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Toxicity of mercuric chloride in cultures of neurons and nonneuronal cells derived from embryonic chick sympathetic ganglia
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
60, 253-262 (1981)
Toxicity of Mercuric Chloride in Cultures of Neurons and Nonneuronal Cells Derived from Embryonic Chick Sympathetic Ganglia PUCKPRINK~ANGDEE,' Department
of Pharmacology,
Received
L. M. PARTLOW,' AND L. G. BUSH
University
of Utah
College
January
8, 1981;
accepted
of Medicine,
March
Salt
Lake
City,
C/t&
84132
24. 1981
Toxicity of Mercuric Chloride in Cultures of Neurons and Nonneuronal Cells Derived from Embryonic Chick Sympathetic Ganglia. SANGDEE, P., PARTLOW, L. M., AND BUSH, L. G. (I 98 1). Toxicol. Appl. Pharmocol. 60,253-262. The effects of mercuric chloride were studied in vitro using both mixed and highly purified cultures of neurons and nonneuronal (glial) cells. Cultures were prepared from the sympathetic ganglia of 1 l-day chick embryos and treated with mercuric chloride (0.1 nM to 100 fiM) for either 2 or 3 days. Acetyl cholinesterase specific activity, [3H]thymidine incorporation per microgram of protein, and [Wlleucine incorporation per microgram of protein were quantified, and the following results were obtained. First, all three biochemical measures either were stimulated or were unaffected by exposure of mixed ganglion cell cultures to 510 pM mercuric chloride for 2 days. In contrast, exposure to 100 PM mercuric chloride for 2 days depressed incorporation of both leucine and thymidine, but stimulated acetyl cholinesterase specific activity. Second, incorporation of both leucine and thymidine rapidly returned to control levels following removal of the mercuric chloride from the culture medium after 2 days of exposure. In contrast, acetyl cholinesterase specific activity remained elevated. Third, all biochemical measures were either inhibited or unaffected by a 3-day exposure of mixed ganglion cell cultures to any concentration of mercuric chloride. Fourth, sympathetic neurons appeared to be much more sensitive to mercuric chloride than homologous nonneuronal cells. Fifth, most of the stimulatory and inhibitory effects of mercuric chloride appeared to result from direct action of the heavy metal on either the neurons or nonneuronal ceils.
Mercury is widely recognized as an extremely hazardous environmental pollutant. Mercury poisoning can result in a wide variety of neurological disturbances including impairment of vision and other sensory inputs, speech and hearing defects, ataxia, and mental disturbances (Takeuchi et al., 1962). Common neuropathological changes resulting from mercury poisoning include neuronal degeneration and glial cell proliferation (Chang and Hartmann, 1972b; ’ Present address: Department of Biopharmaceutical Sciences, Chiang-Mai University, Chiang-Mai, Thailand.
*To whom all correspondence and requests for reprints should be addressed.
Charbonneau et al., 1976; Chang, 1977). A wide variety of biochemical changes have also been described in mercury-intoxicated animals. These include alterations in (a) the content and base composition of RNA in neurons (Chang et al., 1972; Chang and Hartmann, 1972a; Chang, 1977), (b) the rate of protein synthesis (Yoshino et al., 1966; Cavanagh and Chen, 197 1; Brubaker et al., 1973; Syversen, 1977; Verity et al., 1977), and (c) the specific activity of several brain enzymes (Chang et al., 1973). Investigators using in vitro systems have also described a number of mercury-induced biochemical alterations. RNA synthesis is inhibited by both methyl mercury and mer253
0041-008X/81/110253-10$02.00/0 Copyright 0 1981 by Academic Press. Inc All rights of reprcduclion in any form reserved
254
SANGDEE,
PARTLOW,
curie chloride in two cell lines (Nakazawa et al., 1975; Gruenwedel and Cruikshank, 1979). Protein syntesis is inhibited by methyl mercury in HeLa cells (Gruenwedel and Cruikshank, 1979). Activities of several specific enzymes have also been studied in vitro. Methyl mercury inhibits brain succinic dehydrogenase but has little effect on acetyl cholinesterase (Hiisli and Hosli, 1974). Finally, DNA synthesis is inhibited by methyl mercury in a variety of cell lines (Nakazawa et al., 1975; Gruenwedel and Cruikshank, 1979; Nakada and Imura, 1980). In contrast, DNA synthesis has been reported to be both stimulated (Nakada and Imura, 1980) and inhibited (Schuster and Hare, 1970; Nakazawa et al., 1975; Nakada and Imura, 1980) by mercuric chloride. The in vitro system utilized in the present study allows assessment of the effects of heavy metals on essentially pure (298%) primary cultures of separated neurons and nonneuronal (glial) cells derived from embryonic chick sympathetic ganglia (McCarthy and Partlow, 1976a). The nonneuronal cells in these cultures appear to be either satellite cells or Schwann cells, both of which are glial elements, while the neurons are thought to be postganglionic sympathetic neurons. Since mixed cultures of these neurons and nonneuronal cells can also be prepared, it is possible to compare the effects of heavy metals on the biochemistry and morphology of ( 1) neuronal, (2) nonneuronal, and (3) mixed cultures. The studies presented in this paper demonstrate that the responses of neurons and nonneuronal cells to various concentrations of mercuric chloride are quite complex. By appropriate selection of concentration and time of exposure, mercuric chloride could be shown to either stimulate, inhibit, or have no effect on any of three biochemical measures (acetyl cholinesterase specific activity, leucine incorporation per ccg of protein, and thymidine incorporation per pg of protein). Treatment with mercuric chloride for 2 days was generally stimulatory, while treatment
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for 3 days was generally inhibitory. The neurons were much more sensitive to mercuric chloride than the nonneuronal cells. In most cases, the effects of mercuric chloride observed in mixed cultures containing both neurons and nonneuronal cells were ascribable to a direct action on one of these cell types. METHODS Culture medium and salt solutions. L- 15 (Leibovitz) Medium (North American Biologicals, Miami, Fla.) was supplemented (L-15+) so that the final solution contained 10% fetal calf serum (Irvine Scientific, Fountain Valley, Calif.), 0.5% glucose (analytical reagent, Mallinckrodt, St. Louis, MO.), 100 IU/ml penicillin, 100 pg/ml streptomycin (Grand Island, Biological Co., Grand Island, N.Y.), and 15 rig/ml nerve growth factor (Burroughs Welcome Co., Research Triangle Park, N.C.). This medium was then adjusted to 300 mOSM and a pH of 7.15 at 37°C. Puck’s saline G without phenol red (Irvine Scientific) was supplemented with 5 mg/ml glucose. Culture surface. Falcon plastic dishes, 35 mm in diameter (Oxnard, Calif.) were used in all experiments. Both mixed cultures and highly purified nonneuronal cultures were grown directly on the polystyrene surface of these dishes, while highly purified neuronal cultures were grown on surfaces coated with polylysine (Nutritional Biochemical Corp., Irvine, Calif.) according to the procedure of Letourneau (1975). Culture wells. Pyrex tubing with an inner diameter of 13 mm was cut into glass cylinders approximately 5 mm tall. All glass cylinders were boiled in 0.1 M potassium hydroxide, rinsed extensively with water, and autoclaved before use. The cylinders were sealed to the inner surface of culture dishes with Dow Corning high vacuum grease (Midland, Mich.). Preparation of cultures. The thoracolumbar paravertebral ganglia from 1 l-day chick embryos provided the material for all cultures. Both mixed ganglion cell cultures and highly purified neuronal and nonneuronal cultures were prepared according to the method of McCarthy and Partlow (1976a). Both the number of cells (200,000 cells per well) and the volume of medium (200 ~1) were constant in all experiments. Culture wells containing culture medium but lacking cells served as controls for all assays and were incubated and harvested along with the cell-containing cultures. Metal treatment. L-15+ culture medium containing various concentrations of mercuric chloride (Mallinckrodt, analytical reagent) was prepared by addition of a constant proportion (1:19) of a solution of the metallic
TOXICITY
OF MERCURIC
salt in sterile deionized water. Culture medium for untreated control cultures was diluted to the same extent. Addition of mercuric chloride to the L-IS+ culture medium to make final concentrations of 0.1 nM to 10 mM had no effect on either the pH of the medium or the amount of precipitated material in the medium as measured by light scattering. The effects of added chloride (0.2 nM-0.2 IIIM) are assumed to be insignificant as the basal level of this ion in the culture medium was approximately 145 mM. In all experiments, the cultures were incubated in L15+ medium lacking heavy metal for 24 hr in order to allow time for the cells to attach and begin growing (Fig. 1). The normal medium was then removed and replaced with L-15+ medium containing from 0.0001 to 100 pM mercuric chloride, except for the medium in untreated control cultures which never contained mercuric chloride. In all cases, the medium was changed every 24 hr. L-15+ medium containing mercuric chloride bathed the cultures for either 2 (Figs. 1A,C) or 3 days (Fig. la). In some experiments, the medium containing the metallic salt was removed after the cultures were exposed to the heavy metal for 2 days and the cells were allowed to recover (Fig. IC). A large volume of L-15+ medium (3 X 0.4 ml) was used in such recovery experiments in order to rinse away as much of the metallic salt as possible; fresh medium lacking mercuric chloride was then added. Incorporation
of
thymidine
and
leucine.
