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Fullerenol nanoparticles prevents doxorubicin-induced acute hepatotoxicity in rats
Accepted Manuscript Fullerenol nanoparticles prevents doxorubicin-induced acute hepatotoxicity in rats
Vesna Jacevic, Aleksandar Djordjevic, Branislava Srdjenovic, Vukosava Milic-Tores, Zoran Segrt, Viktorija Dragojevic-Simic, Kamil Kuca PII: DOI: Reference:
S0014-4800(17)30005-9 doi: 10.1016/j.yexmp.2017.03.005 YEXMP 4033
To appear in:
Experimental and Molecular Pathology
Received date: Revised date: Accepted date:
4 January 2017 14 February 2017 14 March 2017
Please cite this article as: Vesna Jacevic, Aleksandar Djordjevic, Branislava Srdjenovic, Vukosava Milic-Tores, Zoran Segrt, Viktorija Dragojevic-Simic, Kamil Kuca , Fullerenol nanoparticles prevents doxorubicin-induced acute hepatotoxicity in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yexmp(2017), doi: 10.1016/j.yexmp.2017.03.005
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ACCEPTED MANUSCRIPT Fullerenol Nanoparticles Prevents Doxorubicin-Induced Acute Hepatotoxicity In Rats Vesna Jacevica,b,c*, Aleksandar Djordjevicd, Branislava Srdjenovice, Vukosava Milic-Torese, Zoran Segrtf,b, Viktorija Dragojevic-Simicg,b, Kamil Kucah,c of Experimental Toxicology and Pharmacology, National Poison Control Centre, Military Medical Academy, Belgrade, Serbia bMedical Faculty of the Military Medical Academy, University of Defence, Belgrade, Serbia cDepartment of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic dDepartment of Chemistry, Biochemistry and Environmental Protection, Faculty of Science, University of Novi Sad, Novi Sad, Serbia eDepartment of Pharmacy, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia fDepartment for Treatment, Military Medical Academy, Belgrade, Serbia gCentre for Clinical Pharmacology, Military Medical Academy, Belgrade, Serbia hBiomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
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*Corresponding author at: Department of Experimental Toxicology and Pharmacology, National Poison Control Centre, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia
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E-mail address: [email protected] (Jacevic V.)
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Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAT, catalase; DNA, deoxyribonucleic acid; DOX, doxorubicin; FNP, fullerenol nanoparticle; GSHPx, glutathione peroxidase; GR, glutathione reductase; HDS, hepatic damage score; IND, indomethacin; NADP, nicotinamide adenine dinucleotide phosphate; NSAID, non-steroidal anti-inflammatory drug; O2.-, superoxide anions; OH., hydroxyl radicals; PMNCs, polymorphonuclear cells; PMNL, polymorphonuclear leucocyte; RNA, ribonucleic acid; ROS, reactive oxygen species; SOD, superoxide dismutasae; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances. 1Departamento
de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal (Vukosava Milic-Tores)
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ACCEPTED MANUSCRIPT ABSTRACT
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Doxorubicin (DOX), commonly used antineoplastic agent, affects bone marrow, intestinal tract and heart, but it also has some hepatotoxic effects. Main mechanism of its toxicity is the production of free reactive oxygen species. Polyhidroxilated C60 fullerene derivatives, fullerenol nanoparticles (FNP), act as free radical scavengers in in vitro systems. The aim of the study was to investigate potential FNP protective role against DOX-induced hepatotoxicity in rats. Experiments were performed on adult male Wistar rats. Animals were divided into five groups: (1) 0.9% NaCl (control), (2) 100 mg/kg ip FNP, (3) 10 mg/kg DOX iv, (4) 50 mg/kg ip FNP 30 minutes before 10 mg/kg iv DOX, (5) 100 mg/kg ip FNP 30 minutes before 10 mg/kg iv DOX. A general health condition, body and liver weight, TBARS level and antioxidative enzyme activity, as well as pathohistological examination of the liver tissue were conducted on days 2 and 14 of the study. FNP, applied alone, did not alter any examinated parameters. However, when used as a pretreatment it significantly increased survival rate, body and liver weight, and decreased TBARS level, antioxidative enzyme activity and hepatic damage score in DOX-treated rats. FNP administered at a dose of 100 mg/kg significantly attenuated effects of doxorubicin administered in a single high dose in rats, concerning general condition, body and liver weight, lipid peroxidation level and antioxidative enzyme activity as well as structural alterations of the hepatic tissue.
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Keywords: Doxorubicin; Fullerenol nanoparticles; Hepatic injury; Hepatoprotection; Rats
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ACCEPTED MANUSCRIPT 1. Introduction
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The anthracycline antibiotic doxorubicin (DOX) is an important antineoplastic agent with a high antitumor efficacy in haematological and various solid malignancies (Bonadonna and Valagudda, 1996; Dollery, 1999; Hortobagy, 1997; Gewitz, 1999; Minoti et al., 2004; Mross, 1991). However, therapeutic application of DOX is limited due to its adverse effects on bone marrow, intestinal tract epithelium, heart, liver and kidney, as well as its ability to cause cancer cell resistance during therapy (Chabner et al., 2006; Martindale, 2009). It is well known that DOX is able to interfere with a number of biochemical functions within cells, but precise molecular pathogenesis of DOX cytotoxic properties are still controversial. Doxorubicin is metabolized predominantly by the liver microsomal enzymes and cytoplasmic reductase (Camaggi et al., 1988; Ganey et al., 1988) to the major metabolite doxorubicinol, and several hepatotoxic aglycone metabolites (Ballet et al., 1987; Dodion et al., 1987). Namely, drug hepatotoxicity may ensue through free-radical formation and generation of ROS, such as superoxide anions (O2.-), hydroxyl radicals (OH.) and hydrogen peroxide which induce lipid peroxidation and oxidative damage in cells (Menna et al., 2004). The hepatic cell membrane lipids are also susceptible to DOX-induced oxygen radicals injury. Thereafter, peroxidation continues autocatalytically resulting in structural and functional alterations in the hepatic tissue. Once initiated, oxidative stress induces serious disturbances of DNA synthesis, DNA-dependent RNA synthesis and protein synthesis, which interferes with the regenerative capacity of the organelle. In that case, these irreversible alterations lead to hepatocyte apoptosis or necrosis, and intensive increase of hepatic enzymes in the blood, primarily alanine aminotransferasae (ALA) and aspartate aminotransferasae (AST) (Saad et al., 2001). In experimental animals, hepatic tissue which is oedematous with marked intracellular degeneration has been found in the experimental animals treated with DOX. Almost all hepatocytes had numerous visible intracytoplasmatic vacuoles, and enlarged, hyperchromatic nuclei (Deepa and Varalakshimi, 2003; Deepa et al., 2014). Mild to moderate hepatic functional tests have been generally normalized at least one year after chemotherapy in humans (King and Perry, 2001; Martinel Lamas et al., 2015). It has already been shown that fullerenols nanoparticles (FNP) produces potent antioxidative and antiinflammatory activites in many in vitro and in vivo systems (Deepa et al., 2014; Djordjevic et al., 2016; Grebowski et al., 2013; Grebowski et al., 2014; Injac et al., 2013; Lima et al., 2016; Rochette et al., 2015; Trajkovic et al., 2007). FNP showed excellent antioxidant potency in DOX-induced injury (Bogdanovic et al., 2004; Milic-Torres and Dragojevic-Simic, 2012; Injac et al., 2008a, 2008b, 2009a, 2009b; Labudovic-Borovic et al., 2014; Srdjenovic et al., 2010). Namely, protective effects of fullerenol against DOX-induced cardiotoxicity is a result of its high antioxidative potential, likely by acting as a free radical sponge and/or by removing free iron through the formation of fullerenol-iron complex (Injac et al., 2013; Bogdanovic et al., 2004; Milic-Torres et al., 2010). That disables further cell damage by ROS. FNP also reduced the DOX-induced oxidative stress and exerted beneficial effects in the reduction of the kidneys, lungs, and testes toxicity, as a potentially promising candidate substance for the prevention of nephrotic syndrome, spermatogenesis damage or pulmonary toxicity associated with the anthracycline therapy (Srdjenovic et al, 2010). Since the ionizing radiation produces harmful effects on living organisms by inducing enhanced generation of free-radical species in cells, potential FNP radioprotective effects in vivo were investigated (Trajkovic et al., 2005; Trajkovic et al., 2007; Jacevic et al., 3
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2016). It was found that FNP in a dose of 100 mg/kg ip applied 30 min prior to radiation provided statistically significant prolonged mean lethal time (LT50) and increased survival of mice and rats when they were irradiated with an absolute lethal doses of X-rays. Moreover, it was shown that FNP in a dose of 75 mg/kg had potent anti-inflammatory activity in a model of acute inflammation in rats, comparable to that of indomethacin (Dragojevic-Simicet al., 2011). It might be a consequence of their inhibitory effects on PMNL infiltration and free radical scavenging activity. Therefore, FNP is a valuable candidate for further investigation as an agent for the treatment of various disorders associated with inflammation and production of ROS, including some degenerative one (i.e. intervertebral disk degeneration) (Yang et al., 2014a), but also as a potentially novel tool for bone repair (Yang et al., 2014b). The aim of this study was to examine the effects of fullerenol in the prevention of hepatotoxicity induced by single high dose of DOX in rats. Therefore, measurement of lipid peroxidation, the activity of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase, as well as structural examination of the liver have been performed.
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2. Materials and methods 2.1. Chemicals
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DOX (Adriablastina®) for iv administration was purchased from Pharmacia & Upjohn (Milan, Italy). FNP were made in a two-step synthesis starting from fullerene C60 (purity 99.8%) which was brominated in the presence of a catalyst FeBr3 in order to obtain a symmetric polybrominated derivative C60Br24 (Djordjevic et al., 1988). In the next step, bromine atoms of C60Br24 were substituted with hydroxyl groups in alkaline media (Mirkov et al., 2004). Fullerenol nanoparticles were characterized in the water and saline solutions (Borišev et al., 2016; Ičević et al., 2011). All solutions both for iv and ip administration were prepared ex temporeby dissolving FNP with sonication 10 min, while DOX was dessolved in sterilized and apyrogenic 0.9% NaCl solution in a laminar flow hood.
