Iran Red Crescent Med J. 2015 April; 17(4): e25310.

DOI: 10.5812/ircmj.17(4)2015.25310
Published online 2015 April 25. Research Article

Evaluation of the Protective Effect of Silibinin Against Diazinon Induced

Hepatotoxicity and Free-Radical Damage in Rat Liver
1,* 2 3 4 5
Halil Beydilli ; Nigar Yilmaz ; Esin Sakalli Cetin ; Yasar Topal ; Ozgur Ilhan Celik ; Cem
6 3 7 8
Sahin ; Hatice Topal ; Ibrahim Hakki Cigerci ; Hamdi Sozen
1Department of Emergency Medicine, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
2Department of Medical Biochemistry, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
3Department of Medical Biology, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
4Department of Pediatrics, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
5Department of Medical Pathology, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
6Department of Internal Medicine, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey
7Department of Biology, School of Science, Afyon Kocatepe University, Afyon, Turkey
8Department of Infectious Diseases, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey

*Corresponding Author: Halil Beydilli, Department of Emergency Medicine, School of Medicine, Mugla Sitki Kocman University, Mugla, Turkey. Tel: +90-2522114835,
E-mail: [email protected]

Received: November 12, 2014; Revised: February 28, 2015; Accepted: March 18, 2015

Background: Diazinon (0,0-Diethyl 0-(1-6-methyl-2-isoprophyl 4 pyrimidinyl) phosphorothioate) (DI) is a very effective organophosphate
pesticide, used widely in agriculture. Consequently, data on poisoning cases secondary to DI exposure are important. The DI may affect a
variety of tissues, including liver. Silibinin is a pharmacologically active constitute of Silybum marianum, with documented antioxidant
activity.
Objectives: The aim of our study was to evaluate both histopathologically and biochemically whether silibinin is protective in DI induced
liver damage.
Materials and Methods: Thirty two Wistar albino rats were divided into four groups, as follows: 1) control group - oral corn oil was
given; 2) DI group - rats were administered orally 335 mg/kg in the corn oil solution; 3) Silibinin group - 100 mg/kg/day silibinin was given
alone orally, every 24 hours for 7 days; 4) Silibinin + DI group - DI plus silibinin was given. All rats were sacrificed at the end of experiment.
Superoxide dismutases (SOD), glutathione peroxidase (GPX), nitric oxide (NO) and myeloperoxidase (MPO) were investigated in serum and
liver tissue. In addition, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities were evaluated. The
liver tissue was evaluated histopathologically with Hematoxilin & Eosin dye.
Results: Biochemically, ALT, AST, NO, MPO in serum and NO, MPO in liver tissue were found to be significantly higher in DI group, compared
to control group (P < 0.001). In Group Silibinin + DI, serum AST, ALT, NO, MPO levels were significantly lower (P < 0.01), and both serum and
tissue SOD activities were significantly higher, compared to DI group (P < 0.001). Diazinon induced histopathological changes in liver
tissue were: severe sinusoidal dilatation, moderate disruption of the radial alignment of hepatocytes around the central vein, severe
vacuolization in the hepatocyte cytoplasm, inflammation around central vein and portal region. In rats receiving both DI and silibinin,
the DI induced changes accounted for less sinusoidal dilatation, vacuolization in the hepatocyte cytoplasm and the inflammation around
central vein and portal region (P < 0.05).
Conclusions: The DI was found to induce liver damage by oxidative stress mechanisms. Silibinin reduced the oxidative stress by inducing
antioxidant mechanisms, thereby showing protective effect against DI induced liver damage. Further studies with silibinin should be
performed regarding DI toxicity.

Keywords: Diazinon; Oxidative Stress; Antioxidants; Histopathology; Liver

1. Background
Diazinon (0,0-Diethyl 0-(1-6-methyl-2-isoprophyl 4 py- Diazinon exerts its effects on multiple tissues and cells,
rimidinyl) phosphorothioate) (DI) has been widely used such as blood cells, immune system, hepatocytes, neu-
throughout the world, including Turkey, with applica- ronal cells and renal cells, resulting in increased free
tions in agriculture for controlling the hazardous insects radical production and depletion of tissue antioxidant
and disease vectors (1). The DI is also one of the most wide- mechanism (2-7). Previous studies have reported that
ly known causes of toxicity on many body organs, and DI may cause hepatotoxicity (6, 7). One of the possible
therefore data about its effects on humans are crucial. In mechanism of DI induced hepatotoxicity is the increas-
addition, admission of the patients to the emergency de- ing level of reactive oxygen species (ROS) (4-7). The ad-
partment, with suicide attempt or accidental ingestion ministrations of several antioxidants may attenuate the
of DI, may be possible. DI-induced hepatotoxicity.

