Recombinant human erythropoietin prevents etoposide- and methotrexate-induced toxicity in kidney and liver tissues via the regulation of oxidative damage and genotoxicity in Wistar rats

Abstract

Etoposide (ETO) and methotrexate (MTX) are two effective chemotherapeutic drugs. However, the clinical use of these drugs is limited by its toxicity in normal tissues, especially in kidney and in liver tissues. Recombinant human erythropoietin (rhEPO), erythropoietin hormone, has also been shown to exert tissue protective effects. The purpose of this study was to explore the protective effect of rhEPO against oxidative stress and genotoxicity induced by ETO and MTX in vivo. Adult male Wistar rats were divided into 10 groups (6 animals each): control group, rhEPO alone group, ETO alone group, MTX alone group and rhEPO + ETO/MTX groups. In rhEPO + ETO/MTX groups, three doses of pretreatment with rhEPO were performed: 1000, 3000 and 6000 IU/kg. Our results showed that rhEPO pretreatment protects liver and kidney tissues against oxidative stress induced by the anticancer drugs. The glycoprotein decreased malondialdehyde (MDA) levels, reduced catalase activity and ameliorated glutathione depletion. Furthermore, we showed that rhEPO administration prevented drug-induced DNA damage accessed by comet test. Altogether, our results suggested a protective role of rhEPO, especially at 3000 IU/kg, against ETO- and MTX-induced oxidative stress and genotoxicity in vivo.

Keywords

Etoposide methotrexate recombinant human erythropoietin antioxidant property antigenotoxic property kidney tissue liver tissue

Introduction

Erythropoietin (EPO) is a haematopoietic cytokine, identified as the principal growth factor regulating the proliferation and differentiation of erythroid progenitor cells.^1,2 EPO gene expression is synthesized predominantly by interstitial kidney and liver cells under low oxygen conditions to improve tissue oxygenation.^3,4 Besides, another key physiological role of EPO on general tissue protection was previously confirmed.^5,6 EPO acts through its transmembrane receptor (EPO-R) expressed mainly on red cell precursors but also on different tissues: heart, skeletal muscles, brain and kidney.^7,8 Recombinant human erythropoietin (rhEPO) is indicated for the treatment of anaemia resulting from chronic renal failure or chemotherapy.⁹ rhEPO operates via similar molecular pathways to the endogenous EPO.^10,11

Etoposide (ETO) is an antineoplastic, semi-synthetic podophyllotoxin derivative which has been used for the treatment of a variety of malignancies such as lymphomas, testicular cancer and lung cancer.^12
–14 The toxicity of ETO is caused by the formation of cleavable complexes between DNA-topoisomerase and ETO and by the production of ETO phenoxyl radicals in the redox reaction stimulated by myeloperoxidase.^15,16 Methotrexate (MTX) is an antimetabolite drug which acts by inhibiting dihydrofolate reductase, disrupting purine synthesis and preventing cell division.¹⁷ MTX is widely used in the treatment of breast cancer, lymphoma, stomach cancer and urinary bladder cancer.¹⁸

Chemotherapy using MTX and ETO cancer drugs is one of the most effective methods for tumour treatment but is often associated with several toxicities.¹⁹ The most commonly described side effects of MTX/ETO therapy are myelosuppression hepatotoxicity, mucositis and neurotoxicity.^20

–23 To minimize drug-induced toxicity, antioxidant and antigenotoxic adjunct treatment used in combination with ETO and MTX potentially may be beneficial in improving chemotherapy effects.^24
–26 Thus, the need of developing an efficient protective therapy against drug-induced nephrotoxicity and hepatotoxicity is urgently required. For this, rhEPO may be used as a promising compound in such a therapy.

In the present study, we investigated the potential protective effect of rhEPO against ETO- and MTX-induced oxidative stress and genotoxicity in kidney and liver tissues of rat.

Material and methods

Chemicals

ETO and MTX were purchased from Sigma-Aldrich (Lyon, France). Experiments were performed with a commercially available preparation of rhEPO (Hemax^®; Bio Sidus S.A., Buenos Aires, Argentina). 2,4-Dinitrophenylhydrazine (2,4-DNPH) and guanidine were from VWR International (Fontenay-sous-Bois, France). All other chemicals used were of analytical grade.

