Methotrexate Causes Oxidative Stress in Rat Kidney Tissues

Abstract

Aim. Methotrexate (MTX), a folic acid antagonist, is one of the chemotherapeutic agents widely used in the treatment of some types of cancers. Nephrotoxicity is one of the complications of MTX treatment. The aim of this study was to investigate possible effects of MTX treatment on the oxidant/antioxidant status in rat kidney tissues and enzymatic mechanisms leading to nephrotoxicity. Methods. For this aim, 10 Sprague-Dawley type female rats of 4 weeks old were used in the study. The animals were divided into two groups randomly. Five of them were used as control, and the others were treated with MTX intravenously (60 mg/m² of body surface area per week) for 7 weeks. At the end of this period, they were sacrificed, and kidney tissues were removed to be used in the analyses of malondialdehyde (MDA) levels, antioxidant potential (AOP) values, and superoxide dismutase, catalase, glutathione peroxidase, xanthine oxidase, adenosine deaminase, and 5′ nucleotidase enzyme activities. Results. There was significant increase in the MDA level in the MTX group compared with the control group (1.74 ± 0.23 nmol/mg vs. 1.04 ± 0.30 nmol/mg; p < 0.05, respectively). There were however no meaningful differences between enzyme activities and AOP values of the groups. Conclusion. It has been suggested that MTX leads to oxidative stress in rat kidney tissues, which might be one of the reasons for MTX-induced nephrotoxicity.

• methotrexate (MTX)
• nephrotoxicity
• oxidant stress

Introduction

Methotrexate (MTX), a chemically 4-amino N¹⁰-metil analog of folic acid that functions as a folic acid antagonist, is a chemotherapeutic agent widely used in the treatment of some types of cancers, such as leukemias, lymphomas, osteosarcoma, and several malignant brain tumors. It has also been used in the treatment of some other diseases.[1-3] Renal dysfunction is the most common side effect following MTX therapy.[4] It has been shown that MTX can cause uremia, hematuria, increased serum creatinine levels, and also acute renal failure if administered in high doses.[5] The underlying mechanism of nephrotoxicity caused by the MTX treatment remains unknown. However, in a study it has been reported that NADP malic enzymes are inhibited by MTX, and this could decrease the availability of NADPH in cells.[6] It has been known that NADPH is used by glutathione (GSH) reductase to maintain the reduced GSH.[7] Thus, the reduced GSH content in the cells might render them more sensitive to oxidant stress. In a more recent study, it has been found that MTX administration resulted in increased MDA and decreased GSH levels in some tissues in rats.[8]

The aim of this study was to investigate possible effects of MTX treatment on the oxidant/antioxidant status in rat kidney tissues and to elucidate possible enzymatic mechanisms leading to MTX nephrotoxicity.

Materials and Methods

Ten Sprague-Dawley type female rats of 4 weeks old were used in the study. The animals received animal care, and the study protocol complied with the institution's guidelines of The Health Ministry. The rats weighed 55 ± 5 g at the beginning of the study. They were maintained on a 12-h light/dark cycle at room temperature (23 ± 2°C) and allowed free access to food and water. The animals were divided into two groups randomly. Five of them were used as control, and the others were treated with MTX intravenously (60 mg/m² of body surface area per week) for 7 weeks.[9&10] The animals were fed laboratory diet and water ad libitum during the study period. All the animals were sacrificed at the end of the study. Kidneys of the rats were removed by surgical operation, washed with physiologic saline solution, and cleared of fatty tissue. The tissues were homogenized and prepared for the assays as described previously.[11] The upper clear part of the tissue homogenates was used in the measurements. Protein level of the clear supernatants was studied by the Lowry's method.[12] Malondialdehyde (MDA) levels (nmol/mg), antioxidant potential (AOP) values ([nmol/mg · h]^{− 1}), and superoxide dismutase (SOD) (U/mg), catalase (CAT) (IU/mg), GSH-peroxidase (Px) (mIU/mg), xanthine oxidase (XO) (mIU/mg), adenosine deaminase (ADA) (mIU/mg), and 5′ nucleotidase (5′ NT) (mIU/mg) enzyme activities were measured in the supernatants.