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2.2. Experimental animals
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Experiments were performed on male Wistar rats, 6-8 weeks old (200 to 220g) bred at the Department for Experimental Animals, Military Medical Academy, Belgrade, Serbia. The experimental animals were housed in groups of five in plastic cages (Macrolon® cage type 4, Bioscape, Germany) with sawdust bedding (Versele-Laga, Belgium) certificated as having contaminant levels below toxic concentrations. The environmental conditions were controlled and monitored by a central computer-assisted system with a temperature of 22 ± 20C, relative humidity of 55 ± 15%, 15 - 20 airchanges/h, and artificial lighting of approximately 220 lux (12 hrs light/dark cycle). The experimental animals had free access to food, commercial pellets for rats (Veterinarski Zavod Subotica, Serbia) and tap water from municipal mains, filtered through 1.0 µm filter (Skala Green, Serbia). All above environmental conditions as well as all the procedures adopted for housing and handling of experimental animals were in strict compliance with Guideline for Laboratory Animal Welfare, Ethics Committee for Experiments on Animals of the Military Medical Academy, Belgrade, Serbia who was adopted in complete accordance with the current National Guidelines for Animal Welfare of the Republic of Serbia approved by the European Commission. 4
ACCEPTED MANUSCRIPT The study protocol was approved by the Ethics Committee for Experiments on Animals issued by Military Medical Academy, Belgrade, Serbia (approved study protocol no.: 282-12/2002). 2.3. Experimental design
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Wistar rats were randomly divided into five experimental groups each containing eight individuals. The animals received the following treatments: (1) Control (0.9% NaCl), (2) FNP 100 (100 mg/kg ip FNP), (3) DOX 10 (10 mg/kg iv DOX), (4) DOX 10 + FNP 50 (50 mg/kg ip FNP 30 min before 10 mg/kg iv DOX), and (5) DOX 10 + FNP 100 (100 mg/kg ip FNP 30 min before 10 mg/kg iv DOX).
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2.4. Monitoring of survival rate, general health condition, body and liver weight
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General health conditions were monitored daily through out the whole experimental period. Survival curves were done based on the number of surviving animals on the day 2 and 14 after treatments. The mean values of body weight and liver weight were calculated for each treatment separately on days 2 and 14 of the study. Eight animals were used per experimental group for both time intervals. Survival rate was expressed in percentages, while body and liver weight were expressed in absolute and relative values. 2.5. Biochemical assays
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To measure enzyme activity and lipid peroxidation, the Agilent 8453 UV/VIS spectrophotometer with a thermostated multicell position sample system and biochemical analysis software for assaying enzyme kinetics were used. 2.5.2. Preparation of liver tissue homogenates
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After quickly excising and washing the livers in a 0.9% NaCl solution and removing extraneous fat connective tissue, the organs were submerged in liquid nitrogen for 60 s. After removing the tissue from the liquid nitrogen, the livers were put into TRIS/KCl buffer, pH 7.4, and put in an ultrasonic bath for 2 min in order to obtain a tissue homogenate (10% w/v). Finally, the mixture was homogenized on a Potter-Elvehjem homogenizer. Prior to centrifugation, aliquots for determining thiobarbituric acid reactive substances (TBARS) were taken. The remaining homogenates were centrifuged for 15 min at 15,000x g at 40C. The kinetic measurement of enzymatic activity (superoxide dismutasae (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR)) in the liver tissue was performed immediately after centrifugation of the supernatants. 2.5.3. TBARS assay To measure lipid peroxidation by determining TBARS in the liver tissue, the modified method by Uchiama & Mihara was used (Uchiama and Mihara, 1978). An aliquot of 0.5 ml of 10% tissue homogenate (TRIS/KCl buffer, pH 7.4) was added to a mixture of 3 ml of 1% H3PO4 (JT Baker, USA), and 1 ml 0.6% thiobarbituric acid (TBA) in an aqueous 5
ACCEPTED MANUSCRIPT solution. The mixture was stirred and heated in a boiling bath for 45 min. After cooling, the extraction was carried out with 4 ml of 2-butanol (POCH, Poland) and the organic layer was separated with centrifugation. Optical density of the organic layer was determined with a UV/vis spectrophotometer set at 535 nm. The level of lipid peroxidation was calculated as nmol malondialdehyde/mg protein. Finally, obtained results were expressed in percentage (%), where value of the group I - control was 100%. 2.5.4. Antioxidative enzyme assays
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2.6. Histopathological study
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SOD activity was measured by inhibition of superoxide radical production in a xanthine-xanthine oxidase reaction according to the method described by McCord and Fridovich (1968). One unit of activity was defined as the amount of enzyme necessary to decrease the rate of cytochrome c reduction to 50% at 25°C maximum and pH 7.8. CAT activity was determined by the rate of hydrogenperoxide disappearance measured at 240 nm (Claiborne, 1984). One unit of CAT activity was defined as the amount of enzyme that decomposes one mmol H2O2/min at 250C and pH 7.0. The activity of selenium-containing GSH-Px was determined by the glutathionedependent reduction of t-butyl hydroperoxide. As such, we used a modification of the assay described by Paglia and Valentine (1967). Specifically, the oxidized glutathione formed by the enzymatic action of GSH-Px is instantly and continuously reduced by an excess of GR added to the assay mixture, thereby providing a constant level of reduced glutathione. The concomitant oxidation of NADPH to NADP was monitored spectroscopically at 340 nm. One unit of GSH-Px activity was defined as the amount needed to oxidize one nmol NADPH/min at 250C and pH 7.0. GR activity was determined using the method of Glatzleet al. (1974). This assay is based on NADPH oxidation concomitant with glutathione reduction. One unit of GR activity was defined as the oxidation of 1 nmol NADPH/min at 250C and pH 7.6.
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In order to evaluate the hepatoprotective effects of FNP the animals were sacrificed 14 days after receiving the treatment. Before sacrificing the animals, they were anesthetized with 25% urethane (4 ml/kg) (Sigma, St. Louis, USA), immobilized in a dorsal position and allowed to breathe spontaneously. At necropsy, the dissected liver tissue were carefully spread over a metal tray coated with wax and fixed with 10% neutral buffered formalin solution. Five to seven days after fixation all tissues were divided into 4 portions in order to be prepared for making sections. After process of fixation, all tissue samples were dehydrated in graded alcohol (100%, 96% and 70%), xylol and embedded in paraffin blocks. Finally, 2-μm thick paraffin sections were stained by haematoxylin and eosin (H&E) method. From each slices, whole visual fields magnified by 40x were analyzed by using light microscope according to the 5-point semiquantitative scale, i.e. tissue damage score for hepatic degenerative and vascular alterations - Hepatic Damage Score (HDS) (Table 1). Exact way of HDS calculation is shown in Table 2. Table 1 Tissue damage score for hepatic alterations (HDS - Hepatic Damage Score). Degree Description 0 Normal histological structure. 6
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Mild changes - small groups of hepatocytes with intracellular oedema and normal nucleus. Mild dilatation of blood vessels. Perivascular appearance of polymorphonuclear cells (PMNCs). Moderate damage - micronodular vacuolization of hepatocytes and normal nuclei. Focal oedema, hyperemia and hemorrhage in the sinusoidal spaces. Perivascular accumulation of polymorphonuclear cells (PMNCs). Strong focal damage - macronodular vacuolization of hepatocytes and pycnotic nuclei. Focal tissue accumulation of polymorphonuclear cells (PMNCs). Strong diffuse damage - centrolobular necrosis of hepatocytes and diffuse perivascular and tissue accumulation of polymorphonuclear cells (PMNCs). Massive necrotic fields.
