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 characterising 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, bio-distribution, 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 characterised 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 CCl
4 intoxication is unlikely to occur during physiopathological conditions, CCl4 intoxication 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 oxidative 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 Tunisia planted in the Sahel area. C60 was obtained from USA and used without further purification. 50 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 for 1 h and the supernatant was filtered through a Millipore filter with 0.25 μm 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 (weighing 200–220 g). Rats were housed in individual cages and maintained in an air-conditioned room (22–25 ºC) 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 containing 20 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 = 0) and at 15, 30, 60 min and then at 2, 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
dministration for organ collection (livers, spleens, and brains). Urines were collected at 24 h and 48 h after C60 administration then frozen at -20º Cuntil analysis.
For biodistribution studies, 4 groups of 3 rats (weighing 200 ± 20 g) were treated daily for 7 days either by i.p. administration (2 groups) or oral gavage (2 groups) with the same dose (4 mg/kg bw) of the same C60-oil solution (0.8 mg/ml). At day 1 (D 1), 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 cage once 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 7 days by oral gavages (og groups) or by i.p. injection (ip groups). Groups A (GAog and GAip), 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 C60 in blood, urine, liver, spleen and brain were performed 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 UV – Visible spectra and the traces were recorded at 330 nm. Separations were carried out with a Hypersil 120-5 ODS, 5 μm
cartridge (Macherey–Nagel, Hoerdt, France) protected with a 4.0 mm x 10 mm pre-column packed with the same stationary phase.
For liver and spleen samples, separations were performed at 25 ºC with a flow rate 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 of each sample run. At least 10 column volumes of the initial composition were flushed through the column prior to injecting the sample.
2.5.2. Sample preparation
For whole blood, one hundred ml of sample were diluted in 400 μl of 0.1 M sodium dodecyl sulfate (SDS). After adding 0.5 ml of acetonitrile and shaking for 5 min, C60 was extracted by adding 5 ml of toluene containing 0.2 μg/ml of C70 used as 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 100 μl 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 C18 cartridge (Waters, St Quentin en Yvelines, France) prealably conditioned with 5 ml of a mixture of water/acetonitrile (10/2, v/v). After washing the C18 cartridge with 5 ml of acetonitrile, the retained compounds were eluted with 2 ml of toluene containing 0.2 μg/ml of C70 and evaporated under a stream of nitrogen. The residue was then dissolved in 0.1 ml of toluene and diluted in acetonitrile (1/1, v/v) before injection of 100 μl 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 2.0 μg/ml of IS were added and the mixture was shaken 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 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 of 100 μl 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 1.0 μg/ml under gradient elution (y = 0.5963 x + 0.0006; n = 6; where y is the peak area in AU min and x is the concentration of the injected solution in μg/ml; the relative standard deviations (RSDs, n = 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 was 0.001μg/ml.
Under isocratic conditions, the linearity of the method was checked between 0.01 and 10.0 μg/ml (y = 0.597 x + 0.0098; n = 7; where y is the peak area in AU.min and x is the concentration of the injected solution in μg/ml; the RSDs (n = 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 0.002 μg/ml. The between run (BWR) and between day (BWD) precisions were determined (n = 6) for the lowest and the highest level of each curve of calibration.
Under gradient elution conditions the RSDs were 7.2% and 10.5% for the BWR and 5.3% and 8.4% for the lowest levels and the highest levels, respectively. Under isocratic conditions, the RSDs were 5.6% and 8.5% for the BWR and 3.3% and 6.4% for the lowest levels and the highest levels, respectively.
The recovery of the method was determined for each kind of sample at two levels (n = 3, for each level). For whole blood, urine, and brain samples the recoveries were determined at 0.01 and 0.05 μg/ml or μg/g, respectively and they were 94.3 ± 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 30 μg/g and the recoveries were 97.3 ± 2.8% and 99.1 ± 2.2%, respectively. For spleen samples the levels were 2.0 and 200 μg/g and the 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, and oxidized glutathione/total glutathione (GSSG/TGSH) ratio, where TGSH is the sum of reduced (GSH) and oxidized glutathione (GSSG), were performed as previously described .
