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Toxicology and Applied Pharmacology 240 (2009) 265–272
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Toxicology and Applied Pharmacology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y t a a p
Characterization of the effects of methylmercury on Caenorhabditis elegans
Kirsten J. Helmcke a, Tore Syversen b, David M. Miller III c, Michael Aschner a,d,⁎
Pharmacology Department, Vanderbilt University Medical Center, Nashville, TN, USA
Department of Neuromedicine, Norwegian University of Science and Technology, Trondheim, Norway
Department of Cell and Developmental Biology and Program in Neuroscience, Vanderbilt University Medical Center, Nashville, TN, USA
Pediatrics Department, Vanderbilt University Medical Center, Nashville, TN, USA
a r t i c l e i n f o a b s t r a c t
Article history: The rising prevalence of methylmercury (MeHg) in seafood and in the global environment provides an
Received 9 February 2009 impetus for delineating the mechanism of the toxicity of MeHg. Deleterious effects of MeHg have been widely
Revised 11 March 2009 observed in humans and in other mammals, the most striking of which occur in the nervous system. Here we
Accepted 23 March 2009
test the model organism, Caenorhabditis elegans (C. elegans), for MeHg toxicity. The simple, well-defined
Available online 31 March 2009
anatomy of the C. elegans nervous system and its ready visualization with green fluorescent protein (GFP)
markers facilitated our study of the effects of methylmercuric chloride (MeHgCl) on neural development.
Caenorhabditis elegans Although MeHgCl was lethal to C. elegans, induced a developmental delay, and decreased pharyngeal
Methylmercury pumping, other traits including lifespan, brood size, swimming rate, and nervous system morphology were not
obviously perturbed in animals that survived MeHgCl exposure. Despite the limited effects of MeHgCl on C.
elegans development and behavior, intracellular mercury (Hg) concentrations (≤3 ng Hg/mg protein) in
MeHgCl-treated nematodes approached levels that are highly toxic to mammals. If MeHgCl reaches these
concentrations throughout the animal, this finding indicates that C. elegans cells, particularly neurons, may be
less sensitive to MeHgCl toxicity than mammalian cells. We propose, therefore, that C. elegans should be a
useful model for discovering intrinsic mechanisms that confer resistance to MeHgCl exposure.
© 2009 Elsevier Inc. All rights reserved.
Introduction and occipital lobe damage (Clarkson and Magos, 2006), whereas
younger individuals experience global alterations to the brain,
Mercury (Hg) is a toxicant to which humans are exposed regularly. including microcephaly and inhibition of neuronal migration, leading
Major routes of Hg exposure to humans include inhalation of Hg vapor to distortion of cortical layers, cerebellar abnormalities, alterations in
released from amalgam dental fillings and consumption of seafood glial cells, and alterations in neurotransmitter systems (Clarkson,
containing methylmercury (MeHg) (Clarkson, 2002; Clarkson and 2002; Clarkson and Magos, 2006; Roh et al., 2006). Although MeHg
Magos, 2006). Thimerosal, which contains ethylmercury (EtHg), is possesses high affinity for cysteine, allowing it to bind thiol groups,
used as a preservative in some vaccines and, although some limited the specific molecular targets of MeHg are largely unknown (Kerper
evidence indicates that there may be a link between thimerosal and et al., 1992; Simmons-Willis et al., 2002).
