Effects of methylmercuric chloride, cycloheximide, and colchicine on the reaggregation of dissociated mouse cerebellar cells

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

86,362-37 l(l986)

Effects of Methylmercuric Chloride, Cycloheximide, and Colchicine on the Reaggregation of Dissociated Mouse Cerebellar Cells

A. J. JACOBS,*W. M. MANISCALCO,~ AND J. N. FINKELSTEIN*~~ *Division of Toxicology, Department of Biophysics, and TDivision of Neonatology, Department of Pediatrics, University of Rochester, School ofMedicine and Dentistry, Rochester, New York 14642

Received February 251986; accepted July 11, I986

Effects of Methylmercuric Chloride, Cycloheximide, and Colchicine on the Reaggregation of Dissociated Mouse Cerebellar Cells. JACOBS, A. J., MANISCALCO, W. M., AND RNKELSTEIN, J. N. (1986). Toxicol. Appf. Pharmacol. 86, 362-377. High fetal and neonatal brain levels of methyl mercury (MeHg) have been associated with the abnormal migration of neurons within the cerebellar and cerebral co&es. How MeHg interferes with cellular proliferation, migration, and differentiation is poorly understood. In this study, a cell recognition/cohesion assay based on the ability of dissociated neonatal mouse &rebellar cells to reaggregate was used to test whether MeHg exposure could disrupt cell surface recognition. Reaggregation of dissociated cells was monitored by measuring diameters from low-power photomicrographs. Exposure to 4 mg/kg body wt methylmercuric chloride (MeHgCl) 24 hr prior to the isolation of 3 days postnatal cerebellar cells altered the pattern of reaggregate growth. Between 25 and 5 1 hr in vitro (hiv), the exposed reaggregates grew at a faster rate than controls. Freshly isolated cells exposed in vitro to 0, 0.5, 1.0, and 4.0 pM MeHgCl initially exhibited a dose-related inhibition in reaggregate growth with an IC50 of 1.5 pM at 24 hiv. Following initial inhibition, exposed groups showed a dose-dependent acceleration in reaggregation similar to that found following in vivo exposure. In contrast, in vitro exposure to cycloheximide resulted in only a dose-related inhibition of reaggregation. No acceleration in growth rate was seen. Colchicine exposure produced no initial inhibition but appeared to mimic the long-term effects seen with both in vivo and in vitro MeHgCl exposure. These studies suggest that MeHgCl alters cerebellar cell recognition through a complex mechanism initially involving depressed synthesis of specific proteins followed by alterations in microtubules. Both effects may involve a disruption in the arrangement of specific cell surface recognition molecules. 0 1986 Academic Pm, Inc.

Methyl mercury (MeHg) has been considered an ubiquitous environmental pollutant. Prenatal and early postnatal exposure to MeHg have been shown to produce disturbed brain development in humans (Choi et al., 1978; Chang, 1979) and in laboratory rodents (Reuhl et al., 198 la,b; Sager et al., 1982). High fetal and neonatal brain levels of MeHg are associated with the abnormal migration of neurons to the cerebellar and cerebral cortices and generalized derangement of the cytoarchitecture of the brain (Choi et al., 1978). 0041-008X/86

$3.00

Cop-t Q 1986 by Academic Prm, Inc. All rights of repmduction in any form merved.

In contrast, adult exposure results in focal destructive damage within the CNS (Takeuchi, 1972). The molecular means by which MeHg interferes with cellular proliferation, migration, and differentiation in the developing central nervous system is poorly understood. The majority of effects observed following exposure in vivo or in vitro to MeHg may be attributed to its ability to interfere with sulfiydryl groups, resulting in the poisoning of essential enzymes or changes in the structural or trans362

