Peripheral Blood Lymphocytes' DNA Damage in Different Treatment Outcomes of Chronic Viral C Hepatitis Genotype 4 Infection
Mohammed Abu Elfotouh
Ghada H. El-Arabi
DNA fragmentation in peripheral blood lymphocytes is a reliable marker for oxidative stress occurring in the liver of chronic hepatitis C (CHC) patients which reflects a direct genotoxic effect of the virus and suggests a same genotoxic effect that might operate in the liver. Previous studies have investigated DNA damage in peripheral leukocytes of CHC patients, but none focused on genotype 4. This work aimed at examining DNA damage in different treatment outcomes of genotype 4 infection. The study included 80 viral hepatitis C genotype 4 patients and 80 healthy volunteers. HCV-RNA was detected by real-time PCR, genotype was defined by INNO-LiPA and DNA damage was assayed using alkaline Comet assay. The mean percentage of DNA damage was significantly higher in patients' group than in control group (p<0.01) and in null response (48.75±11.12) and breakthrough response (49.33±1.03) patients than in SVR patients (22.78±13.95) (p<0.05/3). Comet results revealed that all breakthrough and null response patients have DNA damage. Interestingly, 78.5% of SVR patients had DNA damage (64.3% showed mild damage and 14.2% showed marked damage). In conclusion, in genotype 4; despite clearance of serum HCV-RNA and apparent clinical resolution; SVR patients might not experience infection clearance and should be followed up being at suspected risk for virus reactivation.
to cite this article:
Mohammed Mahmoud, Safinaz El-Tokhy, Dalia El-Lebedy, Mohammed Abu Elfotouh and Ghada H. El-Arabi, 2013. Peripheral Blood Lymphocytes' DNA Damage in Different Treatment Outcomes of Chronic Viral C Hepatitis Genotype 4 Infection. Journal of Medical Sciences, 13: 353-359.
Received: March 30, 2013;
Accepted: April 15, 2013;
Published: June 13, 2013
Hepatitis C virus (HCV) infection is prevalent in approximately 2% of the world's
population (Dustin and Rice, 2007). Egypt has the highest
epidemic of HCV in the world, with a recent estimated prevalence of 14.7% (DeWolfe
Millera and Abu-Raddad, 2010) and genotype 4 is the predominant genotype
affecting up to 91% of the patients (El-Ray et al.,
Recently, HCV infection has been characterized by an increased oxidative stress
in the peripheral blood mononuclear cells and liver (La
Vignera et al., 2012). DNA fragmentation in peripheral blood lymphocytes
is a reliable marker for oxidative stress occurring in the liver of chronic
hepatitis C patients (Bolukbas et al., 2006),
it reflects a direct genotoxic effect of the virus and suggests that the same
genotoxic effect may operate in the liver and contribute to hepatocarcinogenesis
(Grossi et al., 2008).
DNA damage assay in the peripheral blood lymphocytes is the least invasive
and low number of cells is required to measure DNA lesions (Loft
and Poulsen, 1999). The Comet assay is one of the useful methods to quantify
DNA damage and has been exploited as a laboratory measure of genotoxicity in
human bio-monitoring and clinical studies (Kassie et
al., 2000; McKenna et al., 2008).
The current standard treatment of chronic HCV infection, a combination of pegylated
interferon and ribavirin, has been reported to have the best overall sustained
response rate in 55% of patients. However, clinical results obtained from several
trials on genotype 4 have been inconsistent (Esmat and
Abdel Fattah, 2009).
Previous clinical studies have investigated DNA damage in peripheral leukocytes
associated with chronic viral C hepatitis with none focused on genotype 4, this
work aimed at studying peripheral blood lymphocytes DNA damage in different
treatment outcomes of chronic viral C hepatitis genotype 4 infection using alkaline
MATERIALS AND METHODS
Subjects: Eighty viral hepatitis C genotype 4 patients, who were enrolled
for interferon therapy protocol, were included in the present study. They were
recruited from hepatology clinics of the General Health Insurance Authority
Polyclinics. Their combined therapy was a fixed weekly dose of 160 μg of
20 KD linear pegylated interferon α-2a and ribavirin in standard and adjusted
doses for 48 weeks (El-Ray et al., 2010; Esmat
and Abdel Fattah, 2009). Patients were fully informed in advance about the
nature of our study and gave informed consents. The study protocol was approved
by the ethical committee of the National Research Center.
