a measure of internal structure/refractive index and protein content38). Two-color suppression circuitry prevented green (CF or FITC) fluorescence from entering the red (PI) channel and vice versa. A helium-neon (HeNe) laser emitting 633-nm (red) light at 0.8 mW was used to obtain the axial light loss signal, a measure of apparent cell volume.3' A 645nm Schott long-pass filter was used to eliminate red 900 light (HeNe laser) scatter from entering the PI fluorescence channel.'3 The flow rate was maintained at less than 200 cells per second and the data were collected, stored, and analyzed in an Ortho 2150 computer system.
Light/Fluorescence Microscopic Analysis Cellular and chromosomal morphology and cellular fluorescence properties were investigated with a Leitz Orthoplan fluorescence/interference microscope.
MeHg Exposure Protocol Methylmercury (11) chloride (Alfa 37123, Danvers, MA) was dissolved in methanol before addition to logarithmically growing MELC. The methanol concentration of the culture medium was 0.1%, which had no effect on cell viability (measured by the CFDA/PI assay) or growth rate. The MeHg concentrations investigated were 0.1, 0.25, 0.5, 1.0,2.5,5.0, 7.5,10, 25, or 50 Amol/l. Duration of exposure was 1, 2, 4, or 6 hours. To investigate recoverability from the effects of MeHg exposure, cells were exposed to
Flow Cytometric Analysis of MeHg Cytotoxicity 1189 AJP November 1990, Vol. 13 7, No. 5
MeHg for 6 hours, washed in prewarmed FBS-supplemented medium, and reincubated for 18 hours.
Progression Assay Colcemid treatment, which inhibits mitosis by blocking microtubule assembly, was used to investigate effects of MeHg on cell-cycle progression by monitoring the rate of accumulation of cells in the G2/M phase. Following 4 hours of exposure to MeHg, each cell culture was divided into two aliquots. One aliquot received 0.2 ,ug/ml Colcemid (Demecolcine, Sigma D7385) from a stock solution of 0.1 mg/ml 95% ethanol; the other (control) received an equivalent amount of 95% ethanol. After 2-hour incubation (6 hours total), the cells were harvested and nuclei prepared for cell-cycle (DNA distribution) analysis. Quantification of the DNA distribution in the Go/Gl, S, and G2/M compartments of the cell cycle, as a function of time, allows estimation of the rate of progression of cells through the cycle (progression assay). In our experiments, exposure of control cells to Colcemid for 2 hours insured accumulation of cells in the G2/M phase of the cycle without total depletion of the GO/G1 population, thereby permitting quantification of the phase distribution of cells by Multicycle, a cell-cycle analysis personal computer software package (Phoenix Flow Systems, San Diego, CA).
Quantification of the Mitotic Fraction of G2/M Nuclei The percentage of G2/M nuclei was determined flow cytometrically by computerized cell-cycle analysis of highresolution DNA histograms obtained from nuclei prepared by detergent-mediated cytolysis in PBS buffer, as described above. To quantify the percentage of cells in the M phase of the cell cycle, nuclei were prepared and analyzed in Pollack's nuclear isolation buffer,39 which allows for flow cytometric discrimination of the M subpopulation.3 Approximately 1 X 1 06 cells/sample were washed twice in ice-cold PBS and suspended in 0.3 ml of Pollack's nuclear isolation buffer (0.5% v/v NP-40, 0.05 mol/l TRIS, 0.05 mol/l NaCI, 1 mmol/l EDTA: pH 7.4) to which 0.3 ml of NaCI-NaHCO3 buffer (0.03 mol/l NaCI, 0.03 mol/l NaHCO3: pH 8.1), 0.3 ml of 70 jig/mI PI in NaCI-NaHCO3 buffer, and 0.06 ml of 3 ,ug/ml FITC were added. Samples were incubated for 30 minutes on ice and then analyzed
atO°C.
PBS by centrifugation (5 minutes, 1 20g). The pellets were resuspended in 10 ml hypotonic (75 mmol/l) KCI solution and fixed in methanol-acetic acid. Chromosome spreads were obtained by centrifuging an aliquot of the fixed cell suspension (-20,000 cells) onto glass slides at 5OOg for 10 minutes at room temperature in Leif cytobuckets (Coulter kit 322, Coulter Electronics, Inc., Hialeah, FL), followed by drying on a slide warmer, staining with 6% Giemsa (Fisher SG-28-100, Raleigh, NC), and mounting with mounting medium (Baxter Scientific M7635-1, McGaw Park, IL). Chromosome morphology was analyzed microscopically and the percentage of cells (nuclei) with normal chromosomes, condensed chromosomes, pulverized chromosomes, and ring formations was obtained from 200 cells. The mitotic index was obtained microscopically from 500 cells. The data represent the mean ± standard deviation of three experiments.
