Update on C60 fullerenes in olive oil

By Vince Giuliano and James P Watson

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More than a year has passed since publication of the November 2012 blog entry Buckyballs, health and longevity – state of knowledge, It will be another year or two before we know whether researchers are capable of reproducing the extraordinary longevity impacts on rats reported in the 2011 publication The prolongation of the lifespan of rats by repeated oral administration of [60] fullerene.  We don’t yet know a number of things we would love to know: 1.  Whether indeed it is verified that “buttered buckyballs” can extend the lives of rats?, 2. if so, can they extend the healthspans and lives of humans?, and 3. exactly what molecular mechanisms would be involved in such lifespan extension?.  Because rats normally live 2+ years, it will be at least another year before we start getting answers to the first question.  Because we live 80+ years it may be decades before we start getting answers to the second question.  But we can continue seriously discussing and speculating about the third question now, and that is the focus of this blog entry.  In particular, a new mechanism is proposed for how C60 can possibly drastically reduce ROS and its pro-aging consequences.

Readers are referred to the earlier blog entry for in-depth background.  Although there have been no further publications empirically relating C60 fullerenes (“buckyballs”) to mammalian longevity, several relevant research publications have appeared since.  Some of these are reviewed here along with several relevant topics not previously well-covered.

1.   C60 might significantly reduce reactive oxygen species produced by leaky mitochondria and associated cell damage, but not by a direct antioxidant effect.

In our minds, this is probably the central new finding.  We have written previously about how “old” and “tired” mitochondria produce damaging ROS(ref)(ref), the Warburg effect and a negative cycle of activities that can lead to cancer.   An important recent paper by David Sinclair and his associates describes how such ROS can be induced by age-related absence of sufficient NAD+ in the cell nucleus leading to a state of pseudohypoxia and insufficient expression of mitochondrially-coded protective genes(ref).  DNA damage triggers the DNA damage response (ddr) which in turn triggers p53 activation which can then inhibit PGC-1b, resulting in the inhibition of gene transcription for nuclear-encoded mitochondrial proteins.  Thus, this mechanism can induce high mitochondrial ROS generation, Warburg-type metabolism, increased oxidative stress leading to further negative effects such as more DNA damage.  DNA damage can then lead to PARP-1 activation, which then can deplete nuclear NAD+, which then will stop SIRT1, SIRT6, and SIRT7 function.  This then leads to a pseudohypoxic state of the nucleus due to HIF-1a stabilization.  This in turn leads to inadequate TFAM, which then results in inadequate expression of mitochondrially-encoded proteins for electron transport.  Thus, mitochondrial dysfunction can be triggered by telomere-dependent, DDR-mediated activation of p53.

It has previously been speculated that C60 buckyballs penetrate the cell and mitochondrial membranes and exercise an antioxidant ROS-quenching effect upon arriving within the mitochondria.  C60 acts as a radical “sponge.”(ref)  This may well be the case, but the cytoprotective effect of the EVOO buckyballs against carbon tetrachloride reported in the 2011 rat longevity study appears to be extraordinary for a simple antioxidant.  Another very interesting hypothesis has been suggested in 2013. That hypothesis is that C60 serves to drastically reduce the production of the superoxide radical via reducing the electric potential across mitochondrial membranes. .Instead of cleaning up a dangerous superoxide radical via an antioxidant, this mechanism, would serve to prevent creation of that radical in the first place.

The argument is laid out in the 2013 publication Possible Mechanisms of Fullerene C60 Antioxidant Action. “Novel mechanism of antioxidant activity of buckminsterfullerene C60 based on protons absorbing and mild uncoupling of mitochondrial respiration and phosphorylation was postulated. In the present study we confirm this hypothesis using computer modeling based on Density Functional Theory. Fullerene’s geroprotective activity is sufficiently higher than those of the most powerful reactive oxygen species scavengers. We propose here that C60 has an ability to acquire positive charge by absorbing inside several protons and this complex could penetrate into mitochondria. Such a process allows for mild uncoupling of respiration and phosphorylation. This, in turn, leads to the decrease in ROS production.”

According to the 2013 publication Feasibility of the C60 Fullerene Antioxidant Properties: Study with Density Functional Theory Computer Modeling: “Fullerene C60 compound was recently found to be a potent anti-oxidant, which may be envisioned as a result of alteration of the inner mitohondria membrane electric potential with protons transport boosted by fullerenes. Here we briefly report on the theoretical test of the very possibility of protons to pass through the surface of C60 fullerene to become confined within latter thus possibly decreasing the transmembrane electric field gradient when fullerene crosses the mitochondria membrane. Quantumchemical calculations within Density Functional Theory are employed as a means of checking described scenario.” — “Fullerene C60 may be a powerful antioxidant demonstrating anti-aging activity. Recently Baati et al. showed that fullerene prolonged rat’s life span approximately twice [1]. Besides, rats treated with fullerene C60 demonstrated high resistance to carbon tetrachloride capable of triggering generation of huge amounts of harmful reactive oxygen species.  Consequently, fullerene C60 was proposed to have high antioxidant activity in vivo. Geroprotective activity of C60 fullerene found experimentally in [1] is much higher than those of the most powerful reactive oxygen species scavengers. Reactive oxygen species may cause oxidative damage.  Geroprotective activity of C60 fullerene found experimentally in [1] is much higher than those of the most powerful reactive oxygen species scavengers.”

As I understand it, the process goes like this:

1.  The outer mitochondrial membrane is charged positively and the inner membrane is charged negatively.   This is because free electrons are spun off in the complexes in the electron transfer chain.  As the chain becomes less efficient and there is decline in expression of mitochondrial antioxidants, the more there is a charge differential. “Accumulation of Skulachev ions in the mitochondria is based on the transmembrane potential difference generated as a result of electron transport chain activity. The outer side of inner membrane of mitochondria has positive charge and the inner side has negative charge.”  The actions of the electron transport chain involve four complexes, and you can get a fair idea how they work by viewing one of these animations.

2.  Superoxide is created as a result of the cross-membrane charge differential, the amount being a nonlinear function of the differential. “The specific feature attributable to the generation of ROS by mitochondria is related to the fact that the higher is the membrane potential (the larger is the difference in the concentration of protons inside and outside the mitochondria), the higher is the level of the superoxide anion production. As it was shown [29], there is steep dependence of mitochondrial superoxide-anion-radical generation on transmembrane potential (Δ). Even a small (10–15%) decline of Δ resulted in tenfold lowering of ROS production rate.”

