34
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National Advisory Board
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Contents Alexandria Journal of Hepatogastroenterology, Volume IVX ( II ), August 2014
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Original Article:
Serum Squamous Cell Carcinoma Antigen Level in
Cirrhotic Chronic Hepatitis C Patients with and without
Hepatocellular Carcinoma
Alaa El Din Mohamed Abdo1; Akram Abd El-Moneim
Deghady2; Essam El Din Saeed Bedewy1 and Walid Ismail
Ellakany1 1Tropical Medicine Department; Clinical and 2Chemical
Pathology Department
------------------------------------------- Original Article:
Standard Endoscopy, Narrow-Band Imaging and
Histopathology in the Diagnosis of Gastro-Esophageal
Reflux Disease
Mohammed M. Shamseya1 and Ayman M. Shamseya2 1Department of Clinical and Experimental Internal Medicine;
Medical Research Institute, 2Department of Internal Medicine;
Faculty of Medicine; University of Alexandria.
------------------------------------------- Original Article:
Study of The Efficacy of Flumazenil Therapy in Different
Clinical Stages of Hepatic Encephalopathy
El Hasafy Youssef Ma*, Hassouna Moustafa Ea, Rady Refaat Ab* a* Internal Medicine Department, Faculty of Medicine,
Alexandria University, Alexandria, Egypt. b* Neuropsychiatry Department, Faculty of Medicine, Alexandria
University, Alexandria, Egypt.
------------------------------------------- Original Article:
Suppression of The Inflammatory Cascade and Oxidative
Stress in Hepatocellular Carcinoma Tumor Bearing Mice:
Effect of Combination of Curcumin, Leflunomide and
Perindopril
Magda Nasr a, Eman Selima a, Omar Hamed b, Amany Kazem c a Department of Pharmacology and Experimental Therapeutics,
Medical Research Institute, Alexandria University, 165 Horreya
Avenue, Alexandria, Egypt b Department of Pharmacology and Toxicology, Faculty of
Pharmacy, Pharos University, Alexandria, Egypt c Department of Pathology, Medical Research Institute,
Alexandria University, Alexandria, Egypt
-------------------------------------------
2
6
13
20
Original Article
Serum Squamous Cell Carcinoma Antigen Level in Cirrhotic Chronic Hepatitis
C Patients with and without Hepatocellular Carcinoma
Alaa El Din Mohamed Abdo1; Akram Abd El-Moneim Deghady2; Essam El Din Saeed Bedewy1 and Walid Ismail
Ellakany1 1Tropical Medicine Department; Clinical and 2Chemical Pathology Department
ABSTRACT
This study will be carried out on 50 personnel divided into five groups : Group A: 10 cases of hepatocellular carcinoma
without interventions. Group B: 10 cases of hepatocellular carcinoma before and 3 months after successful
interventions.Group C: 10 cases of established cirrhosis. Group D: 10 cases with chronic hepatitis C virus infection
without established cirrhosis. Group E: 10 healthy individuals as controls. Patients and Methods: Sera from selected
patients and controls were used for estimation of SCC-Ag using CanAg SCC EIA. Results: high significant increase in
serum SCCA level in patients with HCC (groupA and groupB) when compared to cirrhotic,chronic HCV and control
groups (P < 0.001). Conclusion: Positive significant correlation was found between AFP and serum SCCA level .The
best cut-off value to differentiate HCC patients from cirrhotic patients was 3.2 ng/ml for SCCA yielded with 80%
sensitivity and 90% specificity. When combined sensitivity of both markers were calculated in our study at the best-
chosen cutoff values (SCCA 3.2 ng/ml and AFP 200 ng/ml) sensitivity improved to 93%.
Introduction
Hepatocellular carcinoma (HCC) is a primary
malignancy of the liver. HCC is the fifth most
common malignancy in the world and the third
most common cause of cancer related deaths
worldwide. It is a major health problem in Egypt
with the incidence expected to rise continuously
in the next decade.(1,2). The diagnosis of liver
cancer depends on both screening with alpha-
fetoprotein (AFP) and radiological imaging
studies. Generally, normal levels of AFP are
below 10 ng/ml but AFP greater than 200 ng/ml is
suggestive of HCC . The sensitivity of AFP for
liver cancer is about 67 % ; therefore a normal
AFP does not exclude HCC. Searching another
tumor marker, that together with AFP could
improve the diagnostic utility of HCC.(3).
Squamous cell carcinoma antigen (SCCA), a
member of the high molecular weight family of
serine protease inhibitors named serpins which are
physiologically found in the spinous and granular
layers of normal squamous epithelium but
typically expressed by neoplastic cells of
epithelial origin in a number of different cancers
such as those of the cervix, lung, and head and
neck and so can be used as a clinical marker of
these malignancies.(4). Recently much attention
has been focused on the role of SCCA in HCV
cirrhotic patients suggesting that high levels of
SCCA can assess HCC development.(5). The aim
of this study was to assess the serum level of
squamous cell carcinoma antigen (SCCA) in
cirrhotic chronic HCV patients with and without
hepatocellular carcinoma in relation to alfa feto
protein (AFP).
Aim of the work
The aim of this study was to assess the serum
level of squamous cell carcinoma antigen
(SCCA) in cirrhotic chronic HCV patients with
hepatocellular carcinoma in relation to alfa feto
protein (AFP) .
Patients and Methods
These groups was from both sexes who are
admitted to the inpatient ward and the outpatient
clinic of Tropical Medicine Department, Faculty
of Medicine, Alexandria University. This study
was carried out on : Group A: 10 cases of
hepatocellular carcinoma without interventions.
Group B: 10 cases of hepatocellular carcinoma
before and 3 months after successful
interventions. Group C: 10 cases of established
cirrhosis. Group D: 10 cases with chronic
hepatitis C virus infection without established
cirrhosis. Group E: 10 healthy individuals as
controls. All patients in this study were subjected
to: complete blood picture, liver biochemical
profile serum alanine aminotransferase (ALT),
serum aspartate aminotransferase (AST), serum
alkaline phosphatase, total and direct serum
bilirubin, prothrombin time and activity, serum
albumin blood urea nitrogen(BUN), serum
creatinine. Fasting blood sugar. Serum alpha
fetoprotein (AFP). Determination of squamous
cell carcinoma antigen (SCC-Ag) Sera from
selected patients and controls were used for
estimation of SCC-Ag using CanAg SCC EIA.
The CanAg SCC EIA is a solid phase, non-
competitive immunoassay based upon the direct
sandwich technique. Calibrators and patient
samples are incubated together with biotinylated
Anti-SCC monoclonal antibody in Streptavidin
coated microstrips. After washing buffered
Substrate/Chromogen reagent (hydrogen peroxide
and 3, 3', 5, 5' tetra-methylbenzidine) is added to
each well and the enzyme reaction is allowed to
proceed. During the enzyme reaction a blue
colour will develop if antigen is present. The
intensity of the colour is proportional to the
amount of SCC present in the samples. The colour
intensity is determined in a microplate
spectrophotometer at 620 nm (or optionally at 405
nm after addition of Stop Solution). Calibration
curves are constructed for each calibrator. The
SCC concentrations of patient samples are the
read from the calibration curve.
Results
Table (1) shows a statistical significant difference
between different studied groups regarding alpha
feto protein (P= 0.000).
Table (1): Comparison between different studied groups regarding alpha feto protein
Mean Std. Deviation Minimum Maximum
Gp. A 263.0 96.02 150. 438
Gp. B 209.4 64.7 145. 380.
Gp. C 154.5 48.16 75. 210.
Gp. D 7 1.82574 5 9
Gp. E 1.22 0.27406 0.8 1.6
F
P
38.208
0.000*
Table (2) shows a statistical significant difference between different studied groups regarding SCCA level
(P= 0.000).
Table (2): Comparison between different studied groups regarding SCCA score.
Mean Std. Deviation Minimum Maximum
Gp. A 5.53 2.16 2.5 10.
