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Biomaterials 29 (2008) 3451–3460

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Biomaterials

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Potential hepatoprotective effects of fullerenol C60(OH)24 indoxorubicin-induced hepatotoxicity in rats with mammary carcinomas

Rade Injac a,*, Martina Perse b, Natasa Obermajer a, Vukosava Djordjevic-Milic c, Matevz Prijatelj d,Aleksandar Djordjevic e, Anton Cerar b, Borut Strukelj a

a Faculty of Pharmacy, The Chair of Pharmaceutical Biology, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Sloveniab Institute of Pathology, Medical Experimental Centre, Medical Faculty, University of Ljubljana, Korytkova 2, Ljubljana, Sloveniac Medical Faculty, Department of Pharmacy, University of Novi Sad, Hajduk Veljkova 3, 21000 Novi Sad, Serbiad Faculty of Pharmacy, The Chair of Clinical Biochemistry, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Sloveniae Faculty of Sciences, Department of Chemistry, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia

a r t i c l e i n f o

Article history:Received 16 March 2008Accepted 28 April 2008Available online 27 May 2008

Keywords:AntioxidantChemotherapyCytotoxicityIn vivo testIn vitro testLiver

* Corresponding author. Tel.: þ386 41 964462; fax:E-mail address: injacrade@gmail.com (R. Injac).

0142-9612/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.biomaterials.2008.04.048

a b s t r a c t

The aim of this study was to investigate the potential protective role of fullerenol C60(OH)24 on doxo-rubicin-induced liver toxicity using in vivo (female Sprague–Dawley rats) and in vitro (human hepato-cellular carcinoma – HepG2; colorectal adenocarcinoma cell lines – Caco-2) approaches. The first(healthy control) and second (control with chemically induced mammary carcinomas) group receivedsaline only. The third, fourth and fifth group (all with breast cancer) were injected (i.p.) with a single doseof doxorubicin (8 mg/kg), doxorubicin/fullerenol (100 mg/kg of fullerenol 30 min before administrationof 8 mg/kg doxorubicin) and fullerenol (100 mg/kg), respectively. Two days after treatment, the rats weresacrificed. Results showed that treatment with doxorubicin alone caused significant changes in the se-rum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase(LDH) and a-hydroxybutyrate dehydrogenase (a-HBDH), as well as in the levels of malondialdehyde(MDA), glutathione (GSH), glutathione peroxidase (GSH-Px), total antioxidant status (TAS), glutathionereductase (GR), catalase (CAT) and superoxide dismutase (SOD) in the liver tissue. These effects weresignificantly reduced for all investigated parameters by pre-treatment with fullerenol but not for theMDA and GSH level. The HepG2 and Caco-2 cell lines were continuously treated with fullerenol for 12 h,24 h, 48 h and 96 h at concentrations of 10 mg/mL and 44 mg/mL. With the aim of evaluating the mod-ulating activity of fullerenol on doxorubicin-induced hepatotoxicity, the cell lines were simultaneouslytreated with doxorubicin (1 mM; 5 mM) and fullerenol (10 mg/mL; 44 mg/mL) in different combinations.When the cells are treated with 5 mM doxorubicin along with the fullerenol, we can see a significantimprovement of the cell capability during the entire time-line. We can conclude that fullerenol hascytotoxic effects on HepG2 by itself, but when the oxidative stress is too high the cytotoxic effects offullerenol are overcome by its protective role as a strong antioxidant compound.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Doxorubicin (Dox), an anthracycline antibiotic, is a broad-spectrum antineoplastic agent, which is commonly used in thetreatment of uterine, ovarian, breast and lung cancers, Hodgkin’sdisease and soft tissue sarcomas as well as in several other cancertypes. It was discovered in the early 1960s and represented a con-siderable advancement in the fight against cancer. The Dox anti-tumor effects include mechanisms related to alterations of DNA andthe production of free radicals [1]. The clinical usefulness of Dox isrestricted, since it has several acute and chronic side effects,

þ386 1 425803.

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particularly a dose-depended myocardial injury, which can lead toa potentially congestive heart failure [2]. Other tissues like thekidneys, brain, liver and the skeletal muscles, are also affected byDox [3].

It is believed that oxidative stress and the formation of freeradicals play a crucial role in the mechanism of Dox toxicity. Twodifferent mechanisms have been identified. The first implicates theformation of semiquinone-type free radical molecules, which areproduced via the NADPH-depended reductase enzyme pathway.Derivatives originating from Dox give rise to superoxide radicals byreacting with oxygen. The second pathway includes a non-enzy-matic reaction, which involves a reaction of Dox with iron. Semi-quinone metabolites delocalized Fe2þ Dox from ferritin andgenerate H2O2, hence causing hydroxyl radical formation and oxi-dant injury in cellular systems [4,5].

R. Injac et al. / Biomaterials 29 (2008) 3451–34603452

The disturbance in oxidant–antioxidant systems results in tissueinjury, which is demonstrated with lipid peroxidation and proteinoxidation in the tissue. Several studies have shown that the com-bination of the inflammatory process, free radical oxidative stress,and lipid peroxidation is frequently associated with liver damage,induced by toxic agents such as Dox [5]. Persistent and irreversibleliver damage as a side effect of Dox therapy has been proven and anincrease of the apoptotic processes in liver tissue after a single doseof Dox has been described [6,7]. It was confirmed that the thera-peutic doses of Dox enhance lipid peroxidation in microsomes andmitochondria in the liver, especially in the presence of Fe3þ ions [8].Dox-mediated hepatotoxicity includes focal damage in hepato-cytes, vascular damage and steatosis. Ductules spuriae aroundcentral veins and portocholangial spaces are possible [9]. Sub-cellular hepatic alterations, including polymorphic mitochondria,cytoplasmic vacuolization and lipid droplet accumulation, werealso described [10].

Strategies to attenuate Dox toxicity include dosage optimization,synthesis and the use of analogues or a combined therapy withantioxidants [1]. Clinical and experimental trials have been directedtoward employing various antioxidant agents to ameliorate Dox-induced liver damage. To date, the effect of different compounds onDox-induced hepatotoxicity has been evaluated. For example, vi-tamin E, via its robust free radical scavenging effect, prevents lipidperoxidation and therefore inhibits the hepatotoxic effects of Dox[4]. Other compounds such as erdosteine, cystathionine, and cate-chin might also prevent liver injury induced by oxidative stress[4,5,11,12].