METHYL

MERCURY

AND CELL REAGGREGATION

port components of membranes (Junghans, 1983). Altered levels of protein synthesis, both increased (Brubaker et al., 1973) and decreased (Omata et al., 1980; Syversen, 1982), have been associated with MeHg exposure and may involve the interference with a sulfhydryl-containing regulator of protein synthesis (Cheung and Verity, 1983). MeHg inhibits the polymerization of tubulin in vitro (Peterson et al., 198 1; Vogel et al., 1985) and causes mitotic arrest in cultured glioma cells (Miura et al., 1978). In this study, a cell recognition/cohesion assay was used to test whether exposure to MeHg alters the ability of dissociated neonatal mouse cerebellar cells to reaggregate and to organize into defined histiotypic structures. Enzymatically dissociated morphologically undifferentiated neuroepithelial cells are capable of reaggregating and maturing into histiotypic spheres of differentiated neurons, astrocytes, and oligodendrocytes (Trapp et al., 1979). Our study demonstrates that exposure to MeHg either in vivo, prior to cerebellar cell isolation, or in vitro, during the reaggregation process, causes a complex alteration in the pattern of reaggregate growth. Inhibition in cerebellar cell reaggregation following MeHg exposure is mimicked by in vitro exposure to the protein synthesis inhibitor cycloheximide. Long-term enhancement in reaggregate growth is mimicked by colchitine, a well known disruptor of microtubules. Since the effects of MeHg on both protein synthesis and microtubules have been well documented, it becomes apparent from the results of our study that the mechanism through which MeHg alters cortical development may involve a complex series of insults. Each of these insults may ultimately result in alterations in the arrangement and/or expression of specific cell recognition/adhesion molecules responsible for guiding migrating cells within the developing central nervous system. METHODS Reaggregate cultures. Cerebellar cell reaggmgate cultures were prepared according to a modification of a pro-

363

cedure for fetal rat brain cell reaggregates (Seeds, 197 1). BALB/c mice. obtained from the Inbred Mouse Unit at the University of Rochester were sacrificed by decapitation at 3 days postnatal (dpn). Cerebella were removed and placed in cold, sterile calcium-magnesium-free buffer (CMF), pH 7.4. All buffers and media were filtered prior to use through a 0.22~pm Sterivex-GS filter unit (Millipore Corp., Bedford, Mass.) and maintained at 4°C. All steps were carried out under aseptic conditions within a laminar flow hood. Cerebella were minced into pieces roughly 1 mm3 and subjected to enzymatic digestion for 15 min at 37’C with 0.1% porcine pancreatic trypsin Type. IX (Sigma Chemical Co., St. Louis, MO.) in CMF. The digested tissue was rinsed with cold CMF and triturated using a series of pipets with decreasing bore in a modified Hanks’ buffered saline solution (HBSS) (GIBCO, Grand Island, N.Y.) containing 4% DNAse Type I and 10% Nu-Serum (Collaborative Research, Inc., Lexington, Mass.) until a single cell suspension was obtained. Cell yield and viability were determined using hemocytometer cell counts and trypan blue exclusion. The cells were centrifuged at 800 rpm for 10 min and the pellet was resuspended in a modified minimal essential medium (MEM) (GIBCO Labs) containing 10% FCS. Cells were plated at a density of 7 X IO6 cells/S ml in 60 X 1S-mm petri plates (Falcon 1007). The plates were placed atop a Junior orbit shaker (Lab-line Instruments, Inc., Melrose Park, Ill.), set at 70 rpm within a humid incubator maintained at 37°C in an atmosphere containing 5% CO2 and 95% air. The growth medium was replaced every 2 to 3 days. In vivo exposure to methyl mercury. BALB/c mice at 2 dpn received a single per OSexposure to vehicle control or 4 mg/kg body wt methylmercuric chloride (MeHgCl). Both the vehicle control (5 mM Na2C03) and the dosing solution ( 1.5 mM MeHgCl in 5 mM Na2C03) were loaded into l-cc tuberculin syringes and fitted into a microdosing apparatus. Animals with an average body weight of 2.4 + 0.1 g SE at 2 dpn received a gavage volume of 10.3 pi/g body wt. After 24 hr exposure the mice were sacrificed and the cerebella were removed. Total mercury content within cerebellar and whole brain tissue was determined using cold vapor atomic absorption spectrometry. Cerebellar cells were isolated from both control and exposed animals according to methods previously outlined. Cells were plated at a density of 7 X lo6 cells/5 ml MeHg-free growth medium. In vitro exposure to methyl mercury, cycloheximide, and colchicine. Cerebellar cells derived from 3 dpn mice were plated at a density of 7 X 1O6cells/5 ml in 60 x 15mm petri plates. Within minutes, in vitro exposure to either MeHg, cycloheximide, or colchicine commenced. Reaggregates were exposed to 0, 0.5, 1.0, or 4 PM MeHgCl in 5 mM Na2C03 in a total added volume of 200 ~1.To determine the amount of MeHg accumulated over 24 hr in vitro (hiv) for each exposure group, pellets of reaggregates pooled from each set were obtained after