autism (Geier and Geier, 2006), this link has been largely discredited Despite many years of investigation, numerous questions surround
(Parker et al., 2004; Thompson et al., 2007). The presence of MeHg in the mechanisms of MeHg toxicity in mammals. Investigators have
seafood is caused by global cycling and bioaccumulation of the taken various approaches to study MeHg toxicity using many model
toxicant (Fitzgerald and Clarkson, 1991; Mason et al., 2005). MeHg is systems including rat, mouse, zebrafish, and cell culture. We have
of particular concern due to its ability to pass through the blood-brain adopted an alternative approach of using the model organism, Cae-
and placental barriers where it molecularly mimics methionine and norhabditis elegans (C. elegans), to study MeHg toxicity. C. elegans has
enters cells via the large amino acid transporter, LAT1 (Kerper et al., been used extensively in biological research and provides many
1992; Simmons-Willis et al., 2002; Yin et al., 2008), allowing MeHg to advantages, including its small size, rapid life cycle, self-fertilization,
accumulate in both the brain and the fetus. MeHg has varying effects and ready genetic manipulation; the C. elegans nervous system has
on the nervous system based on age at exposure. Adults exposed to been mapped, and its genome fully sequenced (Sulston and Horvitz,
MeHg experience focal lesions, such as loss of cerebellar granular cells 1977; Sulston, 1983; Sulston et al., 1983; White et al., 1986; Wood,
1988; C. elegans sequencing consortium, 1998). Earlier studies of
⁎ Corresponding author. Vanderbilt University, 11415 MRB IV, 2215 Garland Avenue,
toxicity in C. elegans have revealed high predictive value for
Nashville, TN 37232-0414. Fax: +1 615 936 4080. mammalian systems (Williams and Dusenbery, 1988; National
E-mail address: [email protected] (M. Aschner). Research Council, 2000; Cole et al., 2004; Leung et al., 2008). In
0041-008X/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
266 K.J. Helmcke et al. / Toxicology and Applied Pharmacology 240 (2009) 265–272
addition to measurements investigating effects on the overall health appearance and ability to move in response to poking with a platinum
of C. elegans (lethality, life span, brood size, behavior, etc.), some wire (Bischof et al., 2006; Roh et al., 2007).
assessments included determination of gene induction using reporter
strains and protection afforded by a particular gene through the use of Determination of Hg content. C. elegans larvae were treated with
knockout, over-expression strains, RNAi, or mutagenesis experiments MeHgCl as described above. After 24 h of culture on OP50-containing
(Leung et al., 2008). NGM plates, both live and dead animals were collected and washed
We used C. elegans to study MeHg toxicity and tested several twice with deionized water. For L1 treatments, approximately 10,000
different endpoints including lethality, Hg content, lifespan, brood animals were pooled and assessed, for L4 treatments, approximately
size, body length, overall development, swimming behavior, and 900 animals were pooled and assessed. As expected, protein content
pharyngeal pumping rate. We also used green fluorescent protein was higher in samples treated with lower concentrations of MeHgCl.
(GFP) markers for specific neuronal populations to assess the Average protein content per sample was approximately 110 mg,
development and appearance of the nervous system following ranging from 16 mg to 254 mg. The pelleted pool of live and dead
methylmercuric chloride (MeHgCl) insult. worms was sonicated and a small aliquot was used for protein
Our studies revealed that Hg approached levels (≤3 ng Hg/mg measurement; the remainder of the sample was used for inductively
protein) in C. elegans tissues that are highly toxic to mammals (for coupled plasma-mass spectrometry (ICP-MS) analysis of Hg content.
example, in rat brain, 0.05 ppm resulted in significant structural Although it is possible that some demethylation occurred during the
alterations (Falluel-Morel et al., 2007)). Although exposure to study, it is unlikely that an appreciable amount of inorganic Hg was
MeHgCl induced dose-dependent developmental delay and lethality, formed. This would be an interesting extension of this research,
surviving animals were surprisingly unaffected. The absence of however, due to small sample size, information regarding the
observable defects in development or morphology in the C. elegans potential demethylation of MeHg could not be collected in this
nervous system is particularly noteworthy given the sensitivity of study. Protein content was determined following manufacturer
