Medical Technology SA
| Volume 24 No. 1 | June 2010
peer reviewed ORIGINAL ARTICLE
EFFECTS OF METHOTREXATE ON ANTIOXIDANT ENZYME STATUS IN A
RODENT MODEL
C.L Coleshowers1, O.O Oguntibeju2, M Ukpong1 & E.J Truter2
2
Oxidative Stress Research Centre, Department of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville, South Africa
Department of Biochemistry, Lagos State University, Ojo, Lagos, Nigeria
1
Corresponding author:
Dr O.O Oguntibeju • email: [email protected], [email protected] • tel: +27 21 953 8495 • fax: +27 21 953 8490
Abstract
Methotrexate (MTX), a folic acid antagonist is widely used as a cytotoxic chemotherapeutic agent, however its associated hepatotoxicity is considered to be a major
clinical side-effect. The aim of this study was to investigate the status of antioxidant enzymes during oxidative stress in liver homogenates of rats subjected to oral
methotrexate administration. A total of forty two, 7-week old male Wistar rats with mean weight of 172 g, divided into two groups were used. The first group, control
(n = 6), were fed only standard rat chow as their diet and water ad libitum. The second group (n = 36, subdivided into six sub-groups), fed on the same rat chow
diet, received orally administered methotrexate at a dose of 13.4 mg/kg at weekly intervals for 6 consecutive weeks. Thiobarbituric acid reactive substance (TBARS)
levels and the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) were subsequently determined spectrophotometrically on liver
homogenates of all animals.
It was found that methotrexate caused a significant increase in TBARS levels (an important marker of lipid peroxidation) in the methotrexate administered groups
when compared with the control group (P < 0.05). The activities of superoxide dismutase, catalase, and glutathione reductase were significantly decreased in the
methotrexate groups at weeks 2, 3, 4, 5 and 6 when compared with the control group (P < 0.05). The results therefore indicate that methotrexate causes oxidative
stress by reducing the activities and consequently the effectiveness of the antioxidant enzyme defense system.
Keywords
Methotrexate, oxidative stress, superoxide dismutase, catalase, glutathione reductase, liver
Introduction
Methotrexate (MTX), a folic acid antagonist, is widely used as a cytotoxic
chemotherapeutic agent in the treatment of various malignancies such as acute
lymphoblastic leukaemia as well as in the treatment of various inflammatory
diseases.[1, 2] The efficacy of this agent is often limited by its toxicity which
causes severe side-effects and may lead to conditions such as liver cirrhosis,
fibrosis of the liver, hypertrophy of the hepatocytes, hepatitis, hepato-cellular
necrosis and death.[1, 3] It has also been shown that MTX toxicity has severe
side-effects on the haematopoietic system[4] and liver enzymes in general.[5]
The exact mechanisms of methotrexate-induced toxicity have to date not yet
been elucidated.[6, 7]
Gressier, et al.[8] demonstrated that MTX increases the amount of
hydrogen peroxide and other free radicals which are released by stimulated
polymorphonuclear neutrophils (PMNs), which may lead to toxicity thus
accelerating the rate of cellular damage. It is known that MTX strongly
interferes with the metabolism of homocysteine by reducing the levels of
5-methyltetrahydrofolate and as an indirect result, the levels of homocysteine,
S-adenosylmethionine (SAM) were also found to decrease.[9, 10] MTX has been
shown to lead to a reduction in methionine synthesis, antioxidant enzymes such
as catalase, glutathione peroxidase, superoxide dismutase and a decrease of
SAM (SAM acts as an antioxidant) in cerebrospinal fluid of patients on MTX
treatment.[9, 10, 11] Due to its antioxidant effects, a deficiency of SAM caused
by MTX may be a reason for increased reactive oxygen species (ROS) and it
was shown by Villalobos et al.[12] that administration of SAM in fact caused an
inhibition of lipid peroxidation in a rat model. MTX has also been reported to
affect DNA and RNA synthesis by interfering with the biosynthesis of thymidine
and purines.[9] It is thought that the detrimental effects of MTX is partly due to
its direct toxic action by increasing ROS production. It has further been reported
that MTX administration induces oxidative stress and significantly reduces
antioxidant enzymes such as superoxide dismutase, catalase and glutathione
peroxidase in liver, intestinal mucosa and spinal cord tissues of rats.[1, 13] It is
however known that cells are protected against oxidative stress by the action
of certain enzymes, vitamins, and other substances, collectively known as
antioxidants.[14] As a consequence of the constant oxidative onslaught, cells
have developed antioxidant systems to counter the pro-oxidant fluxes. Included
among these antioxidant mechanisms are enzymes such as superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione
reductase (GR). These enzymes react directly with the oxidizing radicals to
yield non-radical products. When the balance between ROS production and
antioxidant defenses are altered, “oxidative stress” results, which through a
series of events deregulates the cellular functions and could possibly lead to
various pathological conditions.[15]
Few studies have demonstrated that methotrexate administration causes
cell injury and reduction in the activities of antioxidant enzymes [2, 6] however, the
exact duration of administration as well as its resultant effects on antioxidant
enzymes in the liver has not been well investigated.
