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Research Article
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Ameliorating Role of Rutin on Oxidative Stress Induced by Iron Overload in Hepatic Tissue of Rats |
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Samy Ali Hussein Aziza,
Mohammed El-said Azab
and
Soheir Kamal El-Shall
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ABSTRACT
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Iron is an essential element that participates in several
metabolic activities of cells; however, excess iron is a major cause of iron-induced
oxidative stress and several human diseases. Natural flavonoids, as rutin, are
well-known antioxidants and could be efficient protective agents. Therefore,
the present study was undertaken to evaluate the protective influence of rutin
supplementation to improve rat antioxidant systems against IOL-induced hepatic
oxidative stress. Sixty male albino rats were randomly divided to three equal
groups. The first group, the control, the second group, iron overload group,
the third group was used as iron overload+rutin group. Rats received six doses
of ferric hydroxide polymaltose (100 mg kg-1 b.wt.) as one dose every
two days, by intraperitoneal injections (IP) and administerated rutin (50 mg
kg-1 b.wt.) as one daily oral dose until the sacrificed day. Blood
samples for serum separation and liver tissue specimens were collected three
times, after three, four and five weeks from the onset of the experiment. Serum
iron profiles total iron, Total Iron Binding Capacity (TIBC), Unsaturated Iron
Binding Capacity (UIBC), transferrin (Tf) and Transferrin Saturation% (TS%)},
ferritin, albumin, total Protein, total cholesterol, triacylglycerols levels
and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities
were determined. Moreover, total iron in the liver, L-malondialdehyde (L-MDA),
glutathione (GSH), Nitric Oxide (NO) and Total Nucleic Acid (TNA) levels and
glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD)
activities were also determined. The obtained results revealed that, iron overload
(IOL) resulted in significant increase in serum iron, TIBC, Tf, TS% and ferritin
levels and AST and ALT activities and also increased liver iron, L-MDA and NO
levels. Meanwhile, it decreased serum UIBC, total cholesterol, triacylglycerols,
albumin, total protein and liver GSH, TNA levels and Gpx, CAT and SOD activities
when compared with the control group. Rutin administration to iron-overloaded
rats resulted in significant decrease in serum total iron, TIBC, Tf, TS%, ferritin
levels and AST and ALT activities and liver total iron, L-MDA and NO levels
with significant increases in serum UIBC, albumin, total protein and total cholesterol
levels and in liver GSH, CAT and SOD activities compared with the IOL group.
This study provides in vivo evidence that rutin administration can improve the
antioxidant defence systems against IOL-induced hepatic oxidative stress in
rats. This protective effect in liver of iron-loaded rats may be due to both
antioxidant and metal chelation activities.
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INTRODUCTION
Iron is an essential micronutrient for all living organisms and is involved
in fundamental enzymatic reactions, such as oxygen metabolism, electron-transfer
processes and synthesis of DNA and RNA (Galleano et
al., 2004). In the human body excessive amounts of iron may become very
toxic due to it lacks effective mechanisms to protect cells against iron overload
(Siah et al., 2005).
Iron overload is one of the most common metal-related toxicity (Zhao
et al., 2005). It may be caused by: (1) Defects in iron absorption,
as increase in iron absorption from the diet as in hereditary hemochromatosis
(Beutler, 2007), in which genetic defects alter the
regular absorption of iron from the gut or due to iron excess in diet as in
Bantu siderosis (Gordeuk et al., 2003) (2) Parenteral
iron administration in transfusion-dependent anaemias, as β-thalassemia;
(3) Pathological conditions characterized by increases in iron (Crisponi
and Remelli, 2008).
The liver is the main storage organ for iron, in iron overload, free radical
formation and generation of lipid peroxidation (LPO) products, as iron excess
generate oxidative stress through an increase rate of HO● generation
by the Haber-Weiss reaction (Halliwell and Gutteridge, 1984),
may result in progressive tissue injury as fibrosis (Arezzini
et al., 2003) and eventually cirrhosis or hepatocellular carcinoma
(Siah et al., 2005). In hepatocytes, as excessive
iron deposition also lead to further injuries such as hepatocellular necrosis
(Olynyk et al., 1995). Cases of acute iron
toxicity are rare and mostly related to hepatoxicity (Tenenbein,
2001), which in turn cause the oxidation of lipids, proteins, nucleic acids
(Papanastasiou et al., 2000) that may affect
membrane fluidity and permeability and subsequently cell structure, function
and viability (Maaroufi et al., 2011).
