Since 1993 countless studies showed that fullerene (C60) and derivatives exhibit paramount potentialities in several ﬁelds of biology and medicine  mainly including specific DNA cleavage,imaging , UV and radio protection , antiviral, antioxidant, and anti-amyloid activities [1,4e7], allergic response  and angiogen-esis  inhibitions, immune stimulating and anti-tumour effects[10,11], enhancing effect on neurite outgrowth , gene delivery, and even hair-growing activity . However, although several independent research groups conﬁrmed the innocuousness of pristine C60[15e17] the toxicity of this fullerene is still a matter of debate [18,19]. As recently demonstrated, this is mainly due to the lack of characterization of the tested materials [15e19]. Nevertheless, the metabolic fate and the in vivo chronic effects of C60itself still remain unknown. In order to fulfil the potential of C60and derivatives in the biomedical ﬁeld these issues must be addressed.
Aqueous suspensions were previously used to investigate the acute and sub-acute toxicities as well as the in vivo antioxidant properties of pristine C60[20,21]. But, such suspensions are not appropriate for determining toxicity at reiterated doses, because fullerene is active only in soluble form  and because the extremely slow dissolution of C60in biological media prevents controlling accurately the active fraction . This may be the reason for which the chronic toxicity of C60has never been investigated to our knowledge.
C60 is soluble in lipid droplets inside living cells  as well as in fats in general [22,23]. Moreover, C60 can freely cross membrane barriers as observed experimentally  and recently modeled by computer simulations . Thus, C60 interactions with living systems as well as its toxicity should be determined using soluble forms.
Recently, liposomes were used as carriers to study the bio-distribution of unmodiﬁed C60 in rats after tail vein administration. But, as C60 was not detected in blood due to its rapid clearance by tissue-filtration, such formulation was not appropriate for characterizing its pharmacokinetics .
While C60 solubility in vegetable oils [22,23] is not high enough to study its acute toxicity according to institutional recommendations (European Medicines Agency, Evaluation of Medicines for Human Use, 2004) , such solutions should be quite appropriate for studying its chronic toxicity at reiterated doses .
As the in vivo behaviour of soluble forms of C60, including absorption, biodistribution, and elimination was unknown, we determined the in vivo fate of C60 dissolved in olive oil before studying its chronic effects at reiterated doses.
Oily solutions cannot be administered intravenously because of possible vessel obstruction, so we characterized the pharmacokinetics of C60 dissolved in olive oil (0.8 mg/ml) after oral gavage (o.g.)and intra-peritoneal (i.p.) administration to rats (4 mg/kg of bodyweight (mg/kg bw)).
Finally, as C60 is known to be a powerful antioxidant [5,6,21], we checked the effects of C60-olive oil solutions on oxidative stress in a classical model of CCl4 intoxication in rats [28,29]. Although the oxidative stress involved in CCl4 intoxication is unlikely to occur during physiopathological conditions, CCl4intoxication in rats provides an important model for elucidation of the mechanism of action of hepatotoxic effects such as fatty degeneration (steatosis),ﬁbrosis, hepatocellular death, and carcinogenicity involving oxida-tive stress [28,29].
2. Materials and methods
2.1. C60-olive oil solution preparation
Virgin olive oil is obtained from a Chemlali Boughrara cultivar from Tunisiaplanted in the Sahel area. C60 (purity 99.98%) was obtained from Research Corporation (USA) and used without further purification.
Fifty mg of C60 were dissolved in 10 ml of olive oil by stirring for 2 weeks at ambient temperature in the dark. The resulting mixture was centrifuged at 5.000 g for1 h and the supernatant was ﬁltered through a Millipore ﬁlter with 0.25mm porosity.
2.2. Pharmacokinetics and biodistribution studies
All experimental procedures were reviewed and approved by the Animal Experimentation Ethics Committee of Paris XI University.
Pharmacokinetic studies were carried out with male Wistar rats (weighing200e220 g). Rats were housed in individual cages and maintained in an air-conditioned room (22e25C) on a 12 h light/dark cycle with water and food available. The rats were acclimated for 7 days before treatment.
After sodium pentobarbital (20 mg/kg bw in 1.0 ml/kg bw) anaesthesia, a catheter was introduced into the rat right jugular vein, positioned subcutaneously with the tip in the inter-scapular region. The prepared rats were then allowed to recover for 24 h, and the blood catheters were ﬂushed with 0.9% NaCl solution containing20 IU/ml of heparin to avoid possible clot obstruction.
Before C60 administration, the rats were fasted overnight but with access to water. The same single dose of C60(4 mg/kg bw) was delivered orally, through a gavage needle, or intra-peritoneally to two groups of three rats. Blood (0.20 ml)was withdrawn via the canular prior to dosing (t ¼ 0) and at 15, 30, 60 min and then at2, 4, 8,10, 12, 24 and 48 h post-dosing. Antithrombin heparin (20 IU/ml) was added in each blood sample. After each blood collection 0.20 ml of sterile 0.9% NaCl solution were injected to the animal, to avoid hypovolemia. The rats were sacrificed 48 h after C60 administration for organ collection (livers, spleens, and brains). Urines were collected at 24 h and 48 h after C60 administration then frozen at 20C until analysis.
