J. Biomedical Science and Engineering, 2009, 2, 86-89
Published Online April 2009 in SciRes. http://www.scirp.org/journal/jbise JBiSE
Effects of lead exposure on alpha-synuclein and
p53 transcription
Pei-Jun Zuo1, A. Bakr M. Rabie1
1Faculty of Dentistry, the University of Hong Kong, Hong Kong SAR. Correspondence should be addressed to Peijun Zuo (
Received Nov. 1st, 2008; revised Feb. 19th, 2009; accepted Feb. 23rd, 2009
ABSTRACT
Objective: Epidemiological studies have found
that lead exposure increases the risk for Park-
inson’s disease and patients with Parkinson’s
disease have lower odds of developing non-
smoking-related cancer (1). It would be inter-
esting therefore to find the molecular links be-
tween Parkinson’s disease and cancer. To do
this, we studied mRNA expression of alpha-
synuclein gene, a promising genetic marker for
Parkinson’s disease, and expression of the tu-
mor suppressor gene p53 after oxidative stress
induced by lead. Methods: We used ATDC5 cell
line as a model of tumor and treated by lead
nitrate for 0, 2, 4, 16, 24 and 48 hours. The
mRNAs of alpha-synuclein and p53 were quan-
tified by reverse transcriptase polymerase
chain reaction and expressed as mean (±SD)
for 3 samples at each time point. Results: Ex-
pression of both of alpha-synuclein and p53
mRNA increased with increasing exposure of
lead treatment. The levels of alpha-synuclein
and p53 mRNA were correlated with each other
(r=0.9830; P<0.001). Conclusion: We propose
that lead’s neurotoxicity in PD is caused by al-
pha-synuclein expression and aggregation,
which releases the inhibitory influence of al-
pha-synuclein on p53 expression, thereby al-
lowing p53 to act as the cell’s guardian of the
genome and reduce tumorigenic potential.
Treatments that reduce alpha-synuclein aggre-
gation may need to account for a concomitant
reduction in p53’s protective effect.
Keywords: Alpha-synuclein, p53, Real-time
PCR, ATDC5, Aging, Cancer
1. INTRODUCTION
Parkinson’s disease (PD) typically affects people aged
50 years and older, and the risk of disease increases with
age. A promising diagnostic marker for PD is alpha-
synuclein (2), which is the primary structural compo-
nent of inclusion bodies (Lewy bodies) that are found in
the neurons of patients with PD. Interestingly, epidemi-
ological evidence shows that individuals with PD have
reduced odds for many common types of non-smoking-
related cancers (1). However, it is not known if this
finding indicates a direct association between the two
diseases, such as a reduced risk of non-smoking-related
cancer among patients who develop PD, or a reduced
risk of developing PD or other age-related neurodegen-
erative diseases among individuals with non-smoking-
related cancer. It would therefore be interesting to study
the possible biological and molecular links between age-
related neurodegenerative diseases such as PD and can-
cer, especially because cancer is often also regarded as a
disease of aging. The findings would provide important
information on the mechanisms underlying normal and
abnormal developmental and ageing processes. The p53
tumor suppressor protein may be one such link because
hyperactivation of p53 in mice has been shown to in-
crease resistance to spontaneous tumorigenesis while
apparently accelerating aging (3). Cancer and neurode-
generative diseases might also be interrelated at the etio-
logic or environmental level: occupational exposure to
lead is a risk factor for PD (4), while lead can be car-
cinogenic in rodents and genotoxic in fish (5), and it can
also induce cell apoptosis via p53 (6).
To investigate the possible link between lead expo-
sure and expression of alpha-synuclein and p53, we
used a lead-sensitive cell culture model. We selected the
ATDC5 cell line because it is an established mouse em-
bryonic carcinoma-derived cell line that has both car-
cinogenic and chondrogenic properties (7). Not only
does this cell line have chondroprogenitor potential and
produce chondrocyte-specific extracellular matrix when
stimulated by insulin, but it can also proliferate rapidly
in the presence of fetal bovine serum (8). Expression
levels of alpha-synuclein and p53 were thus measured at
the mRNA level after treatment of cells with lead nitrate.