Iron-removal therapy may be achieved by antioxidants, iron chelators and/or
free radical scavenging compounds, as flavonoids (polyphenols) (Blache
et al., 2002), they exert multiple biological effects, including
antioxidant and free radical scavenging activities (Negre-Salvayre
and Salvayre, 1992), in which the metal chelation can be responsible, partly,
both for the antioxidant capacity and some other activities (e.g., inhibition
of lipooxygenases by chelation/reduction of iron in their active sites) (Vasquez-Martinez
et al., 2007). Rutin is a kind of flavonoid glycoside known as vitamin P, a polyphenolic compound
that is widely distributed in vegetables and fruits (Hertog
et al., 1993), it is very effective free radical inhibitor in animal
and human pathological states, such as IOL in rats (Afanasev et al.,
1995), including hepatoprotective (Janbaz et al.,
2002), in which its administration sharply suppressed free radical production
in liver microsomes and by phagocytes in IOL animals (Afanasev et al., 1995) and also could reduce iron content in mouse liver (Gao
et al., 2002). Accordingly, the aim of the present study was to evaluate
the protective role of rutin administration against hepatic oxidative stress
induced in iron-loaded rat models.
MATERIALS AND METHODS
Experimental animals: Sixty white male albino rats of 8-10 weeks old
and weighing 180-220 g were used in the experimental investigation of this study.
Rats were obtained from the Laboratory Animals Research Center, Faculty of Veterinary
Medicine, Benha University, housed in separated metal cages and kept at constant
environmental and nutritional conditions throughout the period of the experiment.
The animals provided with a constant supply of standard pellet diet and fresh,
clean drinking water ad-libitum.
Drug and antioxidants: The drug and antioxidant compounds used in the
present study were:
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Haemojet(R): Haemojet ampoules were produced
by Amriya Pharm. Ind. for European Egyptian Pharma. Ind., Alexandria, Egypt.
Haemojet was obtained as pack of three ampoules of two ml solution. Each
ampoule contains elemental iron (100 mg) as ferric hydroxide polymaltose
complex |
• |
Rutin: Rutin is pale yellow crystalline powder (purity~99%). It
was purchased from EIPICO Egyption Pharmaceutical Industries Company,
10th of Ramadan city, Egypt. Rutin was dissolved in propylene glycol and
administered to animals in daily oral dose of 50 mg kg-1 (b.wt.)
(Fernandes et al., 2010) |
Induction of iron overload: Iron overload was induced by intraperitoneal
injections of six doses (three doses per week) of ferric hydroxide polymaltose
complex of 100 mg kg-1 b.w. (Zhao et
al., 2005).
Experimental design: Rats were randomly divided into three equal groups:
Each group containe twenty rats as follows:
Group I: |
(control group): Received saline
only and served as control for all other groups |
Group II: |
(iron overload): Received six doses (three doses
per week) of 100 mg kg-1 b.wt. of ferric hydroxide polymaltose
administerated as IP-injections |
Group III: |
(iron overload+rutin): Received six doses
(three doses per week) of 100 mg kg-1 b.wt. of ferric hydroxide
polymaltose by IP-injections, followed by daily oral administration of rutin
at a dose level of 50 mg kg-1b.wt. until the sacrificed day |
Sampling: Random blood samples and liver tissue specimens were collected
from all animals groups (control and experimental groups) three times along
the duration of experiment at three, four and five weeks from the onset of rutin
administration.
Blood samples: Blood samples were collected by ocular vein puncture
in dry, clean and screw capped tubes and serum was separated by centrifugation
at 2500 rpm for 15 min. The clean, clear serum was separated and received in
dry sterile samples tube, then kept in a deep freeze at -20°C until used
for subsequent biochemical analysis
Liver tissues: At the end of the each experimental period, rats were
sacrificed by cervical decapitation. The liver specimen was quickly removed
and weighted, then perfused with cold saline to exclude the blood cells and
then blotted on filter study and stored at -20°C. Briefly, half of liver
tissues were cut, weighed and minced into small pieces, homogenized with a glass
homogenizer in 9 volume of ice-cold 0.05 mM potassium phosphate buffer (pH7.4)
to make 10% homogenates. The homogenates were centrifuged at 5,000 rpm for 15
min at 4°C then the supernatant was used for the subsequent biochemical
analysis.
The other half of livers were weighed and putted into glass flask, then 5 volumes
of mixed acid (nitric acid: Perchloric acid, 4:1) were added, heated until large
amount of white vapors could be seen. The volumes of the digested samples were
adjusted to 10 mL with double distilled water, then the obtained solutions were
used to analyze iron contents.
