The prolongation of the lifespan of rats by repeated oral administration of [60]fullerene

Abstract

Countless studies showed that [60]fullerene (C₆₀) and derivatives could have many potential biomedical applications. However, while several independent research groups showed that C₆₀ has no acute or sub-acute toxicity in various experimental models, more than 25 years after its discovery the in vivo fate and the chronic effects of this fullerene remain unknown. If the potential of C₆₀ and derivatives in the biomedical field have to be fulfilled these issues must be addressed. Here we show that oral administration of C₆₀ dissolved in olive oil (0.8 mg/ml) at reiterated doses (1.7 mg/kg of body weight) to rats not only does not entail chronic toxicity but it almost doubles their lifespan. The effects of C₆₀-olive oil solutions in an experimental model of CCl₄ intoxication in rat strongly suggest that the effect on lifespan is mainly due to the attenuation of age-associated increases in oxidative stress. Pharmacokinetic studies show that dissolved C₆₀ is absorbed by the gastro-intestinal tract and eliminated in a few tens of hours. These results of importance in the fields of medicine and toxicology should open the way for the many possible -and waited for- biomedical applications of C₆₀ including cancer therapy, neurodegenerative disorders, and ageing.

Introduction

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 radioprotection [3], antiviral, antioxidant, and anti-amyloid activities [1], [4], [5], [6], [7], allergic response [8] and angiogenesis [9] inhibitions, immune stimulating and antitumour effects [10], [11], enhancing effect on neurite outgrowth [12], gene delivery [13], and even hair-growing activity [14]. However, although several independent research groups confirmed the innocuousness of pristine C₆₀ [15], [16], [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 characterisation of the tested materials [15], [16], [17], [18], [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 biodistribution 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 characterizing 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, 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 body weight (mg/kg bw)).

Finally, as C₆₀ is known to be a powerfull 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, 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].