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2.7. Statistical analysis
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Statistical evaluation was performed using commercial statistical software (Stat for Windows, R.7, Stat Soft, Inc., USA, 2008). All results were statistically analyzed by two-sided ANOVA and Kruskal Wallis test. Kruskal Wallis test was performed to investigate pairwise differences and used for HDS calculation. The difference with values of p < 0.05, p < 0.01 and p < 0.001 were considered significant. 3. Results
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3.1. Survival rate, general health condition, body weight and liver weight of the experimental animals
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Two days after treatments, survival rate was 100% in all experimental groups. After two weeks, the highest percentage of mortality was observed in the rats that received DOX 10 only (35%). In the group of rats protected with FNP 50 percentage of survived animals was 87.5%, while in the other groups it amounted 100% (Fig. 1). The general condition of treated animals was in accordance with these findings. Visible signs of general weakness, with haemorrhagic diarrhoea, adynamia and falling of hairs have been seen in DOX-treated group, only. Also, these animals were extremely anxious to all kind of stimulus, especially 2 days after application of DOX. Fig. 1. [near here]
The body mass of rats treated with 10 mg/kg of DOX only was significantly lower in comparison to the control group 14 days after treatment. On the other hand, two weeks after application, the body weight of rats protected with FNP 50 was significantly lower compared to the control group, but significantly higher than in the group that was treated with DOX 10. During the whole experimental period, a total body mass of the animals protected with FNP 100 did not change significantly compared to the control (Fig. 2). Fig. 2. [near here]
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ACCEPTED MANUSCRIPT Two days after treatment, there were no statistical differences between the relative liver weights in rats concerning all experimental groups. The relative liver weight of rats that received DOX 10 as well as FNP 50 prior to DOX 10 was significantly lower in comparison to the control group (Fig. 3). However, the relative liver weight of rats protected by FNP 100 has not been statistically different compared to the control group. Fig. 3. [near here] 3.2. The effects of FNP on lipid peroxidation in the liver tissue of the experimental animals
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The ip application of FNP 100 did not affect lipid peroxidation intensity in the liver of rats in all experimental groups both two days and 14 days after treatment (Fig. 4). Two days after administration of DOX in a single dose of 10 mg/kg, a significantly increased concentration of TBARS in the liver tissue was observed. However, the intensity of lipid peroxidation in the liver tissue in the experimental groups that were pretreated with FNP 50 and FNP 100 was not significantly different from the control animals during the whole study period. In the group of rats that received only DOX 10, the TBARS level was still significantly higher in comparison to the control group on day 14 of the study (Fig. 4). Taking into account TBARS level, FNP 100 itself did not cause lipid peroxidation in the liver tissue of the experimental animals during the whole study period. Fig. 4. [near here]
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3.3. The effect of FNP on the antioxidative enzymes activity in the liver of the experimental animals
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The levels of SOD, CAT, GR and GSH-Px in the liver rat tissue are shown in Fig. 5A-D. Application of DOX in a single dose of 10 mg/kg significantly elevated the activity of all the examined enzymes compared to the control group. High levels of antioxidant enzymes were observed both two and 14 days after administration of DOX 10. Pretreatment with FNP 50 and FNP 100, respectively, 30 min before the application of DOX 10 successfully attenuated oxidative stress in the liver tissue, since activity of all tested antioxidative enzymes was significantly decreased compared to the values in the group of animals treated with DOX only. On the other hand, two days after the treatment, activity of SOD as well as GSH-Px in both pretreated groups did not reach control level, while on day 14 they were at the control level (except SOD in FNP 50 pretreated group). Activity of GR 14 days after the treatment was decreased in all three FNP treated groups compared to control values. Applied alone FNP in dose of 100 mg has not affected the activity of examined antioxidative enzymes in comparison to the control group during the whole study period. Exception is decreased activity of GR 14 days after the application of FNP alone. Fig. 5. [near here] 3.4. Pathohistological examination of the hepatic tissue of the experimental animals Microscopic examination of the hepatic tissue sections of the control animals has shown normal histological architecture with out changes (Fig. 6A). On the hepatic sections obtained from the rats treated with FNP 100 only mild oedemas in the sinusoidal space 8
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Fig. 6. [near here]
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were seen, especially around the blood vessels in the central lobule. Individual blood vessels were dilated, and surrounded with a small amount of PMNCs (Fig. 6B). On the other hand, hepatic sections of the rats treated with DOX 10 had numerous intracytoplasmatic vacuoles while usual radial arrangements of the liver parenchyma were completely lost. Moderate oedema and hyperemia were expressed in all sinusoidal spaces. A focal hemorrhages were localized primarily perivascularly and partly in the periportal space of the hepatic lobule. Hypertrophic Kupffer's cells could be seen in the sinusoidal spaces. Almost all hepatocytes were oedematous with marked intracellular degeneration. These irregular, round to ovoid cells were characterized by granularity of cytoplasm. In the majority of affected hepatocytes nuclear pleomorphism was present, with large, round to rectangular shapes and prominent nucleoli. In addition, the blood vessels were dilated, with diffuse accumulation of PMNCs (Fig. 6C). In animals given FNP 50 as a pretreatment, the FNP did not succeed in protecting liver tissue against cellular and vascular damages. In this experimental group, radial arrangement of hepatocytes was completely lost in the central part of the lobules. The hepatocytes were oedematous and vacuolarly degenerated, with small, irregular and hyperchromatic nuclei. Massive oedema and hyperemia were present in the sinusoidal spaces, while focal hemorrhages were localized in the central parts of lobules. All Kupffer's cells were hypertrophic and clearly visible in the sinusoidal spaces. Majority of the blood vessels were dilated with discontinued basal membranes surrounded by an accumulation of PMNCs (Fig. 6D). However, the application of FNP 100 prior to DOX 10 significantly attenuates the degenerative and vascular changes caused by this chemotherapeutic agent. In this group, the appearance of small, individual vacuoles was observed in a limited number of hepatocytes in the central part of the lobules, while the radial arrangement of the hepatocytes was largely sustained with the presence of mild oedema and hyperemia. Also, the sinusoidal spaces were slightly extended, with increased number of individual Kupffer's cells. Additionally, all blood vessels were just slightly dilatated without alterations in their basal membranes and were surrounded by individual PMNCs (Fig. 6E).
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3.5. Semiquanitative pathohistological analysis of the livers of the experimental animals
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Semiquantitative pathohistological analysis confirmed that the FNP 100 given as a pretreatment to DOX 10 significantly diminished degenerative and vascular changes in hepatic tissue caused by DOX 10 (Table 2). FNP, applied alone, in a single dose of 100 mg/kg, caused structural alterations defined with HDS expressed as 1.15 ± 0.63, while HDS of control animals was 0.22 ± 0.42. The most intensive degenerative and vascular alterations scored as 2.06 ± 0.62 and 2.06 ± 0.56 were seen in the groups treated with DOX 10 and DOX 10 + FNP 50, respectively. Table 2 The effects of different treatments on the degree of hepatic alterations 14 days after their administration. Hepatic damages score Treatment (mg/kg) (8 livers/group x 4 slices/liver) ± S.D. 0 1 2 3 4 5 1. Control 25 7 0 0 0 0 0.22 0.42 9
ACCEPTED MANUSCRIPT 2. FNP 100 0 30 2 0 0 0 1.15 0.63 a2 b2 3. DOX 10 0 5 20 7 0 0 2.06 0.62 a3 4. DOX 10 + FNP 50 0 22 10 0 0 0 2.06 0.56 a3 c1 d1 5. DOX 10 + FNP 100 0 4 22 6 0 0 1.31 0.47 a3 b2 Statistical analysis was performed using Kruskal Wallis test. a2, a3 - p < 0.01, 0.001 for the results compared with the control group, b2 - p < 0.01 for the results compared with DOX 10 group, c1 - p < 0.05 for the results compared with FNP 100 group, d1 - p < 0.05 for the results compared with DOX 10 + FNP 100 group.
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4. Discussion
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The main finding of present study shows that FNP given in a single dose of 100 mg/kg ip successfully protects rat liver against toxic effects caused by a single high dose of DOX (10 mg/kg ip). Survival rate in DOX-treated group only was 65% 14 days after the beginning of the study, while other authors showed that survival rate was 40% in the same period (De Beer et al., 2002; Dorosshow et al., 1980; Molodykh et al., 2006; Rabelo et al., 2001). On the other hand, the pretreatment with FNP 50 and FNP 100 assured higher survival rate with 87.5% and 100%, respectively. Applied alone, FNP in a dose of 100 mg/kg ip did not cause mortality in rats. When protective effect of FNP was investigated in irradiated mice and rats, it was found that FNP in a dose of 100 mg/kg ip applied 30 min prior to radiation provided statistically significant prolonged mean lethal time (LT50). Also, FNP increased their survival when they were irradiated with an absolute lethal doses of X-rays (Trajkovic et al., 2005; Trajkovic et al., 2007). Its effect was comparable to that of the amifostine, gold standard for radioprotection and cytoprotection, commercially available at the market for oncological patients. Same dose of FNP caused significantly higher body weight in pretreated animals in comparation to DOX-only treated rats. In addition, a significant reduction in the relative liver weight compared to the control group was observed in DOX 10 treated rats, while in rats protected by FNP 100 this parameter has not been statistically different from control animals. Moreover, our experiment showed that FNP expressed a high antioxidant potential which was manifested by the results of measurement of lipid peroxidation intensity and antioxidative enzymes activity in the liver. The intensity of lipid peroxidation was significantly higher in the group treated only with DOX 10 on the day 2 and 14 of the study. However, both doses of FNP (50 mg/kg and 100 mg/kg) significantly attenuated DOX effects and maintained liver lipid peroxidation at a basal level. Applied alone, FNP 100 did not cause any alteration in the TBARS level. The antioxidative activity of SOD, CAT, GR and GSH-Px was significantly higher in the group of animals treated with DOX 10. These results are in accordance with the findings of other authors who reported an elevation of SOD, CAT, GR and GSH-Px activity in the liver tissue after DOX application (Kalendrea et al., 2005). There are findings that DOX indirectly upregulate production of antioxidative enzymes by producing reactive oxygen species (ROS) in different tissues (Deepa and Varalakshimi, 2003; Li and Singal, 2000; Murugan et al., 2002; Yin et al. 1998). In the present study, in the liver of rats pre-treated with FNP in both applied doses, activity of all tested antioxidative enzymes was significantly decreased compared to the values in the group of animals treated with DOX only, indicating the protective effect of FNP in this model of DOX-induced oxidative stress in liver. 10
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Administration of FNP decreased the process of lipid peroxidation caused by DOX and histopathological examination showed that the application of FNP 100 prior to DOX 10 significantly attenuated the degenerative and vascular changes caused by this chemotherapeutic agent. Present findings are in accordance with our previously published results which demonstrated that FNP had reduced oxidative stress caused by DOX in the kidneys, lungs, and testes of experimental rats (Srdjenovic et al., 2010). Two days after the treatment there were still some alterations in antioxidant enzymes activity compared with the control, but free radical scavenging activity of FNP were strong enough to cope with DOX-induced oxidative stress, since the degree of lipid peroxidation was at the control level. Fourteen days after the treatment the activity of all enzymes were like in control animals, except GR that was decreased in comparison to control. It is worth mentioning that FNP applied alone also decreased GR activity what was not the case when the kidneys, lungs, and testes were examined (Srdjenovic et al., 2010). Further investigations are necessary. Pretreatment with 100 mg of FNP exerted better protection in comparison to 50 mg dose, taking into account results from histopathological examination of the liver, as well as results of hepatic antioxidative enzymes activity and survival rate of animals. DjordjevicMilic et al. (2009) already proposed two mechanisms of FNP protection against DOXinduced toxicity in erythrocytes: binding to the hemoglobin sites to keep the site in functional condition and/or making complexes with iron that can diminish free radical reactions. Moreover, when organoprotective potential of FNP was considered, it was supposed that it exerted protection as a free radical sponge and/or by removing free iron through the formation of a FNP-iron complex (Milic-Torres et al., 2010; Srdjenovic et al., 2010). Pretreatment with FNP 100 significantly reduced HDS (hepatic damages score) in comparison to the rats given DOX 10. FNP itself did not cause any irreversible changes in the liver in comparison to the control rats. It was shown that the earliest and most often changes in the rat liver after application of high doses of DOX were the consequences of its direct toxic effect on the hepatocytes and endothelial cells (Molodykh et al., 2006). The current understanding of molecular mechanisms underlying DOX-induced hepatocyte death, both apoptosis and necrosis, still implies excessive toxic and ischemic injuries of the hepatocytes, as well as vascular endothelium in the experimental animals (Octavia et al., 2012; Streba et al., 2013), what was substantiated by our results. Chemotherapeutic agents may cause direct hepatic toxicity and altered liver function that or which may affects drug metabolism and cause an increased risk of non-hepatic toxicity (King and Perry, 2001). In the case of DOX it is especially related to cardiotoxicity. Ischemic liver disease and fulminant hepatitis have been observed in patients treated with DOX who have manifest heart failure and reduced left ventricular ejection fraction caused by hypoxia due to decreased venous flow. It is possible that due to this mechanism DOX also causes damage of the liver, since it is known that the cardiotoxicity with subsequent development of the heart failure is the main dose-limiting toxic effect of this chemotherapeutic agent. In FNP 100 protected animals, as it was previously said, hepatic tissue alterations were significantly less severe than those observed in animals treated only with DOX 10. Ionizing radiation itself produces harmful effects by inducing enhanced generation of freeradical species in cells, like it is the case with chemotherapeutic agent DOX. Therefore, it is no wonder that pathohistological alterations in the hepatic tissue after their application are very similar. Accordingly, protection of the liver tissue from ionizing radiation by the FNP given in the same dose (100 mg/kg) was already shown in our experimental model using rats (Trajkovic et al., 2007; Jacevic et al., 2016). Among other pathohistological findings in 11
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DOX 10 treated rats, we also noted that the majority of the blood vessels were dilated with discontinued basal membranes surrounded by an accumulation of PMNCs. It was not the case in FNP 100 treated group in which all blood vessels were just slightly dilated without alterations in their basal membranes and were surrounded by individual PMNCs. In our previous work we showed that FNP in a dose of 75 mg/kg ip had a potent antiinflammatory activity in a model of acute inflammation in rats, comparable to that of indomethacin (IND), a well-known non-steroidal anti-inflammatory drug (NSAID) (Dragojevic-Simic et al., 2011). It was probably a consequence of its inhibitory effect on PMNL infiltration i.e. inhibition of the main pro-inflammatory mediators production of PMNL, as well as inhibition of the release of PMNL-derived pro-inflammatory mediators, including free radicals. In our previous experiments where FNP showed significant protective effects against DOX toxicity, owing to its unique electrochemical features, antioxidant effects were performed by acting as free radical sponge (scavenger) and/or by removing free iron through the formation of FNP-iron complex, therefore disabling further cell damages by ROS (Milic-Torres et al., 2010).