FIG. 1. Cerebellar cell reaggregates from the (A) control group at 25 hours in vitro (hiv), (B) methyl mercury (MeHg)-exposed group at 25 hiv, (C) control group at 5 1 hiv, (D) MeHg-exposed group at 5 1 hiv, (E) control group at 93 hiv, and(F) MeHg-exposed group at 93 hiv. Bar represents 200 pm.

spinning the petri plate contents through dibutylphthalate:mineral oil (6: 1). From each pellet, the total mercury content was determined using cold vapor atomic absorp tion spectrometry and expressed as nanograms of total mercury per 1O6cells. Additional experiments involved the exposure of freshly isolated cerebellar cells to extracellular concentrations of O-25 pM cycloheximide and O-10 pM colchicine. All in vitro exposures were terminated at 93 hiv when fresh MEM replaced the original growth medium.

Measurement of reaggregate diameters. In order to demonstrate the inhibitory effects of in vitro exposure to both MeHgCl and cycloheximide on cerebehar cell reaggregatioo and to determine an IC50 for both compounds, a simple scoring method based on that described by Kleinschuster (Kleinschuster et al., 1983) was adopted. At 24 hiv five plates from each dose group were examined utilizing a tissue culture microscope and scored for their appearance as compared to the control group. One hundred percent of control represented maggmgates that were iodistinguishable from controls. Seventy-five per-

METHYL

MERCURY

365

AND CELL REAGGREGATION TABLE 1

REACGREGATEDIAMETERSFOLLOWINGIN VIVO EXPOSURE TOMETHYLMERCURIC CHLORIDE 24hr PRIORTOCEREBELLARCELLISOLATION

Exposure group Control n 4 mg/kg MeHgCl n

25 hiv’ 67.5 k 4.6’ 423d 5 64.2 + 6.8 244 5

51 hiv

Pb

N.S.

185.7 + 8.7 484 5 262.9 + 21.2 170 5

93 hiv

P

<0.005

319.9* 7.3 302 5 421.0 f 17.6 111 4

P

<0.005

a hiv = hours in vitro. bp values obtained using Student’s t test represent comparisons with the control group. ’ Values represent the mean reaggregate diameter (pm) within n culture plates f SE. d Combined number of reaggregates measured in all photographic fields. cent represented reaggregates similar in appearance to controls, but with rough edges. A combination of smaller and normal-sized reaggregates with an average size roughly half of normal was ranked at the 50% mark. Plates containing very small reaggregates and clusters of single cells were ranked at 25% of control. The complete inhibition of reaggregation, marked by the presence of dissociated cells, received a score of zero. In all cases, plates within each dose group obtained the same score. For a more complete analysis of cellular reaggregation, photographs randomly taken from each plate were used to measure reaggregate diameters. Living reaggregates from random fields were photographed using Kodachrome color slide him in an Olympus C-35 camera attached to an Olympus IMT-2 tissue culture microscope. Reaggregate diameters were determined after applying the appropriate magnification factors to measured projected images. The data are expressed as the mean of the plate mean diameters for each dose. The total number of reaggregates measured has been reported for each dose of MeHgCl and time point in addition to the number(n) of plates used in the measurement. Differences between the control and exposed groups were compared by Student’s t test for paired samples. The Bonferroni correction for multiple t tests was used when appropriate. Histologicalpreparation of reaggregates. Reaggregates at 8 days in vitro (div) were preserved for light microscopic histological evaluation by fixation for 2 hr in Bouin’s fixative. The fixed tissue was dehydrated in a graded ethanol series and cleared in xylene prior to paraffin embedding. Five-micrometer sections were stained with hematoxylin and eosin according to standard procedures (Humason, 1979). Reaggregates were preserved at 8 div because preliminary results indicated that the typical histiotypic structure of cerebellar reaggregates was evident by this age.

