Page 1
Fe(II)-induced DNA damage in a-synuclein-transfected human
dopaminergic BE(2)-M17 neuroblastoma cells: detection
by the Comet assay
Francis L. Martin, Sally J. M. Williamson, Katerina E. Paleologou, Rebecca Hewitt,
Omar M. A. El-Agnaf and David Allsop
Department of Biological Sciences, I.E.N.S., Lancaster University, Lancaster, UK
Abstract
Lewy bodies in the brains of patients with Parkinson’s disease
(PD) contain aggregates of a-synuclein (a-syn). Missense
mutations (A53T or A30P) in the gene encoding a-syn are
responsible for rare, inherited forms of PD. In this study, we
explored the susceptibility of untransfected human dopamin-
ergic BE(2)-M17 neuroblastoma cells, cells transfected with
vector only, or cells transfected with wild-type a-syn, A30P
a-syn or A53T a-syn to Fe(II)-induced DNA damage in the
form of single-strand breaks (SSBs). DNA SSBs were detec-
ted following 2-h treatments with various concentrations of
Fe(II) (0.01–100.0 lM), using the alkaline single cell-gel
electrophoresis (‘Comet’) assay and quantified by measuring
comet tail length (CTL) (lm). Fe(II) treatment induced signi-
ficant increases in CTL in cells transfected with A30P a-syn or
A53T a-syn, even at the lowest concentrations of Fe(II) tested.
In comparison, untransfected cells, vector control cells or cells
transfected with wild-type a-syn exhibited increases in SSBs
only when exposed to concentrations of 1.0 lM Fe(II) and
above. Even when exposed to higher concentrations (10.0–
100.0 lM) of Fe(II), untransfected cells, vector control cells or
cells transfected with wild-type a-syn were less susceptible to
DNA-damage induction than cells transfected with A30P a-syn
or A53T a-syn. Incorporation of DNA-repair inhibitors,
hydroxyurea and cytosine arabinoside, enhanced the sensi-
tivity of DNA damage detection. Susceptibility to Fe(II)-
induced DNA damage appeared to be dependent on a-syn
status because cells transfected with wild-type a-syn or A53T
a-syn were equally susceptible to the damaging effects of the
mitochondrial respiratory chain inhibitor rotenone. Overall, our
data are suggestive of an enhanced susceptibility to the toxic
effects of Fe(II) in neuroblastoma cells transfected with mutant
a-syn associated with inherited forms of PD.
Keywords: Comet assay, DNA SSBs, Fe(II), neuroblastoma
cells, Parkinson’s disease, a-synuclein.
J. Neurochem. (2003) 87, 620–630.
Parkinson’s disease (PD) is characterised by a progressive
degeneration of the substantia nigra region of the brain.
Although the aetiology of PD remains obscure, various
studies point to a central role of iron (Fe)-induced oxidative
stress mechanisms (Kienzl et al. 1995). Elevated Fe levels
have been localised to degenerate regions of brains from PD
patients, a finding also demonstrated in animal models of the
disease (Thong et al. 1999).
Lesions known as Lewy bodies (LBs) and Lewy neurites
(LNs) constitute the most striking histopathological features in
the brains of patients with PD and dementia with Lewy bodies
(DLB) (Baba et al. 1998; Spillantini et al. 1998). Fibrils
composed of a small protein named a-synuclein (a-syn) arethe main component of LBs and LNs (Spillantini et al. 1998).
Two different mutations (A53Tor A30P) in the gene encoding
a-syn are responsible for rare inherited forms of PD
Received June 12, 2003; revised manuscript received July 4, 2003;
accepted July 11, 2003.
Address correspondence and reprint requests to Dr F. L. Martin,
Department of Biological Sciences, I.E.N.S., Lancaster University,
Lancaster LA1 4YQ, UK. E-mail: [email protected]
Abbreviations used: a-syn, a-synuclein; AEBSF, 4-(2-aminoethyl)
benzenesulfonyl fluoride; ara-C, cytosine arabinoside; BE-M17 cells,
human dopaminergic BE(2)-M17 neuroblastoma cells; CI, confidence
intervals; CNS, central nervous system; CTL, comet tail length; DLB,
dementia with Lewy bodies; DMSO, dimethyl sulfoxide; E-64, N-(trans-
epoxysuccinyl)-L-leucine 4-guanidinobutylamide; EDTA, ethylenedia-
minetetraacetic acid; Fe, iron; FeCl2, Iron(II)chloride; H2O2, hydrogen
peroxide; HRP, horseradish peroxidase; HU, hydroxyurea; LBs, Lewy
bodies; LNs, Lewy neurites; LMP, low melting point; NMP, normal
melting point; NER, nucleotide excision repair; O2–, superoxide anion
radical; ÆOH, hydroxyl radical; PBS, phosphate-buffered saline; PBST,
PBS-Tween-20; PD, Parkinson’s disease; ROS, reactive oxygen species;
SSBs, single-strand breaks.
Journal of Neurochemistry, 2003, 87, 620–630 doi:10.1046/j.1471-4159.2003.02013.x
620 � 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 2
(Polymeropoulos et al. 1997; Kruger et al. 1998). Lesions
similar to those found in PD have been identified in transgenic
animal models expressing high levels of human wild-type or
mutant a-syn gene, and these animals progressively develop
many of the pathological and behavioural features of PD
(Feany and Bender 2000; Masliah et al. 2000; Van der Putten
et al. 2000). The function of a-syn remains to be established;
however, it has been implicated in the regulation of synaptic
plasticity (George et al. 1995), neuronal differentiation
(Kholodilov et al. 1999; Stefanis et al. 2001), regulation of
dopamine synthesis (Abeliovich et al. 2000; Van der Putten
et al. 2000), and it may possess chaperone-like activity (Souza
et al. 2000). More recently, it has been reported that a-synshares some properties with the family of fatty acid binding
proteins, and may thus transport fatty acids between the
aqueous and membrane phospholipid compartments of the
neuronal cytoplasm (Sharon et al. 2001; Sharon et al. 2003).
