Sage Journals: Discover world-class research

Abstract

Mitomycin C (MMC) is an antineoplastic agent used for the treatment of several human malignancies. Nevertheless, the prolonged use of the drug may result in a serious heart and kidney injuries. Recombinant human erythropoietin (rhEPO) has recently been shown to exert an important cytoprotective effect in experimental brain injury and ischemic acute renal failure. The aim of the present work is to investigate the cardioprotective and renoprotective effects of rhEPO against MMC-induced oxidative damage and genotoxicity. Our results showed that MMC induced oxidative stress and DNA damage. rhEPO administration in any treatment conditions decreased oxidative damage induced by MMC. It reduced malondialdehyde and protein carbonyl levels. rhEPO ameliorated reduced glutathione plus oxidized glutathione modulation and the increased catalase activity after MMC treatment. Furthermore, rhEPO restored DNA damage caused by MMC. We concluded that rhEPO administration especially in pretreatment condition protected rats against MMC-induced heart and renal oxidative stress and genotoxicity.

Keywords

rhEPO mitomycin C antioxidant effect antigenotoxic effect

Introduction

Mitomycin C (MMC), an antitumor antibiotic isolated from Streptomyces caespitosus, was used for the treatment of a variety of solid tumors, including cancers of the breast, lung, uterus, and particularly those of gastric, bladder, and colorectal cancer.¹ MMC, an alkylating agent, is activated in vivo to alkylate and crosslink DNA, via G–G interstrand bonds, thereby inhibiting DNA synthesis and transcription.² Despite a broad spectrum of antitumor activity, clinical use of MMC was relatively limited by several adverse reactions including myelosuppression,³ cardiotoxicity,^3,4 pulmonary toxicity,⁵ and nephrotoxicity.^6,7

MMC appears to be toxic through different mechanisms. Oxidative stress was considered to be one of the mechanisms implicated in MMC-induced toxicity.^8

–11 MMC oxygen-dependent toxicity occurs via cyclic 1-electron reduction of the MMC molecule followed by a molecular oxygen oxidation, producing the active MMC molecule and the superoxide radical, an extremely reactive species that produces a variety of toxic radicals and results in several types of damage to biological molecules.^12,13 In fact, reactive oxygen species (ROS) can attack biomolecules, such as DNA, lipids, and thiols in proteins and glutathione, leading to the inactivation of enzymes, genotoxic damage, cell dysfunction, and death.^14,15

Many compounds have been suggested to protect against MMC-induced oxidative damage^10,16; however, the use of recombinant human erythropoietin (rhEPO) has not been evaluated. Erythropoietin (EPO) is primarily a glycoprotein hormone, but not exclusively, synthesized by renal cortical interstitial fibroblasts in response to tissue hypoxia.¹⁷ EPO, used clinically as rhEPO, has been used as a successful treatment for human anemia associated with end-stage renal failure and cancer chemotherapy.^18
–20 Recently, the biological effects of rhEPO have not been limited to the hematopoietic system; many studies have shown that rhEPO is a pleiotropic cytokine that exerts broad tissue-protective effects in diverse nonhematopoietic organs.²¹ Clinical studies have suggested an additional role for EPO in the heart and renal system.^22

–26 In this context, the aim of the present study was to evaluate the possible antioxidant and antigenotoxic effects of rhEPO against MMC-induced oxidative stress and genotoxicity in heart and kidney of rats. For this purpose, we studied the oxidative stress in heart and kidney tissues by the measure of lipoperoxidation and protein carbonyl levels, reduced glutathione plus oxidized glutathione (GSH + GSSG) modulation, and catalase activity. DNA damage involvement was assessed by the comet assay. Our results showed that rhEPO administration, especially in pretreatment condition, protects against MMC-induced oxidative stress and genotoxicity in heart and kidney tissues of rat.

Material and methods

Chemicals

MMC was obtained 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-Dinitro-phenylhydrazine (2,4-DNPH) and guanidine were from VWR International (Fontenay-sous-Bois, France). All the chemicals used were of analytical grade.

