By Vince Giuliano

This and the Part 1 blog entry Adaptogens Part 1 are pursuant to one of the key themes of this blog -  the use of phyto-substances to promote health and longevity.  The Adaptogens Part 1 blog entry discusses adaptogen herbs in general. This Part 2 blog entry is about a particular well studied adaptogen herb – rhodiola.   Rhodiola is treateds from two viewpoints.  First, the viewpoint of a practicing herbalist, Madelon Hope, is conveyed via a video presentation.  Second, the viewpoint of current scientific research is covered in my usual manner: I cite a number of recent research citations relating to rhodiola involving its mechanisms of action and therapeutic potential.      Most of my personal commentary regarding both adaptogens and rhodiola is in the concluding part of this blog entry.

The video features Madelon Hope, Director of the Boston School of Herbal Studies and a long-time practicing herbalist. I asked her for an informal interview which I recorded on a tiny video camera. The first part of the interview, on adaptogens in general, is included in theAdaptogens Part 1 blog entry. The segment of the video interview specific to rhodiola is included here.

MadelonHopePt2 from VinceGiuliano on Vimeo.

Some basics on rhodiola

There are many important subspecies of rhodiola plants, but the best known for health and medicinal purposes is Rhodiola rosea.  Other subspecies mentioned here include Rhodiola Sachalinensis, Rhodiola Crenulataand Rhodiola integrifolia.  Some scholars lump several subspecies under rhodiola rosea, others may see subspecies such as Rhodiola integrifolia as quite separate(ref). To further complexify the matter, the chemical constituents in the plant may vary in different subspecies and vary further due to soil and climate conditions.

According to Wikipedia: “Rhodiola rosea (also Golden Root, Rose Root, Roseroot, Aaron’s Rod, Arctic root, King’s Crown, Lignum Rhodium, Orpin Rose) is a plant in the Crassulaceae family that grows in cold regions of the world. These include much of the Arctic, the mountains of Central Asia, the Rocky Mountains, and mountainous parts of Europe, such as the Alps, Pyrenees, Carpathian Mountains, Scandinavia, Iceland, Great Britain and Ireland. — Rhodiola rosea may be effective for improving mood and alleviating depression. Pilot studies on human subjects^[2][3][4] showed that it improves physical and mental performance, and may reduce fatigue. — Rhodiola rosea’s effects are potentially mediated by changes in serotonin and dopamine levels due to monoamine oxidase inhibition and its influence on opioidpeptides such as beta-endorphin,^[5] although these specific neurochemical mechanisms have not been clearly documented with scientific studies. — Rhodiola is included among a class of plant derivatives called adaptogens which differ from chemical stimulants, such as nicotine, and do not have the same physiological effects. — In Russia and Scandinavia, Rhodiola rosea has been used for centuries to cope with the cold Siberian climate and stressful life.^{[6][citation needed]} Such effects were provided with evidence in laboratory models of stress using the nematodeC. elegans,^[7] and in rats in which Rhodiola effectively prevented stress-induced changes in appetite, physical activity, weight gain and the estrus cycle.^[8] — Rhodiola has been used in traditional Chinese medicine, where it is called hóng jǐng tiān (红景天). — Rhodiola rosea contains a variety of compounds that may contribute to its effects,^[10] including the class of rosavins which include rosavin, rosarin, and rosin. Several studies have suggested that the most active components are likely to be rhodioloside and tyrosol,^[11] with other components being inactive when administered alone, but showing synergistic effects when a fixed combination of rhodioloside, rosavin, rosarin and rosin was used.^[12]” 

Salidroside (Rhodioloside) is “a glucoside of tyrosol found in the plant Rhodiola rosea. It is thought to be one of the compounds responsible for the antidepressant and anxiolytic actions of this plant, along with rosavin.^[1][2] Salidroside may be more active than rosavin,^[3] even though many commercially marketed Rhodiola rosea extracts are standardised for rosavin content rather than salidroside(ref).”