Neuronal cells over-expressing the wild-type a-syn are more
resistant to oxidative stress than untransfected cells (Hashi-
moto et al. 2002; Seo et al. 2002), whereas, cells expressing
A30P a-syn or A53T a-syn, mutations associated with familial
PD and increased oligomerisation of a-syn (El-Agnaf et al.
1998a; Conway et al. 2000), exhibit an increased suscepti-
bility to oxidative stress damage (da Costa et al. 2000; Lee
et al. 2001). Also, exogenous non-aggregated a-syn, at lowconcentrations, protects neuronal cells against cellular stress
conditions such as serum deprivation, oxidative stress, and
excitotoxicity (Seo et al. 2002). In contrast, pre-aggregated a-syn is toxic to neuronal cells (El-Agnaf et al. 1998b) and this
may be due to metal-dependent formation of hydrogen
peroxide by a-syn (Turnbull et al. 2001). This could explain
the fact that cells transfected with a-syn have been reported toshow evidence of oxidative damage (Hsu et al. 2000;
Ostrerova-Golts et al. 2000).
The alkaline single cell-gel electrophoresis (‘Comet’) assay
allows the direct visualisation of DNA single-strand breaks
(SSBs) in individual cell genomes (Martin et al. 1997, 1999).
In the alkaline version of the assay, cells in a single cell
suspension in agarose are lysed at pH 10 to release individual
nuclei. Alkaline conditions facilitate denaturation, unwind-
ing, and the development of alkali-labile DNA SSBs prior to
electrophoresis, where relaxed and broken, negatively
charged DNA migrates at different, size-related rates towards
the anode (Martin et al. 1997, 1999). DNA damage is
proportional to the extent of migration and the assay permits
the detection of overt strand breaks, alkali-labile lesions such
as apurinic sites, and excision-repair-induced strand breaks.
Increased oxidative DNA damage has been reported in the
CNS of patients with PD and chromosomal aberrations and
oxidative DNA damage has been shown in lymphocytes of
patients with untreated PD (Migliore et al. 2002). Increased
levels of oxidative DNA damage have now been shown,
using the Comet assay, in the substantia nigra of aging rats
(Giovannelli et al. 2003) and such damage is also implicated
in the pathology of Alzheimer’s disease (Gabbita et al.
1998). Fenton reaction-mediated generation of hydroxyl
radical (ÆOH) may be responsible for the pathogenesis of cell
death in PD (Tabner et al. 2002). In this study we specifically
explored Fe(II)-induced DNA damage in untransfected
human dopaminergic BE(2)-M17 neuroblastoma (BE-M17)
cells, vector control cells or cells transfected with wild-type
a-syn, A30P a-syn or A53T a-syn, using the Comet assay to
measure levels of DNA SSBs.
Materials and methods
Cell culture and chemicals
All chemicals, including test agents, were obtained from Sigma
Chemical Co. (Poole, UK) unless otherwise stated. Cell culture
constituents were obtained from Invitrogen Ltd. (Paisley, UK)
unless otherwise stated.
From the BE-M17 cell line untransfected cells, eukaryotic
pcDNA3 vector control cells or cells transfected with wild-type
a-syn, A53T a-syn or A30P a-syn, cloned into the NotI site of
pcDNA3 (Ostrerova-Golts et al. 2000), were used.
Cells were grown, at 5% CO2 in air and 37�C in a humidified
atmosphere, in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% foetal calf serum, 100 U/mL penicillin,
100 lg/mL streptomycin and 1% L-glutamine. To maintain selection,
50 lg/mL geneticin (G418) was incorporated into medium.
Western blot analysis of a-syn expression
Cell lysates were collected in 1 mL of CelLytic buffer (Sigma
Chemical Co. Poole, UK), containing a cocktail of protease
inhibitors, including AEBSF, aprotinin, E-64, EDTA and leupeptin
(Calbiochem-Novabiochem Corporation, San Diego, CA, USA),
and centrifuged at 3000 g for 30 min. The supernatant was collected
and then electrophoresed on NuPAGE Bis-Tris 4–12%, 1 mm gels
(Invitrogen Ltd, Paisley, UK). The separated proteins were then
transferred to nitrocellulose membranes (0.45 lm) (Invitrogen Ltd)
and electrophoresed at 125 mA for 45 min. Membranes were boiled
for 5 min in phosphate-buffered saline (PBS), and then blocked with
5% marvel dried skimmed milk, dissolved in PBS-Tween-20
(0.05%) (PBST), for 1 h. The membranes were probed with primary
antibody, 211 mouse monoclonal antibody which recognises a-syn(121–125) (Santa Cruz Biotechnology, Santa Cruz, CA, USA),
overnight at 4�C. The membranes were washed several times with
PBST, followed by incubation with HRP-conjugated goat anti-
mouse secondary antisera (DakoCytomation Ltd, Ely, UK) for
60 min. The protein bands were visualised using ECL reagents
(Pierce, Rockford, IL, USA) as described by the manufacturer.
Cell treatments
Cultured cells were allowed to attain confluence in 12-well multi-
well dishes. Iron(II)chloride (FeCl2) or rotenone, freshly made prior
to experiments, was added in dimethyl sulfoxide (DMSO) (not
exceeding 1% v/v). Treatments were carried out under normal
incubation conditions for 2 h. DNA repair inhibitors, hydroxyurea
(HU) (1 mM) and cytosine arabinoside (ara-C) (120 lM) were
incorporated, as indicated.
Mutant a-syn and Fe(II)-induced SSBs in BE-M17 cells 621
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 3
The Comet assay
Ordinary microscope slides pre-coated with a 1% solution of
normal melting point (NMP) agarose in PBS were employed
(Yared et al. 2002). Post-treatment, cells were disaggregated with
cell dissociation solution (Sigma Chemical Co.) and re-suspended
in 1 mL PBS. To each 1-mL cell suspension in PBS 1 mL of
warm 1% low melting point (LMP) agarose (in PBS) was added,
gently vortexed and 150 lL of the resultant mixture applied to
NMP pre-coated microscope slides. This LMP mixture was then
evenly distributed across the microscope slide following the
application of a coverslip and allowed to set on a cold surface for
5 min prior to removal of the coverslips. Slides were then
submerged in cold lysis solution [2.5 M NaCl, 100 mM EDTA
disodium salt, 10 mM Tris, 1% N-lauroyl sarcosine, adjusted to
pH 10 with NaOH; then made to 1% Triton X-100 and 10%
DMSO prior to use], protected from light and stored at 4�C for at
least 1 h. The slides were then transferred to a tank containing
electrophoresis solution (0.3 M NaOH, 1 mM EDTA, freshly
prepared, pH > 13), and stored for 20 min at 4�C prior to being
transferred to a horizontal electrophoresis tank in a chilled
incubator and covered in fresh electrophoresis solution (pH > 13).
