134

Introduction

Cisplatin (Cisp) is an antineoplastic agent used against various types of animal and human tumors (Loehrer and Einhom, 1984). However, the optimal usefulness of Cisp as an important anticancer drug is usually limited by its secondary effects, especially its nephrotoxicity (Lieberthal et al., 1996; Kintzel, 2001). Earlier studies have reported that Cisp therapy is usually associated with less common toxic effects, such as hepatotoxicity and cardiotoxicity (Chvetzoff et al., 1998; King and Perry, 2001; Pai et al., 2000). Therefore, even though hepatotox-icity and cardiotoxicity are not the most frequent toxic effects of Cisp, it may become a concern under specific conditions.

Mechanisms underlying the side effects induced by Cisp have not been understood clearly until recently. Oxidative stress was considered to be one of the

mechanisms implicated in Cisp-induced toxicity (Koc et al., 2005; Pratibha et al., 2006; Mansour et al., 2006; Iraz et al., 2006). In fact, reactive oxygen species (ROS) can attack biomolecules, such as DNA, lipids, and thiols in proteins and glutathione, leading to enzyme inactivation, genotoxic damage, cell dysfunction, and death. Many compounds have been suggested to protect against Cisp-induced oxidative damage in the liver and heart (Koc et al., 2005; Iraz et al., 2006; Dos Santos et al., 2007; Yüce et al., 2007); however, the use of recombinant human erythropoietin (rhEPO) has not been evaluated.

Erythropoietin (EPO) is a glycoprotein hormone pri-marily, but not exclusively, synthesized by renal cortical interstitial fibroblasts in response to tissue hypoxia (Li et al., 2004). EPO, used clinically as rhEPO, has been used as a successful treatment for human anemia associated with end-stage renal failure and cancer chemotherapy

ReseaRch aRtIcle

Recombinant human erythropoietin prevents cisplatin-induced genotoxicity in rat liver and heart tissues via an antioxidant process

Karima Rjiba-Touati1, Imen Ayed-Boussema1, Anis Belarbia2, Abdelatif Achour2, and Hassen Bacha1

1Laboratory of Research on Biologically Compatible Compounds, Faculty of Dentistry, Monastir, Tunisia and 2Department of Nephrology, Dialysis, and Transplant, University Hospital of Sahloul, Sousse, Tunisia

abstractCisplatin (Cisp) is one of the most effective chemotherapeutic agents. However, at higher doses, liver and heart injuries may occur. Recombinant human erythropoietin (rhEPO) has recently been shown to exert an important cytoprotective effect in many tissues. For that reason, we tried to check the protective effect of rhEPO against Cisp-induced genotoxicity and oxidative stress in liver and heart tissues. Our experiments were performed using six groups of adult male Wistar rats. The control group was treated only with saline solution. The rhEPO group was given a single dose of rhEPO. The Cisp group was given a single injection of Cisp. The rhEPO+Cisp groups were given rhEPO simultaneously, 24 hours before, and 5 days after Cisp injection. Our results clearly showed that Cisp induced noticeable DNA damage in the liver and heart, accompanied by a significant increase in protein carbonyl level, reduced glutathione (GSH) depletion, and a decrease in catalase activity. Rats treated with rhEPO, simultaneously, before, or after Cisp injection, remarkably decreased DNA damage. It decreased also the protein carbonyl level, restored GSH depletion, and enhanced catalase activity. Our results highlight an interesting cytoprotective strategy using rhEPO against Cisp-induced liver and heart injuries.Keywords: Cisplatin, rhEPO, antigenotoxic effect, antioxidant property, liver, heart

Address for Correspondence: Hassen Bacha, Laboratoire de Recherche sur les Substances Biologiquement Compatibles (LRSBC), Faculté de Médecine Dentaire, Rue Avicenne, 5019 Monastir, Tunisia; Fax: +216 73 42 55 50; E-mail: hassen.bacha@fmdm.rnu.tn

(Received 09 February 2011; revised 23 March 2011; accepted 29 March 2011)

Drug and Chemical Toxicology, 2012; 35(2): 134–140© 2012 Informa Healthcare USA, Inc.ISSN 0148-0545 print/ISSN 1525-6014 onlineDOI: 10.3109/01480545.2011.589445

Drug and Chemical Toxicology

2012

35

2

134

140

09 February 2011

23 March 2011

29 March 2011

0148-0545

1525-6014

© 2012 Informa Healthcare USA, Inc.

