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NeuroToxicology xxx (2005) xxxxxx

Mitochondrial Mediated Thimerosal-Induced

Apoptosis in a Human Neuroblastoma
Cell Line (SK-N-SH)
Michelle L. Humphrey a, Marsha P. Cole b, James C. Pendergrass c,
Kinsley K. Kiningham a,*
a

Department of Pharmacology, Joan C. Edwards School of Medicine, Marshall University, 1542 Spring Valley Drive,
Huntington, WV 25704-9388, USA
b
Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40536, USA
c
Affinity Labeling Technologies, Inc., Lexington, KY 40508, USA
Received 6 December 2004

Abstract
Environmental exposure to mercurials continues to be a public health issue due to their deleterious effects on immune,
renal and neurological function. Recently the safety of thimerosal, an ethyl mercury-containing preservative used in
vaccines, has been questioned due to exposure of infants during immunization. Mercurials have been reported to cause
apoptosis in cultured neurons; however, the signaling pathways resulting in cell death have not been well characterized.
Therefore, the objective of this study was to identify the mode of cell death in an in vitro model of thimerosal-induced
neurotoxicity, and more specifically, to elucidate signaling pathways which might serve as pharmacological targets.
Within 2 h of thimerosal exposure (5 mM) to the human neuroblastoma cell line, SK-N-SH, morphological changes,
including membrane alterations and cell shrinkage, were observed. Cell viability, assessed by measurement of lactate
dehydrogenase (LDH) activity in the medium, as well as the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay, showed a time- and concentration-dependent decrease in cell survival upon thimerosal exposure.
In cells treated for 24 h with thimerosal, fluorescence microscopy indicated cells undergoing both apoptosis and oncosis/
necrosis. To identify the apoptotic pathway associated with thimerosal-mediated cell death, we first evaluated the
mitochondrial cascade, as both inorganic and organic mercurials have been reported to accumulate in the organelle.
Cytochrome c was shown to leak from the mitochondria, followed by caspase 9 cleavage within 8 h of treatment. In
addition, poly(ADP-ribose) polymerase (PARP) was cleaved to form a 85 kDa fragment following maximal caspase 3
activation at 24 h. Taken together these findings suggest deleterious effects on the cytoarchitecture by thimerosal and
initiation of mitochondrial-mediated apoptosis.

# 2005 Elsevier Inc. All rights reserved.

Keywords: Mercury; Thimerosal; Mitochondria; Neurotoxicity

INTRODUCTION
Thimerosal (thiomersal, merthiolate, sodium ethylmercury thiosalicylate), an ethyl mercury-containing
* Corresponding author. Tel.: +1 304 696 7314;
fax: +1 304 696 7391.
E-mail address: kiningham@marshall.edu (K.K. Kiningham).

antibacterial and antifungal agent, has been used as an

antiseptic and preservative in various formulations
including paints, topical medications, eye lens cleaners
and cosmetics since the 1930s. Concern has been raised
by the use of thimerosal in more than 30 vaccines
licensed in the United States (Elferink, 1999). With the
addition of several important vaccines over the last
decade and the use of multi-dose vials, exposure to

0161-813X/$ see front matter # 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuro.2005.03.008

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mercury has increased among infants, whose developing nervous systems are vulnerable to mercurial toxicity. Following a report by Ball et al. (2001),
suggesting that cumulative levels of mercury from
vaccinations during the first 6 months of life may
exceed EPA guidelines, efforts have been made to
minimize exposure to thimerosal. While removed from
most pediatric vaccines, thimerosal is still used in
influenza vaccinations that are recommended for
young children, ages 623 months, as well as pregnant
women. Furthermore, many vaccines given to children
in developing countries still contain thimerosal.
Acute effects resulting from thimerosal exposure
include, but are not limited to, hypersensitivity reactions, nephro- and neurotoxicity (Ball et al., 2001).
Using an autoimmune disease-sensitive murine model
(SJL/J), Hornig et al. (2004) reported that genetic
influences may play a role in thimerosal-induced neurotoxicity. Thimerosal-containing ophthalmic medications, when applied topically, lead to mercury
accumulation within the brain (Gasset et al., 1975).
In addition, an increase in inorganic mercury has been
observed in the brains of monkeys following daily
administration of thimerosal (Blair et al., 1975), and
also in the brains of mice following a single injection of
thimerosal (Harry et al., 2004). These findings may be
explained by the fact that organic mercurials can
undergo biotransformation reactions resulting in accumulation of inorganic mercury in the central nervous
system (CNS) (Norseth and Clarkson, 1970; Friberg
and Mottet, 1989; Suda and Takahashi, 1991). While
methyl mercury has a half-life of several days or weeks
in the human brain, the half-life of inorganic mercury
has been estimated to exceed 20 years (Sugita, 1978;
Aschner and Aschner, 1990). Ethyl and methyl mercury distribute similarly upon initial exposure; however, entry into the CNS is thought to occur faster with
methyl mercury as passage across the blood brain
barrier involves an active, rather than a passive, transport process.
Mercuric chloride (HgCl2) and methyl mercuric
chloride (MeHgCl) cause apoptosis in cultured neurons
and astrocytes (Kunimoto, 1994; Monnet-Tschudi,
1998); however, the signaling pathways resulting in
apoptosis have not been clearly elucidated in cells of
either neuronal or glial origin. In vivo studies utilizing
Wistar rats show that MeHg intoxication results in
apoptotic degeneration of cerebellar granule cells characterized by ultrastructural and DNA fragmentation
analysis; however, specific cellular mediators involved
in the programmed cell death pathway have not been
identified (Nagashima et al., 1996).