Data For each cytometric parameter investigated (Pi or FITC fluorescence, 900 light scatter, axial light loss), the distribution or mean of 104 events (cells or nuclei) per condition (dose, duration of exposure) or combination of conditions was determined. Quantification of the distribution of cells (nuclei) across the cycle was obtained with a cell-cycle analysis personal computer software package (Multicycle, Phoenix Flow Systems, San Diego, CA). Following establishment of precise dose (x duration of exposure)/response (viability) data, effects of MeHg on growth rate, the cellular and nuclear biophysical properties obtained by FCM and cytogenetic parameters were established through extensive preliminary investigation. The results
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Figure 1. MELC viability as afunction of MeHg concentration and duration of exposure. Viability was investigated by the P1 exclusion assay. At concentrations s 5 Amol/l, MeHg bad no significant effect on viabilityfor exposures up to 6 bours. At 10 Is mol/l MeHg, decrease in viability was afunction of duration of exposure.
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we report were obtained from two complete repetitions of the entire array of experiments we describe in this communication. The data were consistent both between the repetitions and with those established in preliminary studies. Data derived from cells exposed to MeHg concentrations less than 2.5 Amol/l did not differ from the control condition and are not included.
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Exposure of MELC to 5 ,umol/l MeHg for up to 6 hours has no significant effect on viability (Figure 1), 900 light scatter (Figure 2), or axial light loss (Figure 2) but does inhibit rate of growth (Figure 3) following MeHg washout and reincubation for 18 hours in fresh MeHg-free medium. Following exposure to 10 ,umol/l MeHg, cells lose viability and become permeable to PI (indicating damage to the plasma membrane) in a time-dependent manner (Figure 1). Microscopically (data not shown) these cells exhibit simultaneous PI (red) and CF (green) fluorescence, suggesting persistence of the capacity to convert CFDA to CF, despite plasma membrane damage (PI uptake). Flow cytometry analysis (Figure 2) reveals decreased apparent cell volume (axial light loss) and increased refractive index (90° light scatter), perturbations indicative of cellular
damage.33 The 90° light scatter (refractive index) and FITC fluorescence (protein content) of nuclei obtained (by detergent-mediated cytolysis) from MELC exposed to 5 to 50
50.0
(GLM)
Figure 3. Effect ofMeHg on MELC growth rate. Following 6hours of exposure to 2.5-50,umol/l MeHg, the cells were washed and reincubatedfor 18 hours in MeHg-free medium. MELC doubling time following exposure to 2.5 zmol/l MeHg was essentially equal to that of control cells.
,umol/l MeHg for 6 hours increase as a function of MeHg concentration (Figure 4) independent of DNA distribution across the cell cycle (Figure 5). Fluorescence/interference microscopy (data not shown) reveals that these increases correlate with increasing amounts of proteinaceous (FITC fluorescent) material (cytoplasmic tags) adherent to the nuclei, indicating increased resistance of the plasma membrane/cytoplasm complex to detergent-mediated dissolution.32 Exposure to MeHg alters the cell-cycle distribution of the MELC (Figures 5 and 6). To evaluate the effect of MeHg on the ability of cells to traverse the cycle, MeHgtreated cells were exposed to Colcemid for 2 hours. In the control (untreated) condition, Colcemid blocks microtubule assembly, preventing M-phase cells from dividing
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10. Dose-dependent effects of MeHg on the cell cycle. Figure Nuclei were preparedfrom MELC exposed either to 5.0 or 7.5 itmol/l MeHg for 6 hours, as described in the text. 5 ,umol/l MeHg blocked the cells in mid to late Sphase, whereas exposure
to 7.55mol/l MeHg blocked the cells in the early S phase.
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100
1.
Q), ZD). 0: 0. 0.
0).
Pi Fluorescence tion of dose (Table 1), suggesting that cells leaving G2
become arrested in a premitotic phase in which their nuclei exhibit the same biophysical properties as those of Mphase cells. It is conceivable, also, that exposure to MeHg 2 5.0 ,gmol/l results in increased nuclear fragility and loss of chromosomes during preparation. But fixation of the plasma membrane/cytoplasm complex is observed at MeHg concentrations of more than 5 ,mol/l (Figures 2 and 4), which would argue against increased fragility.
