* Department of Environmental Health, University of Washington, Seattle, Washington 98105;
Gradient Corporation, Mercer Island, Washington 98040;
Zymogenetics Inc., Seattle, Washington 98102;
ICOS Corporation, Bothell, Washington 98021; and
¶ Center for Child Environmental Health Risks Research, Seattle, Washington 98105
Received January 21, 2003; accepted May 5, 2003
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ABSTRACT |
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Key Words: methylmercury; cell cycle; parallelogram; interspecies; stereology; midbrain.
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INTRODUCTION |
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The developing nervous system is highly susceptible to the toxic effects of methylmercury (MeHg). The effects of in utero exposure include both sensory and motor disturbances, with more severe exposures producing mental retardation (Burbacher et al., 1990). Pathology studies of human infants and animals exposed to MeHg during gestation demonstrate alterations in cell number, brain size, cell orientation, and distribution (Chen et al., 1979
; Choi et al., 1978
; Eto et al., 1992
; Rodier et al., 1984
). Burbacher et al. (1990)
noted that cell loss and reduced brain size are consistently observed effects of MeHg exposure across species, from high-dose examples in humans (i.e., Minimata, Iraq) to low-dose examples in rodents (primarily mice). Several studies have indicated that the decreased number of brain cells is a result of the inhibition of proliferation rather than cell death, because decreased proliferation is observed at doses below those that result in cytolethality (Ponce et al., 1994
; Sager et al., 1984
). MeHg-mediated mitotic arrest has been observed in vivo (Choi, 1991
; Choi et al., 1978
; Rodier et al., 1984
) and in vitro (Miura et al., 1978
; Ponce et al., 1994
; Sager, 1988
; Vogel et al., 1986). Disruption of microtubule formation, oxidative stress, altered cellular signaling, gene expression, and protein phosphorlylation have been reported at low MeHg exposure concentrations, i.e., 1 µM or lower (Minnema et al., 1989
; Miura et al., 1984
; Rajanna et al., 1995
; Sarafian and Verity, 1991
; Vogel et al., 1985
; Zucker et al., 1990
). Such effects may contribute independently or jointly to the observed effects of MeHg on mitotic progression.
Ponce et al. (1994) studied the effects of MeHg exposure on micromass cells, that is, primary rat midbrain cells dissociated and cultured at a high density. Via flow cytometry, they observed a clear G2/M phase arrest in cells exposed to 2 to 4 µM of MeHg, which is similar to the type of cell cycle arrest that is observed when cells are exposed to the mitotic spindledisrupting drug colchicine. These results, in a primary, nonimmortalized CNS culture, are consistent with similar effects observed in other cell culture systems (Miura et al., 1984
; Vogel et al., 1986; Wasteneys et al., 1988
; Zucker et al., 1990
) as well as in vivo in mice (Rodier et al., 1984
; Sager et al., 1984
).
The aim of the investigation reported here was to observe the effects of MeHg on cell proliferation in rat midbrain cells in vivo using flow cytometry and quantitative stereology. These observations would provide an in vivo extension of our previous rat in vitro studies (Ponce et al., 1984). A second goal was to develop a quantitative framework that would allow for consideration and comparison of in vitro and in vivo midbrain data. In particular, we were interested not only in the differences in mouse versus rat data, but also in the differences in each species between in vitro and in vivo assessments. These comparisons would provide some indication of the similarity in quantitative response between the in vivo and in vitro systems. Our hypothesis was that, at doses similar to those used in the in vivo mouse studies (i.e., 12 µg/g in the embryonic brain), cell cycle disruption would be observed in the rat.
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MATERIALS AND METHODS |
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Treatment.