Exposure limits for thimerosal, set by the Environmental Protection Agency, are based upon prenatal
exposure to methyl mercury rather than postnatal
exposure to ethyl mercury. Therefore, our objective
was to determine if thimerosal could induce cell death
in an in vitro system consistent with pediatric exposure. We chose to address the question in a model
characterized by an immature neuronal cell type,
resembling the developing nervous system, in order
to define the mode (apoptosis versus necrosis) and
mechanism (mitochondrial dependent versus independent) of thimerosal-mediated cell death. Since
limited studies have been conducted to test actual
mercury levels in infants immediately following
administration of thimerosal-containing vaccines,
we used a range of concentrations to conduct
mechanistic studies in our model. Magos (2001)
identified blood mercury levels of 1.0 mg/mL to be
the lowest level causing symptoms of toxicity upon
exposure to ethyl mercury, with higher levels showing
severe symptoms. The highest concentration (5 mM)
used in our studies is consistent with the lowest toxic
mercury level identified by Magos. However, while
the concentrations in our studies were slightly higher
than those seen by Pichichero et al. (2002) at 328
days following vaccination, they may more closely
reflect the exposure levels immediately following
vaccination.

MATERIALS AND METHODS

Chemicals
Thiosalicylic acid (minimum 95%), thimerosal
(minimum 97% HPLC), sodium pyruvate, 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide,
ethidium bromide, EDTA and other salts were obtained
from Sigma-Aldrich Chemical Corp. (St. Louis, MO).
Hoescht 33342 was obtained from Calbiochem (San
Diego, CA). Cell culture media, fetal bovine serum and
antibiotics were obtained from Invitrogen Corp. (Carlsbad, CA).
Cell Culture
A human neuroblastoma cell line (SK-N-SH)
obtained from American Type Culture Collection
(HTB-11) was maintained in minimal essential medium (MEM) supplemented with 10% heat-inactivated
fetal bovine serum, 1% antibiotics (penicillin/streptomycin/neomycin), 1% non-essential amino acids and

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1 mM sodium pyruvate. Cells were grown at 37 8C in a

humidified atmosphere containing 5% CO2.

0.05  1000 (Loo and Rillema, 1998). Results shown

are representative of three experiments.

Cell Viability Assays

Fluorescent Microscopy

MTT
Cells were seeded in a 96-well flat-bottom plate at
a density of 4  104 cells per well in 100 mL of
medium. After 24 h, thimerosal-containing medium
was added to the cells with final concentrations of 1
5 mM in a total volume of 200 mL. Cells were treated
in replicates of five wells each for every experiment
(6 experiments total), with medium used as a blank.
Cells were treated for either 24 or 48 h. 3-(4,5dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was prepared by dissolving 5 mg/mL in
PBS (pH 7.4) and then sterile filtered. Four hours
before the end of the incubation period 10 mL of
MTT was added to each well. At the end of the
treatment period, 100 mL of 0.1 N isopropanol/HCl
was added to each well and mixed by repeated
pipetting to dissolve granules. Absorbance was measured on a plate reader (Molecular Devices Corp.,
Sunnyvale, CA) at a wavelength of 570 nm (Mosmann, 1983). Results were reported as percentage of
control.

SK-N-SH cells were plated in 6-well plates at a

density of 5  105 cells per well. After 24 h, medium
was removed and replaced with fresh medium containing either thiosalicylate (5 mM) or thimerosal (5 mM).
At the end of the treatment period (24 h), medium was
removed from each well, placed in 50 mL tubes, and
stored on ice. Wells were washed twice with PBS, and
each time the PBS was combined with the corresponding media on ice. After the washes, 2 mL of 1 mM
EDTA (in PBS) were added to each well and allowed to
incubate at room temperature. This suspension was
then added to the appropriate tubes on ice. Tubes were
centrifuged at 200  g for 10 min. The supernatant was
discarded, cells were washed in 1 mL PBS, and centrifuged as previously described. After removal of the
supernatant, cells were resuspended in 100 mL PBS.
Cells were then stained in suspension with Hoechst
33342 (2 mg/mL) and ethidium bromide (5 mg/mL) for
15 min. Cells were examined at 380 nm with a Microphot-SA microscope (Nikon) and Spot RT Software
(Spot Diagnostic Instruments, Inc., Sterling Heights,
MI) (Katsen et al., 1998). Apoptotic cells were identified by nuclear condensation and DNA fragmentation
(Squier and Cohen, 2000). Cells counts were performed on 100 cells on both treated (5 mM) and control
slides at 24 h. Slides shown are representative of
experiments performed at least three times.

