Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats
Aim: To study the effect of silymarin on the levels of tumor markers and MDA (malondialdehyde) – DNA adduct formation during N-nitrosodiethylamine induced hepatocellular carcinoma in male Wistar albino rats. Methods: The levels of AFP, CEA and activities of liver marker enzymes in serum, MDA-DNA imm...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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irk-123456789-1385642018-06-20T03:04:30Z Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats Ramakrishnan, G. Augustine, T.A. Jagan, S. Vinodhkumar, R. Devaki, T. Original contributions Aim: To study the effect of silymarin on the levels of tumor markers and MDA (malondialdehyde) – DNA adduct formation during N-nitrosodiethylamine induced hepatocellular carcinoma in male Wistar albino rats. Methods: The levels of AFP, CEA and activities of liver marker enzymes in serum, MDA-DNA immunohistochemistry were done according to standard procedures in the control and experimental groups of rats. Results: Hepatocellular carcinoma was evidenced from significant (p < 0.05) increases of alpha-fetoprotein, carcinoembryonic antigen, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, acid phosphatase, lactate dehydrogenase, gamma-glutamyltransferase and 5?-nucleotidase in serum and increased MDA-DNA adducts were also observed in the tissue sections of hepatocellular carcinoma. Silymarin treatment significantly attenuated the alteration of these markers and decreased the levels of MDA-DNA adduct formation. Conclusion: Silymarin could be developed as a promising chemotherapeutic adjuvant for the treatment of liver cancer. Цель: изучить влияние силимарина на уровень экспрессии опухолевых и биохимических маркеров и формирование аддуктов малонового диальдегида с ДНК (MDA-DNA) при развитии гепатокариномы у крыс линии истар. Методы: стандартными биохимическими методами определяли активность ферментов в сыворотке крови и проводили иммуногистохимическое определение MDA-DNA в ткани печени крыс. Результаты: показано, что при развитии злокачественной гепатокарциномы в сыворотке крови животных значительно увеличивается количество альфа-фетопротеина, раковоэмбрионального антигена, активность аспартат- и аланинаминотрансферазы, щелочной и кислой фосфатазы, лактатдегидрогеназы, гаммаглутамилтрансферазы и 5-нуклеотидазы. При проведении иммуногистохимического исследования отмечали повышенное образование аддуктов MDA-DNA в ткани печени крыс со злокачественной гепатокариномой. При введении силимарина значительно снижался уровень указанных ферментов в сыворотке крови и формирование аддуктов MDA-DNA в ткани печени. Заключение: применение силимарина может быть эффективно для предупреждения развития злокачественной гепатокарциномы, индуцированной N-нитрозодиэтиламином у крыс, и этот препарат может быть многообещающим химиотерапевтическим адъювантом для лечения рака печени. 2007 Article Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats / G. Ramakrishnan, T.A. Augustine, S. Jagan, R. Vinodhkumar, T. Devaki // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 39-44, . — Бібліогр.: 45 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138564 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Ramakrishnan, G. Augustine, T.A. Jagan, S. Vinodhkumar, R. Devaki, T. Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats Experimental Oncology |
| description |
Aim: To study the effect of silymarin on the levels of tumor markers and MDA (malondialdehyde) – DNA adduct formation during N-nitrosodiethylamine induced hepatocellular carcinoma in male Wistar albino rats. Methods: The levels of AFP, CEA and activities of liver marker enzymes in serum, MDA-DNA immunohistochemistry were done according to standard procedures in the control and experimental groups of rats. Results: Hepatocellular carcinoma was evidenced from significant (p < 0.05) increases of alpha-fetoprotein, carcinoembryonic antigen, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, acid phosphatase, lactate dehydrogenase, gamma-glutamyltransferase and 5?-nucleotidase in serum and increased MDA-DNA adducts were also observed in the tissue sections of hepatocellular carcinoma. Silymarin treatment significantly attenuated the alteration of these markers and decreased the levels of MDA-DNA adduct formation. Conclusion: Silymarin could be developed as a promising chemotherapeutic adjuvant for the treatment of liver cancer. |
| format |
Article |
| author |
Ramakrishnan, G. Augustine, T.A. Jagan, S. Vinodhkumar, R. Devaki, T. |
| author_facet |
Ramakrishnan, G. Augustine, T.A. Jagan, S. Vinodhkumar, R. Devaki, T. |
| author_sort |
Ramakrishnan, G. |
| title |
Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats |
| title_short |
Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats |
| title_full |
Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats |
| title_fullStr |
Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats |
| title_full_unstemmed |
Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats |
| title_sort |
effect of silymarin on n-nitrosodiethylamine induced hepatocarcinogenesis in rats |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2007 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138564 |
| citation_txt |
Effect of silymarin on N-nitrosodiethylamine induced hepatocarcinogenesis in rats / G. Ramakrishnan, T.A. Augustine, S. Jagan, R. Vinodhkumar, T. Devaki // Experimental Oncology. — 2007. — Т. 29, № 1. — С. 39-44, . — Бібліогр.: 45 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT ramakrishnang effectofsilymarinonnnitrosodiethylamineinducedhepatocarcinogenesisinrats AT augustineta effectofsilymarinonnnitrosodiethylamineinducedhepatocarcinogenesisinrats AT jagans effectofsilymarinonnnitrosodiethylamineinducedhepatocarcinogenesisinrats AT vinodhkumarr effectofsilymarinonnnitrosodiethylamineinducedhepatocarcinogenesisinrats AT devakit effectofsilymarinonnnitrosodiethylamineinducedhepatocarcinogenesisinrats |
| first_indexed |
2025-07-10T06:04:08Z |
| last_indexed |
2025-07-10T06:04:08Z |
| _version_ |
1837238793693822976 |
| fulltext |
Experimental Oncology ���� ������� ����� ��arc��� ������ ������� ����� ��arc��� ���arc��� ���� �� ��
Hepatocellular carcinoma �HCC�� is t�e most frequent
primary malignancy of t�e liver and accounts for as
many as one million deat�s worldwide in a year. In some
parts of t�e world it is t�e most common form of internal
malignancy and t�e most common cause of deat� from
cancer [1]. Well-known risk factors of �epatocellular
carcinoma includes �epatitis B virus �HBV���� �epatitis C
virus �HCV���� aflatoxins�� alco�ol and oral contraceptives.
