Rev Cubana Genet Comunit. 2010;4(1):42-47
Oxidative damage and antioxidant enzymes in blood of patients
with Spinocerebellar Ataxia Type 2
Marcadores de daño oxidativo y defensa antioxidante en pacientes
con Ataxia Espinocerebelosa tipo 2
Gretel Riverón Forment,I Olivia Martínez Bonne,II Reinaldo Gutiérrez Gutiérrez,III
Anamarys Pandolfi Blanco,IV Judith Pupo Balboa,V Nayade Pereira,VI Luis Velazquez PerezVII
Abstract
Resumen
Recent evidences suggest that increased oxidative damage as well as deficits in antioxidants defense systems
could be related to the pathogenesis of some hereditary ataxias. The aim of this study was to investigate
some redox status biomarkers in patient with spinocerebellar ataxia type 2. Samples from 33 patients and 22
control subjects, from Holguín province, were used to
determined plasmatic levels of malondialdehyde and enzymatic activities of Cu/Zn superoxide dismutase and
catalase by spectrophotometric methods. Besides, DNA
damage in peripheral blood cells was evaluated using the
Comet assay. Our results evidenced that oxidative damage is higher in spinocerebellar ataxia type 2 Cuban patients in comparison with the control group. In addition,
we found an increase of antioxidant activity of evaluated
enzymes in affected subjects. These findings suggest that
the systemic redox state is altered in patients with spinocerebellar ataxia type 2.
Evidencias recientes sugieren que el aumento del daño
oxidativo, así como deficiencias en los sistemas de defensa antioxidantes podrían estar relacionados a la patogénesis de algunas ataxias hereditarias. El objetivo de
este estudio fue evaluar algunos marcadores del estado
redox en pacientes con ataxia espinocerebelosa tipo 2. En
el estudio se incluyeron, muestras de 33 pacientes y 22
sujetos como controles, todos provenientes de la provincia de Holguín. Se determinaron los niveles plasmáticos
de malondialdehído y las actividades enzimáticas de las
enzimas Cu/Zn superóxido dismutasa y catalasa, por métodos espectrofotométricos. Además, se evaluó el daño
del ADN en las células de sangre periférica mediante el
ensayo Cometa. Los resultados obtenidos demuestran un
aumento del daño oxidativo en los pacientes con ataxia
espinocerebelosa tipo 2 en comparación con el grupo
control. Además, se observa un incremento en las actividades de las principales enzimas antioxidantes, en los
sujetos afectados. Estos hallazgos sugieren que el estado redox a nivel sistémico está alterado en pacientes con
ataxia espinocerebelosa tipo 2.
Keywords: ataxia, antioxidant enzyme, comet assay,
malondialdehyde, neurodegenerative diseases, oxidative
stress.
Palabras clave: ataxia, enzimas antioxidantes, ensayo cometa, malonildialdehído, enfermedades neurodegenerativas, estrés oxidativo.
Máster en Ciencias. Máster en Bioquímica, Licenciada en Bioquímica. Investigador Agregado. Profesor Instructor. Laboratorio de
Estrés Oxidativo. Centro Nacional de Genética Médica. Ciudad de La Habana, Cuba.
II Máster en Ciencias. Técnico de Laboratorio de Estrés Oxidativo. Centro Nacional de Genética Médica. Ciudad de La Habana, Cuba.
III Máster en Genética Médica. Licenciado en Ciencias Farmaceúticas, Investigador Auxiliar. Laboratorio de Estrés Oxidativo. Centro
Nacional de Genética Médica. Ciudad de La Habana, Cuba.
IV Técnico. Laboratorio de Estrés Oxidativo. Centro Nacional de Genética Médica.
V
Doctora en Ciencias. Doctora en Medicina. Especialista de primer grado en Bioquímica. Investigador Agregado. Laboratorio de
Estrés Oxidativo. Centro Nacional de Genética Médica. Ciudad de La Habana, Cuba.
VI
Doctora en Medicina. Especialista de segundo grado en Bioquímica. Investigador Agregado. Profesor Asistente. Centro Nacional
de Genética Médica. Ciudad de La Habana, Cuba.
VII Doctor en Ciencias. Doctor en Medicina. Clínica para Investigación y Rehabilitación de las Ataxias Hereditarias (CIRAH). Holguín,
Cuba.