LDH
Cells were seeded in 2 mL of medium in 6-well
plates. At 8090% confluency, cells were treated with
the following concentrations of thimerosal in replicates of three: 0.1, 1, 2.5, and 5 mM. Cells were
incubated for 24 or 48 h, followed by removal of
medium and centrifugation to pellet floating cells.
The supernatant was removed and placed in a separate tube on ice. Five hundred microliters of 0.2%
Triton X was added to each well, and cells were
scraped and added to the cells pelleted from the
medium. Each well was washed with 500 mL of
water, which was also added to the tube with the
cells. The tubes were vortexed for 1 min, centrifuged,
and placed on ice. b-NADH solution was prepared by
dissolving 2 mg b-NADH in 28.5 mL of 0.1 M potassium phosphate buffer. Fifty microliters of sample
was added to 920 mL b-NADH solution and incubated at room temperature for 20 min. After incubation, 30 mL sodium pyruvate solution was added and
the absorbance was read at a wavelength of 340 nm at
25 8C. Readings were taken every 15 s for 2 min to
determine the absorbance change per min, which was
used to calculate LDH activity with the following
equation: LDH activity (mmol/L) = dA/min/6.22/

Caspase-3 Protease Activity

Activation of caspase 3 was determined with the
ApoAlert assay kit (Clontech Laboratories, Palo Alto,
CA), which fluorometrically detects caspase 3 activity
by proteolytic cleavage of the fluorophore 7-amino-4trifluoromethyl coumarin (AFC) from the substrate
conjugate DEVD-AFC. Cells were plated (6  105)
in 100 mm dishes and treated with 05 mM thimerosal
for 024 h. Cells (2  106) were collected at various
time points after treatment, lysed on ice, and centrifuged (12,000 rpm  3 min), and the supernatants
were collected and stored at 70 8C until further
use. Cell lysates were incubated with DEVD-AFC in
the presence of dithiothreitol for 1 h at 37 8C. Detection of AFC was measured with a 400 nm excitation
filter and a 530 nm emission filter. Caspase 3 activity is
expressed as percent control. Results shown are representative of six individual experiments.

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Subcellular Fractionation
The mitochondrial fraction of SK-N-SH cells was
prepared by washing the cells twice in ice-cold PBS,
followed by resuspension in 5 mL of 0.25 M sucrose,
1 mM EGTA, and 10 mM Tris-HCl (pH 7.4) and
centrifuging at 500  g for 2 min at 4 8C. The supernatant was discarded and the cells were resuspended in
5 mL of the same buffer. The cells were homogenized
in a glass Teflon homogenizer with 10 up-and-down
strokes at 500 rpm. The homogenate was centrifuged at
1500  g for 10 min at 4 8C. The supernatant was
removed and recentrifuged at 10,000  g for 10 min.
The pellet, which contained mitochondria, was resuspended in 50 mL of buffer. The supernatant was recentrifuged at 100,000  g (4 8C, 1 h) to generate the S100 fraction. Protein concentration of both fractions
was determined by a colorimetric assay (Bio-Rad
Laboratories, Hercules, CA).
Western Analysis
All Western analysis experiments were performed at
least three times, with a representative figure shown.

centrifuged (12,879  g) for 3 min and then the supernatants were stored at 70 8C. The protein concentration was determined as described previously. One
hundred micrograms of total cell lysate was resolved
on a 12.5% SDS-PAGE, transferred to nitrocellulose,
and probed with either an affinity-purified rabbit polyclonal antibody raised against a recombinant protein
corresponding to amino acids 315397 mapping within
the carboxy terminus of human caspase 9 (1:1000; Santa
Cruz Biotechnology) or a polyclonal antibody raised
against a synthetic peptide corresponding to residues
surrounding the cleavage site of human caspase 3
(1:1000, Cell Signaling Technology; Beverly, MA).
PARP
Thimerosal-induced apoptosis was examined by
proteolytic cleavage of PARP. One hundred fifty micrograms of nuclear-extracted proteins was loaded and ran
on a 7% SDS-PAGE. The membrane was incubated
with a goat anti-PARP antibody (1:1000; Cell Signaling Technology) raised against a KLH-coupled synthetic peptide mapping at the caspase cleavage site of
poly (ADP-ribose) polymerase.
Statistical Analysis

Cytochrome c
Either 20 mg of mitochondrial proteins or 40 mg of
S-100 fractionated proteins were loaded on a 12.5%
SDS-PAGE (SDS-polyacrylamide gel electrophoresis)
according to the method of Laemmli (1970) and transferred to nitrocellulose. Transfer onto nitrocellulose
membrane was assessed by incubating with 0.1%
Ponceau. The membrane was washed with distilled
water to remove the excess stain and blocked in Blotto
[5% milk, 10 mM Tris-HCl, 150 mM NaCl (pH 8.0),
and 0.05% Tween-20] for 1 h at room temperature. A
monoclonal antibody purchased from PharMingen
(San Diego, CA) was used at a final concentration
of 1 mg/mL. After two washings in TBS (10 mM TrisHCl and 150 mM NaCl)-0.05% Tween-20, the blot was
incubated with an anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa
Cruz, CA) at a 1:5000 dilution in Blotto for 1.5 h at
room temperature. The blot was washed three times
with TBS-0.05% Tween-20 (TBST) and once with
TBS. Protein bands were visualized with the enhanced
chemiluminescence detection system (ECL, Amersham, Little Chalfont, U.K.).
Caspase 9 and Caspase 3
Cells were washed in PBS and solubilized in lysis
buffer (Clontech) for 10 min on ice. Samples were

All experiments were replicated and representative

findings are shown. Statistical analysis was performed
using Tukey analysis of variance with a p-value of 0.05.

