C60 RESEARCH

Since 1993 countless studies showed that [60] fullerene (C60) and derivatives exhibit paramount potentialities in several fields of biology and medicine [1] mainly including specific DNA cleavage, imaging [2], UV and radio protection [3], antiviral, antioxidant, and anti-amyloid activities [1,4–7], allergic response [8] and angiogen-esis [9] inhibitions, immune stimulating and anti-tumour effects [10,11], enhancing effect onneurite outgrowth [12], gene delivery [13], and even hair-growing activity [14]. However, although several independent research groups confirmed the innocuousness of pristine C60 [15–17] 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 [15–19]. Nevertheless, the metabolic fate and the in vivo chronic effects of C60 itself still remain unknown. In order to fulfil the potential of C60 and 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 [21] and because the extremely slow dissolution of C60 in biological media prevents controlling accurately the active fraction [21]. This may be the reason for which the chronic toxicity of C60 has never been investigated to our knowledge.

C60 is soluble in lipid droplets inside living cells [21] as well as in fats in general [22, 23]. Moreover, C60 can freely cross membrane barriers as observed experimentally [21] and recently modelled by computer simulations [24]. 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 [25]. 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 [25].

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) [26], such solutions should be quite appropriate for studying its chronic toxicity at reiterated doses [27].

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].

c60 research – 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.

c60 research – 2.2.Pharmacokinetics and biodistribution studies

All experimental procedures were reviewed and approved by the Animal Experimentation Ethics Committee of Paris XI University.

c60 research – 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 [27] 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.

c60 research – 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.

C60 RESEARCH – 2.5. Chromatographic analyses, sample preparation and method validation

c60 Research – 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 [30].

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 [33] and were stored at -80 ºC until required. The hydroxylation of p-nitrophenol to 4-nitrocatechol was determined by HPLC as described previously [46]. Microsomal protein concentration was determined by the Bradford method [34], 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 [21].

c60 Research – 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).

c60 research – 2.8. Statistics

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.

C60 RESEARCH – 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 [35].

The resulting C60-olive oil solution is purple and contains 0.80 ± 0.02 mg/ml (n = 6) as determined by HPLC [30] 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.

C60 RESEARCH – 3.2. Pharmacokinetics and biodistribution

C60 RESEARCH – 3.3. Chronic toxicity and effects of C60 on lifespan of rats

Fig. 3 shows the animal survival and growth. After five months of treatment (M15) one rat treated with water only exhibited some palpable tumours in the abdomen region. Due to the rapid development of tumours (about 4 cm of diameter) this rat died at M17. As rats are known to be sensitive to gavages, we decided to stop the treatment for all rats and to observe their behaviour and overall survival.

All remaining animals survived with no apparent sign of behavioural trouble until M25 (Fig. 3a). At the end of M25 the animals of the control groups showed signs of ulcerative dermatitis with ageing while C60-treated animals remained normal. As the growths of all surviving animals showed no significant difference until M30 (Fig. 3b) indicating that the treatment did not alter their food intake, we continued observing their survival.

At M38 all water-treated control rats were dead (Fig. 3a). This agrees with the expected lifespan of this animal species that is thirty to thirty six months. At this time 67% of olive-oil-treated rats and 100% of C60-treated rats were still alive.

The survival distributions for C60-olive oil-treated rats and controls were estimated by the non-parametric Kaplane–Meier estimator (Fig. 3) and compared by a log-rank estimated test. The estimated median lifespan (EML) for the C60-treated rats was 42 months while the EMLs for control rats and olive oil-treated rats were 22 and 26 months, respectively. These are increases of 18 and 90% for the olive-oil and C60-treated rats, respectively, as compared to controls.

The log-rank test leads to X2 values (one degree of freedom) of 7.009, 11.302, and 10.454, when we compare water-treated and olive oil-treated rats, water-treated and C60-treated rats, and olive oil-treated and C60-treated rats, respectively. This means that olive oil extends the lifespan of rats with respect to water with a probability of 0.99 while C60-olive oil extends the lifespan of C60-treated rats with a probability of 0.999 and 0.995 with respect to water and olive oil treatments, respectively.