Smoking�� androgenic steroids and diabetes mellitus
are also suspected risk factors [�]. One approac� to
control liver cancer is c�emoprevention — w�en disease
is prevented�� slowed or reversed substantially by t�e
administration of one or more non-toxic naturally occur-
ring or synt�etic agents. In t�is regard�� recently naturally
occurring polyp�enols are receiving increased attention
because of t�eir promising efficacy in several cancer
models [�]. Silymarin is one of suc� naturally occurring
compounds isolated from Silybum marianum�� w�ic� �as
s�own to �ave significant anticancer effect on several
cancers bot� in vitro and in vivo [���].
Classically�� a marker is synt�esized by t�e tumor and
released into t�e circulation�� but it may be produced by
normal tissues in response to invasion by cancer cells
[1�]. A variety of substances�� including enzymes�� �or-
mones�� antigens�� and proteins may be considered as
tumor markers. Analysis of tumor markers can be used
as an indicator of tumor response to t�erapy. Sensitive
and specific liver cancer marker enzymes are used
as indicators of liver injury. Analysis of t�ese marker
enzymes reflects mec�anisms of cellular damage
and subsequent release of proteins and extracellular
turnover [11]. Lipid peroxidation generates a complex
variety of products�� many of w�ic� are reactive electro-
p�iles some of t�ese react wit� protein and DNA and as
a result are toxic and mutagenic [1�]. �alondialde�yde
��DA�� is one of products of lipid peroxidation t�at reacts
wit� DNA to produce �DA-DNA adducts�� w�ic� �ave
been implicated in t�e induction of G→T transversions
and A→G transitions [1�]. T�e ability of �DA-DNA ad-
ducts to induce frame s�ift mutations in sequences for
genetic instability is emerging as a possible direct link
between oxidative stress and �uman cancers [1��� 15].
T�us�� t�e purpose of present study is to evaluate t�e
effect of silymarin on t�e level of tumor markers�� and
�DA-DNA adducts formation during N-nitrosodiet�yl-
amine induced �epatocellular carcinoma in rats.
MATERIALS AND METHODS
Animals. Wistar male rats weig�ing about 15��18� g
were obtained from Tamilnadu Veterinary & Animal Sci-
ence University �TANUVAS���� �ad�avaram�� C�ennai��
India. T�e animals were �oused in cages under proper
environmental conditions and were fed wit� a commer-
cial pelletted diet ��/s Hindustan foods Ltd.�� Bangalore��
India��. T�e animals �ad free access to water.
Chemicals. N-nitrosodiet�ylamine �NDEA�� and
silymarin were manufactured by Sigma c�emical Co.��
�St. Louis�� �O�� USA��. All ot�er c�emicals used were
from SRL ��umbai�� India��.
