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Toxicologic Pathology
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Protective Effect of β-Carotene on Methotrexate−Induced Oxidative Liver Damage
Nigar Vardi, Hakan Parlakpinar, Asli Cetin, Ali Erdogan and I. Cetin Ozturk
Toxicol Pathol 2010 38: 592 originally published online 6 May 2010
DOI: 10.1177/0192623310367806
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Toxicologic Pathology, 38: 592-597, 2010
Copyright # 2010 by The Author(s)
ISSN: 0192-6233 print / 1533-1601 online
DOI: 10.1177/0192623310367806
Protective Effect of ␤-Carotene on
Methotrexate–Induced Oxidative Liver Damage
NIGAR VARDI,1 HAKAN PARLAKPINAR,2 ASLı
CETIN,
1
ALI ERDOGAN,3
AND
I. CETIN OZTURK4
1
Department of Embryology and Histology, Inonu University, Malatya, Turkey
2
Department of Pharmacology, Inonu University, Malatya, Turkey
3
Department of Chemistry, Inonu University, Malatya, Turkey
4
Department of Biochemistry, Inonu University, Malatya, Turkey
ABSTRACT
In this study, the authors aimed to investigate the role of oxidative stress on the hepatic damage caused by methotrexate (MTX) and the
possible protective effects of b-carotene against this damage. The rats were divided into four groups as control, MTX (20 mg/kg ip), b-carotene
(10 mg/kg/day ip) þ MTX, and b-carotene. Histopathologic alterations were evaluated for defining the liver damage. The tissue, malondialdehyde
(MDA), superoxide dismutase (SOD), and glutathione peroxidase (GP-x) contents and serum aspartate aminotransferase (AST) and alanine aminotranferase (ALT) activities were also examined. Histopathologic damage for each group score findings have been determined as control: 0.66 +
0.33; MTX: 7.0 + 0.68; b-carotene þ MTX: 3.3 + 0.42; and b-carotene: 0.5 + 0.3. In the MTX-treated group, MDA, AST, and ALT values were
increased, while SOD and GP-x values were decreased compared with the control group. In the b-carotene þ MTX-treated group, AST and ALT
values significantly decreased, while all other parameters were similar to the control group. This study shows that b-carotene has a protective
effect on MTX-induced oxidative hepatic damage. Consequently, it seems that an antioxidant agents like b-carotene may be useful in decreasing
the side effects of chemotherapy.
Keywords:
antioxidants; b-carotene; methotrexate; liver; oxidative stress.
they contain. Although more than 600 different compounds
have been characterized, until now b-carotene is the most
potent one of the antioxidants (Stahl and Sies 2003). b-carotene
exhibits antioxidant activity by suppressing singlet oxygen,
scavenging peroxide radicals, and directly reacting with peroxy
radicals, thus stabilizing membrane lipids from free radical
attack (Bast, Haenen, and Van den Berg 1998).
This experimental study was designed to clarify the role of
oxidative stress in MTX-induced hepatic damage and the
possible protective effect of b-carotene against it using histological and biochemical parameters.
INTRODUCTION
Methotrexate (MTX), which is a folic acid antagonist, is
used commonly as a cytotoxic agent in the treatment of leukemia and other malignancies as well as in the inflammation diseases such as psoriasis and rheumatoid arthritis in low doses
(Bayram et al. 2005; S¸ener et al. 2006a). With the widespread
use of MTX, although hepatotoxicity is the most important
potential major side effect (West 1997). The toxicity of MTX
in the liver seems to relate to the generation of reactive oxygen
species (ROS) (S¸ ener et al. 2006a; Uraz et al. 2008). However,
the number of laboratory animal studies in which microscopic
and biochemical changes have been evaluated together are
scarce.
There is an abundance of evidence that the regular consumption of fruit and vegetables is associated with a reduced risk of
chronic and degenerative diseases. For this reason, many
researchers have been focused on this subject (Vardi et al.
