determination of dna damage in experimental liver intoxication and role of n-acetyl cysteine
TRANSCRIPT
ORIGINAL PAPER
Determination of DNA Damage in Experimental LiverIntoxication and Role of N-Acetyl Cysteine
Hasan AKSIT • Aysegul BILDIK
� Springer Science+Business Media New York 2014
Abstract The present study aimed at detecting DNA
damage and fragmentation as well as histone acetylation
depending on oxidative stress caused by CCl4 intoxication.
Also, the protective role of N-acetyl cysteine, a precursor
for GSH, in DNA damage is investigated. Sixty rats were
used in this study. In order to induce liver toxicity, CCl4 in
was dissolved in olive oil (1/1) and injected intraperito-
neally as a single dose (2 ml/kg). N-acetyl cysteine appli-
cation (intraperitoneal, 50 mg/kg/day) was started 3 days
prior to CCl4 injection and continued during the experi-
mental period. Control groups were given olive oil and N-
acetyl cysteine. After 6 and 72 h of CCl4 injection, blood
and liver tissue were taken under ether anesthesia. Nuclear
extracts were prepared from liver. Changes in serum AST
and ALT activities as well as MDA, TAS, and TOS levels
showed that CCl4 caused lipid peroxidation and liver
damage. However, lipid peroxidation and liver damage
were reduced in the N-acetyl cysteine group. Increased
levels in 8-hydroxy-2-deoxy guanosine and histone ace-
tyltransferase activities, decreased histone deacetylase
activities, and DNA breakage detected in nuclear extracts
showed that CCl4 intoxication induces oxidative stress and
apoptosis in rat liver. The results of the present study
indicate that N-acetyl cysteine has a protective effect on
CCl4-induced DNA damage.
Keywords Apoptosis � Histone acetylation � Carbon
tetrachloride � N-acetyl cysteine � Oxidative DNA damage �Free radicals
Introduction
The liver is an organ that is often exposed to toxic sub-
stances and various drugs because of their anatomical
localization, and physiological and biochemical roles. The
liver is the major target organ for toxicity following acute
inhalation or ingestion exposure to carbon tetrachloride [1].
Lipid peroxidation develops in cell membranes, which
causes the production of membrane destruction products
like malondialdehyde (MDA). It has been shown that tri-
chloromethyl radical (CCl3), which is formed in the
metabolism of CCl4 via the liver microsomal cytochrome
P450 system, reacts rapidly with molecular oxygen to
produce trichloromethylperoxy radical (OOCCl3). Hydro-
gen atoms are removed from unsaturated fatty acids by
such radical-created carbon-centered lipid radicals. These
lipid radicals quickly add molecular oxygen to form lipid
peroxyl radicals which in turn abstract hydrogen atoms
from other lipid molecules [2, 3].
An important cause of DNA damage concerns the attack
by reactive oxygen species, leading to DNA hydroxylation.
Hydroxylated nucleotides are efficiently removed from
DNA by specific repair enzymes [4, 5]. Oxidizing DNA to
form 8-hydroxy-2-deoxyguanosine (8-OHdG) adducts a
major species of oxidative DNA damage [6].
A remarkably distinct type of cell death called apoptosis
was identified in single cells usually surrounded by healthy
looking neighbors and characterized by cell shrinkage,
blebbing of the plasma membrane, maintenance of orga-
nelle integrity, and condensation and fragmentation of
H. AKSIT (&)
Department of Biochemistry, Faculty of Veterinary Medicine,
University of Balikesir, Balikesir, Turkey
e-mail: [email protected]
A. BILDIK
Department of Biochemistry, Faculty of Veterinary Medicine,
University of Adnan Menderes, Aydin, Turkey
123
Cell Biochem Biophys
DOI 10.1007/s12013-014-0031-4
DNA, followed by the ordered removal through phagocy-
tosis [7].
Histone hypoacetylation plays important roles in the
control of cell cycle progression and apoptosis [8, 9].