Experimental design. T�e experimental animals
were divided into five groups �as s�own in Fig. 1���� 6 ani-
mals per group. Rats from group 1 �normal control��
were fed wit� standard diet and pure drinking water;
in group � �epatocellular carcinoma was induced
by providing �.�1% NDEA t�roug� drinking water
for 15 weeks as described in [16]; rats from group
� were treated wit� 1��� ppm silymarin alone in diet
for 16 weeks; rats from group � were pretreated wit�
1��� ppm silymarin one week before t�e administra-
tion of �.�1% NDEA and received it till t�e end of t�e
experiment �i.e. 16 weeks��; rats from group 5 were post
treated wit� 1��� ppm silymarin for 5 weeks after t�e
administration of NDEA for 1� weeks and received it till
EFFECT OF SILYMARIN ON N-NITROSODIETHYLAMINE INDUCED
HEPATOCARCINOGENESIS IN RATS
G. Ramakrishnan, T.A. Augustine, S. Jagan, R. Vinodhkumar, T. Devaki*
Department of Biochemistry, University of Madras, Tamilnadu, India
Aim: To study the effect of silymarin on the levels of tumor markers and MDA (malondialdehyde) – DNA adduct formation during
N-nitrosodiethylamine induced hepatocellular carcinoma in male Wistar albino rats. Methods: The levels of AFP, CEA and activities
of liver marker enzymes in serum, MDA-DNA immunohistochemistry were done according to standard procedures in the control and
experimental groups of rats. Results: Hepatocellular carcinoma was evidenced from significant (p < 0.05) increases of alpha-fetoprotein,
carcinoembryonic antigen, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, acid phosphatase, lactate
dehydrogenase, gamma-glutamyltransferase and 5´-nucleotidase in serum and increased MDA-DNA adducts were also observed
in the tissue sections of hepatocellular carcinoma. Silymarin treatment significantly attenuated the alteration of these markers and
decreased the levels of MDA-DNA adduct formation. Conclusion: Silymarin could be developed as a promising chemotherapeutic
adjuvant for the treatment of liver cancer.
Key Words: silymarin, N-nitrosodiethylamine, hepatocellular carcinoma, flavonoids, AFP, MDA-DNA.
Received: December 18, 2006.
*Correspondence: Fax: 91-44-22352494
E-mail: devakit@yahoo.co.uk
Abbreviations used: AFP — alpha-fetoprotein; ACP – acid
phosphatase; ALP – alkaline phosphatase; ALT – alanine aminotrans-
ferase; AST – aspartate aminotransferase; CEA – carcinoembryonic
antigen; γ-GT – gamma-glutamyltransferase; HCC – hepatocellular
carcinoma, LDH – lactate dehydrogenase; MDA – malondialdehyde,
5�NT – 5�-nucleotidase; NDEA – N-nitrosodiethylamine.
Exp Oncol �����
���� 1�� �����
�� Experimental Oncology ���� ������� ����� ��arc���
t�e end of experiment. After t�e stipulated experimen-
tal period t�e rats were anaest�etized wit� diet�yl et�er
followed by cervical decapitation. T�e experiments are
performed after t�e approval of t�e Institutional Animal
Et�ics Committee IAEC No. �1/���/�6.
Fig. 1. Experimental protocol
Analysis of alpha-fetoprotein (AFP) & car-
cinoembryonic antigen (CEA). AFP and CEA were
measured in blood serum by c�emiluminescent im-
munoassay �Fully Automated ADVIA Centaur�� Bayer
U.S.A. c�emiluminescence system��.
Biochemical studies. T�e blood samples were
collected from t�e experimental animals�� liver tissue
was removed and was�ed in ice-cooled saline�� and tis-
sues were c�illed in a beaker on cracked ice for 5 min��
and t�en minced wit� s�arp scissors. 1�% �omog-
enate was prepared in ice-cooled �.1 � Tris-HCl buffer
�pH ��.���. Standard procedures were used to assay t�e
various bioc�emical parameters. Protein content was
estimated by Lowry et al. [1��]; activity of enzymes was
valued as described elsew�ere �aminotransferases
�AST�� ALT�� — [18]�� p�osp�atases �ACP�� ALP�� — [1�]��
lactate de�ydrogenase — [��]�� gamma-glutamyltrans-
ferase — [�1]�� 5�-nucleotidase — [��]��.
Immunohistochemical staining of MDA-DNA ad-
ducts. Immuno�istoc�emistry for �DA-DNA adducts
was carried out according to Z�ang et al. [��]. Briefly��
tissue sections were deparraffinized in two c�anges of
xylene at 6� ̊ C and re�ydrated t�roug� a graded series
of alco�ols. T�en t�e slides were was�ed in 1 x PBS��
treated wit� RNase �1�� µl/ml�� at ��� ˚C for 1 ��� was�ed
wit� 1 x PBS�� treated wit� proteinase K �1� µg/ml�� at
room temperature for 1� min and was�ed. To dena-
ture t�e DNA�� t�e slides were incubated wit� � N HCl
for 1� min and t�en was�ed wit� 5� m� Tris base for
5 min�� bot� at room temperature. After was�ing wit� 1 x
PBS�� slides were incubated wit� �.�% H�O� in met�anol
at room temperature for �� min. Non-specific binding
was blocked wit� �% BSA and t�e slides were incubated
overnig�t at � ˚C wit� anti-�DA monoclonal antiserum
number D1�A1 �Dr. P. Srinivasan�� Korea Atomic Energy
Researc� Institute�� Korea��. T�e slides were was�ed in
PBS and t�en incubated wit� anti-mouse HRP labelled
secondary antibody �Genei�� Bangalore�� India�� for 1 � at
room temperature. T�e peroxidase activity was visuali-
zed by treating slides wit� �����-diaminobenzidine tet-
ra�ydroc�loride �SRL�� �umbai�� India���� t�e slides were
slig�tly counterstained wit� �eyer’s �ematoxylin. T�e
labeling index was expressed as number of cells wit�
positive staining per 1�� counted cells in five randomly
selected fields at t�e magnification of objective �� x
under lig�t microscope.