%Viability
Control 2.5 pM 5.0 10.0 25.0
50.0 Colc.
98 98 95 14 3 5 97
%Mitotics 3.1 ±1.0 4.2 ± 2.0 5.0 ± 0.9 4.8 ± 3.0 4.5 ± 0.8 3.9 ± 1.8 41 ± 0
%Normal 92± 6 84 ± 8 74 ± 22 3±4 90 4
Figure 11. Contour cytograms of nuclear 900 scattervs PIfluorescence. Nuclei were preparedfrom cells exposedfor 0, 2.5, 5.0, or 7.5 mol/l MeHg before treatment with Pollack's nuclear isolation buffer.39Mitotic nuclei appear as a distinct subpopulation exhibiting decreased 900 light scatter and PIfluorescence. Following MeHg exposure, the relative percentage of this phase increases with dose.
Exposure to concentrations of MeHg 2 5 ,umol/l had little effect on viability or nuclear FCM parameters (Figures 1, 3, and 4). However morphologic analysis of chromosome preparations reveals a dose-dependent increase in both the mitotic index (although considerably less than that caused by Colcemid) and the percentage of condensed and pulverized chromosomes (Table 1, Figure 12). At a 10-Mmol/l MeHg dose, chromosome spreading was inhibited and the chromosomes of more than one
Chromosomal Aberrations %Condensed %Pulverized
5±2 13 ± 8 22 ± 15 30 ± 34 --10 4
-2 ±2 ±4 ±4 12 ± 9 ---
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34 2±2 55 ±41 100 ± 0 100 ± 0
Table 1. Cytogenetic effects of MeHg. The mitotic index and percentage of chromosomal aberrations were obtainedfrom MELC exposedfor 6 hours to 0 to 50 mol/l MeHg or 0.2 ytg/ml Colcemid. The mitotic index is based on the evaluation of 500 cells. The percentage of chromosomal aberrations is based on the evaluation of 200 mitotic cells. The data represent the combined results of three experiments.
Flow Cytometric Analysis of MeHg Cytotoxicity 1195 AJP November 1990, Vol. 13 7, No. 5
a414
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Figure 12. Effiects ofMeHg on MELC cbromosome morpbology. Pbotomicrograpbs (63OX) of representative cbromosome preparations from cells exposed to MeHg for 6 bours. A: Control; B: 10 /lmoo/ MeHg; C: 25 ,umoo/ MeHg. The percentage of cells exhibiting condensed and pulverized cbromosomes (B) increases as a function of dose. At 10 Almol/ MeHg, the cbromosomes of more than one hagf of the cells in wbicb they are delineated appear in the form of dense ring structures. At bigber doses, the cbromosomes are found exclusively in tbe form of ring structures (C).
half of the mitotic cells appeared in the form of condensed, wreathlike ring structures. Following exposure to 25 or 50 iimol/l MeHg for as little as 1 hour (the shortest time period investigated), all spreads appeared as ring structures.
Discussion The mechanism through which MeHg exerts its toxicity has been postulated to involve binding to sulfhydryl groups and disruption of disulfide bonds. Indeed inhibition of microtubule assembly, disruption of assembled microtubules, and inhibition of mitosis have been attributed to the binding of MeHg to sulfhydryl groups of tubulin.919,40 It is expected that sulfhydryl binding also would affect other cellular functions, including the synthesis, repair and structure of DNA, RNA and protein,8 and chromosome structure.41
In our study, dose-dependent effects of MeHg on logarithmically growing MELC were quantified by FCM. The parameters investigated included cell-cycle kinetics, viability (by the PI exclusion method), apparent cell volume (axial light loss) and refractive index (900 light scatter), and nuclear 900 scatter and FITC fluorescence. Chromosome morphology (of centrifugal cytologic preparations) also was investigated. Two distinct levels of MeHg cytotoxicity, differing in target specificity, are observed. Following exposure to relatively low levels of MeHg (2.5 to 7.5 ,umol/I), most cells exclude PI (ie, are viable), exhibit levels of axial light loss and 900 scatter within the normal (control) range, and