10.3109/01480545.2011.589445

LDCT

589445

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.

Antigenotoxic and -oxidant effect of rhEPO against cisplatin 135

© 2012 Informa Healthcare USA, Inc.

(Kuriyama et al., 1997; Nemoto et al., 2001; Hodges et al., 2007). Recently, the biological effects of rhEPO have not been limited to the hematopoietic system; many stud-ies have shown that rhEPO is a pleiotropic cytokine that exerts broad tissue-protective effects in diverse nonhe-matopoietic organs (Chatterjee, 2005). Clinical studies have suggested an additional role for EPO in the liver and cardiovascular system (Maiese and Chong, 2004, 2005; Schmeding et al., 2007).

In this context, the aim of the present study was to evaluate the possible antigenotoxic and -oxidant effects of rhEPO against Cisp-induced genotoxicity and oxida-tive stress in the liver and heart of rats. For this purpose, we measured DNA damage in liver and heart tissues using the comet assay. Oxidative stress involvement was assessed by the measure of protein carbonyl levels, reduced glutathione (GSH), and catalase activity in the liver and heart of rats.

Methods

ChemicalsCisp (cis-diamminedichloroplatinum II) was purchased from Sigma-Aldrich (Lyon, France). Experiments were per-formed 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 other chemicals used were of analytical grade.

Animal treatmentExperiments were performed on male Wistar rats in the weight range of 120–140 g, kept at controlled environ-mental conditions at room temperature 22 ± 2°C and 12 h light/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 six groups, with 6 animals in each group. All injections were administered by the intraperitoneal (i.p.) route. The control group received a single injection of saline solu-tion (0.9%). The rhEPO group was given only rhEPO, and the Cisp group was given only a single injection of Cisp. To test the effect of rhEPO on Cisp-induced oxidative damage and genotoxicity, three treatment conditions were employed. In a cotreatment group, a single dose of rhEPO was administered simultaneously with Cisp. In a pre-treatment group, a single dose of rhEPO was given 1 day before Cisp. In a post-treatment group, a single dose of rhEPO was given 5 days after Cisp. In each type of treat-ment, rhEPO and Cisp were used, respectively, at 3,000 UL/kg body weight (b.w.) and 6 mg/kg b.w. Experimental design is detailed in Table 1. After animals were sacri-ficed, the liver and heart were immediately removed for subsequent experiments.

Preparation of liver and heart extractsLiver and heart were homogenized with a potter (glass, Teflon) in the presence of 10 mM of Tris-HCl (pH 7.4) at

4°C and centrifuged at 4,000 rpm for 30 minutes at 4°C. The supernatant was collected for analysis, and protein concentrations were determined in liver and heart extract using the protein Bio-Rad assay (Bradford, 1976).

Single-cell gel electrophoresis (the comet assay)Determination of DNA damage by the alkaline comet assay was conducted according to Tice et al. (2000), with minor modifications (Picada et al., 2003). It is of note that these methods have monitored the comet assay after a short time of exposure; however, several other studies (Antunes et al., 2000; Saad et al., 2009; El-Awady et al., 2011), as well as the present work, have investigated the comet assay at a longer time of exposure. Each piece of liver or heart was placed in 0.5 mL of cold phosphate-buffered saline (PBS) and finely minced to obtain a cellular suspension. Liver 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 alka-line 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 (Banath et al., 2002). Slides were then placed in an electrophore-sis alkaline buffer (pH >13), and the embedded cells were exposed to this alkaline solution for 20 minutes to allow for DNA unwinding. Electrophoresis was performed in the same alkaline buffer for 20 minutes 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 con-ducted 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. (1996). 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).

