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Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

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Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Absence of superoxide dismutase activity causes nuclear DNA
fragmentation during the aging process
Khandaker Ashfaqul Muid, Hüseyin Çaglar Karakaya, Ahmet Koc
Izmir Institute of Technology, Department of Molecular Biology & Genetics, 35430 Urla, Izmir, Turkey
article info
Article history:
Received 8 January 2014
Available online 22 January 2014
Keywords:
Oxidative stress
Antioxidant
SOD
Superoxide dismutase
Aging
Longevity
DNA damage
Comet assay
ROS
Reactive oxygen species
abstract
Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organ-
isms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces
cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging
process has been studied extensively in different organisms, analyses of DNA damages has not been per-
formed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles
of SOD1,SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the
single cell comet assay. We observed that extend of DNA damage was not significantly different among
the young cells of wild type, sod1
D
and sod2
D
strains. However, ccs1
D
mutants showed a 60% higher
amount of DNA damage in the young stage compared to that of the wild type cells. The aging process
increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1
D
,sod2
D
, and ccs1
D
mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage con-
tents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal
that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this
process.
Ó2014 Elsevier Inc. All rights reserved.
1. Introduction
Aging may be defined as a multi factorial phenomenon charac-
terized by a time dependent decline in physiological function [1].
The mechanisms of aging, while not fully understood, is clearly
associated with an increase in levels of molecular damage over
the time, which contributes to increasing pathology and mortality
at the organismal level [2,3].
The budding yeast Saccharomyces cerevisiae is one of the most
important model organisms used in aging-related research. Studies
have demonstrated that replicative senescence of yeast cells in-
volves a collapse of the antioxidant defense mechanisms and accu-
mulation of oxidative damage to cellular components [4]. DNA
integrity and stability of an organism is fundamental for survival,
even under the best circumstances DNA is continuously damaged
by endogenous or exogenous genotoxic agents [5,6].
It is estimated that about 1–2% of the total oxygen consumed by
mitochondria is transformed into O2 [7]. SOD enzymes catalyze the
dismutation of superoxide into oxygen and hydrogen peroxide and
they form an important antioxidant defense in nearly all cells ex-
posed to oxygen [8]. SOD enzymes generally present in two forms
inside a eukaryotic cell, Cu/ZnSOD (Sod1) resides in the cytoplasm
and the inner membrane space of mitochondria and MnSOD (Sod2)
mainly present in the mitochondrial matrix [9]. Copper chaperone
Ccs1 is involved in the oxidative stress protection and has been
characterized in S. cerevisiae [10]. It functions as a copper trans-
porter for Sod1 [11]. Although Ccs1 share a significant sequence
homology with Sod1, it has no superoxide dismutase activity.
However, the D200H point mutation converts Ccs1 into a superox-
ide dismutase [12].
In this study, we optimized the yeast comet assay to study the
roles of superoxide dismutases in preserving genomic integrity
during the aging process. We observed that young cells lacking
SOD1 and SOD2 genes have normal levels of ROS and DNA damage;
however, cells without CCS1 gene had a significant increase in both
ROS production and DNA damage rates. Furthermore, the aging
process is a significant factor that increases these rates, and
absence of SOD1,SOD2 and CCS1 genes further exaggerate them.
2. Materials and methods
2.1. Yeast strains, culture and sample preparation
Wild type (WT) strain BY4741 (MATahis3
D
1leu2
D
0met15
D
0
ura3
D
0) and its isogenic mutants (sod1
D
,sod2
D
and ccs1
D
) were
used in the experiments. Cells were grown on either solid or liquid
YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30 °C.
Yeast growth was monitored by optical density at 600 nm(OD
600
).
0006-291X/$ - see front matter Ó2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2014.01.056
Corresponding author. Fax: +90 232 7507303.
E-mail address: ahmetkoc@iyte.edu.tr (A. Koc).
Biochemical and Biophysical Research Communications 444 (2014) 260–263
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
2.2. Elutriation
Cells grown in overnight cultures were transferred to fresh
media and kept growing for 24 h. Then they were directly sub-
jected to the elutriation system (Beckman Coulter USA, Avanti
J-26 XPI) in order to separate the young and the old cells. Cells with
7–10
l
m range were considered as the young cells and 15
l
m and
greater were considered as the old cells.