Copyright © 2015, Iranian Red Crescent Medical Journal. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCom-
mercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial us-
ages, provided the original work is properly cited.
 Beydilli H et al.

Silibinin is a pharmacologically active constitute of Si- 2012/3). The experiments were performed in accordance
lybum marianum (8). Its antioxidant activity may have a with the Guide for the Care and use of Laboratory Ani-
crucial role in the effects of silibinin. It is widely used in mals (DHEW. Publication (NIH) 8523, 1985). Silibinin was
the treatment due to its safety and lack of adverse effects purchased from Sigma/Aldrich Chemicals, USA.
(9). Recent studies have shown that silibinin protects the The animals were randomly divided into four groups:
liver against multiple drugs and chemical induced liver - Group 1: Control group (n = 8); rats were given 0.3 mL
injury (10, 11). Silibinin seems to be a promising protec- corn oil orally;
tive agent for repairing free-radical induced damage in a - Group 2: DI group (n = 8); rats were administered a sin-
variety of pathological conditions (11). gle dose of 335 mg/kg of body weight (BW) of DI (Basudin
Aspartate aminotransferases (AST) and alanine ami- 60 EC, Syngeta Tarım San. ve Tic. AS, Izmir, Turkey) with
notransferases (ALT) are intracellular aminotransferase corn oil (1, 4, 7);
enzymes, present in liver cells. After cell death or dam- - Group 3: Silibinin group (n = 8); silibinin was given
age in liver cells, they are released into the circulation. orally, 100 mg/kg/day every 24 hours, for 7 days (17, 18);
Increased serum transaminases translate a susceptibility - Group 4: DI + Silibinin group (n = 8); rats were given DI
to liver damage (12). single dose of 335 mg/kg BW of DI orally and 100 mg/kg/
Myeloperoxidase (MPO) is the most abundant protein day silibinin was administered orally, every 24 hours for
in neutrophils, catalyzes the conversion of hydrogen per- 7 days.
oxide and chloride ions into hypochlorous acid. It plays a At the end of the experiment, all animals were anesthe-
role in down regulating the inflammatory response (13). tized under intraperiteoneal injection of ketamine/xyla-
Superoxide dismutase (SOD) is regarded as the first line zine (60 mg/kg and 6 mg/kg, respectively). Blood samples
of defense against the detrimental effects of molecular were taken from intracardiac on the sterile tubes, for
oxygen radicals in cells. Superoxide is a crucial source measuring the level of serum ALT, AST, MPO, NO, SOD
of hydroperoxides and free radicals. The activity of SOD and GPx. Blood samples were centrifuged and serum was
inhibits lipid peroxidation by catalyzing the conversion separated. The blood was centrifuged at 2000 × G for 15
of superoxides into hydrogen peroxide and oxygen. The minutes, at 4°C. The top yellow serum layer was pipetted
SOD protects the cells from superoxide toxicity via re- off, without disturbing the white buffy layer. Livers were
moving superoxide free radicals (14). removed immediately and washed with phosphate buf-
Endogenous nitric oxide (NO) is formed from the amino fer solution (PBS) (pH = 7.4) and then frozen promptly in
acid L-arginine with nitric oxide synthase (NOS) enzyme. a deep freezer for biochemical analysis. All samples were
Increasing the level of NO has a crucial role in the modu- protected under -80°C until analysis.
lation of oxidative stress and tissue damage (15). It was re-
ported that oxidative stress results in the increase of the 3.1. Determination of Aspartate Aminotransferase
activity of NO synthase, as a consequence to the elevation and Alanine Aminotransferase Activities
of NO release (16).
Glutathione peroxidase (GPx) is a crucial selenocyste- The activities of AST and ALT were calculated spectro-
ine-containing enzyme, which catalyzes the reduction photometrically in serum, using Beckman Coulter kits by
of hydroperoxides, including hydrogen peroxide, by autoanalyzer (Unicel D × C 800 Synchron, Brea, California,
reduced glutathione and functions to protect the cells USA). The results were expressed as units per liter (U/L).

3.2. Determination of Superoxide Dismutase Activity

from oxidative damage (17).