Acetylation of specific lysine residues on the N-terminal
tail of core histones by histone acetyltransferase (HAT)
results in the uncoiling of the DNA and the increased
accessibility to transcription factor binding. In contrast,
histone deacetylation by histone deacetylase (HDAC)
represses gene transcription by promoting DNA winding,
thereby limiting access to transcription factors. Oxidative
stress also inhibits HDAC activity and in doing so enhan-
ces inflammatory gene expression which leads to a chronic
inflammatory response [10].
The present study, on the activities of HAT and HDAC
enzymes (on transcription indirectly), investigated the
potential effect of oxidative stress by determining whether
there is a relationship between histone acetylation by
apoptosis, DNA damage, and DNA breakage, and whether
there is interest in histone acetylation. Also, whether the
most important intracellular antioxidant glutathione pre-
cursor, N-acetyl cysteine, plays a protective role in the
DNA damage is investigated.
Materials and Methods
Materials
Sixty male Sprague–Dawley rats (350–400 g) were used in
this study. All the animals were kept in standard rat cages,
five rats per cage, and housed in an air-conditioned room
under controlled light (12-h light/dark cycle). All the rats
were allowed free access to water and rat chow.
Methods
All the studies on the animals described in the current study
were reviewed and approved by the University of Adnan
Menderes Institutional Animal Ethic committee (Date:
02.06.2010, No: B.30.2.ADU.0.00.00.00/050.04/2010/
056).
Rats were randomly divided into six groups, each con-
taining ten animals. Rats were subdivided into the fol-
lowing groups:
(1) (CCl4, at 6 h): Rats were intraperitoneally injected
with CCl4 at a dose of 2.0 ml/kg body weight and
olive oil solution at a 1:1 ratio.
(2) (CCl4 ? NAC, at 6 h): Rats were intraperitoneally
injected with NAC (ip 50 mg/kg/day) 3 days before
CCl4 treatment and injected with CCl4 at a dose of
2.0 ml/kg body weight and olive oil solution at a 1:1
ratio.
(3) (CCl4, at 72 h): Rats were intraperitoneally injected
with CCl4 at a dose of 2.0 ml/kg body weight and
olive oil solution at a 1:1 ratio.
(4) (CCl4 ? NAC, at 72 h): Rats were intraperitoneally
injected with NAC (ip 50 mg/kg/day) 3 days before
CCl4 treatment and injected with CCl4 at a dose of
2.0 ml/kg body weight and olive oil solution at a 1:1
ratio.
(5) (Olive oil, at 6 and 72 h): Rats were intraperitoneally
injected with olive oil at a dose of 2.0 ml/kg body
weight.
(6) (Olive oil ? NAC, «): Rats were intraperitoneally
injected with NAC (ip 50 mg/kg/day) 3 days before
olive oil treatment and injected with olive oil at a
dose of 2.0 ml/kg body weight.
The rats were then sacrificed under ether anesthesia at 6
or 72 h after CCl4 treatment, and blood was collected, and
their livers were isolated. The collected blood was sepa-
rated into serum by centrifuging at 2,5009g for 10 min.
The isolated livers were sliced in pieces and washed in ice-
cold 0.1 M KCl, blotted on absorbent paper, and then
weighed as soon as possible. After weighing, the livers
were kept at -80 �C until analysis.
The nuclear extract was prepared from liver homogenate
using a commercially available kit (Cayman, 10009277,
USA). 8-hydroxy-2-deoxyguanosine (EIA Kit, Cayman,
589320), Histone Acetyl Transferase (Kamiya, KT-146),
and Histone Deacetylase (Cayman, 10011563) were mea-
sured in the nuclear extract. Hepatic nuclear extracts were
used according to the manufacturer’s protocol. Total
Antioxidant Status (Rel Assay Diagnostics, RL0017), Total
Oxidant Status (Rel Assay Diagnostics, RL0024), AST
(Archem, A2212), and ALT (Archem, A222) were deter-
mined in serum. Plasma lipid peroxidation (MDA) was
determined using the procedure described by Yoshoiko
et al. [11]. Apoptosis was determined by TUNEL assay in
liver tissue(ApopRag Plus Peroxidase In Situ Apoptosis
Detection Kit, Chemicon, S7101). DNA fragmentation was
determined by ELISA in nuclear extract (Cell Death
Detection ELISA Plus kit, Roche, 11774425001, Mann-
heim, Germany).