[methyl-
‘H]Thymidine (New England Nuclear, Boston, Mass., 6.7 Ci/mmol; 0.05 &i/culture) and t.-[U-%]leucine (New England Nuclear, 325 mCi/mmol; 0.05 rCi/culture) were added to all cultures either 24 (Fig. IA) or 48 hr (Figs. 1B,C) after the addition of culture medium containing mercuric chloride. The incubation was continued for 20 hr before harvesting (vide infra). Amounts of [‘Hlthymidine and [?Z]leucine incorporated into acid-precipitable macromolecules were determined by a paper disc method as described by Partlow and Larrabee (197 1). Since only the nonneuronal cells in these cultures can synthesize new DNA (McCarthy and Partlow, 1976a), thymidine incorporation serves as a marker for the nonneuronal cells. Culture harvesting procedure. Medium was removed from each culture well and attached cells were rinsed three times with 0.4 ml of Puck’s saline G to remove unincorporated labeled substrates. The cells were harvested by sonication in ice-cold distilled water (2 X 60 PI) and the combined sonicate was mixed thoroughly. A portion of each sonicate was mixed with an equal volume of 0.1% albumin and set aside for assay of acetyl cholinesterase. The remaining albumin-free sonicate was used for both protein analysis (Lowry et a/., 1951) and for measurement of incorporated thymidine and leucine. Both portions of the cell sonicate were stored at -80°C. Acetyl chofinesterase. Acetyl cholinesterase (EC
CHLORIDE
IN VITRO
2.55
3.1.1.7, acetylcholine acetylhydrolase) activity was used as a neuronal marker. Enzyme activity was determined by the method of Ellman et al. (1961) using acetylthiocholine (Sigma Chemical Co., St. Louis, MO.) as a substrate. Inhibitors of butyryl cholinesterase (EC 3.1 .1.8, acetylcholine acylhydrolase) were not needed as only acetyl cholinesterase is present in embryonic chick sympathetic ganglia (McCarthy and Partlow. 1976a). Statistics. All measurements are given as means & standard errors of the means. The number of observations (n) is given in parentheses. Differences between means were evaluated by the Student t test.
RESULTS Experimental
Design
Three different experimental designs were employed in this study. First, some mixed sympathetic cultures were incubated for 3 days and exposed to various concentrations of mercuric chloride during the final 2 days in vitro (Zday treatment; Fig. IA). Second, other cultures were incubated for 4 days and exposed to the metallic salt during the final 3 days (3-day treatment; Fig. 1B). Third, some cultures were incubated for 4 days but only exposed to mercuric chloride on the second and third days in vitro (recovery experiment; Fig. 1C). In all cases, thymidine and leucine incorporation were measured during the final 20 hr of incubation. This experimental design made it possible to compare the effects of both 2 and 3 days of exposure to mercuric chloride on sympathetic ganglion cell cultures, and to determine the recovery capacity of these cultures, Untreated
Mixed Ganglion Cell Cultures
The protein content of control cultures increased from 18 + 0.3 pg/culture (n = 6) after 72 hr to 27 f 0.6 pg/culture (n = 6) after 92 hr in vitro. Acetyl cholinesterase specific activity was unchanged in 72- and 92-hr control cultures [ 216 + 16 (n = 6) vs 194 -+ 10 (n = 8) nmol/min/pg protein, respectively]. Protein synthesis as measured by
256
SANGDEE, A.
Two-doy
PARTLOW,
Effects of Mercuric Chloride Sympathetic Cultures
Treatment labels added
24 hr t cells plated
I3
t metal added
Three-day
t’ met01 added
24 hr
t Cd Is plated
t metal odded
Recovery
Experiment
24 hr t cells plated
t cells harvested
Treatment
24 hr
C.
+2% j24hr
24hr
t metal added
t meiol added
24 hr t metal added
AND BUSH
24 hr
20 hr
t metal a lobels odded
24 hr
t cells harvested
20 hr
t metal removed
a
t cells harverted
labels added
FIG. 1. Design of the three types of experiments utilized in the present study. Sympathetic ganglion cell suspensions were plated at zero time. Culture medium was replaced every 24 hr in all experiments. Medium containing mercuric chloride (O-100 PM) was used during the 24-hr periods following the designation “metal added”; thus, mercuric chloride was present in the medium for either 48 (A, C) or 72 hr (B). Labeled thymidine and leucine were only present in the medium for 20 hr following the designation “labels added.” Cultures were harvested immediately after the labeling period in all experiments.
incorporation of labeled leucine per microgram of protein increased slightly during this period [72 f 2 (n = 6) vs 113 t- 2 (n = 8) dpm/pg protein, respectively]. DNA synthesis as measured by incorporation of labeled thymidine per microgram of protein decreased slightly between 72 and 92 hr in vitro [578 -t 7 (n = 6) vs 483 k 10 (n = 8) dpm/pg protein, respectively]. (This decrease in DNA synthesis is most likely due to slowing of glial proliferation as a result of contact inhibition.) Thus, both the neurons and glia in these control cultures were actively growing during this period in vitro.
on Mixed
Cellular morphology was unchanged in both the neurons and nonneuronal cells treated for 2 or 3 days in vitro at any concentration of mercuric chloride from 0.0001 to 100 PM. Similarly, Kasuya (1972) did not observe any morphological change in embryonic chick dorsal root ganglia incubated for 72 hr in concentrations of mercuric chloride as high as 10 PM. The protein content of mixed ganglion cell cultures treated with mercuric chloride ranged from 13 to 20 pg in 72-hr cultures and from 18 to 29 pg in 92-hr cultures. Thus, protein in treated cultures increased during this period by an average of 59 f 7% (n = 7), which is not significantly different from the increase observed for untreated control cultures (50 + 4%, vide supru). Variability in observed protein content probably resulted from differential loss during rinsing prior to harvest (see Methods). To account for this variability in protein content, all biochemical measures are expressed as specific activities (i.e., per pg of protein). Two days of exposure of mixed ganglion cell cultures to mercuric chloride as described in Fig. 1A stimulated all three biochemical measures at one or more concentrations (Fig. 2). Acetyl cholinesterase specific activity was increased by 30-35% at all concentrations between 0.01 and 100 FM. Thymidine incorporation per microgram of protein was stimulated by exposure to 1 or 10 PM mercuric chloride, but was slightly inhibited following exposure to a 100 PM concentration of the same heavy metal (-35%). Leucine incorporation per microgram of protein was elevated following exposure to any concentration of mercuric chloride between 1 RM and 1 PM but was significantly depressed (-18%) following exposure to 100 PM mercuric chloride. Following a 2-day exposure to mercuric chloride, partial recovery was observed after
TOXICITY
OF MERCURIC
a 20-hr incubation in medium lacking the heavy metal (Figs. 1C and 3). Incorporation of both thymidine and leucine returned to control levels, while acetyl cholinesterase specific activity remained elevated for at least 20 hr following exposure to 0.1 nM- 10 /1M mercuric chloride. Three days of exposure of mixed cultures to mercuric chloride as described in Fig. 1B generally inhibited all three biochemical measures (Fig. 4). Acetyl cholinesterase specific activity was slightly, but not significantly, decreased at concentrations I 10 PM and was significantly decreased (- 17%) following exposure to 100 @M mercuric chloride. Thymidine incorporation per microgram of protein was unchanged at any concentration of mercuric chloride I 10 NM but was significantly inhibited at 100 PM. Leucine incorporation per microgram of protein was inhibited at all concentrations of this heavy metal (-48% at 100 I.IM mercuric chloride).
CHLORIDE
IN
2.57
VITRO
350 $ 300 .c 250 s 200 = IS0 I00
RCETYL CHOLINESTERRSE
I
700 .:
600
2B
500-
. 400,---,~Ekb-i&, 300,
-
130 2
1 I t LEUCINE INCORPORRTION
:
110
5at1
-
4
Ii’
12
/ Ii'
I
1
16'
,E8
1, 10'
IO2
MERCURIC CHLORIDE :pM!
FIG. 3. Partial recovery by mixed ganglion cell cultures exposed to mercuric chloride for 2 dcys and then incubated for an additional 20 hr in culture medium lacking the heavy metal. All values represent means & SE (n = 4-8). Control values for untreated mixed cultures are shown as bars extending across the width of the graph. Means of treated cultures which differ significantly from control are marked with either a single asterisk (*) if p ZG0.05 or with two asterisks (**) if p
5 0.01.
Effects of Mercuric Chloride on Purified Neuronal and Nonneuronal Cultures I00 700 ,g 600 ti
500 400 300 130
1 1 1 1 1 LEUCINE INCORPORRTION
1
MERCURIC CHLORIDE WI
FIG. 2. Biochemical changes in mixed ganglion cell cultures exposed to various concentrations of mercuric chloride for 2 days. All values represent means 5 SE (n = 4-8). Control values for untreated mixed cultures are shown as bars extending across the width of the graph. Means of treated cultures which differ significantly from control are marked with either a single asterisk (*) if p 5 0.05 or with two asterisks (**) if p zz 0.01.