Until relatively recently it was thought that salidroside was a relatively inactive ingredient in rhodiola rosea and other key subspecies, probably not responsible for their major health benefits. “Although rosavin, rosarin, rosin and salidroside (and sometimes p-tyrosol, rhodioniside, rhodiolin and rosiridin) are among suspected active ingredients of Rhodiola rosea, these compounds are mostly polyphenols for which no physiological effect in humans is proved to prevent or reduce risk of disease.^[13] (ref).”  Now that view appears to be reversed and much of the recent literature reviewed below is concerned with health benefits of salidroside.

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A number of research publications deal with the capabilities of rhodiola to ameliorate conditions of stress and fatigue.  Almost all the publications are positive with respect to the capabilities of the substance to exercise positive benefits.  From the viewpoint of Western pharmaceutical science, however, the studies may be biased and collectively are probably inconclusive.  More careful studies are needed.

The May 2012 review publication Rhodiola rosea for physical and mental fatigue: a systematic review represents this conservative point of view.  “BACKGROUND: Rhodiola rosea (R. rosea) is grown at high altitudes and northern latitudes. Due to its purported adaptogenic properties, it has been studied for its performance-enhancing capabilities in healthy populations and its therapeutic properties in a number of clinical populations.  OBJECTIVE: To systematically review evidence of efficacy and safety of R.rosea for physical and mental fatigue.  METHODS: Six electronic databases were searched to identify randomized controlled trials (RCTs) and controlled clinical trials (CCTs), evaluating efficacy and safety of R. rosea for physical and mental fatigue. Two reviewers independently screened the identified literature, extracted data and assessed risk of bias for included studies.  RESULTS: Of 206 articles identified in the search, 11 met inclusion criteria for this review. Ten were described as RCTs and one as a CCT. Two of six trials examining physical fatigue in healthy populations report R.rosea to be effective as did three of five RCTs evaluating R. rosea for mental fatigue. All of the included studies exhibit either a high risk of bias or have reporting flaws that hinder assessment of their true validity (unclear risk of bias).  CONCLUSION: Research regarding R.rosea efficacy is contradictory. While some evidence suggests that the herb may be helpful for enhancing physical performance and alleviating mental fatigue, methodological flaws limit accurate assessment of efficacy. A rigorously-designed well reported RCT that minimizes bias is needed to determine true efficacy of R.rosea for fatigue.” 

Personally, looking at many of the specific research publications, I find this conclusion rather harsh.  Using the same logic, one could conclude that there are no clearly documented health benefits of drinking water, and a large scale clinical trial would be needed to clearly establish that there are such health benefits.  My readers are invited to read on below and draw their own conclusions.

Some more-recent rat model studies have looked at actions of rhodiola components, particularly salidroside, on basic biological processes which happen in aging such as as decline in immune function, decline in DNA repair effectiveness, and increase in senescent cells.  The reported results appear to be interesting if not dramatic.

The February 2012 e-publication Rejuvenating activity of salidroside (SDS): dietary intake of SDS enhances the immune response of aged rats reports: “It is well known that immune response decreases with aging. Salidroside (SDS), an antioxidant component isolated from the traditional Chinese medicine roseroot Rhodiola rosea, has been demonstrated to possess potent anti-aging and health-promoting activities. However, the mechanism underlying these activities is poorly understood. In this study, we clearly demonstrated that (1) dietary intake of SDS induced a considerable increase in total T cells (CD3(+)) and T helper cells (CD4(+)) in aged (21 months old) Wistar male rats; (2) SDS supplementation significantly increased the DTH response, a T cell-mediated immune response, in aged rats; and (3) SDS supplementation remarkably promoted the production of total anti-KLH IgG, anti-KLH IgG(1), and anti-KLH IgG(2α) in aged rats without disturbing immune homeostasis. These indicate that SDS is able to counteract immunosenescence, thereby resulting in rejuvenation. Practically, SDS may be used to help the elderly to generate an improved response to vaccine with stronger humoral and cell-mediated immune responses.”  If this finding could be shown to carry over to humans, I believe that would be very exciting.