For biodistribution studies, 4 groups of 3 rats were treated daily for 7 days either by i.p. administration (2 groups) or oral gavage (2groups) with the same dose (4 mg/kg bw) of the same C60-oil solution (0.8 mg/ml).At day 1 (D1), and D8, one group of orally treated and one group of i.p. treated animals were sacrificed for blood and organ collection. Urines were collected daily,then frozen under the same conditions as for pharmacokinetic studies.
2.3. Chronic toxicity and effects of C60 on survival of rats
The rats were housed three per cage and acclimated for 14 days, before dosing. Three groups of 6 rats (10 months old, weighing 465 31 g) were administered daily for one week, then weekly until the end of the second month and then every two weeks until the end of the 7th month, by gavages with 1 ml of water or olive oil or C60 dissolved in olive oil (0.8 mg/ml), respectively.
The rats were weighed before each dosing. Routine observations following ofﬁcial recommendations  were made on all animals inside and outside the cageonce a day throughout the study for signs of departure from normal activity,morbidity and mortality.
2.4. Effects of C60-olive oil solutions on oxidative stress
Sixty rats randomly divided into 10 groups of 6 rats were pre-treated daily for 7days by oral gavages (og groups) or by i.p. injection (ip groups). Groups A (GAog andGAip), received 1 ml of water. Groups B and C (GBog, GCog and GBip, GCip) were pre-treated with 1 ml of olive oil while groups D and E (GDog, GEog and GDip, GEip) were pre-treated with 1 ml of C60-olive oil.
Twenty-four hours before sacrifice, groups GA, GC and GE were i.p. injected with a single dose of CCl4(1 ml/kg bw) while GB and GD, used as controls, were administered with a 0.9% NaCl aqueous solution under the same conditions.
2.5. Chromatographic analyses, sample preparation and method validation
2.5.1. Chromatographic analyses
Chromatographic analyses of C60in blood, urine, liver, spleen and brain wereperformed as described previously  with the following modifications.
HPLC separations were performed using a P4000 multi-solvent delivery system coupled with a UV6000LP photodiode array detector (Thermo Separation Products,Les Ulis, France). Instrument monitoring and data acquisition were performed using ChromQuest Software from the same origin. Peak identifications were based on their UVe Visible spectra and the traces were recorded at 330 nm. Separations were carried out with a Hypersil 120-5 ODS, 5mm cartridge (MachereyeNagel, Hoerdt,France) protected with a 4.0 mm 10 mm pre-column packed with the same stationary phase.
For liver and spleen samples, separations were performed at 25C with a ﬂowrate set at 0.8 ml/min and a mobile phase composed of a mixture of toluene and methanol (35/65, v/v).
For whole blood, urine, and brain samples, separations were performed with 20% of toluene and 80% of methanol for the ﬁrst 5 min, at which time the toluene was increased to 60% for 10 min and then hold constant for the remaining 7 min ofeach sample run. At least 10 column volumes of the initial composition were ﬂushedthrough the column prior to injecting the sample.
2.5.2. Sample preparation
For whole blood, one hundred ml of sample were diluted in 400mlof0.1Msodium dodecyl sulfate (SDS). After adding 0.5 ml of acetonitrile and shaking for5 min, C60was extracted by adding 5 ml of toluene containing 0.2mg/ml of C70usedas internal standard (IS) to the mixture and shaking for 24 h in the dark. After centrifugation (2000 g for 15 min), the supernatant was evaporated under a stream of nitrogen. Then the residue was dissolved in 0.1 ml of toluene and diluted in acetonitrile (50/50, v/v) before injection of 100ml into the chromatograph.
For urine, 1.0 ml of sample were mixed with 0.2 ml of acetonitrile and then loaded into a Sep-pak plus C18cartridge (Waters, St Quentin en Yvelines, France) prealably conditioned with 5 ml of a mixture of water/acetonitrile (10/2, v/v). After washing the C18cartridge with 5 ml of acetonitrile, the retained compounds were eluted with 2 ml of toluene containing 0.2mg/ml of C70and evaporated under a stream of nitrogen. The residue was then dissolved in 0.1 ml of toluene and dilutedin acetonitrile (1/1, v/v) before injection of 100ml into the chromatograph.
For organs, about 1.0 g of liver (right lobe) or brain or 0.2 g of spleen were accurately weighed and then homogenized with 5 ml of 0.1 M SDS and 5 ml of acetonitrile. After shaking for 5 min, 20 ml of toluene containing 2.0mg/ml of IS were added and the mixture was shaken for 24 h in the dark. After centrifugation (2000 gfor 15 min), the supernatant was evaporated under a stream of nitrogen. Then the residue was dissolved in 1 ml of toluene for liver and spleen samples or 0.2 ml of toluene for brain samples, and diluted in acetonitrile (50/50, v/v) before injection of100ml into the chromatograph. Samples exceeding the limit of linearity were reanalyzed after appropriate dilution.
2.5.3. Method validation
For the calibration and the validation of the method, we used whole blood,urine, and organ samples of untreated rats spiked with C60-olive oil solutions (19/1,v/v or m/m).