The protein content of purified neuronal cultures at all concentrations of mercuric chloride was approximately 5 rg, while that of the purified nonneuronal cultures ranged from 7 to 9 pg. Since the same number of cells were added to each type of culture and since neurons at this developmental stage can no longer divide (McCarthy and Parlow, 1976a), the difference in protein content in these 72-hr cultures was probably due to cell division in the nonneuronal cultures. The greater protein contents observed in 72-hr mixed cultures (13-20 pg, vide supru) likely resulted from marked stimulation of nonneuronal cell division due to contact with homologous neurons (McCarthy and Partlow, 1976h). Acetyl cholinesterase specific activities are shown in Table 1 for both mixed gan-
258
SANGDEE,
PARTLOW,
THYHIIlINE INCORPORRTION
138 t
LEUCINE
INCORPORRTION
I
HERCURIC CHLORIDE CpMl
FIG. 4. Biochemical changes in mixed ganglion cell cultures exposed to various concentrations of mercuric chloride for 3 days. All values represent means + SE (n = 4-8). Control values for untreated mixed cultures are shown as bars extending across the width of the graph. Means of treated cultures which differ significantly from control are marked with either a single asterisk (*) if p 5 0.05 or with two asterisks (**) if p 5 0.01.
glion cell cultures and purified neuronal cultures exposed for 2 days to mercuric chloride. Treatment with 0.01 pM mercuric chloride increased the activity of this neuronal enzyme by approximately the same extent in both mixed and neuronal cultures. In contrast, exposure of mixed cultures to a much higher concentration (100 PM) of mercuric chloride increased acetyl cholinesterase activity in mixed cultures but decreased the activity of this enzyme in purified neuronal cultures. Thymidine incorporation by dividing nonneuronal cells was measured in both mixed and purified nonneuronal cultures which had been exposed for 2 days to mercuric chloride (Table 2). Treatment with 1 I.IM mercuric chloride significantly stimulated [‘HIthymidine incorporation by both types of cultures (Table 2). Treatment with 100 PM mercuric chloride inhibited thymidine incorporation by both mixed and nonneuronal
AND BUSH
cultures to the same extent (35 and 3896, respectively). Leucine incorporation was determined both for mixed ganglion cell cultures and for purified cultures of neuronal and nonneuronal cells which had been exposed for 2 days to mercuric chloride (Table 3). Exposure to 100 PM mercuric chloride decreased leucine incorporation by both mixed cultures and purified nonneuronal cultures to approximately the same extent. Similarly, exposure to 1 PM mercuric chloride stimulated leucine incorporation by mixed and purified nonneuronal cultures to the same extent. In contrast, treatment with 0.01 PM mercuric chloride stimulated protein synthesis by the mixed cultures but had no effect on protein synthesis by the purified nonneuronal cultures. TABLE
1
RELATIVE ACETYL CHOLINESTERASE ACTIVITY IN BOTH HIGHLY PURIFIED NEURONALAND MIXED CULTURES OF SYMPATHETIC GANGLION CELLS WHICH WERE EXPOSED TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE FOR 2 DAYS”
Relative acetyl cholinesterase activity (% of control)
0.01 1.oo 100.00
Mixed cultures
Neuronal cultures
130 + 5 (4)b 130 f 7 (6)* 131 + 7 (6)b
141 f 8 (6)b 83 f 8 (5)*
a Both mixed and highly purified neuronal cultures were prepared from the sympathetic ganglia of 1l-day chick embryos, handled as described in Fig. lA, harvested by sonication, and assayed for acetyl cholinesterase activity as described under Methods. All values represent means + SE. Numbers of observations (n) are given in parentheses. The acetyl cholinesterase activities of mixed and neuronal control cultures were 216 + 16 (n = 6) and 280 + 10 (n = 5) nmol/min/~g of protein, respectively. Values for mixed cultures were previously shown in Fig. 2 and are repeated here for comparison. b Values which differ significantly from that of the matched control (p I 0.01).
TOXICITY
OF MERCURIC
Lack of a Direct Effect of Mercuric ride on Acetyl Cholinesterase
DISCUSSION Both the concentration of mercuric chloride and the time of exposure are important 2
RELATIVE AMOUNTS OF [‘HITHYMIDINE INCORPORATED BY BOTH HIGHLY PURIFIED AND MIXEDCULTURES OF SYMPATHETIC NEURONS AND NONNEURONAL CELLS WHICH WERE EX~~SED TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE FOR 2 DAYS“
Relative thymidine incorporation (% of control)
(PM)
Mixed cultures
Nonneuronal cultures
0.01 I .oo 100.00
107 * 4 (4) 120 f 5 (6)* 90 f 4 (6)
102 2 8 (6) 133 + 6 (6)* 79 + 6 (6)6
FWM
IN
’ Both mixed and highly purified cultures were prepared from the sympathetic ganglia of 11-day chick embryos, handled as described in Fig. IA, harvested, by sonication, and assayed for [‘Hlthymidine incorporated into acid-precipitable macromolecules as described under Methods. All values represent means + SE. Numbers of observations (n) are given in parentheses. The amounts of [3H]thymidine incorporated by mixed and nonneuronal control cultures were 578 + 7 (n = 6) and 1235 ? 56 (n = 6) dpm/pg of protein, respectively. Values for mixed cultures were previously shown in Fig. 2 and are repeated here for comparison. b.r Values which differ significantly from that of the control (p 5 0.01 or p s 0.05, respectively).
259
VITRO
TABLE
Chlo-
Dense mixed cultures of sympathetic ganglion cells (475,000 cells/culture) were harvested by sonication. Aliquots of the sonicate were incubated for 1 hr at room temperature with various concentrations of mercuric chloride (0.1 nM- 100 PM) and then assayed as described under Methods. Acetyl cholinesterase activity in the sonicate did not change as a result of exposure to mercuric chloride.
TABLE
CHLORIDE
RELATIVE
AMOUNTS
OF
3 [‘%]LEUCINE
INCORPO-
RATED BY BOTH HIGHLY PURIFIED AND MIXED CULTURES OF SYMPATHETIC NEURONS AND NONNEURONAL CELLS WHICH WERE EXPOSED TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE FOR 2 DAYS’ Relative leucine incorporation (% of control)
lHs’% (PM) 0.01 1.00 1oQ.w
MIxed cultures 138 * 7 (4jb I I I +_ 4 (6)’ 85 t 3 (6Jb
--.
cultures
NeUrOIlal cultures
95 k 6 (6) II6 k 5 (5)’ 97 k 6 (6)
108 2 8 (5) 99 i 7 (61 84 2 5 (5)
NOtlll~U~Oll~l
o Both mixed and highly purified cultures were prepared from the sympathetic ganglia of I l-day chick embryos, handled as described in Fig. IA. harvested by sonication, and assayed for [“Cjleucine incorporated into acid-precipitable macromolecules as described under Methods. All values represent means 2 SE. Numbers of observations (n) are gwen in parentheses. The amounts of [“C]leucine incorporated by mixed, nonncuronal, and neuronal control cultures were 72 + 2 (n = 6). 79 + 3 (n = ht. and 41 zt 2 (n = 5) dpm/pg of protein. respectively. Values for mixed cultures were previously shown in Fig. 2 and are repeated here for compartron. hr Values which differ stgnificantly from that of the control tp - 001 or p 5 0.05. respectively).
in determining the effects of this metallic salt on the biochemistry of sympathetic neurons and nonneuronal cells. Thus, a given biochemical variable might be stimulated by a low concentration of mercuric chloride but inhibited by a higher concentration (Fig. 2). In addition, a concentration which causes stimulation after exposure for 2 days might cause inhibition after exposure for 3 days (cf. Figs. 2 and 4). Because of the complex nature of these effects, each biochemical variable will now be discussed separately. Acetyl Cholinesterase
Activity
Acetyl cholinesterase activity was generally stimulated by exposure of cultures to mercuric chloride for 2 days. Increase acetyl cholinesterase activity in the mixed cultures appears to have resulted from increased synthesis of this enzyme since mercuric chloride had no direct effect on the enzyme in sonicates of mixed sympathetic ganglion cell cul-
260
SANGDEE,
PARTLOW,
tures. This increase in activity of neuronal acetyl cholinesterase occurred both in the presence and absence of nonneuronal cells (Fig. 2 and Table 1) and persisted during a subsequent 20-hr incubation in the absence of the heavy metal (Fig. 3). To our knowledge, the effects of mercuric chloride on acetyl cholinesterase activity have not previously been investigated. However, 1 pM methyl mercury has been reported to either have no effect or to slightly decrease histochemically determined levels of acetyl cholinesterase in primary cultures of nervous tissues (Hosli and H&Ii, 1974). In addition, Christensen (1975) reported that 5 nM methyl mercury decreased the level of acetyl cholinesterase by 36% in chronically exposed baby brook trout.