Animal treatment

Experiments were performed on male Wistar rats weighing 120 –140 g, kept at controlled environmental conditions at room temperature 22 ± 2°C and 12-h light/dark cycles, and allowed free access to food and water. For the time-course experiment, rats were divided randomly into six groups, with six animals in each group. All injections were administered by the intraperitoneal (i.p.) route. The control group received a single injection of saline solution (0.9%). The rhEPO group was given only rhEPO, and the MMC group was given only a single injection of MMC. To test the effect of rhEPO on MMC-induced oxidative damage and genotoxicity, three treatment conditions were used. In the cotreatment group, a single dose of rhEPO was administered simultaneously with MMC. In the pretreatment group, a single dose of rhEPO was given 1 day before MMC. In the posttreatment group, a single dose of rhEPO was given 5 days after MMC. In each type of treatment, rhEPO and MMC were used, respectively, at 3000 IU/kg body weight (b.w.) and 3 mg/kg b.w. Experimental design was detailed in Table 1. After the animals were killed, their kidney and heart were removed immediately for subsequent experiments.

Table 1.

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

	Day 0	Day 1	Day 5	Day 6
Control group	Saline solution injection		Killed
rhEPO group (3000 IU/kg b.w.)	rhEPO injection	Killed
MMC Group (3 mg/kg b.w.)	MMC injection		Killed
Cotreatment group	MMC + rhEPO injection		Killed
Pretreatment group	rhEPO injection	MMC injection		Killed
Posttreatment group	MMC injection		rhEPO injection	Killed

MMC: mitomycin C; rhEPO: recombinant human erythropoietin.

Preparation of heart and kidney homogenates

Heart and kidney were homogenized with a potter (glass, Teflon) in the presence of 10 mM of Tris-hydrochloric acid (Tris-HCl; pH 7.4) at 4°C and centrifuged at 4000g for 30 min at 4°C. The supernatant was collected for analysis, and protein concentrations were determined in kidney and heart homogenates using the protein Bio-Rad assay.²⁷

Protein carbonyl assay

Protein carbonyl content in kidney or heart homogenates, by measuring the reactivity of carbonyl groups with 2,4-DNPH, was determined by a spectrophotometric method, as described by Mercier et al.²⁸ Thus, 200 μl supernatant of kidney or heart were placed in two glass tubes. Then 800 μl of 10 mM DNPH in 2.5 M HCl were added. Tubes were incubated for 1 h at room temperature in the dark. Samples were vortexed every 15 min. Then 1 ml of 20% trichloroacetic acid (TCA) was added to the samples, and the tubes were left in an ice bucket for 10 min and centrifuged for 5 min at 4000g to collect the protein precipitates, and the supernatants were discarded. Next, another wash was performed using 1 ml of 10% TCA, and the protein pellets were mechanically broken 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 hydrochloride (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 the absorbance at 370 nm, applying the molar extinction coefficient of 22.0 mM⁻¹/cm. A range of nanomoles of carbonyls per milliliter was usually obtained for most proteins and was related to the protein content in the pellets.

Evaluation of lipid peroxidation level

The renal malondialdehyde (MDA) levels were quantified by a spectrophotometric method according to Aust et al.²⁹ Briefly, 200 µl of kidney or heart extracts were mixed with 150 µl of Tris-buffered saline (Tris 50 mM and sodium chloride 150 Mm, pH 7.4) and 250 μl TCA-butylated hydroxytoluene. The mixture was vigorously vortexed and centrifuged at 1500g for 10 min. After centrifugation, 400 μl of supernatant were mixed with 280 µl HCl (0.6 N) and 320 μl Tris-thiobarbituric acid (Tris-TBA; Tris 26 mM and TBA 120 mM). The absorbance was measured at 532 nm. The optic density corresponding to the complex formed with the TBA-MDA was proportional to the concentration of MDA and to the lipid peroxide. The concentration of millimoles of MDA per milligram of proteins was 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.

Glutathione assay (GSH and GSSG levels)

GSSG and GSH were measured spectrophotometrically by a described enzyme recycling method³⁰ with some modifications. The kidney or heart tissues of different groups were flash freezed and grounded in liquid nitrogen immediately after excision. Next, 1 mg of kidney or heart powder was taken and mixed with 500 µl of 0.4 M perchloric acid and centrifuged at 10,000g for 10 min at 4°C. To determine GSH, a 50-µl aliquot of the supernatant was combined in a cuvette with 500 µl of buffer (1 mM ethylenediaminetetraacetic acid, 3 mM nicotinamide adenine dinucleotide phosphate, 100 mM potassium phosphate, pH 7.4). Next, 50 µl of 10 mM 5,5-dithiobis-2-nitrobenzoic acid were added. After incubating for 1 min, 100 µl of glutathione reductase (5 U/ml) were added and the absorbance at 412 nm was monitored for 5 min. GSSG was measured in the samples previously neutralized with 100 mg of sodium carbonate and treated with 4 µl of 2-vinylpyridine. After 1 h of incubation in the dark, the samples were centrifuged at 3000g for 5 min, and the supernatant was assayed as described above for GSH determination. A calibration curve submitted to the same procedure was used as reference to calculate GSH and GSSG concentrations.