Statistical analysis. Data were evaluated wit�
SPSS/1� software. Hypot�esis testing met�ods in-
cluded one way analysis of variance �ANOVA�� followed
by least significant difference �LSD�� test. Statistical
significance was defined as P values less t�an �.�5. All
results were expressed as mean ± standard deviation.
RESULTS
T�e significant increase in t�e levels of AFP and
CEA in t�e serum was observed in group � as com-
pared wit� group 1 �Fig. ���. Significant decrease of
t�e level of t�ese tumor markers was demonstrated
in t�e silymarin-treated animals from groups � and 5
as compared wit� group �.
Fig. 2. Effect of silymarin on t�e levels of AFP and CEA in t�e serum
of control and experimental groups of animals �n = 6 per group��. Re-
sults are expressed as mean ± SD. p < �.�5 compared wit� agroup1��
bgroup ��� cgroup ��� dgroup 5. Units: IU/mL for AFP�� ng/mL for CEA
Table. Effect of silymarin on the levels of AST, ALT, ACP, ALP, LDH, GGT and 5�NT in the serum of control and experimental group of animals
Groups AST ALT ACP ALP LDH GGT 5’NT
Group 1 3.90 ± 0.44 25.3 ± 2.85 27.23 ± 2.37 145.6 ± 16.6 1.42 ± 0.16 1.47 ± 0.17 3.09 ± 0.36
Group 2 7.58 ± 0.85acd 47.51 ± 4.99acd 48.8 ± 5.56acd 283.9 ± 32.37acd 2.48 ± 0.28acd 2.92 ± 0.29acd 6.19 ± 0.71acd
Group 3 3.82 ± 0.48 24.91 ± 2.83 26.24 ± 2.25 143 ± 16.3 1.4 ± 0.16 1.45 ± 0.16 3.08 ± 0.35
Group 4 5.3 ± 0.60abd 31.6 ± 3.60abd 33.42 ± 3.69abd 196 ± 22.30abd 1.71 ± 0.19abd 1.91 ± 0.21abd 4.31 ± 0.49abd
Group 5 6.42 ± 0.73abc 38.9 ± 4.43abc 40.7 ± 4.64abc 241 ± 28.4abc 2.09 ± 0.24abc 2.39 ± 0.27abc 5.15 ± 0.57abc
Note: Results are expressed as mean ± SD, (n = 6); p < 0.05 compared with agroup 1, bgroup 2, cgroup 4, dgroup 5. Units: µmoles of pyruvate liberated
mg protein per min for AST, ALT and LDH; µmoles of phenol liberated mg protein per min ACP and ALP; nmoles of p-nitroaniline formed mg protein per min
for GGT; nmoles of Pi liberated mg protein per min for 5�NT.
Experimental Oncology ���� ������� ����� ��arc��� �1���� ������� ����� ��arc��� �1�arc��� �1�� �1 �1
Animals from group � ex�ibited a significant in-
crease in t�e activity of AST�� ALT�� ACP�� ALP�� LDH�� GGT
and 5�NT in blood serum as compared wit� group 1
�Table��. Silymarin-treated animals from groups � and
5 s�owed a significant decrease in t�e levels of t�ese
enzymes w�en compared wit� group � �animals wit�
NDEA-induced �epatocarcinoma��. T�e activity of AST
and ALT was significantly decreased in t�e liver tissue
of animals from group � as compared wit� group 1
�Fig. ����� silymarin-treated animals �groups � and 5��
s�owed a significant increase in t�e level of transami-
nases as compared wit� group �. T�e activity of ACP��
ALP�� LDH�� GGT and 5�NT was significantly increased in
t�e liver tissue of animals from group � as compared
wit� group 1. T�ere was a significant decrease in t�e
activity of t�ese enzymes in silymarin-treated groups
as compared wit� group �.
Fig. 3. Effect of silymarin on t�e activity of AST�� ALT�� ACP�� ALP�� LDH��
GGT and 5’NT in t�e liver of control and experimental groups of
animals �n = 6 per group��. Results are expressed as mean ± SD. p
< �.�5 compared wit� agroup 1�� bgroup ��� cgroup ��� dgroup 5. Units:
µmoles of pyruvate liberated mg protein per min for AST�� ALT and
LDH; µmoles of p�enol liberated mg protein per min for ACP and
ALP; nmoles of p-nitroaniline formed mg protein per min for GGT;
nmoles of Pi liberated mg protein per min for 5�NT
As it was s�own by immuno�istoc�emical analysis
of liver sections�� t�e �DA-DNA adducts were ob-
served in �% of �epatocytes in normal control animals
�Fig. ��� a��. T�e rate of �epatocytes w�ic� were positive
to �DA-DNA adducts was ��%�� �.�6%�� 18% & ��% in
t�e liver sections from NDEA-induced �epatocarcinoma
�Fig. ��� b���� silymarin alone �Fig. ��� c���� silymarin pretreat-
ed group �Fig. ��� d���� and silymarin post-treated animals
�Fig. ��� e�� respectively. T�e grap�ical representation of
% positive cells for �DA-DNA is s�own in Fig. ��� f.