This study was undertaken to investigate the antioxidant enzyme status in
liver homogenates of rats subjected to oral methotrexate administration.
Materials and Methods
Methotrexate (oral) was obtained from the pharmaceutical depot at the Lagos
State University Teaching Hospital (LASUTH), Ikeja, Lagos, Nigeria.
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June 2010 | Volume 24 No. 1 |
Medical Technology SA
Animals
Determination of superoxide dismutase (SOD)
Forty-two male Wistar rats (aged 7 weeks) with a mean weight of 172g were
obtained from the Laboratory Animal Production Unit of the Nigeria Institute
of Medical Research (NIMR), Yaba, Lagos. All animals used in this study were
treated in accordance with the principles of laboratory animal care as indicated
in the Guide for the Care and Use of Laboratory Animals of the National Institute
of Health.[16] Animals were housed in a temperature controlled room (25±20C)
in which a 12 hour light : 12 hour dark cycle was maintained for one week to
condition the animals prior to the start of the experiment. Standard rat chow
and tap water were provided ad libitum.
Estimation of superoxide dismutase was done by an improved
spectrophotometric assay based on epinephrine auto-oxidation [19] and
expressed as I U.
Determination of glutathione reductase (GR)
Activity of GR was determined according to the method of Golderg and
Spooner.[20] The difference in absorbance per minute was used to calculate
the enzyme activity, using a molar absorptivity of NADPH at 6.22 x 103 L mol1
cm-1. The activity of the enzyme is expressed as µmol of NADPH oxidized/min.
Experimental design
Statistical analysis
The animals were divided into two main groups (control group and experimental
group). The experimental group was further subdivided into six subgroups,
making a total of seven subgroups:
Results were expressed as mean ± SD (standard deviation). Using Student’s
t-test, the level of statistical significance was set at P<0.05.
• group i – control group,
• group ii – methotrexate treated rats (13.4 mg/kg body weight
(recommended dosage as prescribed by the manufacturer) for one
week,
• group iii – methotrexate treated rats (13.4mg/kg body weight
recommended for two weeks),
• group iv – methotrexate treated rats (13.4mg/kg body weight for
three weeks),
• group v – methotrexate treated rats (13.4mg/kg body weight for
four weeks),
• group vi – methotrexate treated rats (13.4 mg/kg body weight for
five weeks and
• group vii – methotrexate treated rats (13.4mg/kg body weight for
six weeks).
Animals in the control group (group i) received standard rat chow and
water but no methotrexate for six weeks. Methotrexate (13.4mg/ml) was
administered orally to the animals in the experimental group for one, two,
three, four, five and six weeks respectively at one interval in that order). At the
end of the respective experimental periods of 1, 2, 3 4, 5 and 6 weeks, the
rats were anaesthesized and sacrificed by cervical decapitation. The control
animals were sacrificed at the sixth week. The livers were removed, blotted dry
and immediately transferred to an ice bath.
Preparation of liver homogenates
Livers were homogenized in 0.25M sucrose solution, diluted with 0.9% saline
whilst being kept in an ice bath. The homogenates were then centrifuged at
5000 x g for 30 minutes and the supernatant collected. TBARS, SOD, CAT and
GR levels were determined in the supernatants.
Determination of lipid peroxidation biomarker
Lipid peroxidation as evidenced by the formation of thiobarbituric acid reactive
substances (TBARS) was determined according to the method of Niehaus and
Samuelsson [17] and is expressed as µmol/L
Determination of catalase (CAT)
Catalase activity was determined according to the method of Beers and Sizer
[18]
by measuring the decrease in the H2O2 concentration and reading the
absorbance at 249 nm. The amount of enzyme activity that decomposed 1
µmol of H2O2 per min is expressed as 1U.