Protein synthesis in freshly isolated cells. Protein synthesis was measured in freshly isolated cells according to a modification of the procedure reported by Sarafian (Sarahan et al., 1984). At 2 dpn, animals were exposed by gavage to a vehicle control or to 4 mg/kg MeHgCl as described previously. Twenty-four hours later, cerebellar cells were isolated. Cell incubations (20 mitt, 37°C) were performed in MEM containing 1 ml cell suspension (roughly 7 X lo6 cells), 0.20 ml (10 mCi) [3H]leucine (Amersham Corp., Arlington Heights, Ill.) in MEM, and 25 pl inhibitor (for cycloheximide preparations). Proteins were precipitated from duplicate samples obtained from each preparation. Two separate aliquots of each dissolved pellet were used for determining protein content by the method of Lowry (Lowry et al., 1951). The remaining suspension was subjected to liquid scintillation counting to determine the amount of [‘Hlleucine that had been incorporated into protein during the 20-min incubation period.

RESULTS In Vivo Exposure to Methyl Mercury In vivo exposure to 4 mg/kg MeHgC124 hr prior to cell isolation at 3 dpn had no effect on either cell yield (6.9 rt 0.6 X lo6 cells/animal) or the viability (94.0 f 1.3%) of freshly isolated cerebellar cells. However, cell recognition/cohesion, as indexed by the ability of the isolated cells to reaggregate, was disrupted. Figure 1 and Table 1 present the in vivo methyl mercury data. Through 25 hiv

FIG. 2. Inhibition of cerebellar cell reaggregation by methylmercuric chloride (MeHgCl) within the first 24 hiv. (A) Control reaggregates, and cells exposed to (B) 0.5 pM MeHgCl, (C) 1.0 pM MeHgCl, and (D) 4.0 jtM MeHgCl. Bar represents 200 pm.

the reaggregation of isolated cerebellar cells appeared to be unaffected. Between 25 and 51 hiv, the exposed reaggregates grew at a faster rate than controls and at 51 hiv, attained diameters an average of 40% greater than controls. Between 5 1 and 93 hiv, growth rate approximated that in controls, but exposed reaggregates were much larger due to the earlier growth spurt. All reaggregates in each photographic field were counted. Histologically, exposed reaggregates could not be distinguished from controls except perhaps by their increased size. In some of the largest reaggregates a central necrotic core was observed, indicating an inability of nutrients and/or oxygen to penetrate the misformed reaggregate. Using cold vapor atomic absorption spectrometry, the concentration of total mercury was determined to be 1.42 + 0.09 and 1.49 & 0.01 ng mercury/mg wet tissue wt for the whole brain minus cerebellum and cerebellum, respectively, 24 hr after in viva exposure.

In Vitro Exposure to Methyl Mercury Exposure of isolated cerebellar cells in vitro to MeHgCl resulted in a dosedependent inhibition of reaggregation through 24 hiv as shown in Figs. 2 and 3. Reaggregation was completely blocked by 4 /IM MeHgCl. The concentration causing roughly a 50% inhibition of reaggregation (IC50) was estimated to be 1.5 PM MeHgCl. As demonstrated in Fig. 3, by 95 hiv (at which time the culture medium was replaced with fresh medium containing no MeHgCl) the reaggregates exposed to 1 I.LM MeHgCl had attained a mean diameter of almost twice that seen in controls. At 190 hiv the reaggregates that had been exposed to 0.5 PM from 0 to 95 hiv also were significantly larger than control reaggregates. For the 1 PM group, growth acceleration occurred in the 2% to 95-hiv period as the ratio of diameters compared to controls was equivalent at both the 190- and 95-hiv time points. In contrast, reaggregates exposed to a lower

METHYL

175 150

d F z

125 100

8

75

;

50

:

25

i+i

The scoring method outlined under Methods was used to obtain the inhibition curves. Cycloheximide was a more potent inhibitor with an IC50 of roughly 0.5 PM whereas MeHgCl had an IC50 of 1.5 PM.