Determination of catalase activity

Catalase activity in the kidney and heart extracts was measured by spectrophotometry at 240 nm, 25°C according to Clairbone.³¹ Briefly, 20 μl of 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 the catalase was calculated using the molar extinction coefficient (0.04 mM⁻¹/cm). Results were expressed as micromoles of H₂O₂ per minute per milligram of proteins.

Single-cell gel electrophoresis (the comet assay)

Determination of DNA damage by the alkaline comet assay was conducted according to Tice et al.,³² with minor modifications.³³ Each piece of kidney or heart was placed in 0.5 ml of cold phosphate-buffered saline and finely minced to obtain a cellular suspension. Kidney and heart 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.³⁴ 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 DNA unwinding. Electrophoresis was performed in the same alkaline buffer for 20 min by applying 25 V electric field and adjusting the current to 300 mA. After electrophoresis, the slides were neutralized with 0.4 M 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.³⁵ Total score was evaluated according to the following equation: (% of cells in class 0 × 0) + (% of cells in class 1 × 1) + (% of cells in class 2 × 2) + (% of cells in class 3 × 3) + (% of cells in class 4× 4).

Statistical analysis

Each experiment was carried out separately at least three times. All data were expressed as means ± SD. Statistical significance of the differences among different groups was evaluated by one-way analysis of variance followed by Fisher multiple comparisons test as a post hoc test. Data were analyzed using SPSS statistical program (version 10.0 software, SPSS Inc., Chicago, Illinois, USA). Value of p < 0.05 was considered to be significant. * indicates significant difference from control. ^# indicates significant difference from MMC-treated rats. ^a indicates significant difference from cotreatment group and ^b indicates significant difference from posttreatment group.

Results

Protein carbonyl assay

The formation of protein carbonyls, the most widely used marker of severe protein oxidation, was assayed in kidney and heart homogenates and the results were illustrated in Figure 1. Our study showed that MMC alone significantly generated protein carbonyl formation, as compared to the control group, in both kidney and heart extracts. For kidney extracts, the protein carbonyl level increased significantly from the basal value of 0.61 ± 0.14 nmol/mg of protein in the control group to 3.29 ± 0.22 nmol/mg of protein in the MMC-treated group. Protein carbonyl level in heart extracts increased significantly from a basal value of 0.62 ± 0.05 nmol/mg of protein in the control group to 3.01 ± 0.22 nmol/mg of protein in the MMC-treated group. Besides, our results showed that rhEPO administration (simultaneously, 24 h before and 5 days after MMC exposure) significantly decreased the protein oxidation levels in both kidney and heart extracts, as compared to the MMC group. For example, in kidney extract, protein carbonyl level decreased significantly from the value of 3.29 ± 0.22 nmol/mg of protein in the MMC group to 1.55 ± 0.05, 0.96 ± 0.05, and 1.22 ± 0.13 nmol/mg of protein, respectively, in co-, pre-, and posttreatment conditions. Moreover, our results demonstrated that rhEPO was more effective in the pretreatment condition against MMC-induced protein carbonyl formation either in kidney or heart tissues.

Figure 1.

Effect of rhEPO on protein carbonyl concentrations in rat kidney and heart. rhEPO was administered simultaneously, 24 h before, and 5 days after MMC treatment. *p < 0.05 versus control, ^# p < 0.05 versus MMC, ^a p < 0.05 versus cotreatment condition, and ^b p < 0.05 versus posttreatment condition. MMC: mitomycin C; rhEPO: recombinant human erythropoietin.