RESULTS
Morphological Changes upon Thimerosal
Treatment
In order to determine if thimerosal induced toxicity
in human neuroblastoma, cells (SK-N-SH) were treated with a range of thimerosal concentrations (0
5 mM), visualized and photographed with a Nikon
Phase Contrast microscope. Thiosalicylate (5 mM)
was used as the control in this and all subsequent
experiments. Within 2 h of exposure to thimerosal,
membrane alterations could be visualized, followed
by cell shrinkage and detachment (Fig. 1). Observed
morphological changes upon thimerosal administration were consistent with reports of mercurialmediated cytoskeletal toxicity (Duhr et al., 1993;
Kinoshita et al., 1999). In this model, we showed
cytoskeletal changes at concentrations of thimerosal
significantly below those previously reported by Kim
et al. (2002) in treatment of HeLa S cells (20 mM).

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Fig. 1. Morphological analysis of SK-N-SH cells following treatment with either (A) 5 mM thiosalicylate for 6 h; (B) 5 mM thimerosal for 2 h; (C) 5 mM
thimerosal for 4 h and (D) 5 mM thimerosal for 6 h. No changes in morphology were noted in the thiosalicylate treated cells, whereas thimerosal caused
alterations of membranes (arrow), characteristic of cellular blebbing seen in apoptosis. In addition, cell shrinkage and detachment occurred within 6 h of
treatment. Cells were visualized and photographed using a Nikon Phase Contrast Microscope.

LDH Assay
To further evaluate toxicity, activity of lactate dehydrogenase (LDH), a cytosolic enzyme released into
surrounding media during cell death, was measured. As
the membrane loses integrity during necrosis and late
apoptosis, LDH is released. This assay approximates
cell death rates by measuring enzymatic activity of
LDH as it converts pyruvate and NADH to lactate and
NAD (Loo and Rillema, 1998). The LDH assay indicated a concentration- and time-dependent thimerosalinduced toxicity. At 24 h, LDH activity was significantly higher ( p < 0.001) than control at 2.5 and 5 mM
thimerosal treatments, indicating a loss of membrane
integrity (Fig. 2). Extending the exposure time to 48 h
showed a significant increase in LDH activity with
1 mM thimerosal, as well (Fig. 2).

bility was also significantly decreased at 1 and 2.5 mM

concentrations (Fig. 3). Results from the MTT experiments paralleled those obtained with the LDH assay in
that extended exposures of lower thimerosal concentrations also lead to cytotoxicity.
Fluorescent Analysis of Cell Death
LDH leakage can occur as a result of late stage
apoptosis or oncosis/necrosis. Therefore, we used

MTT Assay
The MTT assay is a colorimetric assay for cytotoxicity. MTT is cleaved to a blue formazan product by
dehydrogenase enzymes of intact mitochondria, allowing for determination of cell viability (Mosmann,
1983). As with the LDH assay, thimerosal treatment
induced toxicity in a concentration- and time-dependent manner in SK-N-SH cells. At 24 h, cell viability
was significantly decreased with 5 mM thimerosal in
comparison to the control (Fig. 3). At 48 h, cell via-

Fig. 2. Enzymatic activity of LDH in medium was measured at 24 and 48 h

in cells treated with a range of thimerosal concentrations (05 mM). LDH
activity increased in a time- and concentration-dependent manner with
significant differences at 2.5 and 5 mM thimerosal at 24 h. At 48 h, there was
also a significant increase in LDH activity of cells receiving 1 mM thimerosal compared to control (5 mM thiosalicylate). Results are expressed as
S.E.M., *p < 0.0001.

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Fig. 3. Cell survival was determined by the MTT assay at 24 and 48 h in

cells treated with a range of thimerosal concentrations (05 mM) compared
to the thiosalicylate control (5 mM). Survival decreased in a time- and
concentration-dependent fashion with significant differences at 5 mM (24 h)
and 1, 2.5 and 5 mM thimerosal at 48 h. Results are expressed as S.E.M.,
*
p < 0.05; +p < 0.001. Results shown are representative experiments of six
experiments at each time point.

fluorescence microscopy to determine if one or both

types of cell death were occurring as a result of
thimerosal exposure. Fluorescent analysis of cell death
as described by Katsen et al. (1998) and Thevenod et al.
(2000) was conducted with both ethidium bromide and
Hoechst 33342. Under ultraviolet epi-illumination
necrotic cells fluoresce pink due to ethidium bromide
uptake, whereas normal and apoptotic cells emit blue
fluorescence due to Hoechst-33342 incorporation.
Apoptotic cells, distinguished from normal cells based
on condensation and fragmentation of nuclei, are
depicted in Fig. 4. In addition, several cells stained
with ethidium bromide, suggesting that a 24 h exposure
to 5 mM thimerosal results in both apoptotic and
oncotic/necrotic morphologies. Cell counts from 100
stained cells showed a three-fold increase in apoptotic
cells (24.7%) over necrotic cells (7.7%) with 5 mM
thimerosal treatment at 24 h (data not shown). Figures
shown are representative of three individual experiments.