The linearity of the method was checked between 0.01 and 1.0mg/ml under gradient elution (y ¼ 0.5963x þ 0.0006; n ¼ 6; where y is the peak area in AU minand x is the concentration of the injected solution inmg/ml; the relative standard deviations (RSDs, n ¼ 5) for the slope and the intercepts were 6.4% and 4.3%,respectively). The limit of detection for a signal to noise ratio equal to 3 was0.001mg/ml.
Under isocratic conditions, the linearity of the method was checked between0.01 and 10.0mg/ml (y ¼ 0.597x þ 0.0098; n ¼ 7; where y is the peak area in AU.min and x is the concentration of the injected solution in mg/ml; the RSDs (n ¼ 5) for the slope and the intercepts were 5.2% and 3.9%, respectively). The limit of detection for a signal to noise ratio equal to 3 was 0.002mg/ml. The between run (BWR) and between day (BWD) precisions were determined (n ¼ 6) for the lowest and thehighest level of each curve of calibration.
Under gradient elution conditions the RSDs were 7.2% and 10.5% for the BWRand 5.3% and 8.4% for the lowest levels and the highest levels, respectively. Underisocratic conditions, the RSDs were 5.6% and 8.5% for the BWR and 3.3% and 6.4% forthe lowest levels and the highest levels, respectively.
The recovery of the method was determined for each kind of sample at two levels (n ¼ 3, for each level). For whole blood, urine, and brain samples the recoveries were determined at 0.01 and 0.05mg/ml ormg/g, respectively and they were94.3 4.9% and 93.8 5.1% and 98.1 2.5% and 96.9 3.5%, respectively. For liver samples the levels were 0.2 and 30mg/g and the recoveries were 97.3 2.8% and99.1 2.2%, respectively. For spleen samples the levels were 2.0 and 200mg/g andthe recoveries and between run precision were 95.3 4.2% and 96.1 3.2%,respectively.
2.6. Biochemical tests and pathological examinations
Tissue and blood sampling, serum alanine amino-transferase (ALT) activity, andoxidized glutathione/total glutathione (GSSG/TGSH) ratio, where TGSH is the sum ofreduced (GSH) and oxidized glutathione (GSSG), were performed as previouslydescribed .
Superoxide-dismutase (SOD) and catalase (CAT) activities were determined aspreviously described [31,32].
Hepatic microsomal fractions were used for measuring the cytochrome P4502E1 (CYP2E1) speciﬁc oxidative activity such as p-nitrophenol hydroxylase. Thehepatic microsomal fractions were prepared by differential centrifugation, asdescribed previously  and were stored at 80C until required. The hydroxyl-ation of p-nitrophenol to 4-nitrocatechol was determined by HPLC as describedpreviously . Microsomal protein concentration was determined by the Bradfordmethod , using bovine serum albumin as a standard.
Pathological examinations and optical microscopy analyses were blindly per-formed by a pathologist ignoring all protocol procedures as well as the purpose ofthe study. The reparation and staining protocols of organ pieces for optical andtransmission electron microscopy (TEM) were performed as described previously.
2.7. Pharmacokinetic analysis
Pharmacokinetic analysis of the individual observed rat plasma data obtainedafter oral and i.p. routes was performed using the WinNonLinÒsoftware (PharsightCorporation, Mountain View, California). A non-compartmental approach was usedto calculate the main pharmacokinetic parameters.
The maximal plasma concentration (Cmax) and the time (Tmax) to reach Cmaxwere obtained directly from experimental observations. The terminal eliminationrate constant (lz) was calculated by linear regression analysis of the natural loga-rithm of the last experimental concentrations and the terminal half-life (t1/2)wascalculated by dividing Ln2 bylz. The area under the plasma concentration-timecurve from zero to inﬁnity (AUCRN0) was the addition of AUC from zero to the lastexperimental concentration (CT), calculated by the trapezoidal rule, and of AUC fromCTto inﬁnity, calculated by dividing CTbylz. The area under the ﬁrst moment curvefrom zero to inﬁnity (AUMCRN0) was the addition of AUMC from zero to the lastexperimental concentration (CT), calculated by the trapezoidal rule, and of AUMCfrom CTto inﬁnity, calculated by [((CT.T)/lz) þ (CT/lz2)]. The mean residence time(MRT) was calculated by dividing AUMCRN0by AUCRN0. The apparent plasmaclearance (Cl/F) was calculated by dividing the dose by AUCRN0, and the apparentvolume of distribution (Vd/F) was calculated by dividing the dose by (AUCRN0RN0.lz).2.8. StatisticsThe normality of data distribution was tested by ShapiroeWilk test. Data arepresented as the mean and standard deviation in the case of normal distributions oras the median and the range. Comparisons with control were performed by usingStudent test, according to the homogeneity of variances determined by Fisher test,or by ManneWhitney test. A value of P < 0.05 was considered statisticallysigniﬁcant.The survival distributions for C60-olive oil-treated and control rats were esti-mated by the non-parametric KaplaneMeier estimator and compared by a log-rankestimated test.3. Results3.1. C60-olive oil preparationThe composition and quality characteristics of olive oil weredetermined as previously described following analytical methodsdescribed in the EEC 2568/91 and EEC 1429/92 European UnionRegulations .
The resulting C60-olive oil solution is purple and contains0.80 0.02 mg/ml (n ¼ 6) as determined by HPLC  afterappropriate dilution in the mobile phase. The chromatographicproﬁle and the extracted spectra of these solutions are similar tothose obtained with a control C60-toluene equimolar solution.