2008; Keen et al. 2005; Ramos et al. 2005). The protective
effects of fruits and vegetables are thought to derive from vitamins and carotenoid, flavonoid, and phenolic composites that
MATERIAL
AND
METHODS
Experimental Conditions
For this study, 24 Wistar albino rats weighing 210–310 g
were obtained from Inonu University Laboratory Animals
Research Center. The rats were kept at a temperature of 21 +
2 C and relative humidity of 60 + 5% in a 12-hour light/dark
cycle. The experiments were carried out according to the
standards of animal research of the National Health Research
Institute and with the approval of the Inonu University Ethical
Committee. Rats were divided into 4 groups randomly, each
group including 6 animals.
Group I (control) rats received the equivalent volumes of
saline intraperitoneally (ip).
Group II (b-carotene) rats received b-carotene (10 mg/kg/
day ip) for 24 days. The dose of b-carotene used in this study
was selected on the basis of a previous related study (Matos
Address correspondence to: Nigar Vardi PhD, Department of Embryology
and Histology, Faculty of Medicine, Inonu University, 44280, Malatya,
Turkey; e-mail: [email protected].
Abbreviations: MTX, methotrexate; MDA, malondialdehyde; SOD,
superoxide dismutase; CAT, catalase; GP-x, glutathione peroxidase; AST,
aspartate aminotransferase; ALT, alanine aminotranferase; ROS, reactive
oxygen species; H-E, hematoxylin-eosin; NADP, nicotinamide adenosine
diphosphate; NADPH, nicotinamide adenosine diphosphate hydrogen; GSH,
glutathione.
592
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ANTIOXIDANT AGAINST LIVER DAMAGE
et al. 2006). b-carotene was obtained from Sigma (St. Louis,
MO, USA).
Group III (MTX) rats were given MTX (David Bull
Laboratories, Mulgrave-Victoria, Australia) as a single ip dose
(in saline, 20 mg/kg) on day 21 of the experiment.
Group IV (b-carotene þ MTX) rats were given b-carotene
by ip injection in vehicle (saline) 10 mg/kg/day for 24 days and
were further administered MTX at a dose level of 20 mg/kg on
day 21 of the experiment.
At the end of the 24th day, all the rats were sacrificed with
cervical dislocation; their blood samples and liver tissues were
excised. Tissue samples were fixed in 10% neutral buffered
formalin during 24 hours and embedded in paraffin. For biochemical analysis, the centrifuged serum from blood (4 cc)
taken from the inferior vena cava was stored at –30 C for
aspartate amino transferase (AST) and alanine amino transferase (ALT) measurements, and the liver tissues that folio covered were stored at –30 C until assay for MDA, SOD, and
GP-x measurements. The sections taken from the paraffin
blocks were examined by Leica DFC-280 microscope following staining with hematoxylin-eosin (H-E) and Masson trichrome staining method.
Histological Analysis
Score of liver damage severity was semi-quantitatively
assessed as follows: disruption in radial arrangement around
central vein, sinusoidal dilatation, and nuclear changes in hepatocytes. The microscopic score of each tissue was calculated as
the sum of the scores given to each criteria. Scores were given
as absent (0), slight (1), moderate (2), and severe (3) for each
criteria. Maximum score is noted as 9.
Biochemical Assessment
After the decomposed liver tissue sections were stored in
deep freeze, 200 mg of liver tissue was homogenized over the
ice particules in the glass and then centrifugation approximately 10 min for assessment of enzyme activities and lipid
peroxidation.
593
of H2O2 per min at 37 C and specific activity corresponding to
transformation of substrate (in mmol) (H2O2) per min per mg
protein.
GP-x Assay
GP-x activity was determined in a coupled assay with glutathione reductase by measuring the rate of NADPH oxidation
at 340 nm using H2O2 as the substrate (Lawrance and Burk
1976). Specific activity is given as the amount of NADPH
(mmol) disappeared per min per mg protein.
Lipid Peroxidation Assay (MDA)
The analysis of lipid peroxidation was carried out as
described by Buege and Aust (1978) with a minor modification. The absorbance of the supernatant was recorded at
532 nm. MDA results were expressed as nmol mg–1 protein
in the homogenate. The protein content of samples was determined using the colorimetric method of Lowry et al. (1951)
using BSA as the standard.
AST and ALT
Determination of the plasma concentrations of the liver
enzymes ALT and AST were measured in serum samples
obtained from all groups of rats. Activities were expressed as
IUL–1. The measurements were done in accordance with the
methods of the diagnostic kits (Biolabo Reagents, Maizy,
France).