The animals were dosed subcutaneously (sc) with MeHgOH (Alpha Aesar, Ward Hill, MA) in the afternoon of GD 11. The control animals received sc doses of sterile saline. Total maternal MeHg doses ranged from 5 to 20 mg/kg. Toxicokinetic data (Lewandowski et al., 2002) indicated that mercury (Hg) concentration in the embryos approached maximum levels 24 h after dosing. Twenty-four or 48 h after MeHg dosing (GD 12, Witschi stage 21; GD 13, Witschi stage 27), short-term (1.5-h) bromodeoxyuridine (BrdU) exposures were carried out. The animals received a single intraperitoneal (ip) injection of BrdU (18 mg/ml dissolved in phosphate-buffered saline (PBS), pH 7.7, total dose = 25 mg/kg) and were sacrificed 1.5 h later. After sacrifice, tissues were collected and processed as described below. A second set of experiments was carried out using 6-h BrdU exposures because previous studies (Miller and Nowakowski, 1991; Miller et al., 1995
) indicate that changes in the fraction of BrdU-labeled cells may be easier to detect with longer labeling times. In these experiments, the animals were dosed with MeHg on GD 11, received an injection of BrdU at 8:00 A.M. on GD 12, and received a second injection 5 h later (1:00 P.M.). The animals were sacrificed approximately 1 h later (total labeling time, 6 h). Previous work indicated that the average duration of the S-phase in these cells was approximately 8 h (Lewandowski et al., 2003
). Thus an interval of 5 h between BrdU injections was sufficient to label all cycling cells (i.e., with BrdU administration every 5 h, no cells would pass completely through the S-phase without being exposed to BrdU). To provide a positive control for MeHg treatment, several animals were also treated with colchicine, a known mitotic spindle inhibitor. Dams were dosed intraperitoneally (ip) with 0.7 or 1.0 mg/kg colchicine (three dams per treatment group) on the morning of GD 12, approximately 30 min prior to the start of BrdU administration. Dosing with colchicine (Sigma-Aldrich, St. Louis, MO) was conducted on GD 12 due to the short half-life of colchicine, reported to be 16 min in the rat (Leighton et al., 1990
). Dosing one day prior to BrdU treatment, as was the case with MeHg, would have resulted in the colchicine being eliminated by the time BrdU labeling occurred.
Tissue collection.
At the appropriate time interval after BrdU injection, the animals were killed using CO2. The uteri were removed, placed on ice, and maintained cold while the embryos were dissected out of the uterine tissues. Dissection on ice has been used to halt BrdU incorporation in embryonic tissues (Miller and Kuhn, 1995) and allows for control of the time of BrdU exposure. After the embryos were obtained from the uteri, the developmental stage was confirmed by measuring the crown rump length, counting somites, and evaluating the overall morphology and comparing these to a standard reference (Altman and Bayer, 1995
). The embryos were examined for gross pathologies (i.e., smaller size or abnormal morphology relative to controls) when removed from the uterine tissue. The embryonic midbrains were then sectioned from the body of the embryo. All of the midbrains obtained from each litter were pooled and dissagregated using 0.25% trypsin/2.5% collagenase and trituration (25°C). The midbrain cell suspensions were then washed with PBS/5% normal goat serum (NGS), and the cells were fixed in cold 70% ethanol (4°C). All work was conducted using dark room lights to minimize BrdU photoactivation.
The embryonic bodies were frozen at -80°C and later analyzed for total Hg content according to the methods described in Lewandowski et al. (2002). The toxicokinetic data (Lewandowski et al., 2002
) indicated that, at this gestational period, embryonic body Hg concentration serves as a reasonable indicator of Hg content in the embryonic brain.
Cell staining.