Protein carbonyl assayProtein carbonyl content was determined, as described by Mercier et al. (2004), in liver and heart homogenates by measuring the reactivity of carbonyl groups with 2,4-dinitrophenylhydrazine (2,4-DNPH). Thus, 200 µL of supernatant of liver or heart were placed in two glass tubes. Then, 800 µL of 10 mM of DNPH in 2.5 M of HCl were added. Tubes were left for 1 hour of incubation at room temperature in the dark. Samples were vortexed every 15 minutes. Then, 1 mL of 20% TCA was added to samples, and the tubes were left in an ice bucket for 10 minutes and centrifuged for 5 minutes at 4,000 rpm to collect the protein precipates, and the supernatants were discarded. Next, another wash was performed using 1 mL of 10% TCA, and protein pellets were broken mechani-cally with the aid of a glass rod. Finally, the pellets were

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.

136 Karima Rjiba-Touati

Drug and Chemical Toxicology

washed with 1 mL of ethanol-ethyl acetate (1:1; v/v) to remove the free DNPH. The final precipitates were dis-solved in 500 µL of guanidine hydrochloride (6 M) and were left for 10 minutes at 37°C with general vortex mix-ing. 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−1 cm−1. A range of nmoles of carbonyls per mL was usually obtained for most proteins and was related to the protein content in the pellets.

Glutathione assayGSH level in liver and heart extract were measured using a colorimetric assay kit, according to the recom-mendations 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-colored product. The reduced compound directly proportional to GSH concentration and its absorbance was measured at 412 nm (Vina et al., 1995; Sian et al., 1997). The liver or heart tissues of different groups were flash-freezed and ground in liquid nitrogen immediately after excision. Next, 0.3 g of liver or heart powder was taken and mixed with 5% 5-sulfosalicylic acid solution for renal protein removal. Aliquots of liver or heart extract were submitted to photometric 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 activityCatalase activity was measured in the liver and heart extract at 240 nm, at 25°C, according to Clairbone (1985). Briefly, 20 µL of the extracts were added in the quartz cuvette containing 80 µL of phosphate buffer (pH = 7) and 200 µL of H

2O

2 (0.5 M). The activity of catalase

was calculated using the molar extinction coefficient (0.04 mM−1 cm−1). Results were expressed as µmol of H

2O

2/min/mg of proteins.

Statistical analysisEach experiment was carried out at least three times. Data were expressed as means ± standard deviation (SD). Statistical comparison between different groups was done using one-way analysis of variance, followed by Fischer’s post-hoc test, to detect the difference between various groups. A value of P < 0.05 was considered to be significant.

Results

Effect of rhEPO on DNA damageThe 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 1. We observed a significant increase of the total DNA damage in rats treated with Cisp only (6 mg/kg i.p.) in both liver and heart extracts. rhEPO treatment (co-, pre-, and postconditions) reduced DNA fragmentation caused by Cisp. The amount of DNA damage decreased by approximately 2-fold with respect to the Cisp value in rats treated with rhEPO in different treatment conditions in heart and liver extracts. The degree of DNA damage seemed to be less noticeable when rats were pretreated with rhEPO 24 hours before Cisp exposure. No specific DNA fragmentation was detected in control and rhEPO-alone groups. Histological examination of liver and heart tissues did not reveal significant tissue damages in the Cisp-treated group, as compared to the control group (data not shown). Thus, we can deduce that in our experimental condition (i.e., tested dose and time expo-sure), DNA damage caused by Cisp was not secondary to tissue damage.

Figure 1. Total DNA damage was measured by the alkaline comet assay in isolated cells of rat liver and heart. DNA fragmentation in liver and heart of rat cells was estimated after a single exposure to Cisp and rhEPO in different treatment conditions: Cisp alone, rhEPO alone, and rhEPO with Cisp in different treatment conditions (rhEPO administration was performed simultaneously, 24 hours before, and 5 days after Cisp treatment). rhEPO (3,000 UL/kg b.w., i.p.) and Cisp (6 mg/kg b.w., i.p.). *P < 0.05 versus control, #P versus Cisp, aP < 0.05 versus cotreatment condition, bP < 0.05 versus post-treatment condition.