3.  In the case of C60, the modeling studies suggest that the fullerenes are initially electrically neutral and penetrate easily to the center of the mitochondrial membranes. “Wong-Ekkabut et al. showed using molecular dynamics simulations [20] that C60 fullerene is capable of penetrating into membrane and accumulates in the middle of lipid bilayer.”

4.  There, a fullerene may pick up as many as six protons, according to the quantum wave-function model.

5.  The electrical gradient then sucks the positively charged fullerene across the inner mitochondrial membrane into the interior of the mitochondria.  “DFT simulations allowed us to propose the following mechanism. C60 fullerene molecules enter the space between inner and outer membranes of mitochondria, where the excess of protons has been formed by diffusion. In this compartment fullerenes are loaded with protons and acquire positive charge distributed over their surface. Such “charge-loaded’’ particles can be transferred through the inner membrane of the mitochondria due to the potential difference generated by the inner membrane, using electrochemical mechanism described in detail by Skulachev et al. [18,24]. In this case the transmembrane potential is reduced, which in turn significantly reduces the intensity of superoxide anion-radical production.”

6.  Once a positively charged fullerene is inside the mitochondria, the charge differential between the inner and the outer membrane is automatically reduced.

7.  A net result is that a relatively small reduction in the charge differential can essentially turn down or turn off the production of superoxide.

8.  This mechanism is completely independent of any antioxidant properties of the fullerene itself.  Basically, the superoxide production is reduced through the C60 being in the role of a charge transporter.

9.  If the fullerene has an antioxidant property once inside the mitochondria, that is an additional mechanism for reduction of ROS.

We note that a similar mechanism of action may explain the effectiveness of MitoQ which is commonly called a “mitochondrial antioxidant.”  MitoQ is designed to be a molecule having a powerful positive charge, consisting of the cation triphenylphosphonium (TPP) covalently bonded to coenzyme Q-10.    The result is a positively charged molecule.  It is thought that the charge is sufficient to propel the molecule through the mitochondrial membrane into the interior of the mitochondria.   Once inside a mitochondrion, as in the case of a C-60 fullerene, a MitoQ molecule reduces the charge differential across the mitochondrial membrane.  This again should result in reduction of superoxide production.  If the Q-10 moiety has an antioxidant function on Complex 2, that too enhances the antioxidant impact.

Similarly, triphenylphosphonium has been used tp target nitroxide radicals to mitochondria to protect against radiation exposure(ref).

So, C60 and MitoQ “antioxidants” don’t have to be antioxidants to work!  If these models are correct, healthily-functioning mitochondria would automatically import less of C60 or MitoQ than mitochondria with leaky electron transport chains because the charge attraction would be less.

On first blush, the mechanism just described can possibly explain what seems to be a strange phenomenon observed in the famed rat study.  EVOO far outlasts the period of supplementation.    In the chronic C60 toxicity study that was done just to evaluate the “toxic” effects of C60 fullerenes, the C60 fullerenes in olive oil were initially administered every day at a dose of 4mg/kg body weight.   However, after 7 days, the dose was reduced to weekly for the next two months.  After that, doses were only given once every two weeks until the first rat died. Then no more C60 fullerenes were given.  This amounted to 17 months of C60 fullerene treatment, then no C60 was administered for the rest of the rat’s lives.  The rats who received C60 fullerene in olive oil lived as long as 66 months, which was 90% longer than the control group, which lived 14-38 months. Thus the C60 fullerenes had a prolonged effect that lasted for years after the last dose of C60 fullerenes was given.  membrane waiting to come to its rescue and, because of its health due to control of ROS, be relatively long lasting.

This is a very nice conjecture except for one quite serious difficulty – which is that the half lives of rat mitochondria themselves are very short – up to a few weeks at most(ref).  So, the explanation would only work if either the fullerenes convey extraordinary longevity to mitochondria or if fullerenes in the membrane of a mitochondrion undergoing mitophagy are recycled into new mitochondria.

2.   There appears to be continuing concern that C60 may induce DNA/RNA damage and other forms of damage

This continues to be an important point though it was initially covered in the earlier blog entry.

The 2012 Publication A large-scale association study for nanoparticle C60 uncovers mechanisms of nanotoxicity disrupting the native conformations of DNA/RNA was quoted on in the previous post as was the 2005 report C60 binds to and deforms nucleotides.  These are among several studies of C60 biological impacts based on theoretical modeling.  I think what actually goes on in-vivo in the case of both studies is unknown.  Nonetheless, such studies point to possible serious long-term negative consequence of C60 supplementation.

An additional 2009 study looked for evidence of damage to DNA as a result of oral consumption of C60, Oxidatively damaged DNA in rats exposed by oral gavage to C60 fullerenes and single-walled carbon nanotubes.  “— BACKGROUND: C60 fullerenes and single-walled carbon nanotubes (SWCNT) are projected to be used in medicine and consumer products with potential human exposure. The hazardous effects of these particles are expected to involve oxidative stress with generation of oxidatively damaged DNA that might be the initiating event in the development of cancer. — OBJECTIVE: In this study we investigated the effect of a single oral administration of C60 fullerenes and SWCNT.  METHODS: We measured the level of oxidative damage to DNA as the premutagenic 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in the colon mucosa, liver, and lung of rats after intragastric administration of pristine C60 fullerenes or SWCNT (0.064 or 0.64 mg/kg body weight) suspended in saline solution or corn oil. We investigated the regulation of DNA repair systems toward 8-oxodG in liver and lung tissue.  RESULTS: Both doses of SWCNT increased the levels of 8-oxodG in liver and lung. Administration of C60 fullerenes increased the hepatic level of 8-oxodG, whereas only the high dose generated 8-oxodG in the lung. We detected no effects on 8-oxodG in colon mucosa. Suspension of particles in saline solution or corn oil yielded a similar extent of genotoxicity, whereas corn oil per se generated more genotoxicity than the particles. Although there was increased mRNA expression of 8-oxoguanine DNA glycosylase in the liver of C60 fullerene-treated rats, we found no significant increase in repair activity.  CONCLUSIONS: Oral exposure to low doses of C60 fullerenes and SWCNT is associated with elevated levels of 8-oxodG in the liver and lung, which is likely to be caused by a direct genotoxic ability rather than an inhibition of the DNA repair system.”