Electrophoresis was carried out at 0.8 V/cm and 300 mA for
24 min. After electrophoresis, slides were neutralised (Tris, 0.5 M,
pH 7.5) and stained with ethidium bromide (20 ng/mL) after
which comet tail length (CTL) (lm) was visualised by epifluo-
rescence using a Leitz Dialux 20 EB microscope. Analyses were
performed using the software package Komet 5.0 (Kinetic
Imaging, Liverpool, UK). A total of 100 digitised images/data
point, 50 from each of two duplicate slides, was measured in each
experiment. CTL measurements obtained from treated-cell popu-
lations were compared with corresponding controls using a non-
parametric (Mann–Whitney) test, a test designed not to make
assumptions about the distribution of the data. All p-values given
are two-tailed.
Results
The Comet assay and a-syn expression
Figure 1(a) shows representative photomicrographs of fluor-
escent images of damaged nuclei isolated from Fe(II)-treated
cells transfected with A30P a-syn. Comet formation occurs
following electrophoretic migration towards the anode in a
manner proportional to the number of breaks in the
negatively charged DNA molecule. At an appropriate
magnification, comparative CTLs are readily apparent. When
the Comet assay is carried out under alkaline conditions,
levels of DNA SSBs can be quantified in lm; the longer the
CTL, the more SSBs. The relative levels of a-syn expression
in the various cells employed in this study are shown in
Fig. 1(b). Clearly, BE-M17 cells transfected with wild-type,
A30P, or A53T a-syn express similarly high levels of the
protein, whereas vector control cells and untransfected cells
express much lower amounts.
The results obtained using the Comet assay may be
presented either as scatter plots which show the levels of
DNA damage induction in each individual nucleus (see
Figs 2 and 3) or as frequency distributions of CTLs (see
Fig. 4). In the latter case, a right-hand shift in distribu-
tion away from zero indicates an increase in CTL. Dose–
response curves (see Fig. 5) are also presented, in which
mean CTL ± 95% CI are plotted as a function of concen-
tration of the test agent. Otherwise, as in Fig. 6, using data
derived from pooled duplicate samples from five separate
(a)
(b) 1 2 3 4 5 6
17 kDa
α-syn
Fig. 1 Human dopaminergic BE(2)-M17 neuroblastoma (BE-M17)
cells employed in this study. (a) Representative photomicrograph of
ethidium bromide-stained nuclei from Fe(II)-treated cells transfected
with A30P a-synuclein (a-syn) following incorporation into the Comet
assay and taken using confocal microscopy. A representative analysis
of electrophoretic migration is shown within the white rectangle: the
‘head’ (within the shorter rectangle) is demarcated from the ‘tail’ (within
the longer rectangle) of the ‘comet’, representing, respectively,
undamaged genomic DNA and migration of damaged DNA in an
electrophoretic field. Analyses, corrected for background fluorescent
levels (within the red rectangle), were performed using the software
package Komet 5.0 (Kinetic Imaging, Liverpool, UK). CTLs (lm) were
measured as described in Materials and methods. (b) Western blot
analysis of a-syn expression levels. Lane number from left to right
represents: (1) recombinant a-syn (control) (0.05 lg); (2) cells trans-
fected with wild-type a-syn; (3) cells transfected with A53T a-syn; (4)
cells transfected with A30P a-syn; (5) vector control cells; (6) un-
transfected BE-M17 cells. Antibodies used: a-syn 211 (1 : 500 dilution)
and anti-mouse HRP (1 : 10000 dilution) as described in Materials
and methods. The amount of protein loaded per well was 12 lg.
622 F. L. Martin et al.
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 4
experiments, a box-whisker plot can be used. Such a graph
shows the median value as indicated by a central vertical
line, the lower and upper quartiles by the corresponding
vertical ends of the box and, the minimum and maximum
values indicated by the extremities.
a-Syn status on susceptibility to Fe(II)-induced DNA
damage
Figure 2(a–e) shows that small increases in comet-forming
activity were observed following treatments in excess of
1.0 lM Fe(II) in untransfected BE-M17 cells. In vehicle
control cells, a median CTL of 9.815 lmwas observed and in
the presence of 0.01, 0.1, 1.0, 5.0, 10.0, 50.0 or 100.0 lMFe(II)
treatment, increases in median CTLs to 12.8 lm, 12.8 lm,
17.06 lm ( p < 0.0001), 13.65 lm ( p < 0.005), 14.95 lm( p < 0.0004), 19.62 lm ( p < 0.0001) and 18.35 lm( p < 0.0001) were induced, respectively. Vector control cells
exhibited a similar susceptibility to Fe(II)-induced comet-
forming activity (Fig. 2b). In vehicle control cells, a median
CTL of 12.8 lm was observed, whilst following treatment
with 0.01, 0.1, 1.0, 5.0, 10.0, 50.0 or 100.0 lM Fe(II)
median CTLs of 11.09 lm, 11.09 lm, 13.23 lm, 15.79 lm( p < 0.02), 16.21 lm ( p < 0.0006), 29.86 lm ( p < 0.0001)
and 33.28 lm ( p < 0.0001) were observed, respectively. Cells
transfected with wild-type a-syn appeared to be similarly
resistant to the toxic effects of Fe(II) (Fig. 2c). In vehicle
control cells transfected with wild-type a-syn, a median CTL
of 12.8 lm was observed and in the presence of 0.01, 0.1,
1.0, 5.0, 10.0, 50.0 or 100.0 lM Fe(II) treatment, median
CTLs of 14.94 lm, 15.79 lm, 17.06 lm( p < 005), 17.06 lm( p < 0.002), 15.79 lm ( p < 0.002), 17.06 lm ( p < 0.009)
and 17.06 lm ( p < 0.02) were induced, respectively.
