Effects of fullerenol nanoparticles and amifostine on ...

G Model JAB 101 No. of Pages 13

Journal of Applied Biomedicine xxx (2016) xxx–xxx

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Effects of fullerenol nanoparticles and amifostine on radiation-induced tissue damages: Histopathological analysis Jacevic Vesnaa,b,c , Jovic Danicad, Kuca Kamilc,e,* , Dragojevic-Simic Viktorijab,f , Dobric Silvab,g , Trajkovic Sanjah , Borisev Ivanac , Segrt Zoranb,i, Milovanovic Zorana , Bokonjic Dubravkob , Djordjevic Aleksandarc a

National Poison Control Centre, Military Medical Academy, Belgrade, Serbia University of Defence in Belgrade, Medical Faculty of the Military Medical Academy, Serbia c Department of Chemistry, Faculty of Science, University of Hradec Kralove, Hradec Kralove, Czech Republic d University of Novi Sad, Faculty of Science, Department of Chemistry, Biochemistry and Environmental Protection, Novi Sad, Serbia e Biomedical Research Center, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic f Centre for Clinical Pharmacology, Military Medical Academy, Belgrade, Serbia g Institute for Scientific Information, Military Medical Academy, Belgrade, Serbia h Alltech, Nicholasville, KY, United States i Department for Treatment, Military Medical Academy, Belgrade, Serbia b

A R T I C L E I N F O

Article history: Received 20 March 2016 Received in revised form 22 May 2016 Accepted 25 May 2016 Available online xxx Keywords: X-ray radiation Tissue damages Radioprotection Fullerenol nanoparticles Amifostine

A B S T R A C T

Fullerenol C60(OH)24 nanoparticles (FNP) are promising radioprotectors in prevention of early and late ionizing radiation injury. The aim of this study was to compare the efficacy of FNP and amifostine (AMI) in protection of rats exposed to whole-body X-ray irradiation (7 or 8 Gy). Both compounds (FNP, 100 mg/kg ip; AMI, 300 mg/kg ip) were given 30 min before irradiation throughout the study. The general radioprotective efficacy of FNP and AMI were evaluated in rats irradiated with an absolutely lethal dose of X-rays (8 Gy) and their survival were monitored during the period of 30 days after irradiation. Both compounds were of comparable efficacy. Tissue-protective effects of tested compounds were assessed in rats irradiated with an sublethal dose of X-rays (7 Gy). For this purpose, the animals were sacrificed on the 7th and 28th day after irradiation. Their lung, heart, liver, kidney, small intestine and spleen were taken for histopathological and semiquantitative analysis. Careful examination of established tissue and vascular alteration revealed better radioprotective effects of FNP compared to those of AMI on the small intestine, lung and spleen, while AMI had better radioprotective effects than FNP in protection of the heart, liver and kidney. Results of this study confirmed high radioprotective efficacy of FNP in irradiated rats that was comparable to that of AMI, a well-known radioprotector. ã 2016 Published by Elsevier Sp. z o.o. on behalf of Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice.

Introduction The acute exposure of living organism to ionizing radiation (IR) results in an increased rate of genetic mutations, cell death or tissue disorganisation. These harmful effects of IR are mainly mediated by free radical formation, such as superoxide (O2) and hydroxyl (OH) radicals, that damage DNA, cytoplasmic organelles and endoplasmic reticulum (Hall and Giaccia, 2011; Prasad, 2005). The successful prevention or treatment of early and late IR effects

* Corresponding author at: Sokolska 581, Hradec Kralove, Czech Republic. E-mail addresses: [email protected], http://www.fnhk.cz/cbv (K. Kamil).

depends on antioxidative potential of different radioprotectors and their ability to react with highly reactive oxygen radical species (Citrin et al., 2010; Koukourakis, 2012; Kuntic et al., 2013; Mettler et al., 2011). Radioprotectors can be of natural or synthetic origin. A variety of natural radioprotectors, such as ginseng extracts, melatonin, antioxidant vitamins etc., have limited potential for reducing the damaging effects of irradiation. On the other hand, the most prominent synthetic radioprotector AMI, a chemical congener of known sulfhydryl radioprotectors like cysteamine and cystamine, has exhibited protective effects in a broad spectrum of normal cells and tissues subjected to radiation, as well as during antineoplastic drug therapy (Hall and Giaccia, 2011). AMI is a phosphorothioate

http://dx.doi.org/10.1016/j.jab.2016.05.004 1214-021X/ ã 2016 Published by Elsevier Sp. z o.o. on behalf of Faculty of Health and Social Sciences, University of South Bohemia in Ceske Budejovice.

Please cite this article in press as: J. Vesna, et al., Effects of fullerenol nanoparticles and amifostine on radiation-induced tissue damages: Histopathological analysis, J. Appl. Biomed. (2016), http://dx.doi.org/10.1016/j.jab.2016.05.004


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J. Vesna et al. / Journal of Applied BiomedicineJ. Appl. Biomed. xxx (2016) xxx–xxx