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.

Antigenotoxic and -oxidant effect of rhEPO against cisplatin 137

© 2012 Informa Healthcare USA, Inc.

Protein carbonyl assayThe formation of protein carbonyls, the most widely used marker of severe protein oxidation, was assayed in liver and heart homogenates, and results are illustrated in Figure 2. We showed that Cisp alone generated pro-tein carbonyl formation, as compared to control groups, in both liver and heart extracts. For liver extracts, the protein carbonyl level increased from the basal value of 0.49 ± 0.02 nmol/mg of protein in the control group to 5.07 ± 0.20 nmol/mg of protein in the Cisp-treated group. The amounts of protein carbonyls in heart extracts were less than those detected in the liver. Indeed, protein carbonyl level increased from a basal value of 0.92 ± 0.11 nmol/mg of protein in control group to 3.39 ± 0.4 nmol/mg of protein in the Cisp-treated group. Further, our results showed that protein oxidation decreased sig-nificantly in the presence of rhEPO in any treatment conditions (simultaneously, 24 hours before, and 5 days after Cisp exposure) in both liver and heart extracts, as compared to the Cisp group. For example, in liver

extract, protein carbonyl level decreased from the value of 5.07 ± 0.20 nmol/mg of protein in the Cisp group to 1.89 ± 0.56, 0.83 ± 0.23, and 1.75 ± 0.45 nmol/mg of protein in groups treated with rhEPO, respectively, in co-, pre-, and post-treatment conditions. Moreover, our results demonstrated that rhEPO was more efficient in the pre-treatment condition against protein carbonyl formation either in liver or heart tissues.

Effect of rhEPO on glutathione depletionThe effect of Cisp on GSH modulation in liver and heart tissues are illustrated in Figure 3. Our data demonstrated that Wistar rats exposed to Cisp alone showed a notice-able depletion of GSH level (P < 0.05), as compared to the untreated group (7.01 ± 0.92 vs. 22.01 ± 1.63 nmoles GSH/g of sample in liver extract and 3.67 ± 1.24 vs. 8.45 ± 0.83 nmoles GSH/g of sample in heart extract). However, a significant increase (P < 0.05) in GSH level was observed in rats treated with Cisp and rhEPO in different treatment conditions (co-, pre-, and post-treatment), as compared

Figure 2. Effect of rhEPO on protein carbonyls concentrations in rat liver and heart. rhEPO was administered simultaneously, 24 hours before, and 5 days after Cisp treatment. rhEPO (3,000 UL/kg b.w., i.p.) and Cisp (6 mg/kg b.w., i.p.). *P < 0.05 versus control, #P < 0.05 versus Cisp, aP < 0.05 versus cotreatment condition, bP < 0.05 versus post-treatment condition.

Figure 3. Effect of rhEPO administration on reduced glutathione level in rat liver and heart. Different treatment conditions were performed: rhEPO (3,000 UL/kg b.w., i.p.) was administered simultaneously, 24 hours before, and 5 days after Cisp (6 mg/kg b.w., i.p.) intoxication. *P < 0.05 versus control, #P < 0.05 versus Cisp, aP < 0.05 versus cotreatment condition, bP < 0.05 versus post-treatment condition.

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.

138 Karima Rjiba-Touati

Drug and Chemical Toxicology

to the Cisp group. Indeed, in liver extracts, GSH levels increased to 14.53 ± 1.65, 20.86 ± 0.4, and 12.99 ± 1.59 nmoles GSH/g of sample, respectively, in co-, pre-, and post-treatment conditions, as compared to the Cisp group (7.01 ± 0.92 nmoles GSH/g of sample). Further, our results demonstrated that the pretreatment condition promoted the best protection against Cisp-induced GSH depletion in liver and heart tissues.