The May 2012 publication Salidroside stimulates DNA repair enzyme Parp-1 activity in mouse HSC maintenance also suggests a rejuvenating role of salidroside related to DNA repair and maintenance of hematopoietic stem cell pools.    “Salidroside is a phenylpropanoid glycoside isolated from the medicinal plant Rhodiola rosea, which has potent antioxidant properties. Here we show that salidroside prevented the loss of hematopoietic stem cells (HSCs) in mice under oxidative stress. Quiescent HSCs were recruited into cell cycling on in vivo challenge with oxidative stress, which was blocked by salidroside. Surprisingly, salidroside does not prevent the production of reactive oxygen species but reduces hydrogen peroxide-induced DNA-strand breaks in bone marrow cells enriched for HSCs. We tested whether salidroside enhances oxidative DNA damage repair in mice deficient for 5 DNA repair pathways known to be involved in oxidative DNA damage repair; we found that salidroside activated poly(ADP-ribose)polymerase-1 (PARP-1), a component of the base excision repair pathway, in mouse bone marrow HSCs as well as primary fibroblasts and human lymphoblasts. PARP-1 activation by salidroside protects quiescent HSCs from oxidative stress-induced cycling in native animals and self-renewal defect in transplanted recipients, which was abrogated by genetic ablation or pharmacologic inhibition of PARP-1. Together, these findings suggest that activation of PARP-1 by salidroside could affect the homeostasis and function of HSCs and contribute to the antioxidant effects of salidroside.”  As suggested in the previous Part 1 blog entry, I very strongly suspect that salidroside in part works by upregulating the expression of Nrf2 thus activating the body’s own natural antioxidant and repair defense systems.

The November 2010 publication Salidroside protects human fibroblast cells from premature senescence induced by H(2)O(2) partly through modulating oxidative status reports “Although salidroside and salidroside-like compounds are considered as most critical constitutes needed and responsible for multiple therapeutic benefits of Rhodiola rosea L., including anti-aging, direct demonstration regarding the role of salidroside in anti-aging process is still deficient. In this study, we selected the H(2)O(2)-induced premature senescence model in human fetal lung diploid fibroblasts to investigate the protection of salidroside against aging in vitro and associated molecular mechanisms. We found that salidroside considerably reversed senescence-like phenotypes in the oxidant challenged model, including alterations of morphology, cell cycle, SA-β-gal staining, DNA damage, as well as related molecules expression such as p53, p21 and p16. The protection occurred in a dose-dependent manner, with 5μM offering best efficacy. The proposed antioxidant property of the compound was confirmed in this cellular system, and thus at least partially accounted for the protection of the compound against premature senescence. Similar protection of salidroside against replicative senescence was observed as well. Interestingly, the regulation of senescence-related molecules by salidroside involved ROS-irrelevant mechanisms in both models. This finding presents salidroside as an attractive agent with potential to retard aging and attenuate age-related diseases in humans”

Multiple recent cell-level studies establish that salidroside definitely exercises cytoprotective effects, in part by inhibiting apoptosis in the presence of oxidative stress.  Again. I suspect the effect is due to promotion of expression of Nrf2.

I cite only a few examples.  For example, the April 2012 publication Salidroside protects human erythrocytes against hydrogen peroxide-induced apoptosis reports “Rhodiola rosea is a commonly used folk medicine for the treatment of high altitude sickness, mountain malhypoxia, and anoxia. Its active ingredient, salidroside [2-(4-hydroxyphenyl)ethyl β-D-glucopyranoside (1)], has been reported to have a broad spectrum of biological effects. However, the protective role of 1 in human erythrocytes remains unclear. This study therefore has investigated the effects of 1 on oxidative stress-induced apoptosis in human erythrocytes (also known as eryptosis or erythroptosis). Compound 1 increased cell survival significantly and prevented human erythrocytes from undergoing eryptosis/erythroptosis mediated by H(2)O(2), as confirmed by the decreased expression of phosphatidylserine on the cell surface and reduced leakage of calcein through the damaged membrane. Mechanistically, 1 was found to exert its protective effects through its antioxidative activity and the inhibition of caspase-3 activation and stress-induced intracellular Ca(2+) rise in a dose-dependent manner. Compound 1 is a protective agent in human erythrocytes against oxidative stress and may be a good adaptogen to enhance the body’s resistance to stress and fatigue.”

