Effects of Methylmercury and Mercuric Chloride on Differentiation and Cell Viability in PC12 Cells

D. K. Parran*, W. R. Mundy{dagger} and S. Barone, Jr.{dagger},1

* Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and {dagger} Cellular and Molecular Toxicology Branch, Neurotoxicology Division, Mail Drop 74-B, National Health and Environmental Effects Research Laboratory (NHEERL), U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711

Received June 8, 2000; accepted October 11, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of methylmercury (CH3Hg) or mercuric chloride (HgCl2) on neurite outgrowth and cell viability were quantified using undifferentiated (unprimed) and differentiated (primed) pheochromocytoma (PC12) cells. In unprimed cells, following 24-h exposure, CH3Hg significantly decreased NGF-stimulated neurite outgrowth at concentrations of 0.3–3 µM. However, HgCl2 significantly increased both neurite outgrowth and the number of branch points, a component of neurite outgrowth. In primed PC12 cells, following 24-h exposure, both CH3Hg and HgCl2 inhibited NGF-stimulated neurite outgrowth with an EC50 of approximately 0.03 µM; however, there was a difference between CH3Hg and HgCl2 effects on the subcomponents of total neurite outgrowth. CH3Hg significantly decreased both the number of branch points (0.3 µM) and fragment length (0.01 µM), while HgCl2 only decreased fragment length (0.03 µM). Cell viability was assessed in the same cultures by trypan-blue exclusion. In unprimed cells, the EC50 for cytotoxicity of CH3Hg in the presence and absence of NGF was 0.21 ± 0.04 and 0.87 ± 0.12 µM, respectively, and for HgCl2 in the presence and absence of NGF was 8.18 ± 1.52 and 5.02 ± 0.74 µM, respectively. In primed cells, the EC50 for cytotoxicity of CH3Hg in the presence or absence of NGF was 1.17 ± 0.38 and 0.73 ± 0.14 µM, respectively, and for HgCl2 in the presence or absence of NGF was 3.96 ± 0.82 and 3.81 ± 0.91 µM, respectively. In the primed PC12 model, cytotoxicity occurred at concentrations that were at least 30-fold higher than the EC50 for neurite outgrowth, suggesting that the mercurial compounds can act selectively on the process of differentiation.

Key Words: neurite outgrowth; developmental neurotoxicity; nerve growth factor (NGF).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methylmercury (CH3Hg) is a neurotoxic compound that is ubiquitous in the environment. Much of the human data concerning CH3Hg neurotoxicity has come from tragic events, which include Minamata Bay and Niigata, Japan (Reuhl and Chang, 1979Go; Takeuchi et al., 1959Go) and Iraq (Amin-Zaki et al., 1974Go; Bakir et al., 1973Go), where large populations were exposed to high-levels of CH3Hg. More recently, the neurotoxic effects of low-level CH3Hg contamination has been studied in the Seychelles Islands (Crump et al., 2000Go; Davidson et al., 1999Go; Myers et al., 1995Go), the Faroe Islands (Grandjean et al., 1998Go, 1999aGo; Weihe et al., 1996Go), New Zealand (Crump et al., 1998Go), and the Amazon Basin (Dolbec et al., 2000Go; Grandjean et al., 1999bGo; Lebel et al., 1998Go). Following high doses of CH3Hg, it was evident that CH3Hg is a potent neurotoxicant and that the developing fetus is more susceptible to CH3Hg poisoning than adults (Choi et al., 1978Go; Matsumoto et al., 1965Go) and that subtle neurobehavioral manifestations, which include decreased motor function and visuospatial performance, were evident following low-level exposure (Dolbec et al., 2000Go; Grandjean et al., 1998Go; Myers et al., 1995Go). Evidence for the susceptibility of the developing organism to the neurotoxic effects of CH3Hg has also been observed in animal studies using primates (Mottet et al., 1987Go; Rice, 1989Go, 1996Go), cats (Khera, 1973Go; Khera et al., 1973, 1974Go), hamsters (Reuhl et al., 1981Go), mice (Chang, 1977Go; Chang et al., 1977Go; Sager et al., 1982Go, 1984Go) and rats (Nonaka, 1969Go; Reuhl and Chang, 1979Go). Following developmental exposure in both humans and animals, cell loss in the cerebellum and cerebrum, atrophic brains, neuronal degeneration, delayed or abnormal development of the cerebellar granule cell layer, and abnormal cortical migration were prominent in the fetus and infant (Burbacher et al., 1990Go; Chang, 1977Go). Interestingly, in humans, the earlier the developmental exposure to CH3Hg, the more widely distributed the neuropathology in the brain (Chang, 1977Go). Animal studies using low doses of CH3Hg have also demonstrated neuropathology similar to that observed in humans exposed to high doses of CH3Hg (Burbacher et al., 1990Go). For example, gestational exposure of pregnant rats to 2 mg/kg of CH3Hg resulted in changes in cell density, cell size, and decreases in the widths of the layers of the posterior neocortex in pups on postnatal days 10 and 21 (Barone et al., 1998Go), suggesting altered cortical differentiation.

There are currently a number of mechanisms that have been proposed to underlie the developmental neurotoxicity of CH3Hg, including brain region-specific changes in neurotrophic factor expression (Lärkfors et al., 1991Go), inhibition of embryonic NCAM conversion to adult NCAM (Lagunowich et al., 1991Go), disassembly of microtubules (Graff et al., 1997Go), increased mRNA expression of Gadd45 and Gadd153 (Ou et al., 1997Go) and decreased expression and/or activity of proteins involved in neurotrophic factor signaling (Barone et al., 1998Go; Haykal-Coates et al., 1998Go; Mundy et al., 2000Go). Because these targets underlie crucial processes in the development of the nervous system (i.e., proliferation, migration, and differentiation), perturbations of these processes by CH3Hg could explain the developmental neurotoxicity observed following in vivo exposure.

In order to examine the effects of CH3Hg on the developing nervous system, a number of studies have used differentiating-cell cultures as a model. In vitro exposure to CH3Hg has been shown to inhibit nerve growth factor (NGF)-induced neurite outgrowth in chick sympathetic and sensory dorsal root-ganglia cultures (Nakada et al., 1981Go; Söderström et al., 1995). One question that arises from these studies is whether the inhibition of NGF-induced neurite outgrowth is due to a selective effect of CH3Hg on differentiation, or is simply the result of cytotoxicity.