Biochemical analysis: Serum iron and total iron binding capacity (TIBC),
ferritin, albumin, total protein, total cholesterol, triacylglycerols levels
and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities
were determined according to the methods described by Makino
(1988), Dawson et al. (1992), Young
(2001), Gendler (1984), Goodman
et al. (1988), Stein (1987) and Young
(2001), respectively.
Moreover, total iron in the liver, L-malondialdehyde (L-MDA), reduced glutathione
(GSH), Nitric Oxide (NO) and Total Nucleic Acid (TNA) levels and Glutathione
Peroxidase (GPx), catalase (CAT) and Superoxide Dismutase (SOD) activities were
determined according to the methods described by Parker
et al. (1967), Esterbauer et al. (1982),
Beutler (1957), Montgomery and Dymock
(1961), Spirin (1958), Gross
et al. (1967), Sinha (1972) and Packer
and Glazer (1990), respectively.
Statistical analysis: The obtained data were statistically analyzed
by one-way analysis of variance (ANOVA) followed by the Duncan multiple test.
All analyses were performed using the statistical package for social science
13.0 software, (SPSS, 2009). Values of p<0.05 were
considered to be significant.
RESULT
The results presented in Table 1 and 2
revealed that, iron overload resulted in significant increase in serum iron,
TIBC, Tf, TS% and ferritin levels and AST and ALT activities and also increased
liver iron, L-MDA and NO levels. Meanwhile, it decreased serum UIBC, total cholesterol,
triacylglycerols, total protein and albumin and liver GSH and TNA levels and
Gpx, CAT and SOD activities, compared with the control group. Rutin administration
to iron-loaded rats resulted in significant decreased in serum iron, TIBC, Tf,
TS%, ferritin levels, AST and ALT activities and liver iron, L-MDA and NO levels,
with significant increases in serum UIBC, total protein, albumin and total cholesterol
levels and in liver GSH level, CAT and SOD activities, compared with the IOL
group.
DISCUSSION
Iron overload in rats is an excellent model to study the in vivo LPO
in which excess iron induced oxidative stress by increasing lipid peroxide levels
in liver and in serum (Reddy and Lokesh, 1996). Subsequently,
MDA and 8-IP adducts that were formed, significantly contributed to liver damage
that is assessed by AST and ALT levels in the iron-supplemented rats (Asare
et al., 2006). Excess hepatic iron may thus cause peroxidation of
membrane lipids and oxidative liver injury (Abel and Gelderblom,
1998) in which IOL enhances liver injury and accelerates the process of
fibrosis (Arezzini et al., 2003). This tissue
injury can be relieved by the administration of an appropriate chelating agent
which can combine with the iron and increase its rate of excretion (Zhao
et al., 2005), as flavonoids that are substances with both chelating
and free radical scavenging properties (Fraga and Oteiza,
2002) thus, rutin may be a very useful medicine for treatment (Reddy
and Lokesh, 1996).
Serum and liver iron and serum TIBC, Tf, TS% and ferritin levels were significantly
elevated in the iron-loaded rats, while serum UIBC was significantly decreased
(Table 1, 2). These results came in accordance
with the data of Silva et al. (2008) that reported,
serum iron and TS% was 75% higher in rats with iron-dextran treatment when compared
with the untreated control group. Also, Junge et al.
(2001) reported that, acute iron overload elicited significant enhancement
in iron levels of rats livers and Nahdi et al. (2010)
observed that, IOL elicited significant enhancement in serum iron and significant
increase (>10-fold) in liver iron in rats, TS was more than 100%, that most
certainly results in the presence of Non Transferrin Binding Iron (NTBI) catalyzing
the formation of reactive radicals (Zhang et al.,
2006). Moreover, Crisponi and Remelli (2008) found
that, when the iron load increases, the Iron Binding Capacity (IBC) of serum
Tf is exceeded and a NTBI fraction of plasma iron appears which generates free
hydroxyl radicals and induces dangerous tissue damage. Additionaly, Theurl
et al. (2005) reported that, liver ferritin levels were increased
with prolonged iron challenge as iron initially accumulated in spleen macrophages
with subsequent increase in macrophage ferroportin and ferritin expression.