Statistical Analysis
The statistical analysis of the differences between groups
was performed using ANOVA and significance of differ-
ences using Duncan’s test. Differences were considered
statistically significant when P \ 0.05 against control
group. All the values were presented as mean ± Sx.
Cell Biochem Biophys
123
Results
As shown in Table 1, compared to control animals, the
mean AST, ALT, and HAT activities as well as MDA,
8-OHdG, TOS, and apoptotic DNA fragmentation levels in
animals treated with CCl4 increased significantly at 6 h
(P \ 0.001). This significant increase was not observed in
NAC-treated rats. Compared to the results measured at 6 h
following NAC administration, levels of MDA were sig-
nificantly higher than that at 72 h. Levels of TAS and
HDAC activities decreased in the CCl4 group compared to
the control animals at 6 h, and the levels of TAS and
HDAC activities were increased again by the addition of
NAC (P \ 0.001). However, there was an increase at 72 h
compared to 6 h. There is a statistically significant dif-
ference between the groups at 72 h (Table 1).
The number of TUNEL-positive cells shown in the CCl4group at 6 h (Fig. 1) was relatively low. The number of
TUNEL-positive cells decreased with the NAC application
(Fig. 2). The number of positive cells decreased thor-
oughly at 72 h (Fig. 5, 6). TUNEL-negative hepatocytes
(Figs. 3, 4, 7, 8) have been identified in the control group.
Discussion
Liver encountering to different toxins, drugs, and chemical
agents is an organ that detoxifies them constantly. The free
radicals are among the most important mechanisms of liver
injury [12, 13]. Among many factors causing the formation
of liver damage, in experimental studies, carbon tetra-
chloride is the most preferred and used [14, 15].
Ariosto et al. [16] applied carbon tetrachloride in lab-
oratory animals and determined ALT levels to be 33 mU/
ml in the control group and 118 mU/ml in the experi-
mental group. Dashti et al. [17] in their study determined
the AST activities to be 1.3 lkat/L in the control group and
4.3 lkat/L in the experimental group. ALT activities were
0.8 lkat/L in the control group and 2.2 lkat/L in the
experimental group.
With the increasing toxicity of carbon tetrachloride on
the liver, MDA levels were increased, and antioxidant
substances applied, such as NAS, were reduced with this
increase [18, 19]. After the evaluation of liver enzymes,
ALT and AST levels were significantly increased in the
carbon tetrachloride group, but these were reduced sig-
nificantly with the application of NAS [20].
Kadiiska et al. [21] exposed rats to 120 mg/kg and
1,200 mg/kg ip dose of CCl4 in their study. The analysis at
7 h showed 8-OHdG levels in the experimental group to be
about 2–6 times higher than the control group; at 16 h, the
8-OHdG levels were found to be higher in the experi-
mental CCl4 group compared to those in the control group ,Ta
ble
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Cell Biochem Biophys
123
but lower than those at 7 h. In this study, the levels of
8-OHdG are higher in the carbon tetrachloride group than
those in the control group at 6 h; the levels of 8-OHdG are
lower at 72 h than those at 6 h, which is consistent with the
findings of the researchers.
In another study, rats were exposed to a single dose of
2 ml/kg ip CCl4 to evaluate the antioxidant activities of
black and green tea [22]. ALT and MDA levels increased
in the group treated with CCl4, while TAS levels
decreased, but opposite results have been identified in the
green and black tea groups. In the NAS-treated groups, for
the antioxidant effects of green and black tea, similar
results were obtained.