Polyhydroxylated fullerenes, including C60(OH)24, have dem-onstrated high antioxidant activity in in vitro and in vivo studiesthat is higher than that of natural antioxidants like ascorbic acidand vitamin E [13–15]. Recent studies show tissue-protective ef-fects from fullerenol (Full) C60(OH)24 in irradiated rats and mice,due to its antioxidative and radical scavenging activity [16,17]. Thein vitro modulation of Dox-induced cytotoxicity from Full C60(OH)24

implicates its potential use in the amelioration of severe side effectsfrom Dox therapy [18]. Recent evidence shows that Full C60(OH)24

has cardioprotective effects in a single dose Dox-induced car-diotoxicity in rats [19,20]. Pathohistological studies also imply itsprotective effects on Dox-induced hepatotoxicity [21].

The aim of this experiment was to investigate the potentialhepatoprotective effects of Full C60(OH)24 on the livers of rats withmammary carcinomas after a single dose of Dox. An in vitro studywas also conducted to further evaluate the results obtained fromthe in vivo experiment.

2. Materials and methods

2.1. In vivo test

2.1.1. Experimental animalsFemale Sprague–Dawley outbred rats (Harlan, Italy) were obtained at 3 weeks of

age, quarantined and housed 3–4 per cage at a 22–23 �C room temperature, 70�10%humidity and a 12 h light/dark cycle. They had free access to a standard laboratorydiet (Altromin, Germany) and water. All experiments were approved by the NationalAnimal Ethical Committee of the Republic of Slovenia (licence number 3440-138/2006) and were conducted in accordance with the European Convention for theprotection of vertebrate animals used for experimental and other scientific purposes(ETS 123).

2.1.2. Drugs and chemicalsFull C60(OH)24 was synthesized and characterized from polybromine derivative

C60Br24 that was synthesized in a reaction of C60 in Br2 with FeBr3 as the catalyst [22].Full C60(OH)24 (Novi Sad, Serbia) was dissolved in a sterilized and apyrogenic 0.9%NaCl:Tween 80 (80:20; w/w) solution (20 mg/mL) inside a laminar flow cabin im-mediately before use. Dox (Adriablastina�) for i.v. administration was obtained fromPharmacia & Upjohn (Milan, Italy). The solution for an i.p. application was dissolvedin a sterilized and apyrogenic 0.9% NaCl solution (3 mg/mL) inside a laminar flowcabin immediately before use. The MNU (1-methyl-1-nitrosourea) was obtainedfrom Sigma (Deisenhofen, Germany). The MNU was always dissolved immediately

before use in a sterilized and apyrogenic 0.9% NaCl solution (14 mg/mL) insidea laminar flow cabin.

2.1.3. Experimental designAll 32 rats received i.p. applications of MNU (50 mg/kg of body weight) (carci-

nogenicity was induced chemically) on the 50th and 113th day of age [23]. On the160th day of age, the rats were treated with Dox and/or Full. The most effective doseof Full was determined in a preliminary study, which had shown significant differ-ences for blood and tissue markers, as well as for pathohistological and oxidativestress after 2, 7 and 14 days of Dox administration at a dose of 100 mg/kg of Full(unpublished data). The animals of both control groups received a sterilized andapyrogenic 0.9% NaCl solution. Two days after the Dox and/or Full application, therats were sacrificed by CO2.

The animals were randomly divided into five groups as follows:

I Untreated control group – rats received saline only;II Cancer control group – rats received MNU and saline;III Dox group – rats received MNU and Dox 8 mg/kg;IV Full/Dox group – rats received MNU and Full 100 mg/kg 30 min before Dox8 mg/kg;V Full group – rats received MNU and Full 100 mg/kg [20].

2.1.4. Estimation of serum AST, ALT, LDH, a-HBDH and haematologicalparameters’ level

In the present study, the liver function was evaluated with serum levels of al-anine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as withlactate dehydrogenase (LDH) and a-hydroxybutyrate dehydrogenase (a-HBDH) ac-tivity, using Tecan Saffire (Tecan UK, Milton Keynes, UK). The assay for ALT, AST, andLDH was carried out according to the methods described in the Chema Diagnostica(Jesi, Italy) commercial kits. a-HBDH was determined by a commercial kit obtainedfrom Dialab (Vienna, Austria). The results for all enzymes were expressed as U/L. Theblood for the analysis was taken by a heart puncture after opening the thoracicregion. The venous blood samples were divided into two portions. 200 mL was putinto eppendorfs with K-EDTA (15%), carefully mixed for 20 min and used for bloodcell counting. The second portion of blood samples (8� 2 mL per animal) was kept ata room temperature for approximately 2 h and then centrifuged. The serums wereused for an analysis of enzymatic activity. Samples were stored at�80 �C until used.

2.1.5. Estimation of hepatic antioxidant level and proteinEach liver was rapidly removed from the sacrificed rat, placed in an ice-cold

solution and trimmed of adipose tissues. Accordingly, each liver was finely mincedand homogenized in a Tris-buffer solution (pH 7.4; organ:buffer 1:10; w/w) anddivided into two portions. One was used for a malondialdehyde (MDA) de-termination, and the other was centrifuged at 13,000�g for 20 min at 4 �C (Beckmanrefrigerated, Ultracentrifuge). Supernatant was used for determination of totalprotein (TP) concentration, glutathione (GSH), glutathione peroxidase (GSH-Px),total antioxidant status (TAS), glutathione reductase (GR), catalase (CAT), and su-peroxide dismutase (SOD). Free GSH and GSSG levels were measured witha Chromsystems Diagnostic commercial kit for HPLC analysis (Munchen, Germany)and were expressed as mg/L. The ratio of free GSH/GSSG was also calculated. AnHPLC Agilent HP 1100-model system, equipped with an autosampler and a fluores-cence detector (Waldbronn, Germany), was used. The Randox commercial systems,Ransod (SOD), Ransel (GSH-Px), total antioxidant status (TAS) and glutathione re-ductase (GR) (Crumlin, United Kingdom) and Sentinel Diagnostics (Milan, Italy),were used to determine the levels of SOD, GSH-Px, TAS, GR and TP, respectively.Catalase activity in the tissue homogenate was quantified according to the methodby Aebi [24] in which H2O2 was reacted with the cell lysates (obtained as describedabove). The initial rate of H2O2 disappearance (0–60 s) was recorded spectropho-tometrically at a wavelength of 240 nm. The results for SOD and CAT were expressedas U/mg TP, for GSH-Px and GR as U/g TP and for TAS as mM. The TAS test measuresthe total antioxidant effect of these three defence systems in circulation. The anti-oxidant system protects tissues from the effects of free radicals. A deficiency in anyantioxidant will result in a reduction in the TAS. Numerous studies have identifiedpatient groups where TAS may prove to be an important diagnostic or prognosticguide [25,26].