maintain growth, albeit at a reduced rate (Figures 1 to 3). Cell-cycle analysis reveals a dose-dependent increase in the percentage of cells in the S compartment, little change in the percentage of cells in the G2/M compartment, compared to the control condition, and depletion of the Go/ G1 compartment (Figures 5 and 10). Depletion of the Go/ G, compartment indicates retardation/inhibition of mitosis. Ordinarily retardation/inhibition of mitosis results in an increase in the size of the G2/M compartment (a colchicinelike effect). That this is not observed indicates retardation of S phase transit, which is supported by the increase in the size of the S compartment (Figures 5 to 8). However these cells exhibit partial, if not complete, recovery from these perturbations following reincubation in MeHg-free medium (Figure 9). To obtain more precise information on cell-cycle effects, we investigated the time course of interaction of MeHg (5 ,mol/l) with logarithmically growing MELC (Figures 7 and 8). The DNA histogram suggests reduction of the rate of DNA synthesis, resulting in retardation of the rates of both S-phase traverse and efflux (Figures 7 and 10). Computerized mathematical analysis42 of the histograms indicates that the percentage of cells in S phase increases with time, reaching a maximum after 2 hours of exposure and remaining constant thereafter (Figures 7 and 8). During the same time period, the percentage of cells in GO/Gl decreases and continues to decrease as a function of exposure up to 4 hours, indicating reduction of the rate of influx of cells into this compartment. The percentage of cells in G2/M changes modestly during the course of the experiment, suggesting retardation of influx
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into and efflux out of this compartment (the size of the G2/M compartment is limited by the relative rates of Sphase efflux and mitosis). Increasing the MeHg dose to 10 ,Amol/I does not increase the size of the G2/M compartment (Figure 6). If the primary target of MeHg were microtubule assembly/ disassembly, it would be expected that increasing dose, below cytotoxic levels, would progressively inhibit mitosis and increase the mitotic index and overall size of the G2/ M compartment. However this is not the case (Figures 5 and 6, Table 1). Indeed cells accumulate in the S compartment. Quantification of the relative contribution of M-phase cells to the G2/M compartment (Figure 11) reveals that, although the overall percentage of cells in G2/M increases modestly as a function of dose (Figure 6), the M subpopulation appears to increase substantially following exposure to MeHg concentrations of more than 2.5 gmol/I. Thus it would appear that, at concentrations of MeHg above which influx into G2 is retarded (more than 2.5 ,umol/ I), efflux out of M (ie, mitosis) is retarded to a greater extent. However morphologic analysis reveals that the mitotic index changes little as a function of MeHg dose (Table 1). This suggests that the apparent increase in the percentage of M-phase nuclei observed using the preparative method of Pollack et al3 results from the cells leaving G2 and becoming arrested in a premitotic phase in which their nuclei exhibit biophysical properties similar to those of M-phase nuclei. This argues against the hypothesis that the mitotic spindle (ie, the microtubule) is the primary target of MeHg,919 as does our observation of only limited accumulation of cells in the G2/M phase of the cell cycle (far less than those seen after treatment with Colcemid, an agent that specifically blocks microtubule assembly). Following exposure to concentrations of MeHg .10 ,umol/l, viability decreases (Figure 1), growth is completely inhibited (Figure 3), and traverse through all phases of the cell cycle is blocked (Figures 5 and 6). Cytotoxicity is manifested flow cytometrically as 1) increased cellular refractive index (90° scatter) and decreased apparent cell volume (axial light loss; Figure 2), 2) simultaneous cellular PI and CF fluorescence, and 3) increased nuclear FITC fluorescence and 900 scatter resulting from inhibition of detergent-mediated cytolysis (Figure 4). These perturbations are an apparent manifestation of irreversible alteration of the plasma membrane/cytoplasm complex resulting from fixation (protein denaturation, cross-linking, and so on'3233Fixation may play a role in MeHg-induced mitotic arrest (eg, see references 5, 6, 15). Cytologic examination reveals that MeHg induces chromosome aberrations (condensation, pulverization) and that the incidence and severity of such effects increase with dose (Table 1, Figure 12). Perturbation of chromosome structure is observed following exposure (6