In BE-M17 cells transfected with A30P a-syn, a control
median CTL of 10.24 lm was observed whilst following
0.01, 0.1, 1.0, 5.0, 10.0, 50.0 or 100.0 lM Fe(II) treatment,
increases in median CTLs to 14.51 lm ( p < 0.0001),
14.08 lm ( p < 0.002), 15.79 lm ( p < 0001), 26.88 lm( p < 0.0001), 23.46 lm ( p < 0.0001), 24.32 lm ( p <
0.0001) and 22.18 lm ( p < 0.0001) were induced, respec-
tively (Fig. 2d). In BE-M17 cells transfected with A53T
a-syn, vehicle control cells exhibited a median CTL of
BE
-M17
cells
0.01
Fe(I
I)
0.1
Fe(I
I)
1.0
Fe(I
I)
5.0
Fe(I
I)
10.0
Fe(I
I)
50.0
Fe(I
I)
100.
0Fe
(II)
0
50
100
150
(a)
µM
Com
etta
ille
ngth
(µm
)
Vec
tor
cont
rol
0.01
Fe(
II)
0.1
Fe(
II)
1.0
Fe(
II)
5.0F
e(II
)
10.0
Fe(
II)
50.0
Fe(
II)
100.
0F
e(II
)
0
50
100
150
(b)
µM
Com
etta
ille
ngth
(µm
)
A30
Pα
-syn
0.01
Fe(I
I)
0.1
Fe(I
I)
1.0
Fe(I
I)
5.0
Fe(I
I)
10.0
Fe(I
I)
50.0
Fe(I
I)
100.
0Fe
(II)
0
50
100
150(d)
µM
Com
etta
ille
ngth
(µm
)
Wil
d-ty
peα
-syn
0.01
Fe(
II)
0.1
Fe(
II)
1.0
Fe(
II)
5.0
Fe(
II)
10.0
Fe(
II)
50.0
Fe(
II)
100.
0F
e(II
)
0
50
100
150
(c)
µM
Com
etta
ille
ngth
(µm
)
A53
Tα
-syn
0.01
Fe(I
I)
0.1
Fe(I
I)
1.0
Fe(I
I)
5.0
Fe(I
I)
10.0
Fe(I
I)
50.0
Fe(I
I)
100.
0Fe
(II)
0
50
100
150(e)
µM
Com
etta
ille
ngth
(µm
)
Fig. 2 Comparison of the DNA-damaging effects of Fe(II), measured
as SSBs in the Comet assay, in human dopaminergic BE(2)-M17
neuroblastoma (BE-M17) cells. Cells were grown to confluence prior to
treatment. From the BE-M17 cell line (a) untransfected cells, (b) vector
control cells, (c) cells transfected with wild-type a-syn, (d) cells
transfected with A30P a-syn or (e) cells transfected with A53T a-syn
were used. In each panel, cell type represents comet-forming activity
in vehicle control cells followed by effects of Fe(II) concentrations:
0.01, 0.1, 1.0, 5.0, 10.0, 50.0 and 100.0 lM, as indicated. Following a
2-h treatment, cells were disaggregated prior to incorporation into the
Comet assay as described in the Materials and methods. CTL (lm)
was used as a measure of DNA damage.
Mutant a-syn and Fe(II)-induced SSBs in BE-M17 cells 623
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 5
14.94 lm and increases in median CTLs to 21.33 lm( p < 0.0001), 19.62 lm ( p < 0.004), 24.74 lm ( p <
0001), 29.86 lm ( p < 0.0001), 39.25 lm ( p < 0.0001),
34.56 lm ( p < 0.0001) and 33.28 lm ( p < 0.0001) were
observed in the presence of 0.01, 0.1, 1.0, 5.0, 10.0, 50.0 or
100.0 lM Fe(II) treatment, respectively (Fig. 2e). Clearly,
these results demonstrate an elevated susceptibility to Fe(II)-
induced comet-forming activity in cells transfected with
mutant forms of a-syn as compared with untransfected cells,
vector control cells or cells transfected with wild-type a-syn.
Effects of repair inhibitors, HU/ara-C, on susceptibility
to Fe(II)
Figure 3(a–e) and Table 1 compare the susceptibilities of
BE-M17 cells to the DNA-damaging effects of Fe(II) in the
absence or presence of the DNA-repair inhibitors, HU/ara-C.
In the absence of HU/ara-C, the cells transfected with A30P
a-syn or A53T a-syn were again more susceptible to the
DNA-damaging effects of Fe(II) as compared with untrans-
fected cells, vector control cells or cells transfected with
wild-type a-syn. Even following the incorporation of HU/
ara-C, only small increases in Fe(II)-induced comet forma-
tion occurred in these latter three cell types. HU/ara-C
appeared to enhance levels of Fe(II)-induced SSBs in cells
transfected with A30P a-syn or A53T a-syn compared with
treatments in their absence (Fig. 3d and e, Table 1).
Table 1 shows that significant increases ( p < 0.0005) in
Fe(II)-induced comet-forming activity were observed at
concentrations equal to or greater than 1.0 lM in untransfected
BE-M17 cells, either in the absence or presence of HU/ara-C.