Catalase activityCatalase is an endogenous antioxidant enzyme that pro-tects cells from the detrimental effects of ROS. Levels of catalase can indicate the magnitude of oxidative stress that occurs. The effect of Cisp and rhEPO on catalase activity is illustrated in Figure 4. Our results showed that Cisp alone induced a marked increase in catalase activity in both liver and heart extracts. Catalase activity increased from the basal value of 32.94 ± 5.47 nmol/min/mg proteins in control group to 553.13 ± 58.14 nmol/mg in the Cisp-treated group in liver extract. In heart extract, catalase activity increased from 39.12 ± 7.36 nmol/min/mg proteins in the control group to 274.22 ± 45.89 nmol/mg in the Cisp group. rhEPO administration simultane-ously, 24 hours before, and 5 days after Cisp treatment produced a decrease of this activity. For example, in heart extract, catalase activity decreased from 274.22 ± 45.89 nmol/mg in the Cisp group to 121.33 ± 17.31, 73.86 ± 3.64, and 128.29 ± 17.32 nmol/mg in groups treated with Cisp and rhEPO, respectively, in co-, pre-, and post-treatment. Thus, the pretreatment condition provided the best pro-tection against Cisp-induced oxidative stress.

Discussion

Cisp is an antitumor agent against several types of cancer and has been widely used in chemotherapy. In spite of its beneficial anticancer action, the dose-related nephrotoxicity, hepatotoxicity, and cardiotoxicity limit its application in clinical oncology (Antunes et al., 2000;

Zicca et al., 2002; Antunes et al., 2001; King and Perry, 2001). Cisp toxicity can be associated to its oxidant property by disturbing the oxidant/antioxidant balance (Fadillioglu et al., 2003; Fadillioglu and Erdogan, 2003). Strategies to protect normal tissues against Cisp toxicity are of clinical interest, and tissue-cytoprotective agents are essential to provide protection against the different Cisp toxicities (Iraz et al., 2006; Ali and Al Moundhri, 2006). In this study, we focused our interest on rhEPO, a glycoprotein hormone essential for the 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 of lipid peroxidation (Solaroglu et al., 2003; Kumral et al., 2004). Thus, the aim of the present study was to evaluate a protective effect of rhEPO against Cisp-induced geno-toxicity and to assess the implication of oxidative stress in the possible antigenotoxic effect of rhEPO in the liver and heart of rats.

To assess to the possible antigenotoxic action of rhEPO, we monitored the comet assay, which is one of the standard methods for assessing DNA damage, including single- and double-strand DNA breaks (Singh et al., 1988). Our results showed that Cisp alone caused a significant increase in DNA fragmentation. rhEPO treat-ment simultaneously, 24 hours before, and 5 days after Cisp administration induced a noticeable decrease in DNA fragmentation in the liver and heart of rats. Further, pretreatment with rhEPO provided the best protective effect against Cisp genotoxicity. Our results are in agree-ment with those of Sun et al. (2004), who showed that rhEPO (300 IU/kg i.p.) caused a reduction in DNA frag-mentation in the rat brain.

It is widely known that macromolecules injuries, espe-cially DNA damage, can be associated with an increase in oxidative stress. For this, we evaluated the eventual pro-tective effect of rhEPO against Cisp-induced oxidative

Figure 4. Effect of rhEPO on Cisp-induced catalase enzyme activity in rat liver and heart. rhEPO (3,000 UL/kg b.w., i.p.) was added simultaneously, 24 hours before, and 5 days after Cisp administration (6 mg/kg b.w., i.p.). *P < 0.05 versus control, #P < 0.05 versus Cisp, aP < 0.05 versus cotreatment condition, bP < 0.05 versus post-treatment condition.

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.

Antigenotoxic and -oxidant effect of rhEPO against cisplatin 139

© 2012 Informa Healthcare USA, Inc.

damage by measuring protein carbonyl levels, GSH, and catalase activity in the liver and heart of rats.