Gp. B 5.3 1.5 3.3 7.6
Gp. C 3.3 1.6 1.2 5.6
Gp. D 0.824 0.15897 0.6 1.05
Gp. E 0.646 0.23172 0.3 0.95
F
P
28.897
0.000*
Also, Positive significant correlation was found between AFP and SCCA in both groups
Table (3): Correlation between AFP and SCCA
AFP
HCC without
intervention
HCC with
intervention
SCCA r 0.629* 0.525*
p
References
1. Lok A, Seeff L, Morgan T. Incidence of
hepatocellular carcinoma and associated risk
factors in hepatitis C related advanced liver
disease.Gastroenterology 2009; 136 :138-48.
2. El-Zayadi A, Badran H. Hepatocellular
carcinoma in Egypt: a single center study over a
decade. World J Gastroenterol 2005; 11:5193–8.
3. Peng SY, Chen WJ, Lai PL , et al. High
alpha-fetoprotein level correlates with high stage,
early recurrence and poor prognosis of
hepatocellular carcinoma: significance of hepatitis
virus infection, age, p53 and beta-catenin
mutations: Int J Cancer 2004; 112:44-50.
4. Giannelli G , Antonaci S.New frontiers in
biomarkers for hepatocellular carcinoma: Dig
Liver Disease; 2011; 38:854-9.
5. Biasiolo A, Chemello L, Quarta S. Squamous
cell carcinoma antigen (SCCA) detection in
patients with HCV infection and rheumatoid
factor seropositivity. J Viral Hepat 2008;15:246-9.
6. Issa H, Awadallah A, Soliman M.
Evaluation of Serum Chromogranin A as a Useful
Tumor Marker for Diagnosis of Hepatocellular
Carcinoma. Journal of American Science 2011; 7:
999-1007.
7. Feng W, Zhi-Biao W, WenChen M, et al.
Extracorporeal High Intensity Focused Ultrasound
Ablation in the Treatment of Patients with Large
Hepatocellular Carcinoma. Surgical Onco 2004;
11:1061-69.
8. Molinari M, Kachuray J, Dixonz E, Suehiro
Y, Morioka H, Fordtran B et al. Transarterial
Chemoembolisation for Advanced Hepatocellular
Carcinoma: Results from a North American
Cancer Centre. Clinical Oncology 2006; 18: 684-
92.
9. Hussein M, Ibrahim A, Abdella H.
Evaluation of serum squamous cell carcinoma
antigen as a novel biomarker for diagnosis of
hepatocellular carcinoma in Egyptian patients.
Indian J Cancer 2008; 45:167-72.
10. El Ezawy H, Shebil N, Mounis A.
Assessment of serum SCCA and KL-6 as tumor
markers and their correlation with tumor size.
Journal of American science 2012; 8:172-9.
11. Bin X, Gansheng F, Shihua L, Kim T,
Takahashi S. SCCA level in Peripheral Blood in
Patients with Hepatocellular Carcinoma Before
and after TACE. J Huazhong Univ Sci Technol
2008; 28:645- 8.
12. Trevisani F, Daniela B, Gianluca F. Serum
SCCA as a predictor of hepatocellular carcinoma
in patients with liver cirrhosis. Open Journal of
Gastroenterology 2012; 2:56-61.
Original Article
Standard Endoscopy, Narrow-Band Imaging and Histopathology in the
Diagnosis of Gastro-Esophageal Reflux Disease Mohammed M. Shamseya1 and Ayman M. Shamseya2 1Department of Clinical and Experimental Internal Medicine; Medical Research Institute, 2Department of Internal
Medicine; Faculty of Medicine; University of Alexandria.
ABSTRACT
Background: Gastroesophageal reflux disease (GERD) is caused by the reflux of gastric contents into the esophagus.
The diagnosis of GERD is based on the combination of clinical symptoms, endoscopic findings and histological
changes. Narrow band imaging (NBI) endoscopic modality facilitates mucosal surface evaluation and may improve the
endoscopic diagnosis of GERD. Aim of the work: In this study we aimed to show the difference between standard
white light endoscopy and the NBI technique in squamo-columnar junction evaluation of patients with GERD and
compare it with mucosal hitopathological findings. Patients and Methods: A total of 60 subjects were recruited; 40
with non-erosive reflux disease (NERD) and 20 with erosive reflux disease (ERD). Patients were subjected to
esophagogastroduodenoscopy (standard white light endoscopy and NBI). In all of them, two mucosal biopsies were
taken 2 cm above the esophagogastric junction for histopathological evaluation (inflammation versus normal mucosa).
Results: NBI was more sensitive than standard white light endoscopy in distinguishing abnormal endoscopic findings.
Histopathological findings were more prevalent than the mucosal changes diagnosed by the standard white light
endoscopy and NBI. Conclusion: NBI is more sensitive than white light endoscopy in detecting inflammation in NERD
patients. However, histopathological evaluation is the most sensitive; therefore taking a biopsy remains useful in
diagnosing GERD.
Introduction
Gastroesophageal reflux disease (GERD) is a
complex disorder caused by the reflux of gastric
contents into the esophagus either with or without
complications (1). Although GERD is widely
reported to be one of the most prevalent diseases
of the gastrointestinal tract, prevalence data is
based primarily on estimates rather than actual
data (2). The incidence of GERD is 10–20% in
Western countries, while in Asia it is about 5% (3-
6). There is no gold standard method for the
diagnosis of GERD (7). Upper gastrointestinal
endoscopy is the standard diagnostic tool for
evaluation and grading of esophagitis and
excluding other esophageal diseases (8). The
sensitivity of endoscopy for GERD is poor, but it
has an excellent specificity of 90% to 95% (8,9).
Reflux esophagitis is defined endoscopically by
the presence of mucosal breaks in the esophagus,
which are the most reliable endoscopic indicators
of reflux esophagitis (9-11). Most patients have no
visible mucosal damage at the time of endoscopy,
referred to as non-erosive GERD, whereas others
have esophagitis, peptic strictures, or Barrett’s
esophagus (12). Narrow band imaging (NBI) is a
novel high resolution endoscopic imaging
technique in which the spectral bandwidth of
filtered light is narrowed(11). In this imaging
technique, blue light with the short wavelength
results in more superficial penetration to the
mucosal surface (11-15). Narrow-band illumination
can enhance the contrast between the esophageal
mucosa and the gastric mucosa. In patients with
GERD, NBI endoscopy may help to improve
diagnostic accuracy compared to conventional
endoscopy(16,17). Narrow band imaging (NBI)
endoscopy system enhances visualization of
microvasculature and mucosal patterns.
Histological abnormalities have been well-defined
for GERD (18). Histology may, therefore, be
considered a diagnostic tool for non-erosive reflux
disease (NERD) by examining simple esophageal
biopsies during upper gastrointestinal endoscopy (19).
Aim of the work:
In this study we aimed to demonstrate the
difference between standard white light
endoscopy and the NBI technique in squamo-
columnar junction evaluation of patients with
GERD and compare it with mucosal
histopathological findings.
Patients and Methods
The study was undertaken at the Hepatology &
Gastrointestinal endoscopy Unit, Medical
Research Institute, & GIT Unit, Faculty of
Medicine, Alexandria University, during the
period from June 2012 to October 2013. The
study was carried out with the approval of the
Medical Research Institute Ethical Committee. A
total of 60 patients with GERD symptoms were
enrolled. Typical GERD symptoms were defined
as at least three episodes of regurgitation and/ or
heartburn per week. Patients who were on
continuous treatment with acid suppression in the
preceding four weeks before endoscopy, who had
undergone previous upper gastrointestinal surgery
such as gastrectomy, fundoplication or distal
esophagectomy, along with those who had had
severe gastroparesis or esophageal varices were
excluded from the study. The distal esophagus
was initially examined by standard white light
endoscopy. Of the sixty patients 40 proved to
have NERD, while the remaining 20 had erosive
reflux disease (ERD). During upper
gastrointestinal endoscopy, the distal 5 cm of the
esophagus mucosal morphology at the squamo-
columnar junction were then visualized by NBI
system using video endoscopes (GIF-H260;
Olympus), video processor (Evis Lucera CV 260
SL; Olympus). During standard white-light
endoscopy and NBI examination erosions,
mucosal breaks and other complications were
graded according to the Los Angeles
classification.(13) We considered the following
index lesions as acid related mucosal changes:
mucosal breaks under standard white-light
endoscopy and mucosal brownish areas under
NBI. Two mucosal biopsies were taken 2 cm
above the esophagogastric junction with Olympus
biopsy forceps. The biopsies were preserved in
10% formalin and transferred for routine
pathology evaluation within 24 hours. The
specimens were visually oriented with slight
magnification during embedding in the paraffin
wax blocks before hardening to ensure vertical
cutting. They were cut and finally stained with
haematoxylin and eosin. Histological esophagitis
was identified by basal cell hyperplasia over
0.15%, increased papillary length over 0.66%, and
infiltration by leukocytes/eosinophils. Apart from
the routine histological reporting, we also
requested a comment on the presence or absence
of inflammation. We termed all histological
differentiations (infiltration with neutrophils and
eosinophils, elongated papillae, and basal zone
thickening) as inflammation.