AND
BUSH
toma cells. Marked stimulation of thymidine incorporation only occurred in a narrow window of cell concentrations ( 1200- 1700 cells/ mm2); our initial cell concentration was approximately 1500 cells/mm2 in all cases. Nakada and Imura (1980) further reported that 50 FM mercuric chloride totally inhibited thymidine incorporation in both cell lines. In contrast, they found that methyl mercury either had no effect on or inhibited thymidine incorporation. Using a variety of different cell lines, earlier investigators reported that mercuric chloride either did not affect DNA synthesis in vitro or inhibited it (Schuster and Hare, 1970, Nakazawa et al., 1975; Fisher, 1976; Potter and Matrone, 1977). Protein Synthesis
DNA Synthesis DNA synthesis by the nonneuronal cells was stimulated by exposure of mixed cultures for 2 days to 1 or 10 PM mercuric chloride but was inhibited by treatment with 100 pM mercuric chloride (Fig. 2). Both effects were also observed in purified cultures of nonneuronal cells and can therefore be explained by a direct action of the metallic salt on the nonneuronal cells (Table 2). Both the stimulatory and inhibitory effects observed after 2 days of treatment disappear following removal of the metallic salt from the culture medium (Fig. 3). Exposure of mixed cultures to mercuric chloride for 3 days inhibited thymidine incorporation at a concentration of 100 pM but had no effect at concentrations of 10 PM or less. Our observation that exposure to mercuric chloride can either stimulate or inhibit thymidine incorporation is supported by a recent study by Nakada and Imura (1980). These investigators reported that l-20 PM mercuric chloride stimulated thymidine incorporation by mouse glioma cells and that l-5 PM mercuric chloride stimulated thymidine incorporation by mouse neuroblas-
Since both neurons and nonneuronal cells incorporate leucine into protein, results obtained with mixed cultures reflect the combined effects of mercuric chloride on both cell types. Leucine incorporation was generally stimulated by exposure for 2 days to mercuric chloride at concentrations 5 10 PM, but was inhibited by exposure to a concentration of 100 pM. These effects were reversible since they were not found in cultures incubated for an additional 20 hr in the absence of the metallic salt (Fig. 3). After 3 days of exposure, leucine incorporation was inhibited at almost all concentrations of mercuric chloride. The stimulatory effects of 1 pM mercuric chloride on mixed cultures are adequately explained by a direct action on the nonneuronal cells and the inhibitory effects observed at 100 pM could be accounted for by a direct action on the neurons (Table 3). However, the stimulatory effects of extremely low concentrations (0.01 PM) of mercuric chloride on leucine incorporation by mixed cultures cannot be explained by direct effects on either cell type (Table 3). It is conceivable that low concentrations of
TOXICITY
OF MERCURIC
mercuric chloride might alter some aspect of the dynamic interactive processes occurring between the neurons and nonneuronal cells (e.g., neuronal-glial interactions). To the authors knowledge, the effects of mercuric chloride on protein synthesis in vitro have not previously been investigated. However, methyl mercury has been reported to either have no effect on or to decrease protein synthesis in a wide variety of in vivo and in vitro systems (Yoshino et al., 1966; Cavanagh and Chen, 1971; Syversen, 1977; Verity et al., 1977; Gruenwedel and Cruikshank, 1979). In contrast, Brubaker et al. ( 1973) reported that methyl mercury increased incorporation of labeled amino acids into protein in vivo. Relative Sensit@ity of Neurons neuronal Cells
and Non-
Neurons appear to be far more sensitive to mercuric chloride than nonneuronal cells. Thus, concentrations as low as 0.0001 PM mercuric chloride significantly increased activity of the neuronal enzyme acetyl cholinesterase (Fig. 3). In contrast, concentrations of mercuric chloride below 1 PM had no effect on DNA synthesis by the glial cells (Figs. 2-4 and Table 2). These results suggest that neurons rather than glia might be the primary site of action of mercuric chloride in the nervous system. Sobkowicz and Murray similarly concluded that mercury exerts its primary action on the neurons rather than the glial cells in primary cultures (Murray, 197 1). In addition, Syversen ( 1977) concluded that neurons are more sensitive than glial cells to methyl mercury by examining protein synthesis in vivo. Toxicological
Significance
CHLORIDE
IN VITRO
261
from lethal heavy-metal intoxication have been reported at 22-50 (Hilmy et al., 1976) and 0.5-124 IAM (Takeuchi et al., 1962). It seems likely that longer-term exposure to such mercury concentrations would produce inhibitory effects. The toxicological significance of the stimulatory effects reported herein as a result of acute exposure to very low concentrations of mercuric chloride is uncertain. Acetyl cholinesterase activity and protein synthesis were both stimulated by concentrations of mercuric chloride as low as 0.1 or 1 BM, respectively (Figs. 2 and 3). Such stimulatory effects would not be expected to persist in mercury-intoxicated nervous tissues because these effects are transient and disappear with continued exposure (compare data for 2- and 3-days exposures in Figs. 2 and 4). In addition, concentrations which stimulate these biochemical measures are generally lower than those found in the brains of normal individuals at autopsy [ 30 IIM (Hilmy et al., 1976), 1.2 pM(Olszewski et al., 1974) or 2.5 PM (Takeuchi et al., 1962)]. ACKNOWLEDGMENTS Supported by a P.O.E. International Peace Scholarship to P.S., U.S. Public Health Service Training Grant GM-00153, and Grant NS-12812 from the National Institute of Neurological Diseases and Stroke.
REFERENCES BRUBAKER, P. E., KLEIN, R., HERMAN, S. P., LUCIER, G. W., ALEXANDER, L. T., AND LONG, M. D. (1973). DNA, RNA, and protein synthesis in brain, liver, and kidneys of asymptomatic methylmercury treated rats. Exp.
Mol.
Pathol.
18, 262-280.
CAVANAGH, J. B., ANDCHEN, F. C. K. (1971). Amino acid incorporation in protein during the “Silent Phase” before organo-mercury and p-bromophenylacetylurea neuropathy in the rat. Acra Neuropathol. 19, 216-224.
All inhibitory effects reported in this paper occurred following a 2 to 3-day exposure to 100 PM mercuric chloride. Concentrations of mercury in brains of patients suffering
CHANG, L. W. (1977). Neurotoxic effects of mercury. Environ.
Res.
14, 329-373.
CHANG, L. W., DESNOYERS, P. A.. AND HARTMANN, H. A. (1972). Quantitative cytochemical studies of RNA in experimental mercury poisoning. I. Changes
262
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in RNA content. J. Neuropathol.
PARTLOW,
Exp. Neurol. 31,
489-501.
CHANG, L. W., AND HARTMANN, H. A. (1972a). Ultrastructural studies of the nervous system after mercury intoxication. I. Pathological changes in the nerve cell bodies. Acta Neuropathol. 20, 122-138. CHANG, L. W., AND HARTMANN, H. A. (1972b). Electron microscopic histochemical study on the localization and distribution of mercury in the nervous system after mercury intoxication. Exp. Neuroi. 35, 122137.
CHANC, L. W., WARE, R. A., AND DESNOYERS, P. A. (1973). A histochemical study on some enzyme changes in the kidney, liver and brain after chronic mercury intoxication in the rat. Food Cosmet. Toxicol.
11, 283-286.
CHARBONNEAU. S. M., MUNRO, I. C., NERA, E. A., ARMSTRONG, F. A. J., WILL&S, R. F., BRYCE, F., AND NELSON, R. F. (1976). Chronic toxicity of methylmercury in the adult cat. Interim report. Taxicology 5, 337-349. CHRISTENSEN, G. M. (1975). Biochemical effects of methylmercuric chloride, cadmium chloride, and lead nitrate on embryos and alevins of the brook trout, Salvelinus fontinalis. Toxicof. Appf. Pharmacol. 32, 191-197. ELLMAN, G. L., COURTNEY, K. D., ANDRES, V., AND FEATHERSTONE, R. M. (1961). A new and rapid colorimetric determination of acetyl cholinesterase activity. Biochem. Pharmacol. 7, 88-95. FISHER, A. B. (1976). Heavy metal toxicity in mammalian cell cultures Zentralbf. Eakteriol. Parasitenkd. Infektionskr. Hyg. Abt. I: Orig. Reihe B. 162, 77-84.