Previous work has not examined the effects of CH3Hg on differentiation and cytotoxicity in a systematic manner. The present study evaluates the developmental neurotoxicity of CH3Hg by examining neurite outgrowth and cell viability in the pheochromocytoma (PC12) cell line, an in vitro model which has been used extensively for the study of neuronal differentiation (reviewed in Fujita et al., 1989). In the presence of NGF, PC12 cells cease to divide and instead differentiate into a neuronal phenotype characteristic of sympathetic neurons. Morphological characteristics indicative of differentiation include an increase in cell size and the extension of neurites (Fujita et al., 1989Go; Greene and Tischler, 1976Go). In contrast to previous studies using qualitative assessments of differentiation, the present work examined the effects of CH3Hg on differentiation quantitatively, using a video-based imaging system. This system allowed for semi-automated quantitation of a number of morphologic measures of differentiation including cell size, neurite branching, and total neurite length (Das and Barone, 1999Go). To directly compare the effects of CH3Hg on differentiation and cytotoxicity, cell viability was determined in the same cultures. The process of differentiation can be divided into at least two phases: neurite initiation and neurite elaboration, which may be subserved by distinct mechanisms (Burstein et al., 1978). Thus, we examined the effects of CH3Hg on PC12 cells that were not differentiated (no previous exposure to NGF; unprimed) to determine effects on neurite initiation, and in PC12 cells that had been exposed previously to NGF (primed) to determine effects on neurite growth. Finally, in order to examine the specificity of CH3Hg, we compared the effects of CH3Hg to inorganic mercury (mercuric chloride; HgCl2). HgCl2 is a toxicant that does not specifically target the developing nervous system, but affects renal development and function (Bartolome et al., 1985Go; Daston et al., 1983Go, 1984Go, 1986Go; Kavlock et al., 1983).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials.
Rat pheochromocytoma cells (PC12 cells) were a gift from Dr. Gordon Guroff (NIH, Bethesda, MD). Human recombinant nerve growth factor-beta (NGF), rat tail collagen type I, HEPES, and sodium bicarbonate were purchased from Sigma Chemical Co. (St. Louis, MO). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), fetal bovine serum (FBS), horse serum and trypan blue solution were purchased from Life Technologies (Grand Island, NY). Twenty-five cm2 tissue culture flasks, 75 cm2 tissue culture flasks and twelve well plates were purchased from Corning Costar Corporation (Cambridge, MA). Mercuric Chloride (HgCl2) was purchased from J.T. Baker Chemical Company (Phillipsburg, NJ). Methylmercury chloride (CH3Hg) was purchased from Pfaltz & Bauer (Waterbury, CT).

Cell culture.
Experiments were performed on pheochromocytoma cells from passage 10 or 11 (passage number after receipt in our laboratory) to minimize interassay variability. The cells were grown in 25 cm2 tissue culture flasks with complete DMEM medium containing 7.5% heat-inactivated FBS, 7.5% horse serum, 2 mM HEPES and 44 mM sodium bicarbonate. Cultures were maintained according to standard protocols (Greene and Tischler, 1976Go) at 37°C in a 95% humidified incubator with 5% CO2. A frozen stock of PC12 cells from passage 8 cells were thawed every week and grown for four days. On the fifth day in culture, the cells were split (passage 9) and replated for the unprimed-cell or primed-cell assay.

Neurite differentiation and morphology.
Passage 9 PC12 cells (1 x 104 cells/well) were replated in 12-well collagen-coated tissue culture plates, in complete DMEM/F12 medium containing 10% FBS and 44 mM sodium bicarbonate. After 2 h, this medium was removed and replaced with DMEM/F12 without FBS, in the presence or absence of NGF and in varying concentrations of CH3Hg or HgCl2. In all these in vitro experiments, cells were exposed and examined from either passage 10 (unprimed) or 11 (primed) only in serum-free DMEM/F12 medium.

In unprimed cells (undifferentiated PC12 cells not exposed to NGF), determinations of the effects of CH3Hg and HgCl2 on NGF-stimulated neurite outgrowth were performed during the initiation phase of neurite outgrowth. These PC12 cells were cultured in serum-free medium for 24 h in the presence or absence of NGF (50 ng/ml) and in combination with either CH3Hg or HgCl2 at doses ranging from 0 to 3 µM. After 24 h of exposure, images from cells were digitized and saved for later analysis to quantify neurite outgrowth (see details below).

In addition to examination of the effects of CH3Hg and HgCl2 on initiation of neurite outgrowth, a primed-cell assay, modified from protocols previously published (Greene and Tischler, 1976Go; Shaughnessy and Barone, 1997Go), was utilized to examine the acute effects of toxicants on NGF-induced neurite outgrowth. Primed PC12 cells are cells that have been exposed to 50 ng/ml of NGF for 7 days. The cells were cultured for one week in serum-free DMEM/F12 medium and fed with fresh NGF (50 ng/ml) on days 3, 5, and 7 following the plating of passage 10. On day 8, primed cells were harvested, washed with NGF-free complete DMEM/F12 medium and centrifuged at 500 rpm for 5 min at 4°C. The collected cells were finally replated (1 x 104 cells/well) in twelve-well collagen-coated tissue culture plates. Fifty ng/ml of NGF, with or without various concentrations of mercury compounds in DMEM/F12 media, was added. Primed PC12 cells were incubated for 24 h at which time neurite outgrowth was measured.

After 24 h of incubation, images of the PC12 cells were captured with phase contrast video microscopy (nominal 200x magnification) and digitized with a MTI-DAGE81 high-resolution black and white camera interfaced with a personal computer. Imaging software (Presage, Advanced Imaging Concepts, Inc., Princeton, NJ) was used to quantify the means of cell size and neurite outgrowth and constituent measures of neurite outgrowth (branch points per cell, fragments per cell, average fragment length per cell, and total neurite outgrowth per cell). This was performed by defining the phase-bright perimeter of the cell bodies based upon threshold values and subtracting that from subsequent images of the total neurite network. The image of the total neurite network was skeletonized to one pixel in width and the total length of neurite outgrowth calculated per cell. The point at which any fibers crossed or branched was determined from this skeletonized image and recorded as the number of branch points. These branch points were subtracted from the image of total neurite outgrowth to derive fiber fragments that were uniquely labeled to determine both number and length of fiber fragments. These measures of neurite outgrowth were quantified for cell clumps ranging in number from 3 to 9 cells. The number of cells/clump was counterbalanced across each experimental condition such that no experimental condition was skewed with only small- or large-cell clumps.

Cytotoxicity assays.
Following 24-h exposure to CH3Hg or HgCl2 and assessment of neurite outgrowth, cytotoxicity was determined for similar concentration-response assessments by the trypan blue-exclusion method. Cells were stained by adding 10 µl of trypan blue (1:100 dilution of 0.4%) into each well and allowed to distribute throughout the well for 5 min at 37°C. Cells with disturbed plasma membrane permeability stained blue, whereas undamaged (viable) cells appeared translucent. Both attached and detached cells were measured for trypan blue staining. Cells were counted with an IMT-2 inverted light microscope at 10x magnification with a mean total cell count of 50 cells per field. Viability results were expressed as a percent of the number of total cells.

Exposure.
CH3Hg and HgCl2 were each dissolved in sterile, distilled water at a stock concentration of 10 mM. Concentrations of 1–1000 µM were prepared by serial dilutions of the stock mercurial solutions. Cells were exposed to either of the mercury solutions, which were added in a volume of 10 µl (1:100 dilution) per well, and exposure lasted for 24 h. Control wells were dosed with an equivalent volume (10 µl) of the vehicle (sterile, distilled water), which did not cause any adverse effects on neurite outgrowth.