Results
The mean value for age of the enrolled patients
was 37.5 ±12.3 years. Gender distribution was:
males 18 (30%); females 42 (70%). The mean age
of males was 38.1 ±16.2 years and that of females
33.7 ±12.7 years (Table 1).
Table (1): Demographic data
Number Percent
Sex
Males 18 30.0
Females 42 70.0
Mean Standard deviation
Age (years)
Males 38.1 ± 16.2
Females 33.7 ± 12.7
Among the 60 symptomatic GERD patients,
examination of the squamo-columnar junction and
esophageal mucosa were defined as normal in 40
cases with standard white-light endoscopy,
whereas NBI guided endoscopic examination
revealed normal mucosal findings in 28 cases
only. NBI was more sensitive than standard
white-light endoscopy in distinguishing abnormal
endoscopic findings (p
Table (2): Endoscopic findings in standard white-light endoscopy and narrow band imaging
Endoscopic
findings
Standard white-light endoscopy NBI P
Males (n) Females (n) Males (n) Females (n)
Normal 10 30 5 23 0.004*
Esophagitis
(Grade-A) 3 5 7 11 0.003*
Esophagitis
(Grade-B) 2 4 3 5 >0.05
Esophagitis
(Grade-C) 2 2 2 2 > 0.05
Esophagitis
(Grade-D) 1 1 1 1 > 0.05
NBI = narrow-band imaging, n = number, Endoscopic findings according to the Los Angeles classification, p
value for comparing between the studied groups, *:statistically significant at p ≤ 0.05.
Figure 1: Standard white-light endoscopic picture of squamo-columnar junction showing GERD Los
Angeles class A
Figure 2: Standard white-light endoscopic picture of squamo-columnar junction showing GERD Los Angeles
class B
Figure 3: Standard white-light endoscopic picture of squamo-columnar junction showing GERD Los Angeles
class C
Figure 4: Narrow Band Imaging (NBI) of squamo-columnar junction showing GERD Los Angeles class A
Normal histopathological findings were observed
only in 17 of the 40 patients who had normal
esophageal mucosa by white-light endoscopy and
17 of the 28 patients who had normal endoscopic
findings with NBI. The histology was abnormal in
all patients with erosive esophagitis that were
diagnosed with standard white-light endoscopy
and NBI (Tables 3,4).
Table (3): Comparison between standard light endoscopy and histopathological findings
Standard light
endoscopy (n=60)
Histopathological
findings (n=60)
P
Normal esophageal mucosa 40 17/40 < 0.001*
Endoscopic esophagitis 20 20 >0.05
n = number, p value for comparing between the studied groups, *:statistically significant at p ≤ 0.05.
Table (4): Comparison between narrow band imaging endoscopy (NBI) and histopathological findings
Narrow Band Imaging
(n=60)
Histopathological
findings (n=60)
P
Normal esophageal mucosa 28 17/28 0.008*
Endoscopic esophagitis 32 32 >0.05
n = number, p value for comparing between the studied groups, *:statistically significant at p ≤ 0.05.
Discussion
GERD is a common disorder that encompasses a
variety of clinical conditions, like regurgitation
and/ or heartburn. Endoscopically, it is classified
into erosive (ERD) and non-erosive (NERD)
subtypes (20). According to population-based
studies, it is estimated that NERD accounts for up
to 60% of patients with GERD (1,21). Endoscopy is
the gold standard for the diagnosis of erosive
GERD (8), and up to 40% of patients with GERD
have positive upper gastro-intestinal endoscopy
findings with standard white-light endoscopy (22).
The Los Angeles classification system is based on
the detection of mucosal breaks in conventional
endoscopy (17). Our findings demonstrated that by
employing conventional endoscopy, esophageal
erosive lesions were encountered in 33% of the
patients with GERD. Application of NBI into the
assessment of the squamo-columnar junction
increased the ERD prevalence to 53% in the same
study population. The ability to detect esophagitis
is therefore improved through the use of NBI
compared to standard white-light endoscopy
(p=0.004). A recent study showed that intra-
observer and inter-observer reproducibility in
determining the esophageal lesions in patients
with GERD can be improved by utilizing NBI
endoscopy compared with conventional
endoscopy (20). Sharma et al assessed the utility of
NBI in patients with GERD symptoms and found
that a significantly higher proportion of patients
with GERD had an increased number of dilatation
and tortuosity of intra-papillary capillary loops
(IPCLs), micro-erosions, and increased
vascularity at the squamo-columnar junction
compared with the control subjects (15). In another
study which included a group of 230 patients with
GERD symptoms, 65.6% of patients were graded
as normal following conventional endoscopy,
whereas 59.1% of patients were graded as normal
following NBI (20). Up to 6% of patients were
reclassified from NERD to grade A esophagitis by
the utilization of NBI. In our work, we observed a
greater shift; 20% of patients diagnosed by
conventional endoscopy as NERD were
reclassified as having ERD after NBI evaluation.
On the other hand, 67% of patients were graded as
normal with standard endoscopy, while only 47%
of patients proved to be normal with NBI.
Histological changes, such as basal cell
hyperplasia and location of the papillae close to
the epithelial surface have been well-documented
in esophageal specimens of GERD patients (23).
Therefore, histological examination of the
esophageal mucosa may provide greater
sensitivity when detecting subtle changes of
mucosa (23). In a previous study, histology was
abnormal in 96% of patients with erosive
esophagitis and in 76% of patients with NERD (24). In our study, 20 patients with standard white-
light endoscopy and 32 patients with NBI showed
reflux esophagitis. The histology was abnormal in
all patients with erosive esophagitis that were
diagnosed with standard white-light endoscopy
and NBI. Zuberi et al showed that in 196 GERD
patients, histological examination revealed
presence of inflammation in 140 patients (71.4%)
patients, while the remaining had normal
histology. In this study, over 50% of patients had
NERD. The severity of clinical symptoms
correlated with the endoscopic findings but not
with histological findings (25). In our study, in 60
symptomatic GERD patients, 40 cases with
standard white light endoscopy and 28 cases with
NBI were diagnosed as NERD. The pathological
examination of esophageal specimens revealed
normal findings in 17 patients. Most patients
involved in our study showed histological changes
in esophageal mucosa, which is concordant with
the results of Zuberi et al. We concluded that,
narrow band imaging upper gastrointestinal
endoscopy is more sensitive than the standard
white-light endoscopy in the detection of
esophageal lesions in GERD patients. However,
histopathological evaluation may provide better
sensitivity for evaluating esophageal mucosa in
non-erosive reflux disease.
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Original Article
Study of The Efficacy of Flumazenil Therapy in Different Clinical Stages of
Hepatic Encephalopathy
El Hasafy Youssef Ma*, Hassouna Moustafa Ea, Rady Refaat Ab* a* Internal Medicine Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt. b* Neuropsychiatry Department, Faculty of Medicine, Alexandria University, Alexandria, Egypt.