GRUENWEDEL, D. W., AND~RUIKSHANK, M. K. (1979). Effect of methyl mercury (11) on the synthesis of deoxyribonucleic acid, ribonucleic acid, and protein in HeLa S3 cells. Biochem. Pharmacol. 28, 651-655. HILMY, M. I., RAHIM,~. A.,ANDABBAS,A. H. (1976). Normal and lethal mercury levels in human beings. Toxicology 6, 155-l 59. H&L], E., AND H%LI, L. (1974). The effects of methyl mercury on morphological and histochemical properties of human and rat spinal cord and cerebellum in tissue culture. Experientia 30, 1300-l 304. KASUYA, M. (1972). Effects of inorganic, aryl, alkyl, and other mercury compounds on the outgrowth of cells and fibers from dorsal root ganglia in tissue culture. Toxicol. Applied Pharmacol. 23, 136-146. LETOURNEAU, P. C. (1975). Possible roles of cell-tosubstratum adhesion in neuronal morphogenesis. Develop. Biol. 44, 77-91. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND
AND
BUSH
RANDALL, R. J. (1951). Protein measurement with Folin-Phenol reagent. J. Biol. Chem. 193, 265-275. MCCARTHY, K. D., AND PARTLOW, L. M. (1976a). Preparation of pure neuronal and non-neuronal cultures from embryonic chick sympathetic ganglia: A new method based on both differential cell adhesiveness and formation of homotypic neuronal aggregates. Brain Res. 114, 391-414. MCCARTHY, K. D., AND PARTLOW, L. M. (1976b). Neuronal stimulation of 3H-thymidine incorporation by primary cultures of highly purified non-neuronal cells. Brain Res. 114, 415-426. MURRAY, M. R. (1971). Nervous tissues isolated in culture. In Handbook of Neurochemistry (A. Lajtha, ed.), Vol. SA, pp, 373-436. Plenum, New York. NAKADA, S., AND IMURA, N. (1980). Stimulation of DNA synthesis and pyrimidine deoxyribonucleoside transport systems in mouse glioma and mouse neuroblastoma cells by inorganic mercury.Toxicoi. Applied Pharmacol. 53, 24-28. NAKAZAWA, N., MAKINO, F., AND OKADA, S. (1975). Acute effects of mercuric compounds on cultured mammalian cells. Biochem. Pharmacoi. 24,489-493. OLSZEWSKI, W. A., PILLAY, K. K. S., GLOMSKI, C. A., AND BRODY, H. (1974). Mercury in the human brain. Acta Neurol. Sand. 50, 581-588. PARTLOW, L. M., AND LARRABEE, M. G. (1971). Effects of nerve-growth factor, embryo age and metabolic inhibitors on growth of fibers and on synthesis of ribonucleic acid and protein in embryonic sympathetic ganglia. J. Neurochem. 18, 2102-2118. POITER, S. D., AND MATRONE, G. (1977). A tissue culture model for mercury-selenium interactions. Toxicol. Appl. Pharmacol. 40, 201-215. SCHUSTER,G. S., AND HARE, J. D. (1970). Some characteristics of an inhibitable thymidine uptake system in mammalian cells. Exp. Cell Res. 59, 163-167. SYVERSEN, T. L. M. (1977). Effects of methyl mercury on in vivo protein synthesis in isolated cerebral and cerebellar neurons. Neuropathol. Appl. Neurobiol. 3, 225-236.
TAKEUCHI, T., MORIKAWA, N., MATSUMOTO, H., AND SHIRAISHI, Y. (1962). A pathological study of Minamata disease in Japan. Acta Neurol. Pathol. 2, 4057.
VERITY, M. A., BROWN, W. J., CHEUNG, M., AND CZER, G. (1977). Methyl-mercury inhibition of synaptosome and brain slice protein synthesis: In vivo and in vitro studies. J. Neurochem. 29, 673-679. YOSHINO, Y., MOZAI, T., AND NAKAO, K. (1966). Biochemical changes in the brain in rats poisoned with an alkylmercury compound, with special reference to the inhibition of protein synthesis in brain cortex slices. J. Neurochem. 13, 1223-1230.
AND
APPLIED
PHARMACOLOGY
60, 253-262 (1981)
Toxicity of Mercuric Chloride in Cultures of Neurons and Nonneuronal Cells Derived from Embryonic Chick Sympathetic Ganglia PUCKPRINK~ANGDEE,' Department
of Pharmacology,
Received
L. M. PARTLOW,' AND L. G. BUSH
University
of Utah
College
January
8, 1981;
accepted
of Medicine,
March
Salt
Lake
City,
C/t&
84132
24. 1981
Toxicity of Mercuric Chloride in Cultures of Neurons and Nonneuronal Cells Derived from Embryonic Chick Sympathetic Ganglia. SANGDEE, P., PARTLOW, L. M., AND BUSH, L. G. (I 98 1). Toxicol. Appl. Pharmocol. 60,253-262. The effects of mercuric chloride were studied in vitro using both mixed and highly purified cultures of neurons and nonneuronal (glial) cells. Cultures were prepared from the sympathetic ganglia of 1 l-day chick embryos and treated with mercuric chloride (0.1 nM to 100 fiM) for either 2 or 3 days. Acetyl cholinesterase specific activity, [3H]thymidine incorporation per microgram of protein, and [Wlleucine incorporation per microgram of protein were quantified, and the following results were obtained. First, all three biochemical measures either were stimulated or were unaffected by exposure of mixed ganglion cell cultures to 510 pM mercuric chloride for 2 days. In contrast, exposure to 100 PM mercuric chloride for 2 days depressed incorporation of both leucine and thymidine, but stimulated acetyl cholinesterase specific activity. Second, incorporation of both leucine and thymidine rapidly returned to control levels following removal of the mercuric chloride from the culture medium after 2 days of exposure. In contrast, acetyl cholinesterase specific activity remained elevated. Third, all biochemical measures were either inhibited or unaffected by a 3-day exposure of mixed ganglion cell cultures to any concentration of mercuric chloride. Fourth, sympathetic neurons appeared to be much more sensitive to mercuric chloride than homologous nonneuronal cells. Fifth, most of the stimulatory and inhibitory effects of mercuric chloride appeared to result from direct action of the heavy metal on either the neurons or nonneuronal ceils.
Mercury is widely recognized as an extremely hazardous environmental pollutant. Mercury poisoning can result in a wide variety of neurological disturbances including impairment of vision and other sensory inputs, speech and hearing defects, ataxia, and mental disturbances (Takeuchi et al., 1962). Common neuropathological changes resulting from mercury poisoning include neuronal degeneration and glial cell proliferation (Chang and Hartmann, 1972b; ’ Present address: Department of Biopharmaceutical Sciences, Chiang-Mai University, Chiang-Mai, Thailand.
*To whom all correspondence and requests for reprints should be addressed.
Charbonneau et al., 1976; Chang, 1977). A wide variety of biochemical changes have also been described in mercury-intoxicated animals. These include alterations in (a) the content and base composition of RNA in neurons (Chang et al., 1972; Chang and Hartmann, 1972a; Chang, 1977), (b) the rate of protein synthesis (Yoshino et al., 1966; Cavanagh and Chen, 197 1; Brubaker et al., 1973; Syversen, 1977; Verity et al., 1977), and (c) the specific activity of several brain enzymes (Chang et al., 1973). Investigators using in vitro systems have also described a number of mercury-induced biochemical alterations. RNA synthesis is inhibited by both methyl mercury and mer253
0041-008X/81/110253-10$02.00/0 Copyright 0 1981 by Academic Press. Inc All rights of reprcduclion in any form reserved
254
SANGDEE,
PARTLOW,
curie chloride in two cell lines (Nakazawa et al., 1975; Gruenwedel and Cruikshank, 1979). Protein syntesis is inhibited by methyl mercury in HeLa cells (Gruenwedel and Cruikshank, 1979). Activities of several specific enzymes have also been studied in vitro. Methyl mercury inhibits brain succinic dehydrogenase but has little effect on acetyl cholinesterase (Hiisli and Hosli, 1974). Finally, DNA synthesis is inhibited by methyl mercury in a variety of cell lines (Nakazawa et al., 1975; Gruenwedel and Cruikshank, 1979; Nakada and Imura, 1980). In contrast, DNA synthesis has been reported to be both stimulated (Nakada and Imura, 1980) and inhibited (Schuster and Hare, 1970; Nakazawa et al., 1975; Nakada and Imura, 1980) by mercuric chloride. The in vitro system utilized in the present study allows assessment of the effects of heavy metals on essentially pure (298%) primary cultures of separated neurons and nonneuronal (glial) cells derived from embryonic chick sympathetic ganglia (McCarthy and Partlow, 1976a). The nonneuronal cells in these cultures appear to be either satellite cells or Schwann cells, both of which are glial elements, while the neurons are thought to be postganglionic sympathetic neurons. Since mixed cultures of these neurons and nonneuronal cells can also be prepared, it is possible to compare the effects of heavy metals on the biochemistry and morphology of ( 1) neuronal, (2) nonneuronal, and (3) mixed cultures. The studies presented in this paper demonstrate that the responses of neurons and nonneuronal cells to various concentrations of mercuric chloride are quite complex. By appropriate selection of concentration and time of exposure, mercuric chloride could be shown to either stimulate, inhibit, or have no effect on any of three biochemical measures (acetyl cholinesterase specific activity, leucine incorporation per ccg of protein, and thymidine incorporation per pg of protein). Treatment with mercuric chloride for 2 days was generally stimulatory, while treatment
AND BUSH