The stability of both oily and control solutions stored at ambienttemperature and in the dark was checked monthly during 48months. No change was recorded under our chromatographicconditions.3.2. Pharmacokinetics and biodistribution
Fig. 1represents the evolution of whole blood C60concentrationsversus time following single dose o.g. and i.p. administration of thesame dose of C60dissolved in olive oil.
Table 1summarizes the main pharmacokinetic parameters. Themaximal concentrations (Cmax) are reached 4 and 8 h after i.p. ando.g. administrations, respectively.
The apparent volume of distribution (Vd/F) of C60after i.p.administration is higher than the blood volume in rats , indi-cating that C60is well distributed in tissues. The value of Vd/F aftero.g. is less signiﬁcant because the administered dose cannot beponderated by the C60bioavailability, which is unknown (Table 1).
The elimination process is slower after i.p. administration thanafter o.g., as illustrated by the elimination half-lifes and the meanresidence times of C60(Table 1).
At day 1 (D1) after administration, C60contents in livers andspleens represent 0.14% and 0.18% of the administered dose by theoral route, respectively, and 4.73% and 1.55% by the i.p. route,respectively (Table 2 ).
After 7 successive days of administration (D8), C60contents inlivers and spleens correspond to 0.39% and 0.51% of the totaladministered dose by the oral route, respectively, and 5.54% and2.39% by the i.p. route, respectively (Table 2).
At D1and D8C60content in brains represents less than 0.01% ofthe administred dose after o.g. while these values are higher than0.12% after i.p. administration (Table 2).
Microscopic examination at D8of the spleen reticulo-endothelial system (RES), where the highest concentrations are observed, shows the presence of some C60aggregates that arelarger and more numerous after i.p. administration (Fig. 2 c and d)than after o.g. (Fig. 2a, b): thus C60concentrations reached the limitof solubility in spleens. In contrast there are no observable depositsinside the livers in all cases indicating that C60concentrations inthese organs are not high enough to trigger precipitation.
While transmission electron microscopy (TEM) at D8after i.p.administration shows numerous spleen macrophages laden C60crystals (Fig. 2e) only some C60crystals were observed inside livermacrophages and very rare crystals in lung (Fig. 2f) and kidney cells(Fig. 2g).
3.3. Chronic toxicity and effects of C60on lifespan of rats
Fig. 3 shows the animal survival and growth. After ﬁve monthsof treatment (M15) one rat treated with water only exhibited somepalpable tumours in the abdomen region. Due to the rapid devel-opment of tumours (about 4 cm of diameter) this rat died at M17.Asrats are known to be sensitive to gavages, we decided to stop thetreatment for all rats and to observe their behaviour and overall survival.
All remaining animals survived with no apparent sign ofbehavioural trouble until M25(Fig. 3a). At the end of M25theanimals of the control groups showed signs of ulcerative dermatitiswith ageing while C60-treated animals remained normal. As thegrowths of all surviving animals showed no signiﬁcant differenceuntil M30(Fig. 3b) indicating that the treatment did not alter theirfood intake, we continued observing their survival.
At M38 all water-treated control rats were dead (Fig. 3a). Thisagrees with the expected lifespan of this animal species that isthirty to thirty six months. At this time 67% of olive-oil-treated ratsand 100% of C60-treated rats were still alive.
The survival distributions for C60-olive oil-treated rats andcontrols were estimated by the non-parametric KaplaneMeier estimator (Fig. 3) and compared by a log-rank estimated test. Theestimated median lifespan (EML) for the C60-treated rats was 42months while the EMLs for control rats and olive oil-treated ratswere 22 and 26 months, respectively. These are increases of 18 and90% for the olive-oil and C60-treated rats, respectively, as comparedto controls.
The log-rank test leads toc2values (one degree of freedom) of7.009, 11.302, and 10.454, when we compare water-treated andolive oil-treated rats, water-treated and C60-treated rats, and oliveoil-treated and C60-treated rats, respectively. This means that oliveoil extends the lifespan of rats with respect to water with a proba-bility of 0.99 while C60-olive oil extends the lifespan of C60-treatedrats with a probability of 0.999 and 0.995 with respect to water andolive oil treatments, respectively.
3.4. Effect of C60-olive oil solutions on oxidative stress
CCl4toxicity with respect to rats is well known [26,27] never-theless, we systematically studied the effects of this halo-alkane onthe animals we used in our experiments in order to avoid misin-terpretations due to inter-strain variability. In addition, to avoiderrors due to inter-individual and inter-season variability, a CCl4-treated control group was included in each experiment.
3.4.1. Animal behaviour and pathological examinations
A few minutes after CCl4injection, the animals showed inac-tivity, lethargy, and pilo-erection. For both GA groups (pre-treatedwith water only) these symptoms persisted during a period of 24 huntil the animals were sacriﬁced for pathological examination. Incontrast, for the animals pre-treated with olive oil or with C60-oil(GC and GD groups) these symptoms completely disappeared about5 h after CCl4 intoxication.
After abdomen incision, 24 h after 0.9% NaCl administration, thelivers of the control groups GBogand GDogorally treated with oliveoil only or C60-oil, respectively, exhibited normal morphology withbrown colour more pronounced for GDoganimals than for GBogones (Fig. 4).