6 | www.smltsa.org.za | ISSN 1011 5528
Results
This study analyzed the effect of administration of methotrexate (MTX) on the
antioxidant enzyme status in the livers of rodents to whom it was given at
different time intervals (in weeks).
The results showed that MTX caused a significant progressive increase in
the level of TBARs (Table 1: groups iv-vii) over the duration of administration.
In the MTX treated groups, the catalase activity was found to show a
decrease for group iv (week 3), followed by group v (week 4), group vi (week 5),
and group vii (week 6) in comparison to group i (control). However, its activity
increased in group ii (week 1) followed by a progressive decrease as can be
seen in group iii to group vii. (Table 2)
Methotrexate administration was shown to cause a significant decrease
in superoxide dismutase activity (groups iv, v, vi, and vii i.e. at week 2, 3, 4,
5 and 6, respectively) (Table 3) and also indicated a significant decrease in
glutathione reductase activity (groups v, vi and vii) in the livers as compared to
the control specimens (Table.4).
Discussion
The study examined the role of oxidative stress in an attempt to suggest a
possible mechanism of MTX-induced hepato-cellular damage or toxicity in
a rodent model. In this study we demonstrated that MTX not only caused
a significant increase in an oxidative stress biomarker (TBARS) but also a
significant decrease in the status of antioxidant enzymes (SOD, CAT and GR) in
the liver following administration of the drug.
It has been suggested that an increase in oxidative stress (caused by ROS)
is linked to the effects of MTX and that MTX-induced toxicity increases lipid
peroxidation in different tissues in rats.[21, 22] The ROS thus formed may lead
to cellular damage by peroxidation of membrane lipids, sulfhydryl enzyme
inactivation, protein cross-linking and DNA breakdown. It has been shown that
treatment with MTX leads to a reduction in the effectiveness of antioxidant
defence systems and that cellular levels of glutathione are reduced by MTX.[21]
TBARS is the breakdown product of the most important chain reactions which
lead to the oxidation of polyunsaturated fatty acids and is believed to serve as a
reliable marker of oxidative stress-mediated lipid peroxidation.[23] Jahovic et al.
[6]
demonstrated in a rat model that MTX administration can result in increased
levels of malondialdehyde. We believe that increased TBARS levels as observed
in our study can be modulated by the likelihood that lipid peroxidation can be
induced by MTX itself or as a result of a possible increase in ROS induced by
MTX. It may also be considered that MTX can inhibit some antioxidant enzymes
which in turn may cause lipid peroxidation to increase due to a reduction in the
activities of protective antioxidant enzymes such as SOD and CAT. Gressier and
Medical Technology SA
| Volume 24 No. 1 | June 2010
Table 1: Effect of administration of methotrexate on TBARS in liver homogenates
Group
TBARS (μmol/L)
T-test
i (control)
2.13 ± 0.61
1.8125
ii (week 1)
2.26 ± 0.92
0.2885
P value
P > 0.05
iii (week 2)
2.30 ± 0.04
0.7739
P > 0.05
iv (week 3)
4.05 ± 1.47*
6.7541
P < 0.05
v (week 4)
4.45 ± 0.54*
6.9755
P < 0.05
vi (week 5)
5.11 ± 0.08*
11.8648
P < 0.05
vii (week 6)
5.83 ± 0.06*
14.7862
P < 0.05
Values are presented as mean ± SD of 6 rats in each group.
* indicates P < 0.05 i.e. mean values being significantly different from those of the control group.
Table 2: Effect of administration of methotrexate on catalase activity in liver homogenates
Group
Catalase (IU)
T-test
i (control)
39.61 ± 1.14
1.8125
ii (week 1)
41.11 ± 1.35*
0.8210
P < 0.05
iii (week 2)
40.37 ± 1.96
2.0794
P > 0.05
P value
iv (week 3)
38.23 ± 1.44*
1.8405
P < 0.05
v (week 4)
32.61 ± 1.25*
10.1352
P < 0.05
vi (week 5)
32.15 ± 1.13*
11.3841
P < 0.05
vii (week 6)
31.25 ± 1.15*
12.7575
P < 0.05
Values are presented as mean ± SD of 6 rats in each group
* indicates P < 0.05 i.e. mean values being significantly different from those of the control group.