2. EXPERIMENTAL PROCEDURES
Cell Culture -The ATDC5 cells were cultured in a 1:1
mixture of Dulbecco’s modified Eagle’s medium and
Ham’s F-12 medium (Flow Laboratories, Irvine, UK)
containing 5% fetal bovine serum (GIBCO BRL,
Gaithersburg, MD), 100 U/mL penicillin, and 100 µg/
mL streptomycin (Biofluids Inc., Rockville, MD, USA)
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P. J. Zuo et al. / J. Biomedical Science and Engineering 2 (2009) 86-89 87
SciRes Copyright © 2009 JBiSE
and then incubated at 37ºC in a humidified atmosphere
containing 5% carbon dioxide. An inoculum of cells
(104 per mL in 30 mL) was transferred to each of 7 Petri
dishes. Lead nitrate was added to the cells. The final
concentration of the lead nitrate per dish was 200
µmol/L. For increasing the solubility of lead nitrate,
glutamic acid was added to medium in equimolar
amounts of the lead nitrate. The cells were harvested at
times of 0, 2, 4, 16, 24 and 48 hours.
Reverse Transcriptase Polymerase Chain Reaction
Analysis- Total RNA was isolated from cells by using
an RNeasy Mini Kit (Qiagen Sciences, Germantown,
MD) according to the manufacturer’s instructions. Re-
verse transcription of the mRNA was performed with
oligo-(dT) primers and MulV reverse transcriptase (Ap-
plied Biosystems, Foster City, CA). Polymerase chain
reaction (PCR) was then done in a StepOne Real-Time
PCR System (Applied Biosystems); each 20-µL sample
contained Power SYBR Green PCR Master Mix (Ap-
plied Biosystems), 40 ng of complementary DNA, and
the pairs of primers listed in Table 1. The cycling pro-
files were as follows: 95ºC for 10 minutes, 95ºC for 15
seconds and 60ºC for 1 minute (for 40 cycles). Samples
were run concurrently with standard curves derived
from PCR products, and serial dilutions were performed
to obtain appropriate template concentrations.
Mouse β-actin was used as an example of a lead-
insensitive house-keeping protein and thus its primer
acted as a negative control to correct for RNA recovery
and reverse transcription efficiency. The mRNA con-
centrations were determined by by optical density at
λ260/280 and were standardized at each time point to
mouse β-actin mRNA concentrations.
Statistical Analysis- Data were expressed as mean ±
SD for 3 or more replicates per sample, in arbitrary units
relative to the mRNA level of β-actin. The student t test
was used to evaluate differences between groups. Differ-
ences were considered significant at a level of p < 0.05.
3. RESULTS
Alpha-synuclein mRNA- Lead exposure induced a 50%
increase in alpha-synuclein mRNA expression in
ATDC5 cells at 2 hours (Figure 1). The mRNA level
further increased by about 20% between 2 hours and 16
hours, remained the same between 16 and 24 hours and
then increased rapidly to 3 times the 24-hour level at 48
hours. The results thus show that the expression of al-
pha-synuclein was significantly increased by the expo-
Table 1. List of primers
Primer Sequence (sense/antisense)
Alpha-synuclein 5'- AGT GGA GGG AGC TGG GAA TA
TAG-3'
5'-TCC TCA CCC TTG CCC ATC T-3'
p53 5'-AGC GCT GCT CCG ATG GT-3'
5'-TTC CTT CCA CCC GGA TAA GA-3'
Mouse β-actin 5'-GGC CAA CCG TGA AAA GAT GA-3'
5'-CAG CCT GGA TGG CTA CGT ACA-3'
Figure 1. Alpha-synuclein mRNA in ATDC5 cells. ATDC5 cells
were treated with 200 µM lead nitrate for duration of 0, 2, 4, 16,
24 and 48 hours. The results as expressed as mean ± SD
Psmc3 mRNA amount relative to beta-actin for three samples.
Figure 2. p53 mRNA in ATDC5 cells. ATDC5 cells were treated
with 200 µM lead nitrate for duration of 0, 2, 4, 16, 24 and 48
hours. The results as expressed as mean ± SD p53 mRNA
amount relative to beta-actin for three samples.
sure time.
p53 mRNA- Expression of p53 mRNA in ATDC5 cells
was also significantly increased by the exposure time. By
2 hours, lead exposure had rapidly increased the p53
mRNA level to more than 3.4 times the basal amount.
(Figure 2) There was a moderate further increase in
mRNA expression between 2 and 24 hours, by about
25%, and then expression decreased very slightly at 48
hours, but the decrease was not statistically significant.
The rising trend in p53 mRNA level with an increase in
exposure time was similar to that of alpha-synuclein
mRNA. The levels of alpha-synuclein and p53 mRNA
were correlated with each other (r=0.9830; P<0.001).