ABSTRACT
Background: hepatic encephalopathy (HE) is a syndrome characterized by depressed level of consciousness, cognitive
impairment, and personality changes induced by circulating neurotoxic substances that have bypassed normal hepatic
detoxification because of diversion of portal blood flow into the systemic circulation. Hepatic encephalopathy most
commonly occurs in the setting of hepatic cirrhosis or in those who have undergone portocaval shunt surgery. Up to two
thirds of patients with cirrhosis may have subclinical hepatic encephalopathy. About 30% of patients with end-stage
liver disease develop clinically significant hepatic encephalopathy. Treatment of hepatic encephalopathy with
flumazenil, a benzodiazepine receptor antagonist, has been described in the literature but has not yet made its way into
routine clinical practice in the Emergency department (ED). Aim of the work: The aim of this study is to evaluate the
efficacy of flumazenil therapy in different clinical stages of hepatic encephalopathy. Patients and Methods: This study
was conducted on sixty patients with clinical HE due to acute liver failure or decompersated liver cirrhosis attending to
the department of emergency medicine, 20 patients were randomized as group not received flumazenil. 40 patients were
randomized as group received flumazenil by direct intravenous route in which 20 patients with grade 1 and 2 HE and 20
patients with grade 3 and 4. Also, 20 age and sex matched healthy subjects with no evidence of live disease were
included as control group. Serum benzodiazepine level, Glasgow come scale (GCS) with grading of HE and arterial
blood ammonia were assessed before and after receiving intravenous flumazeni 0.5 mg. within 6 hours. Results:
clinical improvement was obtained regarding GCS in grade 1.2 HE pre 10.0-15.0 with the mean 12.95 ± 1.47. post: 10.0
– 15.0 with the mean 14.05 ± 10.64. Then control treatment. for grading of HE the best improvement accrued in control
treatment. pre: 1.0 – 3.0 with the mean 2.05 ± 0.76. post: 0.0-3.0 with the mean 1.25 ± 1.12. then HE grade 1.2.
Conclusions: Flumazenil therapy had a beneficial effect on improvement of HE at the end of treatment in early stages
with no effect on serum ammonia or serum benzodiazepine level.
Introduction
Hepatic encephalopathy (HE) is a syndrome
observed in patients with cirrhosis, which is
defined as a spectrum of neuropsychiatric
abnormalities in patients with liver dysfunction,
after exclusion of other known brain disease. It is
characterized by personality changes, intellectual
impairment, and a depressed level of
consciousness. HE is also described in patients
without cirrhosis with either spontaneous or
surgically created protosystemic shunts.(1). Subtle
signs of HE are observed in nearly 70% of
patients with cirrhosis. Symptoms may be
debilitating in a significant number of patients and
are observed in 24-53% of patients who undergo
portosystemic shunt surgery. Approximately 30%
of patients dying of end stage liver disease
experience significant encephalopathy,
approaching coma.(2). HE appears to be result of
both vascular and parenchymal mechanism. The
vascular causes include a serious of pathological
conditions in which toxic metabolites accumulate
in the systemic circulation due to vascular bypass
of the liver. Congenital portocaval shunts are
example of pure vascular diseases. The
parenchymal causes of HE include the conditions
in which the blood supply to the liver is normal
but the population of active hepatocyts is
decreased. An example of pure parenchymal HE
is fulminant hepatic failure due to acetaminophen
overdose.(3). The neurotransmitter rather than
deficit in the cerebral energy metabolism appears
to be involved. The neuronal cell most vulnerable
to liver failure is astrocyte.(4). Also, cerebral
atrophy was reported in brain scans of cirrhotic
patients with chronic recurrent HE. Other
explaination for HE is accumulation of the toxic
substances such as: ammonia, mercaptanes, short
and medium chain fatty acids, manganese, phenol
and dehydrocholate in cases of liver failure which
may increase permeability of the blood brain
barrier (BBB) through different mechanisms.(5,6).
Arterial blood ammonia concentrations are
frequently elevated in patients with all forms of
HE, is provided the results of recent studies using
positron Emission Tomography (PET) scan and
such studies demonstrate an increase in the rate at
which ammonia is taken up and metabolized by
brain.(7-9) . Glutamate is a major CNS excitatory
neurotransmitter in the mammalian brain.
Released from the presynaptic nerve terminal is
inactivated into glutamine via the action of
Glutamine Synthetase (GS). Brain glutamine
concentrations are invariably increased in human
HE, Which is consistent with exposure of brain to
increased concentrations of blood borne ammonia
in liver failure.(13) Neuromuscular abnormalities
including extrapyramidal symptoms such as
tremor and rigidity form part of the clinical
syndrome of HE.(10). The involvement of the
gamma amino butyric acid (GABA) receptors in
HE was first hypothesized by Schafer & Jones.
This theory, as different groups found that GABA
agonists such as barbiturates, benzodiazepines and
muscimol potentiated HE symptoms, while the
administration of the GABA antagonist was found
to improve neurophysiologic changes and
behavioral symptoms of HE.(11). Increase in the
levels of endogenous ligands to the Penzodiazepine
receptors (BZR) have been demonstrated in a wide
range of experiments in animal models and
humans. Benzodiazepine concentrations have been
shown to be increased in the cerebrospinal fluid
and in postmortem brain samples of patients with
HE. BZR binding in the brain of patient with
recurrent HE has also been shown to tbe increased
in PET.(12). it was subsequently demonstrated that
treatment of cultured astrocytes with millimolar
concentrations of ammonia results in increased
binding of BZR ligands. The role of nitric oxide
(NO) which is a free radical that was implicated in
the hyper dynamic circulation in liver cirrhosis
has been emerged also in the pathogenesis of
HE.(13). The possibility of having benzodiazepine
– like substances circulating and producing some
of the symptoms of the HE prompted a great deal
of research. Upon these findings, treatment with
the benzodiazepine antagonist flumazenil was
attempted in HE patients and improvements were
found in some cases. However, the implication of
benzodiazepines in HE has also been criticized.
The grounds for criticism stem from studies
reporting that increased levels of benzodiazepine
ligands seem to accumulate in severe liver disease
independently of the presence of HE, and their
levels are similar to those commonly found in
exogenous benzodiazepine consumers.(14)
Aim of The Work
The aim of the work is to evaluate serum levels of
benzodiazepines and the effect of administration
of flumazenil drug in the different stages of
hepatic encephalopathy.
Subjects
Sixty patients with clinical HE due to acute liver
failure or decompensated liver cirrhosis attending
to the Department of Emergency Medicine were
included in the study. (1) Twenty patients were
randomized as group not received flumazenil,
forty patients were randomized as group receiving
flumazenil by direct intravenous root in which,
Twenty patients in HE grade 1 and 2, Twenty
patients in HE grade 3 and 4. (2) Twenty age and
sex matched healthy subjects with no evidence of
liver disease were included as control group.
Patient and Methods
During period of stabilization all patients will
receive standard treatment - Lactulose 30 ml
orally every 6 hours and, diet of approximately
2000 k. cal. per day. Containing 20 gm proteins.
All patients were subjected to the following:
- Informed consent and complete history were
taken. - Full thorough clinical examination of all
patients and determining. - Glasgow coma scale.
HE grading were done using West Haven
classification.
Laboratory investigations including:
- Arterial blood ammonia level, liver test profile,
arterial blood gases, Serum benzodiazepine
level.(15.16). Forty patients were allocated to receive
flumazenil 0.5 mg amp I.V. by direct intravenous
root and serum Benzodiazepine level was
measured again after giving fiumazenil. HE
grading, arterial serum ammonia, and Glasgow
coma scale were repeated after starting treatment
by six hours.
Results
The main demographic, laboratory and clinical data
(aetiology of liver cirrhosis, child-Pugh scores) of
the studied groups in table I.
Table (1): Demographic and clinical characteristics of the studied groups.
Control
(n = 20)
HEG (1.2)
(n = 20)
HEG (3.4)
(n = 20)
NO % NO % NO %
Age (Years) (Mean ± SD) 56.80 9.57 52.80 10.09 60.0 8.16
Sex (M/F) 13/7 14/6 16/4
Child A score 0 0.0 5 25.0 1 5.0
Child B score 2 10.0 3 15.0 5 25.0
Child C score 18 90.0 12 60.0 14 70.0
Acute liver failure 2 10.0 3 15.0 3 15.0
Liver cirrhosis 18 90.0 17 85.0 17 85.0
Improvement of the neurological status was
observed in patient with HEG 1,2 regarding GCS
pretreatment 10.0-15.0 with the mean 12.95±1.47
post treatment 10.0-15.0 with the mean
14.05±1.64. For serum ammonia level,
improvement in HEG 1,2 was more than in HEG
3,4 patients after treatment 132.0±35.79 than
103.20±38.93. Comparison between pre and post
treatment results in each group according to serum
BZD level the results showed that: No significant
changes occurred in any group.