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5. Conclusions
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Based on these findings it can be concluded that the FNP administered at a dose of 100 mg/kg significantly attenuated effects of DOX administered in a single high dose in rats, concerning general condition, body and liver weight, lipid peroxidation level and antioxidative enzymes activity as well as structural alterations of the hepatic tissue. These results support our hypothesis and previously conducted investigations indicating that FNP possesses high antioxidative and cytoprotective potential. In addition, this study indicates that FNP in dose of 100 mg/kg, not toxic per se, exerts antioxidant effects in vivo, and opens perspectives for its further examination in a variety of other conditions in which increased production of free radicals plays an important role.
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Author disclosure statement
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No competing financial interests exist. Acknowledgments
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This work received partial financial support from the Ministry of Education, Science and Technological Development of the Republic of Serbia [Grant No. III 45005]. This work was also supported by long-term development plan UHK and UHHK of the Czech Republic. References Ballet, F., Vrignaud, P., Robert, J., et al., 1987. Hepatic extraction, metabolism and biliary excretion of doxorubicin in the isolated perfused rat liver. Cancer. Chemother. Pharmacol. 19, 240-245. Bogdanovic, G., Kojic, V., Djordjevic, A., et al., 2004. Modulating activity of fullerenol C60(OH)24 on doxorubicin-induced cytotoxicity. Toxicol. In Vitro. 18, 629-638. Bonadonna, G., Valagudda, P., 1996. Primary chemotherapy in operable breast cancer. Semin. Oncol. 23, 464-474. 12
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Grebowski, J., Kazmierska, P., Krokosz, A., 2013. Fullerenols as a new therapeutic approach in nanomedicine. BioMed. Res. Int. Article. Article ID 751913, 9 pages. https://www.hindawi.com/journals/bmri/2013/751913/ (accessed 18.09.2013.). Grebowski, J., Krokosz, A., Konarska, A., et al., 2014. Rate constants of higlyhydroxylated fullerene C60 interacting with hydroxyl radicals hydrated electrons. Pulse radiolysis study. Radiat. Phys. Chem. 103, 146-152. Hortobagyi, G.N., 1997. Anthracyclines in the treatment of cancer. An Overwiev. Drugs 54, 1-7. Ičević, I., Vukmirović, S., Srđenović, B., et al. 2011. Protective effects of orally applied fullerenol nanoparticles in rats after a single dose of doxorubicin. Hem. Ind. 65, 329-337. Injac, R., Martina, P., Natasa, O., et al., 2008a. Potential hepatoprotective effects of fullerenol C60(OH)24 in doxorubicin-induced hepatotoxicity in rats with mammary carcinomas. Biomaterials. 29, 3451-3460. Injac R., Perse M., Boskovic M., et al., 2008b. Cardioprotective effects of fullerenol C60(OH)24 on a single dose doxorubicin-induced cardiotoxicity in rats with malignant neoplasm. Technol. Cancer. Res. T. 7, 1-11. Injac, R., Radic, N., Govedarica, B., et al., 2009a. Acute doxorubicin pulmotoxicity in rats with malignant neoplasm is effectively treated with fullerenol C60(OH)24 through inhibition of oxidative stress. Pharmacol. Rep. 61, 335-342. Injac, R., Perse, M., Cerne, M., et al., 2009b. Protective effects of fullerenol C60(OH)24 against doxorubicin-induced cardiotoxicity and hepatotoxicity in rats with colorectal cancer. Biomaterials. 30, 1184-1196. Injac, R., Prijatelj, M., Strukelj, B., 2013. Fullerenol nanoparticles: toxicity and antioxidant activity. Meth. Mol. Biol. 1028, 75-100. Jacevic, V., Jovic, D., Kuca, K., 2016. Effects of fullerenol nanoparticles and amifostine on radiation-induced tissue damages: histopathological analysis. J. Appl. Biomed. 14, 285297. Kalendrea, Y., Yelb, M., Kalendrea, S., 2005. Doxorubicin hepatotoxicity and hepatic free radical metabolism in rats. The effects of vitamin E and catechin. Toxicol. 209, 39-45. King, P., Perry, M., 2001. Hpatotoxicity of chemotherapy. Oncologist. 6, 162-176. Labudovic-Borovic, M., Icevic, I., Kanacki, Z., et al., 2014. Effects of fullerenol C60(OH)24 nanoparticles on a single-dose doxorubicin-induced cardiotoxicity in pigs: an ultrastructural study. Ultrastruc. Path. 38, 150-163. Li, T., Singal, P., 2000. Adriamycin induced early changes in myocardial antioxidant enzymes and their modulation by probucol. Circulation. 102, 2105-2110. Lima, R.L., Garcia, Z.M., Avila, T.V., et al., 2016. The reduction of oxidative stress by nanocomposite Fullerol decreases mucositis severity and reverts leukopenia induced by Irinotecan. Pharmacol. Res. 107, 102-110. Martinel Lamas, D.J., Nicoud, M.B., Sterle, H.A., et al., 2015. Selective cytoprotective effect of histamine on doxorubicin induced hepatic and cardiac toxicity in animal models. Cell. Death. Discovery. 1, 150-159. Martindale, M., 2009. The Complete Drug Reference 36 (CD-ROM). Pharmaceutical Press, London. McCord, J.M., Fridovich, I., 1968. The reduction of cytochrome-c by milk xanthine oxidase. J. Biol. Chem. 243, 5753-5760. Menna, P., Salvatorelli, E., Cairo, G., et al., 2004. Anthracyclines: molecular advances and pharmacologic develop-ments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 56, 185-229. 14
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ACCEPTED MANUSCRIPT Fig. 1. Influence of pretreatment with FNP 50 and FNP 100 on survival of rats treated with DOX 10. Fig. 2. Influence of pretreatment with FNP 50 and FNP 100 on body weight of rats treated with DOX 10. The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group.
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Fig. 3. Influence of pretreatment with FNP 50 and FNP 100 on liver weight (%) of rats treated with DOX 10. The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group.
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Fig. 4. Influence of pretreatment with FNP 50 and FNP 100 on TBARS liver levels in rats treated with DOX 10. The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group, b - p < 0.05 for the results compared with DOX 10 group.
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Fig. 5. Influence of pretreatment with FNP 50 and FNP 100 on the antioxidative enzymes activity of rats treated with DOX 10. (A) superoxide dismutase (SOD). (B) catalase (CAT). (C) glutathione reductase (GR). (D) glutathione peroxidase (GSH-Px). The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group, b - p < 0.05 for the results compared with DOX 10 group.
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Fig. 6. Light micrographs of the hepatic tissue of rats 14 days after treatments, H&E stain, magnification 40x. (A) The control group, normal radial arrangement of hepatocytes. (B) The FNP 100 treated group, unclear bubbling of centrolobular hepatocytes and intensive physiological mitosis of hepatocytes. (C) The DOX 10 treated group, pathological mitosis of the vacuolary degenerated hepatocytes and focal accumulation of PMNCI around the dilated blood vessels. (D) The DOX 10 + FNP 50 treated group, individual small vacuoles and oedema of hepatocytes, as well as focal appearance of PMNCs. (E) The DOX 10 + FNP 100 treated group, large groups of degenerated hepatocytes without clearly visible nuclei and hypertrophic Kupffer's cells in the sinusoidal spaces.