C60 RESEARCH – 3.4. Effect of C60-olive oil solutions on oxidative stress

CCl4 toxicity with respect to rats is well known [26, 27] nevertheless, we systematically studied the effects of this halo-alkane on the animals we used in our experiments in order to avoid misinterpretations due to inter-strain variability. In addition, to avoid errors due to inter-individual and inter-season variability, a CCl4-treated control group was included in each experiment.

C60 RESEARCH – 4.1. C60-olive oil solution preparation

It is well known that C60 and derivatives are prone to aggregate even in their best solvents [37]. The C60-olive oil solution used in this study can be considered as free of C60 aggregates because: 1 – its colour is purple that is characteristic of C60 solutions while the colour of C60 aggregate-containing solutions are rather brown, which is true even for water-soluble derivatives [3]; 2 – it is freely and instantaneously soluble in toluene in contrast to C60 aggregate-containing solutions, which slowly dissolve even in the best solvents of C60. Besides, the concentrations of C60 in olive oil as determined by HPLC agree with those previously published by other authors [22].

The stability of C60-olive oil solution determined under our experimental conditions agrees with recently published results showing that the addition of [60] fullerene significantly hampers the peroxide formation thus increasing the stability of the tested oils [38].

C60 RESEARCH – 4.2. Pharmacokinetics and biodistribution

C60 RESEARCH – 4.3. Chronic toxicity and effects of C60 on survival of treated rats

As C60 is absorbed after o.g. we designed a protocol to study its chronic toxicity according to the general guidelines of US FDA [27] with some modifications. C60 has no acute or sub-acute toxicity in rodents [5, 15] as it was further confirmed in various experimental models [16–19]. As it can act as an antioxidant (5, 6, 21), we investigated its chronic toxicity concomitantly with its effects on the survival of rats.

Ten-month old male rats (M10) were chosen instead of young rats as officially recommended [27], in order to avoid possible compensatory effects that can occur during early development [44]. As biodistribution studies after daily gavages showed that C60 accumulates in livers and spleens, in order to avoid the negative effects of prolonged olive oil administration such as obesity, excessive steatosis, liver lipid degeneration, and insulin resistance [45], we treated the rats daily only during 7 days and weekly during the first two months, then every two weeks until one control rat died.

Our results show that while olive oil treatment can lead to an increase of 18% of lifespan of treated rats, C60-olive oil can increase it up to 90%, as compared to controls. The effects of olive-oil on health and ageing are well known [46], and its effect as a function of dose has been thoroughly discussed [45]. But, what is noteworthy is that at M38 all C60-treated rats were still alive. Thus, based on previous investigations [44], C 60 should be the most efficient ever material for extending lifespan.

Significantly weaker similar effects have already been reported in several experimental models but for different hydro soluble C60-derivatives [44, 47]. The effects of C60-derivatives on ageing were attributed to the antioxidant properties and the attenuation of age-associated increases in oxidative stress [4, 44]. Actually, the free-radical scavenging effect remains valid for a number of C60-derivatives with different addends [3, 4, 44, 47–49], which indicates that this property is related to the C60 moiety. Indeed C60 itself is a powerful antioxidant as demonstrated in different experimental models [5, 6, 21].

As our results show that C60 is more efficient than its derivatives [44, 47], they confirm that the effects of C60-derivatives on ageing are mainly due to the C60 moiety, as it has been postulated previously [4].

This is the first investigation of the in vivo chronic effects of a soluble form of C60. The absorbed doses are very low and their efficacy on oxidative stress can be questioned. We already showed that C60 is a powerful antioxidant in a classical in vivo experimental model in rats [21]. But we then used an aqueous suspension and the most efficient doses were about 2500 times higher (2 g/kg bw), which are considerably higher than those observed for its derivatives as well as those used for most biomedical applications. In addition there was a latency period (14 days after administration) to reach the maximum efficiency. It was stated that there was no correlation between the degree of protection and the number of C60 clusters observed in the livers, suggesting that C60 is active only in a soluble form that is when its double bounds are accessible [21].