Induction of lipid peroxidation

MDA is the end product of the major reactions, leading to significant oxidation of polyunsaturated fatty acids in cellular membranes and thus serves as a reliable marker of oxidative stress.^36
–38 To evaluate lipid peroxidation status, the MDA level was measured and the results were illustrated in Figure 2. Our results showed that MMC administration induced a significant increase (p < 0.05) in MDA level in both kidney and heart tissues, when compared with the control group. Therefore, the MDA level, in kidney extract, increased from a basal level of 15.24 ± 0.8 mmol/mg to 36.89 ± 0.7 mmol/mg of protein, the increase in MDA levels was about 2.5-fold as compared to the control group (p < 0.05). On the other hand, rhEPO administration at 3000 IU/kg b.w., simultaneously, before, or after MMC, was associated with a fall in the MDA levels to reach the control group. Thus, MDA levels in heart tissue were 17.51 ± 0.55, 12.44 ± 0.58, and 18.94 ± 0.62 mmol/mg of protein, respectively, in co-, pre-, and posttreatment as compared to 25.46 ± 0.65 mmol/mg of protein, for MMC-treated group. Moreover, as shown in Figure 2, the optimum preventive action of rhEPO was observed in pretreatment condition.

Figure 2.

Lipid peroxidation as determined by MDA level in kidney and heart tissues of Wistar rat. Values are expressed as means ± S.D. *p < 0.05 versus control, ^# p < 0.05 versus MMC, ^a p < 0.05 versus cotreatment condition, and ^b p < 0.05 versus posttreatment condition. MMC: mitomycin C; MDA: malondialdehyde.

Effect of rhEPO on glutathione (GSH + GSSG) modulation

The effect of MMC on GSH and GSSG modulation in kidney and heart tissues was illustrated in Figure 3. Our data demonstrated that Wistar rats exposed to MMC alone showed a noticeable depletion of GSH level (p < 0.05) and a significant increase of GSSG level (p < 0.05), as compared to the untreated group. Thus, GSH levels were 15.35 ± 0.72 versus 25.77 ± 0.63 nmol GSH/g of proteins in kidney extract and 4.42 ± 0.2 versus 7.65 ± 0.28 nmol GSH/g of proteins in heart extract. However, a significant increase (p < 0.05) in GSH level was observed in rats treated with rhEPO in different treatment conditions (co-, pre-, and posttreatment), as compared to the MMC group. Indeed, in heart extracts, the GSH levels increased to 5.12 ± 0.83, 7.46 ± 0.7, and 5.45 ± 0.42 nmol GSH/g of sample, respectively, in co-, pre-, and posttreatment conditions, as compared to the MMC group (4.42 ± 0.2 nmol GSH/mg of protein; Figure 3(a)). Accordingly, the GSSG levels were significantly lower in the groups treated with rhEPO in different treatment condition as compared to the MMC group. Thus, GSSG level in kidney extracts passed from 1.04 ± 0.09 in the MMC-treated group to 0.68 ± 0.04, 0.57 ± 0.02 and 0.83 ± 0.07, respectively, in co-, pre- and posttreatment groups (Figure 3(b)). Furthermore, our results demonstrated that the pretreatment condition promoted the best protection against MMC-induced glutathione modulation and oxidative stress in kidney and heart tissues.

Figure 3.

Effect of rhEPO administration on glutathione (a: GSH and b: GSSG) levels in rat kidney and heart. Different treatment conditions were performed: rhEPO was administered simultaneously, 24 h before, and 5 days after MMC intoxication. *p < 0.05 versus control, ^# p < 0.05 versus MMC, ^a p < 0.05 versus cotreatment condition, and ^b p < 0.05 versus posttreatment condition. MMC: mitomycin C; rhEPO: recombinant human erythropoietin; GSH: reduced glutathione; GSSG: oxidized glutathione.

Catalase activity

Catalase is an endogenous antioxidant enzyme that protects cells from the detrimental effects of ROS. Level of catalase can indicate the magnitude of oxidative stress that occurs. The effect of MMC and rhEPO on catalase activity was illustrated in Figure 4. Our results showed that MMC alone induced a marked increase in catalase activity in both kidney and heart extracts. Catalase activity increased from the basal value of 153.42 ± 6.72 nmol/min/mg proteins in the control group to 428.79 ± 5.9 nmol/mg in the MMC-treated group in kidney extract. In heart extract, catalase activity increased from 130.40 ± 17.02 nmol/min/mg proteins in the control group to 393.41 ± 14.56 nmol/mg in the MMC group. rhEPO administration simultaneously, 24 h before, and 5 days after MMC treatment showed a decreased activity. For example, in heart extract, catalase activity decreased from 393.41 ± 14.56 nmol/mg in the MMC group to 209.76 ± 9.38, 174.08 ± 4.09, and 202.23 ± 3.03 nmol/mg in groups treated with rhEPO, respectively, in co-, pre-, and posttreatment groups. Thus, the pretreatment condition provided the best protection against MMC-induced oxidative stress.