Table 3: Effect of administration of methotrexate on superoxide dismutase activity in liver homogenates
Group
Superoxide dismutase (IU)
T-test
i (control)
6.43 ± 0.41
1.8125
ii (week 1)
6.14 ± 1.49
0.4597
P value
P < 0.05
iii (week 2)
6.94 ± 0.36*
2.2898
P > 0.05
iv (week 3)
5.38 ± 0.59*
3.5798
P < 0.05
v (week 4)
4.32 ± 0.26*
10.6458
P < 0.05
vi (week 5)
4.27 ± 0.31*
10.2935
P < 0.05
vii (week 6)
3.82 ± 0.81*
7.0421
P < 0.05
Values are presented as mean ± SD of 6 rats in each group
* indicates P < 0.05 i.e. mean values being significantly different from those of the normal healthy control group.
Table 4: Effect of administration of methotrexate on glutathione reductase activity in liver homogenates
Group
Glutathione reductase (µmol)
T-test
i (control)
54.02 ± 2.32
1.8125
P value
ii (week 1)
54.16 ± 1.81
0.1165
P < 0.05
iii (week 2)
52.47 ± 2.30
1.1621
P > 0.05
iv (week 3)
52.41 ± 1.64
1.3881
P < 0.05
v (week 4)
43.28 ± 1.69*
9.1655
P < 0.05
vi (week 5)
39.64 ± 1.46*
12.8500
P < 0.05
vii (week 6)
35.88 ± 3.86*
9.8664
P < 0.05
Values are presented as mean ± SD of 6 rats in each group
* indicates P < 0.05 i.e. mean values being significantly different from those of the normal healthy control group (1).
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June 2010 | Volume 24 No. 1 |
Medical Technology SA
co-workers [8] have also indicated that free radicals such as hydrogen peroxide
(H2O2) are implicated in methotrexate-induced lipid peroxidation.
a predictor of haematologic toxicity due to methotrexate therapy.
Arthritis Rheum 1989; 32: 1592-1596.
Catalase, which acts as a preventative antioxidant plays an important role in
protection against the deleterious effects of lipid peroxidation.[24] In the present
study, catalase activity increased in the first 2 weeks of MTX administration,
but on continuous administration, catalase activity decreased significantly
when compared to controls as shown in Table 2, suggesting that peroxidative
damage to tissues probably commenced after 2 weeks of MTX administration.
5. Fries JF, Singh G, Lenert L and Furst DE. Aspirin, hydroxychloroquine
and hepatic abnormalities with methotrexate in rheumatoid arthritis.
Arthritis Rheum 1990; 33: 1611-1619.
Superoxide dismutase (SOD) catalyses the dismutation of oxygen (O-2)
radical anions to hydrogen peroxide (H2O2) and oxygen radicals (O-2).[25] Huang
et al. [26] reported on the importance of SOD in protecting cells against oxidative
stress. Our study showed a progressive decrease in SOD activity after the first
week of MTX administration (Table 3) and this decrease could possibly be
attributed to feedback inhibition or oxidative inactivation of enzyme proteins
due to an excess of reactive oxygen species (ROS) generation.[27] Hydrogen
peroxide is known to slowly but irreversibly inactivate the enzyme SOD.[28]
7. Oktem F. Methotrexate-induced renal oxidative stress in rats: the
role of a novel antioxidant caffeic acid phenethyl ester. Toxicol &
Industr Health 2006; 22(6), 241-247.
The reduction in hepatic glutathione reductase activity from the second
week onwards (as shown in Table 4) indicates that the reduction of oxidized
glutathione to reduced glutathione is impaired.
The findings of this study suggest that oxidative stress commences
approximately after one (1) week of MTX administration and increases
progressively. It has further been shown that free radical mediated damage
is aggravated by a reduction in protective antioxidant enzymes caused by the
MTX.
MTX therapy/administration perturbs the antioxidant system in a time
dependent manner. The net result appears to show that the biological situation
resulting from treatment with MTX leads to a reduction in the effectiveness of
the antioxidant enzyme defense system.
It is proposed that future studies should include the determination of
homocysteine, S-adenosylmethionine, caspases and other liver enzymes
such as alanine amino transferase and aspartate amino transferase. It is also
suggested that a histological examination of the liver should be performed. It is
further proposed that it is necessary to investigate whether the negative effects
of MTX on liver cells can be reversed by supplementation with antioxidants.
Limitations of the study
Our study focused only on the antioxidant enzyme status in the livers of
rodents following methotrexate administration. Further studies on determining
homocysteine and S-adenosylmethionine concentrations including caspases,
alanine amino transferase and aspartate amino transferase are envisaged.
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