Animal treatment

Experiments were performed on male Wistar rats in the weight range of 120–140 g, kept at controlled environmental conditions at room temperature 22 ± 2°C and 12-h light/12-h dark cycles and allowed free access to food and water, but fasted overnight before treatment. For the time-course experiment, rats were divided at random into seven groups, with six animals in each group. All injections were administered by the intraperitoneal route. The control group received a single injection of saline solution (0.9%). The rhEPO group was given only rhEPO, the ETO group was given only a single injection of ETO and the MTX group was given only a single injection of MTX. To test the effect of rhEPO on ETO/MTX-induced oxidative damage and genotoxicity, three doses of pretreatment with rhEPO were employed (1000, 3000 and 6000 IU/kg body weight (b.w.)). In each type of treatment, both ETO and MTX were administrated at 20 mg/kg b.w. Experimental design is detailed in Table 1. After animals were killed, kidney and liver were immediately removed for subsequent experiments.

Table 1.

Animal groups and treatments in the experimental design of this study.

	Day 0	Day 1	Day 3	Day 4
Control group	Saline solution injection			Killed
rhEPO group (6000 IU/kg b.w.)	rhEPO injection	Killed
ETO group (20 mg/kg b.w.)	ETO injection		Killed
MTX group (20 mg/kg b.w.)	MTX injection		Killed
Pretreatment group (1000 IU/kg b.w.)	rhEPO injection	ETO/MTX injection		Killed
Pretreatment group (3000 IU/kg b.w.)	rhEPO injection	ETO/MTX injection		Killed
Pretreatment group (6000 IU/kg b.w.)	rhEPO injection	ETO/MTX injection		Killed

ETO: etoposide; MTX: methotrexate; rhEPO: recombinant human erythropoietin; b.w.: body weight.

Preparation of kidney and liver extracts

Kidney and liver were homogenized with a potter (glass, Teflon) in the presence of 10 mM of Tris-hydrochloride (HCl; pH 7.4) at 4°C and centrifuged at 4000 r/min for 30 min at 4°C. The supernatant was collected for analysis, and protein concentrations were determined in kidney and liver extract using the protein Bio-Rad assay.²⁷

Evaluation of lipid peroxidation level

The renal and liver malondialdehyde (MDA) levels in the extract were quantified according to the method of Aust et al.²⁸ Briefly, 200 ml of kidney or liver extract was mixed with 150 ml of Tris-buffered saline (Tris 50 mmol/l and sodium chloride 150 mmol/l, pH 7.4) and 250 ml of Trichloroacetic acid-Butylated hydroxytoluene (TCA-BHT) (20% TCA and 1% BHT). The mixture was vigorously vortexed and centrifuged at 1500 × g for 10 min; 400 ml of the supernatant was added with HCl 0.6 N and 320 ml Tris-TBA (Tris 26 mmol/l and TBA 120 mmol/l); and the content was mixed and incubated for 10 min at 80°C. The absorbance was measured at 535 nm. The optic density corresponding to the complex formed with the TBA-MDA is proportional to the concentration of MDA and to the lipid peroxide. The concentration of millimoles of MDA per milligram of proteins is calculated from the absorbance at 530 nm using the molar extinction coefficient of MDA 1.56 × 105/mol/l/cm. The results were expressed as millimoles of MDA per milligram of proteins.

Protein carbonyl assay

Protein carbonyl content was determined, as described by Mercier et al.,²⁹ in kidney and liver homogenates by measuring the reactivity of carbonyl groups with 2,4-DNPH. Thus, 200 μl of supernatant of kidney or liver was placed in two glass tubes. Then, 800 μl of 10 mM DNPH in 2.5 M of HCl was added. Tubes were left for 1 h of incubation at room temperature in the dark. Samples were vortexed every 15 min. Then, 1 ml of 20% TCA was added to samples, and the tubes were left in an ice bucket for 10 min and centrifuged for 5 min at 4000 r/min to collect the protein precipitates, and the supernatants were discarded. Next, another wash was performed using 1 ml of 10% TCA, and protein pellets were broken mechanically with the aid of a glass rod. Finally, the pellets were washed with 1 ml of ethanol-ethyl acetate (1:1; v/v) to remove the free DNPH. The final precipitates were dissolved in 500 μl of guanidine HCl (6 M) and were left for 10 min at 37°C with general vortex mixing. Any insoluble materials were removed by additional centrifugation. Protein carbonyl concentration was determined from absorbance at 370 nm, applying the molar extinction coefficient of 22.0/mM/cm. A range of nanomoles of carbonyls per millilitre was usually obtained for most proteins and was related to the protein content in the pellets.