DISCUSSION
During carcinogenesis�� some enzymes can be used
as an bioc�emical indicators of tumor response to
t�erapy [11]. Hepatospecific enzymes were activated
w�en �epatocellular damage gave rise to abnormali-
ties of liver function and t�ese enzymes are remarkably
increased in HCC. AST and ALT activities in blood serum
are generally accepted as an index of liver damage and
t�is tendency is also known to be distinct in rodents [16].
T�ere was a good correlation between t�e activities of
ALT and AST wit� tumor volume during t�erapy. Rocc�i
et al. [��] reported t�at t�ere was an increase in t�e levels
of t�ese transaminases activity in serum of HCC patients.
In concurrent wit� t�e above findings an elevated serum
aminotransferase activities were observed in animals
bearing HCC wit� simultaneous decrease in t�e liver
tissue; silymarin treatment significantly attenuated t�is
alteration t�ereby s�owing its anticarcinogenic activity.
Elevation of alkaline p�osp�atase is one of t�e signs��
suggesting space-occupying lesions in t�e liver. An in-
creased activity of ACP and ALP was seen in blood serum
and liver of animals wit� HCC�� t�is may be due to t�e
disturbance in secretory activity or due to altered gene
expression in t�ese conditions. Development of tumor
results in tissue damage t�at lead to t�e release of ALP
into circulation [�5] and t�is enzyme level �ave been el-
evated in blood serum and liver tissue of t�e tumor-bear-
ing animals and t�is elevation is significantly suppressed
by t�e supplementation of silymarin in diet. GGT �as
been s�own to play an important role in t�e metabolism
of foreign substances and also during cell growt� and
differentiation [�6] and is overexpressed in tumor cells
resistant to t�erapeutic drugs [���]. Experimental stud-
ies �ave s�own t�at GGT was strikingly activated during
t�e course of �epatocarcinogenesis induced by several
�epatocarcinogens in animals [�8]; c�emical carcino-
gens may initiate some systematic effects t�at induce
GGT synt�esis [��]. T�is elevation reflects t�e progress
of carcinogenesis�� since its activity correlates wit� tumor
growt� rate�� differentiation and survival of t�e �ost [��];
in concurrent wit� above findings t�ere was an increase
in t�e levels of GGT in t�e serum and liver of animals
bearing HCC. T�is elevation indicates t�e basic tumor
burden�� and silymarin treatment significantly decreased
t�e elevation of t�e level of t�is enzyme.
5�nucleotidase was found to be elevated in t�e
animals wit� solid tumors [�1]. T�e increased activ-
ity of t�is enzyme seems to �ave originated from t�e
proliferating tumor cells [��]. Elevated activities of
5�nucleotidase in carcinoma of liver and leukemia were
reported [11�� ��]. In our study correlatively increased
activities of 5�nucleotidase were observed in blood
serum and liver of t�e carcinogen administered ani-
mals�� and t�is elevation is significantly in�ibited in t�e
animals treated wit� silymarin.
LDH is a fairly sensitive marker of solid neoplasm
[��] and very �ig� LDH levels correlate wit� treatment
failure [�5]; numerous reports revealed increased
LDH activity in various types of tumors [1��� �6]. T�e
elevated levels of LDH may be due to its overproduc-
tion by tumor cells. Proliferating malignant cells ex�ibit
very �ig� rates of glycolysis�� w�ic� subsequently lead
to elevated LDH activity [���]. T�e results of t�e pres-
ent study are in agreement wit� literature data and
s�ow elevated levels of LDH in blood serum and liver
of t�e NDEA administered rats�� and t�is elevation was
attenuated in silymarin-treated rats.
Elevation of serum AFP levels �as been reported in
several diseases including HCC [�8]. AFP along wit� CEA
is most extensively used in t�e diagnosis of HCC [���� ��].
In our study also t�ere was an increased level of AFP and
CEA in t�e carcinogen administered animals confirming
t�e presence of HCC�� and silymarin treatment signifi-
cantly reduced t�e elevation of bot� AFP and CEA.