Table 1: |
Effect of treatment with rutin on some biochemical blood parameters
in iron overloaded male rats |
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Data are presented as (Mean±SE), SE: Standard error,
Mean values with different superscript letters in the same row are significantly
different at (p<0.05) |
Thus, iron overload treatment suggesting a novel mechanistic link between dopaminergic
GSH depletion and increased Fe levels based on increased translational regulation
of transferrin receptor 1(TfR1) (Kaur et al., 2009),
in which iron deposition and related damages in liver indicate a strong relation
between alterations of cellular redox condtion/increase in ROS generation due
to GSH depletion with altered iron homeostasis in hepatic cell that led to iron
deposition (Tapryal et al., 2010). However, exess
iron induced increase in hepcidin mRNA level that was not sufficient to prevent
increased intestinal iron absorption and onset of IOL compatible with the observation
that serum iron was very high in that condition and TS was more than 100% that
most certainly resulted in the presence of NTBI (Nahdi et
al., 2010). Also, ferritin induction is expected to occur under conditions
of chronic IOL when intracellular iron is abundant, due to lack of binding of
the iron regulatory protein1 (IRP-1) to Iron-Responsive Elements (IRE) and IRP-2
degradation, resulting in significant translation of ferritin mRNA (Kim
and Ponka, 2003). In cases of iron overload, the natural storage and transport
proteins such as ferritin and transferrin become saturated and overwhelmed and
then the iron spills over into other tissues and organs; At the same time, oxidative
stress arises because of the catalytic activity of the metal ion on producing
high reactive oxygen radicals and finally leads to tissue injury (Zhao
et al., 2005).
When rats were administerated with rutin serum and liver iron and serum TIBC,
Tf, TS% and ferritin concentrations were significantly decreased and serum UIBC
was significantly increased than that of iron-loaded group (Table
1, 2). Similarly, Gao et al.
(1999) found that, iron contents were significantly decreased in the liver
of rutin and baicalin fed rat.
Also, Gao et al. (2002) reported that, oral
adminstration of higher doses of rutin in mice can cause a decrease of serum
iron, copper and zinc concentrations. In addition to Gao
et al. (2003) that reported, the iron contents, in the liver of rutin
or baicalin containing diet (1%) fed rats were significantly decreased. Moreover,
Zhao et al. (2005) observed that, when iron-loaded
mice were supplemented with baicalin, there was a decrease of hepatic total
iron, at the same time, serum nonheme iron was significantly increased, indicating
that baicalin could gradually combine with hepatic non-heme iron and finally
excreted it from the body. Moreover, Zhang et al.
(2006) found that, the increased NTBI in quercetin supplemented mice caused
no further oxidation indicating that the increased serum non-heme iron may come
from flavonoids chelated Fe and although serum ferritin level was still higher
than that of normal mouse, it was significantly decreased compared with IOL-mouse.
Furthermore, Kostyuk et al. (2004) observed
that, quercetin effectively released Fe from ferritin and Fe-flavonoid complexes
are demonstrated to be antioxidants. These results can be attributed to metal
chelating effects of rutin, which are involved in the Fenton reaction (Arjumand
et al., 2011) that can be responsible for the documented antioxidant
capacity of flavonoids (Mladenka et al., 2011),
as these chelation effects of flavonoids are structure specific (Gao
et al., 2003) and suggests that the high reducing power and metal
chelating activities mechanisms may play a key role in the inhibition of oxidative
processes (Lue et al., 2010).
Table 2: |
Effect of treatment with rutin on some biochemical liver parameters
in iron overloaded male rats |
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Data are presented as (Mean±SE), SE: Standard error,
Mean values with different superscript letters in the same row are significantly
different at (p<0.05) |
However, although rutin form chelates with Fe ions, it is hydrolyzed by the
intestinal flora to its corresponding aglycone, quercetin (Prince
and Priya, 2010), which is responsible for its in vivo antioxidant activity,
therefore, radical scavenging activity of rutin may be more important than their
metal chelating activity (Kim et al., 2011).
Iron overload resulted in significant increase in serum AST and ALT activities
compared with the normal control rats. The obtained results are nearly similar
to data reported by Asare et al. (2006) that
showed, in the iron-supplemented rats all of the indices of LPO, including AST
and ALT, were increased significantly (~5-fold) compared with the control. Also,
Silva et al. (2008) observed that, administration
of iron dextran to rats increased AST activity, a marker for mitochondrial lesions
and had no effect on ALT. As AST and ALT were used as sensitive indicators of
liver damage (Mahmoud, 2012), the serum enzymes increased
activities in iron-loaded rats can be attributed to the generation of ROS and
oxidative damage by excess hepatic iron that may result in chronic necroinflammatory
hepatic disease, which in turn generates more ROS and causes additional oxidative
damage (Jungst et al., 2004). Rutin administration
in iron-loaded rats decreased serum transaminases activities (Table
1). Similar results were reported by the data of Fernandes
et al. (2010) that recorded, rutin administration to streptozotocin-diabetic
rats decreased serum ALT and AST activities compared to untreated controls.