Glutathione assay

Glutathione (GSH) level in kidney and liver extract was measured using a colorimetric assay kit, according to the recommendations of the manufacturer (Sigma-Aldrich). The method was based on the reduction of 5,5-dithiobis-2-nitrobenzoic acid with GSH to produce a yellow-coloured product. The reduced compound was directly proportional to GSH concentration and its absorbance was measured at 412 nm.^30,31 The kidney or liver tissues of different groups were flash-freezed and grounded in liquid nitrogen immediately after excision. Next, 0.3 g of kidney or liver powder was taken and mixed with 5% 5-sulfosalilyc acid solution for renal protein removal. Aliquots of kidney or liver extract were submitted to photometric total Glutathione (tGSH) determination; DTNB formation rate was monitored at 412 nm and compared with GSH standards (1–20 μM). Each experiment was performed in triplicate.

Determination of catalase activity

Catalase activity was measured in the kidney and liver extract at 240 nm, at 25°C, according to Clairbone.³² Briefly, 20 μl of the extracts were added in the quartz cuvette containing 80 μl of phosphate buffer (pH = 7) and 200 μl of hydrogen peroxide (H₂O₂; 0.5 M). The activity of catalase was calculated using the molar extinction coefficient (0.04/mM/cm). Results were expressed as micromoles of H₂O₂ per minute per milligram of proteins.

Single-cell gel electrophoresis: Comet assay

Determination of DNA damage by the alkaline comet assay was conducted according to Tice et al.,³³ with minor modifications.³⁴ Each piece of kidney or liver was placed in 0.5 ml of cold phosphate-buffered saline and finely minced to obtain a cellular suspension. Kidney and liver cell suspensions (5 μl) were embedded in 60 μl of 1% low melting point agarose and spread on agarose-precoated microscope slides. To lyse cellular and nuclear membranes of the embedded cells and to allow for DNA unwinding in alkaline conditions, the slides were immersed in ice-cold, freshly prepared lysis solution and left at 4°C overnight to improve the efficiency of DNA damage detection.³⁵ Slides were then placed in an electrophoresis alkaline buffer (pH > 13), and the embedded cells were exposed to this alkaline solution for 20 min to allow for DNA unwinding. Electrophoresis was performed in the same alkaline buffer for 20 min by applying a 25-V electric field and adjusting the current to 300 mA. After electrophoresis, the slides were neutralized with 0.4 M of Tris (pH 7.5), and the DNA was stained with 50 μl of ethidium bromide (20 μg/ml). All steps were conducted in darkness to prevent additional DNA damage. A total of 100 comets on each slide were visually scored according to the intensity of fluorescence in the tail and classified by one of five classes, as described by Collins et al.³⁶ Total score was evaluated according to the following equation: (percentage of cells in class 0 × 0) + (percentage of cells in class 1 × 1) + (percentage of cells in class 2 × 2) + (percentage of cells in class 3 × 3) + (percentage of cells in class 4 × 4).

Statistical analysis

Each experiment was carried out at least three times separately. Data are expressed as mean ± standard deviation. Statistical comparison between different groups was done using one-way analysis of variance followed by Fischer post hoc to detect the difference between various groups. Value of p < 0.05 was considered to be significant: ‘*’ indicates significant difference from the control group; ‘#’ indicates significant difference from the ETO/MTX-treated cells; ‘a’ indicates significant difference from the pretreatment with rhEPO at 1000 IU/kg; and ‘b’ indicates significant difference from the pretreatment with rhEPO at 6000 IU/kg.