Yet-another 2011 study only mentioned previously suggests that Non-liposomal (aka non-olive oil encapsulated) C60 fullerenes appear to be toxic and produce damage.  It is In Vitro and In Vivo Genotoxicity Induced by Fullerene (C60) and Kaolin.  “Nanomaterials are being utilized for many kinds of industrial products, and the assessment of genotoxicity and safety of nanomaterials is therefore of concern. In the present study, we examined the genotoxic effects of fullerene (C60) and kaolin using in vitro and in vivo genotoxicity systems. Both nanomaterials significantly induced micronuclei and enhanced frequency of sister chromatid exchange (SCE) in cultured mammalian cells. When ICR mice were intratracheally instilled with these nanomaterials, DNA damage of the lungs increased significantly that of the vehicle control. Formation of DNA adducts in the lungs of mice exposed to nanomaterials were also analyzed by stable isotope dilution LC-MS/MS. 8-Oxodeoxyguanosine and other lipid peroxide related adducts were increased by 2- to 5-fold in the nanomaterial-exposed mice. Moreover, multiple (four consecutive doses of 0.2 mg per animal per week) instillations of C60 or kaolin, increased gpt mutant frequencies in the lungs of gpt delta transgenic mice. As the result of mutation spectrum analysis, G:C to C:G transversions were commonly increased in the lungs of mice exposed to both nanomaterials. In addition, G:C to A:T was increased in kaolin-exposed mice. In immunohistochemical analysis, many regions of the lungs that stained positively for nitrotyrosine (NT) were observed in mice exposed to nanomaterials. From these observations, it is suggested that oxidative stress and inflammatory responses are probably involved in the genotoxicity induced by C60 and kaolin.”

Note that unlike the previously mentioned studies of potential damage, this study involved biological observations.

Studies have also looked at other forms of potential physiologic damage that might be induced by C60.  The newer 2013 publication Human serum albumin interactions with C60 fullerene studied by spectroscopy, small-angle neutron scattering, and molecular dynamics simulations reports: “Concern about the toxicity of engineered nanoparticles, such as the prototypical nanomaterial C60 fullerene, continues to grow. While, evidence continues to mount that C60 and its derivatives may pose health hazards, the specific molecular interactions of these particles with biological macromolecules require further investigation. In this article, we report combined experimental and theoretical studies on the interaction of one of the most prevalent proteins in the human body, human serum albumin (HSA), with C60 in an aqueous environment. The C60–HSA interaction was probed by circular dichroism (CD) spectroscopy, small-angle neutron scattering (SANS), and atomistic molecular dynamics (MD) simulations to understand C60-driven changes in the structure of HSA in solution. The CD spectroscopy demonstrates that the secondary structure of the protein decreases in α-helical content in response to the presence of C60 (0.68 nm in diameter). Similarly, C60 produces subtle changes in the solution conformation of HSA (an 8.0 nm × 3.8 nm protein), as evidenced by the SANS data and MD simulations, but the data do not indicate that C60 changes the oligomerization state of the protein, such as by inducing aggregation. The results demonstrate that the interaction is not highly disruptive to the protein in a manner that would prevent it from performing its physiological function.”

3.  Liposomal encapsulated C60 fullerenes are not toxic and have an anti-cancer effect, whereas non-encapsulated C60 fullerenes do NOT have an anti-cancer effect.

Going back to 2009, the publication Biological safety of liposome-fullerene consisting of hydrogenated lecithin, glycine soja sterols, and fullerene-C60 upon photocytotoxicity and bacterial reverse mutagenicity reported: “Various water-soluble derivatives of fullerene-C60 (C60) have been developed as detoxifiers for reactive oxygen species (ROS), whereas C60 incorporated in liposome (Lpsm) has not been reported yet. We prepared the liposome-fullerene (0.2% aqueous phase, Lpsm-Flln) which was composed of hydrogenated lecithin, glycine soja (soybean) sterols, and C60 in the weight ratio of 89.7:10:0.3, then examined the photocytotoxicity and bacterial reverse mutagenicity, as comparing with the Lpsm containing no C60. Photocytoxicity of Lpsm-Flln or Lpsm was examined using Balb/3T3 fibroblastic cells at graded doses of 0.49-1000 microg/mL under the condition of UVA- or sham-irradiation. The cells were irradiated with UVA (5 J/cm2, 320-400 nm, lambda max = 360 nm) at room temperature for 50 min. The resultant cell viability (% of control) did not decrease dose-dependently to 50% or less regardless of the UVA-irradiation. These results show that Lpsm-Flln or Lpsm does not possess photocytotoxicity to Balb/3T3 fibroblasts, and Lpsm-Flln may not exert a UVA-catalytic ROS-increasing action. A possibility for the reverse mutation by Lpsm-Flln or Lpsm was examined on four histidine-demanding strains of Salmonella typhimurium and a tryptophan-demanding strain of Escherichia coli. As for the dosages of Lpsm-Flln or Lpsm (313-5000 microg/plate), the dose-dependency of the number of reverse mutation colonies of each strain did not show a twice or more difference versus the negative control regardless of the metabolic activation, and, in contrast, marked differences for five positive controls (sodium azide, N-ethyl-N’-nitro-N-nitrosoguanidine, 2-nitrofluorene, 9-aminoacridine, and 2-aminoanthracene). The growth inhibition of bacterial strains and the deposition of Lpsm-Flln or Lpsm were not found. As a result, the bacterial reverse mutagenicity of Lpsm-Flln or Lpsm was judged to be negative under the conditions of this test. Thus, Lpsm-Flln and Lpsm may not give any significant biological toxic effects, such as photocytotoxicity and bacterial reverse mutagenicity.”

Another interesting article related to this point is the 2011 publication Anticancer Effects of Fullerene [C60] Included in Polyethylene Glycol Combined With Visible Light Irradiation Through ROS Generation and DNA Fragmentation on Fibrosarcoma Cells With Scarce Cytotoxicity to Normal Fibroblasts .  As you can see. the PEG C60 solution is really a “nano particle” or “lissome.”  “Fullerene [C60] included in polyethylene glycol (PEG) at a composing ratio of 1:350 w/w was examined for anticancer effects upon photodynamic therapy (PDT). Human connective tissue-derived fibrosarcoma cells HT1080 were decreased for a viability of 50% or 30%, by 3-h administration with PEG-fullerene [C60] at 50 or 100 ppm fullerene [C60] equivalent, respectively, subsequent rinsing out and irradiation with visible light (400‐600 nm, 140 J/cm2:450-fold as intense as in average outdoor), whereas the same tissue type-derived normal fibroblastic cells DUMS16 retained a viability of 93% or 85% under the same conditions. Anticancer effects were dependent on PEG-fullerene [C60] concentrations and irradiation doses, and scarcely exerted by PEG-fullerene [C60] alone, irradiation alone, or by fullerene [C60]-free PEG combined with irradiation, suggesting that the active principle may be fullerene [C60] as small as 0.0028 wt% versus the whole compound. Irradiation with PEG-fullerene [C60] occurred in intracellular DNA fragmentation according to TUNEL assay, and produced reactive oxygen species (ROS) such as hydroperoxides and peroxyl radicals or superoxide anion radicals in HT1080 cells as demonstrated by CDCFH-DA assay or nitroblue tetrazolium assay, respectively. Thus, PEG-fullerene [C60] is expected to be applied to anticancer PDT with scarce side effects on normal cells.”