In the absence or presence of HU/ara-C, vehicle control cells
exhibited a mean CTL ± 95% CI of 10.63 ± 1.28 lm or
14.91 ± 1.80 lm, respectively. Fe(II) treatments of 0.1, 1.0 or
10.0 lM in the absence of HU/ara-C resulted in increases in
mean CTL ± 95% CI to 10.92 ± 1.37 lm, 14.97 ± 1.96 lm( p < 0.0005) and 16.13 ± 2.35 lm ( p < 0.0005), respec-
tively. However, in the presence of repair inhibitors respective
BE
-M17
cells
+H
U/a
ra-C
0.1
Fe(I
I)
+H
U/a
ra-C
1.0
Fe(I
I)
+H
U/a
ra-C
10.0
Fe(I
I)
+H
U/a
ra-C
0
50
100
150(a)
µM
Com
etta
ille
ngth
(µm
)
Vec
tor
cont
rol
+H
U/a
ra-C
0.1F
e(II
)
+H
U/a
ra-
1.0
Fe(I
I)
+H
U/a
ra-CC
10.0
Fe(I
I)
+H
U/a
ra-C
0
50
100
150
(b)
µM
Com
etta
ille
ngth
(µm
)
A30
Pα
-syn
+H
U/a
ra-C
0.1
Fe(I
I)
+H
U/a
ra-C
1.0
Fe(I
I)
+H
U/a
ra-C
10.0
Fe(I
I)
+H
U/a
ra-C
0
50
100
150(d)
µM
Com
etta
ille
ngth
(µm
)
Wil
d-ty
peα
-syn
+H
U/a
ra-C
0.1
Fe(
II)
+H
U/a
ra-C
1.0
Fe(
II)
+H
U/a
ra-C
10.0
Fe(
II)
+H
U/a
ra-C
0
50
100
150(c)
µM
Com
etta
ille
ngth
(µm
)
A53
Tα
-syn
+H
U/a
ra-C
0.1
Fe(I
I)
+H
U/a
ra-C
1.0
Fe(I
I)
+H
U/a
ra-C
10.0
Fe(I
I)
+H
U/a
ra-C
0
50
100
150(e)
µM
Com
etta
ille
ngth
(µm
)
Fig. 3 Comparison of the DNA-damaging effects of Fe(II) in the
absence or presence of DNA-repair inhibitors in human dopaminergic
BE(2)-M17 neuroblastoma (BE-M17) cells. Cells were grown to con-
fluence prior to treatment in the absence or presence of the DNA-
repair inhibtors hydroxyurea and cytosine arabinoside (HU/ara-C,
1 mM/120 lM final concentration). From the BE-M17 cell line (a)
untransfected cells, (b) vector control cells, (c) cells transfected with
wild-type a-syn, (d) cells transfected with A30P a-syn or (e) cells
transfected with A53T a-syn were used. In each panel, cell type rep-
resents comet-forming activity in vehicle control cells followed by
effects of Fe(II) concentrations: 0.1, 1.0 and 10.0 lM, as indicated.
Each treatment was performed in the absence or presence of HU/ara-
C, as indicated. Following 2-h treatments, cells were disaggregated
prior to incorporation into the Comet assay as described in the
Materials and methods. CTL (lm) was used as a measure of DNA
damage.
624 F. L. Martin et al.
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 6
increases to 17.17 ± 2.14 lm, 17.42 ± 2.10 lm and 24.29 ±
3.37 lm ( p < 0.0005) occurred. In vector control cells or
cells transfected with wild-type a-syn only small increases
( p < 0.05) in comet formation were observed following
Fe(II) treatment either in the absence or presence of repair
inhibitors.
Fe(II)-induced effects in BE-M17 cells transfected with
A30P a-syn or A53T a-syn are also shown in Table 1. In the
absence or presence of HU/ara-C, the vehicle control of
cells transfected with A30P a-syn exhibited a mean
CTL ± 95% CI of 15.32 ± 1.78 lm or 18.07 ± 2.07 lm,
respectively. Fe(II) treatment (0.1, 1.0 or 10.0 lM) in the
absence of HU/ara-C resulted in mean CTL ± 95% CI
levels of 26.74 ± 4.17 lm ( p < 0.005), 27.04 ± 4.74 lm( p < 0.0005) and 32.43 ± 4.99 lm ( p < 0.0005) whilst in
their presence increases to 32.00 ± 4.53 lm ( p < 0.0005),
36.96 ± 4.97 lm ( p < 0.0005) and 51.06 ± 6.38 lm ( p <
0.0005) were observed, respectively. In vehicle control cells
transfected with A53T a-syn, a mean CTL ± 95% CI of
16.51 ± 1.51 lm or 23.75 ± 2.19 lm was observed in the
absence or presence of HU/ara-C, respectively. Fe(II)
treatment (0.1, 1.0 or 10.0 lM) in the absence of HU/ara-C
resulted in increases in mean CTL ± 95% CI to 27.43 ±
4.44 lm ( p < 0.005), 26.14 ± 4.08 lm ( p < 0.005) and
31.62 ± 4.46 lm ( p < 0.0005) whilst in their presence
increases to 37.47 ± 4.93 lm ( p < 0.0005), 33.47 ± 4.22 lm
0 50 100 1500
25
50
75(a) Wild-type α-syn
Median = 6.832 µm
0 50 100 1500
25
50
75+ 1 mM HU/120 µM ara-C
Median = 7.180 µm
0 25 50 75 100 125 1500
25
50
75+ 10 µM Fe(II)
Median = 8.530 µm, P <0.03
0 25 50 75 100 125 1500
25
50
75+ HU/ara-C
Median = 9.320 µm, P <0.0005
0 50 100 1500
25
50
75A53T α-syn
Median = 10.670 µm
0 50 100 1500
25
50
75+ HU/ara-C
Median = 11.950 µm
0 50 100 1500
25
50
75+ 10 µM Fe(II)
Median = 17.060 µm,P <0.0001
0 50 100 1500
25
50
75+ HU/ara-C
Median = 27.920 µm,P* <0.0001, P <0.0001
Num
ber
ofob
serv
atio
ns
Comet tail length (µm)
(b)
(c) (d)
(e) (f)
(g) (h)
Fig. 4 Frequency distributions of the levels of DNA single-strand
breaks (SSBs) in cells transfected with wild-type a-synuclein (a-syn) or
A53T a-syn in the absence or presence of 10.0 lM Fe(II) treatment
with or without DNA-repair inhibitors, hydroxyurea and cytosine ara-
binoside (HU/ara-C, 1 mM/120 lM final concentration). BE-M17 cells
were treated as follows: (a) vehicle control cells transfected with wild-
type a-syn; (b) vehicle control cells transfected with wild-type a-syn in
the presence of HU/ara-C; (c) 10.0 lM Fe(II)-treated cells transfected
with wild-type a-syn; (d) 10.0 lM Fe(II)-treated cells transfected with
wild-type a-syn in the presence of HU/ara-C; (e) vehicle control cells
transfected with A53T a-syn; (f) vehicle control cells transfected with
A53T a-syn in the presence of HU/ara-C; (g) 10.0 lM Fe(II)-treated
cells transfected with A53T a-syn; (h) 10.0 lM Fe(II)-treated cells
transfected with A53T a-syn in the presence of HU/ara-C. The vertical
axis represents the number of observations made in the Comet assay;
the horizontal axis represents CTL (lm). p as compared with corres-
ponding control, p* as compared with corresponding treatment in the
absence of HU/ara-C.