200

E

367

AND CELL REAGGREGATION

*

225 fi t; I a

MERCURY

Protein Synthesis in Isolated Cells Exposed to MeHgCl or Cycloheximide

0

0.0

0.5

METHYLMERCURY

1.0 CONC

4.0 (@I

FIG. 3. Effect of in vitro exposure to methylmercuric chloride on the reaggregation of 3 day postnatal mouse cerebellar cells. Open, solid, right-hatched, left-hatched, and cross-hatched bars represent mean reaggregate diameters + SE at 19, 24, 28, 95, and 190 hiv, respectively. Asterisks represent statistically significant (p < 0.017) differences from the control diameters.

dose of MeHgCl (0.5 PM) from 0 to 95 hiv showed a marked acceleration in growth between 90 and 190 hiv. As in the in vivo experiment, reaggregates from the exposed groups could not be histologically distinguished from controls other than by size and the appearance of pyknotic cells in the cores of the largest reaggregates. The mercury content in reaggregates exposed in vitro to MeHgCl as determined by cold vapor atomic absorption spectrometry rose in a dose-related linear fashion. Exposure to 4 PM MeHgCl for 24 hr resulted in a total mercury content of 10.6 ng/ 1O6cells.

A small but significant drop in the incorporation of [3H]leucine into protein occurred 24 hr following an in vivo exposure to 4 mg/kg MeHgCl (Table 2). A similar effect was seen when isolated cells were placed in 50 PM cycloheximide. Combined in vivo exposure to MeHgCl and in vitro exposure to cycloheximide resulted in a drop in [3H]leucine incorporation similar to that seen with each treatment alone. A statistically significant drop in protein content was also seen following exposure to both MeHgCl and cycloheximide but not following exposure to either alone. The average specific activity of the controls was 597.7 cpm mg-’ min-‘. In Vitro Exposure to Colchicine Reaggregate diameters calculated as a percentage of control diameters measured dur-

In Vitro Exposure to Cycloheximide The dose-dependent inhibition of reaggregation seen with in vitro exposure to cycloheximide is demonstrated in Fig. 4. The inhibitory effect continued through 94.5 hiv in contrast to the exposure of isolated cerebellar cells to MeHgCl in vitro. Figure 5 demonstrates the dose-response effect of in vitro exposure to MeHgCl and cycloheximide on cell reaggregation at 24 hiv.

0.0

0.1 0.3 CYCLOHEXIMIOE

0.5

* L*** 2.0

CONC

10.0

(PM)

FIG. 4. Effect of in vitro exposure to cycloheximide on the reaggregation of 3 day postnatal mouse cerebellar cells. Gpen and solid bars represent mean reaggregate diameters + SE at 2 1 and 94.5 hiv, respectively. Asterisks represent statistically significant (p < 0.01) differences from the control diameters.

368

JACOBS, MANISCALCO,

AND FINKELSTEIN

diameters) among exposed reaggregates approximated that of controls. DISCUSSION

CONCENTRATION

(pt4)

FIG. 5. In vitro inhibition of reaggregation of isolated 3 day postnatal mouse cerebellar cells by cycloheximide and methyl mercury. Circles and triangles represent the cycloheximide and methyl mercury data, respectively.

ing exposure to colchicine are shown in Fig. 6. Within the first 21 hiv of exposure of isolated cerebellar cells to colchicine a small but statistically significant inhibition of reaggregation could be seen with doses from 1.0 to 10.0 PM. Between 21 and 93 hiv, the exposed reaggregates grew at a dose-dependent, accelerated rate. At 93 hiv the 10 pM group had attained an average size more than 2.5 times greater than that of the controls. Beyond 93 hiv, after removal of the colchicine from the media, the growth rate (ratio of 26 1 to 93 hiv