I
42
Gretel Riverón Forment
Introduction
Reactive oxygen species (ROS) generated within
cells or, more generally, in a tissue environment,
may easily turn into a source of cell and tissue injury. Aerobic organisms have developed evolutionarily conserved mechanisms and strategies to carefully
control the generation of ROS and other oxidative
stress-related radical or non-radical reactive intermediates. However, a derangement in redox homeostasis, resulting in sustained levels of oxidative stress
and related mediators, can play a significant role in
the pathogenesis of major human diseases including
cancer, diabetes, cardiovascular diseases and neurodegenerative disorders.1
Oxidative stress, manifested by lipid peroxidation,
protein oxidation and DNA oxidation among other
indices, is observed in several neurodegenerative
disorders including Alzheimer and Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), myotonic
dystrophy and hereditary ataxias such as Friedreich’s
ataxia (FRDA), ataxia telangiectasia (AT) and ataxia
with vitamin E deficiency.2-6However, in all cases the
influence of oxidative stress remains unclear. Recent
evidences indicate that loss of neurons in such disorders results from a complex interplay among oxidative injury, excitotoxic stimulation, dysfunction of
critical proteins and genetic factors.7
Redox research outside the Central Nervous System (CNS), particularly in blood cells and plasma of
patient with neurodegenerative diseases, although
controversial, support the presence of peripheral
oxidative damage.8On the other hand, relatively few
studies have considered the effect of oxidative stress
on the pathogenesis of hereditary ataxias. To our
knowledge, no study has so far reported evidenced
of an impairment of antioxidant enzymes and DNA
damage in blood of Spinocerebellar ataxia type 2
(SCA2) patients. Therefore, the aims of this study
were to investigate the levels of antioxidant enzymes
like Superoxide Dismutase and Catalasa and also investigate whether lipid peroxidation and DNA damage is related with the pathological events of SCA2.
Methods
Thirty-three patients with SCA2 (16 women and
17 men; mean age 40,1 ± 12,5 years) and twenty-two
healthy subjects as controls (10 women and 12 men;
mean age 37 ± 10,8 years) were included in the study
after obtaining their informed consent. The study was
performed in accordance with the ethical guidelines
of the World Medical Association Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects.
SCA2's patients were diagnosed in the Clinic for
Investigation and Rehabilitation of Hereditary Ataxias (CIRAH) using PCR amplification for detecting
CAG repeats described by Imbert et al. in 19969. All
patients showed ataxia and related symptoms (slow
saccadic eye movements, dysarthria, hyporeflexia
and adiacosinesia) and could be at clinic stages I, II or
III. For all patient the age at onset was defined as the
onset of motor impairment (mean 27± 10,8 years).
The healthy control subjects were interviewed
about their medical history and physically examined.
They were excluded both if considered to be malnourished and with evidence of significant medical problems or if they were taking antioxidant supplements.
Blood samples were drawn from the cubital median
vein of the subjects and were collected into K2 EDTA
tubes. For Comet Assay ten microliters of whole blood
were used, the rest of blood samples were centrifuged
at 1000 x g for 10 min at 4 °C and stored at -20 °C until their processing (before 10 days). The plasma was
used for the determination of the antioxidant enzymes
activity, as well as to determine the concentration of
malondialdehyde (MDA), product of oxidative damage to lipids.
Biochemical measurements
Determination of Lipid peroxidation (LPO)
Malondialdehyde (MDA), a product of LPO, was
determined by the spectrophotometric method described by Beuge JA and Aust SD.10This assay is
based on the reaction of a thiobarbituric acid with
MDA yielding a stable chromosphore with a maximum
absorbance at 532 nm. Concentration of MDA was
calculated by a calibration curve using 1,1,3,3 tetramethoxypropane (TMP)as a standard in terms of nmol/mL.
Measurement of Superoxide Dismutase Activity
Extracellular Superoxide Dismutase (ec-SOD) activity was assayed according to the method of Marklund and Marklund.11It was based in the ability of
SOD to inhibit auto-oxidation of pyrogallol, considering that one unit of SOD activity is the amount of
the enzyme that inhibits the 50 % auto-oxidation rate
of pyrogallol under standard condition at 420 nm, and
was expressed as Units/mL.
Measurement of Catalase Activity
Catalase (CAT) activity was determined according
to Aebi’s procedure.12The method is based on the capacity of the enzyme to transform H2O2 into H2O and
1/2 O2 in 1 min under standard conditions, decreasing
absorbance at 240 nm. One unit of enzymatic activity
Revista Cubana de Genética Comunitaria 43
Oxidative damage and antioxidant enzymes in blood of patients with Spinocerebellar Ataxia Type 2
was considered as the quantity of enzyme necessary
to transform 1 µmol of H2O2 in 1 minute at 37 oC.