prodrug that is easily converted into its active thiol form (known under code name WR-1065) by the activity of alkaline phosphatase (ALP). WR-1065 easily diffuses into cells and acts as a free-radical scavenger. Although free-scavenging activity is considered to be the main mechanism of AMI radioprotective activity, additional mechanisms of protection have also been reported: tissue hypoxia, hydrogen donation, condensation of DNA, the liberation of endogenous non-protein sulfhydryls (mainly glutathione) from their bond with cells protein, the formation of mixed disulphides in order to protect normal cells (Giambarresi and Jacobs, 1987; Grdina et al., 2002; Murley et al., 2006; Spencer and Goa, 1995), activation of manganese superoxide dismutase (MnSOD) and anti-apoptotic nuclear factor kB (NFkB) (Mihailovic et al., 2009; Utley et al., 1980), induction of acute-phase proteins (Yuhas, 1980) that can provide protection against harmful effects of ionizing radiation and many antineoplastic drugs. Active form of AMI has been shown to penetrate more rapidly and to higher extent into normal tissues than into tumor ones. It is supposed that mechanism of amifostine’s tissue selectivity is related to the different expression of ALP and blood flow in tumor tissues as well as to acidosis of tumors with consequent reduced concentration of WR-1065 in tumor cells compared to normal ones (Smoluk et al., 1988; Spencer and Goa, 1995; Utley et al., 1980; Yuhas, 1980). Because of that, AMI was extensively clinically tested as a radio and chemoprotector. In many of these studies AMI was found to reduce toxicity from radio- and chemotherapy and was approved for clinical use as radio- and chemoprotective drug (Dragojevic-Simic and Dobric, 1996; Spencer and Goa, 1995; Zois et al., 2011). However, AMI produces many adverse effects, some of which are quite serious (e.g. pronounced hypotension, hypersensitive reactions, nausea and vomiting), which consequently led to drug withdrawal (Demiral et al., 2002; Rades et al., 2004; Spencer and Goa, 1995; Vardy et al., 2002). Therefore, search for new radioand chemoprotectors has been continuing. Recently, a new class of compounds, polyhydroxylated derivatives of fullerene C60, called fullerenols (C60(OH)n, n = 2–44), has attracted attention due to their strong free-radical scavenging activity (Cavas et al., 2014; Djordjevic et al., 2004; Krokosz et al., 2014; Mirkov et al., 2004; Yang et al., 2014; Ye et al., 2014). Now, they are considered to be promising agents in therapy of various disorders in which oxidative stress plays an important pathogenic role (Grebowski et al., 2013). One of the most studied fullerenol is C60(OH)24. It has diameter around 1 nm with symmetrically arranged hydroxyl groups on the C60 sphere. Fullerenol is a dark brown amorphous substance,

soluble in water and dimethyl sulfoxide (DMSO). Dissolved in water it forms polyanionnano aggregates (fullerenol nanoparticles FNP) of size mostly between 10 nm and 100 nm (Indeglia et al., 2014). Several earlier studies showed that fullerenol expressed radioprotective and chemoprotective activity in both: in vitro (Bogdanovic et al., 2008; da Rocha et al., 2013; Stankov et al., 2013) and in vivo (Cai et al., 2010; Injac et al., 2008a, 2008b, 2008c, 2009a, 2009b; Shipelin et al., 2013; Thakral and Thakral, 2013; Trajkovic et al., 2005, 2007) experimental models. Moreover, it was shown that fullerenol C60(OH)24 produced radioprotective efficacy comparable to that of AMI in rats irradiated with lethal dose of X-rays (Trajkovic et al., 2007). Having all these results in mind, the aim of this study was to thoroughly compare the efficacy of FNP and AMI in protection of various tissues in irradiated rats. Materials and methods Synthesis of fullerenol C60(OH)24 nanoparticles and amifostine Polyhydroxylated fullerenol C60(OH)24 (Fig. 1A) was synthesized starting from polybrominated derivative C60Br24 in alkaline media by complete substitution of bromine atoms in C60Br24 (Mirkov et al., 2004). Amifostine (Fig. 1B), in the form of dihydrate, was synthesized in the Chemical Department of Military Technical Institute, Belgrade, Serbia, by original procedure based on the method described by Piper et al. (1969). Both compounds were dissolved in aqua pro injectione prior to use. Characterisation of fullerenol C60(OH)24 nanoparticles Results for distribution and zeta-potential of FNP in aqueous solution and normal saline enriched with 20% fetal bovine serum (FBS) were obtained by dynamic light scattering (DLS) using Zetasizer Nano ZS, Malvern. Morphology and structure of FNP in water and normal saline with 20% FBS were evaluated using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Surface topography and phase images were simultaneously acquired by standard AFM tapping mode using a commercial SNC (Solid Nitride Cone) AFM probe (NanoScience-Team Nanotec GmbH), with the tip radius lower than 10 nm. Highly-orientated pyrolytic graphite (HOPG) was used as surface. Multimode quadrex SPM with a NanoscopeIIIa controller (Veeco Instruments, Inc.) operated under ambient conditions. Samples of FNP in saline for

Fig. 1. Structure of fullerenol C60(OH)24 (A) and amifostine (B).




DLS measurements were tempered in dark at 37 C. Measurements were done after: 5 min, 2, 4 and 24 h. Animals Experiments were performed on male Wistar rats, 6–8 weeks old (180–220 g) bred at the Department for Experimental Animals, Military Medical Academy, Belgrade, Serbia. They were housed in plastic cages, under standard laboratory conditions (21–22 C, 12 h light/dark cycle, 30–70% relative humidity) with commercial food and tap water ad libitum. The study protocol was approved by Ethics Committee for Experiments on Animals issued by Military Medical Academy, Belgrade, Serbia (approved study protocol no.: 282-12/2002). Radiation Irradiation procedure was performed as described elsewhere (Trajkovic et al., 2007). Briefly, animals were whole-body irradiated by 8 MeV X-rays at a dose of 7 Gy as well as 8 Gy using a linear accelerator (SL 75–20, Philips, Germany). For whole-body irradiation, rats were treated simultaneously in a well-ventilated Plexiglas box (35 35 7 cm). Plexiglass sheet 2 cm thick, was placed on the top of the box, and the distance from the source to the sheet was 100 cm. Study of FNP and AMI mean lethal dose (LD50) calculation For calculating LD50 values of FNP and AMI, mortality of rats given these compunds were recorded 24 h after their intraperitoneally (ip) administration in increasing doses. LD50 values were then calculted by method of Litchfield and Wilcoxon (1949).

