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Effect of c60oo on Thioacetamide Induced Liver Toxicity in Wistar Rats

c60oo fullerene liver toxicology thioacetamide

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#1 ambivalent

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Posted 07 December 2022 - 05:26 PM

Apologies if the study has appeared before but if it has I haven't seen it, this would appear to be the most important paper on c60oo since Baati ten years ago and perhaps is the type of study we should have expected within a year or two after.


A thanks to the "Ministry of Education, Science and Technological Development of the Republic of Serbia" for funding this research.




A couple of points to note the rats were fed ad libitum except for once a week they were fasted 16 hours. The estimated dietary consumption of olive oil was the human equivalent of 60 grams which for the two c60oo doses is an equivalent of something like 65mg and 13 mg, respectively. The questions as to whether the c60 translates allometrically to the olive oil equivalent as has been queried previously since we're trying to put c60 in the cells of mitochondria rather than scaling up a biological system. 


However, the take home message, of the toxiciology study is, when it comes to c60oo, less is more - as has been discussed before, consistent with Turnbuckle's observations. Unfortunately, there was no c60oo control which would have been great and we only have two c60oo points on a plot subject to a toxic effect and it would seem reasonable to assume optimisitcally that the lower dose isn't the best and concerningly the high dose not the worst.  


No human that we know of has taken several litres a month and the human equivalent of the study wrt c60 might be much higher than that for either dose.


Anyhow, a great study with a comprehensive set of metrics. Some observations:


One of the stand out results, I thought, was the effect of c60oo on the DNA comet tail of the rats. The comet tail of the non-intervention rats increased by 240% compared to controls; olive oil alone resulted in a 100% increase over controls and the high c60oo dose just 10%. However, despite the Thioacetamide, the low dose c60oo group saw a reduction in tail length of around 25 % over the unexposed controls. 


Catalase was another noteworthy metric. In three of the four exposed groups, including the c60oo high dose arm, Catalase expression was reduced but with low dose c60 Catalase was up nearly 30%.


Body mass of all exposed groups were considerably lower than controls. The low dose c60 group, still 15% lower than controls fared the best while the high c60 dose group the worst, down 25%. Water intake saw closeness amongst all exposed groups, well off controls, save the high c60oo dose group, consuming over 10% less than the low dose group.


The sampled images of the livers, which of course likely were not random, were quite revealing - the low dose c60 and control group were similar and the only two appearing healthy - the liver protective effects of c60oo was an observation made from Baati.


There too was indication of increased glycemia which it is posited is due to self-aggregation of c60 leading to pro-oxidation.


C60oo low dose appeared to offer protection against cancer in exposed groups (4 from 8), while high dose c60oo offered none compared to untreated group (6/8), olive oil fared the worst (8/8).


Suggestion of deleterious effects of olive oil on the liver given parenchymal injury, fibrosis, and biliary injury and inflammation were also more prominent in the TAA+O group than all other experimental groups


"Our results showed that in comparison with the control group, treatment with TAA (alone or in combination with VOO) significantly increased the liver’s whole homogenate level of HMGB1 expression (3.2- and 3.3-fold, respectively), and consequently the level of phosphorylated NF-κB p65 (2.2- and 2-3-fold, respectively) . In comparison to the TAA+O rat group, in the TAA+F1 group, the expression level of HMGB1 and p-NF-κB p65 decreased by about 54% and 39%, respectively, while in TAA+F2-treated rats, only p-NF-κB p65 expression level decreased by about 22% . These results suggested that only a lower dose of C60 fullerene has the potential to inhibit activation of NF-κB p65 and, consequently, necrotic events in TAA-induced liver injury."