According to their response to interferon therapy, they were divided into 3
groups. Sustained virologic response (SVR) group including 28 subjects, SVR
was defined by undetectable HCV-RNA in serum 24 weeks after combined therapy
withdrawal (Welker and Zeuzem, 2009). Null Response
group including 26 patients, Null response was defined by <2 log (10)
reduction in HCV-RNA after 12 weeks treatment (Chayama
et al., 2012). The breakthrough group including 26 patients, breakthrough
response is defined when HCV-RNA rebounds and becomes detectable before treatment
is completed (Sherman et al., 2007), all patients
in this group received only 24 weeks of interferon therapy. Eighty age, sex
and culture matched healthy volunteers who were negative for HCV antibodies
were enrolled as a control group. All subjects were evaluated clinically and
exclusion criteria included: history of antioxidants administration for a month
preceding the study, alcohol intake, smoking, chronic diseases and any other
liver diseases or co-infections.
Laboratory methodology: Venous blood samples were withdrawn from all
subjects included in the study. Serum was separated and assayed for liver function
tests: AST (aspartate transaminase), ALT (alanine transaminase), ALP (alkaline
phosphatase), total bilirubin and albumin using Olympus auto-analyzer AU-400
(Olympus Diagnostica, Japan). Prothrombin Time (PT) was assayed and International
Normalized Ratio (INR) was calculated for each subject. Hemoglobin %, total
leukocyte and platelet counts were measured using Hematology auto-analyzer (Abbott
Cell Dyn CD-1700). HCV-RNA was detected by real time-PCR and the genotype was
defined by the reverse line probe assay (INNO-LiPA). Peripheral blood lymphocytes
DNA damage was assayed using alkaline Comet assay (single-cell microgel electrophoresis
Detection of HCV-RNA by real time-PCR and HCV Genotyping: Viral RNA
was extracted from patients' plasma using the QIAamp Viral RNA Kit (Qiagen Hilden,
Germany, Cat No. 52904) according to the manufacturer's protocol. HCV- RNA was
detected by COBAS Amplicor HCV Kit (Roche Diagnostic Systems, NJ, USA) with
a lower limit of detection: 18 IU/mL. HCV genotype was defined by the reverse
line probe assay (INNO-LiPA v.1.0, innogenetics, Ghent, Belgium) according to
the manufacturers instructions.
Assessment of DNA damage by alkaline comet assay
Cell preparation: Peripheral blood lymphocytes were isolated from heparinized
blood samples within a maximum 2 h period after collection by centrifugation
over Ficoll hypaque density gradient (Pharmacia LKB Biotechnology, Piscataway,
NJ, USA). After centrifugation, peripheral blood lymphocytes represented as
a buffy coat were gently aspirated and washed twice by Phosphate Buffered Saline
(PBS) at pH 7.4.
Preparation of cell microgels on slides: All the procedures of the alkaline
Comet assay (Singh et al., 1988; Blasiak
et al., 2003) were done at low temperature to minimize spontaneous
DNA damage. Fully frosted slides were covered with 1% Normal Melting Point (NMP)
agarose (Sigma). After solidification, the gel was scraped off the slide and
cell microgels were prepared on the slides as layers. The first layer of gel
was made by coating the slides with 0.7% NMP agarose (Sigma). When this layer
had solidified at 4°C, a second layer containing the separated peripheral
blood lymphocytes mixed with 0.6% Low Melting Point (LMP) agarose (Sigma) was
placed on the slides. After 10 min solidification on ice, a final layer of 0.6%
LMP agarose was added.
Cell Lysis, DNA unwinding, gel electrophoresis and DNA staining: Afterwards
the slides were immersed for 1 h in ice-cold freshly prepared lysis solution
(2.5M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% Na hydroxide (Sigma), pH 10)
with 1% Triton X-100 (Sigma) and 10% dimethyl sulfoxide (DMSO), added fresh
to lyse cells and allow DNA unfolding.
Then, the slides were placed in a horizontal gel electrophoresis chamber filled
with fresh electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH
13.0) for 20 min at 4°C to allow DNA unwinding and expression of alkali-labile
Denaturation and electrophoresis were performed at 4°C under dim light.
Electrophoresis was carried out for 30 min at 300 mA. After electrophoresis
the slides were rinsed gently three times with a neutralization buffer (0.4
M Tris-HCl, pH 7.5) to remove excess alkali and detergents. Each slide was stained
with ethidium bromide (10 μg mL-1) then cover slipped and stored
at 4°C until analysis.