Figure 4.

Effect of rhEPO on MMC-induced catalase enzyme activity in rat kidney and heart. rhEPO was added simultaneously, 24 h before, and 5 days after MMC administration. ^* p < 0.05 versus control, ^# p < 0.05 versus MMC, ^a p < 0.05 versus cotreatment condition, and ^b p < 0.05 versus posttreatment condition. MMC: mitomycin C; rhEPO: recombinant human erythropoietin.

Effect of rhEPO on DNA damage

The antigenotoxic effect of rhEPO was assessed through the alkaline comet assay. Results of the visual scoring of total basic DNA damage were illustrated in Figure 5. We observed a significant increase in the total DNA damage in rats treated only with MMC (3 mg/kg i.p.) in both kidney and heart extracts. rhEPO treatment (co-, pre-, and postconditions) reduced DNA fragmentation caused by MMC. In kidney extracts, this reduction was about 24.26 ± 3.82%, 42.4 ± 4.32%, and 34.16 ± 3.07% versus MMC in the cotreatment, the pretreatment, and the posttreatment conditions, respectively. In heart extracts, this reduction was about 23.14 ± 4.08%, 41.89 ± 5.3%, and 30.44 ± 3.28% versus MMC, in co-, pre-, and posttreatment conditions, respectively. The degree of DNA damage seemed to be less noticeable when rats were pretreated with rhEPO 24 h before MMC exposure. No specific DNA fragmentation was detected in control and rhEPO alone groups.

Figure 5.

Total DNA damage was measured by the alkaline comet assay in isolated cells of rat kidney and heart. DNA fragmentation in kidney and heart of rat cells was estimated after a single exposure to MMC and rhEPO in different treatment conditions. *p < 0.05 versus control, ^# p < 0.05 versus MMC, ^a p < 0.05 versus cotreatment condition, and ^b p < 0.05 versus posttreatment condition. MMC: mitomycin C; rhEPO: recombinant human erythropoietin.

Discussion

MMC is an antitumor agent against several types of cancer and has been used widely in chemotherapy. In spite of its beneficial anticancer action, the dose-related nephrotoxicity and cardiotoxicity limit its application in clinical oncology.^39

–43 MMC toxicity can be associated with its oxidant property by disturbing the oxidant/antioxidant balance.¹⁰ Strategies to protect normal tissues against MMC toxicity are of clinical interest, and tissue-cytoprotective agents are essential to provide protection against the different MMC toxicities.^10,44 In this study, we focused our interest on rhEPO, a glycoprotein hormone essential for survival, proliferation, and differentiation of the erythrocytic progenitors in the bone marrow. Many studies showed that rhEPO was directly involved in the prevention of oxidative stress with activation of antioxidant enzymes, inhibition of nitric oxide production, and decrease in lipid peroxidation.^45,46 Thus, the aim of the present study was to evaluate a protective effect of rhEPO (3000 IU/kg) against MMC (3 mg/kg)-induced oxidative stress and genotoxicity in the kidney and heart of rats. The selected dose of MMC was in accordance with previous studies showing that MMC administration induced oxidative stress and genotoxicity in rat tissues and bone morrow cells.⁴⁷ Similarly, the selected dose of rhEPO was in agreement with several studies, showing that rhEPO administration to rat protects against renal and heart insults.^48
–50 To assess the eventual protective effect of rhEPO against MMC-induced oxidative damage, we measured protein carbonyl and lipid peroxidation contents as two biomarkers of oxidative stress as well as glutathione modulation (GSH and GSSG) and catalase activity, which are considered as biomarkers of antioxidant defense. Oxidative modification of proteins can often lead to a loss of protein function, which may have lasting detrimental effects on cells and tissues.^51,52 Our results clearly showed that MMC induced a marked increase in protein carbonyl generation in either kidney and heart extracts, which was significantly reduced with rhEPO in different experimental conditions (co-, pre-, and posttreatment; Figure 1). In the present study, although exposure to MMC (3 mg/kg b.w.) induced a noticeable increase in MDA formation in kidney and heart of rats, the administration of rhEPO simultaneously, before, or after MMC exposure provided a significant reduction of this induction, resulting in its drop to the control level (Figure 2). Furthermore, our results showed that rhEPO treatment 24 h before intoxicating the rats with MMC promoted the best protection against the oxidative stress induced by this drug. Glutathione is the most abundant intracellular thiol and plays an important role in the detoxification of ROS and xenobiotics.⁵³ Our results showed that MMC significantly decreased the level of GSH and increased the level of GSSG in both kidney and heart of rat. The amount of GSH and GSSG were clearly ameliorated in the presence of rhEPO in different treatment conditions (simultaneously, before, and after MMC intoxication; Figure 3). Catalase is one of the most effective antioxidant enzymes that are produced naturally in the body to prevent free radical damage. In this study, MMC was found to enhance catalase activity in rat kidney and heart, as an adaptive response to the generated free radicals, evidenced by protein carbonyl content. rhEPO administration, in any treatment condition, significantly reduced catalase activity enhanced by MMC (Figure 4). We have demonstrated that the optimum prevention of rhEPO against the MMC-induced oxidative lesion was observed when rhEPO was administrated 24 h before MMC. Our results were in agreement with other studies, showing that MMC administration was associated with increased formation of free radicals, leading to oxidative damage of proteins, lipids, and nucleic acids.^{8,10,44,54,55}