Obviously there are more observations, but will leave with A&S:




"Thioacetamide (TAA) is widely used to study liver toxicity accompanied by oxidative stress, inflammation, cell necrosis, fibrosis, cholestasis, and hepatocellular carcinoma. As an efficient free radical’s scavenger, C60 fullerene is considered a potential liver-protective agent in chemically-induced liver injury. In the present work, we examined the hepatoprotective effects of two C60 doses dissolved in virgin olive oil against TAA-induced hepatotoxicity in rats. We showed that TAA-induced increase in liver oxidative stress, judged by the changes in the activities of SOD, CAT, GPx, GR, GST, the content of GSH and 4-HNE, and expression of HO-1, MnSOD, and CuZnSOD, was more effectively ameliorated with a lower C60 dose. Improvement in liver antioxidative status caused by C60 was accompanied by a decrease in liver HMGB1 expression and an increase in nuclear Nrf2/NF-κB p65 ratio, suggesting a reduction in inflammation, necrosis and fibrosis. These results were in accordance with liver histology analysis, liver comet assay, and changes in serum levels of ALT, AST, and AP. The changes observed in gut microbiome support detrimental effects of TAA and hepatoprotective effects of low C60 dose. Less protective effects of a higher C60 dose could be a consequence of its enhanced aggregation and related pro-oxidant role."





"In line with the increased interest in the use of nanotechnology for biomedical applications, we investigated the effects of potent free-radical scavenger C60 fullerene in TAA-induced rat liver injury. The obtained data indicated the dose-dependent hepatoprotective effects of C60. In a low dose, C60 fullerene was more efficient in terms of liver antioxidative and anti-inflammatory protection and reduced tissue fibrosis, necrosis, and CCC development. The ability of C60 to upregulate Nrf2 and downregulate HMGB1/NF-kB p65 signalling pathways could contribute to its actions. Our data also implicated that VOO may enhance toxic effects of TAA, which is why the development of water-soluble C60 derivates could be a more promising approach in its therapeutic use."



Edited by ambivalent, 07 December 2022 - 06:19 PM.

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#2 ambivalent

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Posted 17 December 2022 - 10:48 PM

An study on Thioacetamide (TAA) with Tamaraxic Gallica from a few years might explain some of the observations in the c60oo/TAA, when noting the effect of TAA on Hydrogen peroxide, more than doubling vs controls, not measured in the c60oo/TAA study. 


And, a paper on UVA induced DNA damage:


"We investigated the role of different reactive oxygen species (ROS) in ultraviolet A (UVA)-induced DNA damage in a human keratinocyte cell line, HaCaT. UVA irradiation increased the intracellular levels of hydrogen peroxide (H2O2), detected by a fluorescent probe carboxydichlorodihydrofluorescein, and caused oxidative DNA damage, single strand-breaks and alkali-labile sites, measured by alkaline single cell gel electrophoresis (comet assay)."


"In conclusion the conversion of H2O2 to ·OH appears to be the most important step in UVA-induced generation of strand breaks and alkali-labile sites and the bulk H2O2 appears to originate from O2·− generated by UVA irradiation."


So Thioacetamide causes DNA damage, expressed in the comet tail; Thioacetamide increases H202; H202 causes DNA damage as measured by comet assay.


In Table 4  of the C60oo/TAA study Catalase is 30% higher in the c60oo low dose group than controls, with every other group below control levels and from Table 5 comet tails of c60oo low-dose are 25% shorter than controls despite the Thioacetamide exposure, which on its own increased the tail by 200%. The high level of catalase, an H202 antioxidant and the short comet tail might suggest H202 has a low presence - though there still seems much to explain. Why so much catalase, why such a short comet tail - the catalase is above every other group and the comet tail below them? Increase the c60 and the tail lengthens while catalase reduces.


Well, increased H202 concentrations results from higher C60 levels is certainly plausible, H202 is a product of the superoxide/SOD2 interaction and C60 SOD2's mimic, therefore, as c60 concentrations increase so could be expected to increase the number of dismutations of the superoxide and with it levels of H202, and not enough possibly endogenous catalase to cope. So the negative effects of increased c60 might have to do with as the paper suggests, of c60's enhanced aggregation and related pro-oxidant role, but possibly instead it is due to H202 concentrations. 


As for the surprising shortening of the comet tail, despite the DNA damaging Thioacetamide, well this might possibly not be because of c60 but of H202 optimisation.  This study with house flies demonstrates that the 10% of max dose H202 group (and there was a 0%) lived the longest, above controls ,it implies, while all other H202 groups shorten lifespan relative to controls, which bears similarities to the c60oo/TAA study wrt comet tail where one concentration of c60oo shortens the tail over controls while another is longer.  