The livers of i.p. treated control groups GBipand GDipalsoexhibited normal morphology, nevertheless they showed largedeposits of fat due to the accumulation of the administered lipids(Fig. 4). The brown colour of the GDiplivers was more intense thanthat of the orally treated rats (GDog).
As compared to spleens of GB animals treated with olive oil only,spleens of GD animals treated with C60-olive oil exhibited a darkercolour while those of GDipwere hypertrophic (enlarged). Strongereffects in the appearances were observed in previous studies afteradministration of high doses of suspended C60crystals to rodents,without any organ damage or toxic effect [20,21].
Twenty-four hours after CCl4administration, the livers of GAanimals pre-treated with water were pale and looked mottledwhile their lobes were adherent in most cases (5 of 6 rats). Incontrast the livers of GC and GE groups pre-treated with olive oil orC60-oil, respectively, exhibited normal morphology with the samefeatures as those observed for GB and GD control groups (Fig. 4).
At the microscopic scale, the liver sections of both GB and GDcontrol groups treated with olive oil only or C60-oil revealed normalparenchymal architecture without any inﬂammation or ﬁbrosis.These liver sections only showed hepatocytes with clear cytoplasmdue to lipid accumulation (Fig. 4). This phenomenon was moreabundant in liver cells of i.p. treated animals than in those of orallytreated animals. In these groups, C60deposits were detected only inspleens as brown and diffuse clusters inside macrophages withhigher abundances for i.p. treated rats than for orally treated ones(Fig. 2).
At the same time the liver sections of GA and GC animals co-treated with water and CCl4or with olive oil and CCl4, respec-tively, showed important damage including many inﬂammatoryareas as well as large necrotic areas with ballooning necrotic cellsassociated with an important steatosis (Fig. 4). In contrast, micro-scopic examination of the liver sections of GE animals co-treatedwith C60-olive oil and CCl4, revealed few necrotic areas with someballooning cells without apoptosis limited to some cords of hepa-tocytes (Fig. 4).
3.4.2. Biochemical tests
22.214.171.124. C60effects on CCl4induced liver damage. Circulating levelsof alanine amino-transferase activity (ALT), used as a biochemicalmarker of liver injury , conﬁrmed liver-protection by C60.
Twenty-four hours after CCl4injection, the increase of ALT forGA and GC animals (pre-treated with water or olive oil) can reachmore than 14 times and 12 times, respectively, the normal activityobserved for GB control group (Fig. 5). In contrast, in the Eogand Eipgroups pre-treated with C60-oil, the median of ALT activity was only about 5 and 1.2 times higher, respectively, than that observed in thecontrol groups.
126.96.36.199. C60effects on the endogenous antioxidant systems: gluta-thione, superoxidismutase and catalase activities
188.8.131.52.1. Glutathione system. The increase of the GSSG/TGSHratio, used as a gauge for the circulating redox equilibrium [21,29],in the GA and GB groups pre-treated with water and olive oil canreach about 10 and 13 times respectively the GSSG/TGSH of thecontrol group (Fig. 5) thus reﬂecting the intensity of the oxidativestress induced by the metabolism of CCl4.
Oral pre-treatment with C60-oil signiﬁcantly prevents theincrease of the GSSG/TGSH ratio in the GDoggroup. As compared tothe control group, the increase of GSSG/TGSH in the GDoggroupwas about 4 times higher only.
In the GDip group i.p. pre-treated with C60-oil, the GSSG/TGSHwas even signiﬁcantly lower than in the control group. As the liverC60content is signiﬁcantly higher after i.p. administration thanafter o.g. (Table 2), this result conﬁrms the doseeeffect relationship.
It is worthnoting that in the GB animals treated by C60-oilwithout CCl4intoxication the GSSG/TGSH ratio was signiﬁcantlydecreased (about twice as less) as compared to the control group.
184.108.40.206.2. Superoxidismutase (SOD) and catalase (CAT) activi-ties. Animals of GA and GC groups pre-treated with water or oliveoil by oral or i.p. routes adjusted to CCl4intoxication by increasingthe CAT and SOD enzymatic activities in erythrocytes and livers(Fig. 6).
C60-oil pre-treatment led to a signiﬁcant attenuation of theincrease of these activities. In addition this attenuation was moreexpressed in liver where fullerene accumulates than in blood(Table 2).
220.127.116.11. Effects of C60on CCl4metabolism. The microsomal cyto-chrome P450 (CYP2E1) activity determined after microsomeextraction from livers of orally treated animals shows that rats pre-treated by water or olive oil adjusted to CCl4intoxication byenhancing the biosynthesis of CYP2E1 (Fig. 7).
C60-pretreatment signiﬁcantly attenuated the increase ofCYP2E1 activity after CCl4intoxication without inhibitory effect onthis enzyme.
4.1. C60-olive oil solution preparation
It is well known that C60and derivatives are prone to aggregateeven in their best solvents . The C60-olive oil solution used inthis study can be considered as free of C60aggregates because: 1 eits colour is purple that is characteristic of C60solutions while thecolour of C60aggregate-containing solutions are rather brown,which is true even for water-soluble derivatives ;2e it is freelyand instantaneously soluble in toluene in contrast to C60aggregate-containing solutions, which slowly dissolve even in the bestsolvents of C60. Besides, the concentrations of C60in olive oil asdetermined by HPLC agree with those previously published byother authors .