Statistical Analysis
A computer program (SPSS 11.0) was used for statistical
analysis. The results were compared with Kruskal-Wallis variance analysis. Where differences among the groups were
detected, group means were compared using the MannWhitney U (Bonferroni) test. Values of p < .05 were considered
significant. All results were expressed as means + standard
error (SEM).
RESULTS
SOD Assay
Light Microscopic Evaluations
SOD activity was assayed using the nitroblue tetrazolium
(NBT) method of Sun, Oberley, and Li (1988). The samples
were subjected to ethanol-chloroform (62.5/37.5%) extraction
prior to the enzyme activity assay. NBT was reduced to blue
formazan by O2–, which has a strong absorbance at 560 nm.
One unit (U) of SOD is defined as the amount of protein that
inhibits the rate of NBT reduction by 50%. The calculated SOD
activity was expressed as U/mg protein in the tissue.
CAT Assay
CAT activity was measured at 37 C by following the rate of
disappearance of hydrogen peroxide (H2O2) at 240 nm (e240 ¼
40 M–1 cm–1) (Luck 1963). One unit of CAT activity is defined
as the amount of enzyme catalyzing the degradation of 1 mmol
In the livers of rats from the control and b-carotene-treated
rats, hepatocytes showed a normal histological appearance
(Figure 1). In the MTX-treated rats, the normal radial arrangement of hepatocytes from central vein were abnormal
(Figure 2). Foci of apoptopic cells were detected in some slides
inside the lobule (Figure 3). Another conspicuous finding in
this group was the difference in the appearance of the hepatocyte nucleus. Shrinkage and inflection were observed in the
nuclear contour, and condensation in the structure of chromatin
was also observed (Figure 4). In the sinusoid lumen of some
sections, dilatation was observed (Figure 5). In rats given
b-carotene þ MTX, affected to a significantly less degree
than those in the group given MTX alone (Figure 6). The histological appearance of the sinusoidal lumen in the b-carotene
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594
VARDI ET AL.
TOXICOLOGIC PATHOLOGY
FIGURE 1.—Normal histological appearance of liver in control group. H-E X20. FIGURE 2.— Disruption of radial arrangement of hepatocytes in MTX
group. H-E X20. FIGURE 3.— Apoptotic cell (star) and apoptotic body (arrow) distinguished by dense eosinophilic cytoplasm and pyknotic nucleus in
MTX group. H-E X100. FIGURE 4.— Nucleus with irregular contour and dense chromatin structure (arrows) in MTX group. H-E X100. FIGURE 5.— Dilatation in sinusoids (arrows) observed in MTX group. H-E X20. FIGURE 6.— Radial hepatocytes from central vein to periphery in b-carotene þ MTX group.
H-EX20. FIGURE 7.— The appearance of sinusoids in b-carotene þ MTX group (arrows). PV: Portal vein, AH: Hepatic artery, BD: Bile duct. Masson
trichrome staining X40. FIGURE 8.— Hepatocytes whose nucleus (arrows) are euchromatic and its contours regular in b-carotene þ MTX group. H-E X100.
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ANTIOXIDANT AGAINST LIVER DAMAGE
595
TABLE 1.—Comparison of the effect of b-carotene on microscopic damage caused by MTX in liver.
Parameters
b-carotene
Control
Microscopic damage
0.66 + 0.33
0.5 + 0.3
b-carotene þ MTX
MTX
a
3.33 + 0.42b,c
7.0 + 0.68
a
Significantly increased when compared with control group, p ¼ .003.
Significantly decreased when compared with MTX group, p ¼ .007.
c
Significantly increased when compared with control group, p ¼ .004.
b
TABLE 2.—Comparison of the effect of b-carotene on lipid peroxidation caused by MTX and the changes in antioxidant enzyme parameters.
Parameters
b-carotene
Control
1.6 +
38.6 +
180.8 +
14.1 +
MDA (nmol/mg protein)
SOD (U/mg protein)
CAT (U/mg protein)
GP-x (mmol/mg protein)
0.07
2.4
9.8
0.46
1.7 +
43.2 +
129.3 +
15.0 +
0.1
3.0
8.5
0.4
b-carotene þ MTX
MTX
2.1 +
31.2 +
104.3 +
10.2 +
a
0.08
1.5b
4.1b
0.4b
1.7 +
38.4 +
113.2 +
12.7 +
0.09c,e
0.7d,e
5.9f
0.5d,e
a
Significantly increased when compared with control group, p < .05.