To check this hypothesis we studied the effects of C60-olive oil solutions in the same experimental model.

C60 RESEARCH – 4.4. Effects of C60-olive oil solutions on oxidative stress

As we wrote before, four possible mechanisms for C60-liver protection were proposed [21]: (1) C60 can scavenge large numbers of free radicals [5, 6, 21]; (2) it can act as a decomposition catalyst for O2*/H2O2, as it has been postulated for its tris-malonic acid derivatives [4] or (3) as a cytochrome P450 inhibitor as it has been reported for some fullerene derivatives [21]; and (4) it can in activate Kupffer cells (liver resident macrophages) through accumulation and overloading with a large number of C60 aggregates [2].

Biodistribution studies (Table 2) show that C60-liver concentrations after seven successive daily o.g. or i.p. administrations of 4 mg/kg bw of C60-olive oil solution are nearly 7 times lower or 1.5 times higher, respectively, than those observed in previous studies at 14 days, after i.p. injection of large doses (2 g/kg bw) of C60, when the optimum hepatoprotective effect was obtained [21]. Thus, in order to study the effects of C60-oil solutions on CCl4 toxicity, we pre-treated the animals daily for 7 days by i.p. or o.g. administrations before CCl4 treatment.

Pathological examinations show that even at very low doses, 500 times lower than that used previously [21], C60-olive oil solutions effectively protects the livers against CCl4 toxicity. These results are in agreement with those reported for very low doses of water solution of hydrated C60 fullerene in other experimental models [5, 6].

The number of necrotic areas observed in the liver sections of GEip animals was significantly lower than that observed in the GEog group (Fig. 4). As C60 concentrations in the livers of GEip animals are probably about 10 times higher than those of the livers of GEog group (Table 2), these results confirm the dose–effect relationship reported previously [21].

The results obtained for liver injury biomarkers (Fig. 5) are even better than those obtained after administration of a large dose (2 g/kg bw) of C60 suspended in aqueous medium [21]. These results confirm the hypothesis according to which this fullerene is active against oxidative stress only in soluble form [21]. In addition, while the median of ALT in the GDog animals orally treated with C60-oil was equivalent to that of the control group, the activity of this enzyme was even lower in the GDip animals. These results strengthen the dose–effect relationship and confirm that C60 is a powerful liver-protective agent.

As to the involved mechanism, since the doses used in these experiments are very low and since there are no excessive C60 deposits inside Kupffer cells (hepatic macrophages), the hypothesis according to which C60 can inactivate these phagocytes by over-loading them [21] must be discarded.

The initial liver damage after CCI4 administration is mediated through its metabolism by cytochrome P450 2E1 (CYP 2E1) resulting in the formation of the trichloromethyl radical CCl3* [28,29]. This radical can also react with oxygen to form a highly reactive species, the trichloromethyl peroxy radical CCl3OO* which can rapidly initiate the chain reaction of lipid peroxidation [28, 29]. As C60 is able to scavenge in vitro a large number of radicals per molecule [50] including CCl3* and CCl3OO* [51] and because this property can be involved in the mechanism of protection against CCl4 toxicity, we explored the effects of C60 on the antioxidant systems that play a critical role in the defence against oxidative stress, including the circulating levels of reduced (GSH) and oxidised forms (GSSG) of glutathione as well as catalase (CAT) and superoxide- dismutase (SOD) activities in erythrocytes and livers [29].

The results obtained for the glutathione system (Fig. 5) confirm the antioxidant effect of C60 and even its modulating effect on the intracellular redox status even in the absence of CCl4 [21]. The results obtained for the antioxidant enzyme activities (Fig. 6) also confirm the antioxidant effect of C60 against CCl4 toxicity.