hours) to MeHg concentrations as low as 2.5 ,gmol/l (Table 1). At low MeHg concentrations, condensation is the predominant chromosomal aberration observed. Exposure at or above 10 umol/I MeHg results in the induction of wreathlike chromosomal ring structures that appear to be formed by chromosomal fusion. In human leukocytes, Fiskejo28 observed chromosomes clustered into dense ring structures after in vitro exposure for 1 hour to 0.2 to 2 mmol/l MeHg or methoxyethyl mercury (chloride) in balanced salt solution (free of potentially available MeHg-reactive extraneous SH groups). In addition, the cells exhibited increased resistance to squashing compared to control cells, which is indicative of fixation. Rozynkowa and Raczkiewicz17 also described formation of chromosomal ring configurations resulting from 'sticky' chromatin bridges in spreads prepared from PHA-stimulated human lymphocytes exposed in vitro to 40 mg/ml MeHg for 10 minutes at 37°C in Eagle's minimal essential medium supplemented with serum. Although the mechanism of ring structure formation is unknown, direct interaction of MeHg with chromatin and/ or perturbation of the plasma membrane/cytoplasm complex resulting in alteration of the intracellular environment may be involved. Indeed in vitro exposure to MeHg has been shown to affect the membranes of many cell lines. For example, in cultured mouse neuroblastoma cells, Koerker13 reported that exposure to 1 MAmol/l MeHg for 24 to 72 hours at 370C in Ham's F-12 medium supplemented with serum resulted in perturbation of the function of the plasma membrane, lysosomes, mitochondria, and endoplasmic reticulum. Our findings indicate two thresholds (at least) of cytotoxicity following MeHg exposure: at relatively low doses, MeHg specifically perturbs cell-cycle progression into, through, and out of the S and G2 phases (Figures 5 to 1 1). Exposure to higher concentrations, however, results in a fixationlike alteration of the plasma/cytoplasm complex (Figures 2 and 4). The fixation phenomenon is observed flow cytometrically as increased 90° light scatter, decreased axial light loss, simultaneous CF and PI fluorescence, and resistance to detergent-mediated cytolysis (seen as increased nuclear FITC fluorescence and 900 scatter). It is interesting to note that fixation also is observed following exposure of MELC to short-chain trialkyltins (trimethyltin, triethyltin, and tributyltin) at doses specific for each compound.34 This suggests that such severe and nonspecific toxicity may be a common endpoint of exposure to high levels of organometals.
References 1. Atchison WD: Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury. J Pharm Exp Ther 1987, 241:131-139
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2. Olson FC, Massaro EJ: Effects of methylmercury on murine fetal amino acid uptake, protein synthesis and palate closure. Teratology 1977, 16:187-194 3. Slotkin TA, Bartolome J: Biochemical mechanisms of developmental neurotoxicity of methylmercury. Neurotoxicology 1987, 8:65-84 4. Slotkin TA, Kavlock RJ, Cowdry T, Orband L, Bartolome M, Whitmore WL, Bartolome J: Effects of neonatal methylmercury exposure on adrenergic receptor binding sites in peripheral tissues of the developing rat. Toxicology 1986, 41: 95-10 5. Howard JD, Mottet NK: Effects of methylmercury on the morphogenesis of the rat cerebellum. Teratology 1986, 34: 89-95 6. Rodier PM, Aschner M, Sager PR: Mitotic arrest in the developing CNS after prenatal exposure to methylmercury. Neurobehav Tox and Terat 1984, 6:379-385 7. Gruenwedel DW, Glaser JF, Cruikshank MK: Binding of methylmercury (II) by HeLa S3 suspension-culture cells: Intracellular methylmercury levels and their effect on DNA replication and protein synthesis. Chem Biol Int 1981, 36:259-274 8. Verity AM, Cheung M, Sarafian T: Biochemical mechanisms of methyl mercury neurotoxicity. Abstracts of the symposium Methyl Mercury Exposure: Effects on Human Neurotoxicity. National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, July 6-7, 1989 9. Sager PR, Doherty RA, Olmstead JB: Interaction of methylmercury with microtubules in cultured cells and in vitro. Exp Cell Res 1983,146:127-137 10. Sager PR, Syversen TLM: Differential responses to methylmercury exposure and recovery in neuroblastoma and glioma cells and fibroblasts. Exp Neurol 1984, 85:371-382 11. Sager PR, Syverson TLM: Disruption of microtubules by methylmercury. In Clarkson TW, Sager