for 3 days was generally inhibitory. The neurons were much more sensitive to mercuric chloride than the nonneuronal cells. In most cases, the effects of mercuric chloride observed in mixed cultures containing both neurons and nonneuronal cells were ascribable to a direct action on one of these cell types. METHODS Culture medium and salt solutions. L- 15 (Leibovitz) Medium (North American Biologicals, Miami, Fla.) was supplemented (L-15+) so that the final solution contained 10% fetal calf serum (Irvine Scientific, Fountain Valley, Calif.), 0.5% glucose (analytical reagent, Mallinckrodt, St. Louis, MO.), 100 IU/ml penicillin, 100 pg/ml streptomycin (Grand Island, Biological Co., Grand Island, N.Y.), and 15 rig/ml nerve growth factor (Burroughs Welcome Co., Research Triangle Park, N.C.). This medium was then adjusted to 300 mOSM and a pH of 7.15 at 37°C. Puck’s saline G without phenol red (Irvine Scientific) was supplemented with 5 mg/ml glucose. Culture surface. Falcon plastic dishes, 35 mm in diameter (Oxnard, Calif.) were used in all experiments. Both mixed cultures and highly purified nonneuronal cultures were grown directly on the polystyrene surface of these dishes, while highly purified neuronal cultures were grown on surfaces coated with polylysine (Nutritional Biochemical Corp., Irvine, Calif.) according to the procedure of Letourneau (1975). Culture wells. Pyrex tubing with an inner diameter of 13 mm was cut into glass cylinders approximately 5 mm tall. All glass cylinders were boiled in 0.1 M potassium hydroxide, rinsed extensively with water, and autoclaved before use. The cylinders were sealed to the inner surface of culture dishes with Dow Corning high vacuum grease (Midland, Mich.). Preparation of cultures. The thoracolumbar paravertebral ganglia from 1 l-day chick embryos provided the material for all cultures. Both mixed ganglion cell cultures and highly purified neuronal and nonneuronal cultures were prepared according to the method of McCarthy and Partlow (1976a). Both the number of cells (200,000 cells per well) and the volume of medium (200 ~1) were constant in all experiments. Culture wells containing culture medium but lacking cells served as controls for all assays and were incubated and harvested along with the cell-containing cultures. Metal treatment. L-15+ culture medium containing various concentrations of mercuric chloride (Mallinckrodt, analytical reagent) was prepared by addition of a constant proportion (1:19) of a solution of the metallic
TOXICITY
OF MERCURIC
salt in sterile deionized water. Culture medium for untreated control cultures was diluted to the same extent. Addition of mercuric chloride to the L-IS+ culture medium to make final concentrations of 0.1 nM to 10 mM had no effect on either the pH of the medium or the amount of precipitated material in the medium as measured by light scattering. The effects of added chloride (0.2 nM-0.2 IIIM) are assumed to be insignificant as the basal level of this ion in the culture medium was approximately 145 mM. In all experiments, the cultures were incubated in L15+ medium lacking heavy metal for 24 hr in order to allow time for the cells to attach and begin growing (Fig. 1). The normal medium was then removed and replaced with L-15+ medium containing from 0.0001 to 100 pM mercuric chloride, except for the medium in untreated control cultures which never contained mercuric chloride. In all cases, the medium was changed every 24 hr. L-15+ medium containing mercuric chloride bathed the cultures for either 2 (Figs. 1A,C) or 3 days (Fig. la). In some experiments, the medium containing the metallic salt was removed after the cultures were exposed to the heavy metal for 2 days and the cells were allowed to recover (Fig. IC). A large volume of L-15+ medium (3 X 0.4 ml) was used in such recovery experiments in order to rinse away as much of the metallic salt as possible; fresh medium lacking mercuric chloride was then added. Incorporation
of
thymidine
and
leucine.
[methyl-
‘H]Thymidine (New England Nuclear, Boston, Mass., 6.7 Ci/mmol; 0.05 &i/culture) and t.-[U-%]leucine (New England Nuclear, 325 mCi/mmol; 0.05 rCi/culture) were added to all cultures either 24 (Fig. IA) or 48 hr (Figs. 1B,C) after the addition of culture medium containing mercuric chloride. The incubation was continued for 20 hr before harvesting (vide infra). Amounts of [‘Hlthymidine and [?Z]leucine incorporated into acid-precipitable macromolecules were determined by a paper disc method as described by Partlow and Larrabee (197 1). Since only the nonneuronal cells in these cultures can synthesize new DNA (McCarthy and Partlow, 1976a), thymidine incorporation serves as a marker for the nonneuronal cells. Culture harvesting procedure. Medium was removed from each culture well and attached cells were rinsed three times with 0.4 ml of Puck’s saline G to remove unincorporated labeled substrates. The cells were harvested by sonication in ice-cold distilled water (2 X 60 PI) and the combined sonicate was mixed thoroughly. A portion of each sonicate was mixed with an equal volume of 0.1% albumin and set aside for assay of acetyl cholinesterase. The remaining albumin-free sonicate was used for both protein analysis (Lowry et a/., 1951) and for measurement of incorporated thymidine and leucine. Both portions of the cell sonicate were stored at -80°C. Acetyl chofinesterase. Acetyl cholinesterase (EC
CHLORIDE
IN VITRO
2.55
3.1.1.7, acetylcholine acetylhydrolase) activity was used as a neuronal marker. Enzyme activity was determined by the method of Ellman et al. (1961) using acetylthiocholine (Sigma Chemical Co., St. Louis, MO.) as a substrate. Inhibitors of butyryl cholinesterase (EC 3.1 .1.8, acetylcholine acylhydrolase) were not needed as only acetyl cholinesterase is present in embryonic chick sympathetic ganglia (McCarthy and Partlow. 1976a). Statistics. All measurements are given as means & standard errors of the means. The number of observations (n) is given in parentheses. Differences between means were evaluated by the Student t test.
RESULTS Experimental
Design
Three different experimental designs were employed in this study. First, some mixed sympathetic cultures were incubated for 3 days and exposed to various concentrations of mercuric chloride during the final 2 days in vitro (Zday treatment; Fig. IA). Second, other cultures were incubated for 4 days and exposed to the metallic salt during the final 3 days (3-day treatment; Fig. 1B). Third, some cultures were incubated for 4 days but only exposed to mercuric chloride on the second and third days in vitro (recovery experiment; Fig. 1C). In all cases, thymidine and leucine incorporation were measured during the final 20 hr of incubation. This experimental design made it possible to compare the effects of both 2 and 3 days of exposure to mercuric chloride on sympathetic ganglion cell cultures, and to determine the recovery capacity of these cultures, Untreated
Mixed Ganglion Cell Cultures
The protein content of control cultures increased from 18 + 0.3 pg/culture (n = 6) after 72 hr to 27 f 0.6 pg/culture (n = 6) after 92 hr in vitro. Acetyl cholinesterase specific activity was unchanged in 72- and 92-hr control cultures [ 216 + 16 (n = 6) vs 194 -+ 10 (n = 8) nmol/min/pg protein, respectively]. Protein synthesis as measured by
256
SANGDEE, A.
Two-doy
PARTLOW,
Effects of Mercuric Chloride Sympathetic Cultures
Treatment labels added
24 hr t cells plated
I3
t metal added
Three-day
t’ met01 added
24 hr
t Cd Is plated
t metal odded
Recovery
Experiment
24 hr t cells plated
t cells harvested
Treatment
24 hr
C.
+2% j24hr
24hr
t metal added
t meiol added
24 hr t metal added
AND BUSH
24 hr
20 hr
t metal a lobels odded
24 hr
t cells harvested
20 hr
t metal removed
a
t cells harverted
labels added
FIG. 1. Design of the three types of experiments utilized in the present study. Sympathetic ganglion cell suspensions were plated at zero time. Culture medium was replaced every 24 hr in all experiments. Medium containing mercuric chloride (O-100 PM) was used during the 24-hr periods following the designation “metal added”; thus, mercuric chloride was present in the medium for either 48 (A, C) or 72 hr (B). Labeled thymidine and leucine were only present in the medium for 20 hr following the designation “labels added.” Cultures were harvested immediately after the labeling period in all experiments.
incorporation of labeled leucine per microgram of protein increased slightly during this period [72 f 2 (n = 6) vs 113 t- 2 (n = 8) dpm/pg protein, respectively]. DNA synthesis as measured by incorporation of labeled thymidine per microgram of protein decreased slightly between 72 and 92 hr in vitro [578 -t 7 (n = 6) vs 483 k 10 (n = 8) dpm/pg protein, respectively]. (This decrease in DNA synthesis is most likely due to slowing of glial proliferation as a result of contact inhibition.) Thus, both the neurons and glia in these control cultures were actively growing during this period in vitro.