Guidelines for ethical publications

The experimental procedures were carried out according to the American College of Toxicology Statement on the use of animals in toxicology and approved by the local Ethics committee

Results

Induction of lipid peroxidation

To evaluate lipid peroxidation status, MDA level was measured and the results are shown in Figure 1. When compared to control groups, MDA level in the kidney and liver tissues was significantly higher (p < 0.05) in the group treated with ETO/MTX alone at 20 mg/kg b.w. Therefore, in renal tissue, the MDA level increased from a basal level of 10.78 ± 0.67 mmol/mg of protein to 20.08 ± 0.7 mmol/mg of protein in the MTX-treated group, and the increase in MDA levels was about twofold as compared to the untreated group (p < 0.05). Figure 1 showed that ETO caused a more pronounced oxidative damage than MTX. On the other hand, rhEPO administration whatever at 1000, 3000 or 6000 IU/kg b.w., 24 h before ETO/MTX, was associated with a fall in the MDA levels to reach the control group at 3000 IU/kg b.w. For example, MDA level in hepatic tissue was significantly decreased as compared to the MTX groups: 13.63 ± 1.32, 10.56 ± 1.19 and 14.03 ± 1.14 mmol/mg of protein, respectively, at 1000, 3000 and 6000 IU/kg b.w. as compared to 15.75 ± 1.15 mmol/mg of protein, for MTX-treated group. Moreover, as shown in Figure 1, the optimum preventive action of rhEPO pretreatment was shown at 3000 IU/kg b.w.

Protein carbonyl assay

The formation of protein carbonyls, the most widely used marker of severe protein oxidation, was assayed in kidney and liver homogenates, and results are illustrated in Figure 2. We showed that ETO/MTX alone generated protein carbonyl formation, as compared to control groups, in both kidney and liver extracts. For liver extracts, the protein carbonyl level increased from the basal value of 0.97 ± 0.9 nmol/mg of protein in the control group to 4.02 ± 0.83 nmol/mg of protein in the MTX-treated group. The amounts of protein carbonyls in hepatic tissue extracts were less than those detected in the kidney tissue. Indeed for kidney extract, protein carbonyl level increased from a basal value of 0.97 ± 0.9 nmol/mg of protein in control group to 6.01 ± 0.74 nmol/mg of protein in the MTX-treated group. Furthermore, our results showed that protein oxidation decreased significantly in groups pretreated with rhEPO at 1000, 3000 and 6000 IU/kg 24 h before ETO/MTX exposure in both kidney and liver extracts, as compared to the ETO/MTX group. For example, in kidney extract, protein carbonyl level decreased from the value of 9.66 ± 0.43 nmol/mg of protein in the ETO group to 7.09 ± 0.43, 1.88 ± 0.38 and 5.88 ± 0.53 nmol/mg of protein in groups pretreated with rhEPO, respectively, at 1000, 3000 and 6000 IU/kg b.w. Moreover, our results demonstrated that rhEPO was more efficient at the dose of 3000 IU/kg against protein carbonyl formation either in kidney or liver tissues.

Effect of rhEPO on GSH depletion

The effect of rhEPO pretreatment and ETO/MTX exposure on GSH modulation in kidney and liver tissues is illustrated in Figure 3. Our data demonstrated that Wistar rats exposed to ETO/MTX alone showed a noticeable depletion of GSH level (p < 0.05), as compared to the untreated group. In renal extract, GSH level was 6.13 ± 0.45 versus 13.05 ± 0.5 nmol GSH/mg of protein after ETO exposition and 7.98 ± 0.84 versus 13.05 ± 0.5 nmol GSH/mg of protein after MTX exposition. However, a significant increase (p < 0.05) in GSH level was observed in rats pretreated with rhEPO at 1000, 3000 and 6000 IU/kg 24 h before ETO/MTX exposure, as compared to the ETO/MTX group. Indeed, in kidney extracts, GSH levels increased to 9.13 ± 1.19, 12.67 ± 0.52 and 8.87 ± 1.08 nmol GSH/mg of protein, respectively, at 1000, 3000 and 6000 IU/kg b.w., as compared to the MTX group (7.98 ± 0.84 nmol GSH/mg of protein). Further, our results demonstrated that the pretreatment at 3000 IU/kg promoted the best protection against ETO/MTX-induced GSH depletion both in kidney and liver tissues.