Figure (1): Relation between benzodiazepine level and Child Pugh classification in each group.
Benzodiazepine level appears to be related to the degree of the severity of liver disease and child Pugh classification the level
increased with increased severity of liver disease and child Pugh classification.
0
100
200
300
400
500
600
700
800
Control treatment HEG (1.2) HEG (3.4)
Mea
n o
f b
enzo
dia
zeb
ine
Child A
Child B
Child C
Table (2): Comparison between pre and post treatment results in each group according to different Parameter
Control treatment HEG (1.2) HEG (3.4)
Pre Post Pre Post Pre Post
GCS
Range 11.0-15.0 9.0-15.0 10.0-15.0 10.0-15.0 3.0-15.0 3.0-15.0
Mean ± SD 12.35 ± 1.14 13.15 ± 1.87 12.95 ± 1.47 14.05 ± 1.64 8.75 ±0.44 5.40 ±4.16
Median 12.0 13.50 13.0 15.0 12.0 12.0
% of change 6.95 9.11 11.23
p 0.103 0.007* 0.050
HEG
Range 1.0 - 3.0 0.0 - 3.0 1.0 – 2.0 0.0 – 3.0 3.0 – 4.0 2.0 – 4.0
Mean ± SD 2.05 ± 0.76 1.25 ± 1.12 1.55 ± 0.51 0.80 ± 0.83 3.40 ± 0.50 3.35 ± 0.75
Median 2.0 1.0 2.0 1.0 3.0 3.50
% of change 35.83 52.50 1.25
P 0.007*
correlated with the severity of hepatic
encephalopathy.(28) Several other hypotheses have
been proposed: deposition of manganese in the
basal ganglia,(18) deficiency of zinc (responsive to
a decrease in urea cycle enzymes) and
hyperactivity of the GABA-ergic system.
However, as stated by Basile and Jones, 3 the two
main theories for hepatic encephalopathy in
cirrhotic patients, i.e. the ammonia theory and the
GABA-ergic theory, are not mutually exclusive.
The rationale for the latter hypothesis is that
benzodiazepines have a depressant effect on the
central nervous system with a high affinity for
GABA receptors. Endogenous benzodiazepine
agonists, called benzodiazepine-like substances,
have been found in experimental studies,(19,20) and
have been detected in the cerebral tissue,
cerebrospinal fluid and serum of cirrhotic patients
with hepatic encephalopathy. Flumazenil is
usually used for the diagnosis of suspected
poisoning in emergency situations and for the
treatment of benzodiazepine intoxication.(21) This
antagonist property has been proposed in the
treatment of hepatic encephalopathy. Open studies
with flumazenil in humans have shown an
improvement of hepatic encephalopathy.(5-9).
Flumazenil had a significant beneficial effect on
improvement of hepatic encephalopathy at the end
of treatment in early stages only. This may be due
to the presence of many other factors with
advancing HE stage. Flumazenil had no
significant effect on recovery or mortality.
Although we failed to find significant differences
in plasma Benzodiazepine Receptor Ligands
(BZRL) levels between the patients with HE and
those liver cirrhosis, we observed that a
significantly larger proportion of patients in the
HE group than in the liver cirrhosis group had
pharmacologically meaningful levels of
Benzodiazepine in their plasma. This finding
suggests that plasma Receptor Ligands BZRL
begin to accumulate in patients with
decoompensated liver cirrhosis without causing
clinical cognitive impairment. The (BZRL) may
then continue to accumulate reaching their highest
concentrations during HE stages 3 and 4.(22)
Further support for this notion is provided by the
observation that plasma BZRL levels correlate
moderately with the 4 HE stages of severity. The
fact that some patients in the cirrhotic patients
group (20%) had pharmacologically meaningful
plasma levels of BZRL without clinical signs of
encephalopathy and that no BZRL were detected
in approximately one-third of patients with HE
suggests that neuropsychiatric symptoms in HE
cannot be explained by the presence of these
compounds alone. Olasma et al and Basile et al
have reported that significant plasma BZRL levels
were present in approximately 60% of patients
with HE. Additionally, results from a controlled
clinical trial of the efficacy of the benzodiazepine-
receptor antagonist flumazenil in the treatment of
HE showed a significant improvement in only 40
% of patients and symptom reduction of no more
than 50%.(23) These findings suggest that factors
other than the GABA-benzodiazepine complex
receptor and endogenous benzodiazepine
compounds may play a role in the pathogenesis of
HE.(24)
Conclusions
• Benzodiazepine (BZD) level has no correlation
with hepatic encephalopathy grade (HEG). It
appears that the level is more related to the degree
of liver impairment. • Flumazenil therapy had a
significant beneficial effect on improvement
of hepatic encephalopathy at the end of
treatment in early stages only with no
significant effect of adding flumazenil in
treatment of hepatic encephalopathy (HE) in late
stages. • Flumazenil therapy has no effect on
serum ammonia or benzodiazepine level.
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Suppression of The Inflammatory Cascade and Oxidative Stress in
Hepatocellular Carcinoma Tumor Bearing Mice: Effect of Combination of
Curcumin, Leflunomide and Perindopril
Magda Nasr a, Eman Selima a, Omar Hamed b, Amany Kazem c a Department of Pharmacology and Experimental Therapeutics, Medical Research Institute, Alexandria University, 165
Horreya Avenue, Alexandria, Egypt b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Pharos University, Alexandria, Egypt c Department of Pathology, Medical Research Institute, Alexandria University, Alexandria, Egypt
ABSTRACT
Chemoprevention has been considered the best strategy in lowering the current mortality associated with
hepatocellular carcinoma (HCC). Our previous study showed that combination treatment with leflunomide,
perindopril and curcumin resulted in synergistic inhibition of angiogenesis and consequently more effective
chemoprevention of HCC. Aim of the work: In the present study, we examined other underlying
mechanisms of HCC chemoprevention by investigating the effects of curcumin, leflunomide or perindopril
and their combination on oxidative damage and inflammatory markers during diethylnitrosamine (DEN)-
induced hepatocarcinogenesis in mice. Patients and Methods: Six mice groups were used: control, positive
control (DEN-induced HCC), DEN+leflunomide, DEN+perindopril, DEN+curcumin, DEN+combination.
Liver tissues were used for assessment of reduced glutathione (GSH), superoxide dismutase enzyme (SOD),
malondialdehyde, protein carbonyl , tumor necrosis factor (TNF-α) and inducible nitric oxide synthase
(iNOS) expression. Results: There was a significant increase in hepatic lipid peroxidation and protein
carbonyl, a significant decrease in SOD activity and GSH level in DEN-treated animals compared to normal
controls. Elevated expressions of iNOS and increased levels of TNF-α were observed in livers of the same
animals. Curcumin , leflunomide or perindopril and their combination reversed all the aforementioned
markers with more pronounced effects obtained with the combination regimen. Conclusion: The present
study provides evidence that suppression of oxidative stress and inflammatory response could play an
important role in chemoprevention and that the combination has more potent antioxidant and anti-
inflammatory properties than each of the probed agents. The outcome of this study may highlight the use of
this combination in prevention and intervention of human HCC.
Introduction
Considering the limited treatment and grave
prognosis of hepatocelllar carcinoma (HCC),
chemoprevention has been considered the best
strategy in lowering the current morbidity and
mortality associated with this disease (1). Although
the cellular mechanisms contributing to
hepatocarcinogenesis are relatively unknown, a
connection between inflammation and liver cancer
is beginning to be unraveled. The role of
inflammation in the promotion of carcinogenesis
was originally proposed by Virchow in 1863.