Thimerosal Causes Cytochrome c Translocation

from the Mitochondria to the Cytosol
Both inorganic and organic mercurials have been
shown to rapidly accumulate in mitochondria, resulting
in dysfunction. Since the majority of thimerosal treated
cells appeared apoptotic, we chose to investigate if the
cell death cascade was associated with mitochondrial
release of proapoptotic proteins. As shown in Fig. 5, a
rapid increase in the cytosolic content of cytochrome c
was observed after thimerosal administration.

Fig. 4. Cells were viewed by fluorescence microscopy using ethidium

bromide and a Hoechst 33342 counterstain after a 24 h exposure of either
thiosalicylate or thimerosal. Nuclei stain pink in cells upon loss of membrane integrity (oncosis/necrosis). Arrows indicate DNA fragmentation and
condensation in cells receiving 5 mM thimerosal (for interpretation of the
references to color in this figure legend, the reader is referred to the web
version of this article).

Thimerosal Induces Caspase 9 Cleavage in SK-NSH Cells

In the mitochondrial-initiated pathway of apoptotic
cell death, caspase activation is achieved after the
release of cytochrome c from mitochondria. Upon
release of the proapoptotic proteins from the mitochondria, a multimeric protein complex consisting of Apaf1 and cytochrome c forms, followed by recruitment and
activation of procaspase 9. Procaspase enzymes can be
activated after they are cleaved; therefore we examined
the cleavage pattern of caspase 9 in cells treated with
thimerosal. Within 8 h of thimerosal treatment (1
5 mM) the generation of a 34 kDa band (Fig. 6) was
observed in addition to the proform of caspase 9
(46 kDa).
Thimerosal Induces Caspase 3 (CPP-32) Cleavage
in SK-N-SH Cells
Caspase 3 is a downstream target for caspase 9
activity; therefore, we examined by Western analysis
the cleavage patterns of caspase 3 following thimerosal

Fig. 5. Cytochome c was shown to leak from mitochondria of cells treated

with 5 mM thimerosal after 6 h of treatment. Twenty micrograms of
mitochondrial protein or 40 mg of the S-100 cytoplasmic fraction was
loaded onto a 12.5% SDS-PAGE and analyzed by Western analysis.

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Fig. 6. Caspase 9 cleavage was determined by Western analysis in cells

receiving thimerosal (05 mM) or thiosalicylate (5 mM). Procaspase 9
(47 kDa) was cleaved within 8 h to a 38 kDa cleavage product in cells
receiving thimerosal. No cleavage was observed at any time point in cells
receiving thiosalicylate (5 mM).

treatment. After 24 h of treatment, both the 17 and

19 kDa caspase 3 cleavage products were observed
(Fig. 7A).
Caspase 3 activity increases in a concentration- and
time-dependent manner with thimerosal treatment in
SK-N-SH cells
The activity of caspase 3 in thimerosal-induced
apoptosis in SK-N-SH cells was measured with the
ApoAlert assay kit by Clontech. Cells were treated
with 05 mM thimerosal and samples were collected at
various time points from 024 h. Fig. 7B shows a
concentration- and time-dependent increase in caspase
3 activity. At 6 h caspase 3 activity had increased 11.6
fold in the 5 mM treated cells with no increase noted at
1 mM thimerosal. At 24 h both 1 and 5 mM treated cells
had significantly higher caspase 3 activity than control,
15.9- and 22.2-fold, respectively. The increase in caspase 3 activity paralleled the cleavage pattern observed
in Fig. 7A. The use of the fluorescence-based ApoAlert
assay provided greater sensitivity than Western analysis, allowing detection of changes in caspase 3 activa-

Fig. 8. PARP, a 116 kDa downstream target of activated caspase 3, was

analyzed by Western analysis in cells receiving 05 mM thimerosal. Cells
treated with 5 mM thiosalicylate showed minimal cleavage; whereas SK-NSH cells receiving thimerosal contained an abundant 85 kDa cleavage
product after 24 h.

tion at earlier time points. Caspase cleavage was easily

detected by Western analysis at the 24 h time point,
consistent with the fluorescent-based assay.
Caspase 8 can activate caspase 3 independent of
mitochondrial events leading to apoptosis; however, in
our studies we found no activation of caspase 8 (data
not shown), suggesting initiation of cell death signals
by thimerosal at the level of the mitochondria.
Thimerosal Induces PARP Cleavage within
24 h in SK-N-SH Cells
Poly (ADP-ribose) polymerase (PARP), a DNA nick
sensor, is a substrate for cleavage by activated caspase
3. The data shown in Fig. 8 indicates that thimerosal
induces PARP cleavage. At 24 h 5 mM thimerosal
treatment caused degradation of the 116 kDa parental
PARP protein with subsequent generation of an 85 kDa
immunoreactive cleavage product (Fig. 8). No cleavage was observed following 12 h of treatment with
1 mM thimerosal (data not shown).