It is widely known that oxidative stress can be associated with an increase in macromolecules injuries, especially DNA damage. To assess the eventual antigenotoxic action of rhEPO, we performed the comet assay, which is one of the standard methods for assessing DNA damage including single- and double-strand DNA breaks.⁵⁶ Our results showed that MMC alone caused a significant increase in DNA fragmentation. rhEPO treatment simultaneously, 24 h before, and 5 days after MMC administration induced a noticeable decrease in DNA fragmentation in the kidney and heart of rats (Figure 5). Interestingly, pretreatment with rhEPO provided the best protective effect against MMC genotoxicity. Our results were in agreement with those of Sun et al.⁵⁷ who showed that rhEPO (300 IU/kg i.p.) caused a reduction in DNA fragmentation in neonatal hypoxia–ischemia in rat brain.

In conclusion, we have shown that experimental MMC administration increased oxidative stress and genotoxic damage in the kidney and heart of rats. rhEPO administration, especially in pretreatment condition, protected against MMC-induced antioxidative stress and genotoxicity. Thus, our findings would provide a more promising strategy for the prevention of nephrotoxicity and cardiotoxicity in MMC-based chemotherapy.

Footnotes

Author’s Note

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.

Funding

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: LRSBC).”

Declaration of Conflicting Interests

The authors declared no conflicts of interest.

References

Gnad-Vogt

Hofheinz

Saussele

. Pegylated liposomal doxorubicin and mitomycin C in combination with infusional 5-fluorouracil and sodium folinic acid in the treatment of advanced gastric cancer: results of a phase II trial. Anticancer Drugs 2005; 16: 435–440.

Cheung

Rauth

. In vivo efficacy and toxicity of intratumorally delivered mitomycin C and its combination with doxorubicin using microsphere formulations. Anticancer Drugs 2005; 16: 423–433.

Bradner

. Mitomycin C: a clinical update. Cancer Treat Rev 2001; 27: 35–50.

Yeh

. Cardiotoxicity induced by chemotherapy and antibody therapy. Annu Rev Med 2006; 57: 485–498.

Buzdar

Legha

Luna

. Pulmonary toxicity of mitomycin. Cancer 1980; 45: 236–244.

Lameire

Kruse

Rottey

. Nephrotoxicity of anticancer drugs—an underestimated problem? Acta Clin Belg 2011; 66: 337–345.

Shiiki

Dohi

Nishioka

. Glomerular tuft ballooning in mitomycin-C-induced renal impairment. Virchows Archiv A Pathol Anat Histopathol 1992; 420: 545–551.

Komiyama

Kikuchi

Sugiura

. Generation of hydroxyl radical by anticancer quinone drugs, carbazilquinone, mitomycin C, aclacinomycin A and adriamycin, in the presence of NADPH-cytochrome P-450 reductase. Biochem Pharmacol 1982; 31: 3651–3656.

Riggs

. Antitumor antibiotics and related compounds. In: Perry

(ed) The chemotherapy source book. Baltimore, MD: Williams & Wilkins, 1997, pp. 345–385.

10.

Premkumar

Pachiappan

Abraham

. Effect of Spirulina fusiformis on cyclophosphamide and mitomycin-C induced genotoxicity and oxidative stress in mice. Fitoterapia 2001; 72: 906–911.

11.

Turkez

Aydin

Aslan

. Xanthoria elegans (Link) (lichen) extract counteracts DNA damage and oxidative stress of mitomycin C in human lymphocytes. Cytotechnology 2012; 64: 679–686.