Superoxide-dismutase (SOD) and catalase (CAT) activities were determined as previously described [31, 32].
Hepatic microsomal fractions were used for measuring the cytochrome P4502E1 (CYP2E1) specific oxidative activity such as p-nitrophenol hydroxylase. The hepatic microsomal fractions were prepared by differential centrifugation, as described previously  and were stored at -80 ºC until required. The hydroxylation of p-nitrophenol to 4-nitrocatechol was determined by HPLC as described previously . Microsomal protein concentration was determined by the Bradford method , using bovine serum albumin as a standard.
Pathological examinations and optical microscopy analyses were blindly performed by a pathologist ignoring all protocol procedures as well as the purpose of the study. The reparation and staining protocols of organ pieces for optical and transmission electron microscopy (TEM) were performed as described previously .
2.7. Pharmacokinetic analysis
Pharmacokinetic analysis of the individual observed rat plasma data obtained after oral and i.p. routes was performed using the WinNonLin® software (Pharsight Corporation, Mountain View, California). A non-compartmental approach was used to calculate the main pharmacokinetic parameters.
The maximal plasma concentration (Cmax) and the time (Tmax) to reach Cmax were obtained directly from experimental observations. The terminal elimination rate constant (lz) was calculated by linear regression analysis of the natural logarithm of the last experimental concentrations and the terminal half-life (t1/2) was calculated by
dividing Ln2 by lz. The area under the plasma concentration-time curve from zero to infinity (AUC f0) was the addition of AUC from zero to the last experimental concentration (CT), calculated by the trapezoidal rule, and of AUC from CT to infinity, calculated by dividing CT by Àz. The area under the first moment curve from zero to infinity (AUMC f0) was the addition of AUMC from zero to the last experimental concentration (CT), calculated by the trapezoidal rule, and of AUMC from CT to infinity, calculated by [((CT.T)/Àz) + (CT/lz2)]. The mean residence time (MRT) was calculated by dividing AUMC f0 by AUC f0. The apparent plasma clearance (Cl/F) was calculated by dividing the dose by AUC f0, and the apparent volume of distribution (Vd/F) was calculated by dividing the dose by (AUC f0 f0.Àz).
The normality of data distribution was tested by Shapiroe–Wilk test. Data are presented as the mean and standard deviation in the case of normal distributions or as the median and the range. Comparisons with control were performed by using Student test, according to the homogeneity of variances determined by Fisher test, or by Manne–Whitney test. A value of P < 0.05 was considered statistically significant. The survival distributions for C60-olive oil-treated and control rats were estimated by the non-parametric Kaplane–Meier estimator and compared by a log-rank estimated test.
3.1. C60-olive oil preparation
The composition and quality characteristics of olive oil were determined as previously described following analytical methods described in the EEC 2568/91 and EEC 1429/92 European Union Regulations .
The resulting C60-olive oil solution is purple and contains 0.80 ± 0.02 mg/ml (n = 6) as determined by HPLC  after appropriate dilution in the mobile phase. The chromatographic profile and the extracted spectra of these solutions are similar to those obtained with a control C60-toluene equimolar solution.
The stability of both oily and control solutions stored at ambient temperature and in the dark was checked monthly during 48 months. No change was recorded under our chromatographic conditions.
3.2. Pharmacokinetics and biodistribution
Fig. 1 represents the evolution of whole blood C60 concentrations versus time following single dose o.g. and i.p. administration of the same dose of C60 dissolved in olive oil. Table 1 summarises the main pharmacokinetic parameters. The maximal concentrations (Cmax) are reached 4 and 8 h after i.p. and o.g. administrations, respectively.
The apparent volume of distribution (Vd/F) of C60 after i.p.administration is higher than the blood volume in rats , indicating that C60 is well distributed in tissues. The value of Vd/F after o.g. is less significant because the administered dose cannot be ponderated by the C60 bioavailability, which is unknown (Table 1).
Table 1 Pharmacokinetic Paramenters[/caption]The elimination process is slower after i.p. administration than after o.g., as illustrated by the elimination half-lifes and the mean residence times of C60 (Table 1).