The cells were centrifuged (10 min, 700 r.p.m., 25°C for all spins) in 70% ethanol and then resuspended in 1 ml PBS/5% NGS. The cells were allowed to rehydrate at room temperature overnight, as this was found to provide better antibody access to DNA. After the overnight rehydration, the cells were centrifuged and resuspended in 150 µl of PBS/NGS and 450 µl of membrane-shredding solution [100 ml calcium magnesium free (CMF)-PBS, 500 µl NP-40, 20 mg ethylenediamine tetracetic acid (EDTA)]. The samples were placed on ice and vortexed vigorously every 3 min for 20 min to disrupt the cells and gain access to the ethanol-fixed nuclei. The samples were then acidified with 800 µl of 4N HCl and agitated for 45 min at room temperature to denature the double-stranded DNA and allow antibody access. The nuclei were then washed twice with 3 ml 1-M Tris (pH 8.5) and counted using a hemocytometer. The samples were then completely decanted and resuspended with fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antibody (Pharmingen, San Diego, CA) at a ratio of 150 µl antibody to 1E6 cells. The antibody was previously diluted to 1:20 with PBS/NGS/0.5% Tween 20. After 1 h of incubation at 25°C, the samples were washed once with PBS/NGS and resuspended in PBS for flow analysis. Immediately prior to flow analysis, propidium iodide (PI), a DNA staining dye, was added to achieve a final PI concentration of 5 µg/ml in the sample.
Flow cytometry.
Flow cytometry was used to determine the fraction of cells positively labeled with BrdU and the cell cycle profile (i.e., %G1, %G2, and %S). Dual-parameter flow cytometry utilized a Coulter EPICS Elite equipped with a coherent Innova 90 air-cooled argon laser. Excitation occurred at 488 nm for both the FITC and PI. Fluorescence measurements were obtained from approximately 20,000 cells. The data were analyzed using the software package Mplus (Phoenix Flow Systems, San Diego, CA).
Cell cycle kinetic parameter estimation.
The S-phase length (Ts) was determined from the pulse-labeling study by the shift in mean PI fluorescence of the BrdU-labeled cells after 1.5 h of BrdU exposure using the method of Begg et al. (1985) as modified by Terry et al. (1991). The fractions of labeled cells at 1.5 and 6 h of BrdU exposure were determined with adjustments for cells that divided during the labeling period (Nowakowski et al., 1989
).
The effect of embryonic Hg concentration on the cell cycle parameters was evaluated using linear regression and a significance criterion of 0.05. In the case of the 6 h BrdU exposures, the data were also grouped by concentration and analyzed using a t-test or ANOVA.
Experiment 2: Stereology Studies
The design of in vivo stereology studies used to confirm the cell cycle results is summarized in Table 2.
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Tissue preparation.
Following fixation by whole-body immersion in 4% paraformaldehyde (carried in 0.1-N dibasic sodium phosphate buffer, pH 7.2), whole bodies or heads of embryos (depending on age/size) were specifically positioned by embedment in a 3% agar matrix to facilitate handling during subsequent tissue processing and increase histology efficiency by allowing entire sets of embryos to be processed together as a single tissue set. The agar blocks containing the tissue were dehydrated in a graded ethanol series and embedded in glycomethacrylate (Historesin, Cambridge Instruments, Deerfield, IL). The blocks were exhaustively sectioned in the sagittal plane at a thickness of 20 or 30 µm on a JB-4 microtome (Sorvall) using glass knives, and all sections were collected in the order generated. The sections were stained with Giemsa (stock solution, J. T. Baker, Phillipsburg, NJ) using the procedure of Iñiguez et al. (1985) and following the suggestions of Brændgaard et al. (1986
, 1990)
.
Stereology.
Estimates of the number of neurons in the developing rat midbrain were determined by use of the Optical Volume Fractionator (OVF) procedure of Bolender and Charleston (1993) as previously described in Charleston (2000)
. A detailed explanation of this methodology is provided in Lewandowski et al. (2003)
.
Counting was performed on every twelfth section in the set of sections, and 100 to 300 neuroepithelial cells were counted per individual. The boundaries of the midbrain were determined by use of a developmental rat atlas (Altman and Bayer, 1995). The areas considered for cell enumeration included the developing region of the thalamus, thus enabling use of the unambiguous boundary formed by the prominent fissure separating the thalamus from the cerebral cortex. Otherwise, determination of the boundary between the midbrain and the thalamus would have required the use of arbitrary boundaries, resulting in equally arbitrary estimates of the total number of neuroepithelial cells. This area of interest was identified by extending a line between the base of the cerebellar sulcus and the base of the thalamus, thereby transecting the ventricular space (Fig. 1
). This approach was consistent with the cell cycling studies in that the same brain region was evaluated in both sets of experiments.