Statistical analysis.
Neurite outgrowth and cytotoxicity were examined using a split-plot design with subsampling. The main plots were the plates, subplots were the wells, and subsampling occurred with the sampling of 2 images per well. Each well was considered the minimum unit of measure (n) and subsampling of a well was considered a replicate and averaged. Each experiment had at least an "n" of 3 data points per condition and analysis was performed on data compiled from 3 experiments, resulting in at least an n of 9 per condition. The main factors included in the global analysis of variance (ANOVA) were assay condition (unprimed versus primed), acute presence or absence of NGF, concentration of mercury species, and species of mercury (CH3Hg versus HgCl2). The endpoints included in the ANOVA were total neurite outgrowth/cell, branch points/cell, fiber number/cell, fiber length/cell, cell size, and cell viability. The percent viability data was transformed with an arc sin transformation. Following determination of significant interactions between the experimental factors, step-down ANOVAs were performed on each endpoint and differences between the group means were compared using Tukey's Studentized Range Test. Statistically significant differences are reported when p <= 0.05. For simplicity's sake, the focus of the description of the experimental results was based on the step-down ANOVA and post-hoc comparisons of group means. EC50's for neurite outgrowth and cytotoxicity were determined from concentration-response curves for individual experiments using non-linear regression (GraphPad Software, Inc., San Diego, CA) followed by ANOVA of the respective EC50 determined from each experimental condition.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Characterization of the PC12 Cell Model
Initially, a quantitative assessment of morphological measures of differentiation was determined by examining cell-body size, number of branch points, fragment length, and total neurite outgrowth per cell using an image-analysis program. Unprimed PC12 cells were plated at a low density (1 x 104 cells/ml) which allowed for the measurement of total neurite outgrowth of individual groups of cells (groups of 3–9 cells) before the cell culture becomes confluent (Das and Barone, 1999Go; Shaughnessy and Barone, 1997Go). During this differentiation period (7 days exposure to 50 ng/ml NGF), total neurite outgrowth per cell was measured every 24 h. Consistent with observations in previous studies on NGF-induced differentiation (Das and Barone, 1999Go; Greene and Tischler, 1976Go; Shaughnessy and Barone, 1997Go), there was a time-dependent increase in neurite outgrowth, number of branch points, fragment length, and cell size. After 7 days of exposure to NGF, a primed-cell condition is obtained where cells are in the same phase of the cell cycle and a more differentiated state. Upon harvest and replating of these cells, neurites are removed. The time course of new neurite elaboration following replating of primed PC12 cells was determined to establish the progression of neurite outgrowth during an acute exposure period. Total neurite outgrowth, cell body area, branch number, fragment number and fragment length per cell was measured at 0, 3, 6, 12 or 24 h after exposure to 50 ng/ml of NGF. A time-dependent increase in total neurite outgrowth was observed, with a measurable increase in neurite outgrowth occurring as early as 6 h after exposure to NGF, a time when normally no neurite outgrowth is present in unprimed cells. The mean cell size, number of branch points, and fragment lengths also increased in a time-dependent fashion in primed PC12 cells.

Effects of CH3Hg and HgCl2 on the Differentiation of Unprimed PC12 Cells
Unprimed cells, which are naïve to NGF and had not differentiated into a neuronal phenotype, were used to examine the effects of CH3Hg and HgCl2 on the initiation of neurites in the presence or absence of NGF. Neurite outgrowth in unprimed cells in the first 24-h period is limited. Analysis of total neurite outgrowth per cell, measured at 24 h in the presence or absence of both NGF and CH3Hg, indicated that there was an effect of acute NGF exposure and an effect of the concentration of CH3Hg, but no interaction of these 2 exposures. Post-hoc analysis of the data showed that NGF increased neurite outgrowth, and that CH3Hg produced a small but significant inhibition of neurite outgrowth at high concentrations (0.3–3 µM, Fig. 1AGo). Thus, CH3Hg appears to inhibit the early growth (initiation) of neurites at higher concentrations. For other characteristics of neurite outgrowth including number of branch points, there was a main effect of NGF but no effect of CH3Hg (Fig. 1CGo). Neither NGF nor CH3Hg affected fragment length/cell in unprimed cells (Fig. 1EGo). As mentioned above, NGF increased cell size. CH3Hg significantly decreased cell size at concentrations of 0.3 to 3 µM both in the presence and absence of NGF (Fig. 1GGo).



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FIG. 1. Mercury compounds alter neurite outgrowth, number of branch points, fragment lengths and cell body area in unprimed PC12 cells. Unprimed PC12 cells were exposed to different concentrations of either CH3Hg (A, C, E, G) or HgCl2 (B, D, F, H) for 24 h in the presence (•) or absence (o) of NGF (50 ng/ml). Total neurite outgrowth (A, B), number of branch points (C, D), fragment length/cell (E, F), and cell body area (G, H) were measured. The results are expressed as mean ± SE and representative of at least 6 independent measures. In the case of a significant main effect of concentration of mercury (Hg), post hoc analysis was performed on combined data (both the presence and absence of NGF), and points not followed by the same letter (upper case, italic) are significantly different (p < 0.05). In the case of a significant interaction of concentration and NGF, post hoc analysis was performed separately for data in the presence or absence of NGF. In the presence of NGF, points not followed by the same letter (lower case) are significantly different (p < 0.05). In the absence of NGF, points not followed by the same letter (lower case, italic) are significantly different (p < 0.05).

 
Examination of the total neurite outgrowth per cell in unprimed cells at 24 h, in the presence or absence of NGF and HgCl2, indicated that there was an interaction between NGF exposure and HgCl2. In the presence of NGF, HgCl2 increased neurite outgrowth and post hoc analysis revealed that the effect was significant at 0.1 to 3 µM. In the absence of NGF, a significant increase in neurite outgrowth was observed at 3 µM of HgCl2 (Figs. 1B, 2A–2DGoGo). HgCl2 also increased the number of branch points (Fig. 1DGo). Neither NGF nor HgCl2 affected fragment length/cell in unprimed cells (Fig. 1FGo). Analysis of cell size revealed that NGF increased cell size while HgCl2 had no effect, and there was no interaction (Fig. 1HGo).



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FIG. 2. Qualitative presentation of unprimed PC12 cells following exposure to HgCl2. Unprimed PC12 cells were exposed to different concentrations of HgCl2 for 24 h in the presence of NGF (50 ng/ml). Compared to controls (A), HgCl2 exposure resulted in an increase in neurite outgrowth at 0.01 µM of HgCl2 (B), 0.1 µM (C) and 1.0 µM (D).

 
Effects of CH3Hg and HgCl2 on the Differentiation of Primed PC12 Cells
In order to assess the effects of CH3Hg and HgCl2 on NGF-induced neurite elongation, we used PC12 cells that had been previously exposed to NGF prior to replating (primed). Acute exposure of primed PC12 cells to NGF resulted in approximately a 20-fold increase in neurite outgrowth over a 24-h period, which is in contrast to the 7 days it takes unprimed cells to reach this level of elaboration. Analysis of results for total neurite outgrowth per cell in primed cells revealed an interaction NGF and CH3Hg exposure (Fig. 3AGo). Examination of the data indicates that NGF increased neurite outgrowth dramatically in primed cells, and that CH3Hg inhibited NGF-stimulated neurite outgrowth in a concentration-dependent fashion (Figs. 3A, 4A, 4C, 4EGoGo) with a significant decrease at concentrations ranging from 0.01 to 3 µM. The EC50 for the inhibition of neurite outgrowth by CH3Hg was 0.033 ± 0.009 µM. In the absence of NGF, there was very little neurite outgrowth, and no significant effect of CH3Hg (Fig. 3AGo). There was also an interaction of NGF and CH3Hg exposure on the number of branch points (Fig. 3CGo). In the absence of NGF, there were fewer branch points than in the presence of NGF. CH3Hg caused a concentration-dependent decrease in branch points in both the presence and absence of NGF. There was an interaction of NGF and CH3Hg exposure on fragment length/cell in primed cells with CH3Hg, causing a concentration-dependent decrease in fragment length in the presence of NGF (Fig. 3EGo). Analysis of cell size revealed an effect of NGF and CH3Hg, but no interaction between the 2 treatments (Fig. 3GGo). Thus, overall NGF increased cell size, while CH3Hg decreased cell size at the higher end of the concentration response curve (0.1–3 µM) in both the presence and absence of NGF.