12.

Klaunig

Wang

Zhou

. Oxidative stress and oxidative damage in chemical carcinogenesis. Toxicol Appl Pharmacol 2011; 254: 86–99.

13.

Wang

Gray

Mishin

. Distinct roles of cytochrome P450 reductase in mitomycin C redox cycling and cytotoxicity. Mol Cancer Ther 2010; 9: 1852–1863.

14.

Klaunig

Wang

Zhou

. Oxidative stress and oxidative damage in chemical carcinogenesis. Toxicol Appl Pharmacol 2011; 254: 86–99.

15.

Freeman

Parvizi

Della Valle

Steinbeck

. Reactive oxygen and nitrogen species induce protein and DNA modifications driving arthrofibrosis following total knee arthroplasty. Fibrogenesis Tissue Repair 2009; 2: 5.

16.

Mazumdar

Giri

. Role of quercetin on mitomycin C induced genotoxicity: analysis of micronucleus and chromosome aberrations in vivo. Mutat Res 2011; 721: 147–152.

17.

Chong

Maiese

. Erythropoietin on a tightrope. Balancing neuronal and vascular protection between intrinsic and extrinsic pathways. Neurosignals 2004; 13: 265–289.

18.

Kuriyama

Tomonari

Yoshida

. Reversal of anemia by erythropoietin therapy retards the progression of chronic renal failure, especially in nondiabetic patients. Nephron 1997; 77: 176–185.

19.

Nemoto

Yokota

Keane

Rabb

. Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure. Kidney Int 2001; 59: 246–251.

20.

Hodges

Rainey

Lappin

Maxwell

. Pathophysiology of anemia and erythrocytosis. Crit Rev Oncol Hematol 2007; 64: 139–158.

21.

Chatterjee

. Pleiotropic renal actions of erythropoietin. Lancet 2005; 365: 1890–1892.

22.

Liu

Xie

Liu

. Mechanism of the cardioprotection of rhEPO pretreatment on suppressing the inflammatory response in ischemia reperfusion. Life Sci 2006; 78: 2255–2264.

23.

Okada

Sawada

Kubota

. Asialoerythropoietin has strong renoprotective effects against ischemia reperfusion injury in a murine model. Transplantation 2007; 84: 504–510.

24.

Riksen

Hausenloy

Yellon

. Erythropoietin: ready for prime-time cardioprotection. A review. Trends Pharmacol Sci 2008; 29: 258–267.

25.

Ishii

Sawada

Murakani

. Renoprotective effect of erythropoietin against ischaemia-reperfusion injury in a non-human primate model. Nephrol Dial Transplant 2010; 26: 1157–1162.

26.

Chateauvieux

Grigorakaki

Morceau

Dicato

Diederich

. Erythropoietin, erythropoiesis and beyond. Biochem Pharmacol 2011; 82: 1291–1303.

27.

Bradford

. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976; 72: 248–254.

28.

Mercier

Gatellier

Renerre

. Lipid and protein oxidation in vitro, and antioxidant potential in meat from Charolais cows finished on pasture or mixed diet. Meat Sci 2004; 66: 467–473.

29.

Aust

Morehouse

Thomas

CEJ

. Role of metals in oxygen radical reactions. Free Radic Biol Med 1985; 1: 3–25.

30.

Tietze

. Enzimatic method for quantitative determination of nanogram amounts of total and oxidized gluthathione: applications to mammalian blood and others tissues. Anal Biochem 1969; 27: 502–522.

31.

Clairbone

. Catalase activity. Handbook of methods for oxygen radical research. Boca Raton, FL: CRC Press, 1985, pp. 283–284.

32.

Tice

Aqurell

Anderson

. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000; 35: 206–221.

33.

Picada

Flores

Zettler

. DNA damage in brain cells of mice treated with an oxidized form of apomorphine. Brain Res Mol Brain Res 2003; 114: 80–85.

34.

Banath

Kim

Olive

. Overnight lysis improves the efficiency of DNA damage detection in the alkaline comet assay. Radiat Res 2002; 1155: 564–571.

35.

Collins

Dusinska

Gedik

Stetina

. Oxidative damage to DNA: do we have a reliable biomarker? Environ Health Perspect 1996; 104: 465–469.

36.

Draper

Squires

Mahmoodi

. A comparative evaluation of thiobarbituric acid methods for the determination of malondialdehyde in biological materials. Free Radic Biol Med 1993; 15: 353–363.