mammalian neurons to MeHg. Our results indicate that C. elegans instructions for a bicinchoninic acid (BCA) protein assay kit (Pierce,
may exhibit unique mechanisms for detoxifying, trafficking, or Rockford IL). Preparation of the sample for ICP-MS involved addition
metabolizing MeHgCl that render its nervous system resistant or of nitric acid followed by heat digestion and dilution of the samples
inaccessible to MeHg. with water. The samples were digested in PP tubes (352059, BD) in a
block heater after addition of 65% HNO3 (Merck, Suprapur). The
Methods samples were transferred to Teflon tubes and digested in an UltraClave
(Milestone). After digestion the samples were diluted directly in the
C. elegans maintenance. C. elegans were grown on plates containing Teflon tubes with ultrapure water (PURLAB Ultra Analytic, Elga) to
nematode growth medium (NGM) seeded with Escherichia coli strain achieve a final acid concentration of 0.6 mol/L. High Resolution-
OP50 as previously described (Brenner, 1974). Unless otherwise noted, Inductively Coupled Plasma-Mass Spectrometry (HR-ICP-MS) analysis
hermaphroditic wildtype N2 Bristol strain was used for all was performed using a Thermo (Finnigan) model Element 2
experiments. Transgenic lines expressing promoter GFP reporters instrument (Bremen, Germany). The RF power was 1400 W. The
used in this study were: NW1229 F25B3.3∷GFP (a marker of Ras1 sample was introduced using an SC-2 with SC-FAST option auto
guanine nucleotide exchange factor, pan-neuronal GFP expression) sampler (ESI, NE, USA) with a peristaltic pump (pump speed 0.25 mL/
(Altun-Gultekin et al., 2001), LX929 unc-17∷GFP (a marker of a min). The instrument was calibrated using 0.6 mol/L HNO3 solutions
synaptic vesicle acetylcholine transporter, labels cholinergic neurons) of multielement standards at appropriate concentrations. Internal
(Chase et al., 2004), CZ1200 unc-25∷GFP (a marker of glutamic acid standards were not used. To check for possible drift in the instrument,
decarboxylase, labels GABAergic neurons) (Huang et al., 2002), EG1285 a standard solution with known elemental concentrations was
unc-47∷GFP (a marker of a transmembrane vesicular GABA analyzed for every 10 samples. In addition, blank samples (0.6 mol/
transporter, labels GABAergic neurons) (McIntire et al., 1997), TL8 L HNO3, Suprapur) were analyzed for approximately every 10
cat-1∷GFP (a marker of a synaptic vesicular monoamine transporter, samples. The samples were analyzed in random order, and the analyst
labels catecholaminergic neurons) (Colavita and Tessier-Lavigne, was not aware of the identity of the samples. Hg was determined in
2003), GR1333 tph-1∷GFP (a marker of tryptophan hydroxylase, labels the low resolution mode (M / Δm = 300).
serotonergic neurons) (Sze et al., 2000), DA1240 eat-4∷GFP (a marker
of vesicular glutamate transporter, labels glutamatergic neurons) Lifespan and brood size analysis. For lifespan assays, 40 live C.
(Asikainen et al., 2005) (all obtained from the Caenorhabditis Genetics elegans hermaphrodites from each MeHgCl concentration group were
Center, Minneapolis, MN) BY250 dat-1∷GFP (a marker of the dopamine picked to a fresh NGM plate 24 h following treatment. On each
transporter, labels dopaminergic neurons) (Nass et al., 2001), and succeeding day, worms were counted and scored as live or dead. Live
F49H12.4∷GFP (labels PVD neurons) (Watson et al., 2008). C. elegans were picked to fresh plates every day during egg-laying and
every other day once they ceased laying eggs until no live C. elegans
MeHgCl treatments. Animals were treated with an alkaline bleach remained. The experiment was carried out in quadruplicate.
solution to obtain a synchronous population prior to treatment with For brood size analyses, one live C. elegans was placed on each of
MeHgCl (Stiernagle, 1999) and synchronized populations of selected four NGM plates per treatment concentration 24 h after MeHgCl
larval stages (either L1 or L4) were treated. Treatment was conducted exposure. Every 24 h, this animal was transferred to a new NGM plate
by combining larvae (2500 L1s or 300 L4s), concentrated OP50, the until no new progeny were generated in a 24-hour period. The
appropriate volume of MeHgCl dissolved in water, and M9 buffer to a progeny on each of the fresh plates were counted and the experiment
volume of 500 μL in 1.7 mL siliconized tubes. Following the desired was carried out in quadruplicate. This approach allowed the
treatment duration (30 min to 15 h), animals were washed twice with measurement of the overall number of progeny generated and the
deionized water by centrifugation and placed on OP50-containing interval between MeHgCl exposure at different concentrations and
NGM plates. progeny generation.