As shown in Table 5, the olive oil exerts protective effects on the DNA, shortening the comet tail compared to untreated TAA rats. And there is evidence of olive oils DNA protecting effects against H202:  


"The protective effects of the tested extracts or isolated compounds were evaluated from their ability to decrease hydrogen peroxide-induced formation of single strand breaks in the nuclear DNA, while the toxic effects were estimated from the increase of DNA damage when the extracts or isolated compounds were incubated directly with the cells. Significant protection was observed in extracts from olive oil and olive mill waste water. However, above a concentration of 100 microg/ml olive oil extracts exerted DNA damaging effects by themselves in the absence of any H2O2."


The potentially damaging effects of olive oil were observed too in the C60oo/TAA paper: 


"Our data also implicated that VOO may enhance toxic effects of TAA, which is why the development of water-soluble C60 derivates could be a more promising approach in its therapeutic use."


However, the amount of olive oil is the same for all three olive oil groups (zero c60; c60; x5 c60). So if H202 is varying in each group then c60 is playing a role. The paper using C60 in DMSO to intervene in an HD model suggests c60 is homesostatic - with handily measuring C60 "controls" too.  The H202 levels of the control and c60 group, for example, are similar but C60 decreases H202 significantly when pretreated against the HD model. 


 And the paper infers c60's homestatic behaviour:


"We have assumed that C60 influenced GSH recycle via the induction of γ-GCLC and GSTP protein expressions and this is necessary for the recovery of GSH system homeostasis."


And another paper suggesting the right amount of H202 and oxidative damage can extend life.  


"Increased hydrogen peroxide in catalase-deficient cells extends chronological lifespan despite parallel increases in oxidative damage. These findings establish a role for hormesis effects of hydrogen peroxide in promoting longevity that have broad implications for understanding aging and age-related diseases."


It would unlikely to suggest that the benefit of c60 is limited to optimising H202 regulation to perhaps in order to repair DNA, and so shorten comet tails. And guessing still further, c60 might be able to homeostatically correct increasing H202 levels driven by increased concentration c60oo through its SOD2 mimicing on superoxides until some critical point, where the c60 concentrations force another "stable solution" or it just runs away, through some complex ROS feedback.


In the house H202 house fly study, its might have been easy miss the life extending sweet spot amongst the life shortening doses. Whether c60 through its suggested homeostatic regulation of H202 widens the dosing range for life extension would be interesting to know. In the c60/TAA study, there are just two doses one five times the other.


And this may explain why repitition has not been easy, there might be differences across species (SOD2 overexpression, for example, life extending impact appears species dependent) but also perhaps experimental conditions. But also, as Turnbuckle obseved, rats were fasted overnight which may have resulted in increased stem cells supplies with c60 triggering differentation - but too fasting would alter H202 levels 


Going back to Baati, there appears evidence, that fasting increases H202 - with intermittently fasted rats (FIg 6) vastly increased levels of H202 compared to controls. And in trout too:


"In parallel with this difference, in vivo mitochondrial H2O2 levels, estimated from the MitoP/MitoB ratio, were nearly twice as high in the livers of fasted fish compared to fed controls"


Unfortunately, in the C60oo/TAA study there was no c60oo group and so measurement of the comet tail for c60 without TAA - if the comet tail approximated to controls then this would naturally suggest that it is the interaction between Thioacetamide and c60oo shortening the comet tail and this of course could reduce down to the interaction of C60oo and H202. And after looking at the C60 DMSO HD model study, c60 does seem to frequently behave similarly to controls, yet is also homestatically responsive when needed -and so it wouldn't seem too surprising if the comet tails of low dose C60oo w/o TAA and controls were similar.   


And so perhaps in Baati, it is the increased concentrations of H202, induced through fasting, which c60oo regulates, akin to C60oo/TAA study, to repair DNA strands resulting shorter comet tails and so perhaps to extending life. And speculating still further c60 is able to retain this hypothesized H202 induced DNA-repair-state through regulating the production of H202 via its SOD mimetic interactions with superoxide before elimination.


One of the surprising aspects of the Baati study was the that rats lived on for several years after the last dose to double lifespan (I forget exactly when). That would intuitively suggest an upstream benefit is strongly affected for such persisting benefit  - holding back DNA damage a couple of years might seem to fit with that idea.    