A molar extinction coefficient of 43,6 M–1 cm–1 was
used.
All biochemical measurements were carried out at
room temperature using a Spectronic™ GENESYS™
8 UV/VIS spectrophotometer.
Statistical analysis
Data are expressed as mean values ± SD. The comparison between values obtained in patients and controls was performed by Student's t test for unpaired
data. Significance was taken as p< 0,05. The statistical program SPSS v10.0 for Windows was used in all
the analyses.
Measurement of DNA damage in peripheral blood
by Comet Assay
Results
The Comet assay, which is also called the singlecell gel electrophoresis technique, is a simple, sensitive and rapid method that can used to estimate DNA
damage at individual cell level through strand breaks,
open repair sites, cross-links and labile sites.13This
assay was performed as described previously by Collins et al. with slight modifications.14Prepared slides
are examined using the optical microscope. Stained
nuclei with DNA damage exhibit migration of single
stranded DNA toward the anode forming a comet
tail (% of DNA in the tail is related to DNA break
frequency). One hundred nucleoids were scored per
slide and it was classified according to the extent of
tail DNA and given a value 0-4. DNA damage was
expressed in Arbitrary Units (UA).
SCA2 patient showed significantly elevated levels
of LPO in plasma compared to healthy subjects (1,05
± 0,09 vs 0,75 ± 0,09 nmol/L, p=0,012), as illustrated
in figure 1.
We found that the ec-SOD activity in plasma of SCA2
patients was higher than in the control group (19,85 ±
1,50 vs. 9,73 ± 1,64 U/mL, p<0,001) and also found differences in CAT activity between groups (601,59 ± 71,3
vs. 388,22 ± 59,7 U/mL, p=0,038) (Figure 2).
The DNA damage determined in our study is shown
in figure 3. There was a significant difference in DNA
damage between SCA2 patient and controls (246 ±
11, 4 vs. 197 ± 11,3 UA, p=0,005). Interestingly, in
the group of patients, the levels of the DNA damage
increased as the disease progressed (Table 1).
Figure 1. Plasmatic levels of product of lipid peroxidation in
patients with SCA2 and healthy controls. Results given are the
means ± SD. *: p<0.05.
Figure 2. Superoxide Dismutase and Catalase activities in blood of patients with SCA2 and healthy controls. Results given are the
means ± SD. **: p<0.001.
44
Gretel Riverón Forment
Figure 3. In A shows examples of microscopic appearance of comets corresponding to controls and SCA2 patients groups. Control
group with very low amounts of DNA damage, showing almost no comet tail and SCA2 patient’s group shows comet typical of the
highly damaged. In the graph in B, shows comparison of the DNA damage between SCA2 patient and healthy control subjects. The
results are expressed as the mean of arbitrary units (AU). **: p <0.05 Student’s t test.
Table 1. Comparison between DNA damage depends
on the disease progression in SCA2 patients group
Time of Evolution (TE)
TE ≤ 12
TE > 12
N
15
18
DNA damage (AU)
238
252*
AU: Arbitrary Units. Results given are the means. *: p<0.05 Student’s t test.
Discussion
Increased production of ROS can induce oxidative
damage to lipids and as a result of this were formed
lipid peroxidation products such as MDA. Our results
showed that MDA levels were significantly increased
in all SCA2 patients as compared with the control
group. This could be due to an extensive generation
of ROS, thus leading to the uncontrolled progression
of peroxidative damage to lipids and cellular membranes. Oxidative stress increases MDA and the cytotoxic effects of this peroxidation product can induce a
reduction of membrane fluidity, cause DNA damage
and directly inhibit several proteins.15
In this study, increased of MDA levels in the plasma of SCA2 patient was consistent with the finding of
Delgado et al. (2002) in a complementary study with
SCA2 patients.16According with our results, increased MDA concentration has been reported in other
hereditary ataxias. Bradley et al. (2004) reported elevated MDA levels were in the plasma from FRDA
patients.17Reichenbach et al. (2002), who report higher levels of MDA in patients with AT.6 Also elevated
levels of MDA have been observed in other neurodegenerative pathologies, such as PD and ALS, confirming a pathophysiological role of oxidative stress in
these diseases.2, 18, 19
Antioxidant enzymes such as SOD and CAT play
an important role in protecting cells from oxidative
damage; SOD converting superoxide radicals into
hydrogen peroxide, which is then further metabolized
by CAT. The rate of dismutation of the superoxide
anion results in an increase of H2O2 and protection
from this reactive species would be conferred only
by an increased CAT and Glutathione Peroxidase
activities.20Theoretically at least, the balance between
the first and second step antioxidant enzymes may be
critical.21Our data showed a concomitant increase in
SOD and CAT activities in SCA2 patients compared
with healthy control subjects. Considering these results it might be suggested that the ratios of activiRevista Cubana de Genética Comunitaria 45