The stability of C60-olive oil solution determined under ourexperimental conditions agrees with recently published resultsshowing that the addition of fullerene signiﬁcantly hampersthe peroxide formation thus increasing the stability of the testedoils .
4.2. Pharmacokinetics and biodistribution
The results of this pharmacokinetic study show for the ﬁrst timethat C60is absorbed by the gastro-intestinal tract (Fig. 1).
In the case of oily solutions, the drug release rate is controlled bythe partition coefﬁcient of the drug between the oily vehicle andthe tissue ﬂuid and lipophilic drugs may be released concurrentlywith the disappearance of the oily vehicle from the injection site. Thus, in the case of i.p. administration, the delay of 4 h forreaching Cmax(Table 1) can be attributed to the afﬁnity of C60for theoily phase.In the case of highly hydrophobic drugs (Log P > 5) it is wellknown that the absorption of the molecules by the gastro-intestinaltract occurs via the mesenteric lymphatic system after associationwith developing lipoproteins in the enterocytes rather than via theportal blood . Therefore, as the octanol/water partition coefﬁ-cient of C60is estimated to be 6.67 , the absorption of C60occursvia the mesenteric lymphatic system rather than via the portalblood. The longer delay for reaching Cmaxafter o.g. (Table 1) canthen be assigned to the fact that the ﬂow rate of the mesentericlymph in the lamina propria underlying the enterocytes is some 500times lower than that of the portal blood .
Cmaxand the area under the curve (AUC) after o.g. are about 3times and 5 times lower, respectively, than after i.p. administration.Although i.p. administration does not allow assessing the absolutebioavailability, AUCs comparison suggests that a signiﬁcantpercentage of the orally administered dose is absorbed by thegastro-intestinal tract.
The elimination half-lives indicate that C60is completely elim-inated from blood 97 h af ter administration irrespective of theroute of administration. The difference in the elimination half-livescould be attributed to some precipitation of C60in the injection sitefollowed by a slow dissolution of C60crystals in the surroundingtissue ﬂuid. Consequently, the AUC after i.p. administration repre-sents the soluble fraction only. Nevertheless, the precipitatedfraction is likely very weak because the total elimination is onlyslightly delayed. The precipitation phenomenon is unlikely to occurfor the oral route where the absorbed dose is about 5 times smallerand where C60is carried by lipoproteins.
The elimination process follows a non-urinary route becauseunmodiﬁed C60was not detected in urine samples taken up 48 hafter administration. Previous investigations showed that C60ismainly eliminated through the bile ducts  as it has been recently conﬁrmed . Besides, a small increase in C60concen-trations at 12 and 24 h after i.p. administration (Fig. 1) suggests thepresence of an enterohepatic circulation . Furthermore, it hasbeen already shown that C60reacts inside the liver cells withvitamin A following a DielseAlder like reaction both in mice and inrats [21,42]. These two routes may be sufﬁcient for C60elimination,nevertheless, we have to look for other possible biotransformationsand elimination routes, all the more so as the fate of the additionproduct is not known.
4.2.2. Biodistribution studies
As C60and some of its derivatives mainly accumulate in thelivers and spleens of rodents [15,21,42] we studied the bio-distribution of C60in these organs. To investigate its effects at reiterated doses we also studied the accumulation of C60in theseorgans after 7 successive days of administration.
The differences in C60contents in livers and spleens (Table 2)can be obviously assigned to the differences in the absorbed doses.However, the delay of elimination which is somewhat larger afteri.p. administration could also play a non negligible role. The pres-ence of C60crystals inside the cells after i.p. administration (Fig. 2)supports the hypothesis according to which the precipitation ofpart of the administered C60in the injection site may contribute tothe observed delay of elimination after i.p. administration. Never-theless, the weakness of organ concentrations notably at D8after 7daily successive administrations of C60dissolved in olive oil clearlyshows that C60molecules are eliminated from the organs in a fewhours after both oral and i.p. administrations.
Previous results obtained after i.p. administration of large dosesof micronized C60 or intratracheally instilled C60suspensions showed that the clearance of C60from organs can take severaldays. These longer delays of eliminations are likely due to the slowdissolution of C60crystals inside the organ RESs [21,43]. In the caseof tail vein administration  it is difﬁcult to compare the databecause C60was complexed with liposomes. The scarceness of C60crystals inside lung and kidney cells (Fig. 2) conﬁrms the differencein behaviour of C60-liposome complexes, which mainly accumu-lates in lungs after tail vein administration .
As C60contents in lungs and kidneys are likely weaker thanthose in livers and spleens, we only focused on C60content in brainsbecause the issue of its translocation to the brain is still a matter ofdebate [25,43].
Whereas C60particles were not detected in the brain afterintratracheal instillation , the presence of signiﬁcant amountsin the brain 24 hours after both oral and i.p. administrations underour experimental conditions (Table 2) conﬁrms that solubilized C60can cross the bloodebrain barrier .
A complete biodistribution study including intestine, skin, boneand fatty tissue is in progress in our laboratory.