Visualization and analysis of comet: The slides were examined at 400x
magnification using a fluorescence microscope (IX70; Olympus, Tokyo, Japan)
equipped with an excitation filter of 549 nm and a barrier filter of 590 nm,
attached to a digital camera (Olympus) with high resolution. A damaged cell
is visualized as each cell had the appearance of a comet, with a brightly fluorescent
head and a tail to one side formed by the DNA containing strand breaks that
were drawn away during electrophoresis. Normal undamaged cell was visualized
as an intact DNA without any protrusions. Samples were analyzed by counting
the number of damaged cells out of 100 cells per slide to calculate the percentage
(%) of DNA damage.
Interpretation: Genotoxicity is expressed as visual score in a range
of 0-100 that can be classified into groups (Azqueta et
al., 2009; Moller, 2006). In the present study,
we categorized DNA damage into 3 groups (1) mild damage>0 and<25%, (2)
moderate damage 25-50% and marked damage>50%. Comet tail formation was also
documented, the extent of comet tail formation is proportional to DNA damage
present and selected as the best parameter that reflects DNA damage (Collins,
2004; Trzeciak et al., 2008).
Statistical analysis: Data was analysed using the statistical package
for social science (SPSS software version 16, Chicago, Illinois). Data was presented
as Mean±standard deviation (SD). Non-parametric variables were compared
by the Kruskal-wallis one-way analysis of variance with Post Hoc analysis using
a Mann-Whitney U test. Parametric variables were compared using Student's t
test and one-way analysis of variance with Post-Hoc analysis. Differences were
considered significant at 0.05/3 for comparisons made by Kruskal-Wallis one-way
analysis of variance, otherwise at p<0.05.
The demographic and laboratory data of all studied subjects are summarized
in Table 1. There was no statistically significant difference
between different groups with respect to age and sex (p>0.05). HCV-RNA was
detected in all patients of null and breakthrough response, while all SVR subjects
were negative for it.
Serum ALT and AST were significantly higher in patients' group than in control
group (p = 0.03). The SVR patients had lower ALT and AST levels than the non-SVR
patients (p<0.05/3). There was no significant difference regarding the same
parameters on comparing the null response patients to breakthrough response
patients. Serum ALP and total bilirubin levels were significantly higher in
breakthrough response patients than in control group (p = 0.02).
As regards hematological findings, SVR patients had higher platelet count than
the non-SVR patients (p<0.05/3). Breakthrough patients had significantly
lower hemoglobin % and platelet count than control subjects (p = 0.03). No statistical
significant differences were observed regarding the mean levels of albumin,
PT, total leukocyte count among different studied groups.
|| Demographic and laboratory data of different studied groups
|Data presented as Mean±SD, *p<0.05 vs control, §
p<0.05/3 vs. null and breakthrough groups, SVR: Sustained virologic response,
AST: Aspartate transaminase, ALT: Alanine transaminase, ALP: Alkaline phosphatase,
PT: Prothrombin time, INR: International normalized ratio, Hb: Hemoglobin,
TLC: Total leukocyte count, PLT: Platelet count
|| Descriptive analysis of DNA damage in different groups
|SVR: Sustained virologic response
||The mean percentage of peripheral blood lymphocytes DNA damage
in different studied groups
The mean percentage of DNA damage was significantly higher in patients' group
than in control group (p<0.01) and in null response (48.75±11.12)
and breakthrough response (49.33±1.03) patients than in SVR patients
(22.78±13.95) (p<0.05/3) (Fig. 1). However, no statistically
significant difference was found on comparing the null with the breakthrough
groups. Descriptive analysis of DNA damage in different groups is shown in Table
|| DNA damage by Comet assay, Images are visualized by the digital
camera fitted fluorescent microscope, (a) An intact DNA in normal subjects,
(b) High degree of DNA damage clarified by a slightly pointed end due to
the migration of fragmented DNA through electrophoresis (tailed) and (c)
Moderate DNA damage as revealed by less tightly intact DNA due to slight
Comet assay clarified an intact DNA in the control group (Fig.
2a). Meanwhile, all breakthrough and null response patients showed DNA damage;
100% of breakthrough patients had moderate DNA damage, 61.5% of null response
patients had moderate damage while 38.5% had marked damage. Both breakthrough
and null response groups clarified a well defined comet tail formation (Fig.