Significantly decreased when compared with control group, p < .05.
c
Significantly decreased when compared with MTX group, p < .05.
d
Significantly increased when compared with MTX group, p < .05.
e
Not significant when compared with control group, p > .05.
f
Not significant when compared with MTX group, p > .05.
b
TABLE 3.—Comparison of the effect of b-carotene on the changes in AST and ALT enzyme parameters caused by MTX.
Parameters
AST (IU/L)
ALT (IU/L)
a
b
b-carotene
Control
2.5 + 0.2
1.8 + 0.1
b-carotene þ MTX
MTX
a
2.1 + 0.2
1.6 + 0.4
18.8 + 6.7
15.2 + 4.2a
4.6 + 1.2b
3.6 + 0.9b
Significantly increased when compared with control group, p ¼ .004.
Significantly decreased when compared with MTX group, p ¼ .01.
þ MTX group was also similar to that in the control group
(Figure 7). Most of the nuclei of hepatocytes were euchromatic
and normal shape (Figure 8). Apoptotic cells were not observed
in this group. However, b-carotene treatment did not completely alleviate the lesions, and milder degenerative alterations were still present. The microscopic damage score for
each group was determined in the histological section, and
results are given in Table 1.
( p ¼ .26) levels. AST and ALT activities showed increases
in rats given MTX alone in comparison with the levels in
control rats ( p ¼ .004). These values were decreased in the
b-carotene þ MTX group but were still higher than those seen
in control animals. This decrease was found significant when
compared with MTX group ( p ¼ .016).
DISCUSSION
Biochemical Results
Biochemical results are given in Tables 2 and 3.
In the MTX group, the level of MDA, the end product of
lipid peroxidation, significantly increased ( p ¼ .008), while
SOD ( p ¼ .03), CAT ( p ¼ .004), and GP-x ( p ¼ .004) activities significantly decreased compared with the livers of rats in
the control group. When rats were given b-carotene þ MTX,
their biochemical parameters were statistically different from
those of the MTX-alone group. b-carotene administration
caused significant decreases in the hepatic MDA levels ( p ¼
.019) and increases in SOD ( p ¼ .004) and GP-x ( p ¼ .016)
enzyme activities when compared with the levels in the livers
from rats given MTX alone. But b-carotene administration
before MTX had no statistically significant effect on CAT
The results of the studies indicate that MTX causes
oxidative tissue damage by increasing lipid peroxidation in the
liver tissue and decreasing the level of antioxidant enzymes.
Moreover, increased AST and ALT values, biochemical indicators of liver damage, and histopathological findings supported this conclusion. It has also been shown that b-carotene
given for 21 days before the MTX application provided significant protection from the hepatotoxicity of MTX.
Drugs used for cancer chemotherapy are well known to
produce acute toxic side effects in multiple organ systems.
The most common target organs are tissues that contain selfrenewing cell populations such as bone marrow, gastrointestinal tract, mucosal membranes, and hair follicles (Kim, Kim,
and Chung 1999). It has been reported that liver damage may
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596
VARDI ET AL.
occur as well in particular high doses or following chronic
administration of MTX (S¸ ener et al. 2006b; Uraz et al. 2008).
The conversion of MTX to its major extracellular metabolite, 7-hydroxymethotrexate, takes place in the liver, where it
is oxidized by a soluble enzymatic system. Inside cells, MTX
is stored in a polyglutamated form. Long-term drug administration can cause accumulation of MTX polyglutamates and
decreased folate levels. The presence of higher levels of polyglutamates causes a longer intracellular presence of the drug,
and this has been suggested as a mechanism for MTX
hepatotoxicity (Johovic et al. 2003). Moreover it is known that
oxidative stress plays a role in the tissue damage caused by
MTX (S¸ ener et al. 2006b; Johovic et al. 2003; Cetinkaya
et al. 2006). It is demonstrated that the cytosolic nicotinamide
adenosine diphosphate (NADP)–dependent dehydrogenases
and NADP malic enzyme are inhibited by MTX, suggesting
that the drug could decrease the availability of NADPH (nicotinamide adenosine diphosphate hydrogen) in cells (Johovic
et al. 2003). Under normal conditions, NADPH is used by
glutathione reductase to maintain the reduced state of cellular
(GSH) glutathione, an important cytosolic antioxidant. Thus,
the significant reduction in glutathione (GSH) levels promoted
by MTX could lead to a reduction of effectiveness in the antioxidant enzyme defense system, sensitizing the cells to ROS
(Babiak et al. 1998). In this study, it has been shown that MTX
administration increases the level of MDA in the liver and
decreases the enzyme activities of SOD and GP-x capacity.