4.   One of the primary ways in which C60-EVOO may exercise its longevity effects on rats may be via promotion of autophagy in cancer cells, killing them. 

This pathway was briefly discussed in the earlier blog entry on buckyballs and longevity. Cancer is normally a primary cause of death in rats.  Cancer cells have evolved numerous mechanisms to resist cell death via apoptosis. They are subject to death via another set of mechanisms, however, mechanisms associated with autophagy. See the this list of previous blog entries related to autophagy, in particular the 2013 blog entry  Autophagy – the housekeeper in every cell that fights aging.  “Autophagy (“self eating”) is an old, evolutionarily conserved stress response that is present in all living cells. Like apoptosis, autophagy is a programmed response and has several sub-pathways.  Unlike apoptosis, autophagy promotes life rather than death.  Recent discoveries have shown that almost every genetic, dietary, and pharmacologic manipulation proven to extend lifespan activates autophagy as part of its mechanism of action. — Autophagy is the way your cells “clean house” and “recycle the trash”.  Along with the ubiquitin proteasome system, autophagy is one of the main methods that cells use to clear dysfunctional or misfolded proteins.  Autophagy can clear any kind of trash: intracellular viruses, bacteria, damaged proteins, protein aggregates and subcellular organelles. Although autophagy has long been known to exist, only recently has there been a clear understanding of the genes and pathways related to it.  This recent evidence suggests that the declining efficacy of autophagy may be a driver of many of the phenotypic phenomena of aging.  This blog entry explores the “evidence for the autophagy theory of aging” and builds a strong case that defective autophagy is a central driver for age-related diseases and aging itself.”

Autophagy is increasingly being investigated as an anticancer therapy.  The 2012 publication Autophagy as a target for cancer therapy: new developments reports: “Autophagy is an evolutionarily conserved lysosomal degradation pathway that eliminates cytosolic proteins, macromolecules, organelles, and protein aggregates.  Activation of autophagy may function as a tumor suppressor by degrading defective organelles and other cellular components.  However, this pathway may also be exploited by cancer cells to generate nutrients and energy during periods of starvation, hypoxia, and stress induced by chemotherapy.  Therefore, induction of autophagy has emerged as a drug resistance mechanism that promotes cancer cell survival via self-digestion.  Numerous preclinical studies have demonstrated that inhibition of autophagy enhances the activity of a broad array of anticancer agents.  Thus, targeting autophagy may be a global anticancer strategy that may improve the efficacy of many standard of care agents. These results have led to multiple clinical trials to evaluate autophagy inhibition in combination with conventional chemotherapy.  In this review, we summarize the anticancer agents that have been reported to modulate autophagy and discuss new developments in autophagy inhibition as an anticancer strategy.”

The 2013 report Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment highlights the importance of utilizing autophagy for getting around cancer resistance to chemotherapies. “Induction of cell death and inhibition of cell survival are the main principles of cancer therapy. Resistance to chemotherapeutic agents is a major problem in oncology, which limits the effectiveness of anticancer drugs. A variety of factors contribute to drug resistance, including host factors, specific genetic or epigenetic alterations in the cancer cells and so on. Although various mechanisms by which cancer cells become resistant to anticancer drugs in the microenvironment have been well elucidated, how to circumvent this resistance to improve anticancer efficacy remains to be defined. Autophagy, an important homeostatic cellular recycling mechanism, is now emerging as a crucial player in response to metabolic and therapeutic stresses, which attempts to maintain/restore metabolic homeostasis through the catabolic lysis of excessive or unnecessary proteins and injured or aged organelles. Recently, several studies have shown that autophagy constitutes a potential target for cancer therapy and the induction of autophagy in response to therapeutics can be viewed as having a prodeath or a prosurvival role, which contributes to the anticancer efficacy of these drugs as well as drug resistance. Thus, understanding the novel function of autophagy may allow us to develop a promising therapeutic strategy to enhance the effects of chemotherapy and improve clinical outcomes in the treatment of cancer patients.”

You can also see these publications relating autophagy, cancer and survival.   Cell death and autophagy: cytokines, drugs, and nutritional factors (2008), Autophagy: Can it become a potential therapeutic target?(2010), To die or not to die: that is the autophagic question(2008), Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer(2010),

The actions of autophagy in cancer can be either pro-oncogenic or anti-oncogenic in a context-dependent manner.  Cancer cells may utilize autophagy-related pathways for their survival. Some of the complexities involved are described in the recent publications:

Getting back to the central issue here, for some time, it has been known that water-based C60 solutions have a capability to kill cancer cells via induction of autophagy.

This capability was briefly  discussed in the earlier blog entry, mainly in the context of using C60 preparations in conjunction with cancer chemotherapy.  One publication with abstract there is Autophagy-mediated chemosensitization in cancer cells by fullerene C60 nanocrystal (2009).  Another publication mentioned there was Pristine C 60  Fullerenes Inhibit The Rate Of Tumor Growth And Metastasis (2011).

We recall that in the experiment where C60-EVOO extended the lifespan of rats by 90%, a remarkable observation was that none of the treated rats had developed any cancers.

A newer (2013) hypothesis is that induction of autophagy may be a generic property of many nanomaterials. What appears to be involved is activation of one or more stress pathways.