�� Experimental Oncology ���� ������� ����� ��arc���
�DA is a �ig�ly reactive electrop�ile�� capable of
interacting wit� DNA to form �DA-DNA adducts [�1]��
t�at induce frame s�ift and base-pair substitution muta-
tions [1�]. T�e level of �DA-DNA adducts is found to be
increased in several cancers [����5]. Recent evidence
suggests t�at oxidative stress may contribute to genetic
instability and promote tumor progression [1��� 15]. In
t�e present study t�e levels of �DA-DNA adducts are
increased in HCC-bearing animals�� and pre- and post-
silymarin treatment significantly reduced t�e formation
of �DA-DNA adduct. So�� we can conclude t�at silymarin
could be developed as a promising c�emot�erapeutic
adjuvant for t�e treatment of liver cancer.
ACkNOwLEDGEMENTS
T�e aut�ors like to t�ank Dr. P. Srinivasan�� Korea
Atomic Energy Researc� Institute�� Sout� Korea for
providing �DA-DNA primary antibody.
Fig. 4. Immuno�istoc�emical staining of liver sections for �DA-DNA adducts: a — group 1; b — group �; c — group �; d — group
�; e — group 5; �f�� representative grap� of % �DA-DNA positive cells. �agnification �� x
Experimental Oncology ���� ������� ����� ��arc��� ������ ������� ����� ��arc��� ���arc��� ���� �� ��
REFERENCES
1. Befler AS, Di Bisceglie AM. Hepatocellular carcinoma: di-
agnosis and treatment. Gasteroenterology 2002; 122: 1609–19.
2. Chang CK, Astrakianakis G, Thomas DB, Seixas NS,
Ray RM, Gao DL, Wernli KJ, Fitzgibbons ED, Vaughan TL,
Checkoway H. Occupational exposure and risks of liver cancer
among Shanghai female textile workers – a case cohort study.
Int J Epidemiol 2006; 35: 361–9.
3. Tyagi AK, Agarwal C, Singh RP, Shroyer KR, Glode LM,
Agarwal R. Silibinin down-regulated surviving protein and
mRNA expression and causes caspases activation and apop-
tosis in human bladder transtitional-cell papilloma RT4 cells.
Biochem Biophys Res Comm 2006; 312: 1178–84.
4. Singh RP, Dhanalakshmi S, Tyagi AK, Chan DC,
Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits
advance human prostate carcinoma growth in athymic nude
mice and increases plasma insulin-like growth factor-binding
protein-3 levels. Cancer Res 2003; 62: 3063–9.
5. Zi X, Feyes DK, Agarwal R. Anticarcinogenic effect of a
flavonoid antioxidant, silymarin, in human breast cancer cells
MDA-MB 468: induction of G1 arrest through an increase in
Cip1/p21 concomitant with a decrease in kinase activity of
cyclin-dependent kinases and associated cyclins. Clin Cancer
Res 1998; 4: 1055–64.
6. Chatterjee ML, Katiyar SK, Mohan RR, Agarwal R.
A flavonoid antioxidant silymarin, affords exceptionally high
protection against tumor promotion in the SENCAR mouse
skin tumorigenesis model. Cancer Res 1999; 59: 622–32.
7. Kohno H, Tanaka T, Kawabata K, Hirose Y, Sugie S,
Tsuda H, Mori H. Silymarin, a naturally occurring polyphenolic
antioxidant flavonoid, inhibits azoxymethane induced colon car-
cinogenesis in male F344 rats. Int J Cancer 2002; 101: 461–8.
8. Yanaida Y, Kohno H, Yoshida K, Hirose Y, Yamada Y,
Mori H, Tanaka T. Dietary silymarin suppresses 4-nitroquino-
line 1-oxide-induced tongue carcinogenesis in male F344 rats.
Carcinogenesis 2002; 23: 787–94.
9. Vinh PQ, Sugie S, Tanaka T, Hara A, Yamada Y, Ka-
tayama M, Deguchi T, Mori H. Chemopreventive effects of a
flavonoid antioxidant silymarin on N-butyl-N- (4-hydroxy-
butyl) nitrosamine-induced urinary bladder carcinogenesis in
male ICR mice. Jpn J Cancer Res 2002; 93: 42–9.
10. Thangaraju M, Rameshbabu J, Vasavi H, Ilanchezian S,
Vinitha R, Sachdanandam P. The salubrious effect of tamoxifen on
serum marker enzymes, glycoproteins, lysosomal enzyme level in
breast cancer women. Mol Cell Biochem 1998; 185: 85–94.
11. Thirunavukkarasu C, Sakthisekaran D. Sodium selenite
modulates tumour marker indices in N-nitrosodiethylamine
initiated and phenobarbital-promoted rat liver carcinogenesis.
Cell Biochem Funct 2003; 21: 147–53.
12. Marnett LJ. Lipid peroxidation-DNA damage by
malondialdehyde. Mut Res 1999; 424: 83–95.
13. Benamira M, Johnson K, Chaudhary A, Bruner K,
Tibbetts C, Marnett LJ. Induction of mutations by replication
of malondialdehyde-modified M13 DNA in Escherichia coli:
determination of the extent of DNA modification, genetic
requirements for mutagenesis, and types of mutations induced.
Carcinogenesis 1995; 16: 93–9.