4.3. Chronic toxicity and effects of C60on survival of treated rats
As C60is absorbed after o.g. we designed a protocol to study itschronic toxicity according to the general guidelines of US FDA with some modiﬁcations. C60has no acute or sub-acute toxicity inrodents [5,15] as it was further conﬁrmed in various experimentalmodels [16e19]. As it can act as an antioxidant (5, 6, 21), weinvestigated its chronic toxicity concomitantly with its effects onthe survival of rats.
Ten-month old male rats (M10) were chosen instead of young ratsas ofﬁcially recommended , in order to avoid possiblecompensatory effects that can occur during early development .As biodistribution studies after daily gavages showed that C60accumulates in livers and spleens, in order to avoid the negativeeffects of prolonged olive oil administration such as obesity, exces-sive steatosis, liver lipid degeneration, and insulin resistance ,we treated the rats daily only during 7 days and weekly during theﬁrst two months, then every two weeks until one control rat died.
Our results show that while olive oil treatment can lead to anincrease of 18% of lifespan of treated rats, C60-olive oil can increaseit up to 90%, as compared to controls. The effects of olive-oil onhealth and ageing are well known , and its effect as a function ofdose has been thoroughly discussed . But, what is noteworthy isthat at M38all C60-treated rats were still alive. Thus, based onprevious investigations ,C60should be the most efﬁcient evermaterial for extending lifespan.
Signiﬁcantly weaker similar effects have already been reportedin several experimental models but for different hydrosoluble C60-derivatives [44,47]. The effects of C60-derivatives on ageing wereattributed to the antioxidant properties and the attenuation of age-associated increases in oxidative stress [4,44].
Actually, the free-radical scavenging effect remains valid fora number of C60-derivatives with different addends [3,4,44,47e49],which indicates that this property is related to the C60moiety.Indeed C60itself is a powerful antioxidant as demonstrated indifferent experimental models [5,6,21].
As our results show that C60is more efﬁcient than its derivatives[44,47], they conﬁrm that the effects of C60-derivatives on ageing are mainly due to the C60moiety, as it has been postulated previ-ously .
This is the ﬁrst investigation of the in vivo chronic effects ofa soluble form of C60. The absorbed doses are very low and theirefﬁcacy on oxidative stress can be questioned. We already showedthat C60is a powerful antioxidant in a classical in vivo experimentalmodel in rats . But we then used an aqueous suspension and themost efﬁcient doses were about 2500 times higher (2 g/kg bw),which are considerably higher than those observed for its deriva-tives as well as those used for most biomedical applications. Inaddition there was a latency period (14 days after administration)to reach the maximum efﬁciency. It was stated that there was nocorrelation between the degree of protection and the number of C60clusters observed in the livers, suggesting that C60is active only ina soluble form that is when its double bounds are accessible .
To check this hypothesis we studied the effects of C60-olive oilsolutions in the same experimental model.
4.4. Effects of C60-olive oil solutions on oxidative stress
As we wrote before, four possible mechanisms for C60-liverprotection were proposed : (1) C60can scavenge large numbersof free radicals [5,6,21]; (2) it can act as a decomposition catalyst forO2/H2O2, as it has been postulated for its tris-malonic acidderivatives  or (3) as a cytochrome P450 inhibitor as it has beenreported for some fullerene derivatives ; and (4) it can inacti-vate Kupffer cells (liver resident macrophages) through accumu-lation and overloading with a large number of C60aggregates .
Biodistribution studies (Table 2) show that C60-liver concen-trations after seven successive daily o.g. or i.p. administrations of4 mg/kg bw of C60-olive oil solution are nearly 7 times lower or 1.5times higher, respectively, than those observed in previous studiesat 14 days, after i.p. injection of large doses (2 g/kg bw) of C60, whenthe optimum hepatoprotective effect was obtained . Thus, inorder to study the effects of C60-oil solutions on CCl4toxicity, wepre-treated the animals daily for 7 days by i.p. or o.g. administra-tions before CCl4treatment.
Pathological examinations show that even at very low doses,500 times lower than that used previously ,C60-olive oil solu-tions effectively protects the livers against CCl4toxicity. These results are in agreement with those reported for very low doses ofwater solution of hydrated C60fullerene in other experimentalmodels [5,6].
The number of necrotic areas observed in the liver sections ofGEipanimals was signiﬁcantly lower than that observed in the GEoggroup (Fig. 4). As C60concentrations in the livers of GEipanimals areprobably about 10 times higher than those of the livers of GEoggroup (Table 2), these results con ﬁrm the doseeeffect relationshipreported previously .
The results obtained for liver injury biomarkers (Fig. 5) are evenbetter than those obtained after administration of a large dose (2 g/kg bw) of C60suspended in aqueous medium . These resultsconﬁrm the hypothesis according to which this fullerene is activeagainst oxidative stress only in soluble form . In addition, whilethe median of ALT in the GDoganimals orally treated with C60-oilwas equivalent to that of the control group, the activity of thisenzyme was even lower in the GDipanimals. These resultsstrengthen the doseeeffect relationship and conﬁrm that C60isa powerful liver-protective agent.
As to the involved mechanism, since the doses used in theseexperiments are very low and since there are no excessive C60deposits inside Kupffer cells (hepatic macrophages), the hypothesisaccording to which C60can inactivate these phagocytes by over-loading them  must be discarded.