Oxidative modification of proteins can often lead to a loss of protein function, which may have lasting detrimen-tal effects on cell and tissues (Dalle-Donne et al., 2003a, 2003b). Our results clearly showed that Cisp induced a marked increase in protein carbonyl generation in either liver and heart extracts, which was significantly reduced with rhEPO in the different experimental conditions (co-, pre-, and post-treatment) (Figure 2).

Glutathione is the most abundant intracellular thiol and plays an important role in the detoxification of ROS and xenobiotics (Meister and Anderson, 1983). Our results showed that Cisp significantly decreased the level of GSH either in the liver and heart of rats. The amount of GSH was clearly enhanced in the presence of rhEPO in different treatment conditions (simulta-neously, before, and after Cisp intoxication) (Figure 3). Catalase is one of the most efficient antioxidant enzymes that are produced naturally within the body to prevent free radical damage. In this study, Cisp was found to enhance catalase activity in rat liver and heart. This elevation was because of the adaptive response to the generated free radicals, as evidenced by protein carbonyl content. rhEPO administration, in any treat-ment condition, significantly reduced catalase activity enhanced by Cisp (Figure 4). We have demonstrated that the optimum prevention of rhEPO against the Cisp-induced oxidative lesion was observed when rhEPO was administrated 24 hours before Cisp. Our results are in agreement with other studies showing that Cisp administration was associated with increased forma-tion of free radicals, leading to oxidative damage of proteins, lipids, and nucleic acids (Antunes et al., 2000, 2001; Mora et al., 2003; Packer and Landvik, 1990). Thus, our findings suggested that the antigenotoxic effect of rhEPO was the consequence of inhibition of oxidative stress induced by Cisp.

conclusion

In conclusion, we have shown that experimental Cisp administration increased genotoxic and oxidative dam-age in the liver and heart of rats. rhEPO administration, especially in pretreatment condition, protects against Cisp-induced genotoxicity by an antioxidant process. Thus, our findings would provide a more promising strat-egy for prevention of hepatotoxicity and cardiotoxicity in Cisp-based chemotherapy.

Declaration of interest

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).

ReferencesAli, B. H., Al Moundhri, M. S. (2006). Agents ameliorating or

augmenting the nephrotoxicity of cisplatin and other platinum compounds: a review of some recent research. Food Chem Toxicol 44:1173–1183.

Antunes, L. M. G., Darin, J. D. C., Bianchi, M. L. P. (2000). Protective effects of vitamin C against cisplatin-induced nephrotoxicity and lipid peroxidation in adult rats. Pharmacol Res 41:405–411.

Antunes, L. M. G., Darin, J. D. C., Bianchi, M. L. P. (2001). Effects of the antioxidants curcumin or selenium on cisplatin-induced nephrotoxicity and lipid peroxidation in rats. Pharmacol Res 43:145–150.

Banath, J. P., Kim, A., Olive, P. (2002). Overnight lysis improves the efficiency of DNA damage detection in the alkaline comet assay. Radiat Res 1155:564–571.

Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.

Chatterjee, P. K. (2005). Pleiotropic renal actions of erythropoietin. Lancet 365:1890–1892.

Chvetzoff, G., Bonotte, B., Chauffert, B. (1998). Anticancer chemotherapy. Prevention of toxicity. Presse Med 27:2106–2112.

Clairbone, A. (1985). Catalase activity. In: Handbook of methods for oxygen radical research (pp. 283–284). Boca Raton, Florida, USA: CRC Press.

Collins, A. R., Dusinska, M., Gedik, C. M., Stetina, R. (1996). Oxidative damage to DNA: do we have a reliable biomarker? Environ Health Perspect 104:465–469.

Dalle-Donne, I., Giustarini, D., Colombo, R., Rossi, R., Milzani, A. (2003a). Protein carbonylation in human diseases. Trends Mol Med 9:169–176.