Oxidative damage and antioxidant enzymes in blood of patients with Spinocerebellar Ataxia Type 2
ty on the first and second steps antioxidant enzymes
were increased in order to protect the organism from
oxidative damage and could be a compensatory mechanism by the body to prevent tissue damage. Our
results are in agreement with previous reports in other
hereditary ataxias. Tozzi et al. reported evidence of an
impairment of enzymatic antioxidants in FRDA, supporting a relevant role of free radical damage in the
pathophysiology of neurodegeneration.5On the other
hand, Aksoy et al., found significantly higher SOD
and Catalase activities of erythrocytes in AT patients,
compared to healthy control subjects and they suggest
that increased antioxidant activities of erythrocytes in
these patients may be an indication of the presence
of constant oxidative stress22. Myotonic Dystrophy
type 1 (DM1) is a disease with genetic basis similar
to SCA2, that consists of a mutational expansion of a
repetitive trinucleotide sequence. Previous study has
revealed that DM1’s patients showed significantly
higher levels of SOD than normal controls.23
Cellular DNA is continuously exposed to insults
from exposure to endogenous and exogenous electrophilic agents and oxidative stress.24ROS also induce
oxidative damage of DNA, including strand breaks
and base and nucleotide modifications, particularly in
sequences with high guanosine content.25Our study is
the first attempt to investigate peripheral cell blood
DNA damage in patients with SCA2 using the Comet assay. We found that peripheral blood cell from
patients with SCA2 exhibited higher levels of DNA
damage than those from controls.
The consequences of DNA damage are manifold,
in addition to mutagenic lesions, blocks of transcription or replication leading to altered gene regulation
and expression are probable.26 A major source of damage could be the genotoxic action of lipid peroxidation compounds. These are, however, very complex
and heterogeneous.27Our studies presented here have
shown that the DNA endogenous damage could be
due to actions of lipid peroxidation products.
On the other hand, the degree of DNA damage is
based on the genotoxic or oxidative stress prevailing
in a given tissue, the levels of endogenous detoxifying systems, and the DNA repair capacity.26 Therefore, we suggested that DNA damage observed in
these patients could be a consequence of an imbalance between lesion inductions from radical processes
and repair. It has been reported that reduced repair
will result in elevated lesions and an increased risk
of disease.28
In a number of genetically determined disorders,
collectively known as the chromosome breakage
syndromes or DNA-repair disorders, such as Ataxia
Telangiectasia, Xeroderma Pigmentosum, Tricho46
thiodystrophy, and the Cockayne syndrome, neurological symptoms as primary feature of their phenotypes have been described. They are characterized by
spontaneous or induced susceptibility to chromosomal breakage and represent a well-known connection
between DNA damage and neurodegeneration.8.One
possibility to account for this neurodegeneration is
the potential genotoxicity of endogenous ROS, generated as byproducts of cellular metabolism; in repaircompromised individuals, these agents could exert a
genotoxic role in the nervous system.29-31
The relationship between DNA damage and increasing disease duration suggested that DNA damage
increased as the disease progressed. It was suggested
that oxidative damage may play a more important role
as the disease progresses and antioxidant therapy may
help to alleviate this facet of disease progression. The
absence of any correlation with CAG size or age at onset suggested the severity of the genetic abnormality
was less influential in determining the extent of DNA
damage (data not shown). Similar findings have been
obtained previously by means of different methods on
peripheral DNA damage, among neurodegenerative diseases such as AD, PD, ALS, FRDA and AT.2, 7, 27, 32, 33
In conclusion, this study provides evidence of oxidative damage to different molecules and its impact
on antioxidant system in SCA2 patients. Our findings
show evidence of an increased sensitivity to oxidative stress in these patients as compared with healthy
people, suggesting that oxidative stress could be participating in the SCA2 pathogenesis, at least partially.
Furthermore, the methods used are relatively inexpensive, easily performed and require non- time-consuming procedures. The biochemical tests evaluated
could contribute to an integral overview in SCA2 patients and could be used as indices of treatment efficacy. Nowadays, there isn’t an effective treatment of
SCA2 and we found that oxidative stress is implicated
in the progression of the disease; perhaps antioxidant
consumption could ameliorate the symptoms. Further
studies, particularly, clinical trials will be needed to
probe this hypothesis.
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