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Study of general radioprotective efficacy In the second set of experiments FNP (100 mg/kg) and AMI (300 mg/kg) were given intraperitoneally (ip) 30 min before X-irradiation in an absulutely lethal dose of 8 Gy. Control animals (unprotected ones) were injected by 1 mL/kg ip of physiological solution also 30 min before irradiation. Experimental groups consisted of 10 animals each. Survival were monitored during the period of 30 days after irradiation. Percentage of survival and mean lethal time (LT50) of both irradiated and previously treated animals were calculated (Litchfield and Wilcoxon, 1949). Light microscopy study In order to thoroughly evaluate protective effects of FNP on several tissues and compare it with amifostine second part of the experiment was carried out on rats irradiated with a sublethal dose of 7 Gy. FNP was given in a dose of 100 mg/kg ip, AMF in a dose of 300 mg/kg ip, both 30 min before irradiation. Experimental groups consisted of 6 animals each. Animals were sacrificed on day 7 and 28 after irradiation. At necropsy, the dissected organs (heart, lung, small intestine, spleen, liver, and kidneys) 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 6 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-mm-thick paraffin sections were stained by Haematoxylin and Eosin (H&E) method. From each specimens, whole visual fields magnified by 20 x were analyzed by using light microscope according to the 5-point semiquantitative scale, i.e. tissue damage score for degenerative and vascular changes as described previously (Trajkovic et al., 2007).

Fig 2. AFM analysis of FNP in saline after 24 h at 370 C in the dark. (A) Fullerenol nanoparticle of 95 nm formed of several smaller nanoparticles of radius within 16–42 nm. (B) Cross-section of fullerenol nanoparticles formed out of particles with following widths: 19 nm, 39 nm, 31 nm, and heights: 3.5 nm, 5.5 nm and 3.9 nm, respectively. (C) Largescale image 1200 1200 nm2. (D) 3D image of FNP on the HOPG surface.



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J. Vesna et al. / Journal of Applied BiomedicineJ. Appl. Biomed. xxx (2016) xxx–xxx Table 1 Mean lethal doses of fullerenol nanoparticles (FNP) and amifostine (AMI) in rats.

Fig. 3. TEM analysis of FNP. Cross-section of fullerenol nanoparticles and agglomerates with following widths: 2.9 nm, 3.2 nm, 3.4 nm, 4.7 nm, 5.2 nm, 7.1 nm, 8.9 nm and 18.6 nm.

Statistical analysis Statistical evaluation was performed using commercial statistical software (Stat for Windows, R.7, Stat Soft, Inc., USA, 2008). Data are presented as means standard deviations (SD). Evaluation of general radioprotective efficacy (comparison of cumulative survival times among treatments) was done by using Kaplan-Meier analyzis (post hoc Log rank test). All results were statistically analyzed by two-way ANOVA and posthoc Tukey test was performed to investigate pairwise differences. The level of significance was set at 2alpha = 0.05. Results AFM analysis of FNP in saline and saline with 20% FBS In Fig. 2 and Fig. 3 results of AFM and TEM analysis of physiological solution of FNP are shown. The samples were tempered at 37 C during 24 h in the dark. In Fig. 4 distribution of fullerenol nanoparticles in physiological solution and saline with 20% FBS is shown.

Tested compound

LD50(95% confidence limits) (mg/kg)

FNP AMI

349.05 (282.34–431.53) 645.35 (566.56–763.82)

AFM images of the analyzed samples showed that FNP dissolved in saline were not homogeneous (Fig. 2C). The particles had a diameter of 29 nm to 109 nm (Fig. 2A). The larger particles (109 nm) consisted of smaller particles having a size of approximately 16–42 nm (Fig. 2B). The 3-D image of fullerenol nanoparticles of 109 nm was presented in Fig. 2D. In the TEM image (Fig. 3) results showed agglomerates of fullerenol nanoparticles with different sizes in saline that tempered at 220 C during 24 standing in the dark. Dominant particles were in the range of 10 nm to 30 nm. The agglomerates of fullerenol nanoparticles with size ranged from 10 nm to 20 nm were formed from smaller particles with size of 1.56 nm and 4.44 nm. After 24 h at 37 C in dark no significant influence of 20% FBS on size distribution of FNP in saline was detected (Fig. 4). Zeta potential of FNP in saline was 7.5 mV (result not presented). Study of acute toxicity of FNP and AMI For calculating LD50 values in rats, FNP and AMI were given at a dose of 200, 300, and 400 mg/kg ip, and 400, 600, and 800 mg/kg ip, respectively. LD50 values are given in Table 1. Mortality of animals treated by FNP occured after giving dose of 200 mg/kg (2 out of 6), and 400 mg/kg (4 out of 6). At necroscopy animals were found to have multiple organ haemorrhages (lungs, hearth, and mesentery), ascites and pulmonary oedema. Study of general radioprotective efficacy In this part of the study comparative radioprotective efficacy of FNP and AMF were examined. FNP given at a dose of 100 mg/kg ip resulted in good protection and the effect was comparable to that of AMI at a dose 300 mg/kg ip (Fig. 5). FNP in that dose, as well as AMF, provided LT50 longer than 28 days. Single dose of FNP provided 60% survival rate on the day 28 after irradiation, while the

Fig 4. Distribution of FNP in saline and saline with 20% FBS by number at 370 C after 24 h in dark (green

saline + FNP; red

saline + 20% FBS + FNP).




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survival rate of animals protected by AMI was 80%. There is no statistically significant difference between AMI and FNP treated groups. Study of tissue-protective efficacy of FNP andAMI

Fig. 5. Influence of pretreatment with FNP (100 mg/kg ip) and AMI (300 mg/kg ip) on survival of rats irradiated with 8 Gy of X-rays.