Dalle-Donne, I., Rossi, R., Giustarini, D., Milzani, A., Colombo, R. (2003b). Protein carbonyl groups as biomarkers of oxidative damage in human disease. Clin Chim Acta 329:23–38.

Dos Santos, N. A., Martins, N. M., Curti, C., Pires Bianchi Mde, L., Dos Santos, A. C. (2007). Dimethylthiourea protects against mitochondrial oxidative damage induced by cisplatin in liver of rats. Chem Biol Interact 170:177–186.

El-Awady, E. S. E., Moustafa, Y. M., Abo-Elmatty, D. M., Radwan, A. (2011). Cisplatin-induced cardiotoxicity: mechanisms and cardioprotective strategies. Eur J Pharmacol 650:335–341.

Fadillioglu, E., Erdogan, H., Sogut, S., Kuku, I. (2003). Protective effects of erdosteine against doxorubicin-induced cardioomyopathy in rats. J Appl Toxicol 23:71–74.

Fadillioglu, E., Erdogan, H. (2003). Effects of erdosteine treatment against doxorubicin-induced toxicity through erythrocyte and plasma oxidant/antioxidant status in rat. Pharmacol Res 47:317–322.

Table 1. Animal groups and treatments in the experimental design of this study. Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6Control group saline solution injection sacrifice rhEPO group rhEPO injection sacrifice Cisp Group Cisp injection sacrifice Co-treatment group Cisp-rhEPO injection sacrifice Pre-treatment group rhEPO injection Cisp injection sacrificePost-treatment group Cisp injection rhEPO injection sacrifice

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.

140 Karima Rjiba-Touati

Drug and Chemical Toxicology

Hodges, V. M., Rainey, S., Lappin, T. R., Maxwell, A. P. (2007). Pathophysiology of anemia and erythrocytosis. Crit Rev Oncol Hematol 64:139–158.

Iraz, M., Ozerol, E., Gulec, M., Tasdemir, S., Idiz, N., Fadillioglu, E., et al. (2006). Protective effect of caffeic acid phenethyl ester (CAPE) administration on cisplatin-induced oxidative damage to liver in rat. Cell Biochem Funct 24:357–361.

King, P. D., Perry, M. C. (2001). Hepatotoxicity of chemotherapy. Oncologist 6:162–176.

Kintzel, P. E. (2001). Anticancer drug-induced kidney disorders. Drug Saf 24:19–38.

Koc, A., Duru, M., Ciralik, H., Akcan, R., Sogut, S. (2005). Protective agent, erdosteine, against cisplatin-induced hepatic oxidant injury in rats. Mol Cell Biochem 278:79–84.

Kumral, A., Baskin, H., Gokmen, N., Yilmaz, O., Genc, K., Genc, S. (2004). 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 85:51–54.

Kuriyama, S., Tomonari, H., Yoshida, H., Hashimoto, T., Kawaguchi, Y., Sakai, O. (1997). Reversal of anemia by erythropoietin therapy retards the progression of chronic renal failure, especially in nondiabetic patients. Nephron 77:176–185.

Li, F., Chong, Z. Z., Maiese, K. (2004). Erythropoietin on a tightrope: balancing neuronal and vascular protection between intrinsic and extrinsic pathways. Neurosignals 13:265–289.

Lieberthal, W., Triaca, V., Levine, J. (1996). Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs necrosis. Am J Physiol 240:700–708.

Loehrer, P. J., Einhom, L. H. (1984). Cisplatin. Ann Intern Med 100:704–713.

Maiese, K., Li, F., Chong, Z. Z. (2004). Erythropoietin in the brain: can the promise to protect be fulfilled? Trends Pharmacol Sci 25:577–583.

Maiese, K., Li, F., Chong, Z. Z. (2005). New avenues of exploration for erythropoietin. JAMA 293:90–95.

Mansour, H. H., Hafez, H. F., Fahmy, N. M. (2006). Silymarin modulates cisplatin induced oxidative stress and hepatotoxicity in rats. J Biochem Mol Biol 39:656–561.