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FIG. 3. Mercury compounds alter neurite outgrowth, number of branch points, fragment lengths, and cell-body area in primed PC12 cells. Primed cells were replated and exposed to different concentrations of either CH3Hg (A, C, E, G) or HgCl2 (B, D, F, H) for 24 h in the presence (•) or absence (o) of NGF (50 ng/ml) (see methods). Total neurite outgrowth (A, B), number of branch points (C, D), fragment length/cell (E, F), and cell body area (G, H) were measured. The results are expressed as mean ± SE and representative of at least 6 independent measures. In the case of a significant main effect of concentration of mercury (Hg), post-hoc analysis was performed on combined data (both the presence and absence of NGF), and points not followed by the same letter (upper case, italic) are significantly different (p < 0.05). In the case of a significant interaction of concentration and NGF, post hoc analysis was performed separately for data in the presence or absence of NGF. In the presence of NGF, points not followed by the same letter (lower case) are significantly different (p < 0.05). In the absence of NGF, points not followed by the same letter (lower case, italic) are significantly different (p < 0.05).

 


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FIG. 4. Qualitative presentation of primed PC12 cells following mercury exposure. Unprimed PC12 cells were exposed to 50 ng/ml of NGF for 7 days, after which, the primed cells were replated and exposed to different concentrations of either CH3Hg (A, C, E) or HgCl2 (B, D, F) for 24 h in the presence of NGF (50 ng/ml). Compared to controls (A, B), exposure to either CH3Hg (C) or HgCl2 (D) resulted in a concentration-dependent decrease in neurite outgrowth with an EC50 of approximately 0.03 µM, with almost total inhibition of neurite outgrowth at 0.1 µM of either CH3Hg (E) or HgCl2 (F).

 
Analysis of the effects of HgCl2 on total neurite outgrowth in primed cells showed similar results to those obtained with CH3Hg. There was a significant interaction of NGF with HgCl2 exposure (Fig. 3BGo). NGF increased neurite outgrowth in primed cells, and HgCl2 inhibited NGF-stimulated neurite outgrowth in a concentration-dependent fashion (Figs. 3B, 4B, 4D, 4FGoGo) with significant decreases at concentrations of 0.03–3 µM. The EC50 for the inhibition of neurite outgrowth by HgCl2 was 0.026 ± 0.006 µM. In the absence of NGF, there was little neurite outgrowth, and no effect of HgCl2 (Fig. 3BGo). For number of branch points, there was a significant increase produced by NGF exposure, which was not altered by HgCl2, unlike CH3Hg (Fig. 3DGo). There was an interaction of NGF with HgCl2 for fragment length/cell in primed cells (Fig. 3FGo), with HgCl2 causing a concentration-dependent decrease in fragment length only in the presence of NGF. Analysis of cell size revealed an effect of NGF and HgCl2 but no interaction between these two treatments (Fig. 3HGo). NGF increased cell size, and post hoc analysis revealed that HgCl2 decreased cell size only at 3 µM, and in both the presence and absence of NGF.

Effects of CH3Hg and HgCl2 on Cell Viability
To determine the effects of mercury compounds on cell survival, unprimed PC12 cells were exposed for 24 h to various concentrations of either CH3Hg or HgCl2 in the presence or absence of NGF, as described above, and cell viability was examined using trypan-blue exclusion. CH3Hg exposure resulted in a concentration-dependent decrease in cell viability (Fig. 5AGo) with a significant interaction of NGF and CH3Hg. The EC50 for cytotoxicity was lower in the absence of NGF (EC50 = 0.21 ± 0.04 µM) than in the presence of NGF (EC50 = 0.87 ± 0.12 µM) (Table 1Go). HgCl2 produced a concentration-dependent decrease in cell viability (Fig. 5BGo), and statistical analysis revealed a significant effect of HgCl2 and no interaction with NGF. EC50 values of 5.02 ± 0.74 and 8.18 ± 1.52 µM were obtained in the presence or absence of NGF, respectively (Table 1Go). In total, the cell viability data indicate that CH3Hg is more cytotoxic than HgCl2, and that cytotoxicity was greatest following CH3Hg exposure in the absence of NGF (Table 1Go).



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FIG. 5. Mercury compound's effects on cell viability. Unprimed and primed PC12 cells were exposed to different concentrations of either CH3Hg (A, C) or HgCl2 (B, D) for 24 h in the presence (•) or absence (o) of NGF (50 ng/ml). Cell viability was determined by trypan-blue exclusion. The results are expressed as mean ± SE and representative of at least 6 independent measures. In the case of a significant main effect of concentration of mercury (Hg), post hoc analysis was performed on combined data (both the presence and absence of NGF), and points not followed by the same letter (upper case, italic) are significantly different (p < 0.05). In the case of a significant interaction of concentration and NGF, post hoc analysis was performed separately for data in the presence of absence of NGF. In the presence of NGF, points not followed by the same letter (lower case) are significantly different (p < 0.05). In the absence of NGF, points not followed by the same letter (lower case, italic) are significantly different (p < 0.05).

 

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TABLE 1 EC50's for Cytotoxicity of CH3HgCl2 in Unprimed and Primed PC12 Cells
 
The results of the trypan-blue exclusion cell viability assay in primed PC12 cells revealed an interaction of exposure to NGF and CH3Hg. There was a concentration-dependent decrease in cell viability (Fig. 5CGo). EC50 values of 1.17 ± 0.38 and 0.73 ± 0.14 µM were obtained in the presence or absence of NGF, respectively (Table 1Go). Analysis of the concentration-response curves for HgCl2 revealed a significant effect of HgCl2 only. Regardless of NGF exposure, HgCl2 significantly affected cell viability above 0.3 µM in primed PC12 cells (Figs. 5B, 5DGo). EC50 values of 3.91 ± 0.91 µM in the presence of NGF and 3.96 ± 0.82 µM in the absence of NGF were obtained (Table 1Go). As observed with unprimed cells, CH3Hg is more cytotoxic than HgCl2 in primed cells and CH3Hg-induced cytotoxicity was greatest in the absence of NGF (Table 1Go).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we used an in vitro culture system as a model to examine the effects of CH3Hg and HgCl2 on neurotrophin-mediated neuronal differentiation and survival. The differentiation of PC12 cells was examined by measuring a number of endpoints of neurite outgrowth with a video-based imaging system, which allowed for semi-automated quantitation of differentiation and cell viability in the same cultures. The results indicated that decreased neurite outgrowth was a more sensitive index of toxicity for both mercury species than overt cytotoxicity.

The PC12 cell line, derived from a rat adrenal medullary pheochromocytoma tumor, has been used extensively as a model for investigating biomolecular events involved in neuronal differentiation. In the presence of NGF, proliferating PC12 cells cease to divide and differentiate into a neuronal phenotype, which includes the elaboration of neurites (Das and Barone, 1999Go; Greene and Tischler, 1976Go; Shaughnessy and Barone, 1997Go). In unprimed PC12 cells, following stimulation with NGF, total neurite outgrowth increased in a time-dependent manner. Other measures of differentiation, such as cell-body area, number of branches, and fragment lengths, also increased in a time-dependent manner. These measures of differentiation were used to determine the effects of mercury compounds on the initial events of differentiation. The increase in NGF-stimulated total neurite outgrowth in unprimed PC12 cells was robust only after 48 h. In contrast, the primed-cell system provided the ability to quantify the acute effects of mercury compounds on neurite outgrowth in a fully differentiated cell.

Neurite Outgrowth in Unprimed Cells
Evaluation of neurite outgrowth in unprimed PC12 cells provides a measure of early events in nervous system differentiation after neurotrophic-factor stimulation. The lack of robust effects of 24-h exposure to CH3Hg on neurite outgrowth in unprimed PC12 cells suggests that CH3Hg does not selectively alter early events of differentiation. In the presence of NGF, there was a decrease in total neurite outgrowth at the higher doses of CH3Hg.