MDA level was measured by the thiobarbituric acid reactive substances method.[13] AOP value was established as described here: after the samples were preincubated with fish oil and xanthine-xanthine oxidase system at room temperature for 1 h, MDA level was determined. Because MDA level was negatively correlated with the antioxidant potential value, one unit of AOP value was expressed as (nmol MDA/mg protein/h)^{− 1}.[14] Superoxide dismutase activity was measured as described previously.[15] One unit for SOD activity was expressed as the enzyme protein amount causing 50% inhibition in nitroblue tetrazolium reduction rate. CAT activity was determined by measuring absorbance decrease of hydrogen peroxide (H₂O₂) at 240 nm.[16] GSH-Px activity was measured by following changes in NADPH absorbance at 340 nm.[17] XO activity was determined by measuring uric acid formation from xanthine at 293 nm.[18] In the activity calculations, extinction coefficients of uric acid, H₂O₂, and NADPH were used for XO, CAT, and GSH-Px enzymes, respectively. ADA and 5′ NT activities were measured as described, respectively.[19&20]

Statistics

In the statistical evaluation of the results, the Mann-Whitney U test was used. P values lower than 0.05 were judged as significant.

Results

The results are given in the Table 1. As shown in the Table 1, there was significant increase in the MDA level (nmol/mg) in the MTX group compared with the control group (1.74 ± 0.23 vs. 1.04 ± 0.30; p < 0.05, respectively). In the enzyme activities and AOP values, there were however no meaningful differences between the groups.

Table 1. Malondialdehyde levels, antioxidant potential values, and enzyme activities in the kidney tissues of the groups (mean ± SD)

	MTX group (n = 5)	Control group (n = 5)
MDA (nmol/mg)	1.74 ± 0.23	1.04 ± 0.30
AOP (nmol/mg/h)^− 1	0.121 ± 0.004	0.135 ± 0.022
SOD (U/mg)	64.87 ± 3.26	59.72 ± 5.92
CAT (IU/mg)	127.0 ± 13.1	132.0 ± 13.7
GSH-Px (mIU/mg)	14.15 ± 1.64	16.73 ± 2.17
XO (mIU/mg)	0.158 ± 0.016	0.150 ± 0.019
ADA (mIU/mg)	71.17 ± 5.75	69.21 ± 15.53
5′ NT (mIU/mg)	28.94 ± 5.00	21.53 ± 7.51

*p < 0.05; Mann-Whitney U test.

Discussion

MTX is used in the treatment of some cancers. High doses of MTX can cause acute renal failure and elevation of serum creatinine levels, uremia, and hematuria.[5], [21&22] In the study performed by Jahovic et al., MTX administration resulted in increased MDA and decreased GSH levels in some organs and tissues in rats, and an antioxidant substance like melatonin prevented these changes.[8] Although the nephrotoxic potential of MTX treatment has been known, the underlying mechanism(s) remains undefined.

In this study, we have found that MTX treatment caused significant increase in MDA level in the kidney tissue. Even though AOP value was found to be lower and XO activity higher in the MTX-treated group, they were statistically not meaningful. Slightly lowered AOP might be one of the reasons leading to oxidant stress, and slightly increased XO activity was also one of the factors in the formation of oxidant stress due to MTX treatment in rat kidney tissue. Increased XO activity indicates excessive superoxide radical production, which can cause oxidant stress and peroxidation in the cells. It is possible that decreased AOP value and increased XO activity both might be responsible of the oxidant stress and peroxidation in the kidney tissue from MTX-treated animals.

In conclusion, we suggest that MTX leads to oxidative stress and peroxidation in rat kidney tissues. This might be one of the reasons for MTX nephrotoxicity. If this is the case, it means antioxidant therapy might give helpful results in the prevention of MTX nephrotoxicity.