Meister, A., Anderson, M. E. (1983). Glutathione. Ann Rev Biochem 52:711–760.

Mercier, Y., Gatellier, P., Renerre, M. (2004). Lipid and protein oxidation in vitro, and antioxidant potential in meat from Charolais cows finished on pasture or mixed diet. Meat Sci 66:467–473.

Mora, L. O., Antunes, L. M., Francescato, H. D., Bianchi, M. I. (2003). The effects of oral glutamine on cisplatin-induced nephrotoxicity in rats. Pharmacol Res 47:517–522.

Nemoto, T., Yokota, N., Keane, W. F., Rabb, H. (2001). Recombinant erythropoietin rapidly treats anemia in ischemic acute renal failure. Kidney Int 59:246–251.

Packer, L., Landvik, S. (1990). Vitamin E in biological systems. In: Emerit, I., Packer, L., Auclair, C. (Eds.), Antioxidants in therapy and preventive medicine (pp. 93–104). New York: Plenum.

Pai, V. B., Nahata, M. C. (2000). Cardiotoxicity of chemotherapeutic agents: incidence, treatment, and prevention. Drug Saf 22:263–302.

Picada, J. N., Flores, D. G., Zettler, C. G., Marroni, N. P., Roesler, R., Henriques, J. A. P. (2003). DNA damage in brain cells of mice treated with an oxidized form of apomorphine. Brain Res Mol Brain Res 114:80–85.

Pratibha, R., Sameer, R., Rataboli, P. V., Bhiwgade, D. A., Dhume, C. Y. (2006). Enzymatic studies of cisplatin induced oxidative stress in hepatic tissue of rats. Eur J Pharmacol 532:290–293.

Saad, A. A., Youssef, M. I., El-Shennawy, L. K. (2009). Cisplatin induced damage in kidney genomic DNA and nephrotoxicity in male rats: the protective effect of grape seed proanthocyanidin extract. Food Chem Toxicol 47:1499–1506

Schmeding, M., Neumann, U. P., Boas-Knoop, S., Spinelli, A., Neuhaus, P. (2007). Erythropoietin reduces ischemia-reperfusion injury in the rat liver. Eur Surg Res 39:189–197.

Sian, J., Dexter, D. T., Cohen, G., Jenner, P. G., Marsden, C. D. (1997). Comparison of HPLC and enzymatic recycling assays for the measurement of oxidized glutathione in rat brain. J Pharmacol 49:332–335.

Singh, N., McCoy, M., Tice, R., Schneider, E. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191.

Solaroglu, I., Solaroglu, A., Kaptanoglu, E., Dede, S., Haberal, A., Beskonakli, E. (2003). Erythropoietin prevents ischemia-reperfusion from inducing oxidative damage in fetal rat brain. Childs Nerv Syst 19:19–22.

Sun, Y., Zhou, C., Polk, P., Nanda, A., Zhang, J. H. (2004). Mechanisms of erythropoietin-induced brain protection in neonatal hypoxia-ischemia rat model. J Cereb Blood Flow Metab 24:259–270.

Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, Y., et al. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221.

Vina, J., Sastre, M., Asensi, L., Packer, L. (1995). Assay of blood glutathione oxidation during physical exercise. Meth Enzymol 251:237–243.

Yüce, A., Atessahin, A. A., Çeribasi, A. O., Aksakal, M. (2007). Ellagic acid prevents cisplatin-induced oxidative stress in liver and heart tissue of rats. Basic Clin Pharmacol Toxicol 101:345–349.

Zicca, A., Cafaggi, S., Mariggiò, M. A., Vannozzi, M. O., Ottone, M., Bocchini, V., et al. (2002). Reduction of cisplatin hepatotoxicity by procainamide hydrochloride in rats. Eur J Pharmacol 442:265–272.

Dru

g an

d C

hem

ical

Tox

icol

ogy

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

Yor

k U

nive

rsity

Lib

rari

es o

n 10

/11/

13Fo

r pe

rson

al u

se o

nly.