37.

Kim

Mun

Lee

. Oxidative damage in renal transplant patients. Transplant Proc 2000; 32: 1777–1778.

38.

Dotan

Lichtenberg

Pinchuk

. Lipid peroxidation cannot be used as a universal criterion of oxidative stress. Prog Lipid Res 2004; 43: 200–227.

39.

Verweij

Funke-Kupper

Teule

. A prospective study on the dose dependency of cardiotoxicity induced by mitomycin C. Med Oncol Tumor Pharmacother 1988; 5: 159–163.

40.

Wen

Chen

. Mitomycin c-induced renal insufficiency. A case report. J Med Sci 2003; 19: 317–321.

41.

Panjrath

Jain

. Monitoring chemotherapy-induced cardiotoxicity: role of cardiac nuclear imaging: a review. J Nucl Cardiol 2006; 13: 415–426.

42.

Naughton

. Drug-induced nephrotoxicity: a review. Am Fam Physicians 2008; 78: 743–750.

43.

Senkus

Jassem

. Complications of treatment cardiovascular effects of systemic cancer treatment. Cancer Treat Rev 2011; 37: 300–311.

44.

Siddique

Ara

Beg

. Assessment of cell viability, lipid peroxidation and quantification of DNA fragmentation after the treatment of anticancerous drug mitomycin C and curcumin in cultured human blood lymphocytes. Exp Toxicol Pathol 2010; 62: 503–508.

45.

Solaroglu

Kaptanoglu

. Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain. Childs Nerv Syst 2003; 19: 19–22.

46.

Kumral

Baskin

Gokmen

. Selective inhibition of nitric oxide in hypoxic-ischemic brain model in newborn rats: is it an explanation for the protective role of erythropoietin? Biol Neonate 2004; 85: 51–54.

47.

Mazumdar

Giri

. Role of quercetin on mitomycin C induced genotoxicity: analysis of micronucleus and chromosome aberrations in vivo . Mut Res 2011; 721: 147–152.

48.

Yang

Jung

. Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney. FASEB J 2003; 10: 1096–1191.

49.

Mihov

Bogdanov

Grenacher

. Erythropoietin protects from reperfusion-induced myocardial injury by enhancing coronary endothelial nitric oxide production. Eur J Cardiothorac Surg 2009; 35: 839–846.

50.

Johnson

Vesey

Gobe

. Erythropoietin protects against acute kidney injury and failure. Open Drug Discov J 2010; 2: 8–17.

51.

Dalle-Donne

Giustarini

Colombo

. Protein carbonylation in human diseases. Trends Mol Med 2003; 9: 169–176.

52.

Dalle-Donne

Rossi

Giustarini

. Protein carbonyl groups as biomarkers of oxidative damage in human disease. Clin Chim Acta 2003; 329: 23–38.

53.

Meister

Anderson

. Glutathione. Annu Rev Biochem 1983; 52: 711–760.

54.

Nakano

Sugioka

Nakano

. Importance of Fe²⁺-ADP and the relative unimportance of OH in the mechanism of mitomycin C-induced lipid peroxidation. Biochim Biophys Acta 1984; 796: 285–293.

55.

Pagano

. Mitomycin C and diepoxybutane action mechanisms and FANCC protein functions: further insights into the role for oxidative stress in Fanconi’s anaemia phenotype. Carcinogenesis 2000; 21: 1067–1068.

56.

Singh

McCoy

Tice

Schneider

. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184–191.

57.

Sun

Zhou

Polk

. Mechanisms of erythropoietin-induced brain protection in neonatal hypoxia ischemia rat model. J Cereb Blood Flow Metab 2004; 24: 259–270.

Effect of recombinant human erythropoietin on mitomycin C-induced oxidative stress and genotoxicity in rat kidney and heart tissues

Abstract

Keywords

Introduction

Material and methods

Chemicals

Animal treatment

Preparation of heart and kidney homogenates

Protein carbonyl assay

Evaluation of lipid peroxidation level

Glutathione assay (GSH and GSSG levels)

Determination of catalase activity

Single-cell gel electrophoresis (the comet assay)

Statistical analysis

Results

Protein carbonyl assay

Induction of lipid peroxidation

Effect of rhEPO on glutathione (GSH + GSSG) modulation

Catalase activity

Effect of rhEPO on DNA damage

Discussion

Footnotes

Author’s Note

Funding

Declaration of Conflicting Interests

References