The 2013 publication Nanomaterials and autophagy: new insights in cancer treatment reports: “Autophagy represents a cell’s response to stress. It is an evolutionarily conserved process with diversified roles. Indeed, it controls intracellular homeostasis by degradation and/or recycling intracellular metabolic material, supplies energy, provides nutrients, eliminates cytotoxic materials and damaged proteins and organelles. Moreover, autophagy is involved in several diseases. Recent evidences support a relationship between several classes of nanomaterials and autophagy perturbation, both induction and blockade, in many biological models. In fact, the autophagic mechanism represents a common cellular response to nanomaterials. On the other hand, the dynamic nature of autophagy in cancer biology is an intriguing approach for cancer therapeutics, since during tumour development and therapy, autophagy has been reported to trigger both an early cell survival and a late cell death. The use of nanomaterials in cancer treatment to deliver chemotherapeutic drugs and target tumours is well known. Recently, autophagy modulation mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anti-cancer agent. Herein, we summarize the effects of nanomaterials on autophagic processes in cancer, also considering the therapeutic outcome of synergism between nanomaterials and autophagy to improve existing cancer therapies.”

5.    The autophagy-inducing properties of C60 may also be useful for treating neurodegenerative diseases like Alzheimer’s Disease where accumulation of beta amloid plays a major role.

The 2011 publication C60 fullerene-pentoxifylline dyad nanoparticles enhance autophagy to avoid cytotoxic effects caused by the β-amyloid peptide reports: “Many studies have focused on the neuroprotective effects of C(60) fullerene-derived nanomaterials. The peculiar structure of C(60) fullerene, which is capable of “adding” multiple radicals per molecule, serves as a “radical sponge,” and it can be an effective antioxidant by reducing cytotoxic effects caused by intracellular oxidative stress. In this study, PEG-C(60)-3, a C(60) fullerene derivative incorporating poly(ethylene glycol), and its pentoxifylline-bearing hybrid (PTX-C(60)-2) were investigated against β-amyloid (Aβ)(25-35)-induced toxicity toward Neuro-2A cells. PEG-C(60)-3 and PTX-C(60)-2 significantly reduced Aβ(25-35)-induced cytotoxicity, with comparable activities in decreasing reactive oxygen species and maintaining the mitochondrial membrane potential. Aβ(25-35) treatment elicited adenosine monophosphate-activated protein kinase-associated autophagy. Cytoprotection by PEG-C(60)-3 and PTX-C(60)-2 was partially diminished by an autophagy inhibitor, indicating that the elicited autophagyand antioxidative activities protect cells from Aβ damage. PTX-C(60)-2 was more effective than PEG-C(60)-3 at enduring the induced autophagy. Our results offer new insights into therapeutic drug design using C(60) fullerene-PTX dyad nanoparticles against Aβ-associated diseases. —

FROM THE CLINICAL EDITOR: The neuroprotective effects of C60 fullerene-derived nanomaterials are known and thought to be related to their capacity of “absorbing” multiple free radicals. In this study, another interesting property is presented: they may enhance autophagy of beta-amyloid peptide, which could minimize the damaging effects of this peptide.”

While there appeared to be several research publications relating nano materials and C60 in particular to autophagy and the inhibitions of cancer, none of these publications deals explicitly with the C60-EVOO concoction used in the rat longevity experiments. Therefore it remains a conjecture that a major factor in the longevity of the rats was activation of autophagy pathways.  As a conjecture, however, I believe it is a fairly good one.

6.  There is a possibility that C60 fullerenes can be used to treat arthritis, mast cell and peripheral blood basophil-associated diseases.

From a patent application;  Use of fullerenes for the treatment of mast cell and basophil-mediated disease (2006):  “Mast cell (MC) and peripheral blood basophil (PBB)-associated diseases are treated or prevented, or their symptoms are alleviated by the administration of water soluble fullerenes (buckeyballs) to the individual under conditions sufficient to inhibit MC and PBB responses. MC and PBB responses are associated with, for example, various allergies including Type 1 hypersensitivity initiated by IgE-antigen, arthritis, multiple sclerosis, urticaria, atopic dermatitis, heart disease, etc. The treatment regimen can be enhanced using Chimeric fullerenes that specifically home to and inhibit MC and PBB cells. These molecules, for example, comprise fullerenes to which are attached IgE Fc or stem cell factor (SCF) peptides that bind to receptors specifically on MC and PBB cells. Additional molecules which may be used in the processes include IgE Fc or SCF peptides with several fullerenes covalently attached.”  Also quoted in the earlier blog entry was the 2007 publication Fullerene nanomaterials inhibit the allergic response.

7.    There is an increasing interest in liposomal C60 formulations

Item 3 above outlines what appear to be low-toxicity and high effectiveness advantages of liposomal C60 formulations.  The 2013 publication Liposome Formulation of Fullerene-Based Molecular Diagnostic and Therapeutic Agents reports: “Fullerene medicine is a new but rapidly growing research subject. Fullerene has a number of desired structural, physical and chemical properties to be adapted for biological use including antioxidants, anti-aging, anti-inflammation, photodynamic therapy, drug delivery, and magnetic resonance imaging contrast agents. Chemical functionalization of fullerenes has led to several interesting compounds with very promising preclinical efficacy, pharmacokinetic and safety data. However, there is no clinical evaluation or human use except in fullerene-based cosmetic products for human skincare. This article summarizes recent advances in liposome formulation of fullerenes for the use in therapeutics and molecular imaging.”

8.  Liposomal C60 fullerenes may protect against UVA skin damage

Again, it can be confusing but we think that these bucky balls may be both antioxidants and pro-oxidants.

The 2009 publication Fullerene-C60/liposome complex: Defensive effects against UVA-induced damages in skin structure, nucleus and collagen type I/IV fibrils, and the permeability into human skin tissue reports: “We previously reported biological safety of fullerene-C60 (C60) incorporated in liposome consisting of hydrogenated lecithin and glycine soja sterol, as Liposome-Fullerene (0.5% aqueous phase; a particle size, 76nm; Lpsm-Flln), and its cytoprotective activity against UVA. In the present study, Lpsm-Flln was administered on the surface of three-dimensional human skin tissue model, rinsed out before each UVA-irradiation at 4 J/cm(2), and thereafter added again, followed by 19-cycle-repetition for 4 days (sum: 76 J/cm(2)). UVA-caused corneum scaling and disruption of epidermis layer were detected by scanning electron microscopy. Breakdown of collagen type I/IV, DNA strand cleavage and pycnosis/karyorrhexis were observed in vertical cross-sections of UVA-irradiated skin models visualized with fluorescent immunostain or Hoechst 33342 stain. These skin damages were scarcely repressed by liposome alone, but appreciably repressed by Lpsm-Flln of 250 ppm, containing 0.75 ppm of C60-equivalent to a 1/3300-weight amount vs. the whole liposome. Upon administration with Lpsm-Flln [16.7 microM (12 ppm): C60-equivalent] on human abdomen skin biopsies mounted in Franz diffusion cells, C60 permeated after 24h into the epidermis at 1.86 nmol/g tissue (1.34 ppm), corresponding to 0.3% of the applied amount and a 9.0-fold dilution rate, but C60 was not detected in the dermis by HPLC, suggesting no necessity for considering a toxicity of C60 due to systemic circulation via dermal veins. Thus Lpsm-Flln has a potential to be safely utilized as a cosmetic anti-oxidative ingredient for UVA-protection.”