Our findings support the results obtained from the
research of other investigators. Oxidative stress can induce
damage to biological molecules such as protein, lipid, and
DNA [23–25]. Lim et al. [26] reported cases that led to an
increase of oxidative stress on the early oxidative damage
to DNA molecules.
Apoptosis was observed especially in non-parenchy-
mal fibrotic cells. Fibrosis can be reduced by increasing
apoptosis [27]. Cholestasis results in the accumulation
of toxic bile salts in the liver, which induce Fas-med-
iated apoptosis by oligomerization of the Fas death
receptor, activation of Fas-associated death proteins,
and activation of the caspase cascade. Researcher
reported that apoptosis increased depending on ligation
and apoptosis reduced with honey treatment in hepato-
cytes. TUNEL (?) staining was increased in the liga-
tion group compared to that in the control group in
hepatocytes [28].
N-Acetyl cysteine decreases apoptosis, prolongs the cell
life by regulating the activities of various proteins, and
reduces endothelial dysfunction [29].
Fig. 1 At 6 h in the CCl4 apoptotic cell group, TUNEL (?) (arrow
marks)
Fig. 2 At 6 h in the group: CCl4 ? NAC apoptotic cells, TUNEL
(?) (arrow marks)
Fig. 3 At 6 h in the control group (olive oil), normal cells TUNEL
(-)
Fig. 4 At 6 h in the control group (olive oil ? NAC), normal cells
TUNEL (-)
Cell Biochem Biophys
123
Rats were exposed to a single dose of 0.3 ml/kg intra-
peritoneal CCl4, and apoptotic DNA fragments were
determined by TUNEL assay and gel electrophoresis. The
amount of apoptotic hepatocytes in the experimental group
started to increase at 3 h after CCl4 injection and peaked at
6 h, and then began to decline [30]. TUNEL assay also
confirms the previous findings obtained due to the change
in the number of apoptotic cells. Our findings are consistent
with results obtained in the studies by other investigators.
HDAC inhibitor, valproic acid, leads to apoptotic cell
death with changes in cell morphology and increases cas-
pase-3 expression [31].
The balance between acetylation and deacetylation of
histones provides HAT and HDAC families. Recent studies
have shown that compounds which inhibit HDAC stopped
cell division, and induced apoptosis and differentiation
[32].
Increased TOS levels were detected in the CCl4-treated
groups, while decreased TAS levels were measured in the
same group. However, the opposite results were obtained
when N-acetyl cysteine was used as an antioxidant. Results
show that oxidative stress is caused by carbon tetrachloride
and N-acetyl cysteine as an antioxidant plays an important
role in protecting against oxidative stress.
Another aim of the study was to investigate the rela-
tionship between apoptosis and histone acetylation during
the oxidative stress in DNA. Regulation of apoptosis is a
process that starts with the p53 gene expression and con-
tinues until the caspases. With the increase in the levels of
8-OHdG and high levels of DNA fragmentation in both the
groups, parallel studies confirm that oxidative stress may
lead to apoptosis. Enzyme activities play a role in histone
acetylation, and have shown apoptotic cells in the experi-
mental groups; the acetylation of histone proteins in DNA
Fig. 5 At 72 h in the CCl4 apoptotic cell group, TUNEL (?) (arrow
marks)
Fig. 6 At 72 h in the group CCl4 ? NAC apoptotic cells, TUNEL
(?) (arrow marks)
Fig. 7 At 72 h in the control group (olive oil), normal cells TUNEL
(-)
Fig. 8 At 72 h in the control group (olive oil ? NAC), normal cells
TUNEL (-)
Cell Biochem Biophys
123
has become appropriate for transcription. In this regard, the
p53 gene that regulates apoptosis may have relationship
with the levels of 8-OHdG. This is supportive of our
opinion that NAC applications change activities of
enzymes that perform histone acetylation, and that the
number of apoptotic cells is lower in the control group than
that in the experimental group. At the same time, research
results show that CCl4 intoxication was tolerated by the
organism with respect to time, and the application of NAC
as an antioxidant in a healthy control group is not
important.
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