2. Objectives The tissue was homogenized at 16000 rpm on ice, in 5

- 10 mL cold buffer, 20 mM HEPES buffer, pH 7.2, contain-
The present investigation aimed to evaluate the anti-
ing 1mM EGTA, 210 mM mannitol and 70 mM sucrose per
oxidant and protective efficacies of silibinin against DI
g tissue. The mixture was centrifuged at 1500 × G for 5
induced hepatotoxicity in rats by evaluation of NO, MPO,
minutes, at 4°C. The supernatant was removed. Serum
SOD, GPx, AST, ALT and histological values.
should be diluted 1:5 with sample buffer. The SOD activ-

3. Materials and Methods

ity was measured in the supernatant and serum. The SOD
was determined via Cayman’s Superoxide Dismutase
Thirty two adult female 12 week-old Wistar albino rats assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) in
(Suleyman Demirel University Experimental Research Bio-Tek ELx-800 (Winooski, USA). The detection of super-
Centre, Isparta, Turkey) weighing 170 - 220 g were used oxide radicals were generated by xanthine oxidase and
for the present experiment. The animals were housed in hypoxanthine. One unit of SOD is defined as the amount
quiet rooms (20 - 25°C; 50 - 60% relative humidity) on a 12 of enzyme required to exhibit 50% dismutation of the su-
hour light/dark cycle (7 a.m. - 7 p.m.) and allowed a com- peroxide radical. The results were expressed as units per
mercial standard rat diet (Abalioglu Yem Sanayi, Deni- mg protein (U/mg) tissue, for liver tissue, and units per
zli, Turkey) and water ad libitum. All animal procedures milliliter (U/mL) for serum. The dynamic range of the kits
were approved by the university Ethics Committee (No: is 0.005 - 0.05 U/mL SOD. Recommended by the company

2 Iran Red Crescent Med J. 2015;17(4):e25310

 Beydilli H et al.

for measuring formulation, the SOD was calculated by ap- three grades (grade 1: 1-5 points, grade 2: 6 - 10 points and
plying SOD values. grade 3: 11-15 points) (12, 13).
Six parameters of liver damage were evaluated:
3.3. Determination of Glutathione Peroxidase Ac- 1. Sinusoidal dilatation;
tivity 2. Distortion radial alignment around central vein;
3. Vacuolization in hepatocytes;
The tissue was homogenized in 5-10 mL cold buffer (50 4. Inflammation in the portal area and around central
mM Tris-HCl, pH 7.5, 5 mM EDTA and 1 mM DTT (Dithio- vein;
threitol) per tissue. Then, it was centrifuged at 10000 × 5. Hepatocellular necrosis;
G for 15 minutes, at 4°C. The supernatant was removed 6. Eosinophils infiltration of in the periportal field or
after centrifugation. The blood was centrifuged at 700 - around central vein.
1000 × G for 10 minutes, at 4°C. The serum was removed.
The GPx activity was measured in liver tissue and serum
3.7. Statistical Analysis
samples. The GPx activity was determined via Cayman’s
GPx assay kit (Cayman Chemical Co., Ann Arbor, MI, USA) Data were analyzed using a commercially available
in Bio-Tek ELx-800. The GPx activity was measured indi- statistics software package (SPSS Statistics for Windows,
rectly by a coupled reaction with glutathione reductase. Version 20.0. IBM Corp., Armonk, NY, USA). All data were
The oxidized glutathione was produced upon reduction presented as the mean ± SD for comparisons. Compari-
of hydroperoxide by GPx. The results were expressed as sons between groups were performed using the Kruskal-
units per mg protein (U/mg) tissue, for liver tissue, and Wallis analysis of variance for unpaired comparisons,
units per milliliter (U/mL), for serum. The dynamic range followed by the Mann Whitney U test. The P < 0.05 was
of the assay is only limited by the accuracy of the absor- considered significant.

4. Results
bance measurement.

3.4. Determination of Nitric Oxide Level

The tissue was homogenized in PBS (pH 7.4) and centri- 4.1. Biochemical Results
fuged at 10000 × G for 20 minutes to create the superna- The levels of NO and MPO in serum and liver tissue
tant. Total NO assay was performed by spectrophotom- were found to be significantly increased in the DI group,
etry at 540 nm using nitrate/nitrite colorimetric assay kit compared to control group (P < 0.0001) (Tables 1 and 2).
(Cayman, Ann Arbor, Michigan USA) in Bio-Tek ELx-800. The activity of ALT and AST were found to be significant-
The assay was based on nitrate and nitrite determina- ly increased in DI group, compared to control group (P
tions. The nitrate and nitrite are the stable end products < 0.001) (Table 1). The levels of NO and MPO in serum
of the reaction of NO with molecular oxygen. The total were found to be significantly decreased in DI + silibinin
accumulation of nitrate and nitrite in serum and liver group, compared with DI group (P < 0.01 and P < 0.001).
tissue was measured. The results were expressed as µm/g When comparing DI + silibinin group to DI group the
protein. activities of AST and ALT were found decreased (Tables