The initial liver damage after CCI4administration is mediatedthrough its metabolism by cytochrome P450 2E1 (CYP 2E1) resultingin the formation of the trichloromethyl radical CCl3[28,29].Thisradical can also react with oxygen to form a highly reactive species,the trichloromethyl peroxy radical CCl3OOwhich can rapidlyinitiate the chain reaction of lipid peroxidation [28,29].AsC60is ableto scavenge in vitro a large number of radicals per molecule including CCl3and CCl3OO and because this property can beinvolved in the mechanism of protection against CCl4toxicity, weexplored the effects of C60on the antioxidant systems that playa critical role in the defence against oxidative stress, including thecirculating levels of reduced (GSH) and oxidised forms (GSSG) ofglutathione as well as catalase (CAT) and superoxide-dismutase(SOD) activities in erythrocytes and livers .
The results obtained for the glutathione system (Fig. 5) conﬁrmthe antioxidant effect of C60and even its modulating effect on theintracellular redox status even in the absence of CCl4. Theresults obtained for the antioxidant enzyme activities (Fig. 6) alsoconﬁrm the antioxidant effect of C60against CCl4toxicity.
The suppression of CYP2E1 activity can result in a reduction inthe level of the resulting CCl4reactive metabolites, and, corre-spondingly, a decrease of tissue injuries. Therefore, a possiblemechanism for the liver-protection by C60may involve CYP2E1inhibition. As C60is insoluble in the usual biological systems used tostudy the in vitro activity of this enzyme, we used the hepaticmicrosomal fractions of orally treated rats to assay the CYP2E1-speciﬁc oxidative activity p-nitrophenol hydroxylase  in orderto check this hypothesis. C60-pretreatment signiﬁcantly attenuatedthe increase of CYP2E1 activity after CCl4intoxication withoutinhibitory effect on this enzyme (Fig. 7). The absence of inhibitoryeffects is reﬂected in the presence of a residual activity signiﬁcantlyhigher than that of the control group. Besides, the activity of thecontrol group treated with C60only is not signiﬁcantly different from the control group treated with water only. Therefore thehypothesis of CYP2EI inhibition by C60must also be discarded.
The results of the present study, notably the prevention of GSHdepletion, rather suggest that the protective effect involves a free-radical scavenging mechanism.
It has been recently reported that administration of C60sus-pended (not dissolved) in corn oil by oral gavage can increase thehepatic level of 8-oxodG whereas corn oil per se generated moregenotoxicity than the particles . Surprisingly, the authors didnot conclude that C60may prevent the genotoxicity of the usedvehicle.
It is to be stressed that dissolved C60appears hundred timesmore active than when it is in suspension . In fact the action ofsoluble C60is immediate while that of suspended C60is delayedbecause it has to be dissolved to act. In all cases, based on C60-livercontent (Table 2), these results prove that this fullerene is active atthe nano molar level. However, the involved mechanism remains tobe established.
For the time being the hypothesis of free-radical scavenging ofCCl3Oor CCl3COOremains possible. C60could act as a free-radicalscavenger as it has been widely demonstrated in vitro, but up tonow no resulting C60adduct has been observed in vivo. The onlyin vivo reaction ever observed for C60is a DielseAlder like reactionwith retinol and retinyl-esters inside the liver cells .Wearepresently trying to detect some C60-adducts resulting froma possible radical addition.
C60could also act as a superoxide-dismutase mimetic as it hasbeen modelized in silico and experimented in vitro for one of itswater-soluble derivatives . However, our results show that this isunlikely for pristine C60because the increase of H2O2concentrationdue to such activity should induce a correlated increase of catalaseactivity, which is not the case under our conditions (Fig. 6). Alternatively, C60could act as superoxide/catalase mimetics in vitro, but this is not the case in vivo.
Another possible mechanism has been also proposed for watersolutions of hydrated C60fullerene . It suggests that the struc-tured water layer around C60can be able to deactivate hydroxylradicals by allowing recombination to hydrogen peroxide. Onceagain, this mechanism remains to be conﬁrmed by means of otherexperiments.
The effect of pristine C60on lifespan emphasizes the absence ofchronic toxicity. These results obtained with a small sample ofanimals with an exploratory protocol ask for a more extensivestudies to optimize the intestinal absorption of C60as well as thedifferent parameters of the administration protocol: dose, posol-ogy, and treatment duration. In the present case, the treatment wasstopped when a control rat died at M17, which proves that theeffects of the C60treatment are long-lasting as the estimatedmedian lifespan for C60-treated rats is 42 months. It can be thoughtthat a longer treatment could have generated even longer lifespans.Anyway, this work should open the road towards the developmentof the considerable potential of C60in the biomedical ﬁeld,including cancer therapy, neurodegenerative disorders and ageing.Furthermore, in the ﬁeld of ageing, as C60can be administeredorally and as it is now produced in tons, it is no longer necessary toresort to its water-soluble derivatives, which are difﬁcult to purifyand in contrast to pristine C60may be toxic.
This work was partially supported by the CMCU grant (Ref. N:ST/AM/GM/4C5 001/n1233. Cote: 6.2.2).
We thank Prof. Stephen R Wilson for his valuable comments.
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