2.1.6. Estimation of hepatic thiobarbituric acid reactive substances (TBARS)The hepatic TBARS level, an index of malonyldialdehyde (MDA) production, was

determined by a Chromsystems Diagnostic commercial kit for HPLC analysis(Munchen, Germany) and expressed as mg/L.

2.1.7. Coefficients of organsAfter weighing the body and tissues, the coefficients of liver to body weight

were calculated as the ratio of tissues (wet weight, mg) to body weight (g).

2.1.8. Histological analysis of liver and fat tissuesThe liver and abdominal fat (ventral side of mesenteric fat) tissue was fixed in

buffered formalin and the sections cut were stained with haematoxylin and eosinand observed under a light microscope. The effect of Full was evaluated on

Fig. 1. Macroscopic changes: abdomen after 100 mg/kg of fullerenol i.p.administration.

R. Injac et al. / Biomaterials 29 (2008) 3451–3460 3453

inflammatory and necrotic changes with respect to the untreated and cancer controlgroups.

2.1.9. Statistical analysisThe values are expressed as mean� standard deviation (SD). ANOVA, followed

by a post hoc test (Student’s t-test), was used to compare the groups, and the valuesof p< 0.05 were considered as statistically significant (a: p< 0.05; b: p< 0.001; c:p< 0.0001).

2.2. In vitro test

2.2.1. Cell linesThe cell lines used in the study were: HepG2 (human hepatocellular carcinoma,

ATCC HB-8065) and Caco-2 (human colorectal adenocarcinoma, ATCC HTB-37).HEPG2 cells were grown in William’s medium, supplemented with 4 mM glutamine,Sigma (St. Louis, MO), 15% fetal bovine serum (FCS), HyClone (Logan, UT) and anti-biotics (Sigma, St. Louis, MO). Caco-2 cells were grown in a modified Eagle’s medium(MEM) with 2 mM glutamine, 10% FCS and antibiotics. Cells were grown at 37 �C ina humidified atmosphere containing 5% CO2. Prior to use in an assay, the cells weredetached from the culture flasks with 0.05% trypsin and 0.02% EDTA in PBS, pH 7.4.The viability of cells in the experiments was at least 90%, as determined by a stainingwith nigrosin.

2.2.2. Cytotoxicity assayCell viability was evaluated by a CellTiter 96� AQueous One Solution Cell Pro-

liferation Assay (MTS colorimetric assay), Promega (Madison, WI) [27] on HepG2and Caco-2 cells. The cells were harvested, counted by nigrosin exclusion and platedinto 96-well microtiter plates (Costar) at an optimal seeding density of 2�104 forHepG2 cells and 1.5�104 for Caco-2 cells. The cells were plated in a volume of100 mL per well and pre-incubated in a complete medium at 37 �C for 24 h. Fulldissolved in a growth medium was added in different concentrations to HepG2 andCaco-2 cells, except to the controls; in second experiment Full and/or Dox, dissolvedin the growth medium, were added to HepG2 cells (seeded as previously described)in different concentrations. All the microplates were incubated for 12 h, 24 h, 48 hand 96 h. Two hours before the end of the incubation period, 15 mL of MTS wasadded. Absorbance was measured on a microplate reader at 492 nm. The wellswithout cells containing a complete medium and MTS only acted as blanks. Cellviability was expressed as a percentage of a control, and was calculated according tothe formula:

ðAtest cells=Acontrol cells � 100Þ:

2.2.3. Flow cytometryFor an analysis of mitochondria with flow cytometry, 2�105 HepG2 cells were

seeded in a 6-well cell culture plate (Costar) and Full was added in different con-centrations. The control cells were cultured in the absence of Full. After, 24 h, 48 hand 96 h, the cells were washed with PBS and labeled with a 100 nM MitoTracker(Molecular Probes) for 40 min. Afterwards, the cells were washed twice with PBS, pH7.4, harvested, and a flow cytometry analysis was performed on a FACScalibur(Becton Dickinson, Inc.).

2.2.4. Fluorescence microscopyHepG2 cells were plated on a glass slide in a volume of 300 mL per well. They

were incubated in a complete medium at 37 �C for 24 h. Full C60(OH)24, dissolved ina complete growth medium, was added in different concentrations, except to thecontrols. After 24 h of incubation, the cells were stained with Hoechst 33342�

(Molecular Probes�, Invitrogen) and MitoTracker Red CMXRos� (MolecularProbes�, Invitrogen), according to standard protocols recommended by the man-ufacturer. The cells were examined with a fluorescence microscope system CellR

(Olympus), in order to asses the binding of MitoTracker Red CMXRos� dye to themitochondrial membranes of the cells, treated with Full C60(OH)24.

2.2.5. Statistical analysisAll data were expressed as a mean� SD, and evaluated using a two-tailed Stu-

dent’s t test. The level of significance was 95% (p< 0.05).