2.3. Formation of spheroplasts by cell wall degradation
Pellets were suspended in ice cold S buffer (1 M sorbitol, 25 mM
KH
2
PO
4,
pH 6.5) to a density of 1.2 10
7
cells/ml and then centri-
fuged 2 min at 4000 rpm at 4 °C. The cells were washed twice with
the same volume of ice-cold deionized water and diluted in 1 ml
ice cold S-buffer. Then cells were pelleted by centrifugation for
2 min at 4000 rpm at 4 °C. Pellets were resuspended in lyticase
buffer (3 mg/ml lyticase, 500
l
l2S buffer, 300
l
l deionized
water and 50 mM b-mercaptoethanol) and incubated at 37 °Cin
water bath for 30 min in order to get a good number of
spheroplasts. At the end, spheroplasts were collected by centrifu-
gation at 14,000 rpm for 2 min.
2.4. Comet assay
Many difficulties for yeast comet assay have been reported pre-
viously due to small nucleus and presence of the cell wall. We fol-
lowed the procedures described by Azevedo et al. [13] with some
modifications. A good number of spheroplast with degraded cell
wall was obtained by applying 3 mg/ml lyticase at 37 °C in water
bath for 30 min. We also changed the voltage and duration of elec-
trophoresis to view the complete migration of the damaged DNA.
In brief, 1.5% Low Melting Point Agarose (LMPA) (w/v in S buffer)
and 0.5% Normal Melting Agarose (NMA) (w/v in distilled water)
were prepared. These were heated by microwave oven and while
NMA was hot, slides were dipped up to one-third of the frosted
area. Then the slides were air dried and stored at room tempera-
ture until needed. Pelleted spheroplasts were resuspended in
80
l
l LMA (1.5%) at 37 °C and quickly applied the mixure onto pre-
viously prepared 0.5% NMA coated slides and covered with cover
slips. The cover-slips were then removed and the slides immersed
in a freshly prepared lysis solution (30 mM NaOH, 1 M NaCl, 0.5%
w/v laurylsarcosine, 50 mM EDTA, 10 mM Tris–HCl, pH 10–10.5)
for a minimum of at least 1 h and a maximum of two, at 4 °C. Then
the slides were placed side by side on a horizontal gel box near one
end, as close together as possible. The buffer reservoirs were filled
with freshly made electrophoresis buffer (300 mM NaOH, 10 mM
EDTA, pH > 12) until the liquid level completely covered the slides.
The slides were taken in the alkaline buffer for 3 20 min to allow
DNA to unwind. Power supply was turn onto 20 V and 250 mA by
raising or lowering the buffer level. The slides were then electro-
phoresed using agarose gel electrophoresis unit for 12 min. Then
the slides were coated drop wise with Neutralization Buffer:
(0.4 M Tris was added to 800 ml dH
2
O and pH was adjusted to
7.5 with concentrated (>10 M) HCl: q.s. to 1000 ml with dH
2
O)
for at least 5 min and repeated twice. The samples were fixed, first
with 75% and then 100% ethanol. After successive dehydration the
slides were dried in the air at RT or in a laminar flow chamber. The
samples were then stained by pipeting 100
l
lofa10
l
g/ml stock
solution of Ethidium bromide directly onto each slide and
incubated for 20 min. The stained slides were then rinsed in
400 ml distilled water to remove excess stain and the cover slips
were placed over the slides.
2.5. Confocal microscopy and evaluation of DNA damage
To visualize DNA damage, comets were observed and counted
under a confocal fluorescence microscope (1000) using 488 nm
band-pass filter following the staining of DNA with the fluorescent
dye ethidium bromide. Pictures were taken with a CCD camera
(Andor technology). Three independent repetitions of the experi-
ments were performed. In each experiment, three hundred
Young cell’s
nucleoids
Old cell’s
nucleoids
WT
sod1Δ
+
+
+
+
+ +
+ +
sod2Δ
ccs1Δ
0
20
40
60
80
100
120
140
WT sod1Δ sod2Δ ccs1Δ
DNA damage score
Young cells
Old cells
A
B
Fig. 1. Comet assay for wild type and SOD deficient cells. Cells were grown in YPD
media, young and old cells were separated by an elutriation system and DNA
damage rates were assessed by the yeast comet assay and tails were counted for
300 comets for each sample. (A) Confocal fluorescence microscopic analyses of
comets at 1000magnification. The symbol and + represent cathode and anode
respectively and arrows show the comet tails. (B) Evaluation and comparison of the
comet frequencies based on the damage score among the young and old yeast
strains (see Table 1). Error bars show standard deviation from three repetitive
experiments.
K.A. Muid et al. / Biochemical and Biophysical Research Communications 444 (2014) 260–263 261
nucleoids were analyzed using visual classification based on the
migration of DNA fragments of the nucleus.