0 2 4 6 8 10
0
20
40
60
80
100
120
*********
**
[Fe(II)] (µM, 2 h)
Mea
nC
TL
(µm
)±95
%C
I
0 2 4 6 8 10
0
20
40
60
80
100
120 Wild-type α-syn
A53T -syn
****** ***
* *** *
[Rotenone] (µM, 2 h)
Mea
nC
TL
(µm
)±95
%C
I
α
Fig. 5 Mean CTL ± 95% CI versus concentration of Fe(II) or the
mitochondrial respiratory chain inhibitor rotenone, as indicated, in cells
transfected with wild-type a-synuclein (a-syn) or A53T a-syn. Cells
were grown to confluence prior to treatment. Following 2-h treatment,
cells were disaggregated prior to incorporation into the Comet assay
as described in the Materials and methods. CTL (lm) was used as a
measure of DNA damage. *p < 0.01, **p < 0.001, ***p < 0.0001 as
compared with corresponding control.
Mutant a-syn and Fe(II)-induced SSBs in BE-M17 cells 625
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 7
( p < 0.0005) and 48.54 ± 7.21 lm ( p < 0.0005) were
observed, respectively.
Comparative susceptibilities
The relative susceptibilities of BE-M17 cells transfected with
either wild-type a-syn or A53T a-syn to the effects of
10.0 lM Fe(II) treatment in the presence or absence of HU/
ara-C are compared in Fig. 4(a–h). Wild-type transfected
cells appeared to be somewhat resistant to 10.0 lM Fe(II)
both in the absence or presence of HU/ara-C (Fig. 4c and d).
However, 10.0 lM Fe(II) treatment of cells transfected with
A53T a-syn resulted in an appreciable increase in CTL either
in the absence or presence of HU/ara-C (Fig. 4g and h).
Median CTL levels (vehicle control vs. 10.0 lM Fe(II)
treatment) were 6.83 lm versus 8.53 lm ( p < 0.03) in cells
transfected with wild-type a-syn and 10.67 lm versus
17.06 lm ( p < 0.0001) in cells transfected with A53T a-syn in the absence of HU/ara-C. In the presence of repair
inhibitors median CTL levels (vehicle control vs. 10.0 lM
Fe(II) treatment) were 7.18 lm versus 9.32 lm( p < 0.0005) in cells transfected with wild-type a-syn and
11.95 lm versus 27.92 lm ( p < 0.0001) in cells transfected
with A53T a-syn. Levels of SSBs were significantly greater
( p < 0.0001) in cells transfected with A53T a-syn treated
with Fe(II) in the presence of HU/ara-C than in the absence
of these DNA-repair inhibitors (Fig. 4g and h).
A comparison with the mitochondrial respiratory chain
inhibitor, rotenone
The effects of rotenone (1.0, 5.0 or 10.0 lM) in BE-M17 cells
transfected with either wild-type a-syn or A53T a-syn are
Unt
rans
fect
ed 0.1
1.0
10.0
Vec
tor
cont
rol
0.1
1.0
10.0
Wild
type
α-s
yn 0.1
1.0
10.0
A30
Pα-
syn
0.1
1.0
10.0
A53
Tα-
syn
0.1
1.0
10.0
0
50
100
150
Fe(II) treatment (µM, 2 h)
Com
etta
ille
ngth
(µm
)
Fig. 6 Box-whisker plot of the DNA-damaging effects of Fe(II),
measured as SSBs in the Comet assay, in human dopaminergic
BE(2)-M17 neuroblastoma (BE-M17) cells. From the BE-M17 cell line
untransfected cells, eukaryotic pcDNA3 vector control cells or cells
transfected with wild-type a-syn, A53T a-syn or A30P a-syn were
used, as indicated. Each individual box-whisker plot represents data
pooled from five separate experiments performed in duplicate, i.e.
individual analyses were derived from 10 different slides. These show
the median value for each experimental group indicated by a central
vertical line, lower and upper quartiles by the corresponding vertical
ends of each box and, minimum and maximum values indicated by the
extremities. Following a 2-h treatment, cells were disaggregated prior
to incorporation into the Comet assay as described in the Materials
and methods. CTL (lm) was used as a measure of DNA damage.
p-values for individual treatments, as compared with corresponding
vehicle control cells, are shown in Table 1.
Table 1 The effects of DNA-repair inhibitors on Fe(II)-induced comet formation
BE-M17 cells
(–/+DNA repair inhibitors)
lM Fe(II) (mean ± 95% CI)
0 0.1 1.0 10.0
Untransfected – 10.63 ± 1.28 10.92 ± 1.37 14.97 ± 1.96*** 16.13 ± 2.35***
cells + 14.91 ± 1.80 17.17 ± 2.14 17.42 ± 2.10 24.29 ± 3.37***
Vector – 14.97 ± 1.36 17.88 ± 1.59* 16.89 ± 1.99 19.30 ± 2.69
control + 19.25 ± 1.63 22.17 ± 2.27 23.84 ± 2.61* 23.43 ± 2.61*
Wild-type – 13.34 ± 1.69 12.10 ± 1.55 13.74 ± 1.72 15.90 ± 1.76*
a-syn + 16.36 ± 1.93 16.78 ± 2.00 19.03 ± 2.23* 20.86 ± 2.56*
A30P – 15.32 ± 1.78 26.74 ± 4.17** 27.04 ± 4.74*** 32.43 ± 4.99***
a-syn + 18.07 ± 2.07 32.00 ± 4.53*** 36.96 ± 4.97*** 51.06 ± 6.38***
A53T – 16.51 ± 1.58 27.43 ± 4.44** 26.14 ± 4.08** 31.62 ± 4.46***
a-syn + 23.75 ± 2.19 37.47 ± 4.93*** 33.47 ± 4.22** 48.54 ± 7.21***
Mean CTL ± 95% CI versus Fe(II) concentration in the absence or presence of the DNA-repair inhibitors hydroxyurea and cytosine arabinoside
(HU/ara-C, 1 mM/120 lM final concentration), in the Comet assay, in human dopaminergic BE(2)-M17 neuroblastoma (BE-M17) cells. Cells were
grown to confluence prior to treatment in the absence or presence of HU/ara-C, as indicated. Following 2-h treatment, cells were disaggregated
and incorporated into the Comet assay as described in the Materials and methods. CTL (lm) was used as a measure of DNA damage. *p < 0.05,