3. Results

3.1. In vivo results

3.1.1. Macroscopic changesThe experimental hepatopathy induced with Dox (histopatho-

logical changes of the liver caused by Dox in the rat) was describeda few decades ago. The macroscopic changes of organs for the invivo studies in rats with malignant neoplasm for Dox and Full ad-ministration have never been described in literature. A very high

volume of exudative liquid in the chest and abdomen after the i.p.application of Dox was found. The exudative liquid amounted toapproximately 2� 0.2 mL for the I and II groups, 22� 2.1 mL for theIII group and 12� 0.8 mL for the IV and V groups. It is a typicalphenomenon, actually a side effect of Dox as an antineoplasticagent. The level of exudates in the abdomen and chest was thus thehighest in the Dox-treated group, but it has also been found thatFull could have harmful influence on some of the organs after i.p.administration because of its physical properties. After i.p. appli-cation of Full in a dose of 100 mg/kg, about 20% of Full has not beenabsorbed because of its size of particles and pure solubility andconsequently remained in the ventral side of abdomen, especiallyon the ventral surface of the liver, pancreas and spleen where actedas foreign body (Fig. 1). Black and brown particles could be seen inthe abdomen, particularly in the mesenteric fat area, surroundingthe liver, pancreas and spleen.

3.1.2. Coefficients of organsDuring the period of the breast cancer development in the an-

imal model, the body weight of each rat was checked every 7 days.The final body weights of the MNU-treated animals (II–V groups)were approximately 17% less when compared to the untreatedcontrol group of rats. Significant differences (Table 1) between eachgroup were found only at the beginning of the first application ofthe MNU for the untreated and cancer control groups (p< 0.05).After sacrificing, all groups with an induced breast cancer hadlower body weight than the untreated control group. A significantdifference was found only between the untreated control and Fullgroup (p< 0.05).

After 2 days of applying saline, Dox and/or Full, the rats weresacrificed and the weight of the body and various tissues/organswere collected. Table 1 shows the coefficients of liver expressed asmg (wet weight of tissues)/g (dead body weight). For the cancercontrol group measured in comparison to the untreated controlgroup (I), the coefficient is significantly higher (p< 0.05), and for

Table 1Body weight of rats and coefficients of liver after sacrificing (p< 0.05)

Group Body weight (g) Liver (mg/g)

1st MNU application 2nd MNU application After sacrificing

I 192.0� 7.7 256.3� 12.5 271.2� 12.8 31.4� 2.2II 201.5 ± 6.41 252.1� 9.0 262.5� 22.0 39.2 ± 6.91

III 195.9� 11.3 246.1� 13.6 257.0� 15.8 33.6 ± 2.21,2

IV 196.6� 9.4 240.6� 16.1 262.0� 13.8 32.9 ± 2.51,2

V 198.8� 6.9 245.3� 16.1 251.5 ± 14.41 33.6 ± 2.71,2

Superscript numbers 1,2,3 represents significant difference from the correspondinggroup.

R. Injac et al. / Biomaterials 29 (2008) 3451–34603454

the Dox, Full/Dox and Full groups measured in comparison to thecontrol cancer group (II), the coefficients of the liver are signifi-cantly lower (p< 0.05).

3.1.3. Effect of fullerenol on liver and fat tissue histologyAn i.p. injection of Dox caused histological changes in the rat

liver, consisting of leukocytic inflammation, centrilobular necrosisand vacuolation. Co-treatment with Full did not produce significantattenuation of the inflammatory and necrotic changes in the acutephase. The potential protective effect of Full was not proven on thehistological level. We found that in the acute phase, the fat tissuenecrosis with inflammation is more significant for the treatedgroups in comparison to both control groups (I and II). A differencebetween Dox and Full/Dox groups was not found (Fig. 2).

3.1.4. Haematological parametersThe results of measuring the white blood cell (WBC) number,

red blood cells (RBC), haemoglobin (Hb), and hematocrite (Ht), inthe animals sacrificed after 2 days of Dox and/or Full application,showed statistically significant changes of some haematologicalparameters (Table 2). Changes in RBC and Hb concentrations didnot show statistically significant difference between the cancergroups and the untreated group. However, taking into account bothparameters, there was a statistically significant difference betweenthe Dox and the Dox/Full groups compared to the cancer controlgroup.

Determining the WBC number showed a statistically significantdecrease of the number of leukocytes in the Dox and the Dox/Fullgroups compared to the untreated and the cancer control groups. Asignificant increase of neutrophils and a significant decrease oflymphocytes in the Dox, Dox/Full and Full-treated groups com-pared to both control groups (I and II) were detected. The per-centage of neutrophils was approximately five times higher in theDox group compared to the untreated group (57.8� 13.6% versus8.7�6.5%). Conversely, the level of lymphocytes was almost twicelower for the Dox group compared to both control groups(38.8� 12.8% versus 80.0� 7.4% and 88.6� 28.8%).

Fig. 2. Fat necrosis (lower half of the picture) in contrast to the vital fat tissue (upper half), inDox-treated group.

3.1.5. Effect of fullerenol on serum ALT, AST, LDH and a-HBDH levelsALT, AST, LDH and a-HBDH were measured in the serum as

markers of cellular injury. We also calculated the liver statusaccording to the ALT/AST and a-HBDH/LDH ratio.

In the female rats with breast cancer, there were significantchanges in the enzymes of AST and ALT (p< 0.05), after an i.p. ad-ministration of Dox/Full and Full. AST significantly increased in theFull/Dox and Full groups compared to the untreated and cancercontrol groups, as well as ALT values for Full/Dox and Full groupscompared to the untreated control group (Table 2). According to theresults for AST and ALT, the ALT/AST ratio significantly decreased(Table 2) for all groups compared with the untreated control group,but on the other hand a significant difference was found in the Doxgroup, where the ALT/AST ratio was increased, compared to thecontrol cancer group. The ALT/AST ratio is lower than 0.40 for the allcancer (II–V) groups. An increase of AST, two to three times (Full/Dox and Full groups) compared with the untreated control group,and a slight increase of ALT in serum were confirmed.

It is well known that both the LDH and a-HBDH are often used asmarkers of cardiovascular damage. Their elevated levels could in-dicate Dox related impairment of the heart function (Table 3). Therewas a significant increase in LDH levels (p< 0.05) in the Dox-treated group 48 h after Dox injection. The level in LDH release wassignificantly reduced in the groups pretreated with Full (Full/Doxand Full compared with Dox group). An increase of the circulatinglevels of LDH is an index of myocardial infarction, renal failure,hepatitis, anemia, malignancies, and affections of skeletal muscles.Even with induced breast cancer in the all cancer groups (cancercontrol, Dox, Full/Dox and Full), there was no significant differencebetween the untreated control group and other groups, except theDox group, calculating on the LDH level in serum.