Catalase activity

Catalase is an endogenous antioxidant enzyme that protects cells from the detrimental effects of reactive oxygen species (ROS). Levels of catalase can indicate the magnitude of oxidative stress that occurs. The effect of ETO/MTX and rhEPO on catalase activity is illustrated in Figure 4. Our results showed that ETO/MTX alone induced a marked increase in catalase activity in both renal and liver extracts. Catalase activity increased from the basal value of 76.51 ± 21.43 nmol/min/mg of protein in control group to 678.32 ± 25.56 and 502.62 ± 32.88 nmol/mg in groups treated with ETO and MTX in liver extract. In kidney extract, catalase activity increased from 83.85 ± 12.03 nmol/min/mg of protein in the control group to 706.43 ± 43.44 nmol/mg in the ETO group and to 671.02 ± 30.31 in the MTX-treated group. rhEPO administration 24 h before ETO/MTX treatment produced a decrease of this activity. For example, in renal extract, catalase activity decreased from 671.02 ± 30.31 nmol/mg in the MTX group to 343.65 ± 41.55, 264.51 ± 33.27 and 590.6 ± 49.68 nmol/mg in groups pretreated with rhEPO, respectively, at 1000, 3000 and 6000 IU/kg. Thus, the pretreatment at 3000 IU/kg provided the best protection against ETO/MTX-induced oxidative stress in renal and hepatic tissues.

Effect of rhEPO pretreatment on DNA damage induced by ETO and MTX

The antigenotoxic effect of rhEPO was assessed through the alkaline comet assay. Results of the visual scoring of total basic DNA damage are illustrated in Figure 5. We observed a significant increase of the total DNA damage in rats treated with ETO/MTX only in both kidney and liver extracts. rhEPO pretreatment at 1000, 3000 and 6000 IU/kg reduces DNA fragmentation caused by ETO/MTX exposure. The degree of DNA damage seemed to be less noticeable when rats were pretreated with rhEPO at the dose of 3000 IU/kg. The amount of DNA damage decreased by approximately 2.5-fold with respect to the ETO value and by twofold with respect to the MTX value in rats pretreated with rhEPO at 3000 IU/kg in kidney and liver extracts. No specific DNA fragmentation was detected in negative control and in rhEPO alone groups.

Discussion

rhEPO is a haematopoietic cytokine, which has been used to treat anaemia.¹ Binding to its receptor (EpoR) on the surface of erythroid progenitors, rhEPO promotes cell proliferation and differentiation and protects cells from apoptosis.³⁷ Recently, many researchers reported that rhEPO can protect organs, tissues and cells against different adverse effects.^38,39 ETO and MTX were widely used for many cancer therapies.^22,40,41 However, the clinical use of these drugs was usually limited by several harmful effects especially hepatic fibrosis and renal insufficiency.^23,42,43 For this, protective strategies such as the use of rhEPO against ETO and MTX toxicities may be requisite. The molecular mechanism of rhEPO action against ETO- and MTX-induced organ damage is poorly understood so, in the present study, the possible antioxidant and antigenotoxic effect of rhEPO against ETO and MTX toxicity in kidney and liver tissues was explored.

Oxidative stress generation caused oxidation of polyunsaturated fatty acids to produce lipid peroxyl radicals and lipid hydroperoxides, a process called to lipid peroxidation.⁴⁴ MDA is the end product of the major reactions, which serves as a reliable marker of oxidative stress.^45
–47 Therefore, to evaluate the oxidative damage induced by ETO/MTX and the eventual antioxidant property of rhEPO in kidney and liver tissues, the MDA level was examined. In the present study, while exposure to ETO and to MTX at 20 mg/kg b.w. induced a noticeable increase in MDA formation in kidney and liver tissues of rat, rhEPO pretreatment especially at 3000 IU/kg provided a significant reduction of this induction which dropped to the control level. Our results are in accordance with those of Heba et al.,⁴⁸ who demonstrated that MTX enhances MDA level in hepatic and renal tissues of rats and with those of Ademola et al.,²⁶ who demonstrated that MTX exposure increased the MDA level in rat liver. Our data were also agree with Oztürk et al.,⁴⁹ who demonstrated that rhEPO reduced lipid peroxidation by decreasing both nitric oxide (NO) synthesis and xanthine oxidase activity in rat brain injured tissue.