In contrast to the effects of CH3Hg on unprimed PC12 cells, there was an increase or induction of neurite outgrowth following HgCl2 exposure, principally in the presence of NGF. At concentrations between 0.1 and 3 µM, there was an increase in total neurite outgrowth in the presence of NGF and an increase at 3 µM in the absence of NGF. This HgCl2-induced increase in total neurite outgrowth was due to an increase in the number of branch points and fiber branches (data not shown) with no significant effect on fragment length. These data suggest that HgCl2 was increasing initiation of neurites versus having an effect on neurite elongation. Similar findings were observed previously using this neurite outgrowth assay after exposure to lead acetate, which also stimulated neurite initiation at low doses (Crumpton et al., 2000Go). This increase in initiation of neurite outgrowth may be associated with the ability of HgCl2 to stimulate basal diacylglycerol production and activate protein kinase C (Nicotera et al., 1992Go). During NGF-induced differentiation, there is an increase in calcium flux (Schubert, 1978Go) and cytosolic calcium concentrations (Pandiella-Alonso et al., 1986Go). HgCl2 has been shown to increase intracellular calcium, a process that could be prevented by pretreatment with calcium entry blockers (Rossi et al., 1991Go). The HgCl2-induced increase in intracellular calcium concentration, along with the increase induced by NGF, may stimulate proteins including PLC-{gamma} (Nicotera et al., 1992Go; Vignes et al., 1993Go) which could account for this increase in neurite outgrowth.

Neurite Outgrowth in Primed Cells
The primed PC12 cell assay may be considered an in vitro approximation of dendritic elaboration following repeated stimulation with neurotrophins in vivo. In the present model, total neurite outgrowth is analogous to dendritic elaboration, while the components of neurite outgrowth (number of branch points and fragment length) may be counterparts to the number of branches and branch length in vivo. In the primed PC12 cell assay, exposure of cells to either CH3Hg or HgCl2 inhibited NGF-stimulated neurite outgrowth in a concentration-dependent fashion. Significant inhibition was observed at approximately 0.03 µM for both CH3Hg and HgCl2, a concentration that did not result in cytotoxicity (approximately 90% cell viability). The EC50's for the inhibition of neurite outgrowth by either CH3Hg or HgCl2 were approximately 35–150 times lower than the EC50's for cytotoxicity. This suggests that inhibition of neurite outgrowth is a sensitive measure of toxicity, and is not a consequence of general toxicity of mercury compounds to PC12 cells. The current findings of mercury-induced inhibition of neurite outgrowth is consistent with previous findings in other test systems (Abdulla et al., 1995Go; Nakada et al., 1981Go; Pendergrass et al., 1997Go; Söderström and Ebendal, 1995Go; Windebank, 1986Go). However, these investigations did not attempt to address the question of whether mercury preferentially affects differentiation versus overt cytotoxicity. Using other models to examine neurite outgrowth, significant differences in the potency of the 2 mercury compounds were observed. In chick embryonic sensory ganglia, the inhibitory effect of CH3Hg (EC50 = 2 µM) on neurite outgrowth was about 25 times more potent than HgCl2 (EC50 = 50 µM; Nakada et al., 1981Go). In contrast, there was no significant difference between the EC50 of CH3Hg and HgCl2 on total neurite outgrowth per cell in our primed cell system. However, differences were noted between the effects of the 2 forms of mercury on the components of neurite outgrowth (i.e., number of branch points per cell and fragment length per cell). CH3Hg caused significant decreases in the number of branch points per cell at 0.3 µM while HgCl2 did not have any significant effects. Also, CH3Hg significantly decreased fragment length per cell at a concentration of 0.01 µM; HgCl2 decreased fragment length per cell at 0.03 µM. Analogous effects of CH3Hg on differentiation have been observed in vivo. Early postnatal exposure of mice to CH3Hg has been shown to diminish dendritic elaboration in Purkinje cells (Choi et al., 1981Go) as well as decreased laminar widths of the neocortex in rats prenatally exposed to CH3Hg (Barone et al., 1998Go). From these studies, it can be hypothesized that CH3Hg may interfere with trophic factor-induced elaboration of neuronal dendrites in vivo.

Although not examined in the present study, there are several possible mechanisms by which mercury compounds could alter NGF-induced neurite outgrowth. One possible mechanism is that the mercury compounds are disrupting neurotrophin signaling by directly affecting proteins of the neurotrophin signal-transduction cascade. Inhibition of neurotrophin signaling has been shown to block differentiation and neurite outgrowth (Altin et al., 1992Go; Coleman and Wooten, 1994Go; Cowley et al., 1994Go; Obermeier et al., 1994Go; Pang et al., 1995Go; Tsukada et al., 1994Go; Stephens et al., 1994Go). Since many of the proteins in the neurotrophin-signaling cascade contain significant amounts of sulfhydryl groups, they may be targets for these mercury compounds. This alteration in neurotrophin signaling may then lead to a disruption in differentiation. Previously, we have shown that gestational exposure to methylmercury affects a number of steps in the neurotrophin signal-transduction cascade, including a decrease in the neurotrophin receptor, trk, (Barone et al., 1998Go), alterations in PKC isoform expression and activity (Haykal-Coates et al., 1998Go) and neurotrophin- and carbachol-stimulated phosphatidylinositide hydrolysis (Mundy et al., 2000Go) in a regionally- and temporally-specific manner.

Cell Viability
While differences in the effects of exposure to species of mercury were evident when examining neurite outgrowth in unprimed cells, the differential effect of the mercury species was even more evident when we examined cell viability. In unprimed cells, CH3Hg was approximately 6 times more toxic than HgCl2 in the presence of NGF, and 40 times more toxic than HgCl2 in the absence of NGF. Similar results were obtained in the primed cells, with CH3Hg being more toxic than HgCl2 in both the presence and absence of NGF. CH3Hg is probably a more potent toxicant than HgCl2, because HgCl2 is less efficient at penetrating the plasma membrane than CH3Hg (Lakowicz and Anderson, 1980Go; Nakada et al., 1982).

The effects of CH3Hg were also dependent on the condition of the cells. In the absence of NGF, CH3Hg was more cytotoxic in unprimed PC12 cells compared to primed PC12 cells (0.21 and 0.73 µM, respectively). This pattern was similar to that observed by Kunimoto et al., (1992), except that EC50 values of 1.3 and 4.8 µM were obtained in unprimed and primed PC12 cells, respectively. In that study, the EC50's were approximately 6 times higher than in the present study. The increased potency of CH3Hg in this study as compared to previous work could be due to differences in cell culturing conditions. Kunimoto et al. (1992) cultured their PC12 cells in medium that contained 5% horse serum and 5% newborn calf serum, which is in contrast to serum-free culture medium used in this study. This differential sensitivity of primed versus unprimed cells might result from state-dependent differences that relate to the degree of differentiation and the history of exposure to NGF. For example, certain enzyme systems are induced in PC12 cells by NGF, including catalase and glutathione peroxidase (Sampath et al., 1994Go), which are responsible for decreased sensitivity of primed PC12 cells. NGF has also been shown to protect against apoptosis following exposure to various compounds (Kamata et al., 1996Go; Nakajima et al., 1994Go; Satoh et al., 1996Go; Spear et al., 1997Go). Thus, NGF can provide protection against cytotoxicity in PC12 cells.

Acute exposure to NGF also affected the sensitivity of PC12 cells to CH3Hg-induced cytotoxicity, since PC12 cells that did not have any NGF during the 24-h exposure period were more sensitive than PC12 cells exposed to NGF. Again, this may be attributed to NGF's ability to promote survival as well as regulate and modulate differentiation in the PC12 cells (Jackson et al., 1990Go) and some investigators have suggested that NGF acts to help stabilize the reorganization of cytoskeletal elements of cells (Heidemann et al., 1985Go).