**p < 0.005, ***p < 0.0005 as compared with corresponding control.
626 F. L. Martin et al.
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 8
compared with Fe(II)-induced effects in Fig. 5. At all
concentrations (0.01, 0.1, 1.0 or 10.0 lM) of Fe(II)-tested
significant increases in comet-forming activity ( p < 0.001
with 0.01 lM, p < 0.0001 with higher concentrations) as
compared with background vehicle control levels were
observed in cells transfected with A53T a-syn. In contra-
distinction, cells transfected with wild-type a-syn appeared tobe more resistant to the DNA-damaging effects of Fe(II).
Rotenone was observed to induce significant increases
( p < 0.01 with 1.0 or 10.0 lM, p < 0.0001 with 5.0 lM) in
comet-forming activity in cells transfected with wild-type a-syn whilst in cells transfected with A53T a-syn similarly
significant increases ( p < 0.0001) were observed with all
concentrations tested.
Susceptibility to Fe(II)-induced DNA damage: pooled
experiments
To confirm the enhanced susceptibility to Fe(II) of BE-M17
cells transfected with either A30P a-syn or A53T a-syn in
comparison with untransfected cells, vector control cells or
cells transfected with wild-type a-syn, data derived from five
separate experiments, each performed in duplicate, were
examined (Fig. 6 and Table 2). Figure 6 displays the median
and two measures of spread, the range and the interquartile
range. Over the concentration range (0.1, 1.0 or 10.0 lM)
employed, the cells used in this study showed clear
differences in susceptibility to Fe(II). Whilst Fe(II)-induced
increases in DNA damage were observed in untransfected
cells, vector control cells or cells transfected with wild-type
a-syn, these elevations in SSBs do not appear to be
profound. However, in cells transfected with either A30P
a-syn or A53T a-syn, increases in Fe(II)-induced comet-
forming activity were apparent over the entire concentration
range employed, along with profound dose-related effects
(Fig. 6 and Table 2). These results are exemplified in
numerical fashion in Table 2; in untransfected cells, vector
control cells or cells transfected with wild-type a-syn, highlysignificant ( p < 0.0001) increases in SSBs were observed
only following treatment with 10.0 lM Fe(II). No differences
in DNA damage levels were observed between 0.1 lM Fe(II)
treatment groups or corresponding controls in these cells. In
comparison, highly significant ( p < 0.0001) increases in
DNA damage levels following treatment with 0.1 lM Fe(II)
were apparent in cells transfected with either A30P a-syn or
A53T a-syn as compared with corresponding controls
(Table 2).
Discussion
PD is a progressive neurological disorder with important
implications for ageing populations (Zhang et al. 2000;
Shastry 2001). The majority of cases of PD appear to be
sporadic, but approximately 5% of patients do exhibit a
family history, and there is now strong evidence that a
combination of genetic predisposition factors and putative
environmental factors initiates the pathobiology of this
disease (Mizuno et al. 1999). Mutations in the gene encoding
a-syn are responsible for some rare, early onset forms of
inherited PD, and these mutations increase the propensity of
the protein to form toxic oligomers (El-Agnaf et al. 1998a;
Conway et al. 2000). An important environmental factor
could be exposure to metals, including Fe, which can induce
Table 2 The result of pooled data sets to validate differential susceptibility to Fe(II)
BE-M17 cells
[number (n) of separate experiments]
lM Fe(II) (mean ± 95% CI)
0 0.1 1.0 10.0
Untransfected n ¼ 5 16.43 ± 1.16 15.32 ± 0.96 17.76 ± 1.03 20.24 ± 1.17
cells p-value – 0.6280 0.0048 < 0.0001
Vector n ¼ 5 13.30 ± 0.62 13.37 ± 0.62 14.44 ± 0.71 18.23 ± 0.96
control p-value – 0.8633 0.0753 < 0.0001
Wild-type n ¼ 5 10.97 ± 0.66 11.29 ± 0.65 12.42 ± 0.75 14.13 ± 0.88
a-syn p-value – 0.3481 0.0050 < 0.0001
A30P n ¼ 5 14.47 ± 0.86 23.81 ± 1.44 27.04 ± 1.77 33.62 ± 2.21
a-syn p-value – < 0.0001 < 0.0001 < 0.0001
A53T n ¼ 5 18.93 ± 1.05 25.72 ± 1.41 32.57 ± 1.83 43.70 ± 2.3
a-syn p-value – < 0.0001 < 0.0001 < 0.0001
Mean CTL ± 95% CI vs. Fe(II) concentration from five separate experiments carried out in duplicate for each experimental treatment, in
human dopaminergic BE(2)-M17 neuroblastoma (BE-M17) cells. Cells were grown to confluence prior to treatment, as indicated. Fol-
lowing 2-h treatment, cells were disaggregated and incorporated into the Comet assay as described in the Materials and methods. CTL
(lm) was used as a measure of DNA damage. p-values (two-tailed) are as compared with corresponding controls.