4. DISCUSSION
We have demonstrated that expression of alpha-
synuclein and p53 mRNA increased with increasing
duration of lead exposure in ATDC5 cells and that there
was a correlation between alpha-synuclein and p53
mRNA expression after lead treatment. Although we did
not examine the expression or localization of the corre-
sponding proteins, our findings suggest that alpha-
synuclein and p53 expression are stimulated by lead
treatment in a coordinated way, which may reflect a
shared cellular response to biological effects of lead
exposure such as neurotoxicity, genotoxicity and oxida-

88 P. J. Zuo et al. / J. Biomedical Science and Engineering 2 (2009) 86-89
SciRes Copyright © 2009 JBiSE
tive stress. In this study, alpha-synuclein was assumed
to be a marker of increased likelihood of PD develop-
ment and p53 was a marker of decreased cancer poten-
tial. Our results thus suggest a molecular mechanism for
the inverse epidemiological association observed be-
tween PD and cancer (1).
Alpha-synuclein aggregation appears to play a major
role in Lewy body formation and PD (9) (10), and occu-
pational exposure to lead is a risk factor for PD (4). In
addition, lead can form inclusion bodies in renal cells of
poisoned humans or animals (11) and lead is a known
potent neurotoxicant. Evidence indicates that lead expo-
sure early in life may later on cause neurodegenerative
disease such as Alzheimer’s disease (12). Indeed, Alz-
heimer’s disease is similar to PD, in that both are caused
by a progression of amyloidogenesis in the brain. The
link between alpha-synuclein and p53 expression on
lead exposure demonstrated in this study suggests that
alpha-synuclein protein expression and aggregation in
cells can be stimulated by lead and contribute to inclu-
sion body formation seen in cells of PD patients, while
p53 protein accumulates to prevent cellular and genetic
damage due to increased oxidative stress.
Because the p53 transcription factor regulates the cell
cycle and apoptosis, it has been described as “the guard-
ian of the genome,” referring to its tumor suppressor
role in conserving genomic stability by preventing the
accumulation of mutations (13). An increase in p53 ex-
pression during lead exposure would result in an in-
creased likelihood of cell cycle arrest to allow for ge-
nomic repair, or apoptosis if the genome is irreparable,
and a similar mechanism might explain the possible
lowered risk of tumorigenesis among PD patients. In-
deed, lead can induce cell apoptosis via p53 in culture
cells (6), and doing so would decrease the genotoxic
potential of lead and hence the likelihood of cancer
transformation. Another known role of p53 is to induce
differentiation, which is another potential mechanism
for preventing tumorigenesis. Interestingly, lead ion
(Pb2+)-induced neurotoxicity may also be partially me-
diated through p53-independent apoptosis that is en-
hanced by glutamate (14), thereby again lowering tu-
morigenic potential.
A link between alpha-synuclein and p53 function has
been previously demonstrated. In neuronal cell cultures,
alpha-synuclein reduced the ability of cells to apoptose
with and without the apoptotic trigger of staurosporine,
and also reduced p53 expression and transcriptional
activity (15). Both of the p53 expression and transcrip-
tional activity was tested 48 hours after transfection.
However, the dopamine-derived drug 6-hydroxy-
dopamine reversed these effects and increased alpha-
synuclein aggregation. The present data indicated p53
transcriptional activity was increased in 48 hours as well
as alpha-synuclein transcriptional activity after lead
treatment. Although we did not test for apoptotic status
and immunoreactivity in our study, it is conceivable that
alpha-synuclein has an effect on p53 expression and
function and that lead treatment promotes alpha-
synuclein aggregation, thereby resulting in p53 expres-
sion and related downstream cellular events. This series
of events may explain lead’s neurotoxicity and parallels
the proposed role of the natural toxin 6-
hydroxydopamine in alpha-synuclein aggregation in the
etiology of PD (15).
The biological mechanism connecting alpha-
synuclein, p53, lead exposure, PD and cancer does not
appear to be simple. Epidemiological studies have
shown that the odds of only non-smoking-related, but
not smoking-related, cancer were lowered among pa-
tients with PD (1). The paradox is that smoking in-
creases a human’s intake of lead. Yet, there is a weak
association between stomach and lung cancer frequency
and an individual’s exposure to lead (16), and a small
but statistically significant increase in mortality has
been found among employees at lead battery plants and
lead smelters (17). In contrast, these employees’ mortal-
ity from kidney cancer, bladder cancer, cancer of the
central nervous system, lymphatic cancer and hemato-
poietic cancer was not increased (17). Finally, although
both PD and cancer can be viewed as diseases of aging,
laboratory studies have reported that p53 can uncouple
cancer and aging by regulating both in a mutually exclu-
sive way-namely, p53 hyperactivation in mice reduced
the risk of spontaneous cancer but accelerated organis-
mal aging (3). Organismal aging may be related by
p53’s ability to induce apoptosis and, on prolongued
activation, to induce cellular terminal cell cycle block or
“senescence,” and may be controlled by subcellular lo-
calization (18). We will use of a neuronal cell line to do
assays for protein expression, phenotypes such as apop-
tosis, differentiation, cell division, cell cycle, etc give
better information.