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Experiment 3: Evaluation of Hg Concentrations in Vitro
To permit comparison of previous in vitro data with results of our in vivo study described above, a common dose metric was required. In vitro studies generally report chemical exposures as media concentrations (e.g., µM), whereas in vivo studies typically report tissue concentrations (e.g., µg/g). To establish a means of comparison, micromass (primary embryonic rat midbrain) and P19 cells (murine embryonic carcinoma cells) were used to measure the mercury uptake from the culture media. For micromass cultures, the culture medium was Hamss F12 supplemented with 10% fetal bovine serum, penicillin (50 IU/ml), streptomycin (5 µg/ml), and L-glutamine (5.8 mg/ml). For P19 cultures, the medium was the same, except that F12 was replaced with Dulbeccos Modified Eagle Medium (DMEM). The cultures were grown on Falcon Primaria dishes (Beckton-Dickinson Labware, Lincoln Park, NJ) possessing a hydrophillic polystyrene culture surface. The cells were incubated at 37°C in a 5% CO2/95% air atmosphere with 100% relative humidity for 2 h to allow for attachment prior to treatment. The media was then removed and replaced with an MeHg-containing media (0.5, 1.0, and 2.0 µM), and the cells were further incubated for 6 h. This was believed to be sufficient time for the MeHg to equilibrate between the cells and the media (Furukawa et al., 1982). After treatment, the culture medium was removed and the cells were rinsed twice with CMF-PBS. The cells were then incubated for 5 min at 37°C in 500-µl CMF-PBS containing 0.05% trypsin and 0.02% ETDA. Following incubation, 500 µl of mercury-free culture medium was added to each dish, and the cells were collected into 15-ml polystyrene centrifuge tubes. The cells were centrifuged at 800 r.p.m. for 20 min at 4°C, the supernatant was decanted, and the cells were stored at -80°C until analyzed for mercury. Approximately 0.15 g of the cellular material was collected per experiment. One to two experiments were conducted at each dose level/cell type. Analysis for total cellular Hg was conducted using an atomic fluorescence spectrometer (PSA Ltd., Kent, UK), as described in Lewandowski et al. (2002)
. The media concentrations were identical to those used by Ponce et al. (1994)
, and the cell culture methods were similar to those employed in that study. During collection, the cell suspensions were washed several times to remove any extracellular Hg that might have been present in the media or loosely associated with the cell membranes. A linear regression was used to determine the relationship between the intracellular Hg content and the Hg in the culture media.
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RESULTS |
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Experiment 3: Evaluation of Hg Concentrations in Vitro
The results for the analysis of Hg content in cells exposed in vitro to MeHg are shown in Figure 7. The increase in cellular Hg concentration was linear across the range tested (i.e., 0.52 µM). The relationship between the intracellular Hg (µg/g) and the culture media Hg (µM) was approximately 3 to 1 (r2 = 0.99), indicating that a media concentration of 1 µM of MeHg results in a cellular concentration of 3 µg Hg/g tissue. A very similar relationship was observed using P19 cells.
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DISCUSSION |
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A lesser sensitivity of the rat to MeHg developmental effects, as compared to the mouse, has been documented (e.g., Fuyuta et al., 1978). To a large degree, this differential sensitivity has been attributed to the different toxicokinetics of MeHg in these two species, with the rat sequestering MeHg more avidly in the blood than any other species, thereby lessening the levels distributed to other organs, such as the brain. According to this rationale, it should be possible to produce the same effects in rats and mice if the maternal loading of MeHg was adjusted to produce equivalent organ concentrations. Toxicodynamic differences between the two species, although postulated, have not been demonstrated. The work of Rodier (1995)
and Sager (1988)
has shown that mice exposed in vivo to MeHg demonstrated cell cycle effects in the developing brain at MeHg levels of 23 µg/g. Data in the rat are more limited but suggest a higher threshold of sensitivity. For example, Chen et al. (1979)
observed that the rate of DNA synthesis in the brains of rat embryos exposed to MeHg in utero were only 36% of the control rat values. The fetal Hg levels in the Chen et al. study were 10 µg/g, five times higher than those reported in the mouse studies. Although cell proliferation data in the embryonic rat have not been reported by other authors, other studies with the rat do suggest a lesser sensitivity of the developing rat brain relative to the mouse (e.g., Fredriksson et al., 1993
).