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ACCEPTED MANUSCRIPT Highlights Investigating potential FNP protective role against DOX-induced hepatotoxicity in Monitoring of lipid peroxidation level, antioxidative enzymes activity and
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hepatoprotection in rats. FNP (100 mg/kg ip) significantly attenuated toxic effects of doxorubicin (10 mg/kg iv).
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Vesna Jacevic, Aleksandar Djordjevic, Branislava Srdjenovic, Vukosava Milic-Tores, Zoran Segrt, Viktorija Dragojevic-Simic, Kamil Kuca PII: DOI: Reference:
S0014-4800(17)30005-9 doi: 10.1016/j.yexmp.2017.03.005 YEXMP 4033
To appear in:
Experimental and Molecular Pathology
Received date: Revised date: Accepted date:
4 January 2017 14 February 2017 14 March 2017
Please cite this article as: Vesna Jacevic, Aleksandar Djordjevic, Branislava Srdjenovic, Vukosava Milic-Tores, Zoran Segrt, Viktorija Dragojevic-Simic, Kamil Kuca , Fullerenol nanoparticles prevents doxorubicin-induced acute hepatotoxicity in rats. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Yexmp(2017), doi: 10.1016/j.yexmp.2017.03.005
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ACCEPTED MANUSCRIPT Fullerenol Nanoparticles Prevents Doxorubicin-Induced Acute Hepatotoxicity In Rats Vesna Jacevica,b,c*, Aleksandar Djordjevicd, Branislava Srdjenovice, Vukosava Milic-Torese, Zoran Segrtf,b, Viktorija Dragojevic-Simicg,b, Kamil Kucah,c of Experimental Toxicology and Pharmacology, National Poison Control Centre, Military Medical Academy, Belgrade, Serbia bMedical Faculty of the Military Medical Academy, University of Defence, Belgrade, Serbia cDepartment of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic dDepartment of Chemistry, Biochemistry and Environmental Protection, Faculty of Science, University of Novi Sad, Novi Sad, Serbia eDepartment of Pharmacy, Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia fDepartment for Treatment, Military Medical Academy, Belgrade, Serbia gCentre for Clinical Pharmacology, Military Medical Academy, Belgrade, Serbia hBiomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic
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aDepartment
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*Corresponding author at: Department of Experimental Toxicology and Pharmacology, National Poison Control Centre, Military Medical Academy, Crnotravska 17, 11000 Belgrade, Serbia
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E-mail address: [email protected] (Jacevic V.)
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Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; CAT, catalase; DNA, deoxyribonucleic acid; DOX, doxorubicin; FNP, fullerenol nanoparticle; GSHPx, glutathione peroxidase; GR, glutathione reductase; HDS, hepatic damage score; IND, indomethacin; NADP, nicotinamide adenine dinucleotide phosphate; NSAID, non-steroidal anti-inflammatory drug; O2.-, superoxide anions; OH., hydroxyl radicals; PMNCs, polymorphonuclear cells; PMNL, polymorphonuclear leucocyte; RNA, ribonucleic acid; ROS, reactive oxygen species; SOD, superoxide dismutasae; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances. 1Departamento
de Química e Bioquímica, Faculdade de Ciências da Universidade de Lisboa, Lisboa, Portugal (Vukosava Milic-Tores)
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ACCEPTED MANUSCRIPT ABSTRACT
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Doxorubicin (DOX), commonly used antineoplastic agent, affects bone marrow, intestinal tract and heart, but it also has some hepatotoxic effects. Main mechanism of its toxicity is the production of free reactive oxygen species. Polyhidroxilated C60 fullerene derivatives, fullerenol nanoparticles (FNP), act as free radical scavengers in in vitro systems. The aim of the study was to investigate potential FNP protective role against DOX-induced hepatotoxicity in rats. Experiments were performed on adult male Wistar rats. Animals were divided into five groups: (1) 0.9% NaCl (control), (2) 100 mg/kg ip FNP, (3) 10 mg/kg DOX iv, (4) 50 mg/kg ip FNP 30 minutes before 10 mg/kg iv DOX, (5) 100 mg/kg ip FNP 30 minutes before 10 mg/kg iv DOX. A general health condition, body and liver weight, TBARS level and antioxidative enzyme activity, as well as pathohistological examination of the liver tissue were conducted on days 2 and 14 of the study. FNP, applied alone, did not alter any examinated parameters. However, when used as a pretreatment it significantly increased survival rate, body and liver weight, and decreased TBARS level, antioxidative enzyme activity and hepatic damage score in DOX-treated rats. FNP administered at a dose of 100 mg/kg significantly attenuated effects of doxorubicin administered in a single high dose in rats, concerning general condition, body and liver weight, lipid peroxidation level and antioxidative enzyme activity as well as structural alterations of the hepatic tissue.
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Keywords: Doxorubicin; Fullerenol nanoparticles; Hepatic injury; Hepatoprotection; Rats
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ACCEPTED MANUSCRIPT 1. Introduction
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The anthracycline antibiotic doxorubicin (DOX) is an important antineoplastic agent with a high antitumor efficacy in haematological and various solid malignancies (Bonadonna and Valagudda, 1996; Dollery, 1999; Hortobagy, 1997; Gewitz, 1999; Minoti et al., 2004; Mross, 1991). However, therapeutic application of DOX is limited due to its adverse effects on bone marrow, intestinal tract epithelium, heart, liver and kidney, as well as its ability to cause cancer cell resistance during therapy (Chabner et al., 2006; Martindale, 2009). It is well known that DOX is able to interfere with a number of biochemical functions within cells, but precise molecular pathogenesis of DOX cytotoxic properties are still controversial. Doxorubicin is metabolized predominantly by the liver microsomal enzymes and cytoplasmic reductase (Camaggi et al., 1988; Ganey et al., 1988) to the major metabolite doxorubicinol, and several hepatotoxic aglycone metabolites (Ballet et al., 1987; Dodion et al., 1987). Namely, drug hepatotoxicity may ensue through free-radical formation and generation of ROS, such as superoxide anions (O2.-), hydroxyl radicals (OH.) and hydrogen peroxide which induce lipid peroxidation and oxidative damage in cells (Menna et al., 2004). The hepatic cell membrane lipids are also susceptible to DOX-induced oxygen radicals injury. Thereafter, peroxidation continues autocatalytically resulting in structural and functional alterations in the hepatic tissue. Once initiated, oxidative stress induces serious disturbances of DNA synthesis, DNA-dependent RNA synthesis and protein synthesis, which interferes with the regenerative capacity of the organelle. In that case, these irreversible alterations lead to hepatocyte apoptosis or necrosis, and intensive increase of hepatic enzymes in the blood, primarily alanine aminotransferasae (ALA) and aspartate aminotransferasae (AST) (Saad et al., 2001). In experimental animals, hepatic tissue which is oedematous with marked intracellular degeneration has been found in the experimental animals treated with DOX. Almost all hepatocytes had numerous visible intracytoplasmatic vacuoles, and enlarged, hyperchromatic nuclei (Deepa and Varalakshimi, 2003; Deepa et al., 2014). Mild to moderate hepatic functional tests have been generally normalized at least one year after chemotherapy in humans (King and Perry, 2001; Martinel Lamas et al., 2015). It has already been shown that fullerenols nanoparticles (FNP) produces potent antioxidative and antiinflammatory activites in many in vitro and in vivo systems (Deepa et al., 2014; Djordjevic et al., 2016; Grebowski et al., 2013; Grebowski et al., 2014; Injac et al., 2013; Lima et al., 2016; Rochette et al., 2015; Trajkovic et al., 2007). FNP showed excellent antioxidant potency in DOX-induced injury (Bogdanovic et al., 2004; Milic-Torres and Dragojevic-Simic, 2012; Injac et al., 2008a, 2008b, 2009a, 2009b; Labudovic-Borovic et al., 2014; Srdjenovic et al., 2010). Namely, protective effects of fullerenol against DOX-induced cardiotoxicity is a result of its high antioxidative potential, likely by acting as a free radical sponge and/or by removing free iron through the formation of fullerenol-iron complex (Injac et al., 2013; Bogdanovic et al., 2004; Milic-Torres et al., 2010). That disables further cell damage by ROS. FNP also reduced the DOX-induced oxidative stress and exerted beneficial effects in the reduction of the kidneys, lungs, and testes toxicity, as a potentially promising candidate substance for the prevention of nephrotic syndrome, spermatogenesis damage or pulmonary toxicity associated with the anthracycline therapy (Srdjenovic et al, 2010). Since the ionizing radiation produces harmful effects on living organisms by inducing enhanced generation of free-radical species in cells, potential FNP radioprotective effects in vivo were investigated (Trajkovic et al., 2005; Trajkovic et al., 2007; Jacevic et al., 3
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2016). It was found that FNP in a dose of 100 mg/kg ip applied 30 min prior to radiation provided statistically significant prolonged mean lethal time (LT50) and increased survival of mice and rats when they were irradiated with an absolute lethal doses of X-rays. Moreover, it was shown that FNP in a dose of 75 mg/kg had potent anti-inflammatory activity in a model of acute inflammation in rats, comparable to that of indomethacin (Dragojevic-Simicet al., 2011). It might be a consequence of their inhibitory effects on PMNL infiltration and free radical scavenging activity. Therefore, FNP is a valuable candidate for further investigation as an agent for the treatment of various disorders associated with inflammation and production of ROS, including some degenerative one (i.e. intervertebral disk degeneration) (Yang et al., 2014a), but also as a potentially novel tool for bone repair (Yang et al., 2014b). The aim of this study was to examine the effects of fullerenol in the prevention of hepatotoxicity induced by single high dose of DOX in rats. Therefore, measurement of lipid peroxidation, the activity of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase, as well as structural examination of the liver have been performed.