on Mixed
Cellular morphology was unchanged in both the neurons and nonneuronal cells treated for 2 or 3 days in vitro at any concentration of mercuric chloride from 0.0001 to 100 PM. Similarly, Kasuya (1972) did not observe any morphological change in embryonic chick dorsal root ganglia incubated for 72 hr in concentrations of mercuric chloride as high as 10 PM. The protein content of mixed ganglion cell cultures treated with mercuric chloride ranged from 13 to 20 pg in 72-hr cultures and from 18 to 29 pg in 92-hr cultures. Thus, protein in treated cultures increased during this period by an average of 59 f 7% (n = 7), which is not significantly different from the increase observed for untreated control cultures (50 + 4%, vide supru). Variability in observed protein content probably resulted from differential loss during rinsing prior to harvest (see Methods). To account for this variability in protein content, all biochemical measures are expressed as specific activities (i.e., per pg of protein). Two days of exposure of mixed ganglion cell cultures to mercuric chloride as described in Fig. 1A stimulated all three biochemical measures at one or more concentrations (Fig. 2). Acetyl cholinesterase specific activity was increased by 30-35% at all concentrations between 0.01 and 100 FM. Thymidine incorporation per microgram of protein was stimulated by exposure to 1 or 10 PM mercuric chloride, but was slightly inhibited following exposure to a 100 PM concentration of the same heavy metal (-35%). Leucine incorporation per microgram of protein was elevated following exposure to any concentration of mercuric chloride between 1 RM and 1 PM but was significantly depressed (-18%) following exposure to 100 PM mercuric chloride. Following a 2-day exposure to mercuric chloride, partial recovery was observed after
TOXICITY
OF MERCURIC
a 20-hr incubation in medium lacking the heavy metal (Figs. 1C and 3). Incorporation of both thymidine and leucine returned to control levels, while acetyl cholinesterase specific activity remained elevated for at least 20 hr following exposure to 0.1 nM- 10 /1M mercuric chloride. Three days of exposure of mixed cultures to mercuric chloride as described in Fig. 1B generally inhibited all three biochemical measures (Fig. 4). Acetyl cholinesterase specific activity was slightly, but not significantly, decreased at concentrations I 10 PM and was significantly decreased (- 17%) following exposure to 100 @M mercuric chloride. Thymidine incorporation per microgram of protein was unchanged at any concentration of mercuric chloride I 10 NM but was significantly inhibited at 100 PM. Leucine incorporation per microgram of protein was inhibited at all concentrations of this heavy metal (-48% at 100 I.IM mercuric chloride).
CHLORIDE
IN
2.57
VITRO
350 $ 300 .c 250 s 200 = IS0 I00
RCETYL CHOLINESTERRSE
I
700 .:
600
2B
500-
. 400,---,~Ekb-i&, 300,
-
130 2
1 I t LEUCINE INCORPORRTION
:
110
5at1
-
4
Ii’
12
/ Ii'
I
1
16'
,E8
1, 10'
IO2
MERCURIC CHLORIDE :pM!
FIG. 3. Partial recovery by mixed ganglion cell cultures exposed to mercuric chloride for 2 dcys and then incubated for an additional 20 hr in culture medium lacking the heavy metal. All values represent means & SE (n = 4-8). Control values for untreated mixed cultures are shown as bars extending across the width of the graph. Means of treated cultures which differ significantly from control are marked with either a single asterisk (*) if p ZG0.05 or with two asterisks (**) if p
5 0.01.
Effects of Mercuric Chloride on Purified Neuronal and Nonneuronal Cultures I00 700 ,g 600 ti
500 400 300 130
1 1 1 1 1 LEUCINE INCORPORRTION
1
MERCURIC CHLORIDE WI
FIG. 2. Biochemical changes in mixed ganglion cell cultures exposed to various concentrations of mercuric chloride for 2 days. All values represent means 5 SE (n = 4-8). Control values for untreated mixed cultures are shown as bars extending across the width of the graph. Means of treated cultures which differ significantly from control are marked with either a single asterisk (*) if p 5 0.05 or with two asterisks (**) if p zz 0.01.
The protein content of purified neuronal cultures at all concentrations of mercuric chloride was approximately 5 rg, while that of the purified nonneuronal cultures ranged from 7 to 9 pg. Since the same number of cells were added to each type of culture and since neurons at this developmental stage can no longer divide (McCarthy and Parlow, 1976a), the difference in protein content in these 72-hr cultures was probably due to cell division in the nonneuronal cultures. The greater protein contents observed in 72-hr mixed cultures (13-20 pg, vide supru) likely resulted from marked stimulation of nonneuronal cell division due to contact with homologous neurons (McCarthy and Partlow, 1976h). Acetyl cholinesterase specific activities are shown in Table 1 for both mixed gan-
258
SANGDEE,
PARTLOW,
THYHIIlINE INCORPORRTION
138 t
LEUCINE
INCORPORRTION
I
HERCURIC CHLORIDE CpMl
FIG. 4. Biochemical changes in mixed ganglion cell cultures exposed to various concentrations of mercuric chloride for 3 days. All values represent means + SE (n = 4-8). Control values for untreated mixed cultures are shown as bars extending across the width of the graph. Means of treated cultures which differ significantly from control are marked with either a single asterisk (*) if p 5 0.05 or with two asterisks (**) if p 5 0.01.
glion cell cultures and purified neuronal cultures exposed for 2 days to mercuric chloride. Treatment with 0.01 pM mercuric chloride increased the activity of this neuronal enzyme by approximately the same extent in both mixed and neuronal cultures. In contrast, exposure of mixed cultures to a much higher concentration (100 PM) of mercuric chloride increased acetyl cholinesterase activity in mixed cultures but decreased the activity of this enzyme in purified neuronal cultures. Thymidine incorporation by dividing nonneuronal cells was measured in both mixed and purified nonneuronal cultures which had been exposed for 2 days to mercuric chloride (Table 2). Treatment with 1 I.IM mercuric chloride significantly stimulated [‘HIthymidine incorporation by both types of cultures (Table 2). Treatment with 100 PM mercuric chloride inhibited thymidine incorporation by both mixed and nonneuronal
AND BUSH
cultures to the same extent (35 and 3896, respectively). Leucine incorporation was determined both for mixed ganglion cell cultures and for purified cultures of neuronal and nonneuronal cells which had been exposed for 2 days to mercuric chloride (Table 3). Exposure to 100 PM mercuric chloride decreased leucine incorporation by both mixed cultures and purified nonneuronal cultures to approximately the same extent. Similarly, exposure to 1 PM mercuric chloride stimulated leucine incorporation by mixed and purified nonneuronal cultures to the same extent. In contrast, treatment with 0.01 PM mercuric chloride stimulated protein synthesis by the mixed cultures but had no effect on protein synthesis by the purified nonneuronal cultures. TABLE
1
RELATIVE ACETYL CHOLINESTERASE ACTIVITY IN BOTH HIGHLY PURIFIED NEURONALAND MIXED CULTURES OF SYMPATHETIC GANGLION CELLS WHICH WERE EXPOSED TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE FOR 2 DAYS”
Relative acetyl cholinesterase activity (% of control)
0.01 1.oo 100.00
Mixed cultures
Neuronal cultures
130 + 5 (4)b 130 f 7 (6)* 131 + 7 (6)b
141 f 8 (6)b 83 f 8 (5)*
a Both mixed and highly purified neuronal cultures were prepared from the sympathetic ganglia of 1l-day chick embryos, handled as described in Fig. lA, harvested by sonication, and assayed for acetyl cholinesterase activity as described under Methods. All values represent means + SE. Numbers of observations (n) are given in parentheses. The acetyl cholinesterase activities of mixed and neuronal control cultures were 216 + 16 (n = 6) and 280 + 10 (n = 5) nmol/min/~g of protein, respectively. Values for mixed cultures were previously shown in Fig. 2 and are repeated here for comparison. b Values which differ significantly from that of the matched control (p I 0.01).
TOXICITY
OF MERCURIC
Lack of a Direct Effect of Mercuric ride on Acetyl Cholinesterase
DISCUSSION Both the concentration of mercuric chloride and the time of exposure are important 2
RELATIVE AMOUNTS OF [‘HITHYMIDINE INCORPORATED BY BOTH HIGHLY PURIFIED AND MIXEDCULTURES OF SYMPATHETIC NEURONS AND NONNEURONAL CELLS WHICH WERE EX~~SED TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE FOR 2 DAYS“
Relative thymidine incorporation (% of control)
(PM)
Mixed cultures
Nonneuronal cultures
0.01 I .oo 100.00
107 * 4 (4) 120 f 5 (6)* 90 f 4 (6)
102 2 8 (6) 133 + 6 (6)* 79 + 6 (6)6
FWM
IN
’ Both mixed and highly purified cultures were prepared from the sympathetic ganglia of 11-day chick embryos, handled as described in Fig. IA, harvested, by sonication, and assayed for [‘Hlthymidine incorporated into acid-precipitable macromolecules as described under Methods. All values represent means + SE. Numbers of observations (n) are given in parentheses. The amounts of [3H]thymidine incorporated by mixed and nonneuronal control cultures were 578 + 7 (n = 6) and 1235 ? 56 (n = 6) dpm/pg of protein, respectively. Values for mixed cultures were previously shown in Fig. 2 and are repeated here for comparison. b.r Values which differ significantly from that of the control (p 5 0.01 or p s 0.05, respectively).