Fig. 7. A: Caspase 3, a downstream target of caspase 9, is cleaved to a 17 and 19 kDa fragment upon activation. At 24 h both cleavage products were detected in
cells treated with 5 mM thimerosal. Although cleavage products were not detectable until 24 h following treatment, caspase 3 activity was elevated within 6 h in
cells receiving 5 mM thimerosal (7B). With the ApoAlert fluorescence detection assay, caspase 3 activity was increased in both 1 and 5 mM thimerosal receiving
cells after 24 h; however, those receiving 1 mM showed a lower caspase activation.

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DISCUSSION
In the current study we demonstrate a role for
mitochondria in thimerosal-induced apoptosis in the
human neuroblastoma cell line, SK-N-SH. The model
was chosen due to cellular characteristics representative of those in an immature nervous system. The SKN-SH cell line, comprised of a neuroblast cell type,
retains the capacity to differentiate in vitro, rather than
representing a model of terminal differentiation. In
addition, neuroblastoma models have been reported
to be sensitive to mercurial toxicity (Sager and
Syversen, 1984; Stoiber et al., 2004; Toimela and Tahti,
2004). Several studies have indicated that both inorganic and organic mercurials induce apoptosis in vitro
as well as in vivo. Comparison studies of inorganic
(HgCl2) and organic mercurial (MeHgCl) toxicity have
been conducted in T lymphocytes. Findings suggest
that both types of mercurials disrupt mitochondrial
function, increase cellular oxidative stress and cause
apoptosis. However, the specific cell death pathway
(mitochondrial dependent versus independent) varies
with mercurial form (Guo et al., 1998; Shenker et al.,
1999, 2000); inorganic mercury induces apoptosis in a
mitochondrial
independent
manner,
whereas,
MeHgCl-mediated cell death is associated with cytochrome c mobilization.
Release of proapoptotic factors such as cytochrome c
and apoptosis inducing factor (AIF) are characteristics
of mitochondrial-mediated cell death. It has been well
established that release of cytochrome c from the mitochondria triggers the formation of a caspase 9 complex,
which recruits additional factors, including Apaf-1 and
ATP (Li et al., 1997). Collectively this complex has been
termed the apoptosome. Formation of the complex
leads to activation of caspase 9, which then processes
and activates other caspases, including caspase 3. In
contrast, release of AIF from mitochondria results in
caspase-independent cell death.
In the present study, exposure to thimerosal resulted
in an apoptotic cascade more closely resembling the
effects of organic mercurials than those of HgCl2. First,
we saw evidence of mitochondrial involvement as
thimerosal-treated cells showed release of cytochrome
c. Additionally, a concentration- and time-dependent
loss of mitochondrial viability was observed with the
MTT assay. Release of cytochrome c was followed by
cleavage of caspase 9. This is the first study to identify
caspase 9 as a component of mercurial-induced cell
death, and more specifically that of thimerosal exposure. The reactivity of Hg with sulfhydryl groups is
thought to occur nonspecifically; however, in this

model we show selectivity in regard to caspase activation, with thimerosal specifically activating the mitochondrial caspase 9 pathway, without activating
mitochondrial-independent caspase 8.
We next saw an increase in caspase 3 cleavage at
24 h with 5 mM thimerosal treatment. With the ApoAlert assay, which is more sensitive than Western blotting, an increase in caspase 3 activity was seen as early
as 6 h with the same treatment. Evidence of the activation of the apoptotic cascade at this earlier time point is
consistent with the morphological changes observed in
the SK-N-SH cells; phase contrast microscopy
revealed membrane alterations, cell shrinkage, and
detachment beginning to occur within this time frame.
By 24 h, as seen by fluorescence microscopy, apoptotic
cells were numerous, but some cells also exhibited a
loss of membrane integrity, as evidenced by the uptake
of ethidium bromide. This loss of membrane integrity,
which is confirmed with the LDH studies, is most
frequently associated with oncosis/necrosis. However,
Baskin et al. (2003) suggests that these membrane
changes may not be a necrotic process, but may be
caused by a direct effect of thimerosal on the cell
membrane itself. Furthermore, Baskin et al. (2003)
suggests that free radicals may be involved in thimerosal-induced damage to the cell membrane.
Inorganic and organic mercurials, including thimerosal, have been shown to increase reactive oxygen
species (ROS) and induce a state of oxidative stress
in numerous cell types; however, it is unknown whether
this is a direct effect of the metal. Studies have shown
that thimerosal causes release of calcium from intracellular stores followed by an influx of extracellular
calcium (Gericke et al., 1993; Elferink, 1999), which
can lead to an increase in reactive oxygen and nitrogen
species (RNS) production in the mitochondria. Both
ROS and RNS have been shown to cause apoptotic cell
death (McGowan et al., 1996; Szabo, 1996; Cai and
Jones, 1998; Chung et al., 2001). Reactive oxygen
species and RNS can directly interact with macromolecules such as proteins and nucleic acids, leading to
perturbations of vital cellular functions which may
result in cell death. Additionally, Brawer et al.
(1998) reported mitochondrial accumulation of iron
in astrocytes cultured with HgCl2. LeBel et al. (1992)
reported that pretreatment with the iron chelator deferoxamine inhibited Hg-induced generation of ROS in
the brain. Taken together these studies suggest a role
for iron-catalyzed ROS production within mitochondria of mercury-exposed tissues.
Finally, in our studies we see a differential processing of PARP. Processing of PARP by cysteine pro-