Also, Mahmoud (2012) observed that, rutin administration
significantly decreased the levels of AST and ALT activities in hyperammonemic
rats, suggesting protection by preserving the structural integrity of the hepatocellular
membrane against ammonium chloride. Rutin also scavenged free radicals (Cillard
et al., 1990) and inhibiting LPO process (Karthick
and Prince, 2006), in which the iron-rutin complexe studied not only retained
the antioxidant properties of rutin, but in many cases exhibited enhanced free
radical-scavenging activity (Ostrakhovitch and Afanas'ev,
2001).
The obtained results revealed that, iron overload resulted in significant decrease
in serum total protein and albumin levels. The results agree well with those
recorded by Asare et al. (2006) that found,
iron accumulation disrupts the cell redox balance and geneates chronic oxidative
stress, which damages DNA, lipids and protein in hepatocytes leading to both
necrosis and apoptosis. That can be explained by protein oxidation that is known
to give rise to alterations in both the backbone and side chains of the molecule,
leading to the denaturation and loss of biological activities of various important
proteins and cell death (Zhang et al., 2006),
in which ROS may also cause oxidative damage to polyunsaturated fatty acids
of membrane phospholipids, LPO enhanced by high-iron diet (Lafay
et al., 2005), releasing cytotoxic and reactive aldehyde metabolites
as MDA (Asare et al., 2006). Those cytotoxic
products may impair cellular functions, including nucleotide and protein synthesis
(Cheeseman, 1993). This suggestion was confirmed also
by Youdim et al. (2005) that reported, ROS are
capable of oxidizing cellular proteins, nucleic acids and lipids.
Rutin administration to iron-overloaded rats induced significant increases
in serum total protein and albumin concentrations compared with the IOL-group.
Similar results were reported by Zhang et al. (2006)
that recorded ring the treatment with baicalin and quercetin in IOL-induced
mice liver injury, the reduction of liver protein oxidation can be considered
as a sign of protection under IOL. Kamalakkannan and Prince
(2006) also observed that, oral administration of rutin to diabetic rats
lead to significant increase in the plasma total protein and albumin concentrations
when compared with the diabetic control. The antioxidant activity of rutin in
Fenton reaction (Caillet et al., 2007) may explain
the reduction of protein oxidation, as a mechanism of action preventing protein
oxidation, in which the inhibitive effects of flavonoids on protein oxidation
may come from the combination of both iron eliminating and direct free radical
scavenging activities (Zhang et al., 2006).
Serum total cholesterol and triacyglycerols levels were significantly decreased
in the iron loaded rats. These obtained results may be explained by Silva
et al. (2008) that reported, hepatic injury triggered by iron excess
may increase the concentration of secondary serum metabolites, such as cholesterol,
triacylglycerols and glucose and also recorded that, treatment with Fe-dextran
in male rats increased serum triacylglycerols level, but had no effect on the
cholesterol evel. However, Turbino-Ribeiro et al.
(2003) reported that, absence of alteration in serum cholesterol in rabbits
receiving Fe-dextran injections.
When rats were administerated with rutin, serum total cholesterol and triacyglycerols
levels were significantly increased than that of iron loaded control group.
Contradictory results were obtained by Fernandes et al.
(2010) reported that, serum total cholesterol and Low Density Lipoprotein
(LDL)-cholesterol levels were lowered in the rutin-treated diabetics rats group.
Also, Park et al. (2002) recorded that, supplementation
of 0.1% rutin and tannic acid significantly lowered both plasma total cholesterol
and triacylglycerols compared with control. These results may be explained by
the lose of the amphiphilic properties of rutin, that be less capable of scavenging
free radicals from the most lipophilic regions of the LDL particle (cholesterol
esters and triacylglycerols) (Lue et al., 2010).
In which the decrease in cholesterol level was due exclusively to the LDL and
VLDL fraction (Aggarwal and Harikumar, 2009). On the
other hand, Jiang et al. (2007) showed a dose-response
effect of rutin in inhibiting LDL peroxidation and the reduced CAT activity
in the rutin and tannic acid treatment might lead to less cholesteryl ester
being available for VLDL packing, thereby resulting in a reduction in its secretion
from the liver (Carr et al., 1992).