At concentrations which produced cytotoxicity (0.3 to 3 µM), CH3Hg exposure resulted in a decrease in cell-body area, a morphological characteristic that would suggest apoptosis. Apoptosis has also observed in vitro in organotypic cultures of cerebellar slices (Kunimoto and Suzuki, 1997Go), cerebellar granule cell cultures (Bulleit et al., 1998) and in vivo (Nagashima et al., 1996Go) following exposure to high concentrations of CH3Hg (>3 µM). Future experiments could determine the commitment pathways by which PC12 cells undergo CH3Hg-induced cell death. Interestingly, the effects of HgCl2 on cell viability in the presence or absence of NGF were not significantly different, suggesting that NGF exposure and signaling may not play a significant role in the commitment to cell death produced by HgCl2.

Summary
Data from the present study indicated that significant morphological changes occurred in PC12 cells following acute exposures at relatively low concentrations to either CH3Hg or HgCl2. These changes in total neurite outgrowth occurred in a concentration range of 0.01–0.1 µM (0.002–0.02 ppm), which was significantly lower than the concentration range that resulted in overt cytotoxicity (1–10 µM; 0.25–2.5 ppm). Much of the description of neuropathological effects in humans following developmental CH3Hg poisoning has come from high-level exposures (12–20 ppm brain concentrations) but morphological changes were observed at much lower brain concentrations (< 3 ppm) in animal models (Burbacher et al., 1990Go; Khera et al., 1974Go; Nonaka, 1969Go). In both humans and animal models, the morphological changes observed included decreases in brain size, loss of neurons and myelin in the cerebellum and cortex, and neurobehavioral alterations (Burbacher et al., 1990Go). Because of the obvious differences that exist between in vivo and in vitro exposures, it is often difficult to interpret the potential in vivo significance of an in vitro result, particularly with mercury species that are highly reactive compounds. However the present results provide a characterization of the concentration response for differentiation and cytotoxicity that may provide some mechanistic insight to the effects observed previously following low-level exposures to mercury during critical windows of mammalian neural development. In addition, the results from this test system provide a foundation for future studies examining the role of mercury-induced alterations in cell-signaling pathways that regulate differentiation and apoptosis.


    ACKNOWLEDGMENTS
 
The authors are grateful for the technical assistance of Dr. Kaberi Das and Ms. Connie Meacham, statistical advice from Mr. Dennis House, and photographic assistance from Mr. Keith Tarpley. In addition, the critical suggestions of Dr. Timothy J. Shafer and Dr. Stephen G. Chaney on a previous draft of this manuscript are much appreciated. DKP received support from NIEHS Training Grant T32ES07126.


    NOTES
 
This manuscript was reviewed by the NHEERL, U.S. Environmental Protection Agency, and approved for publication. Opinions expressed or implied in this manuscript do not necessarily reflect U.S. EPA agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed. Fax: (919) 541–4849. E-mail: barone.stan{at}epa.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abdulla, E. M., Calaminici, M., and Campbell, I. C. (1995). Comparison of neurite outgrowth with neurofilament protein subunit levels in neuroblastoma cells following mercuric oxide exposure. Clin. Exp. Pharmacol. Physiol. 22, 362–363.[ISI][Medline]

Altin, J. G., Wetts, R., Riabowol, K. T., and Bradshaw, R. A. (1992). Testing the in vivo role of protein kinase C and c-fos in neurite outgrowth by microinjection of antibodies into PC12 cells. Mol. Biol. Cell 3, 323–333.[Abstract]

Amin-Zaki, L., Elhassani, S., Majeed, M. A., Clarkson, T. W., Doherty, R. A., and Greenwood, M. (1974). Intra-uterine methylmercury poisoning in Iraq. Pediatrics 54, 587–595.[Abstract]

Bakir, F., Damluji, S. F., Amin-Zaki, L., Murtadha, M., Khalidi, A., al-Rawi, N. Y., Tikriti, S., Dahahir, H. I., Clarkson, T. W., Smith, J. C., and Doherty, R. A. (1973). Methylmercury poisoning in Iraq. Science 181, 230–241.[ISI][Medline]

Barone, S., Jr., Haykal-Coates, N., Parran, D. K., and Tilson, H. A. (1998). Gestational exposure to methylmercury alters the developmental pattern of trk-like immunoreactivity in the rat brain and results in cortical dysmorphology. Brain Res. Dev. Brain Res. 109, 13–31.[ISI][Medline]

Bartolome, J., Grignolo, A., Bartolome, M., Trepanier, P., Lerea, L., Weigel, S., Whitmore, W., Michalopoulos, G., Kavlock, R., and Slotkin, T. (1985). Postnatal methyl mercury exposure: Effects on ontogeny of renal and hepatic ornithine decarboxylase responses to trophic stimuli. Toxicol. Appl. Pharmacol. 80, 147–154.[ISI][Medline]

Bulleit, R. F., and Cui, H. (1998). Methylmercury antagonizes the survival-promoting activity of insulin-like growth factor on developing cerebellar granule neurons. Toxicol. Appl. Pharmacol. 153, 161–168.[ISI][Medline]

Burbacher, T. M., Rodier, P. M., and Weiss, B. (1990). Methylmercury developmental neurotoxicity: A comparison of effects in humans and animals. Neurotoxicol. Teratol. 12, 191–202.[ISI][Medline]

Burstein, D. E., and Greene, L. A. (1978). Evidence for RNA synthesis-dependent and -independent pathways in stimulation of neurite outgrowth by nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 75, 6059–6063.[Abstract]

Chang, L. W. (1977). Neurotoxic effects of mercury—a review. Environ. Res. 14, 329–373.[ISI][Medline]

Chang, L. W., Reuhl, K. R., and Lee, G. W. (1977). Degenerative changes in the developing nervous system as a result of in utero exposure to methylmercury. Environ. Res. 14, 414–423.[ISI][Medline]

Choi, B. H., Kudo, M., and Lapham, L. W. (1981). A Golgi and electron-microscopic study of cerebellum in methylmercury-poisoned neonatal mice. Acta Neuropathol. 54, 233–237.[ISI][Medline]

Choi, B. H., Lapham, L. W., Amin-Zaki, L., and Saleem, T. (1978). Abnormal neuronal migration, deranged cerebral cortical organization, and diffuse white matter astrocytosis of human fetal brain: A major effect of methylmercury poisoning in utero. J. Neuropathol. Exp. Neurol. 37, 719–733.[ISI][Medline]

Coleman, E. S., and Wooten, M. W. (1994). Nerve growth factor-induced differentiation of PC12 cells employs the PMA-insensitive protein kinase C-zeta isoform. J. Mol. Neurosci. 5, 39–57.[ISI][Medline]

Cowley, S., Paterson, H., Kemp, P., and Marshall, C. J. (1994). Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells. Cell 77, 841–852.[ISI][Medline]

Crump, K. S., Kjellstrom, T., Shipp, A. M., Silvers, A., and Stewart, A. (1998). Influence of prenatal mercury exposure upon scholastic and psychological test performance: Benchmark analysis of a New Zealand cohort. Risk Anal. 18, 701–713.[ISI][Medline]

Crump, K. S., Van Landingham, C., Shamlaye, C., Cox, C., Davidson, P. W., Myers, G. J., and Clarkson, T. W. (2000). Benchmark concentrations for methylmercury obtained from the Seychelles Child Development Study. Environ. Health Perspect. 108, 257–263.[ISI][Medline]

Crumpton, T. L., Atkins, D., Zawia, N., and Barone, S., Jr. (2000). Lead exposure in pheochromocytoma (PC12) cells alters neural differentiation and Sp1 DNA-binding. Neurotoxicology (in press).