PR, Syversen TLM, eds. The Cytoskeleton: A Target for Toxic Agents. New York, Plenum, 1986, pp 97-116 12. Vogel DG, Margolis RL, Mottet NK: The effects of methylmercury binding to microtubules. Toxicol Appl Pharmacol 1985, 80:473-486 13. Koerker RL: The cytotoxicity of methylmercuric hydroxide and colchicine in cultured mouse neuroblastoma cells. Toxicol Appl Pharmacol 1980, 53:458-469 14. Ramel C: Methylmercury as a mitosis disturbing agent. J Japan Med Assoc 1969, 61:1072-1081 15. Miura K, Inokawa M, Imura N: Effects of methylmercury and some metal ions on microtubule networks in mouse glioma cells and in vitro tubulin polymerization. Toxicol Appl Pharmacol 1984, 73:218-231 16. Miura K, Suzuki K, Imura N: Effects of methylmercury on mitotic mouse glioma cells. Environ Res 1978, 17:453-471 17. Rozynkowa D, Raczkiewicz B: Destructive effect of methylmercury chloride on human mitoses in living cells in vitro. Mutat Res 1977, 56:185-191 18. Abe T, Haga T, Kurokawa M: Blockage of axoplasmic transport and depolymerization of reassembled microtubules by methyl mercury. Brain Res 1975, 86:504-508 19. Sager PR: Selectivity of methylmercury effects on cytoskeleton and mitotic progression in cultured cells. Toxicol App Pharmacol 1988, 94:473-486
20. Vogel DG, Rabinovitch PS, Mottet NK: Methylmercury effects on cell cycle kinetics. Cell Tissue Kinet 1986, 19:227-242 21. Costa M, Cantoni 0, Demars M, Swartzendruber DE: Toxic metals produce an S-phase specific cell cycle block. Res Comm Chem Pathol Pharmacol 1982, 38:405 22. Bienvenue E, Boudou A, Desmazes JP, Gavach C, Georgescauld D, Sandeaux J, Sandeaux R, Seta P: Transport of mercury compounds across biomolecular lipid membranes: Effect of lipid composition, pH and chloride concentration. Chem Biol Interactions 1984, 48:91-101 23. LeBlanc RM, July LP, Paiement J. pH-dependent interaction between methyl mercury chloride and some membrane phospholipids. Chem Biol Interactions 1984, 48:237-241 24. Goodman DR, Fant ME, Harbison KD: Perturbation of aminoisobutyric acid transport in human placental membranes: Direct effects by HgCI2, CH3HgCI, and CdCI2. Teratogen Carcinogen Mutagen 1983, 3:89-100 25. Peckham NH, Choi BH: Surface charge alterations in mouse fetal astrocytes due to methyl mercury: An ultrastructural study with cationized ferntin. Exp Mol Pathol 1986, 44:230-234 26. Verschaeve L, Kirsch-Volders M, Hens L, Susanne C: Comparative in vitro cytogenetic studies in mercury-exposed human lymphocytes. Mutat Res 1985, 157:221-226 27. Verschaeve L, Kirsch-Volders M, Susanne C, Groetenbriel C, Haustermans R, LeComte A, Roossels D: Genetic damage induced by occupationally low mercury exposure. Environ Res 1976, 12:306-316 28. Fiskejo G: The effect of two organic mercury compounds on human leukocytes in vitro. Hereditas 1970, 64:142-146 29. Traxinger DL, Atchison WD: Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release. J Pharm Exp Ther 1987, 240:451-459 30. Shapiro H: Practical Flow Cytometry, Second Edition. New York, Alan R. Liss, 1988 31. Cambier JC, Monroe JG: Flow cytometry as an analytical tool for studies of neuroendocrine function. In Conn PM, ed. Methods in Enzymology, Vol. 103. New York, Academic Press, 1983, pp 227-245 32. Zucker RM, Elstein KH, Easterling RE, Ting-Beall HP, Allis JW, Massaro, EJ: Effects of tributyltin on biomembranes: Alteration of flow cytometric parameters and inhibition of Na+, K+-ATPase two-dimensional crystallization. Toxicol Appl Pharmacol 1989, 96:393-403 33. Zucker RM, Elstein KH, Easterling RE, Massaro EJ: Flow cytometric analysis of the cellular toxicity of tributyltin. Toxicol Lett 1988, 43:201-218 34. Zucker RM, Elstein KH, Easterling RE, Massaro EJ: Flow cytometric comparison of the effects of trialkyltins on the murine erythroleukemic cell. Toxicology 1989, 58:107-119 35. Rotman B, Papermaster BW: Membrane properties of living cells as studied by enzymatic hydrolysis of fluorogenic esters. Proc Natl Acad Sci USA 1966, 55:134-141 36. Crissman HA, Steinkamp JA: Rapid, simultaneous measurement of DNA, protein, and cell volume in single cells from large mammalian cell populations. J Cell Biol 1973, 59:766771 37. Crissman HA, Darzynkiewicz Z, Tobey RA, Steinkamp JA: Correlated measurements of DNA, RNA and protein in in-
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