Lethality. Following MeHgCl treatment and washing, animals were Measurement of size and developmental progress. Following
transferred (approximately 300 per plate) to 60 mm NGM plates treatment and washing, C. elegans were imaged on a Nikon Eclipse
seeded with OP50 and allowed to grow for 24 h. Animals were then 80i microscope. Body length was measured using Nikon Element
counted and scored as dead or alive. Viability was scored based on software to trace the body contour from the posterior bulb of the
K.J. Helmcke et al. / Toxicology and Applied Pharmacology 240 (2009) 265–272 267
stage and the progeny of those worms treated at the L4 stage were
observed using a Nikon Eclipse 80i microscope. Quantitative analysis
of dat-1∷GFP worms involved counting the number of head neurons
(4 CEPs and 2 ADEs), projections from CEP neurons to the tip of the
nose, and neurons in the C. elegans body (2 PDEs). Quantitative
analysis of unc-25∷GFP worms involved counting the number of head
neurons (4 RMEs), the number of neurons along the ventral nerve cord
(13 VDs and 6 DDs), the number of commissures traveling across the
body, and whether there were any breaks in the commissures or the
nerve cord. Other GFP strains (F25B3.3∷GFP, unc-17∷GFP, unc-47∷GFP,
cat-1∷GFP, tph-1∷GFP, eat-4∷GFP, F49H12.4∷GFP) were examined to
assess for obvious changes in overall structures of the labeled neurons.
Statistics. GraphPad Prism 4 was used to assess significance. For
dose–response, Hg content, brood size, pharyngeal pumping rate,
thrashing rate, body length, and neuronal quantification, ANOVA with
Bonferroni's Multiple Comparison Test was applied. For lifespan, log
rank test was applied. When p-values were lower than 0.05, groups
were considered significantly different, higher than 0.05 were not
considered significantly different.
C. elegans larvae are sensitive to MeHgCl
Dose–response curves were generated to test for dose-dependent
toxicity of MeHg to C. elegans. L1 and L4 larval stages were selected to
coincide with developmental processes in the worm (L1) and of the
germ line of the worm (L4). Worms treated for 30 min with MeHg at
the L1 stage [LC50 = 1.08 mM, n = 10 (throughout document, each ‘n’
is one separate experiment, usually conducted at least in triplicate)]
were significantly (p b 0.001) more sensitive to MeHg compared with
Fig. 1. Dose–response curve of lethality of MeHgCl to C. elegans. Worms treated at L1
(LC50 = 1.08, n = 10 p b 0.001) were more sensitive to the toxicant than worms treated worms treated at the L4 stage (LC50 = 4.51 mM, n = 6) (Fig. 1A).
at the L4 stage (LC50 = 4.51, n = 6) (A). Toxicity increased as exposure duration Additionally, increasing the duration of MeHg exposure in L4 worms
increased, L4 worms were treated for 15 h (LC50 = 0.33, n = 9), for 6 h (LC50 = 0.57, from 30 min to 6 h (LC50 = 0.57 mM, n = 6) and 15 h (LC50 = 0.33 mM,
n = 6), and for 30 min (LC50 = 4.51, n = 6) (B).
n = 9) statistically significantly (p b 0.001) increased the toxicity to C.
elegans, indicating that longer exposures are more lethal to C. elegans
pharynx to the anus. Twenty worms per treatment were also assessed (Fig. 1B).
for their development through the larval stages using the following
criteria: L1s had 4 or fewer gonadal cells, L2s had more than 4 gonadal Hg accumulates in a dose-dependent manner in animals treated with
cells and the gonad had begun to extend along the length of the MeHgCl
animal, L3 worms had undergone further extension of the gonad and
vulval morphogenesis had begun to occur, L4s displayed dorsal Hg content was measured for selected exposures for different
rotation of the gonad, and adults had observable eggs. treatments, including L1 treatment for 30 min and L4 treatment for
Behavioral analysis: pharyngeal pumping and thrashing rates.
Pharyngeal pumping rate was assessed using a Leica MZ16FA
microscope following MeHgCl treatment and washing. Pumps per
minute were manually counted following treatment with MeHgCl. To
test thrashing rates, C. elegans were placed in 10 μL of water on a Pyrex
Spot Plate and their behavior was videotaped through a microscope
for 3 min, as previously described (Matthies et al., 2006). Briefly, AVI
movies were generated using a frame grabber Piccolo graphics card
(Ingenieur Helfrich) and VidCap32 AVI capture application (Microsoft,
Redmond, CA). The movies were analyzed using a script written in
MatLab 7.0.1 (MathWorks, Natick, MA) to determine the position of
the worm in each frame using motion detection and selection of a
pixel designating the centroid of the worm (available upon request).