Intestinal lesions All of the irradiated rats had lesions throughout the intestinal tract, which were least severe on the day 28 (Fig. 6A and D). There were no differences in the severity of the lesions among these segments, although grossly the distal jejunum and ileum were more congested than other segments. Also, lesions were noted in the three major components of the tunica mucosa. There was segmental-to-extensive necrosis and exfoliation of the surface epithelium with villous atrophy in the small intestine. Focal necrosis and exfoliation of the crypt epithelial cells were observed, too. Congestion of the blood vessels and variable numbers of inflammatory cells were found in the lamina propria. Irradiation of the Payer's patch caused severe lymphocytolysis throughout the follicles and parafollicular zones, but infiltration of macrophages containing nuclear debris were observed on the day 28. The severity of these intestinal alterations statistically significant increased with time over a period of 1–4 weeks (Table 2). Also, these values were statistically significant different from irradiated rats protected with FNP or AMI during the whole experimental period. By seventh day, mild intestinal alteration was found in both irradiated groups of animals protected with FNP or AMI (a mean TDS and VDS were no higher than 1.33 and 1.50, respectively) (Fig. 6B and C). Mild to moderate intensity of the pathohistological

Fig 6. Light micrographs of the intestinal lesions of rats. H&E stain. magnification 20. (A, B, C; 7 days after treatments) and 20 (D, E, F; 28 days after treatments). (A) The IR group, focal necrosis and exfoliation of the crypt epithelial cells; (B) The FNP-treated group, mild oedema and hyperaemia in the Tunica submucosa; (C) The AMI-treated group, segmental lose of the superficial epithelium; (D) The IR group, extensive necrosis and exfoliation of the surface epithelium with complete villous atrophy, and severe lymphocytolysis in the Payer's patch; (E) The FNP-treated group, segmental collapse of the mucosal epithelium, and haemorrhagic foci associated with accumulation of inflammatory cells in the Lamina propria and the Tunica submucosa; (F) The AMI-treated group, severe and focal ulcerations, haemorrhage and secondary inflammatory cell infiltrations in the intestinal wall.



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Table 2 Intestinal damage score – IDS (frequency and severity of the intestinal lesions in irradiated rats only or irradiated rats treated with FNP–100 mg/kg or AMI–300 mg/ kg). X SD

Treatment Days after treatment TDS 0 1+ 2+ 3+ 4+ 5+ IR IR + FNP IR + AMI

7 28 7 28 7 28

0 0 0 0 0 0

0 0 4 0 3 0

1 0 2 4 3 0

3 0 0 2 0 3

2 2 0 0 0 3

0 4 0 0 0 0

3.17 0.72 4.67 0.52 a 1.33 0.52 a b 2.33 0.52 b c 1.50 0.55 a b 3.50 0.55 a b d X SD

Treatment Days after treatment VDS 0 1+ 2+ 3+ 4+ 5+ IR IR + FNP IR + AMI

7 28 7 28 7 28

0 0 0 0 0 0

0 0 4 3 3 0

1 0 2 3 3 3

3 0 0 0 0 3

2 3 0 0 0 0

0 3 0 0 0 0

3.17 0.72 4.50 0.55 a 1.33 0.52 a b 1.50 0.55 a b 1.50 0.55 a b 2.50 0.55 a b d

Statistical evaluation was performed using Tukey test. TDS – tissue damage score; VDS – vascular damage score; a – statistically significant vs. IR (7th day); b – statistically significant vs. IR (28th day); c – statistically significant vs. IR + FNP (7th day); d – statistically significant vs. IR + AMI (7th day).

changes could be seen only in irradiated rats treated with FNP during the rest period of the study (statistically significant as compered with IR + AMI) (Fig. 6E and F). Pulmonary lesions Irradiation of the lungs caused degeneration and inflammation (Fig. 7A). In the acute phase, the vacuolization of capillary endothelial and alveolary epithelial cells were observed, with subsequent flooding of alveoli with serofibrinous exudate. Majority of injured alveoli were lined by an increased number of vacuolated alveolar epithelial cells. This was followed by leukocytic infiltration of both the alveolar lumina and the pulmonary interstitium. Described histological alterations resulted in an increased in both TDS (3.67 0.52) and VDS (2.50 0.55) scores, seven days after irradiation (Table 3). Single application of FNP or AMI statistically significant decreased the frequency and severity of pulmonary changes, but less pronounced individual scores were established when FNP was used in pretreatment of irradiated-rats (Fig. 7B and C). The later phase of pulmonar injury, 28 days after treatments, was characterized by intraalveolar accumulation of macrophages, proliferation of alveolar type II cells, accumulations of lymphoid cells, fibroblast proliferation, and collagen deposit. Moderate haemorragic foci could be seen in the pulmonary interstitium, too. The bronchial epithelium was lined with enlarged mucous cells and mucus fluid. Extensive tissue destruction, so-called “honeycomb” lung, were observed in all irradiated lungs (Fig. 7D), and several pulmonal samples of irradiated animals protected with AMI (Fig. 7F). In these experimental groups, degenerative and vascular changes were

Fig. 7. Light micrographs of the pulmonary lesions of rats. H&E stain. magnification 20. (A, B, C; 7 days after treatments) and 20 (D, E, F; 28 days after treatments). (A) The IR group, serofibrinous exudate seen in alveoli lined by vacuolated epithelial cells; (B) The FNP-treated group, the macrophage's infiltration of the pulmonary interstitium; (C) The AMI-treated group, haemorrhagic foci seen in the pulmonary interstitium; (D) The IR group, extensive pulmonary tissue destruction; (E) The FNP-treated group, moderate infiltration of the pulmonary interstitium with mononuclear cells and heamorrhages; (E) The AMI-treated group, severe interstitial fibrosis and a moderate quantity of serofibrinous exudate in the alveoli.



J. Vesna et al. / Journal of Applied BiomedicineJ. Appl. Biomed. xxx (2016) xxx–xxx Table 3 Pulmonary damage score – PDS (frequency and severity of the pulmonary lesions in irradiated rats only or irradiated rats treated with FNP–100 mg/kg or AMI–300 mg/ kg). X SD

Treatment Days after treatment TDS

IR IR + FNP IR + AMI

Treatment


0

1+

2+ 3+

4+

5+

7 28 7 28 7 28

0 0 0 0 0 0

0 0 4 0 0 0

0 0 2 4 2 0

4 2 0 0 0 4

0 4 0 0 0 0

Days after treatment

VDS

7 28 7 28 7 28

2 0 0 2 4 2

3.67 0.52 4.67 0.52 a 1.33 0.52 a b 2.33 0.52 a b c 2.67 0.52 a b 3.67 0.52 b d X SD