The a-HBDH levels in serum of all groups were very similar andwithout statistically important differences (Table 2). For differen-tiation between liver and heart diseases, the a-HBDH/LDH ratio canbe calculated. As is shown in Table 2, a statistically significantdifference between all groups compared with the untreated con-trol group was confirmed. There is also a significant variation be-tween all treated groups (Dox and/or Full) compared to the cancercontrol group. A comparison of all groups with the untreatedcontrol group brings the conclusion that in the case of a lower a-HBDH/LDH ratio liver damage is stronger (<0.25). On the otherhand, the groups pretreated with Full have an almost normal levelof a-HBDH/LDH ratio calculating from �10% of the untreatedcontrol group.

3.1.6. Effect of fullerenol on hepatic antioxidants and TBARS levelIn order to evaluate the effect of Full on Dox-induced oxidative

stress, the tissue levels of GSH, GSSG, MDA and the activities ofantioxidant enzymes SOD, GSH-Px, TAS, GR and CAT were mea-sured. Administration of Dox by i.p. route produced a marked

between is an acute inflammatory reaction (100�); (A) Dox-treated group and (B) Full/

Table 2Effect of fullerenol on haematological parameters and serum enzyme levels in rats with doxorubicin-induced hepatotoxicity

Group WBC(�109/L)

RBC(�1012/L)

Hb (g/dL) Ht (%) Neutrophile(%)

Lymphocyte(%)

ALT (U/L) AST (U/L) ALT/AST LDH (U/L) a-HBDH (U/L) a-HBDH/LDH

I 6.9� 2.4 6.7� 2.1 13.2� 4.6 37.7� 12.3 8.7� 6.5 88.6� 28.8 29.1� 6.4 52.1� 5.2 0.56 861.5� 132.0 265.9� 26.8 0.31II 14.1� 5.3 5.1� 1.7 11.0� 3.3 31.9� 9.8 17.0 ± 3.21 80.0� 7.4 16.2� 3.7 57.5� 12.9 0.281 817.4� 253.6 207.2 ± 44.91 0.251

III 4.3 ± 0.71,2 6.8 ± 0.42 14.7 ± 0.22 39.4 ± 1.22 57.8 ± 13.61,2 38.8 ± 12.81,2 20.9 ± 7.21 52.9� 12.7 0.401,2 1127.5 ± 107.21,2 243.3� 48.3 0.221

IV 4.3 ± 1.11,2 6.8 ± 0.52 14.8 ± 1.12 39.7� 3.0 52.7 ± 19.11,2 44.7 ± 19.71,2 34.6 ± 4.82,3 120.7 ± 20.51,2,3 0.291,3 818.7 ± 223.53 272.0� 89.9 0.312,3

V 10.6� 4.3 7.2� 1.5 13.9� 2.9 36.1� 8.5 41.2 ± 8.91,2,3 48.7 ± 21.71,2 32.8 ± 3.82,3 126.2 ± 14.31,2,3 0.261,3 789.5 ± 155.13 254.8� 74.6 0.322,3

Superscript numbers 1,2,3 represents significant difference from the corresponding group (p< 0.05).

R. Injac et al. / Biomaterials 29 (2008) 3451–3460 3455

increase in the liver MDA level and GSH-Px, GR, CAT, SOD and TASactivity, compared to both control groups. This was accompaniedby an increase in the GSSG level of Dox-treated groups compared tothe cancer control group and a non-significant change of GSH levelcompared to both control groups. Pre-treatment with Full at a doseof 100 mg/kg produced a marginal change in the oxidative stressparameters. The activities of GSH-Px, GR, CAT, SOD and TAS for Full/Dox and/or Full-treated groups are significantly lower in contrast tothe Dox group, and in some cases over both control groups (Table3). The MDA level significantly increased in the Full/Dox groupcompared to the Dox and both control groups, while the free GSHlevel for the Full/Dox group decreased in comparison to the samegroups.

3.2. In vitro results

3.2.1. Effects of fullerenol on the growth of cell linesHuman hepatocellular carcinoma (HepG2) and colorectal ade-

nocarcinoma (Caco-2) cell lines were continuously treated with Fullfor 12 h, 24 h, 48 h and 96 h at concentrations 10 mg/mL and 44 mg/mL. Concentration of 44 mg/mL was determined to be a saturatedsolution of Full in a growth medium [28]. Full inhibited the growthof the Caco-2 cell line time and dose dependently. The effect couldnot be observed after the first 12 h, but afterwards, it graduallyincreased for the total range of concentrations up to 96 h. In theHepG2 cells, however, the effect was not as pronounced as for Caco-2 cells. Cell viability reached 52.23% for a maximal soluble con-centration of Full after 48 h, compared to 38.74% for Caco-2 cells inthe same period. Lower concentrations were also significantly lesstoxic for HepG2 cells as compared to the Caco-2 cell line (Fig. 3).

3.2.2. Flow cytometryThe intensity of the fluorescence of MitoTracker demonstrates

the activity of the mitochondria and, presumably, their disruption.Flow cytometry analysis of the mitochondria revealed that in theHepG2 cells, the fluorescence intensity for the control cells and thecells treated with 10 mg/mL or 44 mg/mL Full was significantly lowerafter 24 h, 48 h and 96 h of incubation (Fig. 4).