Worm oscillation over time was displayed following calculation of
movement in Hz. Four worms per treatment were tested in each
behavioral analysis.
Microscopic observation of neurons. GFP reporter strains were
Fig. 2. Hg content in C. elegans following MeHgCl exposure. Hg content was measured
treated with MeHg as described above (30-minute treatment of L1 as a function of sample protein content (n = 3). Hg content significantly increased as
and 15-hour treatment of L4 animals followed by washing and culture the duration of exposure to MeHgCl increased and as the MeHgCl treatment
on OP50-containing NGM plates). C. elegans treated at the L1 or L4 concentration increased.
268 K.J. Helmcke et al. / Toxicology and Applied Pharmacology 240 (2009) 265–272
significantly higher Hg content than control-treated worms
(⁎p b 0.05). Additionally, animals treated with 1 and 10 mM MeHgCl
contained significantly more Hg than those treated with 0.1 or
0.4 mM MeHg (⁎p b 0.05). Following a 6-hour treatment, L4 worms
treated with 0.4 mM MeHg contained significantly more Hg than the
control-treated worms (⁎p b 0.05). Following 15-hour treatment at L4
stage, control worms (0 mM MeHgCl) contain an average of 0.02 ng
Hg/mg protein, whereas those treated at 0.1 and 0.4 mM MeHgCl
contain an average of 0.45 and 3.34 ng Hg/mg protein, respectively
(p b 0.001 vs. controls). As duration of exposure increases, Hg
accumulation in C. elegans significantly increased in a time-
dependent manner (Fig. 2). For instance, when L4s were treated
for 30 min at 0.4 mM MeHgCl, the average Hg content was 0.29 ng
Hg/mg protein; when the duration of exposure increased to 6 h and
Fig. 3. Body length of C. elegans was shorter following treatment with MeHgCl. After 15 h, average Hg content increased to 0.81 and 3.34 ng Hg/mg
growth for 24 or 48 h, animals treated at either L1 or L4 stages with the toxicant were
significantly (⁎⁎⁎p b 0.001, n = 4) shorter than control animals, as measured using the
protein, respectively (p b 0.01). A comparison of the Hg content of L1s
Nikon Element software to measure their length in pixels (arbitrary units) according to and L4s treated for 30 min revealed that L1s had significantly lower
their body contour from the posterior bulb of the pharynx to the anus. levels of Hg (p b 0.01). This finding indicates that L1s may be more
sensitive to Hg than the dose–response curves (Fig. 1) revealed, as
they are killed at lower levels of internal Hg than are L4 animals with
30 min, 6 h, and 15 h (n = 3 for each treatment). Exposures tested comparable Hg content.
were selected to represent a range of doses that corresponded to a
low concentration (LC0), at least one medium concentration (LC20– MeHgCl does not alter lifespan or brood size of C. elegans
LC80), and at least one high concentration (LC100) for each of the
conditions tested. Hg content was not tested when dose–response For animals that survive exposure to MeHgCl, longevity did not
curves indicated death of all worms. The resulting values indicate seem to correlate with exposure dose (Supplemental Fig. 1). For
that there is an increase in Hg content with increased MeHgCl example, average lifespan following a 30-minute treatment of L1 C.
exposure (Fig. 2). Comparing the animals treated for 30 min and for elegans (Supplemental Fig. 1A) or a 15-hour treatment of L4 C.
15 h at L4, Hg content was significantly higher following a treatment elegans was 13–15 days (Supplemental Fig. 1B). Additionally, we
at 0.1 and 0.4 mM MeHgCl (p b 0.05) with the longer exposure. As the tested the lifespan of the progeny of L4 C. elegans treated for 15 h,
MeHgCl concentration to which the worms were exposed increased, which had an average lifespan of 15–17 days (Supplemental Fig. 1C).