0

1+

2+

3+

4+

5+

0 0 0 0 0 0

0 0 3 0 0 0

3 0 3 3 4 0

3 0 0 3 2 4

0 2 0 0 0 2

0 4 0 0 0 0

2.50 0.55 4.67 0.52 a 1.50 0.55 a b 2.50 0.55 b c 2.33 0.52 3.33 0.52 b d


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statistically significant higher than those obtained in irradiatedgroup protected with FNP (Fig. 7E). Splenic lesions The first observable splenic alterations, depletion of lymphocytes in the white pulp were detected in all irradiated rats 7 days after the treatment. Histologically, this depletion gave the impression of an inverted splenic architectures, with higher lymphocytes density in outer rather than in the inner parts of the lymphatic nodules (Fig. 8A). In later phase, diffuse lymphocyte depletion was associated with phagocytosis by macrophages leading to a “starry sky” appearance. Polymorphs were seen and abnormal cells, mainly dead and dying lymphocytes, were present in the white pulp. Histological abnormalities, especially in the endothelial cells of the central arterioles, with vacuolisation and the highest incidence of picnotic nuclei could be seen. Vascular alterations, focal and massive haemorrhage, which were associated with a mild to moderate fibrosis were presented in the red pulp, too (Fig. 8D). The severity of splenic alterations were the highest in the control group of animals and animals protected with amifostine, which were sacrificed 4 weeks after irradiation (Table 4). Their TDS and VDS values were statistically significant different from those established in the irradiated group protected with FNP. Also, pretreatment with FNP statistically significant reduced intensity of splenic damages in comparison with the AMI treated groups throught the whole study period (compare Fig. 8B with C, and E with F, respectively). Cardiac lesions Prominent myocardial alterations were seen only in irradiated animals. Seven days after the treatment, structural changes were

Fig 8. Light micrographs of the splenic lesions of rats. H&E stain. magnification 20. (A, B, C; 7 days after treatments) and 20 (D, E, F; 28 days after treatments). (A) The IR group, focal lymphocyte depletion in the lymphatic nodules; (B) The FNP-treated group, higher lymphocytes density in outer parts of the lymphatic nodules; (C) The AMItreated group, diffuse lymphocyte depletion associated with oedema, hyperaemia and haemorrhage. (D) The IR group, complete loss of the white pulp architectures; (E) The FNP-treated group, phagocytosis of single dead and dying lymphocytes; (E) The AMI-treated group, the highest lymphocytes density associated with various numbers of the plasma cells.



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Table 4 Splenic damage score SDS (frequency and severity of the splenic lesions in irradiated rats only or irradiated rats treated with FNP–100 mg/kg or AMI–300 mg/ kg). X SD

Treatment


0

1+

2+

3+

4+

5+

IR

7 28 7 28 7 28

0 0 0 0 0 0

0 0 3 0 0 0

0 0 3 3 4 0

3 0 0 3 2 0

3 2 0 0 0 2

0 4 0 0 0 4


VDS

IR + FNP IR + AMI

Treatment


7 28 7 28 7 28

TDS

3.50 0.55 4.67 0.52 a 1.33 0.52 a b 2.50 0.55 a b c 2.33 0.52 a b 4.67 0.55 a d X SD

0

1+

2+

3+

4+

5+

0 0 0 0 0 0

0 0 4 0 0 0

0 0 2 2 4 0

3 0 0 4 2 0

3 2 0 0 0 1

0 4 0 0 0 5

3.50 0.55 4.67 0.52 a 1.33 0.52 a b 2.67 0.52 a b c 2.33 0.52 a b 4.33 0. 52 b d


characterized by sarcoplasmatic vacuolisation and nuclear polymorphism, as well as the presence of focal haemorrhages and inflammatory cell infiltrates in the vicinity of the blood vessels (Fig. 9A). These affected areas were mostly observed in all three anatomical layers (epicardium, myocardium and endocardium), and mean TDS and VDS scores were 2.50 0.55. The frequency and

severity of cardiac lesions statistically significant increased with time over a period of 7–28 day (Table 5 and Fig. 9D). After administration of FNP, histopathological changes in irradiated rats were qualitatively similar, but less intensive throughout the whole experimental period (compare Fig. 9B and E). Animals treated with AMI showed almost normal myocardial morhology during the first week of the study (a mean TDS, as well as VDS, was 0.33 0.52). Semiquantitative assessment of cardiac lesions in the animals protected with AMI, showed on Fig. 9C and F, demonstrated the lowest scores for degenerative and vascular changes when compared with the other treated groups (statistically significant as compered with IR and IR + FNP, respectively). Hepatic lesions In the early post-irradiation phase, the affected liver areas were abnormal in colour, frequently pale, and appeared slightly swollen. The main structural changes were characterized by mild vacuolar degeneration with nuclear polymorphism, and the presence of single inflammatory cell. In this study period, the hepatic injuries were the least severe in the amifostine-treated rats (a mean TDS, and VDS were 1.33 0.52, respectively) (compare Fig. 10C with Fig. 10A and B, respectively). These values were statistically significant different from FNP-treated and unprotected group (Table 6). 28 days after the treatment, hepatic lesions consisted of multifocal vacuolar degeneration and necrosis of a single or groups of hepatocytes, which were associated with inflammatory cells infiltrates. In all irradiated rats, liver necrosis was characterized by necrosis of the entire liver lobule. The extensive necrosis affected the entire lobular structure of multiple lobules, multilobular necrosis (Fig. 10D) with the highest TDS (4.50 0.55), and VDS (4.33 0.52). Necrosis of single hepatocytes or small groups of cells, sometimes accompanied by mild neutrophilic infiltration,

Fig. 9. Light micrographs of the myocardial lesions of rats. H&E stain. magnification 20. (A, B, C; 7 days after treatments) and 20 (D, E, F; 28 days after treatments). (A) The IR group, focal interstitial bleedings and sarcoplasmatic vacuolisation; (B) The FNP-treated group, extensive lack of cross-striations of myofibrils; (C) The AMI-treated group, focal myocardial oedema; (D) The IR group, diffuse myocardial degeneration and haemorrhagic infiltration; (E) The FNP-treated group, diffuse sarcoplasmatic vacuolisation and numerous haemorrhagic foci; (F) The AMI-treated group, single endocardial cells with sarcoplasmatic vacuolization.