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teases during apoptosis results in an 85 kDa cleavage

product, and causes loss of its nick sensing function,
thereby preventing activation of cellular repair programs. PARP cleavage in a necrotic cellular demise,
mediated by lysosomal proteases, results in a multitude
of fragments ranging in size from 35 to 89 kDa (Shah
et al., 1996; Casiano et al., 1998). Based on the
fragmentation pattern of PARP at 24 h after thimerosal
treatment in the SK-N-SH cells, our results further
suggest a predominantly apoptotic cell death program,
as only the 85 kDa PARP cleavage product could be
detected in SK-N-SH cells upon thimerosal treatment.
In summary, we have shown that thimerosal can
cause mitochondrial-mediated apoptosis in a human
neuroblastoma cell line. To our knowledge, this is the
first study to chronologically show the mitochondrial,
cytosolic and nuclear events associated with thimerosal-mediated toxicity in a non-differentiated neuronal
system. Although thimerosal has been shown to alter
redox status, further evaluation will be required to
determine the effect, if any, these alterations have on
the cascade of events reported in this study.

ACKNOWLEDGEMENTS
This work was supported by the National Institutes
of Health Grant R15, ES12209-01, to Dr. Kelley
Kiningham. Marsha P. Cole is the recipient of a training
grant (DK07778-02) in the Graduate Center for Nutritional Sciences at the University of Kentucky. The
authors wish to thank Kris Grimes, Brittany Ray and
Carla Cook for technical contributions to the manuscript, as well as Dr. Daret St. Clair for helpful suggestions in preparation of the manuscript.

REFERENCES
Aschner M, Aschner JL. Mercury neurotoxicity: mechanisms of
blood-brain barrier transport. Neurosci Biobehav Rev
1990;14(2):16976.
Ball LK, Ball R, Pratt RD. An assessment of thimerosal use in
childhood vaccines. Pediatrics 2001;107(5):114754.
Baskin DS, Ngo H, Didenko VV. Thimerosal induces DNA breaks,
caspase-3 activation, membrane damage, and cell death in
cultured human neurons and fibroblasts. Toxicol Sci
2003;74(2):3618.
Blair AMJN, Clark B, Clarke AJ, Wood P. Tissue concentrations of
mercury after chronic dosing of squirrel monkeys with thiomersal. Toxicology 1975;3(2):1716.
Brawer JR, McCarthy GF, Gornitsky M, Frankel D, Mehindate K,
Schipper HM. Mercuric chloride induces a stress response in

cultured astrocytes characterized by mitochondrial uptake of

iron. Neurotox 1998;19(6):76776.
Cai J, Jones DP. Superoxide in apoptosis. Mitochondrial generation
triggered by cytochrome c loss. J Biol Chem
1998;273(19):114014.
Casiano CA, Ochs RL, Tan EM. Distinct cleavage products of
nuclear proteins in apoptosis and necrosis revealed by autoantibody probes. Cell Death Differ 1998;5(2):18390.
Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as
a bioregulator of apoptosis. Biochem Biophys Res Commun
2001;282(5):10759.
Duhr EF, Pendergrass JC, Slevin JT, Haley BE. HgEDTA complex
inhibits GTP interactions with the E-site of brain beta-tubulin.
Toxicol Appl Pharmacol 1993;122(2):27380.
Elferink JG. Thimerosal: a versatile sulfhydryl reagent, calcium
mobilizer, and cell function-modulating agent. Gen Pharmacol
1999;33(1):16.
Friberg L, Mottet NK. Accumulation of methylmercury and inorganic mercury in the brain. Biol Trace Elem Res 1989;21:2016.
Gasset AR, Itoi M, Ishii Y, Ramer RM. Teratogenicities of
ophthalmic drugs II. Teratogenicities and tissue accumulation
of thimerosal. Arch Opthalmol 1975;93(1):525.
Gericke M, Droogmans G, Nilius B. Thimerosal induced changes
of intracellular calcium in human endothelial cells. Cell Calcium 1993;14(3):2017.
Guo TL, Miller MA, Shapiro IM, Shenker BJ. Mercuric chloride
induces apoptosis in human T lymphocytes: evidence of mitochondrial dysfunction. Toxicol Appl Pharmacol 1998;153(2):
2507.
Harry GJ, Harris MW, Burka LT. Mercury concentrations in brain
and kidney following ethylmercury, methylmercury and Thimerosal administration to neonatal mice. Toxicol Lett
2004;154(3):1839.
Hornig M, Chian D, Lipkin WI. Neurotoxic effects of postnatal
thimerosal are mouse strain dependent. Mol Psychiatry
2004;9(9):83345.
Katsen AD, Vollmar B, Mestres-Ventura P, Menger MD. Cell
surface and nuclear changes during TNF-alpha-induced apoptosis in WEHI 164 murine fibrosarcoma cells. A correlative
light, scanning, and transmission electron microscopical study.
Virchows Arch 1998;433(1):7583.
Kim E, Kim JH, Kim HS, Ryu SH, Suh PG. Thimerosal stimulates
focal adhesion kinase and cytoskeletal changes by redox modulation. Biochim Biophys Acta 2002;1593(1):915.
Kinoshita Y, Ohnishi A, Kohshi K, Yokota A. Apparent diffusion
coefficient on rat brain and nerves intoxicated with methylmercury. Environ Res 1999;80(4):34854.
Kunimoto M. Methylmercury induces apoptosis of rat cerebellar
neurons in primary culture. Biochem Biophys Res Commun
1994;204(1):3107.
Laemmli UK. Cleavage of structural proteins during the assembly
of the head of bacteriophage T4. Nature 1970;227(5259):6805.
LeBel CP, Ali SF, Bondy SC. Deferoxamine inhibits methyl
mercury-induced increases in reactive oxygen species formation in rat brain. Toxicol Appl Pharmacol 1992;112(1):1615.
Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M,
Alnemri ES, Wang X. Cytochrome c and dATP-dependent
formation of Apaf-1/caspase-9 complex initiates an apoptotic
protease cascade. Cell 1997;91(4):47989.
Loo DT, Rillema JR. Measurement of cell death. Methods Cell Biol
1998;57:25164.