2.6. ROS measurement by flow cytometry
Endogenous ROS levels of wild type and mutant cells
(OD
600
= 0.2) were measured by a flow cytometer (FACS-BD) using
5
l
M2
0
,7
0
- DCF-DA (Invitrogen) fluorescence dye (excitation/emis-
sion = 488/525) according to manufacturer’s protocol.
2.7. Statistical analysis
Data were presented as the frequency of damaged cells, class
distribution and damage scores. Comets were grouped into four
groups (0, 1, 2 and 3; representing no, little, medium and high
damage respectively). Damage score was calculated as the sum
of nucleoids in each group multiplied by the number of groups
(0–3). Statistical analysis was performed using the 2 tailed paired
Ttest.
3. Results and discussion
It has been shown that deletion of yeast SOD1,SOD2 and CCS1
genes dramatically decreases the replicative lifespan [14].InCae-
norhabditis elegans, deletion of SOD genes leads to unexpected re-
sults; absence of SOD2 gene causes an increase and simultaneous
deletion of all five SOD genes causes no shortage in the lifespan
[15]. It has been reported that Drosophila null mutations for either
the cytoplasmic Cu/ZnSOD or the mitochondrial MnSOD greatly
decreases viability and the life span [16], however in mice only
SOD2 mutation has a severe effect [17,18]. Thus, regulation of the
life span by SODs is a controversial issue in different organisms
and further characterization of SOD deficient cells needs to be per-
formed to understand the ambiguities regarding their regulatory
roles in the life span.
In order to understand the mechanisms how SOD genes affect
the life span in yeast, we collected young and old cells lacking
SOD1,SOD2 and CSS1 genes and analyzed their genomic integrity
by the whole cell comet assay. We analyzed 300 nucleoids for each
sample and evaluated the results by statistical methods as de-
scribed in the Section 2.
Fig. 1A shows representative pictures for comet assays for wild
type and mutant strains. As seen in left panel (Fig. 1A) young
nucleoids had no tails and consisted of only head regions, but,
old cells (right panel) displayed nucleoidal tails (white arrows)
indicating the occurrence of DNA damage in these cells.
When we analyzed young cells for their DNA damage contents,
indicated as DNA damage scores in Fig. 1B and Table 1, we did not
observe any significant differences among the wild type, sod1
D
and
sod2
D
mutants (p> 0.05). However, young ccs1
D
cells showed a
64% higher DNA damage score compared to that of the wild type
cells indicating that, absence of CCS1 gene creates more profound
phenotype than in the absence of either SOD1 or SOD2 genes. Using
mitochondrial protein carbonylation levels as a measure of oxida-
tive protection, O’Brien et al. (2004) showed that logarithmically
growing sod1
D
and sod2
D
cells have similar protein carbonylation
levels to that of the wild type cell [19]. We could consider logarith-
mically growing cells as young cells, since 97% of the cells in a gi-
ven population is made of cells divided 4 times or less. Thus, their
results also suggest that the absence of SOD1 and SOD2 does not
make young cells vulnerable to oxidative damage.
Replicatively old cells harbored substantial amount of DNA
damages (Fig. 1B, grey bars) when compared to their young coun-
terparts (dark bars) (p< 0.05). Aging process caused more than 3-
fold higher level of DNA damage in the wild type, and approxi-
mately 5-fold higher level of DNA damage in sod1
D
,sod2
D
and
ccs1
D
mutants.
In order to test the idea that absence of SOD genes leads to ROS
accumulation which damages DNA and ultimately causes aging,
we analyzed the ROS levels of the young and the old cells by a flow
cytometric approach after staining the cells with fluorescein (FITC)
(Fig. 2). Interestingly, deletion of the SOD genes or CCS1 resulted in
approximately 20% higher level of ROS accumulation in young cells
when compared to that of the wild type (Fig. 2B, left panel). Nev-
ertheless, young cells did not contain oxidative DNA damages that
could be detected by the comet assay (Fig. 1A). However, old cells
harbored significantly higher amount of ROS, and the extend of this
increase was 67% for sod1
D
, 88% for sod2
D
, and 92% for ccs1
D
cells.
The higher level of ROS was consistent with the higher level of DNA
damage in old cells.
Our analyses showed that the ccs1
D
mutants harbor the highest
amount of ROS and DNA damage among the mutants. Ccs1 is
known as the sole cupper provider for Sod1p in yeast [20], but ab-
sence of CCS1 gene clearly generates more damaging conditions
than absence of SOD1. Consistent with this observation, ccs1
D
mu-
tants were previously shown to have more oxidative damages than
sod1
D
and sod2
D
mutants [21]. It is not clear why ccs1
D
cells are
more vulnerable to oxidative damage. It could be possible that
Ccs1 has other functions or interactions apart from being copper
chaperone for Sod1 and thus deletion of CCS1 may cause a pleo-
tropic effect on the life span and ROS accumulation.