J. Vesna et al. / Journal of Applied BiomedicineJ. Appl. Biomed. xxx (2016) xxx–xxx Table 5 Cardiac damage score – CDS (frequency and severity of the cardiac lesions in irradiated rats only or irradiated rats treated with FNP–100 mg/kg or AMI–300 mg/ kg). X SD

Treatment


0

1+

2+

3+

4+

5+

IR

7 28 7 28 7 28

0 0 0 0 4 0

0 0 4 0 2 4

3 0 2 4 0 2

3 3 0 2 0 0

0 3 0 0 0 0

0 0 0 0 0 0

Treatment


VDS

IR

7 28 7 28 7 28

IR + FNP IR + AMI

IR + FNP IR + AMI

TDS

2.50 0.55 3.50 0.55 a 1.33 0.52 a b 2.50 0.55 b c 0.33 0.52 a b 1.33 0.52 a b d X SD

0

1+

2+

3+

4+

5+

0 0 0 0 4 0

0 0 4 0 2 4

3 0 2 3 0 2

3 3 0 3 0 0

0 3 0 0 0 0

0 0 0 0 0 0

2.50 0.55 3.50 0.55 a 1.33 0.52 a b 2.33 0.52 b c 0.33 0.52 a b c 1.33 0.52 a b d


9

was noted in the liver of irradiated-rats protected with FNP. In some hepatic sections, not every lobule was equally affected (Fig. 10E). Moderate to severe destruction of the lobular architecture were not found only in group of irradiated-rats treated with AMI (Fig. 10F). Semiquantative analysis of these lesions demonstrated lower scores for degenerative and vascular changes when compared to irradiated rats only (statistically significant as compered with IR). Renal lesions One week after irradiation there were moderate vacuolar changes in renal tubular epithelial cells, extensive vascular changes and atrophy of some glomeruli (Fig. 11B). These lesions progressed to degeneration and reduction of tubular epithelial cells 4 weeks after irradiation. However, with renal tubular epithelium decrease there was a corresponding increase in interstitial connective tissue. The initial signs of perivascular fibrosis were seen in some tissue samples. Marked thickening of the juxtaglomerular arteries was associated with loss of some glomeruli at the end of the study (Fig. 11D). This was followed by intensive inflammatory cells infiltration of the renal cortex and the renal medula. Described histological alterations resulted in an increase of both TDS (4.33 0.52) and VDS (4.50 0.55) scores, 28 days after irradiation (Table 7). Pretreatment with FNP statistically significant decreased the frequency and severity of renal alterations through the whole study period (Fig. 11B and E), however less pronounced individual scores (no higher than 2.33 0.52) were established when AMI was used in protection of irradiated rats (Fig. 11C and F).

Fig 10. Light micrographs of the hepatic lesions of rats. H&E stain. magnification 20. (A, B, C; 7 days after treatments) and 20 (D, E, F; 28 days after treatments). (A) The IR group, vacuolar degeneration with nuclear polymorphism; (B) The FNP-treated group, moderate cytoplasmatic vacuolisation of the hepatocytes, mild oedema and hyperaemia seen in the sinusoidal spaces; (C) The AMI-treated group, numerous necrotic hepatocytes are surrounded with haemorrhage and inflammatory cells; (D) The IR group, complete multilobular necrosis, and massive accumulation of inflammatory cells on affected blood vessel wall; (E) The FNP-treated group, fragmented fibers are surrounded by massive oedema and hyperaemia, and severe and focal inflammatory cell infiltrates; (F) The AMI-treated group, fragmented fibers are surrounded by massive oedema and hyperaemia, and severe and focal inflammatory cell infiltrates.



10


Table 6 Hepatic damage score – HDS (frequency and severity of the hepatic lesions in irradiated rats only or irradiated rats treated with FNP–100 mg/kg or AMI–300 mg/ kg). X SD

Treatment


0

1+

2+

3+

4+

5+

IR

7 28 7 28 7 28

0 0 0 0 0 0

0 0 0 0 4 0

0 0 4 0 2 4

4 0 2 4 0 2

2 3 0 2 0 0

0 3 0 0 0 0

Treatment


VDS

IR

7 28 7 28 7 28

IR + FNP IR + AMI

IR + FNP IR + AMI

TDS

3.50 0.55 4.50 0.55 a 2.33 0.52 a b 3.33 0.52 a b c 1.33 0.52 a b 2.33 0.52 a b d X SD

0

1+

2+

3+

4+

5+

0 0 0 0 0 0

0 0 0 0 4 0

0 0 4 0 2 0

4 0 2 4 0 4

2 4 0 2 0 2

0 2 0 0 0 0

3.33 0.52 4.33 0.52 a 2.33 0.52 a b 3.33 0.52 b c 1.33 0.52 a b 3.33 0.41 b d


Discussion Results of our study confirmed the previously published ones of Trajkovic et al. (2007) on high radioprotective efficacy of FNP in irradiated rats that was comparable to that of AMI, a well-known radio- and chemoprotector. Moreover, FNP produce radioprotection in a dose that is approximately 1/3 of their LD50, while a radioprotective dose of AMI is almost 1/2 of its LD50 in rats. Bearing in mind that the higher dose of radioprotectors provides greater protective effect (Giambarresi and Jacobs, 1987), this finding suggests better safety profile of FNP in comparison to that of AMI. Relatively low toxicity of FNP has been shown in the study of Cai et al. (2010) performed in mice, in which mice were given FNP at a dose of 40 mg/kg/day for two weeks and whole-body irradiated by a lethal dose of 8 Gy. Although animals received cumulatively 560 mg/kg of FNP, none of them died before irradiation. Results of our study also showed that components of the serum did not alter the behavior of FNP aggregates that were not larger than 100 nm, which is proved to be physiologically eligible (Grebowski et al., 2013; Indeglia et al., 2014). Our results on FNP efficacy in protection of several main organs in irradiated rats, obtained by semiquantitative histopathological analysis, are in agreement with the results of biodistribution of FNP in animal tissues showing that radiolabelled FNP were detected in most tissues in the first hour after injecting (Djordjevic et al., 2011). After comparing results for size distribution of FNP by number, it can be concluded that in sample containing physiological solution with 20% FBS the contribution of larger particles increases, but not in a time-dependent manner, except for sample measured