3.2.3. Fluorescence microscopyIn order to assess changes in the cell morphology and to confirm

the results obtained using flow cytometry, the cells were incubatedwith Full in a concentration of 10 mg/mL or 44 mg/mL and dyed with

Table 3Effect of fullerenol on various parameters of oxidative stress in rats with doxorubicin-in

Group Free GSH (mg/L) GSSG (mg/L) Free GSH/GSSG

MDA (mg/L) SODprot

I 85.3� 11.7 113.2� 13.5 1.9� 0.5 970.7� 107.1 56.II 69.5� 13.3 80.0� 15.1 1.8� 0.3 2546.1 ± 722.11c 71.III 77.8� 11.6 101.2� 10.2 1.9� 0.1 5484.0 ± 1517.21c,2b 100.IV 46.6 ± 12.01b,2a,3b 67.8 ± 11.11a,2a,3c 1.7� 0.3 8663.3 ± 1377.21c,2c,3a 62.V 63.3 ± 10.61a,3a 95.3 ± 8.01a,3a 1.6 ± 0.23a 5930.9 ± 2610.31b,2a 63.

Superscript numbers 1,2,3 represents significant difference from the corresponding grou

nuclei- and mitochondria-specific dyes. The fluorescence picturestaken after 48 h of incubation revealed no substantial morpholog-ical changes of the HepG2 cells (such as increased levels of apo-ptotic nuclei caused by Full toxicity) (Fig. 5). On the other hand, theresults obtained by FACS were confirmed, since the intensity of theMitoTracker dye florescence is lowered in a dose-dependent man-ner, indicating the drop of the mitochondrial membrane potentialas a result of an incubation with Full.

3.2.4. Protective effects of fullerenol in in vitro-induced doxorubicintoxicity

In order to evaluate the modulating activity of Full on Dox-in-duced hepatotoxicity, the cell lines were simultaneously treatedwith Dox (1 mM, 5 mM) and Full (10 mg/mL, 44 mg/mL) in differentcombinations. The cell growth was evaluated after 12 h, 24 h, 48 hand 96 h. As seen in Fig. 6, Dox-induced cytotoxicity on the HepG2cell line is time and dose-dependent. When co-added with Dox at5 mM concentration, Full, with its antioxidant activity, significantlyincreased the cell survival rate for almost twofold within the first24 h (Fig. 6A). After 24 h of Dox and/or Full treatment, the survivalfractions of the HepG2 cells were as follows: 28.68, 61.47, and71.86% (Dox, Dox/Full 10 mg/mL, and Dox/Full 40 mg/mL, re-spectively). On the other hand, when the cells were treated withDox in 1 mM concentration, Full exhibited its protective antioxidantactivity only after 96 h and not before. After 96 h of Dox/Fulltreatment (5 mM/44 mg/mL or 5 mM/10 mg/mL), the survival rate ofthe HepG2 cells was almost seven times higher among the Dox/Full-treated cells, compared to Dox alone (Fig. 6B). The level ofcytotoxicity of the Dox/Full combination was similar to or lowerthan the cytotoxicity induced by the Full alone.

4. Discussion

The present study was designed to evaluate pre-treatment withFull C60(OH)24, which would have a hepatoprotective effect on Dox-induced liver necrosis. It has already been reported that Full couldprotect against the progression of Dox-induced hepatic injury inhealthy Wistar rats [21]. However, the in vivo hepatoprotectiveactivity of Full on Dox-induced hepatotoxicity in rats with malig-nant neoplasm remains unknown, as well as the in vitro results forthe hepatic human cell line treated with Dox and/or Full.

A difference in the coefficient of the liver, which was found forall Dox and/or Full-treated groups (III–V), might be caused by a very

duced hepatotoxicity

(U/mgein)

GSH-Px (U/gprotein)

GR (U/gprotein)

CAT (U/mgprotein)

TAS (mM)

5� 14.3 927.6� 116.7 79.9� 38.2 356.1� 46.6 1.7� 0.74� 15.2 735.5 ± 70.81a 89.3� 27.5 339.9� 75.0 2.1� 0.64 ± 15.91a,2a 1066.6 ± 138.52b 106.8� 28.6 407.9� 42.6 3.2� 0.49 ± 11.73b 708.3 ± 145.41a,3a 76.9� 31.6 175.9 ± 60.31c,2b,3c 2.3 ± 0.43a

9 ± 12.73b 747.3 ± 88.51a,3b 45.7 ± 24.52a,3a 190.3 ± 31.31c,2a,3c 1.9 ± 0.53a

p; a: p< 0.05; b: p< 0.001; c: p< 0.0001.

Fig. 3. Viability (%) of (A) HepG2 and (B) Caco-2 cells treated with 10 mg/mL and 44 mg/mL of Full over a 96 h time period.

R. Injac et al. / Biomaterials 29 (2008) 3451–34603456

high volume of exudative liquid in the chest and abdomen after ani.p. application of Dox [1,2]. Experimental hepatopathy and myo-cardiopathy induced with Dox were described a few decades ago.Macroscopic changes of organs in in vivo studies in rats with ma-lignant neoplasm for Dox and Full administration have never beendescribed in the literature. Goodwin et al. [29] investigated singledose toxicity of hepatic intra-arterial infusion of Dox, coupled toa novel magnetically targeted drug carrier in a swine model. They

B

A

0

20

40

60

80

100

0 24 48 96hours

Mito

Tra

ck

er

flo

ure

sc

en

ce

(%

)

MitoTracker

96 h

100 101 102 103 104

Co

un

ts

0

50

100

150

200

250

Co

un

ts

MitoTracker

24 h

0100 101 102 103 104

50

100

150

200

250

Fig. 4. Concentration-depended effect of Full on MitoTracker fluoresce

found hepatic necrosis and microscopic damage of the liver aftera high single dose of Dox. Granulomatous inflammation or neu-trophilic inflammation in the spleens was also described. In ourstudy, it was found that Dox and Dox/Full have a very strong in-fluence on the balance in intra and extra-cellular compartments.On the other hand, we have also found that Full could have a sideeffects on some of the abdominal organs after an i.p. administrationbecause of its physical properties. Full is a hydrophilic derivative of

10 µg/mL Full44 µg/mL Fullcontrol

unmarked

10 µg/mL Fullcontrol

44 µg/mL Full

Co

un

ts

MitoTracker

48 h

100 101 102 103 1040

50

100

150

200

250

nce in human hepatocellular carcinoma cells, evaluated by FACS.