It is unfortunate that we were unable to achieve higher brain concentrations using the acute dosing procedure, because this prevented us from defining the threshold for MeHgs cell cycling effects. Higher concentrations may have been achievable using a multiday dosing regimen (Chen, 1979; Lee and Han, 1995
). The basis for our choice of acute dosing was twofold: Our interest was in performing an in vivo study that would parallel the in vitro study of Ponce et al. (1994)
, allowing us to draw a potential in vitroin vivo comparison. In addition, we were interested in quantifying the effects of MeHg on cell cycle kinetics. Chronic dosing would have complicated the interpretation of our results; if we observed effects on GD 13, it would have been difficult to determine if these were attributable to the tissue burden on GD 13 or to the initial exposures on GD 7. A more invasive approach, such as delivering MeHg directly to the embryos via injection to the utero-placental circulation, might allow for acute delivery of higher MeHg doses to the embryo, but the effects of the surgery on cell cycle patterns would likely be considerable. Alternatively, whole embryo cultures would allow for more direct control of the dosing, although this would require additional assumptions when drawing conclusions regarding the effects in intact animals.
Our data using colchicine demonstrated that colchicine clearly had a negative impact on the cell cycle. The effects of the colchicine on brain development have been well studied (Kalter, 1968). The drug blocks dividing cells at metaphase, causing a cessation of cell proliferation (Dalu et al., 1998
). Colchicine has also been shown to decrease BrdU labeling in the brain of developing rats when administered postnatally (Carbajo-Perez and Watanbe, 1990
). The positive results that we obtained using colchicine demonstrate that our analytical method is capable of detecting changes in cell cycling such as might be expected from MeHg treatment.
Using the data in Figure 7 relating in vitro media concentration and intracellular Hg content, we were able to compare in vivo and in vitro studies using a common dose metric of tissue Hg concentration (i.e., µg Hg/g cellular material). Figure 8
presents in vivo and in vitro data on the effects of MeHg on cell proliferation. Where applicable, the No Observed Adverse Effect Level (NOAEL), Lowest Observed Effect Level (LOAEL), and highest dose from each study are indicated. The y-axes are mercury concentration in µg/g or µM in culture media that are aligned and equated using the relationship shown in Figure 7
. As shown in Figure 8
, intraspecies differences in MeHg effects on proliferation in the developing brain are not as noticeable using in vitro systems as they are in vivo. For example, Wasteneys et al. (1988)
, using a murine carcinoma cell line, noted effects on microtubule stability at 0.33 µM (1 µg/g) and decreases in cell growth at 1 µM (3 µg/g). Similarly, Ponce et al. (1994)
, studying rat midbrain cells in vitro, observed decreases in cell cycle progression at 1 µM (3 µg/g). Similar data may be considered from Miura et al. (1978
; mouse glioma cells), Zucker et al. (1990
; mouse leukemia cells), and Vogel (1985
; human fibroblasts). These in vitro data suggest a quantitative similarity in response to MeHg between species. However, in our in vivo study we observed that, at brain concentrations of approximately 3 µg/g, no effects on cell cycling occurred in the rat midbrain. Thus, in vitro systems may not be able to fully quantitatively replicate interspecies differences in isolation and without detailed analyses.
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ACKNOWLEDGMENTS |
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This work was funded by grants from the U.S. Environmental Protection Agency.
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NOTES |
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