Our analyses suggest that SOD function is dismissible in young
stage and support the idea that SODs are required for antioxidant
protection during the aging process. Moreover, short lifespan of
superoxide dismutase mutants in yeast and occurrence of exten-
sive DNA damages during the aging process in these cells
support the idea that oxidative stress is a modulator of the life
span. Increased ROS production results in an increased rate of
DNA damage and mutagenesis, thus causing a vicious cycle of
increasing oxidative damage which eventually culminates in
aging and death.
Table 1
Analyses of comet assay results.
Cells Ages Nucleoids analysed Comets (%) Comet classes DNA damage score
0 (no damage) 1 (little) 2 (Med) 3 (High)
WT Young 300 7.0 ± 1.5 93.0 ± 1.5 2.5 ± 0.7 2.0 ± 0 2.5 ± 0.7 14 ± 2.5
Old 300 23.5 ± 0.7 76.5 ± 0.7 9.0 ± 4.2 5.5 ± 2.1 9 ± 2.8 47 ± 9.8
sod1
D
Young 300 9.0 ± 0 91.0 ± 0 3.5 ± 0.7 3.0 ± 1.4 2.5 ± 0.2 17 ± 2.0
Old 300 44.5 ± 1.4 55.5 ± 1.4 19 ± 1.4 11 ± 1.4 14.5 ± 0.7 84.5 ± 13.3
sod2
D
Young 300 11.5 ± 0.7 88.5 ± 0.7 5.0 ± 0 4.0 ± 0 2.5 ± 0.7 18.0 ± 1.7
Old 300 48.5 ± 2.0 51.5 ± 2.0 25.5 ± 2.1 9.5 ± 0.7 15.5 ± 2.1 91 ± 14.3
ccs1
D
Young 300 13.0 ± 1.5 87.0 ± 1.5 5.5 ± 0.7 4.5 ± 0.7 3 ± 0 23.5 ± 2.0
Old 300 60.5 ± 0.7 39.5 ± 0.7 21 ± 1.4 20.5 ± 3.5 19 ± 1.4 119 ± 18.0
262 K.A. Muid et al. / Biochemical and Biophysical Research Communications 444 (2014) 260–263
Acknowledgment
The authors are thankful to Dr. Alper Arslanoglu, Dr. Serdar
Ozcelik and Biological Mass Spectrometry (Izmir Institute of
Technology) facilities for their instrumental help. This work was
supported by grant 2008K120730 from the State Planning Organi-
zation of Turkey. K.A. Muid was supported by a pre-doctoral fel-
lowship from the Scientific and Technological Research Council of
Turkey (TUBITAK-BIDEP).
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WT
sod1Δ
sod2Δ
ccs1Δ
Young Cells Old cells
0
5
10
15
20
25
30
35
40
45
WT sod1Δ sod2Δ ccs1Δ
%log fluorescence
Young cells
Old cells
A
B
Fig. 2. Flow cytometric analyses of cellular ROS contents. (A) Representative
histograms showing the endogenous ROS in young and old stages of cells using
2
0
,7
0
-DCF-DA (5
l
M) fluorescent dye (excitation/emission = 488/525) by a flow
cytometer (FACS-BD). (B) Relative levels of endogenous ROS detected by flow
cytometer. Error bars show the standard deviations of three repetitive experiments.
Y stands for young cells and O stands for old cells.
K.A. Muid et al. / Biochemical and Biophysical Research Communications 444 (2014) 260–263 263
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... En esta Tesis, sin embargo se hallaron cambios en la enzima SOD, pero no en CAT. En este sentido ha sido reportado que la actividad de SOD es esencial para preservar la integridad genómica en procesos que involucran un aumento de EROs como el envejecimiento (Muid et al., 2014). En Argentina, el trabajo de Bernardi et al. (2015), demostró el aumento de la frecuencia de micronúcleos (otro marcador de daño genotóxico) en un grupo de niños expuestos ambientalmente a xenobióticos. ...
... Yeast cells accumulate DNA damage during the aging process. In this way, the antioxidant defense plays an important role in preserving the genomic integrity (Muid et al. 2014). Mitochondrial dysfunction is associated with increased levels of nuclear DNA damage and thus it could be the cause of aging and aging related diseases. ...
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