DTD 5

10

M.L. Humphrey et al. / NeuroToxicology xxx (2005) xxxxxx

Magos L. Review on the toxicity of ethylmercury, including its

presence as a preservative in biological and pharmaceutical
products. J Appl Toxicol 2001;21(1):15.
McGowan AJ, Ruiz-Ruiz MC, Gorman AM, Lopez-Rivas A, Cotter
TG. Reactive oxygen intermediate(s) (ROI): common mediator(s) of poly (ADP-ribose) polymerase (PARP) cleavage and
apoptosis. FEBS Lett 1996;392(3):299303.
Monnet-Tschudi F. Induction of apoptosis by mercury compounds
depends on maturation and is not associated with microglial
activation. J Neurosci Res 1998;53(3):3617.
Mosmann T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Methods 1983;65(12):5563.
Nagashima K, Fujii Y, Tsukamoto T, Nukuzuma S, Satoh M, Fujita
M, Fujioka Y, Akagi H. Apoptotic process of cerebellar degeneration in experimental methylmercury intoxication of rats.
Acta Neuropathol 1996;91(1):727.
Norseth T, Clarkson TW. Studies on the biotransformation of
203
Hg-labeled methyl mercury chloride in rats. Arch Environ
Health 1970;21(6):71727.
Pichichero ME, Cernichiari E, Lopreiato J, Treanor J. Mercury
concentrations and metabolism in infants receiving vaccines
containing thiomersal: a descriptive study. Lance 2002;360
(9347):173741.
Sager PR, Syversen TL. Differential responses to methylmercury
exposure and recovery in neuroblastoma and glioma cells and
fibroblasts. Exp Neurol 1984;85(2):37182.
Shah GM, Shah RG, Poirier GG. Different cleavage pattern for
poly(ADP-ribose) polymerase during necrosis and apoptosis in
HL-60 cells. Biochem Biophys Res Commun 1996;229(3):838
44.

Shenker BJ, Guo TLOI, Shapiro IM. Induction of apoptosis in

human T-cells by methyl mercury: temporal relationship
between mitochondrial dysfunction and loss of reductive
reserve. Toxicol Appl Pharmacol 1999;157(1):2335.
Shenker BJ, Guo TL, Shapiro IM. Mercury-induced apoptosis in
human lymphoid cells: evidence that the apoptotic pathway is
mercurial species dependent. Environ Res 2000;84(2):8999.
Stoiber T, Degen GH, Bolt HM, Unger E. Interaction of mercury(II)
with the microtubule cytoskeleton in IMR-32 neuroblastoma
cells. Toxicol Lett 2004;151(1):99104.
Squier MK, Cohen JJ. Assays of apoptosis. Methods Mol Biol
2000;144:32737.
Suda I, Takahashi H. Degradation of methyl and ethyl mercury into
inorganic mercury by oxygen free radical-producing systems:
involvement of hydroxyl radical. Arch Toxicol 1991;65(2):129
34.
Sugita M. The biological half-time of heavy metals. The existence
of a third slowest component. Int Arch Occup Environ Health
1978;41(1):2540.
Szabo C. DNA strand breakage and activation of poly-ADP
ribosyltransferase: a cytotoxic pathway triggered by peroxynitrite. Free Radic Biol Med 1996;21(6):85569.
Thevenod F, Friedmann JM, Katsen AD, Hauser IA. Up-regulation
of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from
cadmium- and reactive oxygen species-induced apoptosis. J
Biol Chem 2000;275(3):188796.
Toimela T, Tahti H. Mitochondrial viability and apoptosis induced
by aluminum, mercuric mercury and methylmercury in cell
lines of neural origin. Arch Toxicol 2004;78(10):56574.