Hg content also increased. After treatment of L1 animals for 30 min, None were significantly altered when using the log rank test to
worms treated at 1 mM MeHgCl contained significantly more Hg compare the control and MeHg-treatment groups (n = 5). In
than control-treated worms (⁎p b 0.05). After treatment of L4 worms measuring brood size, the same three populations (L1 30-minute
for 30 min, worms treated at all MeHgCl concentrations had treatment, L4 15-hour treatment, and progeny of L4 treatment) were
Fig. 4. C. elegans larvae were developmentally delayed following exposure to MeHgCl. Animals treated at higher concentrations of MeHgCl took longer to develop through the larval
stages and into adults following a 30-minute exposure at L1 stage (A–C) or a 15-hour exposure at L4 stage.
K.J. Helmcke et al. / Toxicology and Applied Pharmacology 240 (2009) 265–272 269
tested (Supplemental Fig. 2). Animals that underwent 15-hour L4
treatment had an overall decrease in brood size (progeny generation
of L1 30-minute treated worms at 0 mM MeHgCl was 279 ± 14, of L4
15-hour treated worms was 221 ± 11, and of progeny of L4 15-hour
treated worms was 243 ± 13). However, the only significant MeHg-
dependent alteration in brood size occurred when L1 30-minute
treated worms were exposed to 1 mM MeHgCl (187 ± 21 progeny
generated compared to 279 ± 14 progeny generated under control
conditions, p b 0.001). There were no other statistically significant
alterations in brood size (n = 6).
MeHgCl treatment retards C. elegans larval development
Following treatment with MeHgCl, C. elegans length was altered
in a dose-dependent manner, with higher MeHgCl doses correlating
with shorter length (Fig. 3). This observation prompted an
investigation into a potential developmental delay of C. elegans
following MeHgCl treatment. This study detected a corresponding
dose-dependent developmental delay (Fig. 4, Supplemental Table 1,
n = 5 experiments). Under normal conditions at 20 °C, C. elegans
embryogenesis takes 14 h, and then the worm undergoes a series of
molts at 29, 38, 47, and 59 h post fertilization (Hope, 1999).
Retarded development occurred in both the worms treated at the L1
stage for 30 min and those treated at L4 for 15 h. After growth for
24 h, control-treated L1 larvae had all reached the L2 stage while
many worms treated at higher concentrations of MeHgCl remained
L1s (Fig. 4A). This trend continued 48 (Fig. 4B) and 72 (Fig. 4C)
hours after treatment, when most worms had reached the adult
stage. This trend also occurred in animals treated for 15 h at the L4
stage (Fig. 4D–E). Many control-treated animals reached the adult
stage 24 h after treatment while those treated at higher MeHgCl had
remained L4s (Fig. 4D). At 48 h after treatment, all control-treated
worms had reached the adult stage, while only some of those
treated with higher MeHgCl concentrations had reached the adult
Fig. 5. Pharyngeal pumping rates of C. elegans decrease following MeHgCl exposure.
stage (Fig. 4E) (n = 5 experiments). Number of pharyngeal pumps per minute significantly decreased in a dose-dependent
manner following 30 minute MeHgCl exposure of L1 worms 48 h following treatment at
Pharyngeal pumping decreases following MeHgCl exposure, thrashing is 0.75 and 1 mM MeHgCl (⁎⁎p b 0.01, n = 12) and 72 h following treatment at 1 mM
unaffected MeHgCl (⁎p b 0.05, n = 12). Exposure of L4 worms for 15 h induced a decrease in
pharyngeal pumping rate 24 h following exposure at 0.4, 0.6, and 0.75 mM MeHg
(⁎p b 0.05, n = 11). No alteration in pharyngeal pumping rate was noted in progeny of
Pharyngeal pumping rates were significantly decreased in a dose- L4-treated animals (n = 8).