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2. Materials and methods 2.1. Chemicals
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DOX (Adriablastina®) for iv administration was purchased from Pharmacia & Upjohn (Milan, Italy). FNP were made in a two-step synthesis starting from fullerene C60 (purity 99.8%) which was brominated in the presence of a catalyst FeBr3 in order to obtain a symmetric polybrominated derivative C60Br24 (Djordjevic et al., 1988). In the next step, bromine atoms of C60Br24 were substituted with hydroxyl groups in alkaline media (Mirkov et al., 2004). Fullerenol nanoparticles were characterized in the water and saline solutions (Borišev et al., 2016; Ičević et al., 2011). All solutions both for iv and ip administration were prepared ex temporeby dissolving FNP with sonication 10 min, while DOX was dessolved in sterilized and apyrogenic 0.9% NaCl solution in a laminar flow hood.
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2.2. Experimental animals
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Experiments were performed on male Wistar rats, 6-8 weeks old (200 to 220g) bred at the Department for Experimental Animals, Military Medical Academy, Belgrade, Serbia. The experimental animals were housed in groups of five in plastic cages (Macrolon® cage type 4, Bioscape, Germany) with sawdust bedding (Versele-Laga, Belgium) certificated as having contaminant levels below toxic concentrations. The environmental conditions were controlled and monitored by a central computer-assisted system with a temperature of 22 ± 20C, relative humidity of 55 ± 15%, 15 - 20 airchanges/h, and artificial lighting of approximately 220 lux (12 hrs light/dark cycle). The experimental animals had free access to food, commercial pellets for rats (Veterinarski Zavod Subotica, Serbia) and tap water from municipal mains, filtered through 1.0 µm filter (Skala Green, Serbia). All above environmental conditions as well as all the procedures adopted for housing and handling of experimental animals were in strict compliance with Guideline for Laboratory Animal Welfare, Ethics Committee for Experiments on Animals of the Military Medical Academy, Belgrade, Serbia who was adopted in complete accordance with the current National Guidelines for Animal Welfare of the Republic of Serbia approved by the European Commission. 4
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Wistar rats were randomly divided into five experimental groups each containing eight individuals. The animals received the following treatments: (1) Control (0.9% NaCl), (2) FNP 100 (100 mg/kg ip FNP), (3) DOX 10 (10 mg/kg iv DOX), (4) DOX 10 + FNP 50 (50 mg/kg ip FNP 30 min before 10 mg/kg iv DOX), and (5) DOX 10 + FNP 100 (100 mg/kg ip FNP 30 min before 10 mg/kg iv DOX).
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2.4. Monitoring of survival rate, general health condition, body and liver weight
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General health conditions were monitored daily through out the whole experimental period. Survival curves were done based on the number of surviving animals on the day 2 and 14 after treatments. The mean values of body weight and liver weight were calculated for each treatment separately on days 2 and 14 of the study. Eight animals were used per experimental group for both time intervals. Survival rate was expressed in percentages, while body and liver weight were expressed in absolute and relative values. 2.5. Biochemical assays
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To measure enzyme activity and lipid peroxidation, the Agilent 8453 UV/VIS spectrophotometer with a thermostated multicell position sample system and biochemical analysis software for assaying enzyme kinetics were used. 2.5.2. Preparation of liver tissue homogenates
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After quickly excising and washing the livers in a 0.9% NaCl solution and removing extraneous fat connective tissue, the organs were submerged in liquid nitrogen for 60 s. After removing the tissue from the liquid nitrogen, the livers were put into TRIS/KCl buffer, pH 7.4, and put in an ultrasonic bath for 2 min in order to obtain a tissue homogenate (10% w/v). Finally, the mixture was homogenized on a Potter-Elvehjem homogenizer. Prior to centrifugation, aliquots for determining thiobarbituric acid reactive substances (TBARS) were taken. The remaining homogenates were centrifuged for 15 min at 15,000x g at 40C. The kinetic measurement of enzymatic activity (superoxide dismutasae (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GR)) in the liver tissue was performed immediately after centrifugation of the supernatants. 2.5.3. TBARS assay To measure lipid peroxidation by determining TBARS in the liver tissue, the modified method by Uchiama & Mihara was used (Uchiama and Mihara, 1978). An aliquot of 0.5 ml of 10% tissue homogenate (TRIS/KCl buffer, pH 7.4) was added to a mixture of 3 ml of 1% H3PO4 (JT Baker, USA), and 1 ml 0.6% thiobarbituric acid (TBA) in an aqueous 5
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2.6. Histopathological study
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SOD activity was measured by inhibition of superoxide radical production in a xanthine-xanthine oxidase reaction according to the method described by McCord and Fridovich (1968). One unit of activity was defined as the amount of enzyme necessary to decrease the rate of cytochrome c reduction to 50% at 25°C maximum and pH 7.8. CAT activity was determined by the rate of hydrogenperoxide disappearance measured at 240 nm (Claiborne, 1984). One unit of CAT activity was defined as the amount of enzyme that decomposes one mmol H2O2/min at 250C and pH 7.0. The activity of selenium-containing GSH-Px was determined by the glutathionedependent reduction of t-butyl hydroperoxide. As such, we used a modification of the assay described by Paglia and Valentine (1967). Specifically, the oxidized glutathione formed by the enzymatic action of GSH-Px is instantly and continuously reduced by an excess of GR added to the assay mixture, thereby providing a constant level of reduced glutathione. The concomitant oxidation of NADPH to NADP was monitored spectroscopically at 340 nm. One unit of GSH-Px activity was defined as the amount needed to oxidize one nmol NADPH/min at 250C and pH 7.0. GR activity was determined using the method of Glatzleet al. (1974). This assay is based on NADPH oxidation concomitant with glutathione reduction. One unit of GR activity was defined as the oxidation of 1 nmol NADPH/min at 250C and pH 7.6.
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In order to evaluate the hepatoprotective effects of FNP the animals were sacrificed 14 days after receiving the treatment. Before sacrificing the animals, they were anesthetized with 25% urethane (4 ml/kg) (Sigma, St. Louis, USA), immobilized in a dorsal position and allowed to breathe spontaneously. At necropsy, the dissected liver tissue were carefully spread over a metal tray coated with wax and fixed with 10% neutral buffered formalin solution. Five to seven days after fixation all tissues were divided into 4 portions in order to be prepared for making sections. After process of fixation, all tissue samples were dehydrated in graded alcohol (100%, 96% and 70%), xylol and embedded in paraffin blocks. Finally, 2-μm thick paraffin sections were stained by haematoxylin and eosin (H&E) method. From each slices, whole visual fields magnified by 40x were analyzed by using light microscope according to the 5-point semiquantitative scale, i.e. tissue damage score for hepatic degenerative and vascular alterations - Hepatic Damage Score (HDS) (Table 1). Exact way of HDS calculation is shown in Table 2. Table 1 Tissue damage score for hepatic alterations (HDS - Hepatic Damage Score). Degree Description 0 Normal histological structure. 6
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Mild changes - small groups of hepatocytes with intracellular oedema and normal nucleus. Mild dilatation of blood vessels. Perivascular appearance of polymorphonuclear cells (PMNCs). Moderate damage - micronodular vacuolization of hepatocytes and normal nuclei. Focal oedema, hyperemia and hemorrhage in the sinusoidal spaces. Perivascular accumulation of polymorphonuclear cells (PMNCs). Strong focal damage - macronodular vacuolization of hepatocytes and pycnotic nuclei. Focal tissue accumulation of polymorphonuclear cells (PMNCs). Strong diffuse damage - centrolobular necrosis of hepatocytes and diffuse perivascular and tissue accumulation of polymorphonuclear cells (PMNCs). Massive necrotic fields.