Das, K. D., and Barone, S., Jr. (1999). Neuronal differentiation in PC12 cells is inhibited by chlorpyrifos and its metabolites: Is acetylcholinesterase inhibition the site of action? Toxicol. Appl. Pharmacol. 160, 217–230.[ISI][Medline]

Daston, G. P., Gray, J. A., Carver, B., and Kavlock, R. J. (1984). Toxicity of mercuric chloride to the developing rat kidney: II. Effect of increased dosages on renal function in suckling pups. Toxicol. Appl. Pharmacol. 74, 35–45.[ISI][Medline]

Daston, G. P., Kavlock, R. J., Rogers, E. H., and Carver, B. (1983). Toxicity of mercuric chloride to the developing rat kidney: I. Postnatal ontogeny of renal sensitivity. Toxicol. Appl. Pharmacol. 71, 24–41.[ISI][Medline]

Daston, G. P., Rehnberg, B. F., Hall, L. L., and Kavlock, R. J. (1986). Toxicity of mercuric chloride to the developing rat kidney: III. Distribution and elimination of mercury during postnatal maturation. Toxicol. Appl. Pharmacol. 85, 39–48.[ISI][Medline]

Davidson, P. W., Myer, G. J., Shamlaye, C., Cox, C., Gao, P., Axtell, C., Morris, D., Sloane-Reeves, J., Cernichiari, E., Choi, A., Palumbo, D., and Clarkson, T. W. (1999). Association between prenatal exposure to methylmercury and developmental outcomes in Seychellois children: Effect modification by social and environmental factors. Neurotoxicology 20, 833–841.[ISI][Medline]

Davis, L. E., Kornfeld, M., Mooney, H. S., Fiedler, K. J., Haaland, K. Y., Orrison, W. W., Cernichiari, E., and Clarkson, T. W. (1994). Methylmercury poisoning: Long-term clinical, radiological, toxicological, and pathological studies of an affected family. Ann. Neurol. 35, 680–688.[ISI][Medline]

Dolbec, J., Mergler, D., Sousa-Passos, C. J., Sousa, de Morais, S., and Lebel, J. (2000). Methylmercury exposure affects motor performance of a riverine population of the Tapajos river, Brazilian Amazon. Int. Arch. Occup. Environ. Health 73, 195–203.[ISI][Medline]

Fujita, K., Lazarovici, P., and Guroff, G. (1989). Regulation of the differentiation of PC12 pheochromocytoma cells. Environ. Health Perspect. 80, 127–142.[ISI][Medline]

Graff, R. D., Falconer, M. M., Brown, D. L., and Reuhl, K. R. (1997). Altered sensitivity of post-translationally modified microtubules to methylmercury in differentiating embryonal carcinoma-derived neurons. Toxicol. Appl. Pharmacol. 144, 215–224.[ISI][Medline]

Grandjean, P., Budtz-Jorgensen, E., White, R. F., Jorgensen, P. J., Weihe, P., Debes, F., and Keiding, N. (1999a). Methylmercury exposure biomarkers as indicators of neurotoxicity in children aged 7 years. Am. J. Epidemiol. 150, 301–305.[Abstract]

Grandjean, P., Weihe, P., White, R. F., and Debes, F. (1998). Cognitive performance of children prenatally exposed to "safe" levels of methylmercury. Environ. Res. 77, 165–172.[ISI][Medline]

Grandjean, P., White, R. F., Nielsen, A., Cleary, D., and de Oliveira Santos, E. C. (1999b). Methylmercury neurotoxicity in Amazonian children downstream from gold mining. Environ. Health Perspect. 107, 587–591.[ISI][Medline]

Greene, L. A., and Tischler, A. S. (1976). Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. U.S.A. 73, 2424–2428.[Abstract]

Haykal-Coates, N., Shafer, T. J., Mundy, W. R., and Barone, S., Jr. (1998). Effects of gestational methylmercury exposure on immunoreactivity of specific isoforms of PKC and enzyme activity during post-natal development of the rat brain. Brain Res. Dev. Brain Res. 109, 33–49.[ISI][Medline]

Heidemann, S. R., Joshi, H. C., Schechter, A., Fletcher, J. R., and Bothwell, M. (1985). Synergistic effects of cyclic AMP and nerve growth factor on neurite outgrowth and microtubule stability of PC12 cells. J. Cell. Biol. 100, 916–927.[Abstract]

Jackson, G. R., Apffel, L., Werrbach-Perez, K., and Perez-Polo, J. R. (1990). Role of nerve-growth factor in oxidant-antioxidant balance and neuronal injury: I. Stimulation of hydrogen peroxide resistance. J. Neurosci. Res. 25, 360–368.[ISI][Medline]

Kamata, H., Tanaka, C., Yagisawa, H., and Hirata, H. (1996). Nerve growth factor and forskolin prevent H2O2-induced apoptosis in PC12 cells by glutathione independent mechanism. Neurosci. Lett. 212, 179–182.[ISI][Medline]

Kavlock, R. J., and Gray, J. A. (1983). Morphometric, biochemical, and physiological assessment of perinatally induced renal dysfunction. J. Toxicol. Environ. Health 11, 1–13.[ISI][Medline]

Khera, K. S. (1973). Teratogenic effects of methylmercury in the cat: Note on the use of this species as a model for teratogenicity studies. Teratology 8, 293–303.[ISI][Medline]

Khera, K. S., Iverson, F., Hierlihy, L., Tanner, R., and Trivett, G. (1974). Toxicity of methylmercury in neonatal cats. Teratology 10, 69–76.[ISI][Medline]

Khera, K. S., and Tabacova, S. A. (1973). Effects of methylmercuric chloride on the progeny of mice and rats treated before or during gestation. Food Cosmet. Toxicol. 11, 245–254.[ISI][Medline]

Kunimoto, M., and Suzuki, T. (1997). Migration of granule neurons in cerebellar organotypic cultures is impaired by methylmercury. Neurosci. Lett. 226, 183–186.[ISI][Medline]

Lagunowich, L. A., Bhambhani, S., Graff, R. D., and Reuhl, K. R. (1991). Cell adhesion molecules in the cerebellum: Targets of methylmercury toxicity? Soc. for Neuroscience 17, 715.

Lakowicz, J. R., and Anderson, C. J. (1980). Permeability of lipid bilayers to methylmercuric chloride: Quantification by fluorescence quenching of a carbazole-labeled phospholipid. Chem. Biol. Interact. 30, 309–323.[ISI][Medline]

Lärkfors, L., Oskarsson, A., Sundberg, J., and Ebendal, T. (1991). Methylmercury induced alterations in the nerve growth factor level in the developing brain. Brain Res. Dev. 62, 287–291.[ISI][Medline]

Lebel, J., Mergler, D., Branches, F., Lucotte, M., Amorim, M., Larribe, F., and Dolbec, J. (1998). Neurotoxic effects of low-level methylmercury contamination in the Amazonian Basin. Environ. Res. 79, 20–32.[ISI][Medline]

Matsumoto, H., Koya, G., and Takeuchi, T. (1965). Fetal Minamata disease: A neuropathological study of two cases of intrauterine intoxication by a methyl mercury compound. J. Neuropathol. Exp. Neurol. 24, 563–574.[ISI][Medline]

Mottet, N. K., Shaw, C., and Burbacher, T. M. (1987). The pathological lesions of methyl mercury intoxication in monkeys. In The Toxicity of Methyl Mercury. (C.U. Eccles and Z. Annau, Eds.), pp. 73–103. Johns Hopkins University Press, Baltimore, MD.