Our findings provide some insight into the association
between PD and cancer, and the behavior of cells in
response to lead exposure. We propose that lead’s neu-
rotoxicity in PD is caused by alpha-synuclein expression
and aggregation, which releases the inhibitory influence
of alpha-synuclein on p53 expression and allows p53 to
act as the cell’s guardian of the genome, thereby reduc-
ing tumorigenic potential. It is possible that in the ab-
sence of lead, other triggers of alpha-synuclein expres-
sion and aggregation are involved in promoting p53
expression in the etiology of PD and other age-related
neurodegenerative diseases. The results also suggest that
treatments for PD based on preventing alpha-synuclein
aggregation need to take into account the possible side
effect of reducing p53 expression and function, and in-
creasing tumorigenic potential. Treatments for cancer
based on p53 expression also need to take into account
the side effect of aging if p53 is allowed to be hyperac-
tivated.
5. DISCLOSURE STATEMENT

P. J. Zuo et al. / J. Biomedical Science and Engineering 2 (2009) 86-89 89
SciRes Copyright © 2009 JBiSE
The authors have declared that no competing interests
exist.
ACKNOWLEDGMENTS
The authors thank Dr. Trevor Lane for critical evaluation of this
manuscript. This research was supported by research grant of Profes-
sor Dr. A. Bakr M. Rabie.
REFERENCES
[1] A. B. West, V. L. Dawson and T. M. Dawson, (2005) Trends
Neurosci 28, 348-352.
[2] O. M. El-Agnaf, S. A. Salem, K. E. Paleologou, M. D. Curran,
M. J. Gibson, J. A. Court, M. G. Schlossmacher and D. Allsop,
(2006) FASEB J 20, 419-425.
[3] S. D. Tyner, S. Venkatachalam, J. Choi, S. Jones, N. Ghebrani-
ous, H. Igelmann, X. Lu, G. Soron, B.Cooper, C. Brayton, S.
Hee Park, T.Thompson, G. Karsenty, A. Bradley, and L. A.
Donehower, (2002) Nature 415, 45-53.
[4] S. Coon, A. Stark, E. Peterson, A. Gloi, G. Kortsha, J. Pounds, D.
Chettle and J. Gorell, (2006) Environ Health Perspect 114,
1872-1876.
[5] A. G. Osman, I. A. Mekkawy, J. Verreth, S. Wuertz, W. Kloas,
and F. Kirschbaum, (2008) Environ Toxicol.
[6] J. Xu, L. D. Ji and L. H. Xu, (2006) Toxicol Lett 166, 160-167.
[7] T. Atsumi, Y. Miwa, K. Kimata and Y. Ikawa, (1990) Cell Differ
Dev 30, 109-116.
[8] C. Shukunami, C. Shigeno, T. Atsumi, K. Ishizeki, F. Suzuki and
Y. Hiraki, (1996) J Cell Biol 133, 457-468.
[9] P. Jenner and C. W. Olanow, (1998) Ann Neurol 44, S72-84.
[10] W. Zhou and C. R. Freed, (2004) J Biol Chem 279, 10128-
10135.
[11] W. Qu, B. A. Diwan, J. Liu, R. A. Goyer, T. Dawson, J. L. Hor-
ton, M. G. Cherian and M. P. Waalkes, (2002) Am J Pathol 160,
1047-1056.
[12] L. D. White, D. A. Cory-Slechta, M. E. Gilbert, E. Tiffany-
Castiglioni, N. H. Zawia, M. Virgolini, A. Rossi-George, S. M.
Lasley, Y. C. Qian and M. R. Basha, (2007) Toxicol Appl Phar-
macol 225, 1-27.
[13] S. Bates, A. C. Phillips, P. A. Clark, F. Stott, G. Peters, R. L.
Ludwig and K. H. Vousden, (1998) Nature 395, 124-125.
[14] J. Loikkanen, K. Chvalova, J. Naarala, K. H. Vahakangas and K.
M. Savolainen, (2003) Toxicol Lett 144, 235-246.
[15] C. Alves Da Costa, E. Paitel, B. Vincent and F. Checler, (2002) J
Biol Chem 277, 50980-50984.
[16] H. Fu and P. Boffetta, (1995) Occup Environ Med 52, 73-81.
[17] O. Wong and F. Harris, (2000) Am J Ind Med 38, 255-270.
[18] J. Wesierska-Gadek and G.Schmid, (2005) Cell Mol Biol Lett 10,
439-453.