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2.7. Statistical analysis
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Statistical evaluation was performed using commercial statistical software (Stat for Windows, R.7, Stat Soft, Inc., USA, 2008). All results were statistically analyzed by two-sided ANOVA and Kruskal Wallis test. Kruskal Wallis test was performed to investigate pairwise differences and used for HDS calculation. The difference with values of p < 0.05, p < 0.01 and p < 0.001 were considered significant. 3. Results
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3.1. Survival rate, general health condition, body weight and liver weight of the experimental animals
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Two days after treatments, survival rate was 100% in all experimental groups. After two weeks, the highest percentage of mortality was observed in the rats that received DOX 10 only (35%). In the group of rats protected with FNP 50 percentage of survived animals was 87.5%, while in the other groups it amounted 100% (Fig. 1). The general condition of treated animals was in accordance with these findings. Visible signs of general weakness, with haemorrhagic diarrhoea, adynamia and falling of hairs have been seen in DOX-treated group, only. Also, these animals were extremely anxious to all kind of stimulus, especially 2 days after application of DOX. Fig. 1. [near here]
The body mass of rats treated with 10 mg/kg of DOX only was significantly lower in comparison to the control group 14 days after treatment. On the other hand, two weeks after application, the body weight of rats protected with FNP 50 was significantly lower compared to the control group, but significantly higher than in the group that was treated with DOX 10. During the whole experimental period, a total body mass of the animals protected with FNP 100 did not change significantly compared to the control (Fig. 2). Fig. 2. [near here]
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ACCEPTED MANUSCRIPT Two days after treatment, there were no statistical differences between the relative liver weights in rats concerning all experimental groups. The relative liver weight of rats that received DOX 10 as well as FNP 50 prior to DOX 10 was significantly lower in comparison to the control group (Fig. 3). However, the relative liver weight of rats protected by FNP 100 has not been statistically different compared to the control group. Fig. 3. [near here] 3.2. The effects of FNP on lipid peroxidation in the liver tissue of the experimental animals
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The ip application of FNP 100 did not affect lipid peroxidation intensity in the liver of rats in all experimental groups both two days and 14 days after treatment (Fig. 4). Two days after administration of DOX in a single dose of 10 mg/kg, a significantly increased concentration of TBARS in the liver tissue was observed. However, the intensity of lipid peroxidation in the liver tissue in the experimental groups that were pretreated with FNP 50 and FNP 100 was not significantly different from the control animals during the whole study period. In the group of rats that received only DOX 10, the TBARS level was still significantly higher in comparison to the control group on day 14 of the study (Fig. 4). Taking into account TBARS level, FNP 100 itself did not cause lipid peroxidation in the liver tissue of the experimental animals during the whole study period. Fig. 4. [near here]
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3.3. The effect of FNP on the antioxidative enzymes activity in the liver of the experimental animals
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The levels of SOD, CAT, GR and GSH-Px in the liver rat tissue are shown in Fig. 5A-D. Application of DOX in a single dose of 10 mg/kg significantly elevated the activity of all the examined enzymes compared to the control group. High levels of antioxidant enzymes were observed both two and 14 days after administration of DOX 10. Pretreatment with FNP 50 and FNP 100, respectively, 30 min before the application of DOX 10 successfully attenuated oxidative stress in the liver tissue, since activity of all tested antioxidative enzymes was significantly decreased compared to the values in the group of animals treated with DOX only. On the other hand, two days after the treatment, activity of SOD as well as GSH-Px in both pretreated groups did not reach control level, while on day 14 they were at the control level (except SOD in FNP 50 pretreated group). Activity of GR 14 days after the treatment was decreased in all three FNP treated groups compared to control values. Applied alone FNP in dose of 100 mg has not affected the activity of examined antioxidative enzymes in comparison to the control group during the whole study period. Exception is decreased activity of GR 14 days after the application of FNP alone. Fig. 5. [near here] 3.4. Pathohistological examination of the hepatic tissue of the experimental animals Microscopic examination of the hepatic tissue sections of the control animals has shown normal histological architecture with out changes (Fig. 6A). On the hepatic sections obtained from the rats treated with FNP 100 only mild oedemas in the sinusoidal space 8
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Fig. 6. [near here]
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were seen, especially around the blood vessels in the central lobule. Individual blood vessels were dilated, and surrounded with a small amount of PMNCs (Fig. 6B). On the other hand, hepatic sections of the rats treated with DOX 10 had numerous intracytoplasmatic vacuoles while usual radial arrangements of the liver parenchyma were completely lost. Moderate oedema and hyperemia were expressed in all sinusoidal spaces. A focal hemorrhages were localized primarily perivascularly and partly in the periportal space of the hepatic lobule. Hypertrophic Kupffer's cells could be seen in the sinusoidal spaces. Almost all hepatocytes were oedematous with marked intracellular degeneration. These irregular, round to ovoid cells were characterized by granularity of cytoplasm. In the majority of affected hepatocytes nuclear pleomorphism was present, with large, round to rectangular shapes and prominent nucleoli. In addition, the blood vessels were dilated, with diffuse accumulation of PMNCs (Fig. 6C). In animals given FNP 50 as a pretreatment, the FNP did not succeed in protecting liver tissue against cellular and vascular damages. In this experimental group, radial arrangement of hepatocytes was completely lost in the central part of the lobules. The hepatocytes were oedematous and vacuolarly degenerated, with small, irregular and hyperchromatic nuclei. Massive oedema and hyperemia were present in the sinusoidal spaces, while focal hemorrhages were localized in the central parts of lobules. All Kupffer's cells were hypertrophic and clearly visible in the sinusoidal spaces. Majority of the blood vessels were dilated with discontinued basal membranes surrounded by an accumulation of PMNCs (Fig. 6D). However, the application of FNP 100 prior to DOX 10 significantly attenuates the degenerative and vascular changes caused by this chemotherapeutic agent. In this group, the appearance of small, individual vacuoles was observed in a limited number of hepatocytes in the central part of the lobules, while the radial arrangement of the hepatocytes was largely sustained with the presence of mild oedema and hyperemia. Also, the sinusoidal spaces were slightly extended, with increased number of individual Kupffer's cells. Additionally, all blood vessels were just slightly dilatated without alterations in their basal membranes and were surrounded by individual PMNCs (Fig. 6E).
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3.5. Semiquanitative pathohistological analysis of the livers of the experimental animals
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Semiquantitative pathohistological analysis confirmed that the FNP 100 given as a pretreatment to DOX 10 significantly diminished degenerative and vascular changes in hepatic tissue caused by DOX 10 (Table 2). FNP, applied alone, in a single dose of 100 mg/kg, caused structural alterations defined with HDS expressed as 1.15 ± 0.63, while HDS of control animals was 0.22 ± 0.42. The most intensive degenerative and vascular alterations scored as 2.06 ± 0.62 and 2.06 ± 0.56 were seen in the groups treated with DOX 10 and DOX 10 + FNP 50, respectively. Table 2 The effects of different treatments on the degree of hepatic alterations 14 days after their administration. Hepatic damages score Treatment (mg/kg) (8 livers/group x 4 slices/liver) ± S.D. 0 1 2 3 4 5 1. Control 25 7 0 0 0 0 0.22 0.42 9
ACCEPTED MANUSCRIPT 2. FNP 100 0 30 2 0 0 0 1.15 0.63 a2 b2 3. DOX 10 0 5 20 7 0 0 2.06 0.62 a3 4. DOX 10 + FNP 50 0 22 10 0 0 0 2.06 0.56 a3 c1 d1 5. DOX 10 + FNP 100 0 4 22 6 0 0 1.31 0.47 a3 b2 Statistical analysis was performed using Kruskal Wallis test. a2, a3 - p < 0.01, 0.001 for the results compared with the control group, b2 - p < 0.01 for the results compared with DOX 10 group, c1 - p < 0.05 for the results compared with FNP 100 group, d1 - p < 0.05 for the results compared with DOX 10 + FNP 100 group.
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4. Discussion
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The main finding of present study shows that FNP given in a single dose of 100 mg/kg ip successfully protects rat liver against toxic effects caused by a single high dose of DOX (10 mg/kg ip). Survival rate in DOX-treated group only was 65% 14 days after the beginning of the study, while other authors showed that survival rate was 40% in the same period (De Beer et al., 2002; Dorosshow et al., 1980; Molodykh et al., 2006; Rabelo et al., 2001). On the other hand, the pretreatment with FNP 50 and FNP 100 assured higher survival rate with 87.5% and 100%, respectively. Applied alone, FNP in a dose of 100 mg/kg ip did not cause mortality in rats. When protective effect of FNP was investigated in irradiated mice and rats, it was found that FNP in a dose of 100 mg/kg ip applied 30 min prior to radiation provided statistically significant prolonged mean lethal time (LT50). Also, FNP increased their survival when they were irradiated with an absolute lethal doses of X-rays (Trajkovic et al., 2005; Trajkovic et al., 2007). Its effect was comparable to that of the amifostine, gold standard for radioprotection and cytoprotection, commercially available at the market for oncological patients. Same dose of FNP caused significantly higher body weight in pretreated animals in comparation to DOX-only treated rats. In addition, a significant reduction in the relative liver weight compared to the control group was observed in DOX 10 treated rats, while in rats protected by FNP 100 this parameter has not been statistically different from control animals. Moreover, our experiment showed that FNP expressed a high antioxidant potential which was manifested by the results of measurement of lipid peroxidation intensity and antioxidative enzymes activity in the liver. The intensity of lipid peroxidation was significantly higher in the group treated only with DOX 10 on the day 2 and 14 of the study. However, both doses of FNP (50 mg/kg and 100 mg/kg) significantly attenuated DOX effects and maintained liver lipid peroxidation at a basal level. Applied alone, FNP 100 did not cause any alteration in the TBARS level. The antioxidative activity of SOD, CAT, GR and GSH-Px was significantly higher in the group of animals treated with DOX 10. These results are in accordance with the findings of other authors who reported an elevation of SOD, CAT, GR and GSH-Px activity in the liver tissue after DOX application (Kalendrea et al., 2005). There are findings that DOX indirectly upregulate production of antioxidative enzymes by producing reactive oxygen species (ROS) in different tissues (Deepa and Varalakshimi, 2003; Li and Singal, 2000; Murugan et al., 2002; Yin et al. 1998). In the present study, in the liver of rats pre-treated with FNP in both applied doses, activity of all tested antioxidative enzymes was significantly decreased compared to the values in the group of animals treated with DOX only, indicating the protective effect of FNP in this model of DOX-induced oxidative stress in liver. 10
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Administration of FNP decreased the process of lipid peroxidation caused by DOX and histopathological examination showed that the application of FNP 100 prior to DOX 10 significantly attenuated the degenerative and vascular changes caused by this chemotherapeutic agent. Present findings are in accordance with our previously published results which demonstrated that FNP had reduced oxidative stress caused by DOX in the kidneys, lungs, and testes of experimental rats (Srdjenovic et al., 2010). Two days after the treatment there were still some alterations in antioxidant enzymes activity compared with the control, but free radical scavenging activity of FNP were strong enough to cope with DOX-induced oxidative stress, since the degree of lipid peroxidation was at the control level. Fourteen days after the treatment the activity of all enzymes were like in control animals, except GR that was decreased in comparison to control. It is worth mentioning that FNP applied alone also decreased GR activity what was not the case when the kidneys, lungs, and testes were examined (Srdjenovic et al., 2010). Further investigations are necessary. Pretreatment with 100 mg of FNP exerted better protection in comparison to 50 mg dose, taking into account results from histopathological examination of the liver, as well as results of hepatic antioxidative enzymes activity and survival rate of animals. DjordjevicMilic et al. (2009) already proposed two mechanisms of FNP protection against DOXinduced toxicity in erythrocytes: binding to the hemoglobin sites to keep the site in functional condition and/or making complexes with iron that can diminish free radical reactions. Moreover, when organoprotective potential of FNP was considered, it was supposed that it exerted protection as a free radical sponge and/or by removing free iron through the formation of a FNP-iron complex (Milic-Torres et al., 2010; Srdjenovic et al., 2010). Pretreatment with FNP 100 significantly reduced HDS (hepatic damages score) in comparison to the rats given DOX 10. FNP itself did not cause any irreversible changes in the liver in comparison to the control rats. It was shown that the earliest and most often changes in the rat liver after application of high doses of DOX were the consequences of its direct toxic effect on the hepatocytes and endothelial cells (Molodykh et al., 2006). The current understanding of molecular mechanisms underlying DOX-induced hepatocyte death, both apoptosis and necrosis, still implies excessive toxic and ischemic injuries of the hepatocytes, as well as vascular endothelium in the experimental animals (Octavia et al., 2012; Streba et al., 2013), what was substantiated by our results. Chemotherapeutic agents may cause direct hepatic toxicity and altered liver function that or which may affects drug metabolism and cause an increased risk of non-hepatic toxicity (King and Perry, 2001). In the case of DOX it is especially related to cardiotoxicity. Ischemic liver disease and fulminant hepatitis have been observed in patients treated with DOX who have manifest heart failure and reduced left ventricular ejection fraction caused by hypoxia due to decreased venous flow. It is possible that due to this mechanism DOX also causes damage of the liver, since it is known that the cardiotoxicity with subsequent development of the heart failure is the main dose-limiting toxic effect of this chemotherapeutic agent. In FNP 100 protected animals, as it was previously said, hepatic tissue alterations were significantly less severe than those observed in animals treated only with DOX 10. Ionizing radiation itself produces harmful effects by inducing enhanced generation of freeradical species in cells, like it is the case with chemotherapeutic agent DOX. Therefore, it is no wonder that pathohistological alterations in the hepatic tissue after their application are very similar. Accordingly, protection of the liver tissue from ionizing radiation by the FNP given in the same dose (100 mg/kg) was already shown in our experimental model using rats (Trajkovic et al., 2007; Jacevic et al., 2016). Among other pathohistological findings in 11
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DOX 10 treated rats, we also noted that the majority of the blood vessels were dilated with discontinued basal membranes surrounded by an accumulation of PMNCs. It was not the case in FNP 100 treated group in which all blood vessels were just slightly dilated without alterations in their basal membranes and were surrounded by individual PMNCs. In our previous work we showed that FNP in a dose of 75 mg/kg ip had a potent antiinflammatory activity in a model of acute inflammation in rats, comparable to that of indomethacin (IND), a well-known non-steroidal anti-inflammatory drug (NSAID) (Dragojevic-Simic et al., 2011). It was probably a consequence of its inhibitory effect on PMNL infiltration i.e. inhibition of the main pro-inflammatory mediators production of PMNL, as well as inhibition of the release of PMNL-derived pro-inflammatory mediators, including free radicals. In our previous experiments where FNP showed significant protective effects against DOX toxicity, owing to its unique electrochemical features, antioxidant effects were performed by acting as free radical sponge (scavenger) and/or by removing free iron through the formation of FNP-iron complex, therefore disabling further cell damages by ROS (Milic-Torres et al., 2010).