In previous studies (Vardi et al. 2008, 2009), we have observed
similar effects of MTX on intestinal and testicular tissue. This
finding is in agreement with several reports demonstrating that
MTX induces oxidative stress in tissues as demonstrated by
increasing MDA levels (Uraz et al. 2008; Johovic et al. 2003;
Cetinkaya et al. 2006; Cetin et al. 2008) and decreasing SOD
activities (Cetinkaya et al. 2006; Cetin et al. 2008). The oxidative damage increasing with MTX in liver tissue was prevented
by dosing b-carotene for 21 days prior to MTX administration.
It plays an important role in protecting cell membrane against
oxidative damage because of its property of scavenging lipid
and peroxyle radicals (El-Demerdash et al. 2004; Sergio and
Russel 1999). Berryman et al. (2004) reported that in the liver
of diabetic rats, MDA levels significantly increased when compared a control group, whereas the addition of b-carotene
returned levels back to normal. In this same study, the addition
of b-carotene also increased the activity of GP-x. Similar to our
findings, b-carotene is reported to increase antioxidant
enzymes in tissues, or prevent their decrease in a series of
different experiment models (Vardi et al. 2008, 2009;
El-Demerdash et al. 2004; Kheir-Eldin et al. 2001). Under the
current experimental conditions, pretreatment with b-carotene
may have protected the hepatocyte membrane integrity by preventing the peroxidation of fatty acids by free radicals and inhibiting an important mechanism that causes liver damage. In our
study, after MTX application, AST and ALT levels, known
indicators of hepatic damage, had increased 7 fold compared
with the control group. It is known that in the use of drugs causing liver toxicity transaminase levels increase in the plasma
TOXICOLOGIC PATHOLOGY
depending upon the degree of hepatic damage. Kuvandik
et al. (2008) reported that the levels of AST and ALT in plasma
serum have increased significantly in acetaminophen hepatotoxicity; and Uraz et al. (2008) reported that MTX also
increased ALT levels. In our study; the level of AST and ALT
in the group supplied with b-carotene was statistically lower
than in those animals given MTX alone. Sugiura et al.
(2006), reported that carotenoids may prevent increases of
serum aminotransferase increase in the early periods of liver
damage in hyperglycemic patients. In our microscopic investigations in the livers of rats given MTX, hepatic nuclei were
larger and karyolemma contours were irregular; and in some
areas, apoptopic hepatocytes could be seen. MTX acts as a
dihydrofolic acid analogue that binds to the dihydrofolic acid
reductase enzyme by inhibiting the synthesis of tetrahydrofolate, which is required for DNA synthesis (Babiak et al.
1998). De novo inhibition of purine and pyrimidine synthesis
leads to DNA defects, which results in apoptosis (Tsurusawa,
Saeki, and Fujimoto 1997). Increased oxidative stress may
cause shape and structural changes of the nucleus by causing
DNA fragmentation and denaturation, which play a critical role
in the initiation of apoptosis (Prahalathan, Selvakumar, and
Varalakshmi 2005). It has been shown that production of ROS
may mediate a signal for apoptotic cell death (Vardi et al.
2009). The current study has shown that b-carotene þ MTX
group nucleus changes were limited, and apoptosis was not
observed. b-carotene may have protected cells from DNA
changes and apoptosis with the ability of connecting singlet
oxygen radicals and scavenging peroxyle radicals.
As a result, b-carotene probably protects from
MTX-induced liver damage by scavenging of free radicals.
Therefore, b-carotene might be an effective therapy in protecting the liver in patients given MTX treatment. Although further
experimental and clinical studies are required to confirm these
findings.
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