9.   Hydroxylated C60 fullerences protect against radiation injury

The 2009 article The polyhydroxylated fullerene derivative C60(OH)24 protects mice from ionizing-radiation-induced immune and mitochondrial dysfunction reports:  “These results suggest that the polyhydroxylated fullerene derivative C60(OH)24 protects against ionizing-radiation-induced mortality, possibly by enhancing immune function, decreasing oxidative damage and improving mitochondrial function.”

10.  C60 fullerenes can generate ROS in the presence of UV light and induce bacterial killing as well as kill cancer cells

They do this via the generation of radicals directly and by inhibiting anti-oxidant enzymes, such as glutathione reductase, glutathione peroxidase, glutathione s transferase, catalase, and superoxide dismutase.  Thus, c60 fullerenes are actually “ROS generators”  The 2011 publication Photodynamic therapy with fullerenes in vivo: reality or a dream? relates: “The fullerene molecule with its unique structure of 60 carbon atoms arranged in a soccer ball structure is a molecule of great potential for a variety of applications and has drawn the attention of lots of physicists, chemists and engineers. Recently these nanostructures have also been studied for their biological activities with a view towards using them for biomedical applications. One of the therapies for which fullerenes may have a medical application is the light-based therapy called photodynamic therapy (PDT) [1], which is a nonsurgical, minimally invasive approach that has been used in the treatment of solid tumors and many nonmalignant diseases [2]. PDT is a nonthermal photochemical reaction, which requires the simultaneous presence of a photosensitizing drug (photosensitizer [PS]), oxygen and visible light (Figure 1). It is a two-step procedure that involves the administration of a PS, followed by activation of the drug with the appropriate wavelength of light [35]. The photoactivation of the drug generates singlet oxygen and other reactive oxygen species (ROS), which cause a lethal oxidative stress and membrane damage in the treated cells and in the case of tumors, leads to cell death by direct cytotoxicity and a dramatic antivascular action that impairs blood supply to the area of light exposure [6]. It is known that depending on the parameters involved, in vitro PDT can kill cancer cells via apoptosis, necrosis or autophagy. The direct killing effect of PDT on malignant cancer cells that has been studied in detail in vitro also clearly applies in vivo, but in addition, two separate in vivo mechanisms leading to PDT-mediated tumor destruction have been described. They are the vascular shutdown effect mentioned above [7], and the PDT-induced activation of the host immune system [8]. In case of antimicrobial PDT Gram-positive bacteria are found to be more susceptible as compared with Gram-negative bacteria. This observation is explained by the difference in the structure of their cell walls (Figure 2) [9].  In this vein, the earlier blog entry quotes from the 2012 publication Water-Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo- and Photodynamic Therapeutic Actions.

Image and legend source “Jablonski diagram – Initial absorption of a photon (or two photons of twice the energy) by the ground state of the singlet fullerene, which gives rise to the short-lived excited singlet state. This can lose energy by fluorescence (negligible in the case of fullerenes), internal conversion to heat or by intersystem crossing to the long-lived triplet state. Fullerene triplet states are efficiently quenched by molecular oxygen (a triplet state) to give type II (singlet oxygen) and type I (superoxide and hydroxyl radical) reactive oxygen species. In the absence of oxygen fullerene triplet states lose energy by phosphorescence.”

11.               Fullerenes are receiving increasing attention for their medical therapeutic potentials

From the 2013 publication Application of fullerenes in nanomedicine: an update:  “Fullerenes are carbon spheres presently being pursued globally for a wide range of applications in nanomedicine. These molecules have unique electronic properties that make them attractive candidates for diagnostic, therapeutic and theranostic applications. Herein, the latest research is discussed on developing fullerene-based therapeutics as antioxidants for inflammatory diseases, their potential as antiviral/bacterial agents, utility as a drug delivery device and the promise of endohedral fullerenes as new MRI contrast agents. The recent discovery that certain fullerene derivatives can stabilize immune effector cells to prevent or inhibit the release of proinflammatory mediators makes them potential candidates for several diseases such as asthma, arthritis and multiple sclerosis. Gadolinium-containing endohedral fullerenes are being pursued as diagnostic MRI contrast agents for several diseases. Finally, a new class of fullerene-based theranostics has been developed, which combine therapeutic and diagnostic capabilities to specifically detect and kill cancer cells.”

The 2010 publication Fullerenes as unique nanopharmaceuticals for disease treatment reports “As unique nanoparticles, fullerenes have attracted much attention due to their unparalleled physical, chemical and biological properties. Various functionalized fullerenes with -OH, -NH2, -COOH, and peptide modifications were developed. It summarized the biological activities of fullerenes derivatives in cancer therapy with high efficiency and low toxicity, as reactive oxygen species scavenger and lipid peroxidation inhibitor, to inhibit human immunodeficiency virus and to suppress bacteria and microbial at low concentration. In addition, the mechanism for fullerene to enter cells and biodistribution of fullerene in vivo was also discussed. This research focuses on the current understanding of fullerenes-based nanomaterials in the potential clinical application as well as biological mechanism of fullerenes and its derivatives in disease therapy.”

The proposed mechanism of action is the generation of ROS.  See Molecular Docking Analysis of Fullerene (C60) with Human Antioxidant Enzymes: Implications in Inhibition of Enzymes (2013): “Fullerenes are a new class of carbon allotropes with unique physicochemical and biological properties for which they are widely used in biomedical technology. This leads to over exposure of fullerene and its detrimental effects on living organisms are not well characterized. Recent studies demonstrated that fullerene (C60) can generate free radicals that disrupt normal cellular function through lipid peroxidation and inducing cell death. It is not well understood whether fullerene induces the free radical generation by itself or the induction of oxidative stress is due to inhibition of enzymes associated with conversion of free radicals into water molecules. Hence forth, research is still evolving to understand the mechanism of fullerene toxicity on enzymes. In the present study we report the docking studies of fullerene with key antioxidant enzymes viz., glutathione reductase, glutathione peroxidase, glutathione s transferase, catalase and superoxide dismutase that scavenge free radicals. Docking was performed using Autodock 4.0 to examine the interactions of fullerene with all the five enzymes. Docking studies of fullerene with glutathione reductase, glutathione peroxidase, glutathione s transferase, catalase and superoxide dismutase demonstrated that it can bind with the active site residues of these enzymes through van der Waals interactions. Hence, we speculate that fullerene inhibit the activities of these enzymes with respective substrates which might lead to accumulation of reactive oxygen intermediates.”