Mutant a-syn and Fe(II)-induced SSBs in BE-M17 cells 627
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 9
cell damage via reactive oxygen species (ROS)-induced
oxidative stress mechanisms. It has been shown previously
that the addition of Fe(II) to pre-incubated solutions of a-synresults in the generation of ÆOH (Tabner et al. 2001; Turnbull
et al. 2001; Tabner et al. 2002). These ÆOH appear to be
generated from hydrogen peroxide (H2O2), by means of
Fenton chemistry. Furthermore, the addition of Fe(II) to
a-syn-transfected cultured cells has been reported to induce
the intracellular aggregation of a-syn, with this effect being
much more pronounced with the A53T mutant form of the
protein (Ostrerova-Golts et al. 2000). Thus, metals, partic-
ularly Fe(II), could play an important role in a-syn-mediated
cell death. This could have important implications as Fe(II)
or Fe(III) is the most abundant transition metal in the brain
(Thompson et al. 2001) and there is good evidence that ROS,
such as H2O2, superoxide anion radical (O2–) or ÆOH, play a
major role in nigral neuronal death observed in PD (Thong
et al. 1999; Obata 2002).
This study examined the susceptibility of dopaminergic
BE-M17 neuroblastoma cells expressing various forms of
a-syn to Fe(II)-induced genomic damage in the form of
SSBs. Addition of Fe(II) to these same cells (particularly
those expressing the a-syn A53T mutant) induces a-synaggregation (Ostrerova-Golts et al. 2000). The Comet assay
is a sensitive microelectrophoretic technique for the direct
visualisation of DNA damage, in the form of SSBs, in single
cells (Martin et al. 1997, 1999). Untransfected cells, vector
control cells or cells transfected with wild-type, A30P or
A53T a-syn were treated with various concentrations of
Fe(II) over a large concentration range (0.01–100.0 lM).
Fe(II) treatment for 2 h highlighted clear differences in
susceptibility to DNA damage induction (Fig. 2a–e). In the
presence of lower Fe(II) exposures (0.01–10.0 lM) untrans-
fected cells, vector control cells and cells transfected with
wild-type a-syn exhibited slight, albeit sometimes signifi-
cant, increases in CTL. However, cells transfected with
A30P a-syn or A53T a-syn (Fig. 2d and e) showed
significant increases in susceptibility to these lower expo-
sures in comparison with the former three cell types
(Fig. 2a–c). Cells transfected with A53T a-syn appeared to
be the most susceptible to Fe(II) toxicity at these concen-
trations (Fig. 6 and Table 2). Higher concentrations (50.0
and 100.0 lM) of Fe(II) resulted in a similar pattern of DNA
damage induction. Vector control cells were more suscept-
ible than untransfected cells or cells transfected with wild-
type a-syn (Fig. 2a–c). In cells transfected with A30P a-synor A53T a-syn highly significant increases ( p < 0.0001) in
comet formation as measured by CTL were observed
following either 50.0 or 100.0 lM Fe(II) treatment (Fig. 2d
and e). In all cases, the highest levels of SSBs were observed
in cells transfected with A53T a-syn, suggesting that these
cells are the most susceptible employed in this study.
Overall, our results are consistent with previous reports
showing that cells transfected with mutant a-syn exhibit an
enhanced susceptibility to various toxic insults (da Costa
et al. 2000; Lee et al. 2001).
One possible interpretation for the effects of the A30P and
A53T mutations is that, in the presence of Fe(II), both mutant
forms of a-syn aggregate into toxic oligomers more rapidly
than wild-type a-syn (Ostrerova-Golts et al. 2000). These
oligomers could then induce oxidative damage to the cells,
via the direct formation of ROS (Tabner et al. 2001; Turnbull
et al. 2001; Tabner et al. 2002). There may already be
ongoing intracellular damaging processes in cells expressing
mutant a-syn, and further treatment with Fe(II) may exacer-
bate this already compromised state. This idea is supported
by the fact that cells transfected with A53T a-syn do appear
to exhibit consistently higher levels of pre-existing SSBs
compared with other cell types (Tables 1 and 2). Treatment
with Fe(II) would induce further damage, via an a-syn-mediated mechanism, or through a different mechanism
whereby Fe(II)-induced damage occurs independently of
a-syn.In order to investigate the role of repair, DNA-repair
inhibitors (HU/ara-C) were used. These are inhibitors of
nucleotide excision repair (NER) and have been previously
shown to enhance the sensitivity of the Comet assay by
allowing the accumulation of repair-induced SSBs (Martin
et al. 1997, 1999). DNA lesions generated by a Fe(II)/H2O2
system have been shown to possess chromatographic
characteristics similar to DNA adducts formed by bulky
carcinogens; such adducts would be removed by NER
(Lloyd et al. 1997; Lloyd and Phillips 1999). At all
concentrations tested, higher levels of DNA damage
induction in cells transfected with A30P a-syn or A53T
a-syn were observed in the presence of HU/ara-C as
compared with levels of SSBs following treatments in the
absence of these repair inhibitors (Fig. 3 and Table 1).
Differences in the susceptibility of the cells employed in
this study are highlighted in Fig. 4 where the effects of
10.0 lM Fe(II) on cells transfected with either wild-type
a-syn or A53T a-syn are compared. In the presence or
absence of HU/ara-C, cells transfected with A53T a-synwere clearly more susceptible to the comet-forming effects
of Fe(II). This susceptibility appears to be a redox metal-
specific effect because less profound differences in the
levels of DNA SSBs were observed following rotenone
treatment of cells transfected with either wild-type a-syn or
A53T a-syn (Fig. 5). Rotenone is believed to enhance
mitochondrial ROS production (Li et al. 2003) and hence
would generate such damage in a manner independent of
redox metal-mediated mechanisms.
In conclusion, the results of this study using the Comet
assay demonstrate the enhanced susceptibility of BE-M17
cells expressing mutant forms of the a-syn to redox metal-
mediated DNA damage. Additional studies are in progress to
further investigate the underlying molecular mechanisms
responsible for these effects.
628 F. L. Martin et al.
� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 87, 620–630
Page 10
Acknowledgements
RH is supported by a grant (CR 582) from the North-West Cancer
Research Fund, UK. OMAE and KEP are supported by The
Parkinson’s Disease Society, UK. We thank John Dent for assistance
with the confocal microscopy. The authors also thank Dr Mark
Cookson (National Institutes of Health) for gifting us the various
human neuroblastoma dopaminergic cells used in this study.
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