To more accesses to the antioxidant propriety of rhEPO against ETO and MTX toxicity, protein carbonyl generation was examined. Our results clearly demonstrate that ETO/MTX induced a marked increase in protein carbonyl generation in kidney and liver extracts which was significantly reduced with rhEPO pretreatment. Statistics analysis of different treatment conditions showed that the pretreatment with rhEPO at 3000 IU/kg promotes the best protection against ETO and MTX toxicity (Figure 2). Our findings were in accordance with those of Dang et al.,⁵⁰ showing a protective effect of rhEPO through an antioxidant mechanism in renal tubular cell.

It is widely known that oxidative stress may be generated by an overproduction of ROS or by the depletion of antioxidant defenses.⁵¹ For this, the effects of rhEPO on oxidative damage caused by ETO and MTX were also investigated through assessing of reduced GSH level. GSH is the most abundant intracellular thiol and plays an important role in the detoxification of ROS and xenobiotics.⁵² Our results obviously showed that ETO/MTX decreased significantly the level of GSH in kidney and liver tissues. The amount of GSH was clearly restored in the presence of rhEPO pretreatment at 1000, 3000 and 6000 IU/kg 24 h before ETO/MTX administration. Moreover, we showed that anticancer drugs enhanced catalase activity and this activation was significantly reduced when rats were pretreated with rhEPO at the different pretreatment doses. Our results clearly demonstrated that the optimum protective action of rhEPO against ETO- and MTX-induced oxidative damage was observed at the dose of 3000 IU/kg. Our study is in agreement with those of Ademola et al.,²⁶ who demonstrated that MTX exposure decreases the amount of GSH in liver of rats and with those of Ahmed et al.,⁵³ who demonstrated that MTX treatment induced the reduction of GSH levels and a marked increase of catalase and superoxide dismutase activities on both buccal and lingual mucosae. Moreover, our results are in accordance with several other studies, showing that rhEPO was directly involved in the prevention of oxidative stress with activation of antioxidant enzymes, inhibition of NO production and decrease of lipid peroxidation.^50,54
–56 According to these data, we can suggest that rhEPO pretreatment especially at 3000 IU/kg can be used as a good therapy to protect liver and kidney tissues against oxidative stress induced by these anticancer drugs.

It is widely known that oxidative stress can attack biomolecules, such as DNA, leading to genotoxic damage.⁵⁷ For this, the antioxidant action of rhEPO can inhibit the genotoxic effect of ETO/MTX. To assess the possible antigenotoxic action of rhEPO, we monitored the comet assay which is one of the standard methods for assessing DNA damages including single- and double-strand DNA breaks.⁵⁸ Our results showed that ETO/MTX alone caused a significant increase in DNA fragmentation as compared to the control group. Our results are agree with those of Ahmed at al.,⁵³ who demonstrated that MTX-treated rats showed an increased level of DNA fragmentation, and with those of Fortune and Osheroff,⁵⁹ showing that ETO causes cell mortality by induction of DNA double-strand breaks. However, rats pretreated with rhEPO (1000, 3000 and 6000 IU/kg) 24 h before ETO/MTX administration induced a noticeable decrease in DNA fragmentation as compared either to ETO or to MTX groups. Furthermore, pretreatment with rhEPO at 3000 IU/kg provided the best protective effect against ETO and MTX genotoxicity as compared to groups pretreated with rhEPO at 1000 or 6000 IU/kg. Our results are in agreement with those of Sun et al.,⁶⁰ who showed that pretreatment of rats with rhEPO 24 h before the insult caused a reduction in DNA fragmentation. Our study could contribute in the understanding of the mechanism by which rhEPO inhibits ETO/MTX toxicity in kidney and liver tissues and point towards rhEPO clinical implications in cancer therapies.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was supported by the Ministère Tunisien de l’Enseignement Supérieur et de la Recherche Scientifique et de la Technologie (Laboratoire de Recherche sur les Substances Biologiquement Compatibles).

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