14. Valko M, Izkovic M, Mazur M, Rhodes CJ, Telser J.
Role of oxygen radicals in DNA damage and cancer incidence.
Mol Cell Biochem 2004; 266: 37–56.
15. Zienolddiny S, Ryberg D, Haugen A. Induction of
microsatellite mutations by oxidative agents in human lung
cancer cell lines. Carcinogenesis 2000; 21: 1521–6.
16. Ha WS, Kim CK, Sung SH, Kang CB. Study on the
mechanism of multistep hepatotumorigenesis in rat: develop-
ment of hepatotumorigenesis. J Vet Sci 2001; 2: 53–8.
17. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ.
Protein measurement with the Folin-phenol reagent. J Biol
Chem 1951; 193: 265–75.
18. King J. The transferase-alanine and aspartate transami-
nase. In: Practical Clinical Enzymology. King J, ed. London:
D Van Nostrand Company Ltd. 1965; 121–38.
19. King J. The phosphohydrolases-acid and alkaline
phosphatase. In: Practical Clinical Enzymology. King J, ed.
London: D Van Nostrand Company Ltd. 1965; 191–208.
20. King J. The dehydrogenases or oxido reductase-lactate
dehydrogenase. In: Practical Clinical Enzymology. King J, ed.
London: D Van Nostrand Company Ltd. 1965; 83–93.
21. Rosalki SB, Rao D. Serum glutamyl transpeptidase
activity in alcoholism. Clin Chim Acta 1972; 39: 41–7.
22. Luly P, Barnabei O, Tria E. Hormonal control in vitro
of plasma membrane bound Na+, K+-ATPase of rat liver.
Biochim Biophys Acta1972; 282: 447–52.
23. Zhang Y, Chen SY, Hsu T, Santella RM. Immunohis-
tochemical detection of malondialdehyde-DNA adducts in
human oral mucosa cells. Carcinogenesis 2002; 23: 207–11.
24. Rocchi E, Seium Y, Camellini L, Casalgrandi G,
Borghi A, D’Alimonte P, Cioni G. Hepatic tocopherol content
in primary hepatocellular carcinoma and liver metastases.
Hepatology 1997; 26: 67–72.
25. Iqbal J, Minhajuddin M, Beg ZH. Suppression of
diethylnitrosamine and 2-acetylaminoflurene induced hepa-
tocarcinogenesis in rats by tocotrienol rich fraction isolated
from rice bran oil. Eur J Can Prev 2004; 13: 515–20.
26. Thusu N, Raina PN, Johri RK. γ-Glutamyl transpep-
tidse activity in mice: age dependent changes and effectof
cortisol. Ind J Exp Biol 1991; 29: 1124–6.
27. Bailey D, Mujoo K, Blake ME, Arntxen CJ, Gutter-
man JU. Avicins: triterpenoid saponins from Acacia victoriae
(Bentham) induce apoptosis by mitochondrial perturbation.
PNAS USA 2001; 98: 5821–6.
28. Fiala S, Fiala ES. Activation by chemical carcinogen of
gamma glutamyl transpeptidase in rat and mouse liver. J Natl
Cancer Inst 1973; 51: 151–8.
29. Vanisree AJ, Shyamaladevi CS. Effect of therapeutic
strategy established by N-acetyl cystine and vitamin C on the
activities of tumour marker enzymes in vitro. Ind J Pharmacol
1998; 31: 275–8.
30. Koss B, Greengurd O. Effect of neoplasma on the
content and activity of alkaline phosphatase and gamma glu-
tamyl transpeptidase in uninvolved host tissues. Cancer Res
1982; 42: 2146–51.
31. Ip C, Daw T. Alterations in serum glycosyl trasnferase
and 5´-nucleotidase in breast cancer patients. Cancer Res
1978; 38: 723–8.
32. Dao TL, Ip C, Pater J. Serum sialyl transferase and
5´nucleotidase as reliable biomarkers in cancer. J Natl Cancer
Inst 1980; 65: 529–34.
33. Rosi F, Tabucchi A, Carlucci F, Galieni P, Lauria F,
Zanoni L, Guerranti R, Marinello E, Pagani R. 5´nucleotidase
activity lymphocytes from patients affected with B-cell lym-
phocytic leukemia. Clin Biochem 1998; 31: 269–72.
34. Lipport M, Papadopoulos N, Javadpour NR. Role of
lactate dehydrogenase isoenzymes in testicular cancer. Urol-
ogy 1981; 18: 50–3.
35. Pui CH, Dodge Pk, Dahl GV, Rivera G, Look AT, Kalwin-
sky D, Bowman WP, Ochs J, Abromowitch M, Mirro J. Serum
lactic dehydrogenase levels has prognostic value in childhood
acute lymphoblastic leukemia. Blood 1985; 66: 778–82.
36. Engan T, Hannisdal E. Blood analysis as a prognostic
factor in primary lung cancer. Acta. Oncol 1990; 29: 151–4.