3.5. Determination of MPO Activity

1 and 2).
There was no significant statistical difference between
The quantitative detection of MPO was used by an the tissue or serum GPx activities, for all groups. The serum
enzyme-linked immunosorbent assay (ELISA) kit (MPO and tissue SOD activity was found increased in DI + silibinin
Instant Elisa, eBioscience, Vienna, Austria) in Bio-Tek ELx- group, when compared to DI group (P < 0.001) (Table 1).
800. The results were expressed as ng/mL protein.
4.2. Histopathological Results
3.6. Histopathology of Liver Tissue Hepatocytes of control group (Figure 1 A) and silibinin
The liver tissue was also removed for histopathological group (Figure 1 B) were observed to have a normal struc-
investigation. The specimens were fixed in 10% formalin ture (Figure 1). It was determined histopathologically
subsequent overnight and then were dehydrated by im- that the liver tissue intoxicated by DI was significantly
mersion in a series of alcohol solutions of various con- damaged (Grade 3).
centrations, cleared in xylene and paraffin embedded In histopathological examination, rats administered DI
tissue sections. The tissue samples were then infiltrated showed severe sinusoidal dilatation, moderate disrupt
with paraffin as blocks, sectioned (5 µm-thick slides). The radial alignment of hepatocytes, severe vacuolization of
prepared samples were examined under a light micro- hepatocyte cytoplasm, and centrilobular necrosis (P <
scope according to the severity of the lesions modified 0.05) (Figure 2). In contrast, rats in DI + Silibinin group
from Yehia et al. (19). Each parameter was scored between exhibited these changes significantly, especially inflam-
0 and 3 (0: normal, 1: mild, 2: moderate and 3: severe) and mation around the central vein and portal space (P <
according to the point total, lesions were classified into 0.05) (Figure 3).

Iran Red Crescent Med J. 2015;17(4):e25310 3

 Beydilli H et al.

Figure 1. A, Control Group (H & E × 40); B, Silibinin Group (H & E × 40), Normal Liver Morphology

Figure 2. Diazinon Group

A, Dilatation in sinusoids (arrows) (H & E × 40); B, Cellular disruption (stars) around central vein (arrow) (H & E × 40); C, Vacuolation (arrows) (H & E × 200);
D, Mononuclear inflammatory cells (stars) among hepatic cells, around the central vein (arrow) and portal space (arrow head) (H & E × 40); E, Hepatocel-
lular necrosis (stars) (H & E × 40).

Figure 3. Diazinon + Silibinin Group

A, Mild dilatation in sinusoid (arrows) (H & E × 100); B, Minimal vacuolation (arrows) (H & E × 200); C: Mild inflammation (stars) around the central vein
(arrow) and portal space (arrow head) (H & E × 100).

4 Iran Red Crescent Med J. 2015;17(4):e25310

 Beydilli H et al.

Table 1. Biochemical Serum Values of Four Groups of Rats a, b

Groups AST, U/L ALT, U/L SOD, U/mL GPx, U/mL NO, µm/g MPO, ng/mL
Control group 107.63 ± 22.37 55.13 ± 13.04 0.1 ± 0.02 3.48 ± 0.98 1.43 ± 0.73 1.51 ± 1.02
DI group 80.00 ± 13.9 90.00 ± 10.18 0.09 ± 0.02 3.74 ± 2.77 7.13 ± 2.73 4.83 ± 1.60
Silibinin group 90.5 ± 11.25 44.88 ± 3.22 3.69 ± 2.58 7.95 ± 2.68 1.26 ± 0.72 1.19 ± 0.53
DI + Silibinin group 105 ± 23.34 54.25 ± 9.99 0.52 ± 0.34 c 7.481 ± 2.43 2.54 ± 1.35 d 0.81 ± 0.33 e
a Abbreviations: ALT, Alanine aminotransferase; AST, Aspartate aminotransferase; GPx, Glutathione peroxidase; MPO, Myeloperoxidase; NO, Nitric
oxide; SOD, Superoxide dismutases.
b Data are presented as Mean ± SD for n = 8.
c P < 0.05 SOD, DI + silibinin compared with DI group.
d P < 0.05 NO, DI + silibinin compared with DI group.
e P < 0.05 MPO, DI + silibinin compared with DI group.