Fig. 5. Fluorescent pictures (A), (B), and (C) and fluorescence intensity profiles (a), (b), and (c) of human hepatocellular carcinoma cells: (A/a) control, (B/b) treated with 10 mg/mL ofFull and (C/c) 44 mg/mL Full. Nuclei were stained with Hoechst (blue) and mitochondria with MitoTracker (red).

R. Injac et al. / Biomaterials 29 (2008) 3451–3460 3457

C60, but the level of C60(OH)24 solubility is still very low [28].Consequently about 20% of Full remains as black and brown par-ticles in the abdomen, especially on the ventral surface of the liver,pancreas and spleen, particularly in the ventral mesenteric fat areasurrounding the liver (Fig. 1). Full as foreign body could havea harmful effect on the peritoneal compartments (peritonitis),which could produce secondary changes of the liver tissue.

In a more than 12 weeks old healthy Sprague–Dawley femalerat, 80% of all WBC are lymphocytes, followed by 14% of neutro-phils, 3% of eosinophils, 2% of monocytes and 1% of basophiles[30]. Full is xenobiotic for rats, just as Dox is, but the level ofproducing neutrophils is lower in the Full group. Therefore, Full

Fig. 6. Viability of HepG2 cells treated with (A) 1 mM of Dox (alone or in combination with 1044 mg/mL of Full) over a 96 h time period.

might have a positive influence on decreasing the neutrophils’count after treatment with Dox. Conditions, which result in lym-phocyte reduction, include steroid treatment, uraemia, systemiclupus erythematosus, Legionnaire’s disease, marrow infiltration,AIDS, post chemotherapy or radiotherapy [31]. It is obvious that inour case a post-chemotherapeutic treatment causes a decrease oflymphocytes. The production level of lymphocytes is higher in theFull group, therefore Full might have a positive influence onincreasing the lymphocyte count after treatment with Dox [19].The influence of Dox and/or Full on some other haematologicalparameters is well described in our previously publishedpaper [19].

mg/mL or 44 mg/mL of Full) or (B) 5 mM of Dox (alone or in combination with 10 mg/mL or

R. Injac et al. / Biomaterials 29 (2008) 3451–34603458

In female rats with mammary carcinomas, there are significantchanges for both liver-specific enzymes (ALT, AST) after an i.p. ad-ministration of Dox/Full and Full. In infectious hepatitis and otherinflammatory conditions affecting the liver, ALT is typically as highas or higher than AST, and the ALT/AST ratio, which normally and inother conditions is less than one, becomes greater than one.According to our results, we have had just the opposite situationthan that pointed out for hepatitis. One of the reasons for this couldbe the high number of malign tumours and metastases developedin rats. On the other hand, an increase of AST without significantchanges in ALT is also typical of heart injury, which has been con-firmed [19]. ALT is a more liver-specific enzyme. Measurement ofboth AST and ALT has some value in distinguishing hepatitis fromother parenchymal lesions. After a myocardial infarction, increasedAST activity appears in serum, as can be expected from the rela-tively high AST concentration in the heart muscle. The peak valuesof AST activity are roughly proportional to the extent of cardiacdamage. In some of our previous work [19,20] we confirmed,according to biochemical and histopathology results, that Dox hada strong cardiotoxicity effect in the acute phase. We also found thatFull in combination with Dox decreased the cardiac damage.Therefore, the results for the levels of AST and ALT/AST ratio can notbe explained only by a myocardial infarction, which is typical forthe Dox group. According to all of the results, we conclude thatprobable problems related to the solubility and consequently de-livery of Full after an i.p. administration, can induce liver damage,and peritonitis, which is consequently reflected on liver status.Increased levels of AST in the serum, two to three times (Full/Doxand Full groups) compared to the untreated control group, andslightly increased levels of ALT in serum confirm our findings [32].

It is well known that both LDH and a-HBDH are often used asmarkers of cardiovascular damage, while the a-HBDH/LDH ratiocan be calculated for a differentiation between liver and heartdiseases. Comparing all groups with the untreated control group,bring the conclusion that in the case of lower a-HBDH/LDH ratio,parenchymal liver damage is stronger. On the other hand, groupspretreated with C60(OH)24 have almost normal level of a-HBDH/LDH ratio, compared to the untreated control group. Full is alsoliver-protective in some cases [21], for example radioprotective[16,17], but low solubility and lower distribution after an i.p. ad-ministration have secondary harmful effects on the abdominal or-gans. Hepatocytes are the likely targets of reactive oxygen species(ROS) attacks in the failing liver. It is conceivable that free radicalscause damage at their formation. Consequently, as a major source ofROS production, mitochondria could also be major targets suscep-tible to ROS attacks [4–12]. The defects in the mitochondrialstructural design would lead to the adaptation of mitochondrialmetabolism, resulting in decreased activity of mitochondrial en-zymes in the Dox intoxicated liver, hence becoming a key con-tributor to intrinsic cell dysfunction [6–8]. Mitochondrialdegeneration and dysfunction has been reported in Dox adminis-tration [1,4–12,17,18]. The increased levels of oxidative stress en-zymes (SOD, GSH-Px, GR, CAT, TAS) were observed and confirmedin Dox-induced rats [19]. The impaired mitochondrial function,which may contribute to the pathogenesis of Dox-induced toxicity[19,20], might be a reason for the observed change in the activitiesof the typical markers for oxidative status in damaged liver tissue.

In this study, it was found that oxidative stress increased insome cases. Damage of the liver tissue after Dox administration wasfound to be the same as it was presented in our previously pub-lished paper for the cardiotoxicity of Dox [19]. Changes in the SOD,GSH-Px, GR, CAT and TAS levels have the same trend, therefore theyverify, in Full groups, a potential hepatoprotective influence of Fullas a pre-treatment agent in high single dose Dox-induced hepa-totoxicity in the acute phase. Accordingly, it could be confirmedthat Full is a potential hepatoprotector against Dox-induced

hepatotoxicity. The hepatoprotective influence of Full has alreadybeen investigated in X-ray irradiated mice and rats [16,17]. Withpresent results, it is confirmed that intracellular Full protects thehepatocytes against Dox toxicity, but the levels of MDA and dif-ferent glutathione forms show very significant cellular damage(Table 3). Thus we may ask ourselves, what the real picture of liverstatus in vivo after pre-treatment with 100 mg/kg Full in a single8 mg/kg dose of Dox in rats with mammary carcinomas is and whythe MDA and glutathione show hepatic injury in Dox and/or Fullgroups in spite of the results for typical enzymes of oxidativestatus?