Fig. 11. Light micrographs of the renal lesions of rats. H&E stain. magnification 20. (A) The IR group, 24 h after treatment, atrophy of some glomeruli and haemorrhagic foci; (B) The FNP-treated group, mild degeneration and a reduction of tubular epithelial cells associated with oedema and hyperaemia; (C) The AMI-treated group, moderate degeneration and a reduction of tubular epithelial cells associated with oedema and hyperaemia; (D) The IR group, degeneration and a reduction of glomeruli and tubular epithelial cells; (E) The FNP-treated group, focal cortical and medular cells degeneration; (F) The AMI-treated group, massive cortical and medular inflammatory cells infiltration.



J. Vesna et al. / Journal of Applied BiomedicineJ. Appl. Biomed. xxx (2016) xxx–xxx Table 7 Nephritic damage score – NDS (frequency and severity of the nephritic lesions in irradiated rats only or irradiated rats treated with FNP–100 mg/kg or AMI–300 mg/ kg). X SD

Treatment


0

1+

2+

3+

4+

5+

IR

7 28 7 28 7 28

0 0 0 0 0 0

0 0 2 0 5 0

2 0 4 2 1 5

4 2 0 4 0 1

0 4 0 0 0 0

0 0 0 0 0 0

Treatment


VDS

IR

7 28 7 28 7 28

IR + FNP IR + AMI

IR + FNP IR + AMI

TDS

3.33 0.52 4.33 0.52 a 2.33 0.52 a b 3.33 0.52 b c 1.17 0.41 a b 1.83 0.41 a b X SD

0

1+

2+

3+

4+

5+

0 0 0 0 0 0

0 0 2 0 4 0

0 0 4 2 2 4

3 0 0 4 0 2

3 3 0 0 0 0

0 3 0 0 0 0

3.50 0.55 4.50 0.55 a 1.67 0.52 a b 2.67 0.52 a b c 1.33 0.52 a b 2.33 0.52 a b d


after 24 h at 37 C in dark. Results for size distribution of particles were approximately equal for examined samples. After incubation at 37 C in dark in a 24-h experiment, the presence of saline with 20% FBS did not affect the size distribution of FNP by number. Reorganisation of water molecules surrounding FNP, changes in particles’ charge and size, as well as interaction with proteins play key role in radioprotective potential of FNP. Furthermore, watersoluble derivatives of fullerene are known to localise close to mitochondria (Grebowski et al., 2013). Mitochondria are wellknown for their great susceptibility towards oxidative stress (Djordjevic et al., 2011), therefore the most prominent effect of radioprotectors would be maintaining homeostasis of mitochondria (Stankov et al., 2013). Results of the research that dealed with biodistribution of polyhydroxylated fullerenes in in vivo models point out that nanoparticles were widely distributed in all tissues, especially in liver, spleen, heart, salivary glands and bones, while they got excreted through renal and bile pathways (Foley et al., 2002; De Grey, 2000; Quingnuan et al., 2002). The presence of nanoparticles is known to induce production of reactive oxygen species (ROS) either by direct or indirect pathway thus causing disruption of cell homeostasis (Beranova et al., 2011; Johnson-Lyles et al., 2010; Yan et al., 2013), however, nanoparticles of fullerenols, exhibit high anti-oxidative and anti-inflammatory potential (Grebowski et al., 2013; Narayanan and Park, 2013; Ye et al., 2014). Our results, as well as the ones previously published (Koukourakis, 2012; Kuntic et al., 2013; Mettler et al., 2011), support the statement that the pathogenesis of radiation injury is complex, for example high doses of X-rays can directly alter DNA strands. Moreover, exposure of living cells to dose of ionizing radiation, less than 8 Gy, release reactive oxygen species (ROS) and free radicals that damage both DNA and the cytoplasmic organelles and endoplasmic reticulum (Koukourakis, 2012). Also, the intensity of different cytomorphological manifestation correlates with duration of exposure and tissue sensitivity (Kuntic et al., 2013; Mettler et al., 2011). In our experiment, the prominent signs of cellular degeneration, congestion of the blood vessels, lymphocytes depletion and inflammation could be found in all tissues irradiated with 8 Gy observed on the day 7. A mild to moderate intensity of damages were found in all examinated tisssue samples (a mean TDS and VDS were in the range within 2.50–3.67). This acute or early effects of radiation injury consists of 2 phases. During