Table 2. GPx, SOD, NO and MPO Liver Tissue Values of Four Groups of Rats a, b

Groups GPx, U/mg SOD, U/mg NO, µm/g MPO, ng/mL

Control group 0.89 ± 0.21 1.6 ± 0.47 2.86 ± 1.84 3.51 ± 2.40
DI group 0.76 ± 0.23 2.70 ± 1.71 9.45 ± 2.71 7.54 ± 3.32
Silibinin group 0.96 ± 0.29 3.84 ± 1.48 2.2 ± 0.01 4.56 ± 2.26
DI + Silibinin group 0.18 ± 0.11 2.82 ± 0.83 5.9 ± 1.14 c 3.73 ± 1.11 d
a Abbreviations: GPX, Glutathione peroxidase; MPO, Myeloperoxidase; NO, Nitric oxide; SOD, Superoxide dismutases.
b Data are presented as Mean ± SD for n = 8.
c P < 0.05 NO, DI + Silibinin, compared with DI group.
d P < 0.05 MPO, DI + Silibinin, compared with DI group.

5. Discussion
The levels of ALT, AST, NO and MPO in serum were found oxidative stress increased nitrate and nitrite. The MPO,
increased in DI group, when compared with control which is a peroxidase enzyme that synthetizes hypochlo-
group. We suggest that DI induced a significant liver rous acid from H2O2 and chloride, plays an important
damage. Silibinin reduced the levels of AST, ALT, NO, MPO role, as a powerful oxidant, which utilizes free radicals (13,
in silibinin + DI group, compared with DI group. Silib- 23-25). In this study, DI exposure could induce oxidative
inin, given to rats with DI, showed a significant protective stress by the increased NO, MPO concentrations, which
activity against liver damage induced by DI. In addition, should induce membrane lipid peroxidation, resulting
the level of SOD in serum and liver tissue increased via in liver injury. These results were correlated with previ-
silibinin in silibinin+DI group (Table 2). In histopatholog- ous reports of Messarah et al. who showed DI might gen-
ical examination, DI caused severe sinusoidal dilatation erate ROS (26). It was reported that silibinin has antioxi-
and severe vacuolation, inflammation around the portal dant effects (27). To the best our knowledge, the current
area and central vein and disrupted the radial alignment study is the first to investigate the silibinin antioxidant
around the central vein in hepatocytes. Silibinin signifi- effects on DI induced hepatotoxicity. Oxidative stress in-
cantly reversed the DI-induced sinusoidal dilatation, se- duced by DI administration is also demonstrated by a
vere vacuolization and inflammation around the central highly significant increase in the activities NO and MPO
vein in hepatocytes (P < 0.05) (19, 20). and our results are in agreement with previous reports
The liver is a very crucial organ for the detoxification (26, 27). The DI affects the mitochondrial membrane
processes and oxidative stress is thought to be a key transportation in rat liver (18). Diazinon binds extensive-
mechanism of hepatocellular injury. The liver tissue was ly to biological membranes, especially to the phospholip-
the major site of DI metabolism, by assembling a great ids bilayers (28). Silibinin acts on the polar head group of
quantity of its metabolites (21). In the present study, we phospholipids of the cellular membrane. It was reported
suggest that DI increased the reactive oxygen species that silibinin act as an excellent protective agent against
(ROS) in liver tissue and silibinin carries out free-radical- lipid peroxidation on cellular membrane (29). Previous
eliminating activity and extensive antioxidant effect. studies have reported the protective role of silibinin via
Similarly, in previous studies, it was been shown that DI scavenging free radicals and antioxidant properties (10).
caused increases in lipid peroxidation (4, 22). It was re- Silibinin is membranotropic in nature and it has been
ported that DI exposure has been implicated in inducing found to bind firmly to the hepatocellular membrane.

Iran Red Crescent Med J. 2015;17(4):e25310 5

 Beydilli H et al.