See also Photosensitizers for targeted photodynamic therapy. a 2012 patent mentioned earlier.

In summary, C60 binds to the active sites of these enzymes via van der Waals interactions

12.   Water soluble C60 fullerences can treat low back pain

This is amazing!  See the 2013 publication A Novel Treatment of Neuroinflammation against Low Back Pain by Soluble Fullerol Nanoparticles.  “Results: Fluorescence staining results indicated that TNF-α markedly increased the production of intracellular ROS and the number of apoptotic cells. Under fullerol treatment cellular apoptosis was reduced along with concomitant suppression of ROS. The expression of inflammatory cytokines IL-1 β, IL-6, COX-2, and PGE2, was also inhibited by fullerol in a dose-dependent manner. Furthermore, fullerol-treated cells exhibited up-regulation of anti-oxidative enzyme genes SOD2 and catalase. – Conclusion: The results obtained from this study clearly suggest that fullerol treatment suppresses the inflammatory responses of DRG and neurons, as well as cellular apoptosis by decreasing the level of ROS and potentially enhancing anti-oxidative enzyme gene expression. Therefore, fullerol has potential to serve as a novel therapeutic agent for low back pain treatment.”

Note that while several studies mentioned above suggest that fullerenes work to kill cancer cells and produce other beneficial effects  by promoting ROS, that this and other studies suggest that fullerenes work to suppress inflammation by decreasing the expression of ROS.  These seemingly contradictory conclusions require more detailed explanation since both of these observations could well be correct.   For example, production of ROS could be a cellular response to C60, and above we related how C60 might suppress the generation of ROS by inhibiting the formation of superoxide as well as by antioxidant effects.

The 2013 publication Fullerol nanoparticles suppress inflammatory response and adipogenesis of vertebral bone marrow stromal cells-a potential novel treatment for intervertebral disc degeneration reports:  PURPOSE: To investigate the potential of a free radical scavenger, fullerol nanoparticles, to prevent vertebral bone marrow lesion and prevent disc degeneration by inhibiting inflammation and adipogenic differentiation of vertebral bone marrow stromal cells (vBMSCs).  STUDY DESIGN/SETTING: Fullerol nanoparticle solutions were prepared to test their in vitro suppression effects on mouse vBMSC inflammation and adipogenic differentiation compared with non-fullerol-treated groups.  METHODS: With or without fullerol treatment, vBMSCs from Swiss Webster mice were incubated with 10 ng/mL interleukin-1 β (IL-1 β). The intracellular reactive oxygen species (ROS) were measured with fluorescence staining and flow cytometry. In addition, vBMSCs were cultured with adipogenic medium (AM) with or without fullerol. Gene and protein expressions were evaluated by real-time polymerase chain reaction and histologic methods.  RESULTS: Fluorescence staining and flow cytometry results showed that IL-1 β markedly increased intracellular ROS level, which could be prevented by fullerol administration. Fullerol also decreased the basal ROS level to 77%. Cellular production of matrix metalloproteinase (MMP)-1, 3, and 13 and tumor necrosis factor alpha (TNF-α) induced by IL-1 β was suppressed by fullerol treatment. Furthermore, adipogenic differentiation of the vBMSCs was retarded markedly by fullerol as revealed by less lipid droplets in the fullerol treatment group compared with the adipogenic group. The expression of adipogenic genes PPARγ and aP2 was highly elevated with AM but decreased on fullerol administration.  CONCLUSIONS: These results suggest that fullerol prevents the catabolic activity of vBMSCs under inflammatory stimulus by decreasing the level of ROS, MMPs, and TNF-α. Also, fat formation in vBMSCs is prevented by fullerol nanoparticles, and, therefore, fullerol may warrant further in vivo investigation as an effective biological therapy for disc degeneration.

 

 13.  Water soluble C60 fullerenes up regulate the production of SOD2 and catalase

Again, see the 2013 publication A Novel Treatment of Neuroinflammation against Low Back Pain by Soluble Fullerol Nanoparticles.  Also again, this is getting more and more confusing, but it makes sense if you read the above and following article and the one in #14:

Fullerene nanoparticles and their anti-oxidative effects: a comparison to other radioprotective agents (2012) “Radiation therapy occupies an important position in the treatment of malignant diseases in spite of the  existence of radiation side effects on normal tissues. Thus, substances are being developed which are designed to reduce both the acute and long term radiation effects on healthy tissues. Currently a sulphur-containing compound amifostine (WR2721, ethyol) is used in clinical practice as a radioprotectant. However, it itself has considerable side effects including hypotension (found in 62% of patients), hypocalcaemia, diarrhoea, nausea, and vomiting. Carbon nanospheres, known as fullerenes, and their water soluble derivatives (e.g. C60(OH)24, dendrofullerene DF-1) exert anti-oxidative properties and reduce damage to the DNA in irradiated cells. Water soluble fullerenes are low-toxic substances and thus, are attractive in terms of their use as radioprotectants.”

Again, we seem to have contradictory findings between this and other reports:  fullerenes induce ROS and  oxidative stress and fullerenes protect against oxidation, fullerenes reduce DNA damage and fullerenes induce DNA damage, water-soluble fullerenes have low toxicity and water soluble fullerenes have potentially high toxicity.  Again, attention has to be paid to the nature of the study involved (e.g. quantum effect modeling study vs. cell-level biochemical effects study vs observation study of impact on oxidative stress parameters on rats), the particular kind of fullerene involved (e.g. water soluble vs lipid encased, etc.), and exact experimental conditions.   The devil is in the details.

14. Liposomal (aka olive oil encapsulated) C60 Fullerenes may make a key difference in animal health, but figuring out how may require thinking “out of the box.”

As indicated above, the famed rat longevity study could be in complete concordance with what both of us believe about stress, stress signaling, and the difference between “good ROS” (signaling ROS within a hormetic dose and duration range) and “bad ROS” (damage-inducing ROS, of dose or duration outside of the hormetic range), and about non-antioxidant mechanisms that drastically diminish ROS.   Here is yet-another factor (Jim) has learned:

By complexing with catalase, C60-EVOO fullerenes PRODUCE hydrogen peroxide and singlet oxygen, but inhibit the hydroxyl radical production.   Thus, they quench “bad ROS damage” but produce “good ROS signaling.”  See:

There is probably much more to be told to complete the C60 health story.  We will continue to follow it.