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the type I early radiation effects (within a few days after radiation injury) cellular death and cellular depletion followed by a proliferative response of stem cells are seen. In late phase or type II early radiation effects (up to 1 week after radiation injury) such as vascular permeability and tissue oedema increased the activation of macrophages and polymorphonuclears (PMNLs) and promoted released ROS free radicals, nitric oxide and cytokines (TNF-a and IL-6). If the irradiation induces irreverisible damages of fibroblasts, endothelial cells and other cells of connective origin, type II early radiation effects can progress to type III late radiation effects (Haschek and Rousseaux, 1998; Koukourakis, 2012). It was noticed in our experiment by using semiquantitaive histopathological analysis (28th day). Irreversible celular disorganisation and disfunction, followed by oedema, haemorrage, neutrophil infiltration, proliferation of fibroblasts and blood vessels were found. Severe damage of the vascular component led to necrosis in all examinated tissue samples. The calculated values of TDS and VDS were in the range of 3.50–4.67. This is a result of continuous cycle of growth factor and cytokine production by fibroblasts, endothelium and macrophages and other white cells, and it led to radiation inflammation (Narayanan and Park, 2013). Keeping this in mind, it can be supposed that the antiinflammatory effects of FNP and AMI, are related to inhibition of the main pro-inflammatory mediators, production or PMNL infiltration as well as to inhibition of the release of PMNL-derived mediators, including free radicals (Ma and Liang, 2010; Trajkovic et al., 2007). Also, both compounds protected all examined tissues. The most prominent reduction in TDS and VDS were noticed in the small intestine, lung and spleen of irradiated rats protected by a single dose of FNP (100 mg/kg). Also, we found that calculating scores in these tissues were lowest on day 7 (a mean IDS, PDS and SDS were in the range of 1.33–1.50). Later on, calculated IDS, PDS and SDS were increased but their values were statistically significant lower than in the IR and IR + AMI groups, respectively. There are just few studies showing the influence of fullerenols on leukocyte, epithelial and endothelial cells recruitment in the inflammation site. Roursgaard et al. (2008) showed that when mice were pretreated with fullerenol C60OH202, the quartzinduced neutrophilic lung inflammation was attenuated. Pretreatment reduced neutrophilic response by 50%. It has already been demonstrated that ROS are increasingly produced by inflammatory cells as a response to stimulation by cytokines such as TNF-a, IL-1, IL-6 and IL-17 and play an important role as messengers of the intracellular signaling pathway (Dragojevic-Simic et al., 2011). It was suggested that ROS, in turn, activate inflammatory cells that have part in the progression of inflammation. Therefore, targeting of ROS may have a therapeutic value as a strategy to reduce the development of inflammation. In our experiments FNP, given in dose of 100 mg/kg, was successful in protection of the small intestine, lungs and spleen of rats against harmful effects of ionizing radiation. This dose was used in previous studies, as well (Ma and Liang, 2010; Trajkovic et al., 2007). On the other hand, pretreatman with AMI also statistically significant reduced the irradiation-induced inflammation in rats, but more effective than fullerenol in protection of the heart, liver and kidney. The minimal CDS, HDS and NDS were observed on day 7 (0.33, 1.33 and 1.17, respectively) and statistically significant lower than that of IR and IR + FNP groups. Also, AMI had the lowest TDS and VDS in the heart, liver and kidney of IR rats on day 28. The maximal calculated values of CDS, HDS and NDS were in the range of 1.33–3.33, and statistically significant lower than that of FNP. It is considered that WR-1065, the active metabolite of AMI with free thiol group, acts as a potent scavenger of ROS that are result of interaction of ionizing radiation and water molecules in the cells (Dragojevic-Simic et al., 2011; Zois et al., 2011). AMI is a negative charged thiol which accumulates within the mitochondria and



12


around DNA. These facts explain higher protective potential of AMI compared with that of neutral or positive charged thiols. On the other hand, several lines of evidence suggest that AMI is presumably modified by membrane-bound alkaline phosphatase which is highly expressed in the endothelium and transferred into WR-1065, which then, quickly penetrates into cells, and acts as free-radical scavenger protecting cells from oxidative damage (Giambarresi and Jacobs, 1987). Besides, marked elevation of the expression of antioxidant enzyme MnSOD gene in human microvascular endothelial cells following their exposure to a WR-1065 can result in elevated resistance to the cytotoxic effects of ionizing radiation. Namely, MnSOD is nuclear-encoded mitochondrial enzyme that scavenges O2 in mitochondrial matrix, and has been shown to be highly protective against radiationinduced ROS (Babbar and Casero, 2006; Spencer and Goa, 1995). According to Kuntic et al. (2013), after ionizing radiation, ROS by inducing lipid peroxidation produce cytotoxic aldehydes resulting in inflammatory reactions. This eventually leads to increased synthesis of cytokines, infiltration of mononuclear cells and cellular death (Jamalludin et al., 2007; Zabbarova and Kanai, 2008). In accordance with this, in our experiment the presence of mononuclear cells and fibroblasts was decreased in AMI-protected rats, while necrotic cells in the heart, liver and kidney were rare compared with IR-only treated group. Conclusion Our results have indicated that FNP (100 mg/kg) expressed better radioprotection of the small intestine, lung and spleen, while these effects of AMI (300 mg/kg) were more prominent in the heart, liver and kidney. FNP examined in this model by comparing with AMI, potent cytoprotector with antiinflamatory effects, is potentially a valuable candidate for further investigation as an agent for the prevention and/or treatment of various disorders associated with inflammation. Acknowledgments 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. We would especially like to thank dr Zlatko Rakocevic for participation in AFM measurements and useful discussion. References Babbar, N., Casero, R.A., 2006. Tumor necrosis factor-a increases reactive oxygen species by inducing spermine oxidase in human lung epithelial cells: a potential mechanism for inflammation-induced carcinogenesis. Cancer. Res. 66, 11125– 11130. Beranova, E., Klouda, K., Zeman, K., 2011. C60 Fullerene derivative: influence of nanoparticle size on toxicity and radioprotectivity of water soluble fullerene derivative. Mat. Sci. Eng. A Struct. 1, 948–956. Bogdanovic, V., Stankov, K., Icevic, I., Zikic, D., Nikolic, A., Solajic, S., Djordjevic, A., Bogdanovic, G., 2008. Fullerenol C60(OH)24 effects on antioxidative enzymes activity in irradiated human erythroleukemia cell line. J. Radiat. Res. 49, 321– 327. Cai, X., Hao, J., Zhang, X., Yu, B., Ren, J., Luo, C., Li, Q., Huang, Q., Shi, X., Li, W., Liu, J., 2010. The polyhydroxylated fullerene derivative C60(OH)24 protects mice from ionizing-radiation-induced immune and mitochondrial dysfunction. Toxicol. Appl. Pharmacol. 243, 27–34. Cavas, T., Cinkilic, N., Vatan, O., Yilmaz, D., 2014. Effects of fullerenol nanoparticles on acetamiprid induced cytoxicity and genotoxicity in cultured human lung fibroblasts. Pestic. Biochem. Phys. 114, 1–7. Citrin, D., Cotrim, A.P., Hyodo, F., Baum, B.J., Krishna, M.C., Mitchell, J.B., 2010. Radioprotectors and mitigators of radiation induced normal tissue injury. Oncologist 15, 360–371. De Grey, A.D., 2000. The reductive hotspot hypothesis: an update. Arch. Biochem. Biophys. 373, 295–301.

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