As an aside, an observation, it is worth noting that in the two Thioacetamide (TAA) studies both on Wistar rats, standalone TAA reduces SOD in the Tamarix Gallica study but increases it in C60oo. 


Another aside, it is interesting to note that Sinclair was recently quoted as taking c60 fused to oleic acid. Well, oleic acid raises H202 - so perhaps this rather cleverly combines the H202 ROS stress that c60 is ready and able to then regulate into reducing DNA damage.


Briefly looking at Turnbuckles protocols to see if anything in it raises H202 - there is some suggestion of it here and here. It would be interesting to find out the protocol could raises H202 when C60oo is administered. 


Edited by ambivalent, 17 December 2022 - 11:19 PM.

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#3 ambivalent

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Posted 18 December 2022 - 02:43 PM

Maybe it was the palmitric acid implcit in Turnbuckle's protocol which led to H202 production and the DNA repair response in the C60oo/TAA study.


Two studies demonstrating palmitric acid elevating H202, albeit in vitro.




"Palmitic acid induced H2O2 formation in the peroxisomes of insulin-producing cells"


"After a 24 h incubation with palmitic acid the hydrogen peroxide production increased 10-fold in comparison to untreated cells while oleic acid treatment did not result in a significant increase in hydrogen peroxide production"


And in another paper "Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: Effect of catalase overexpression"


"The hydrogen peroxide was elevated after palmitic acid treatment by ~ 31, 36, and 20% compared to control, at the periods of 24, 48 and 72 h, respectively. This effect was abolished when palmitic acid treatment was combined with catalase overexpression at 24, 48 and 72 h." 


So again Palmitric Acid linked with raised H2O2 - also noteworthy that in the low, DNA repairing, c60 dose, catalse expression was extremely high suggesting, perhaps, that c60 enabled catalase to reduce H2O2 to a level where, perhaps, it was optimal to repairing DNA.




"This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids."


This may explain so many of the immediate benefits experienced with c60oo: c60 regulates or optimises H2O2 so that it in turn regulates mitochnodrial respiration. Given glycaemia in the TAA group was only raised 1% above controls and similar to TAA+OO and low dose TAA+C60 perhaps suggested elevated H2O2 levels of c60 are not the cause of raised glycaemia in high dose c60 in the C60oo/TAA study. 


It is a shame there was no H2O2 levels measured in the C60oo/TAA study and too standalone c60oo low dose and high dose groups, then it at least could be firmly concluded that c60 is reducing DNA damage through its direct or indirect response to TAA and so to some effect caused by it, very possibly H2O2.


There is though strong evidence pointing towards c60's possible regulation of H2O2 for reducing DNA damage.


There should be an attempt to Turnbuckle's reduction damage through consuming c60 in the context of this study. And we only know that C60oo reduces control-comparative DNA damage in the presence of a toxin, and not without it, and not with the toxin present at higher doses of c60 - and we do know of at least one part of the protocol that promotes H2O2 (in vitro), palmitric acid, while it is also known that TAA raises H2O2, and fasting too, per Baati rats and that in the right doses H2O2 is thought to life-extension in some species as well as regulate mitochondrial respiration.       





Edited by ambivalent, 18 December 2022 - 03:25 PM.

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#4 Empiricus

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Posted 20 December 2022 - 11:29 AM

Another aside, it is interesting to note that Sinclair was recently quoted as taking c60 fused to oleic acid. Well, oleic acid raises H202 - so perhaps this rather cleverly combines the H202 ROS stress that c60 is ready and able to then regulate into reducing DNA damage.


Any idea what Sinclair talking about here?  Olive oil consists of 55-83% oleic acid.  So what makes his c60 more "fused" than the way everyone else takes it? Ultrasonics?

#5 ambivalent

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Posted 20 December 2022 - 02:35 PM

I know as much as is contained within snippet of the Sinclair interview on the forum. He refers to a paper, though I don't know which. He, from the comments, appears to be refencing Ian Mitchells from WIzard Sciences - I haven't looked further than that currently. 

Also tagged with one or more of these keywords: c60oo, fullerene, liver, toxicology, thioacetamide

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