Silibinin has a role in metabolic and cell-regulating ac- Lipid peroxidation starts as a consequence of ROS-
tions via antioxidative mechanism, which is regarded as induced isolation of hydrogen from polyunsaturated
a major hepatoprotective effect. fatty acids (PUFAs) from the cellular membrane, which
In addition, ALT and AST enzymes activities represent a results in the formation of relatively stable compounds,
marker of hepatic function when determining hepato- like NO. Increasing the level of NO has a crucial role in
toxicity. The DI exposure resulted in the increase of the the modulation of oxidative stress and tissue damage
activities of serum AST and ALT. The phenomenon may (15). It was reported that oxidative stress results in in-
occur due to disturbing the transport function of the he- creasing the activity of NO synthase, as a consequence
patocytes. In a previous study, silibinin repair function in of the elevation of NO release (16). The DI induced the
hepatotoxicity was reported thorough the reduction in secretion of excess NO reaction with the superoxide an-
the serum levels of ALT and AST enzymes (30). Probably, ion to generate the peroxynitrite radical involved in the
increases of serum ALT and AST enzymes activities are one toxification process. Silibinin treatment significantly re-
of the important markers for the diagnosis of liver dam- duced lipid peroxidation, as an antioxidant. Therefore,
age. In addition, by increasing MPO, NO levels, DI plays silibinin treatment repaired the excess NO reaction.
role in pathogenesis of hepatic toxicity via oxidative stress Treatment with silibinin effectively decreased the lev-
mechanism. The MPO, which is the most abundant protein els of NO. It has been reported that silibinin scavenges
in neutrophils and catalyzes the conversion of hydrogen nitrogen species. Similarly, Prabu et al. found that the
peroxide and chloride ions into hypochlorous acid, plays level of NO decreased as a result of silibinin treatment
a role in down-regulating the inflammatory response (13). against arsenic induced toxicity (11).
However, in non-infectious diseases, MPO that was found The GPx is a crucial selenocysteine-containing enzyme,
increased was associated with strong oxidative activity. which catalyzes the reduction of hydroperoxides, includ-
The activity of MPO in the oxidation of DI was reported ing hydrogen peroxide, by reduced glutathione and func-
previously (31). Silibinin counteracted the inflamma- tions to protect the cell from oxidative damage (17). It was
tory process by decreasing the MPO pathway and also by reported that DI induced oxidative toxicity through oxi-
preventing free radical production. Liver injury, marked dation of GPx (35). In another study, the GPx activity val-
as centrilobular necrosis and neutrophilic infiltration ues were found to be non-significantly different between
around centrilobular area, could be seen in H & E stained the DI group and DI + N-acetyl cysteine group. Similarly,
rat liver cells. In this study, the activity of MPO and histo- in our study, GPx activity was not found significantly dif-
pathological results are correlated about hepatotoxicity. ferent between DI group and DI + silibinin group.
Silibinin has been intensively studied in vitro, in vivo In conclusion, our present results demonstrate that
and also, in clinical trials. Van Wenum et al. reported that silibinin exerts hepatoprotective, antioxidant, free radi-
silibinin used in the treatment of cirrhosis, hepatitis and cal scavenging effects against DI induced hepatotoxic-
alcohol-induced liver disease, is usually connected with ity. It may be suggested that silibinin is convenient as a
antioxidant action (32). Silibinin is extensively applied therapeutic agent for the amelioration of DI induced
due to its safety and lack of adverse effects. We found an hepatotoxicity. However, further studies are required in
increased activity of plasma antioxidant enzymes, name- order to understand and quantify the beneficial effects
ly SOD in DI + silibinin group. The SOD is assumed to be of silibinin in DI induced hepatotoxicity and its possible
the most effective antioxidant (33). Therefore, it is regard- clinical use.

Acknowledgements
ed as the first line of defense against the detrimental ef-
fects of molecular oxygen radicals in cells. Superoxide is
a crucial source of hydroperoxides and free radicals. The The authors thank the heads and staffs of the Suleyman
activity of SOD inhibits lipid peroxidation by catalyzing Demirel University Experimental Research Center, Ispar-
the conversion of superoxides into hydrogen peroxide ta, Turkey and Mugla Sitki Kocman University Scientific
and oxygen (14). The SOD protects the cells from super- Research Projects, Mugla, Turkey. Also, our thanks go to
oxide toxicity via removing superoxide free radicals. the Coordination Unit for their financial and technical
Silibinin also restored SOD activity. The increased he- supports.

Authors’ Contributions
patic SOD activity we observed in the group treated with
silibinin. Accordingly, the co-administration of silibinin,
after DI exposure, increased SOD levels and ameliorated Study concept and design: Halil Beydilli, Nigar Yilmaz;
the oxidative system. The co-administration of silibinin animal procedures and sampling: Halil Beydilli, Esin
reduced the detrimental effects of DI by possibly scav- Sakalli Cetin, Hamdi Sozen; analysis and interpretation of
enging or neutralizing ROS. These results showed that data: Yasar Topal, Hatice Topal; drafting of the manuscript:
silibinin might have a beneficial role in lowering DI toxic- Halil Beydilli, Cem Sahin; critical revision of the manu-
ity. Furthermore, a protective effect of silibinin has also script for important intellectual content: Nigar Yilmaz,
been reported against N-nitrosodimethylamine induced Halil Beydilli; statistical analysis: Cem Sahin; study super-
oxidative stress. Silibinin caused increases of SOD in liver vision: Halil Beydilli, Ibrahim Hakki Cigerci; histopatho-
tissue against N-nitrosodimethylamine (34). logic evaluation: Ozgur Ilhan Celik.