The suppression of CYP2E1 activity can result in a reduction in the level of the resulting CCl4 reactive metabolites, and, correspondingly, a decrease of tissue injuries. Therefore, a possible mechanism for the liver-protection by C60 may involve CYP2E1 inhibition. As C60 is insoluble in the usual biological systems used to = study the in vitro activity of this enzyme, we used the hepatic microsomal fractions of orally treated rats to assay the CYP2E1-specific oxidative activity p-nitrophenol hydroxylase [33] in order to check this hypothesis. C60-pretreatment significantly attenuated the increase of CYP2E1 activity after CCl4 intoxication without inhibitory effect on this enzyme (Fig. 7). The absence of inhibitory effects is reflected in the presence of a residual activity significantly higher than that of the control group. Besides, the activity of the control group treated with C60 only is not significantly different from the control group treated with water only. Therefore the hypothesis of CYP2EI inhibition by C60 must also be discarded.

The results of the present study, notably the prevention of GSH depletion, rather suggest that the protective effect involves a free-radical scavenging mechanism. It has been recently reported that administration of C60 suspended (not dissolved) in corn oil by oral gavage can increase the hepatic level of 8-oxodG whereas corn oil per se generated more genotoxicity than the particles [52].

Surprisingly, the authors did not conclude that C60 may prevent the genotoxicity of the used vehicle. It is to be stressed that dissolved C60 appears hundred times more active than when it is in suspension [21]. In fact the action of soluble C60 is immediate while that of suspended C60 is delayed because it has to be dissolved to act. In all cases, based on C60-liver content (Table 2), these results prove that this fullerene is active at the nano molar level. However, the involved mechanism remains to be established.

For the time being the hypothesis of free-radical scavenging of CCl3O* or CCl3COO* remains possible. C60 could act as a free-radical scavenger as it has been widely demonstrated in vitro, but up to now no resulting C60 adduct has been observed in vivo. The only in vivo reaction ever observed for C60 is a Dielse–Alder like reaction with retinol and retinyl-esters inside the liver cells [42]. We are presently trying to detect some C60-adducts resulting from a possible radical addition. C60 could also act as a superoxide-dismutase mimetic as it has been modelized in silico and experimented in vitro for one of its water-soluble derivatives [4]. However, our results show that this is unlikely for pristine C60 because the increase of H2O2 concentration due to such activity should induce a correlated increase of catalase activity, which is not the case under our conditions (Fig. 6).

Alternatively, C60 could act as superoxide/catalase mimetics in vitro [4], but this is not the case in vivo. Another possible mechanism has been also proposed for water solutions of hydrated C60 fullerene [6]. It suggests that the structured water layer around C60 can be able to deactivate hydroxyl radicals by allowing recombination to hydrogen peroxide. Once again, this mechanism remains to be confirmed by means of other experiments.

The effect of pristine C60 on lifespan emphasises the absence of chronic toxicity. These results obtained with a small sample of animals with an exploratory protocol ask for a more extensive studies to optimise the intestinal absorption of C60 as well as the different parameters of the administration protocol: dose, posology, and treatment duration. In the present case, the treatment was stopped when a control rat died at M17, which proves that the effects of the C60 treatment are long-lasting as the estimated median lifespan for C60-treated rats is 42 months. It can be thought that a longer treatment could have generated even longer lifespans. Anyway, this work should open the road towards the development of the considerable potential of C60 in the biomedical field, including cancer therapy, neurodegenerative disorders and ageing. Furthermore, in the field of ageing, as C60 can be administered orally and as it is now produced in tons, it is no longer necessary to resort to its water-soluble derivatives, which are difficult to purify and in contrast to pristine C60 may be toxic.

Acknowledgements

This work was partially supported by the CMCU grant (Ref. No: ST/AM/GM/4C5 001/nº1233. Cote: 6.2.2).

We thank Prof. Stephen R Wilson for his valuable comments. Article Link:

https://www.researchgate.net/publication/224004891_The_prolongation_of_the_lifespan_of_rats_by_repeated_oral_ administration_of_60fullerene

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