An increase in the hepatic MDA concentration was detected, asan index of oxidative liver damage, in Dox and/or Full-treated rats.We did not observe a significant reduction of the MDA concentra-tion by Full, as we did for the heart tissues from the same rats [19].Despite good results for the cellular oxidative enzymes, the MDAlevel is significantly higher (60%) in the Dox/Full group whencompared to the Dox and Full and more than two times higher inthe Dox and Full when compared to the control cancer group.Previous study has shown that Full protected cells from lipid per-oxidation [19], hence, there has to be some other explanation forthe liver status. Low levels of glutathione (Table 3) were observedduring an increase in oxidative stress caused by Dox administration[1–3,5–12]. GSH is essential in maintaining the reduced status ofthe cell/tissue [33], and its severe depletion is reported to lead toliver injury [34].

According to previously published papers [35,36], the admin-istration of Full, preventively and therapeutically, diminished theincrease of tissue MDA concentration, originating from the ische-mia–reperfusion of the small intestine in dogs [35,36]. Therefore, itis not possible, that Full exhibits hepatotoxic effects after an i.v.administration, but in our case the low solubility and the higheffective (cardioprotective dose) i.p. administration [19,20] causedperitonitis and fat necrosis in the ventral mesenteric fat areaaround the liver surface (Figs. 1 and 2). Consequently, mediators ofinflammation and non-soluble particles caused lipid peroxidationand an increase of AST and ALT, as well as a decrease of GSH. On theother hand, the subcellular protection of hepatocytes is still work-ing as is confirmed by the SOD, GSH-Px, GR, CAT and TAS activities.

Several studies have reported cytotoxic effect of fullerene C60

and its derivatives to several cell lines [37–45]. Our results dem-onstrated the growth inhibition activity in the human colon ade-nocarcinoma cell line and human hepatocellular carcinoma cells.The effect was concentration- and time-dependent. The anti-proliferative effect and their enzyme inhibition can be attributed tothe antioxidative property [46,47]. Knowing that low levels of freeoxygen species promote cell proliferation and that redox alterationsplay a significant role in a signal transduction pathway, importantfor cell growth regulation [48,49], it is reasonable to propose thatFull, as antioxidant and free radical scavenger, might influence theHepG2 and Caco-2 cell redox state, leading to decreased cell pro-liferation [50]. Such observation could explain the milder effect ofFull on growth inhibition for the human hepatocellular carcinomacells in comparison to adenocarcinoma cells. For the cells to protectthemselves against oxidative stress and to regenerate after injury,there is a high requirement for enzymes to regulate cellular pro-cesses [51]. A water-soluble fullerene derivative was shown topreferentially bind to the mitochondria [52]. Fullerols are good freeradical trappers in biological systems [35,36,53] and Ueng et al. [54]reported that fullerols can suppress the activities of mitochondrialoxidative phosphorilation in vitro. In this regard, we decided toevaluate the mitochondrial status in hepatocellular carcinoma cellsand demonstrate that Full does disturb the mitochondrial activitysince a significant dose-depended drop of fluorescence after in-cubation with Full was seen in both the FACS experiment as well asin the analysis of fluorescent pictures taken by a microscope. This

R. Injac et al. / Biomaterials 29 (2008) 3451–3460 3459

indicates the disturbance of the mitochondrial integrity and loss ofmembrane potential resulting in the smaller intake of MitoTrackerdye. Since the increased appearance of apoptotic nuclei was notseen, this could again indicate the high mitochondrial capacity andregeneration properties of the HepG2 cells.

In order to demonstrate that the antioxidant properties of Fullare beneficial during Dox treatment in vitro, time-course and dose-related experiments were performed. The results, previouslyobtained from the in vivo experiment, indicating that Full can act asa hepatoprotector due to its free radical scavenging properties,were confirmed. When cells are treated with 5 mM of Dox along withthe Full, we can see a significant improvement of the cell viabilityduring the entire time-line. In the first 24 h, Dox-induced cyto-toxicity is being prevented at almost 100%, since the cell viability issimilar to that of Full alone. When cells are treated with 1 mM Dox,Full does not prevent Dox-induced toxic effects, but rather con-tributes to the loss of cell viability. This indicates, as was also pre-viously reported [4,5,54], that the cytotoxic mechanisms of Full andDox are different. We can conclude that Full has cytotoxic effects onHepG2 by itself, but when the oxidative stress is too high, the cy-totoxic effects of Full are overcome by its protective role as a strongantioxidant compound.

5. Conclusions

The results of this study indicate that doxorubicin generated invitro–in vivo hepatotoxicity by inducing the formation of ROS, whilethe fullerenol is not equally effective in all cases, in terms of itseffects on liver status in doxorubicin-induced hepatotoxicity. Full-erenol may play a hepatoprotective role in doxorubicin-inducedhepatotoxicity via its antioxidant properties. Current in vivo resultsconfirm that intracellular fullerenol protects hepatocytes againstdoxorubicin toxicity, but the levels of MDA and different glutathi-one forms show very significant cellular injury. Therefore, the pro-oxidant influence of fullerenol on healthy hepatocytes in vivo or invitro, as well as the in vivo irritability of the peritoneum and ab-dominal tissue caused by very low fullerenol solubility, could givedark light on the potential cardio- [19,20] and hepatoprotector. Onthe other hand, when oxidative stress is too high and induced withvery aggressive pro-oxidant such as doxorubicin, the cytotoxic ef-fects of fullerenol are overcome by its protective role as a strongantioxidant compound. These results propose that fullerenol mightbe a potential hepatoprotector in doxorubicin-induced hepatotox-icity, but only after an improvement of fullerenol solubility. Furtherchronic investigation is strongly recommended.

Acknowledgments

This work received partial financial support from the Ministry ofScience, Belgrade, Serbia, Grant no. 142076 and from the SlovenianResearch Agency, Ljubljana, Slovenia, Grant no. P4-0127.

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