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5. Conclusions
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Based on these findings it can be concluded that the FNP administered at a dose of 100 mg/kg significantly attenuated effects of DOX administered in a single high dose in rats, concerning general condition, body and liver weight, lipid peroxidation level and antioxidative enzymes activity as well as structural alterations of the hepatic tissue. These results support our hypothesis and previously conducted investigations indicating that FNP possesses high antioxidative and cytoprotective potential. In addition, this study indicates that FNP in dose of 100 mg/kg, not toxic per se, exerts antioxidant effects in vivo, and opens perspectives for its further examination in a variety of other conditions in which increased production of free radicals plays an important role.
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Author disclosure statement
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No competing financial interests exist. Acknowledgments
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This work received partial financial support from the Ministry of Education, Science and Technological Development of the Republic of Serbia [Grant No. III 45005]. This work was also supported by long-term development plan UHK and UHHK of the Czech Republic. References Ballet, F., Vrignaud, P., Robert, J., et al., 1987. Hepatic extraction, metabolism and biliary excretion of doxorubicin in the isolated perfused rat liver. Cancer. Chemother. Pharmacol. 19, 240-245. Bogdanovic, G., Kojic, V., Djordjevic, A., et al., 2004. Modulating activity of fullerenol C60(OH)24 on doxorubicin-induced cytotoxicity. Toxicol. In Vitro. 18, 629-638. Bonadonna, G., Valagudda, P., 1996. Primary chemotherapy in operable breast cancer. Semin. Oncol. 23, 464-474. 12
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Borišev, M.K., Borišev, I.Đ., Župunski, M.D., et al., 2016. Drought impact is alleviated in sugar beets (Beta vulgaris L.) by foliar application of fullerenol nanoparticles. PLoSONE. 11, 11: e0166248. Camaggi, C.M., Comparsi, R., Srtocchi, E., et al., 1988. Epirubicin and doxorubicin comparative metabolism and pharmacokinetics. Cancer. Chemother. Pharmacol. 21, 221228. Chabner, B.A., Alegra, C.J., Curt, G.A., et al., 2006. Chemotherapy of neoplastic diseases, in: Brunton L.L., Lazo J.S., Parker K.L. (Eds.), Goodman & Gilman’s the Pharmacological Basis of Therapeutics. McGraw-Hill, New York, pp. 1315-1404. Claiborne, A., 1984. Catalase activity, in: Greenwald, R.A., (Eds.), CRC Handbook of Methods for Oxygen Radical Research. CRC Press Inc., Boca Raton, pp. 283-284. De Beer, E.L., Bottone, A.E., Van Rijk, M.C., et al., 2002. Dexrazoxan pre-treatment protect skinned rat cardiac trabeculae against delayed docorubicin-induced impairement of crossbridge kinetics. Br. J. Pharmacol. 135, 1707-1714. Deepa, P.R., Varalakshimi, P., 2003. Protective effect of low molecular weight heparin on oxidative injury and cellular abnormalities in adriamycin induced cardiac and hepatic toxicity. Chem. Biol. Interact. 146, 201-210. Deepa, K.I., Mandlikb, K.M., Suresh, R.N., 2014. Models of hepatotoxicity and the underlying cellular, biochemical and immunological mechanism(s): A critical discussion. Environ. Toxicol. Pharmcol. 37,118-133. Djordjevic, A., Vojinovic-Miloradov, M., Petranovic N., et al., 1988. Catalytic preparation and characterization of C60Br24. Fuller. Sci. Technol. 6, 689-694. Djordjevic, A., Srdjenovic, B., Seke, M., et al., 2015. Review of syntesis and antioxidant potential of fullerenol nanoparticles. J. Nanomater. Article. ID 567073, 15 pages. http://www.hindawi.com/journals/jnm/2015/567073/ref/ (accessed 11.04.2015.). Djordjevic-Milic, V., Stankov, K., Injac, R., et al., 2009. Activity of antioxidative enzymes in erythrocytes after a single dose administration of doxorubicin in rats pretreated with fullerenol C60(OH)24. Toxicol. Mech. Methods. 19, 24-28. Dodion, P., Bernstein, A.L., Fox, B.M., et al., 1987. Loss of fluorescence by anthracycline antibiotics: effects of xanthine oxidase and identification of the nonfluorescent metabolites. Cancer. Res. 47, 1036-1039. Dollery, C., 1999. Cyclophosphamide, in: Dollery C. (Ed.), Therapeutic drugs. Churchill Livingstone, Edinburgh, pp. 349-354. Dorosshow, J.H., Locker, G.Y., Myers, C.E., 1980. Enzymatic defences of the mouse heart against reactive oxygen metabolites. J. Clin. Ivest. 65, 128-135. Dragojevic-Simic, V., Jacevic, V., Dobric, S., et al., 2011. Anti-inflammatory activity of fullerenol C60(OH)24 nano-particles in a model of acute inflammation in rats. Dig. J. Nanomater. Bios. 6, 819-827. Ganey, P.E., Kauffman, F.C., Thurman, R.G., 1988. Oxigen dependent hepatotoxicity due to doxorubicin: role of reducing equivalent supply in perfused rat liver. Mol. Pharmacol. 34, 695-701. Gewitz, D.A., 1999. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem. Pharmacol. 57, 727-741. Glatzle, D., Vuilleumier, J.P., Weber, F., et al., 1974. Glutathione reductase test with whole blood - a convenient procedure for the assessment of riboflavine status in humans. Experientia. 30, 565-638. 13
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ACCEPTED MANUSCRIPT Fig. 1. Influence of pretreatment with FNP 50 and FNP 100 on survival of rats treated with DOX 10. Fig. 2. Influence of pretreatment with FNP 50 and FNP 100 on body weight of rats treated with DOX 10. The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group.
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Fig. 3. Influence of pretreatment with FNP 50 and FNP 100 on liver weight (%) of rats treated with DOX 10. The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group.
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Fig. 4. Influence of pretreatment with FNP 50 and FNP 100 on TBARS liver levels in rats treated with DOX 10. The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group, b - p < 0.05 for the results compared with DOX 10 group.
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Fig. 5. Influence of pretreatment with FNP 50 and FNP 100 on the antioxidative enzymes activity of rats treated with DOX 10. (A) superoxide dismutase (SOD). (B) catalase (CAT). (C) glutathione reductase (GR). (D) glutathione peroxidase (GSH-Px). The results are expressed as a percentage related to the control group. a - p < 0.05 for the results compared with the control group, b - p < 0.05 for the results compared with DOX 10 group.
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Fig. 6. Light micrographs of the hepatic tissue of rats 14 days after treatments, H&E stain, magnification 40x. (A) The control group, normal radial arrangement of hepatocytes. (B) The FNP 100 treated group, unclear bubbling of centrolobular hepatocytes and intensive physiological mitosis of hepatocytes. (C) The DOX 10 treated group, pathological mitosis of the vacuolary degenerated hepatocytes and focal accumulation of PMNCI around the dilated blood vessels. (D) The DOX 10 + FNP 50 treated group, individual small vacuoles and oedema of hepatocytes, as well as focal appearance of PMNCs. (E) The DOX 10 + FNP 100 treated group, large groups of degenerated hepatocytes without clearly visible nuclei and hypertrophic Kupffer's cells in the sinusoidal spaces.
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ACCEPTED MANUSCRIPT Highlights Investigating potential FNP protective role against DOX-induced hepatotoxicity in Monitoring of lipid peroxidation level, antioxidative enzymes activity and
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hepatoprotection in rats. FNP (100 mg/kg ip) significantly attenuated toxic effects of doxorubicin (10 mg/kg iv).
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