6 Iran Red Crescent Med J. 2015;17(4):e25310

 Beydilli H et al.

Funding/Support 17. Arthur JR. The glutathione peroxidases. Cell Mol Life Sci.
2000;57(13-14):1825–35.
The research received funding from the Mugla Sitki Koc- 18. Nakagawa Y, Moore G. Role of mitochondrial membrane perme-
man University Scientific Research Projects Coordination ability transition in p-hydroxybenzoate ester-induced cytotoxic-
ity in rat hepatocytes. Biochem Pharmacol. 1999;58(5):811–6.
Unit, Mugla, Turkey. 19. Yehia MA, El-Banna SG, Okab AB. Diazinon toxicity affects histo-
physiological and biochemical parameters in rabbits. Exp Toxicol

References
Pathol. 2007;59(3-4):215–25.
20. Alp H, Aytekin I, Esen H, Basarali K, Kul S. Effects of caffeic acid
1. Shah MD, Iqbal M. Diazinon-induced oxidative stress and renal phenethyl ester, ellagic acid, sulforaphane and curcumin on diazi-
dysfunction in rats. Food Chem Toxicol. 2010;48(12):3345–53. non induced damage to the lungs, liver and kidneys in an acute
2. Galloway T, Handy R. Immunotoxicity of organophosphorous toxicity rat model. Kafkas Univ Vet Fak Derg. 2011; 17(6):927–33.
pesticides. Ecotoxicology. 2003;12(1-4):345–63. 21. Giray B, Gurbay A, Hincal F. Cypermethrin-induced oxidative
3. Zarichi Baghlani K, Saberi M, Rasooli Vani J. Protective effects of stress in rat brain and liver is prevented by vitamin E or allopuri-
guanosine on Diazinon induced oxidative stress in neuralgia nol. Toxicol Lett. 2001;118(3):139–46.
U373MG cell line. Trauma Mon. 2011;16(3):157–61. 22. Akturk O, Demirin H, Sutcu R, Yilmaz N, Koylu H, Altuntas I. The
4. Yilmaz N, Yilmaz M, Altuntas I. Diazinon-induced brain tox- effects of diazinon on lipid peroxidation and antioxidant en-
icity and protection by vitamins E plus C. Toxicol Ind Health. zymes in rat heart and ameliorating role of vitamin E and vita-
2012;28(1):51–7. min C. Cell Biol Toxicol. 2006;22(6):455–61.
5. Giordano G, Afsharinejad Z, Guizzetti M, Vitalone A, Kavanagh TJ, 23. Mehta A, Verma RS, Srivastava N. Chlorpyrifos induced altera-
Costa LG. Organophosphorus insecticides chlorpyrifos and diazi- tions in the levels of hydrogen peroxide, nitrate and nitrite in
non and oxidative stress in neuronal cells in a genetic model of rat brain and liver. Pesticide Biochem Physiol. 2009;94(2-3):55–9.
glutathione deficiency. Toxicol Appl Pharmacol. 2007;219(2-3):181–9. 24. Klebanoff SJ. Myeloperoxidase: friend and foe. J Leukoc Biol.
6. Shadnia S, Dasgar M, Taghikhani S, Mohammadirad A, Kho- 2005;77(5):598–625.
rasani R, Abdollahi M. Protective Effects of alpha-Tocopherol 25. Heinecke JW, Li W, Francis GA, Goldstein JA. Tyrosyl radical gener-
and N-Acetyl-Cysteine on Diazinon-Induced Oxidative Stress and ated by myeloperoxidase catalyzes the oxidative cross-linking of
Acetylcholinesterase Inhibition in Rats. Toxicol Mech Methods. proteins. J Clin Invest. 1993;91(6):2866–72.
2007;17(2):109–15. 26. Messarah M, Amamra W, Boumendjel A, Barkat L, Bouasla I, Ab-
7. Teimouri F, Amirkabirian N, Esmaily H, Mohammadirad A, Ali- dennour C, et al. Ameliorating effects of curcumin and vitamin E
ahmadi A, Abdollahi M. Alteration of hepatic cells glucose me- on diazinon-induced oxidative damage in rat liver and erythro-
tabolism as a non-cholinergic detoxication mechanism in coun- cytes. Toxicol Ind Health. 2013;29(1):77–88.