About Vince Giuliano

Being a follower, connoisseur, and interpreter of longevity research is my latest career, since 2007. I believe I am unique among the researchers and writers in the aging sciences community in one critical respect. That is, I personally practice the anti-aging interventions that I preach and that has kept me healthy, young, active and highly involved at my age, now 93. I am as productive as I was at age 45. I don’t know of anybody else active in that community in my age bracket. In particular, I have focused on the importance of controlling chronic inflammation for healthy aging, and have written a number of articles on that subject in this blog. In 2014, I created a dietary supplement to further this objective. In 2019, two family colleagues and I started up Synergy Bioherbals, a dietary supplement company that is now selling this product. In earlier reincarnations of my career. I was Founding Dean of a graduate school and a full University Professor at the State University of New York, a senior consultant working in a variety of fields at Arthur D. Little, Inc., Chief Scientist and C00 of Mirror Systems, a software company, and an international Internet consultant. I got off the ground with one of the earliest PhD's from Harvard in a field later to become known as computer science. Because there was no academic field of computer science at the time, to get through I had to qualify myself in hard sciences, so my studies focused heavily on quantum physics. In various ways I contributed to the Computer Revolution starting in the 1950s and the Internet Revolution starting in the late 1980s. I am now engaged in doing the same for The Longevity Revolution. I have published something like 200 books and papers as well as over 430 substantive.entries in this blog, and have enjoyed various periods of notoriety. If you do a Google search on Vincent E. Giuliano, most if not all of the entries on the first few pages that come up will be ones relating to me. I have a general writings site at www.vincegiuliano.com and an extensive site of my art at www.giulianoart.com. Please note that I have recently changed my mailbox to vegiuliano@agingsciences.com.
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8 Responses to Update on C60 fullerenes in olive oil

  1. Jim Watson has bought an additional piece of research to my attention suggesting that an additional health producing function of C-60 could be the enhancement of mitophagy. In the article above I pointed out how depolarization of the mitochondrial membrane is likely to be an impact of C-60, and this can lead to decrease or elimination of the production of superoxide with all its damaging impacts. The following publication suggests that a second benefit of such depolarization could be an enhancement of mitophagy. If this applies, C-60 might have three different positive effects: 1. direct antioxidant effect once it enters a mitochondrion, 2. reductionof the production of superoxide, and 3. enhancement of mitophagy. See Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin at
    http://www.ncbi.nlm.nih.gov/m/pubmed/21173115/?i=4&from=/23620051/related

    As explained in the blog entry, the same benefits might be realized from MitoQ.

  2. Michael says:

    Thanks for this comprehensive update. My question is whether there is any evidence that the intermittent dosing of the c60 made a crucial difference in the rat study results, and whether continuous dosing might have produced different results. Also, there has been some discussion on the Longecity site with some speculation that c60 might have some effect on stem cell proliferation or replication. I was wondering of there is any evidence of this.

  3. Pingback: Digital health – health and fitness wearables, Part 3: Heart Rate Variability: Principles and Science and Practical Measuring Devices | AGING SCIENCES – Anti-Aging Firewalls

  4. Hildegard says:

    Greetings Vince,
    Are you familiar with the work of Bob Greska? He’s the sole producer of single molecule (60 atom) Carbon 60 and does not need or use use a solvent (hexane or benzene) as there is no waste in his production method. All the other Carbon 60 produced is in clumps thereby reducing the effectiveness. I would appreciate it greatly if you and the brainy types who frequent this blog would have a listen to the two interviews at the following link and let me know what you think. At approx. 17:55 of the Neil Swartz interview he goes into the difference between Bob’s and the other methods of extraction. http://jamesmartinezmedia.net/c60-buckminsterfullerene-3/
    http://c-60.com/#oid=1001_1
    Thank you!
    ‘Hildegard’

  5. Hildegard says:

    Regarding point 2 above: …”C60 fullerenes or SWCNT (0.064 or 0.64 mg/kg body weight) suspended in saline solution or corn oil.” “Suspension of particles in saline solution or corn oil yielded a similar extent of genotoxicity, whereas corn oil per se generated more genotoxicity than the particles.”

    If the corn oil isn’t organic then genetically modified corn oil is likely.

    “In immunohistochemical analysis, many regions of the lungs that stained positively for nitrotyrosine (NT) were observed in mice exposed to nanomaterials. From these observations, it is suggested that oxidative stress and inflammatory responses are probably involved in the genotoxicity induced by C60 and kaolin.”

    Pardon me as I’m a lay person here but how can C60 cause oxidative stress when C60 has been proven to be an extremely powerful antioxidant?

    I would like to know exactly who is conducting these studies and why.

  6. justin says:

    I’m as puzzled as Hildegard. There eseem to be conflicting studies re C60. I bought C60 from the Vaughter Wellness site. The site is sober and interacts with and rejects bogus and scam claims. It also responds to claims that C60 is toxic and quotes Professor Moussa, leader of the rat study team, that C60 is totally non-toxic. On the strength of this I bought 6 bottles. But having since seen the cautions on Vince’s site (for which I’m grateful) and having also now done a PubMed search on C60 and toxicity I am confused. There are multiple claims of its benefits but also a large number warning of toxicity. Perhaps it will turn out to be safe but there are so many conflicting studies. The jury is out. I don’t want to be a guinea pig so I’ve decided to stop taking C60. A pity. I had hoped it would be beneficial but I’m not prepared to take the risk. Anyone who thinks I’m too cautious I’m prepared to be dissauded but my PubMed search has really made me hesistant to go down the C60 road!

  7. Federer says:

    Any update on C-60?

  8. bobio says:

    In a wishfully blind effort to ride the brake on aging/health I volunteered .

    Ascendance Biomedical Is involved in a voluntary trial ,for a one-month period. The Bucky-balls are 99.95% purity combined with olive oil.

    Daily dosages and regimen protocols appear specifically designed to have the optimal result. You’ll learn more about the daily/weekly dosages should you choose to enroll.

    Blood testing: cholesterol, lipid panel, and comprehensive blood count.
    Our DNA methylation analysis service measures the age-related epigenetic change that occurs in the promoter region of two separate genes to get a biological estimate of age.

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