The ability of methyl mercury to interfere with cellular proliferation, migration, and differentiation in the developing central nervous system may involve direct cellular damage, changes in nutrient availability, or disruption of specific cell-cell interactions required for proper architectural and biochemical development. The complexity of the central nervous system makes the systemic investigation of these processes difficult. The purpose of this study has been to investigate the effects of MeHg on cell-cell recognition and cohesion using a neonatal mouse cerebellar cell reaggregate culture model. Cell populations derived from early postnatal mouse cerebellum consist primarily of proliferating granule cells from the external granular layer along with glial elements including oligodendrocytes and astrocytes (Lindner and Schachner, 1982). The reaggregation of dissociated cells can serve as an index for their ability to form appropriate contacts during a critical developmental period. A disruption in cell-cell recognition or cohesion during the sensitive premigratory stage in granule cell development could produce

TABLE 2 EFTECT OFIN

METHYLMERCURYEXPOSUREAND INVITRO CYCLOHEXIMIDEEXPOSUREONTHE INCORP~RATIONOF[~H]LEUCINEINTOCEREBELLARCELLPROTEIN

VIVO

Exposure group

?I

dose or concn

Control Methyl mercury Cycloheximide Methyl mercury and cycloheximide

5 2 5 7

4 mp/kp 50 pM 4 mdkg 50 vM

cpm/ 1O6cells 2959.9 2419.4 2708.1 2538.3

f 22.6’ k 5 1.O f 76.0 IL 68.2

P”

10.005
“p values obtained using Student’s t test represent comparisons with the control group. According to the Bonferroni correction, statisticahy significant dilferences occur when p < 0.0 17. b Values represent the mean of n duplicate samples f SE.

METHYL

0.0

0.1

0.5

COLCHICINE

1.0

MERCURY

2.0 CONC

5.0

AND CELL REAGGREGATION

10.0

(/JM)

FIG. 6. Effect of in vitro exposure to colchicine on the reaggregation of 3 day postnatal mouse cerebellar cells. Open, solid, and right hatched bars represent mean reaggregate diameters + SE at 21, 93, and 26 1 hiv, respectively. Asterisks represent statistically significant (p < 0.0083) differences from the control diameters.

those pathological defects observed following MeHg intoxication, specifically abnormal migration of cortical neurons and generalized cortical derangement. The results of our study indicate that the pattern of reaggregation of isolated neonatal mouse cerebellar cells is altered following in vivo (4 mg/kg body wt) and in vitro (IC50 = 1.5 pM) exposure to MeHgCl. In vivo treatment 24 hr prior to cell isolation produced an enhancement in reaggregation alter 24 hiv. During in vitro exposure commencing at the time the cells were first placed in culture, we observed an inhibitory response to increasing doses of MeHgCl through 24 hiv. Reaggregation was completely blocked by 4 PM MeHgCl, an observation similar to that reported by Kleinschuster and co-workers for embryonic chick neural retinal cells (KleinSchuster et al., 1983). A dose-related enhancement in cerebellar cell reaggregation followed the initial inhibitory phase in the MeHgCl-treated cultures. In vivo exposure to MeHgCl failed to cause inhibition in cell reaggregation presumably due to the 24-hr delay between exposure and cell isolation. To test the hypothesis that the initial inhibitory effect on cell recognition/cohesion is re-