259
VITRO
TABLE
Chlo-
Dense mixed cultures of sympathetic ganglion cells (475,000 cells/culture) were harvested by sonication. Aliquots of the sonicate were incubated for 1 hr at room temperature with various concentrations of mercuric chloride (0.1 nM- 100 PM) and then assayed as described under Methods. Acetyl cholinesterase activity in the sonicate did not change as a result of exposure to mercuric chloride.
TABLE
CHLORIDE
RELATIVE
AMOUNTS
OF
3 [‘%]LEUCINE
INCORPO-
RATED BY BOTH HIGHLY PURIFIED AND MIXED CULTURES OF SYMPATHETIC NEURONS AND NONNEURONAL CELLS WHICH WERE EXPOSED TO VARIOUS CONCENTRATIONS OF MERCURIC CHLORIDE FOR 2 DAYS’ Relative leucine incorporation (% of control)
lHs’% (PM) 0.01 1.00 1oQ.w
MIxed cultures 138 * 7 (4jb I I I +_ 4 (6)’ 85 t 3 (6Jb
--.
cultures
NeUrOIlal cultures
95 k 6 (6) II6 k 5 (5)’ 97 k 6 (6)
108 2 8 (5) 99 i 7 (61 84 2 5 (5)
NOtlll~U~Oll~l
o Both mixed and highly purified cultures were prepared from the sympathetic ganglia of I l-day chick embryos, handled as described in Fig. IA. harvested by sonication, and assayed for [“Cjleucine incorporated into acid-precipitable macromolecules as described under Methods. All values represent means 2 SE. Numbers of observations (n) are gwen in parentheses. The amounts of [“C]leucine incorporated by mixed, nonncuronal, and neuronal control cultures were 72 + 2 (n = 6). 79 + 3 (n = ht. and 41 zt 2 (n = 5) dpm/pg of protein. respectively. Values for mixed cultures were previously shown in Fig. 2 and are repeated here for compartron. hr Values which differ stgnificantly from that of the control tp - 001 or p 5 0.05. respectively).
in determining the effects of this metallic salt on the biochemistry of sympathetic neurons and nonneuronal cells. Thus, a given biochemical variable might be stimulated by a low concentration of mercuric chloride but inhibited by a higher concentration (Fig. 2). In addition, a concentration which causes stimulation after exposure for 2 days might cause inhibition after exposure for 3 days (cf. Figs. 2 and 4). Because of the complex nature of these effects, each biochemical variable will now be discussed separately. Acetyl Cholinesterase
Activity
Acetyl cholinesterase activity was generally stimulated by exposure of cultures to mercuric chloride for 2 days. Increase acetyl cholinesterase activity in the mixed cultures appears to have resulted from increased synthesis of this enzyme since mercuric chloride had no direct effect on the enzyme in sonicates of mixed sympathetic ganglion cell cul-
260
SANGDEE,
PARTLOW,
tures. This increase in activity of neuronal acetyl cholinesterase occurred both in the presence and absence of nonneuronal cells (Fig. 2 and Table 1) and persisted during a subsequent 20-hr incubation in the absence of the heavy metal (Fig. 3). To our knowledge, the effects of mercuric chloride on acetyl cholinesterase activity have not previously been investigated. However, 1 pM methyl mercury has been reported to either have no effect or to slightly decrease histochemically determined levels of acetyl cholinesterase in primary cultures of nervous tissues (Hosli and H&Ii, 1974). In addition, Christensen (1975) reported that 5 nM methyl mercury decreased the level of acetyl cholinesterase by 36% in chronically exposed baby brook trout.
AND
BUSH
toma cells. Marked stimulation of thymidine incorporation only occurred in a narrow window of cell concentrations ( 1200- 1700 cells/ mm2); our initial cell concentration was approximately 1500 cells/mm2 in all cases. Nakada and Imura (1980) further reported that 50 FM mercuric chloride totally inhibited thymidine incorporation in both cell lines. In contrast, they found that methyl mercury either had no effect on or inhibited thymidine incorporation. Using a variety of different cell lines, earlier investigators reported that mercuric chloride either did not affect DNA synthesis in vitro or inhibited it (Schuster and Hare, 1970, Nakazawa et al., 1975; Fisher, 1976; Potter and Matrone, 1977). Protein Synthesis
DNA Synthesis DNA synthesis by the nonneuronal cells was stimulated by exposure of mixed cultures for 2 days to 1 or 10 PM mercuric chloride but was inhibited by treatment with 100 pM mercuric chloride (Fig. 2). Both effects were also observed in purified cultures of nonneuronal cells and can therefore be explained by a direct action of the metallic salt on the nonneuronal cells (Table 2). Both the stimulatory and inhibitory effects observed after 2 days of treatment disappear following removal of the metallic salt from the culture medium (Fig. 3). Exposure of mixed cultures to mercuric chloride for 3 days inhibited thymidine incorporation at a concentration of 100 pM but had no effect at concentrations of 10 PM or less. Our observation that exposure to mercuric chloride can either stimulate or inhibit thymidine incorporation is supported by a recent study by Nakada and Imura (1980). These investigators reported that l-20 PM mercuric chloride stimulated thymidine incorporation by mouse glioma cells and that l-5 PM mercuric chloride stimulated thymidine incorporation by mouse neuroblas-
Since both neurons and nonneuronal cells incorporate leucine into protein, results obtained with mixed cultures reflect the combined effects of mercuric chloride on both cell types. Leucine incorporation was generally stimulated by exposure for 2 days to mercuric chloride at concentrations 5 10 PM, but was inhibited by exposure to a concentration of 100 pM. These effects were reversible since they were not found in cultures incubated for an additional 20 hr in the absence of the metallic salt (Fig. 3). After 3 days of exposure, leucine incorporation was inhibited at almost all concentrations of mercuric chloride. The stimulatory effects of 1 pM mercuric chloride on mixed cultures are adequately explained by a direct action on the nonneuronal cells and the inhibitory effects observed at 100 pM could be accounted for by a direct action on the neurons (Table 3). However, the stimulatory effects of extremely low concentrations (0.01 PM) of mercuric chloride on leucine incorporation by mixed cultures cannot be explained by direct effects on either cell type (Table 3). It is conceivable that low concentrations of
TOXICITY
OF MERCURIC
mercuric chloride might alter some aspect of the dynamic interactive processes occurring between the neurons and nonneuronal cells (e.g., neuronal-glial interactions). To the authors knowledge, the effects of mercuric chloride on protein synthesis in vitro have not previously been investigated. However, methyl mercury has been reported to either have no effect on or to decrease protein synthesis in a wide variety of in vivo and in vitro systems (Yoshino et al., 1966; Cavanagh and Chen, 1971; Syversen, 1977; Verity et al., 1977; Gruenwedel and Cruikshank, 1979). In contrast, Brubaker et al. ( 1973) reported that methyl mercury increased incorporation of labeled amino acids into protein in vivo. Relative Sensit@ity of Neurons neuronal Cells
and Non-
Neurons appear to be far more sensitive to mercuric chloride than nonneuronal cells. Thus, concentrations as low as 0.0001 PM mercuric chloride significantly increased activity of the neuronal enzyme acetyl cholinesterase (Fig. 3). In contrast, concentrations of mercuric chloride below 1 PM had no effect on DNA synthesis by the glial cells (Figs. 2-4 and Table 2). These results suggest that neurons rather than glia might be the primary site of action of mercuric chloride in the nervous system. Sobkowicz and Murray similarly concluded that mercury exerts its primary action on the neurons rather than the glial cells in primary cultures (Murray, 197 1). In addition, Syversen ( 1977) concluded that neurons are more sensitive than glial cells to methyl mercury by examining protein synthesis in vivo. Toxicological
Significance
CHLORIDE
IN VITRO
261
from lethal heavy-metal intoxication have been reported at 22-50 (Hilmy et al., 1976) and 0.5-124 IAM (Takeuchi et al., 1962). It seems likely that longer-term exposure to such mercury concentrations would produce inhibitory effects. The toxicological significance of the stimulatory effects reported herein as a result of acute exposure to very low concentrations of mercuric chloride is uncertain. Acetyl cholinesterase activity and protein synthesis were both stimulated by concentrations of mercuric chloride as low as 0.1 or 1 BM, respectively (Figs. 2 and 3). Such stimulatory effects would not be expected to persist in mercury-intoxicated nervous tissues because these effects are transient and disappear with continued exposure (compare data for 2- and 3-days exposures in Figs. 2 and 4). In addition, concentrations which stimulate these biochemical measures are generally lower than those found in the brains of normal individuals at autopsy [ 30 IIM (Hilmy et al., 1976), 1.2 pM(Olszewski et al., 1974) or 2.5 PM (Takeuchi et al., 1962)]. ACKNOWLEDGMENTS Supported by a P.O.E. International Peace Scholarship to P.S., U.S. Public Health Service Training Grant GM-00153, and Grant NS-12812 from the National Institute of Neurological Diseases and Stroke.
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