Along this line, compelling evidence has
accumulated which provides an insight into the
role of inflammation in initiation, promotion and
progression of HCC (2). Hepatic inflammation, due
to exposure to infectious agents (hepatotropic
viruses) as well as toxic compounds, may
represent an early step in the development of
malignancy with genetic and epigenetic events
occurring as a later manifestation of a prolonged
inflammatory process (3-4). It has been shown that
HCC almost always develops on a background of
chronic liver injury including chronic hepatitis
and cirrhosis, both considered to be preneoplastic
stages of hepatocellular tumor development(5). An
expanding body of evidence suggests that
inflammation-mediated processes, including the
production of cytokines, chemokines, reactive
oxygen and nitrogen species, and mediators of
inflammatory pathways may contribute to hepatic
neoplasia(6-8). Oxidative stress, through generation
of reactive oxygen species (ROS) acts as a
predisposing factor to hepatocarcinogenesis and is
the common driving force of HCC in chronic liver
diseases (6,9). This stressful condition is known to
play a major role in cancer development, mainly
by enhancing DNA damage and modifying some
key cellular processes, such as cell proliferation,
apoptosis, and cell motility cascades by
superoxide radicals and hydrogen peroxides (10). It
is well known that inflammation is one of the
biological responses driven by oxidative stress.
Thus, attenuation of oxidative damage as well as
inflammation may have an important role in
chemoprevention against hepatocarcinogenesis.
We have previously reported, for the first time,
that combination treatment with curcumin,
leflunomide and perindopril affords
chemoprevention against diethylnitrosamine
(DEN)-induced hepatocarcinogenesis in a mouse
model (11). Although the molecular mechanisms of
liver tumor inhibitory effects of such combination
have not been completely elucidated, we have
found that synergistic angiopreventive effect is
implicated in their prevention of DEN-induced
HCC (12-14). In fact, ample studies have shown that
curcumin exhibits a potent anti-inflammatory and
antioxidant activities in vivo in chemically-
induced hepatocarcinogenesis (15-19). In addition,
leflunomide and perindopril have been reported to
exert different inhibitory effects on inflammation (20-23). In view of these studies and as abnormal
cell proliferation, inflammation and reactive
oxygen species play crucial roles in angiogenesis [20-23], we hypothesized that combination of these
drugs might have a potent anti-inflammatory and
antioxidant effect, which may be linked to its
previously observed antiangiogenic and
antihepatocarcinogenic effect. Thus, the objective
of the present study was to investigate the
combined effect of curcumin, leflunomide and
perindopril on inflammation and oxidative stress
during DEN-induced hepatocarcinogenesis in
mice.
Material and methods
Chemicals
DEN and Curcumin powder (95% pure) were
purchased from Sigma-Aldrich (St. Louis, MO,
USA). Leflunomide and perindopril were
generously supplied by EVA Pharm Co. (Cairo,
Egypt) and Servier Co. (France), respectively. All
other chemicals and reagents used were purchased
from Chematech Co (Alexandria, Egypt).
Animals
Male Albino mice, 6-8 weeks old were purchased
from the Animal House of the Medical Research
Institute, Alexandria University. They were
housed in stainless-steel mesh cages in groups of
eight, under controlled conditions of light
illumination, relative humidity and temperature.
Animals were allowed access to food and tap
water ad libitum throughout the acclimatization
and experimental periods. All animal procedures
were performed according to approved protocols
and in accordance with the standard
recommendations for the proper care and use of
laboratory animals.
Experimental design
Liver samples utilized in the present study were
harvested from our previously reported
chemopreventive study following established
DEN-induced hepatocarcinogenesis protocol (11).
Mice were randomly divided into six groups (n=8
in each group) except the DEN group which was
comprised of 12 animals. While the negative
control group served as the vehicle control, mice
in the other groups received an intraperitoneal
(i.p) injection of 75 mg/kg of DEN weekly for 3
weeks, then 100 mg/kg weekly for another 3
weeks. Mice in DEN group did not receive any
additional treatment and served as positive
controls. Four mice from the DEN group were
sacrificed starting from the 6th week following
administration of the hepatocarcinogen DEN, at
weekly intervals till the end of the 8th week, to
monitor the appearance of HCC by
histopathological examination of livers. Animals
in leflunomide and perindopril groups were
treated with 10 mg/kg/day of leflunomide and 2
mg/kg/day of perindopril by oral gavage,
respectively. Mice in curcumin group were treated
with 100 mg/kg of curcumin three days a week
intraperitoneally. The combination treatment of
leflunomide, perindopril and curcumin group was
designated as combination group. All drug
treatments were given two weeks prior to DEN
administration and for eight consecutive weeks.
After 10 weeks, animals of all groups were
sacrificed by cervical dislocation. Livers were
excised and cut into small sections.The results on
HCC incidence have already been reported (11). In
the current study, liver sections were used for the
preparation of liver homogenate to be used for
tissue biochemical analysis and enzyme linked
immunosorbent assay (ELISA) analysis.
Lipid peroxidation
The Thiobarbituric acid (TBA) test procedure
described by Uchiyama and Mihara (1978) (24) is
the most frequently used index of lipid
peroxidation in various animal tissue
homogenates. The TBARs is frequently referred
to as malondialdehyde (MDA), which is generated
by autoxidation of the polyunsaturated fatty acids
PUFAs or esters containing three or more double
bonds.
Reduced glutathione (GSH):
The GSH was measured according to Murphy’s
method. (25) This method is based on the reductive
cleavage of Ellman’s reagent (5,5’-dithiobis-2-
nitrobenzoic acid) (DTNB), by SH group of
glutathione to yield a yellow color with a
maximum absorbance at 412 nm.
Superoxide dismutase (SOD) activity:
The activity of SOD was determined by
pyrogallol method of Marklund and Marklund.(26)
This method depends on the spontaneous
autoxidation of pyrogallol in alkaline pH,
resulting in the production of superoxide anion
radicals (O·2¯), which in turn enhance
autoxidation of pyrogallol. Autoxidation is
manifested as an increase in absorbance at 420
nm.
Protein carbonyl contents PCC):
Protein carbonyl content (PCC) was determined
spectrophotometrically by a method based on the
reaction of the carbonyl group with 2,4-
dinitrophenylhydrazine to form 2,4-
dinitrophenylhydrazone (27) (values are expressed
as nanomoles carbonyl/mg protein). Protein
concentration was estimated using the Bio-Rad
bovine serum albumin assay kit, following the
manufacturer’s protocol.
Tumor necrosis factor-α (TNF-α):
Tumor necrosis factor- α (TNF- α) was assayed in
crude liver tissue. This was done by ELISA(28)
using kits commercially available from
eBioscience® Inc., San Diego, USA and according
to the manufacturer’s instructions.
Immunohistochemical examination
Immunohistochemical detection of iNOS in ∼10-
μm-thick liver sections was done by standard
immunohistochemical techniques(29). Briefly, the
sections were incubated for 10 minutes at 80°C in
10 mmol/L sodium citrate buffer (pH 6.0) for
antigen retrieval. Following a 5-minute wash with
PBS, the endogenous peroxidases were blocked
by 1% H2O2 in PBS for 5 minutes. The sections
were washed as before and blocked for 1 hour in
PBS containing 5% normal goat serum. The slides
were washed and then incubated overnight with
primary antibodies (1:100) at 4°C in a humidified
chamber. After washing with PBS, the sections
were incubated with HRP-conjugated secondary
antibody (goat anti-rabbit, 1:200 dilution) for 30
minutes at 37°C. The chromogenic reaction was
developed with 3,3′-diaminobenzidine
tetrahydrochloride solution. Negative control
sections were processed similarly with the
omission of the primary antibodies. All sections
were viewed under a light microscope; 1,000
hepatocytes were analyzed per animal; and results
were expressed as percentage of positive cells.
Statistical Analysis
Quantitative data were described using median as
well as mean ± SD. Comparisons between groups
were performed with the nonparametric Mann–
Whitney test in the case of abnormally distributed
continuous variables, while with Fisher's exact
test for categorical variables. Significance of the
obtained results was judged at the 5% level.
Results
Antioxidant effects during
hepatocarcinogenesis:
Lipid peroxidation in mouse liver
The extent of lipid peroxidation in hepatic tissue
was determined by measuring MDA.