369

lated to a depression in protein synthesis, a known protein synthesis inhibitor, cycloheximide, was added to isolated neonatal cerebellar cells. As with MeHgCl exposure, reaggregation was inhibited in a dose-related fashion through 24 hiv. By 93 hiv, exposed reaggregates had diameters comparable, but not larger than, controls. These results suggest that depressed protein synthesis may result in a delayed reincorporation of cell surface recognition or adhesion molecules lost during trypsinization. A small reduction (8.5%) in [3H]leucine incorporation into cellular protein followed exposure to 50 PM cycloheximide, a dose well above that required to completely inhibit cell reaggregation. Therefore, only a minor disruption in the rate of protein synthesis appears to be capable of producing the inhibitory phase in cerebellar cell reaggregation. Cycloheximide was a more effective inhibitor of cerebellar cell reaggregation than MeHgCl as seen by comparing the ICSOs (0.5 and 1.5 PM, respectively). Protein synthesis was depressed 16% 24 hr after in vivo exposure to 4 mg/kg MeHgCl. At this time, no effect on cerebellar cell reaggregation was observed, further supporting the conclusion that cycloheximide was a more potent inhibitor of reaggregation. Combined in vivo treatment with MeHgCl and in vitro exposure to cycloheximide produced an effect on protein synthesis not significantly greater than that following either treatment alone, suggesting that both act to depress protein synthesis through a common mechanism. Cycloheximide is known to inhibit the peptidyl transferase activity of the 60s ribosomal subunit in eucaryotes, thereby inhibiting protein synthesis in the elongation phase of translation (Stryor, 198 1). Cheung and Verity (1983) have localized sites of MeHg action to components of the pH 5 enzyme fraction from whole brain extracts which includes transfer RNAs, tRNA synthetase enzymes, and various translational initiation and elongation factors. It therefore seems likely that both work to inhibit peptide elongation.

370

JACOBS. MANISCALCO.

Depressed protein synthesis appears to be unrelated to the enhancement in cerebellar cell cohesion caused by MeHgCl seen after 24 hiv. To test the hypothesis that microtubular disruption would produce altered cell recognition/cohesion we added colchicine to freshly isolated cerebellar cells and monitored the growth of reaggregates. The enhancement in cerebellar cell reaggregation observed after the first 24 hr of MeHgCl exposure was mimicked by colchicine while the inhibitory effects seen immediately following either MeHgCl or cycloheximide treatment were not observed. These results demonstrate that neither the inhibition of protein synthesis nor the disruption in microtubules alone is sufficient to yield the characteristic response seen in our study following MeHgCl exposure. The molecular nature of cell surface recognition or adhesion molecules affected by MeHgCl intoxication is as yet unknown. A disruption in the appearance and/or arrangement of cell surface recognition molecules at appropriate developmental ages could produce those pathological defects observed following MeHg intoxication, specifically abnormal migration of cortical neurons and generalized cortical derangement. Supporting this hypothesis is a study by Rebel and coworkers (1983) which has demonstrated changes in the relative concentrations of specific gangliosides within mouse neuroblastoma cells exposed in vitro to MeHgCl. In conclusion, MeHgCl has been demonstrated to alter cell-cell recognition and/or cohesion in reaggregating cultures of dissociated neonatal mouse cerebellar cells in a complex manner. The identity of the cell recognition/adhesion molecules disrupted following MeHgCl exposure remains to be investigated. ACKNOWLEDGMENTS We thank the Environmental Health Sciences core analytical facility at the University of Rochester for determining the mercury content in tissue and cell samples. We also thank Dr. M. F. D. Notter of the Department

AND FINKELSTEIN of Neurobiology and Anatomy for use of her lab for the preparation of histological specimens. This work was supported by NIEHS grants ES07026 and ES0 1248.

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mouse neuroblastoma cells in culture. Lipids 18,664661.

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aggregating brain cell cultures. Proc. Nat. Acad. Sci. CJSA68,1858-1861. STRYOR, L. (198 1). Biochemistry. Freeman, San Francisco. SYVERSEN, T. L. M. (1982). Changes in protein and RNA synthesis in rat brain neurons after a single dose of methylmercury. Toxicol. Lett. 10,3 l-34. TAKEUCHI, T. (1972). Biological reactions and pathological changes in human beings and animals caused by organic mercury contamination. In Environmental Mercury Contamination (R. Hartung and B. D. Dinman, eds.), pp. 247-289. Ann Arbor Science Pub., Ann Arbor, Mich. TRAPP, B. D., HONECCER, P., RICHELSON, E., AND WEBSTER, H., DEF. (1979). Morphological differentiation of mechanically dissociated fetal rat brain in aggregating cell cultures. Brain Res. 160, 117- 130. VOGEL, D. G., MARGOLIS, R. L., AND MOTTET, N. K. (1985). The effects of methylmercury binding to microtubules. Toxicol. Appl. Pharmacol. 80,473-486.