Mundy, W. R., Parran, D. K., and Barone, S., Jr. (2000). Gestational exposure to methylmercury alters the developmental pattern of neurotrophin- and neurotransmitter-induced phosphoinositide (PI) hydrolysis. Neurotoxicity Research 1, 271–283.

Myers, G. J., Marsh, D. O., Davidson, P. W., Cox, C., Shamlaye, C. F., Tanner, M., Choi, A., Cernichiari, E., Choisy, O., and Clarkson, T. W. (1995). Main neurodevelopmental study of Seychellois children following in utero exposure to methylmercury from a maternal fish diet: Outcome at six months. Neurotoxicology 16, 653–664.[ISI][Medline]

Nagashima, K., Fujii, Y., Tsukamoto, T., Nukuzuma, S., Satoh, M., Fujita, M., Fujioka, Y., and Akagi, H. (1996). Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats. Acta Neuropathol. 91, 72–77.[ISI][Medline]

Nakada, S., and Imura, N. (1982). Uptake of methylmercury and inorganic mercury by mouse glioma and mouse neuroblastoma cells. Neurotoxicology 3, 249–258.[ISI][Medline]

Nakada, S., Saito, H., and Imura, N. (1981). Effect of methylmercury and inorganic mercury on the nerve growth factor-induced neurite outgrowth in chick embryonic sensory ganglia. Toxicol. Lett. 8, 23–28.[ISI][Medline]

Nakajima, M., Kashiwagi, K., Ohta, J., Furukawa, S., Hayashi, K., Kawashima, T., and Hayashi, Y. (1994). Nerve growth factor and epidermal growth factor rescue PC12 cells from programmed cell death induced by etoposide: Distinct modes of protection against cell death by growth factors and a protein-synthesis inhibitor. Neurosci. Lett. 176, 161–164.[ISI][Medline]

Nicotera, P., Dypbukt, J. M., Rossi, A. D., Manzo, L., and Orrenius, S. (1992). Thiol modification and cell signalling in chemical toxicity. Toxicol. Lett. 64–65, 563–567.

Nonaka, I. (1969). An electron microscopic study on the experimental congenital Minamata disease in the rat. Kumamoto Med. J. 22, 27–40.[Medline]

Obermeier, A., Bradshaw, R. A., Seedorf, K., Choidas, A., Schlessinger, J., and Ullrich, A. (1994). Neuronal differentiation signals are controlled by nerve growth factor receptor/Trk binding sites for SHC and PLC gamma. EMBO J. 13, 1585–1590.[Abstract]

Ou, Y. C., Thompson, S. A., Kirchner, S. C., Kavanagh, T. J., and Faustman, E. M. (1997). Induction of growth arrest and DNA damage-inducible genes Gadd45 and Gadd153 in primary rodent embryonic cells following exposure to methylmercury. Toxicol. Appl. Pharmacol. 147, 31–38.[ISI][Medline]

Pandiella-Alonso, A., Malgaroli, A., Vicentini, L. M., and Meldolesi, J. (1986). Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells. FEBS Lett. 208, 48–51.[ISI][Medline]

Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995). Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J. Biol. Chem. 270, 13585–13588.[Abstract/Free Full Text]

Pendergrass, J. C., Haley, B. E., Vimy, M. J., Winfield, S. A., and Lorscheider, F. L. (1997). Mercury vapor inhalation inhibits binding of GTP to tubulin in rat brain: Similarity to a molecular lesion in Alzheimer diseased brain. Neurotoxicology 18, 315–324.[ISI][Medline]

Reuhl, K., and Chang, L. W. (1979). Effects of methylmercury on the development of the nervous system: A review. Neurotoxicology 1, 21–55.[ISI]

Reuhl, K. R., Chang, L. W., and Townsend, J. W. (1981). Pathological effects of in utero methylmercury exposure on the cerebellum of the golden hamster: II. Residual effects on the adult cerebellum. Environ. Res. 26, 307–327.[ISI][Medline]

Rice, D. C. (1989). Delayed neurotoxicity in monkeys exposed developmentally to methylmercury. Neurotoxicology 10, 645–650.[ISI][Medline]

Rice, D. C. (1996). Evidence for delayed neurotoxicity produced by methylmercury. Neurotoxicology 17, 583–596.[ISI][Medline]

Rossi, A., Manzo, L., Orrenius, S., Vahter, M., and Nicotera, P. (1991). Modifications of cell signalling in the cytotoxicity of metals. Pharmacol. Toxicol. 68, 424–429.[ISI][Medline]

Sager, P. R., Aschner, M., and Rodier, P. M. (1984). Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure. Brain Res. 314, 1–11.[Medline]

Sager, P. R., Doherty, R. A., and Rodier, P. M. (1982). Effects of methylmercury on developing mouse cerebellar cortex. Exp. Neurol. 77, 179–193.[ISI][Medline]

Sampath, D., Jackson, G. R., Werrbach-Perez, K., and Perez-Polo, J. R. (1994). Effects of nerve growth factor on glutathione peroxidase and catalase in PC12 cells. J. Neurochem. 62, 2476–2479.[ISI][Medline]

Satoh, T., Sakai, N., Enokido, Y., Uchiyama, Y., and Hatanaka, H. (1996). Free radical-independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide-triggered apoptosis. J. Biochem. 120, 540–546.[Abstract]

Schubert, D. (1978). NGF-induced alterations in protein secretion and substrate-attached material of a clonal nerve cell line. Brain Res. 155, 196–200.[ISI][Medline]

Shaughnessy, L. W., and Barone, S. J. (1997). Damage to the NBM leads to a sustained lesion-induced increase in functional NGF in the cortex. Neuroreport 8, 2767–2774.[ISI][Medline]

Söderström, S., and Ebendal, T. (1995). In vitro toxicity of methyl mercury: Effects on nerve growth factor (NGF)-responsive neurons and on NGF synthesis in fibroblasts. Toxicol. Lett. 75, 133–144.[ISI][Medline]

Spear, N., Estevez, A. G., Barbeito, L., Beckman, J. S., and Johnson, G. V. (1997). Nerve growth factor protects PC12 cells against peroxynitrite-induced apoptosis via a mechanism dependent on phosphatidylinositol 3-kinase. J. Neurochem. 69, 53–59.[ISI][Medline]

Stephens, R. M., Loeb, D. M., Copeland, T. D., Pawson, T., Greene, L. A., and Kaplan, D. R. (1994). Trk receptors use redundant signal transduction pathways involving SHC and PLC-gamma 1 to mediate NGF responses. Neuron 12, 691–705.[ISI][Medline]

Takeuchi, T., Kambara, T., Marikawa, N., Matsumoto, H., Shiraishi, Y., and Ito, H. (1959). Pathological observation of the Minamata Disease. Acta Pathol. Jpn. 9, 768–783.

Tsukada, Y., Chiba, K., Yamazaki, M., and Mohri, T. (1994). Inhibition of the nerve growth factor-induced neurite outgrowth by specific tyrosine kinase and phospholipase inhibitors. Biol. Pharm. Bull. 17, 370–375.[ISI][Medline]

Vignes, M., Guiramand, J., Sassetti, I., and Recasens, M. (1993). Effect of thiol reagents on phosphoinositide hydrolysis in rat brain synaptoneurosomes. Eur. J. Neurosci. 5, 327–334.[ISI][Medline]

Weihe, P., Grandjean, P., Debes, F., and White, R. (1996). Health implications for Faroe islanders of heavy metals and PCBs from pilot whales. Sci. Total Environ. 186, 141–148.[ISI][Medline]

Windebank, A. J. (1986). Specific inhibition of myelination by lead in vitro; comparison with arsenic, thallium, and mercury. Exp. Neurol. 94, 203–212.[ISI][Medline]