Liver L-MDA concentrations were significantly elevated in the iron-loaded rats
as compared with control group. The obtained results are nearly similar to those
of Kokoszko et al. (2008) who obseved that,
the injection of FeCl or FeSO4 (3 mg Fe2+/100 g b.w.)
significantly increased LPO products in the rat liver. Also, Nahdi
et al. (2010) reported that, IOL in rats was accompanied by enhancement
in LPO as shown by a significant increase in all tissue MDA concentration in
iron supplemented group except the spleen and added that, in the liver there
was a perfect correlation between MDA level and tissue iron content, suggesting
production of oxidative stress. LPO generated by ROS was measured in terms of
MDA (Arjumand et al., 2011), that is a secondary
end product of the oxidation of polyunsaturated fatty acids (Yang
et al., 2008). Focusing on the liver organ, the cytotoxic degradation
products as MDA and 4-hydroxy-2-non-enal (4-HNE) (Esterbauer
et al., 1991) can form covalent adducts with proteins, phospholipids
and DNA (Guichardant et al., 1998), in which
the formation of liver microsomal MDA protein adducts, during IOL in mice and
the microsomal function impairment may alter protein function and might lead
to cellular injury and Fe-associated hepatotoxicity (Valerio
and Petersen, 1998). When rats were administerd with rutin hepatic L-MDA
concentrations were significantly decreased than that of iron-loaded control
group (Table 2). The obtained results agree with the data
of Ostrakhovitch et al. (1995) that reported,
rutin administration in IOL-rats sharply decreased microsomal LPO in the liver
and spontaneous oxygen radical production by peritoneal macrophages. Also, Gao
et al. (2003) reported that, LPO level in the liver of the rutin fed
group was significantly decreased in comparison to the control group, As iron
overload led to enhancement in LPO (Nahdi et al.,
2010), some flavonoids can be both antioxidants and iron chelators; it means
that flavonoids will be good candidates for curing IOL related diseases, as
they can play a double role in reducing the rate of oxidation, one act as iron
chelator (Borsari et al., 2001) and the other
act as radical trap (Van Acker et al., 1998). Afanasev
et al. (1995) also found that, rutin administration sharply suppressed
free radical production in liver microsomes by phagocytes in iron overload rats. This antioxidant activity
due to the inactive iron-rutin complexes of rutin that may be a good in vivo
antioxidant and may be more effective free radical scavengers compared to the
parent rutin (Ostrakhovitch and Afanas'ev, 2001). The
sharp decrease in L-MDA in IOL-rats may be also attributed to the free radical
scavenging property of rutin, in which IOL in rats induces the oxidative stress
that was characterized by oxygen radical overproduction in liver microsomes,
peritoneal macrophages and blood neutronphils (Mahmoud,
2012).
The obtained results revealed that, IOL resulted in significant decrease in
liver GSH levels when compared with the control group. The obtained data are
nearly similar to those reported by Poli et al.
(2004) that observed, the increase in protein carbonylation and reduction
in GSH content as well as in the GSH/GSSG ratio of the liver were observed after
6 weeks of treatment probably induced by iron-generated free radical activity
(Pietrangelo, 2003). Also, Jagetia
and Reddy (2011) reported that, introduction of Fe into mouse liver mitochondrial
fraction caused a time dependent depletion in GSH (~2-fold) lower than control
at 30 min post treatment. GSH is a major non-enzymatic tripeptide multifunctional
intracellular antioxidant (Morimoto et al., 2008)
that was often used as an estimation of the redox environment of the cell (Schafer
and Buettner, 2001). Iron deposition and related damages in liver indicate
a strong relation between alterations of cellular redox condtion/increase in
ROS, e.g., Fe-induced free radical (Pietrangelo, 2003)
due to GSH depletion, suggesting a novel mechanistic link between dopaminergic
GSH depletion and increased iron levels (Kaur et al.,
2009); In conclusion, reduced GSH functions intracellularly to reduce numerous
oxidizing compounds, including ROS (Gong et al.,
2010).
Rutin administration to iron-overloaded rats resulted in significant increase
in liver GSH level compared with the IOL-group. Similarly, Korkmaz
and Kolankaya (2010) observed that, rutin pretreatment significantly protected
against the severe depletion of GSH content and MnSOD activity in the I/R-induced
damage in rat kidney. Moreover, Arjumand et al.