Diethylnitrosamine treatment exhibited a
significant increase (P < 0.05) in the generation of
MDA in mice liver when compared to the control
group. Curcumin (100 mg/kg, i.p), leflunomide
(10 mg/kg, orally) or perindopril (2 mg/kg, orally)
alone and their combination inhibited oxidative
damage during DEN hepatocarcinogenesis as
evidenced from their abilities to prevent DEN-
induced hepatic lipid peroxidation in mice.
Curcumin not only suppressed DEN-induced lipid
peroxidation but also brought the levels more than
normal control group. (Fig. 1-A)
Protein carbonyls in mouse liver
To explore the effects of the different regimens on
the oxidative modifications of hepatic proteins
during DEN hepatocarcinogenesis, we measured
the carbonyl contents of proteins in several
experimental groups. DEN administration
produced a significant (P < 0.01) increase in
protein carbonyls compared with normal animals
(Fig.1-B). Treatment with curcumin (100 mg/kg,
i.p), leflunomide (10 mg/kg, orally) or perindopril
(2 mg/kg, orally) alone and their combination
caused a reduction in DEN-induced increment in
protein carbonyl. Remarkably, the level of protein
carbonyl in the combination group was found to
be slightly less than that of the normal group.
Reduced glutathione (GSH) level in mouse
liver
Reduced GSH level (μ.mole/ g tissue) estimated
in the liver of the different treated groups is
illustrated in (Fig.1-C). A significant decrease in
GSH level was observed in mice treated with
DEN only compared to normal controls. All tested
regimens including leflunomide (10 mg/kg - oral),
curcumin (100 mg/kg - i.p) and perindopril (2
mg/kg - oral) alone and their combination showed
a significant increase in hepatic GSH level
compared to the DEN- treated group. Noticeably,
the combination treated group showed a marked
increase in hepatic GSH that was significantly
different from that of leflunomide, perindopril or
even curcumin which induced the highest
elevation in GSH level among other
monotherapies.
Superoxide dismutase (SOD) activity in mouse
liver
Results concerned with the effect of DEN
administration alone or with the different
preventive regimens on SOD activity in mice
livers, estimated as U/ml, are presented in (Fig.1-
D). The DEN- treated group showed a significant
decrease in SOD activity compared to the control
group. A significant increase in the activity of
hepatic SOD was detected with administration of
curcumin (100 mg/kg, i.p), leflunomide (10
mg/kg, orally) or perindopril (2 mg/kg, orally)
alone as compared to the DEN group. Combined
leflunomide, curcumin and perindopril treatment,
two weeks prior to DEN administration and for 8
consecutive weeks, caused a significant rise in
hepatic SOD activity compared to the DEN group.
The degree of elevation in hepatic SOD activity
caused by the combination treatment was
significantly more than that of perindopril,
leflunomide and even curcumin which showed the
highest increase among other monotherapies.
http://cancerpreventionresearch.aacrjournals.org/content/3/6/753.long#F1
Fig.(1) Effects of leflunomide(10 mg/kg/day, oral), perindopril(2 mg/kg/day, oral) , curcumin (100 mg/kg/every other day, i.p)
and their combination on DEN-induced alterations in the liver of male Albino mice (A) lipid peroxidation (MDA) (B) hepatic
protein carbonyl (C) reduced glutathione (GSH) (D) Superoxide dismutase (SOD) activity. Mice were sacrificed 10 weeks
following the commencement of the study. Each column represents mean ± SD (n = 8 livers). *P < 0.05 as compared to DEN
group; # P < 0.05 as compared to combination group
Anti-inflammatory effects during
hepatocarcinogenesis:
Level of TNF-α in mouse liver
The levels (pg/ml) of TNF-α expressed in mice
livers tissues in different experimental groups are
illustrated in Fig.2. Administration of either
leflunomide (10 mg/kg - oral) , curcumin (100
mg/kg- i.p) or perindopril (2 mg/kg - oral)
resulted in a significant decrease in the level of
hepatic TNF-α in mice compared to the DEN-
treated group. A significant reduction in the level
of hepatic TNF-α expression was noticed in mice
treated with combined curcumin, leflunomide and
perindopril compared to the DEN group.
Although leflunomide showed the marked
decrease in TNF-α level compared to other
monotherapies, its co-administration with
curcumin and perindopril showed a characteristic
significant reduction in hepatic TNF-α, as
measured 8 weeks following DEN administration.
Fig.(2) Effects of leflunomide (10 mg/kg/day, oral),
perindopril(2 mg/kg/day, oral), curcumin (100
mg/kg/every other day, i.p) and their combination on
DEN-induced alterations in the level of tumor necrosis
factor alpha (TNF-α) during hepatocarcinogenesis in
mouse liver. Mice were sacrificed 10 weeks following the
commencement of the study. Each column represents
mean ± SD (n = 8 livers). *P < 0.05 as compared to DEN
group; # P < 0.05 as compared to combination group
0
10
20
30
40
50
60
Control DEN LEF
+ DEN
PE
+ DEN
CUR
+ DEN
Combination
+ DEN
MD
A (µ
mol
/mg
pro
tein
)
*
*#
*#
*# *
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Control DEN LEF
+DEN
PE
+DEN
CUR
+DEN
Combination
+ DEN
Pro
tein
ca
rb
on
yls
(n
an
om
o/m
g p
ro
tein
)
0
1
2
3
4
5
6
7
Control DEN LEF
+ DEN
PE
+ DEN
CUR
+ DEN
Combination
+ DEN
Mea
n G
SH
in li
ver
(μ.m
ole/
g ti
ssu
e)
*
*#*#
*#
*
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Control DEN LEF
+ DEN
PE
+ DEN
CUR
+ DEN
Combination
+ DEN
Mea
n S
OD
in li
ver(
U/m
l)
*
*#
*#
*#
*
0
50
100
150
200
250
300
Control DEN LEF
+ DEN
PE
+ DEN
CUR
+ DEN
Combination
+ DEN
Mea
n h
epa
tic
TN
F –
α (
Pg
/ml)
*
*
*#
*#
*#
*
*# *#
*#
*
A
A
A B
C D
iNOS induction during hepatocarcinogenesis
Minimal hepatic iNOS expression was observed
in normal animals (Fig. 3). Treatment with
curcumin (100 mg/kg, i.p), leflunomide (10
mg/kg, orally) or perindopril (2 mg/kg, orally)
alone and their combination reduced the hepatic
iNOS protein expression in DEN-treated group.
The degree of supression of iNOS over expression
caused by the combination treatment was
significantly more than that of perindopril,
curcumin and even leflunomide which showed the
highest increase among other monotherapies.
A
Fig. (3A) Effects of of leflunomide(10 mg/kg/day, oral), perindopril(2 mg/kg/day, oral) , curcumin (100 mg/kg/every other
day, i.p) and their combination on hepatic iNOS expression during DEN-induced hepatocarcinogenesis in male Albino mice.
A, representative immunohistochemical localization of iNOS (magnification, 100×). Mice were sacrificed 10 weeks following
the commencement of the study, and immunohistochemistry was done to detect iNOS. Arrows indicate immunohistochemical
staining of iNOS. (a) Absence of immunostaining in normal liver, (b) intense immunoreactivity in DEN control liver, (c)
marked decrease iNOS expression in the 100 mg/kg leflunomide group, (d) mild decrease iNOS expression in the 2 mg/kg
perindopril group, (e) moderate decrease iNOS expression in the 2 mg/kg curcumin , and (f)almost normal–appearing liver of
the combination group. Each column represents mean ± SD (n = 8 livers). *P < 0.05 as compared to DEN group; # P < 0.05 as
compared to combination group
a b
c d
e f
Fig. (3B) Effects of leflunomide(10 mg/kg/day, oral), perindopril(2 mg/kg/day, oral) , curcumin (100 mg/kg/every other day,
i.p) and their combination on DEN-induced alterations in hepatic iNOS expression during heptaocarcinogenesis in mice. Mice
were sacrificed 10 weeks following the commencement of the study. Each column represents mean ± SD (n = 8 livers). *P <
0.05 as compared to DEN group; # P < 0.05 as compared to combination group
Discussion
Our previous study showed that combination
treatment with leflunomide, perindopril and
curcumin significantly inhibited DEN-induced
hepatocarcinogenesis using a mouse model (11). It
offered an advantage of more pronounced and
effective chemoprevention, than single agent
treatment, due to distinct complementary
angiopreventive mechanisms. Nevertheless,
additional studies were required to reveal other
mechanisms implicated in the chemopreventive
efficacy of such combination other than
angioprevention. Two main findings are presented
here. First, modulation of oxidative damage and
suppression of inflammation are believed to be
important means of protecting against
hepatocarcinogenesis. Second, the data
corroborate with our previous study suggesting
that combination therapy is superior to single
agent treatment, as it possesses additional
antioxidant and anti-inflammatory properties,
which might play an essential role in protecting
the liver against carcinogen-induced neoplasia.