2b). Interestingly, only 21.5% of SVR patients showed intact DNA, while
78.5% showed DNA damage; of which in 64.3% it was mild and in 14.2% it was marked,
with no comet tail formation (Fig. 2c).
HCV infection is implicated in the development of hepatocellular carcinoma
(HCC). The virus induces double-strand DNA breaks and enhances the mutation
frequency of proto-oncogenes and tumor suppressors (Machida
et al., 2010) which may lead to inflammation-related carcinogenesis
(Murata et al., 2012).
An increased oxidative stress in chronic hepatitis C patients due to the continuous
generation of Reactive Oxygen Species (ROS) and reactive nitrogen species in
Kupffer cells and polymorphonuclear cells in the liver as well as systemic oxidative
stress secondary to hepatic oxidative stress has been reported (La
Vignera et al., 2012). The use of antioxidant and antiviral therapies
can reverse these deleterious effects of HCV in part by inhibiting ROS induction
by HCV and restoring the function of the DNA repair enzymes, respectively (Pal
et al., 2010).
Although hepatocytes are the primary sites of viral replication, HCV is potentially
lymphotropic, invading and propagating in lymphocytes which are differentially
implicated in the active forms of CHC (Bhargava et al.,
2011). DNA fragmentation in peripheral blood lymphocytes of CHC patients
is a reliable marker for oxidative stress occurring in the liver (Bolukbas
et al., 2006).
In this study, we investigated the occurrence of peripheral blood lymphocytes
DNA fragmentation in different clinical outcomes of interferon therapy in patients
with chronic viral hepatitis genotype 4. In order to quantify the degree of
DNA damage, we used the alkaline Comet assay which is very sensitive well-established
genotoxicity test and a good biomarker of induced DNA damage (Kassie
et al., 2000; McKenna et al., 2008).
Our results showed that SVR group had lower ALT levels, AST and higher platelet
count than the non-SVR group. Similar findings were reported by Sanefuji
et al. (2009). Comet analysis revealed that CHC patient groups had
a significant higher DNA damage than the control group. Both the Null and the
breakthrough response groups which represent the failure of the interferon therapy
in our patients showed higher DNA damage than the SVR group which represents
the clinical resolution of virus. Both breakthrough and Null response patients
had high degrees of DNA damage with comet tail formation, coinciding with their
studied liver function tests which denote activity of CHC.
Our SVR patients had a significant DNA damage compared to control subjects.
In SVR group, 64.3% had mild DNA damage and 14.2% had marked damage. These findings
point strongly that the interferon therapy in SVR patients did not provide complete
In a previous study done by Radkowski et al. (2005),
HCV- RNA has been detected in the livers of patients who have achieved a sustained
virologic response to antiviral therapy. Also, Castillo
et al. (2006) reported that HCV persists and replicates in the liver
and peripheral blood mononuclear cells of most SVR responders for years after
normalization of liver enzyme levels and clearance of serum HCV-RNA. A plausible
explanation may be the newly reported issue of occult hepatitis C infection
(Bhargava et al., 2011), in which both hepatocytes
and lymphocytes are differentially implicated. Though it's an occult state,
it induces DNA damage (Deng et al., 2008) and
is associated with increased risk of developing HCC (Bhargava
et al., 2011).
Radkowski et al. (2005) reported that HCV RNA
may persist in liver or macrophages and lymphocytes in patients with SVR and
this continuous viral presence could result in persistence of humoral and cellular
immunity for many years after therapy and could present a potential risk for
Furthermore, Sanefuji et al. (2009) declared
that recurrent HCC still developed after the curative hepatectomy, even if viral
elimination had been successful. In 12.5% of cases with SVR, HCV-RNA was detected
in the non-cancerous tissue and immunohistochemistry revealed overexpression
of p53 in 100% of HCCs from SVR patients. He suggested that molecular alterations
in hepatocarcinogenesis of SVR patients might be different from those of CHC
In conclusion, CHC genotype 4 patients who are non responders to combined pegylated
interferon/ribavirin therapy, namely the null and the breakthrough responders,
have a high degree of DNA damage. Also, SVR patients have DNA damage but to
a lesser degree. Suggesting that despite clearance of serum HCV- RNA and apparent
clinical disease resolution, SVR patients might not experience HCV infection
clearance and should be followed up being at suspected risk for virus reactivation.
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