(2011) reported that, rutin treatment in cisplatin administered rats showed
significant improvement in GSH concentration, suggesting its role in scavenging
the free radicals generated by cisplatin-induced renal inflammation and apoptosis.
The antioxidant imbalance was compensated by the prophylactic treatment of rutin
as excessive LPO can cause increased GSH consumption (La
Casa et al., 2000), this inhibitory effects of rutin on in vivo free
radical production in IOL-rats are probably explained by its ability to form
inactive iron-rutin complexes (Afanas'ev et al.,
1989).
The obtained results revealed that, iron overload resulted in significant decrease
in liver Gpx, CAT and SOD activities when compared with the control group. Iron
overload can destruct the balance between prooxidants and antioxidants, leading
to severe loss of total antioxidant status level. This phenomenon can be seen
in most iron overload animal models (Dabbagh et al.,
1994) as well as iron overload disease such as hereditary haemochromatosis
(Young et al., 1994) and thalassemia (Livrea
et al., 1996).
Catalase is an iron-containing antioxidant enzyme, it was reported that under
iron overload, there was a significant decrease of catalase activity in rat
liver (Galleano and Puntarulo, 1997). Likewise, Zhao
et al. (2005) who showed that, Fe-dextran injection in mouse caused
a significant decrease in hepatic CAT activity. Moreover, Valko
et al. (2006) reported that, GPx significantly competes with CAT
for H2O2 substrate and it is the major source of protection
against low levels of oxidative stress-induced cancer. These results can be
explained as the ROS generated during normal cellular processes are immediately
detoxified by endogenous antioxidants like GSH, CAT, GPx, glutathione reductase,
glutathione-S-transferase, etc. (Kim et al., 2006).
SOD work in conjunction with CAT and GPX (Michiels
et al., 1994), preventing its interaction with Fe and therefore formation
of the highly toxic ●OH, in which SOD and GPX are
supportive enzyme system of the first line cellular defense against oxidative
injury (Kalpravidh et al., 2010). GPx decomposes
peroxides, that initiate a chain of free radical formation (Jagetia
and Reddy, 2011) to H2O (or alcohol) while simultaneously oxidizing
GSH; Significantly, GPx competes with CAT for H2O2 substrate,
as it is the major source of protection against low levels of oxidative stress
(Valko et al., 2006) and the increase of SOD/GPx
ratio in Fe treated cells compared to control cells indicated that Fe-induced
oxidative injury appeared, that might be indicative of ROS increase due to unefficient
scavenging by enzymes (Formigari et al., 2007).
Rutin administration to iron-loaded rats resulted in significant increases
in liver CAT and SOD activities compared with the IOL group (Table
2). As natural antioxidants, flavonoids intake may increase total antioxidant
status level in living body, supplementation of baicalin or another flavonoid,
rutin, could increase hepatic total antioxidant status level in rats and mice
(Gao et al., 2003; Zhao
et al., 2005). This effect may come from the chelation of free iron
ion with stopping iron-catalyzed oxidative reaction and the direct increasing
antioxidant status by baicalin and its metabolites that acte as strong antioxidant.
Thus, the baicalin supplementation introduced a new source of antioxidant and
could partly inhibit peroxidation-induced heme destruction and then provided
a protection on catalase (Zhao et al., 2005).
The significant increases in liver antioxidant enzymes in rutin treated iron-loaded
rats are in conformity with the data reported by Park et
al. (2002) that recorded, dietary rutin and tannic acid have a significant
effect on SOD and GPx in rats. As a concomitant increase in CAT and/or GPx activity
is essential if a beneficial effect from the high SOD be expected. Mahmoud
(2012) found that, hyper-ammonemic rats pretreated with rutin significantly
increased liver CAT and GPx. Chronic iron administration induced adaptive responses
involving stimulation of the antioxidant defenses. Rutin significantly inhibited
LPO in IOL-microsomes (Afanasev et al., 1995)
that may be due to the free radical scavenging property (Mahmoud,
2012). The three enzymes can prevent damage by detoxifying ROS. The significant
elevation of SOD activity also suggests that its free-radical scavenging activity
is only effective when it is accompanied by an increase in activity of CAT and/or
GPx activity, because SOD generates H2O2 as a metabolite
(Park et al., 2002), which is more toxic than
oxygen radicals in cells and needs to be scavenged by CAT or GPx (Pigeolet
et al., 1990).