dependent manner following 15-hour treatment of L4 C. elegans with
MeHgCl (control-treated worms pumped at a rate of 230 ± 6 pumps
per minute 24 h following treatment while worms treated at 0.1 and
0.4 mM MeHgCl pumped at 168 ± 9 and 69 ± 11 pumps per minute, Alterations in neuronal morphology were not observed in worms that
respectively, p b 0.001, Fig. 5). Other researchers have demonstrated survived MeHgCl exposure
that at the L4 stage, C. elegans typically pump at a rate of 150–200
pumps per minute. The rate increases as they mature into adults and GFP markers were used to observe cholinergic, glutamatergic,
peaks 2 days later at 300–350 pumps per minute before declining as serotonergic, dopaminergic, and GABAergic neuronal populations for
the worm ages (Huang et al., 2004). Since the pumping rates potential alterations following MeHgCl insult. Animals were treated
observed in our experiments were lower than expected even for L4 with 0, 0.1, 0.4, and 1 mM MeHgCl for a 30-minute treatment at the L1
C. elegans, we do not attribute this decrease to the developmental stage, and a 15-hour treatment at the L4 stage. Live worms treated at
delay. A similar trend was observed in animals treated at the L1 stage the L1 stage were observed 24, 48, and 72 h following treatment and
for 30 min, and significant differences were noted between control worms treated at the L4 stage were observed 24 and 48 h following
worms and those treated at 0.4 and 1 mM MeHgCl (p b 0.05). The treatment. Additionally, progeny of L4-treated animals were observed
decreased pumping rate induced by MeHgCl could contribute to the once they reached the L4 stage. No obvious phenotypes were observed
decreased rate of development in worms. No alterations were seen in in these neuronal populations under any of the treatment paradigms.
the pumping rate of the progeny of C. elegans treated for 15 h at the L4 Due to ease of measurement because of a low cell number and
stage at any concentration tested (0.1, 0.2, 0.3, and 0.4 mM MeHgCl, readily available GFP markers, dopaminergic and GABAergic neuronal
n = 7). populations were quantitatively investigated. Analysis of the dopami-
Thrashing data showed no trends in MeHgCl-dependent altera- nergic system revealed no alteration in cell number [6 head neurons
tions on the swimming behavior of C. elegans (Supplemental Fig. 3). (Supplemental Fig. 4A) and 2 PDEs (Supplemental Fig. 4B)] or ability of
[There was one outlier among worms treated as L4s for 15 h at 0.1 mM projections to travel from the nerve ring to the tip of the nose
MeHgCl 24 h following treatment. Mean thrashing rate was 0.27 (Supplemental Fig. 4C) in worms surviving MeHgCl insult (Figs. 6A, B).
(p b 0.05) while thrashing means for all other groups ranged from 0.38 GABAergic analysis also revealed no alteration in cell number in the
to 0.65 and were not statistically significantly different from each head (Supplemental Fig. 4D) or nerve cord (Supplemental Fig. 4E),
other (n = 6, data not shown)]. ability of projections to pass across the body (Supplemental Fig. 4F), or
270 K.J. Helmcke et al. / Toxicology and Applied Pharmacology 240 (2009) 265–272
Fig. 6. Representative dopaminergic and GABAergic C. elegans neurons following MeHgCl insult. Dopaminergic (A, B) and GABAergic (C, D) cells and projections are identical under
control (A and C) and MeHgCl treatment (B and D) conditions (at all concentrations observed, 0.1, 0.4, and 1 mM MeHg) following treatment at L1 for 30 min or L4 for 15 h (n = 4).
Progeny of worms treated at L4 for 15 h were also unaffected (n = 4).
number of breaks in the commissures (Supplemental Fig. 4G) of C. registering Hg levels of 0–8 ppm, depending on dosage and duration
elegans surviving MeHgCl treatment (Figs. 4C, D). (Newland et al., 2006) and mice treated with MeHgCl having 0–3 ppm
when pregnant mice were exposed and their pups tested at various
Discussion postnatal days (Stringari et al., 2008). A single dose of 5 μg/g body-
weight MeHgCl in rat pups, resulting in brain Hg levels of
Here we describe our first experiments to probe the neurotoxicity approximately 0.05 ppm, produced extensive alterations in the
of MeHgCl in the model organism, C. elegans. No neuronal alterations brain, including reduced hippocampal size and cell number as well
were observed upon MeHgCl exposure, indicating that the C. elegans as deficits in learning (Falluel-Morel et al., 2007). Alterations in the C.
nervous system may possess unique mechanisms for dealing with the elegans nervous system would have been expected due to the body of
insult of this toxicant. However, the possibility does exist that MeHg is literat

Use: 0.0286