Data of the present study showed that DEN
administration in mice increased biomarker of
lipid peroxidation (MDA), protein carbonyls, and
reduced antioxidant capacity (GSH content and
SOD activity) in liver homogenates. These
findings, which are consistent with previous
reports(29-32) implicate oxidative stress in DEN-
induced HCC deterioration during
hepatocarcinogenesis in mouse liver. Reactive
oxygen species (ROS) capable of producing lipid
peroxidation and oxidation of DNA and other
cellular macromolecules has a role in the
initiation, promotion, and progression of
hepatocarcinogenesis (33). DEN confers its
hepatocarcinogenicity through metabolic
activation in hepatic microsomes, resulting in the
release of ethylcarbonium ions that bind to DNA,
producing adducts and generating superoxide
radicals through lipid peroxidation of
phospholipid membrane fatty acids(34).
Malondialdehyde (MDA), a product of lipid
peroxidation of polyunsaturated fatty acid
metabolism and degradation, has been established
as a mutagenic and carcinogenic entity (31).
Further,it has been established that ROS could
modify the chemical structure of proteins with
formation of protein carbonyls due to oxidative
cleavage of the main peptide backbone or by
0
20
40
60
80
100
120
140
160
180
200
Control DEN LEF
+ DEN
PE
+ DEN
CUR
+ DEN
Combination
+ DEN
iNO
S p
rote
in e
xp
ress
ion
*
*#
*#
*#
*
oxidation of amino acids, including arginine,
lysine, proline, and threonine (35). Protein carbonyl
content has been the most commonly used marker
of protein oxidation. It was reported that oxidative
stress elevates the protein carbonyl content in
plasma of HCC patients (36). Our results showed
that pretreatment of DEN-induced HCC mice with
curcumin, perindopril or leuflunomide reduced
elevated levels of MDA and protein carbonyl and
increased GSH content and SOD activity.
Curcumin, a dietary polyphenol, has been
reported to possess anti-inflammatory and
antioxidant properties (37). Supporting our
observations, Singh et al.(38) has shown that
curcumin increases glutathione S-transferase
activity which is involved in the synthesis of
GSH; suggesting that curcumin increases de novo
synthesis of glutathione. Similarly, curcumin has
been reported (39, 40) to cause a beneficial increase
in hepatic SOD activity. Accumulating evidence
has shown that curcumin counteracted ROS by
increasing ornithine decarboxylase, glutathione,
antioxidant enzymes, and phase II metabolizing
enzymes and thus protected the liver from
oxidative damage induced by DEN
administration(41). Curcumin inhibited superoxide
anion generation in xanthine– xanthine oxidase
system as well as hydroxyl ion generation (42). It
has been reported that curcumin possesses both
neuroprotective and anti-aging effects (43). One of
the most important cellular defense mechanisms
against oxidative stress or electrophiles is
mediated by the transcription factor Nrf2 (43).
Thus, curcumin can counteract the effect of
oxidative stress through this pathway and help in
avoiding an oxidative damage. Therefore, our data
are in line with previous studies signifying that
potent attenuation of oxidative stress could be
involved in the observed chemopreventive action
of curcumin in our previous study (11). As regards
perindopril, our results provide an experimental
evidence of the antioxidant potential of
perindopril which could play an important role in
the inhibition of HCC in mice. Published data
with regards to the antioxidant properties of of
angiotensin-converting enzyme inhibitors are
controversial. Perindopril not only lowers blood
pressure in elderly patients but also restores pro-
and anti-oxidative homeostasis in order to
increase anti-oxidative defenses (44). By contrast, it
was found that only captopril, among ACEIs, that
demonstrated antioxidant activity[23]. Published
data highlight the antioxidative effect of
leflunomide. Several studies demonstrated that
leflunomide decreased the production of MDA
and PC and increased antioxidant activity in liver
injury induced by ischemia reperfusion or biliary
obstruction (45, 46). Similarly, Manna et al. (47)
showed that leflunomide suppressed TNF-induced
reactive oxygen intermediate generation and lipid
peroxidation. Yao et al. (48, 49) demonstrated that
leflunomide significantly decreased
proinflammatory cytokines and MDA and
increased antioxidant activity in immunological-
and CCl4- induced liver injury, respectively. The
antioxidant activity of leflunomide could be
attributed, at least in part, to its anti-inflammatory
effect and its ability to inhibit dihydroorotate
dehydrogenase enzyme. This explanation is
supported by the ability of teriflunomide (non-
enzymatic metabolite of leflunomide) to inhibit
mitochondrial oxygen consumption and ROS
production in premalignant and malignant prostate
epithelial cells due to inhibition of dihydroorotate
dehydrogenase enzyme (50). Further studies are
needed to explain detailed mechanism of its
antioxidant activity. Remarkably, pretreatment
with the combination of leflunomide, perindopril
and curcumin restored surrogate measures of the
oxidative stress to near- control levels. Such
potent antioxidant activity would presumably be
related to the antioxidant effect of curcumin
which had been the greatest among the probed
drugs. Oxidative and inflammatory insults are
intimately connected with each other in multistage
carcinogenesis. During recent years, compelling
evidence strongly implicates the role of
inflammation in initiation, promotion, and
progression of HCC (2). Inflammatory
environment is one that would support tumor
development and is consistent with that observed
in tumor sites (52, 53). It is believed that
inflammation acts as a key regulator in the
promotion of initiated hepatocytes during
hepatocarcinogenesis, possibly by providing them
with proliferating signals and/or preventing
apoptosis (54, 55). Two major contributors of
chronic inflammatory reactions are NO and TNF-
α. Nitric oxide is produced by hepatic
parenchymal as well as nonparenchymal cells
from l-arginine by iNOS. Oxidative stress is
known to increase iNOS gene transcription and
promoter activity in hepatocytes (56). Mounting
evidence underlines the important role that iNOS
plays in the development and progression of HCC
as this enzyme has been found to be
overexpressed in rodents as well as human HCC (57, 58). Calvisi et al. (58) have shown that
suppression of iNOS by aminoguanidine, a
selective iNOS inhibitor, leads to suppression of
HCC growth, suggesting that iNOS signaling
could be an important target for prevention and
treatment of human HCC. In the present study, we
have observed an elevated level of hepatic iNOS
expression in DEN-treated animals, confirming
the results of prior studies (57, 59). Tumor necrosis
factor-α acts as a master switch in establishing an
intricate link between inflammation and cancer. It
is also produced by tumors and can act as an
endogenous tumor promoter(60). The hepatic level
of TNF-α was significantly increased in mice
receiving DEN only compared to normal controls.
This result revealed the role of TNF-α as an
inflammatory mediator in DEN-induced
hepatocarcinogenesis, where DEN administration
contributes to acute hepatitis, a pronounced
Kupffer cell response, hepatocyte proliferation
and DNA damage, ultimately leading to HCC
(61.62). Data of the present study showed that
pretreatment with leflunomide, perinopril or
curcumin caused a significant reduction of TNF-α
and iNOS expression in mice liver compared to
the DEN animals. Remarkably, the decrease in
TNF-α and iNOS expression as more pronounced
in the leflunomide group. Similar effects have
been previously reported, where leflunomide
significantly suppressed hepatic TNF-α mRNA
expression in vivo(63) and inhibited TNF-α
released in Kupffer cells, following liver inj