�� Experimental Oncology ���� ������� ����� ��arc���
ВЛИЯНИЕ СИЛИМАРИНА НА ГЕПАТОКАНЦЕРОГЕНЕЗ,
ИНДУЦИРОВАННЫЙ У КРЫС NN-НИТРОЗОДИЭТИЛАМИНОМ
Цель: изучить влияние силимарина на уровень экспрессии опухолевых и биохимических маркеров и формирование аддуктов
малонового диальдегида с ДНК (MDA-DNA) при развитии гепатокар�иномы у крыс линии �истар.MDA-DNA) при развитии гепатокар�иномы у крыс линии �истар.-DNA) при развитии гепатокар�иномы у крыс линии �истар.DNA) при развитии гепатокар�иномы у крыс линии �истар.) при развитии гепатокар�иномы у крыс линии �истар. Методы: стандартными
биохимическими методами определяли активность ферментов в сыворотке крови и проводили иммуногистохимическое
определение MDA-DNA в ткани печени крыс.MDA-DNA в ткани печени крыс.-DNA в ткани печени крыс.DNA в ткани печени крыс. в ткани печени крыс. Результаты: показано, что при развитии злокачественной гепатокар�и-
номы в сыворотке крови животных значительно увеличивается количество альфа-фетопротеина, раковоэмбрионального
антигена, активность аспартат- и аланинаминотрансферазы, щелочной и кислой фосфатазы, лактатдегидрогеназы, гамма-
глутамилтрансферазы и 5�-нуклеотидазы. При проведении иммуногистохимического исследования отмечали повышенное
образование аддуктов MDA-DNA в ткани печени крыс со злокачественной гепатокар�иномой. При введении силимаринаMDA-DNA в ткани печени крыс со злокачественной гепатокар�иномой. При введении силимарина-DNA в ткани печени крыс со злокачественной гепатокар�иномой. При введении силимаринаDNA в ткани печени крыс со злокачественной гепатокар�иномой. При введении силимарина в ткани печени крыс со злокачественной гепатокар�иномой. При введении силимарина
значительно снижался уровень указанных ферментов в сыворотке крови и формирование аддуктов MDA-DNA в тканиMDA-DNA в ткани-DNA в тканиDNA в ткани в ткани
печени. Заключение: применение силимарина может быть эффективно для предупреждения развития злокачественной
гепатокар�иномы, инду�ированной N-нитрозодиэтиламином у крыс, и этот препарат может быть многообещающим хи-
миотерапевтическим адъювантом для лечения рака печени.
Ключевые слова: MDA-DNA, альфафетопроеин, злокачественная гепатокар�инома,MDA-DNA, альфафетопроеин, злокачественная гепатокар�инома,-DNA, альфафетопроеин, злокачественная гепатокар�инома,DNA, альфафетопроеин, злокачественная гепатокар�инома,, альфафетопроеин, злокачественная гепатокар�инома, N-нитрозодиэтиламин, силимарин,
флавоноиды.
Copyright © Experimental Oncology, 2007
37. Doa TL, Ip C, Rater J. Serum sialyl transferase and
5´ nucleotidase as reliable biomarkers in women with breast
cancer. J Natl Cancer Inst 1980; 65: 529.
38. Abeleb GI. Production of embryonal serum alpha
globulin by hepatomas: review of experimental and clinic data.
Cancer Res 1986; 28: 1344–50.
39. Banker DD. Viral hepatitis (Part-IV). Ind J Med Sci
2003; 57: 511–7.
40. Stroescu C, Herlea V, Dragnea A, Popescu I. The di-
agnostic value of cytokeratins and carcinoembryonic antigen
immunostaining in differentiating hepatocellular carcinomas
from intrahepatic cholangiocarcinoma. J Gastrointestin Liver
Dis 2006; 15: 9–14.
41. Marnett LJ. Oxyradicals and DNA damage. Carcino-
genesis 2000; 21: 361–70.
42. Wang MY, Dhingra K, Hittelman WN Liehr JG, de
Andrade M, Li DH. Lipid peroxidation induced putative malo-
ndialdehyde-DNA adducts in human breast tissues. Cancer
Epidemiol Biomark Prev 1996; 5: 705–10.
43. Munnia A, Amasion ME, Peluso M. Exocyclic malo-
ndialdehyde and aromatic DNA adducts in larynx tissues. Free
Radical Biol Med 2004; 37: 850–8.
44. Munnia A, Bonassi S, Verna A, Quaglia R, Pelucco D,
Ceppi M, Neri M, Buratti M, Taioli E, Garte S, Peluso M. Bron-
chial malondialdehyde DNA adducts, tobacco smoking and
lung cancer. Free Radical Biol Med 2006; 41: 1499–505.
45. Bartsch H, Nair J, Owen RW. Exocyclic DNA adducts
as oxidative stress markers in colon carcinogenesis: potential
role of lipid peroxidation, dietary fat and antioxidants. Biol
Chem 2002; 383: 915–21.
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