teracting diazinon-induced oxidative stress. Hum Exp Toxicol. 27. Sozmen M, Devrim AK, Tunca R, Bayezit M, Dag S, Essiz D. Protec-
2006;25(12):697–703. tive effects of silymarin on fumonisin B(1)-induced hepatotoxic-
8. Trappoliere M, Caligiuri A, Schmid M, Bertolani C, Failli P, Vizzutti ity in mice. J Vet Sci. 2014;15(1):51–60.
F, et al. Silybin, a component of sylimarin, exerts anti-inflamma- 28. Lee AG, Malcolm East J, Balgavy P. Interactions of insecticides
tory and anti-fibrogenic effects on human hepatic stellate cells. J with biological membranes. Pesticide Sci. 1991;32(3):317–27.
Hepatol. 2009;50(6):1102–11. 29. Erlejman AG, Verstraeten SV, Fraga CG, Oteiza PI. The interaction
9. Trouillas P, Marsal P, Svobodova A, Vostalova J, Gazak R, Hrbac J, et of flavonoids with membranes: potential determinant of flavo-
al. Mechanism of the antioxidant action of silybin and 2,3-dehy- noid antioxidant effects. Free Radic Res. 2004;38(12):1311–20.
drosilybin flavonolignans: a joint experimental and theoretical 30. Cacciapuoti F, Scognamiglio A, Palumbo R, Forte R, Cacciapuoti
study. J Phys Chem A. 2008;112(5):1054–63. F. Silymarin in non alcoholic fatty liver disease. World J Hepatol.
10. Gazak R, Walterova D, Kren V. Silybin and silymarin--new and 2013;5(3):109–13.
emerging applications in medicine. Curr Med Chem. 2007; 31. Lazarević-Pašti T, Čolović M, Savić J, Momić T, Vasić V. Oxidation of
14(3):315–38. diazinon and malathion by myeloperoxidase. Pesticide Biochem
11. Prabu SM, Muthumani M. Silibinin ameliorates arsenic induced Physiol. 2011;100(2):140–4.
nephrotoxicity by abrogation of oxidative stress, inflammation 32. van Wenum E, Jurczakowski R, Litwinienko G. Media effects on
and apoptosis in rats. Mol Biol Rep. 2012;39(12):11201–16. the mechanism of antioxidant action of silybin and 2,3-dehydro-
12. Andreoli TE, Carpenter CCJ, Cecil RLF. Cecil Essentials of Medicine. silybin: role of the enol group. J Org Chem. 2013;78(18):9102–12.
3rd edMichigan: Saunders; 1995. 33. Sirmali M, Solak O, Tezel C, Sirmali R, Ginis Z, Atik D, et al. Com-
13. Klebanoff SJ. Myeloperoxidase. Proc Assoc Am Physicians. 1999; parative analysis of the protective effects of caffeic acid pheneth-
111(5):383–9. yl ester (CAPE) on pulmonary contusion lung oxidative stress
14. Beyer W, Imlay J, Fridovich I. Superoxide dismutases. Prog Nucleic and serum copper and zinc levels in experimental rat model. Biol
Acid Res Mol Biol. 1991;40:221–53. Trace Elem Res. 2013;151(1):50–8.
15. Tutanc M, Arica V, Yilmaz N, Nacar A, Zararsiz I, Basarslan F, et al. 34. Ezhilarasan D, Karthikeyan S, Vivekanandan P. Ameliorative effect
Effects of erdosteine on cyclosporin-A-induced nephrotoxicity. of silibinin against N-nitrosodimethylamine-induced hepatic fi-
Hum Exp Toxicol. 2012;31(6):565–73. brosis in rats. Environ Toxicol Pharmacol. 2012; 34(3):1004–13.
16. Peresleni T, Noiri E, Bahou WF, Goligorsky MS. Antisense oligode- 35. Shadnia S, Ashrafivand S, Mostafalou S, Abdollahi M. N-acetyl-
oxynucleotides to inducible NO synthase rescue epithelial cells cysteine a Novel Treatment for Acute Human Organophosphate
from oxidative stress injury. Am J Physiol. 1996;270(6 Pt 2):F971–7. Poisoning. Int J Pharmacology. 2011;7(6).

Iran Red Crescent Med J. 2015;17(4):e25310 7