Iron overload resulted in significant increase in liver NO levels when compared
with the control group. Similar results were recorded by Cornejo
et al. (2001) that found, increased NO generation was evidenced in
the liver under conditions of acute IOL. Moreover, Vadrot
et al. (2006) explored that, increased production of NO by nitric
oxide synthase-2 was demonstrated in patients with hepatic cell carcinoma complicating
hereditary haemochromatosis. That can be explained as NO is an inorganic reactive
nitrogen species synthesized in liver by inducible nitric oxide synthase (iNOS)
found in hepatocytes, Kupffer cells and endothelial cells (Alderton
et al., 2001), whose expression is controlled by the redox-sensitive
transcription factor, nuclear factor-kappa B (Kleinert
et al., 2004) and the complex interrelationships between Fe and NO
(Galleano et al., 2004) can result in changes
in in vivo NO production (Kagan et al., 2001).
Also the increase in rat liver NOS activity due to chronic iron overload (Cornejo
et al., 2007) is related to upregulation of iNOS expression (Cornejo
et al., 2005). Rutin administration to iron-overloaded rats resulted
in significant decreased liver NO levels compared with the IOL group. The data
obtained are in harmony with Chen et al. (2001a,
b) and Shen et al. (2002)
that showed, rutin inhibit lipopolysaccharide-induced NO production. NO was
proposed to act as a pro-oxidant at high conc. (Morand
et al., 2000), or when it reacts with O2●B,
forming the highly reactive peroxynitrite (Radi et al.,
2001) that is suppressed by flavonoids by direct scavenging (Haenen
et al., 1997).
Iron overload resulted in significant decreased in liver TNA levels when compared
with the control group. Similarly, Youdim et al.
(2005) explored that, iron is a major generator of ROS that lead to damage
of lipids, proteins carbohydrates and nucleic acids. Park
and Park (2011) reported that, Ferric-nitrilotriacetate markedly induced
DNA damage in human leukocytes in vitro and rat leukocytes in vivo. The
decrease in total nuclic acid level can be attributed to the primary generation
of H2O2 and ●OH, due to Fe-induced oxidative
stress, that damages DNA and other biomolecules (Huang,
2003). As ●OH may damage the nucleotide bases themselves,
resulting in oxidized base products such as 8 oxo-guanine and fragmented or
ring-opened derivatives (Lu et al., 2001). Iron
also is thought to be involved in β-cleavage of lipid hydroperoxides, producing
biogenic aldehydes that interact with DNA to form exocyclic products (Kew,
2009) that trigger free radical-mediated chain reaction including LPO, DNA
damage and protein oxidation (Gutteridge and Halliwell,
2000).
Rutin administration to iron-overloaded rats, non significantly increased liver
TNA concentration compared with the IOL-group. The obtained results are of the
same harmony with the data of Undeger et al. (2004)
that reported, rutin can prevent damage to DNA as it may modulate the enzymes
necessary for activation of carcinogens. Also, Gong et
al. (2010) observed that, rutin (50 μM) blocked H2O2-induced
apoptosis in human umbilical vein endothelial cells and thus protecting DNA
damage. Moreover, Omololu et al. (2011) reported
that, rutin protect the stability of the genome. This protection against DNA
damage is particularly important since oxidative damage to DNA, especially strand
breaks, is highly dependent on the amount of Fe bound to DNA (Diaz-Castro
et al., 2010), in which polyphenols, due to their ability to coordinate
iron, are one large class of antioxidants that has been extensively examined
for treatment and prevention of conditions associated with iron-generated ROS
and oxidative stress (Perron and Brumaghim, 2009),
as iron chelation was responsible for prevention of nuclear DNA damage by quercetin
(Sestili et al., 1998).
CONCLUSION
From the obtained results it could be concluded that, the natural flavonoids,
rutin inhibited the adverse effect of ferric ion induced oxidative stress by
reducing protein and DNA oxidation and inhibition of lipid peroxidation in liver
tissue due to its marked hepatoprotective role in the experimental rats. Rutin
treatment improved the cytoprotective enzymatic and non-enzymatic antioxidants
as revealead by elevated the GSH concentration, GPx, CAT and SOD activities
and thus, might protect the cellular environments from iron-induced free radical
damage. The results indicate that, rutin may have potential effects in inhibiting
the iron-induced oxidative stress in human. The protective effect of rutin on
livers of iron-overloaded rats may be due to its high antioxidant activity,
including both its radical scavenging and iron chelation activities. So we recommended
using rutin-enriched food regularly with additional research work for medicine
manufacturing for protection against the bad complications of IOL-induced oxidative
stress.
|
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