The July 2012 publication Salidroside and tyrosol from Rhodiola protect H9c2 cells from ischemia/reperfusion-induced apoptosis reports: “AIMS: Heart disease is the leading cause of death worldwide. Ischemia-reperfusion injury can lead to apoptotic death of heart cells and subsequently heart failure. Rhodiola is an herbal medicine with two main bioactive compounds – salidroside (SAL) and tyrosol (TYR). This study aimed to investigate whether these two compounds can prevent ischemia/reperfusion-induced apoptosis in H9c2 cells.  MAIN METHODS: Assays for total phenolics assay and Oxygen Radical Absorbance Capacity showed high antioxidant capacity of SAL and TYR. H9c2 cells were subjected to simulated ischemia/reperfusion (IR) in the presence and absence of SAL and/or TYR, and nuclei condensation, caspase-3 activity, cytochrome c release and JNK phosphorylation were determined.  KEY FINDINGS: In H9c2 cells, IR can lead to a 5-fold increase in p-JNK level. Apoptotic nuclei condensation, caspase-3 activity and cytochrome c release were markedly elevated, indicating the occurrence of apoptosis. SAL and TYR caused a dose-dependent inhibition of nuclear condensation. Furthermore, SAL and TYR, separately and in combination, significantly reduced caspase-3 activity, cytochrome c release and JNK activation. The anti-apoptotic effect of the combination was markedly higher than that of SAL or TYR alone.  SIGNIFICANCE: The inhibition of the JNK signaling pathway is the key mechanism for the cytoprotective effect of SAL and TYR in IR-induced apoptosis.”

The August 2012 publication Protective effects of salidroside from Rhodiola rosea on LPS-induced acute lung injury in mice reports: “Salidroside is a major component extracted from Rhodiola rosea. In this study, we investigated protective effects of salidroside on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. In the mouse model, we found that pretreatment with a single 120 mg/kg dose of salidroside prior to the administration of intratracheal LPS induced a significant decrease in the W/D ratio and mouse myeloperoxidase activity of lung, reduction protein concentration, the number of total cells, neutrophils and macrophages in the bronchoalveolar lavage fluid. In addition, salidroside also inhibited the production of several inflammatory cytokines, including tumor necrosis factor-α, interleukin-6 (IL-6) and IL-1β, and the NF-κB DNA-binding activation after LPS challenge. These results indicated that salidroside possess a protective effect on LPS-induced ALI in mice.”

The 2011 publication Salidroside promotes erythropoiesis and protects erythroblasts against oxidative stress by up-regulating glutathione peroxidase and thioredoxin relates: “ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola rosea is commonly used in China and Tibet folk medicine for the treatment of high altitude sickness, anoxia and mountain malhypoxia.  AIM OF STUDY: Salidroside (SDS) is an active ingredient of Rhodiola rosea. This study attempted to examine the potential erythropoiesis-stimulating and anti-oxidative effect of SDS in TF-1 erythroblasts.  MATERIALS AND METHODS: The erythropoiesis-promoting effect was determined by treating human TF-1 cells, one of the popular in vitro models for studying erythropoiesis, with SDS in the presence and absence of erythropoietin (EPO) through the measurement of the expression of a series of erythroid markers such as glycophorin A (GPA), transferrin receptor (CD71) and hemoglobin (Hb). The potential protective effect of SDS against H(2)O(2)-induced apoptosis and its underlying mechanism in TF-1 erythroblasts were examined by flow cytometry and Western blot analysis.  RESULTS: SDS promotes erythropoiesis in the EPO-treated cells and it also reduces the number of apoptotic cells in TF-1 erythroblasts after H(2)O(2) treatment probably through the up-regulation of protective proteins thioredoxin-1 (Trx1) and glutathione peroxidase-1 (GPx1). CONCLUSION: Our study provides evidence to explain the ethnopharmacological role of SDS and Rhodiola rosea in Chinese medicine. Our findings also support the use of SDS as an erythropoiesis-adjuvant agent to correct anemia and malhypoxia.”  Again, we have here the familiar themes of protection against oxidative stress, prevention of apoptosis and stimulating stem cell differentiation.

The October 2011 publication Salidroside protects against hydrogen peroxide-induced injury in cardiac H9c2 cells via PI3K-Akt dependent pathway reports: “Oxidative stress induces serious tissue injury in cardiovascular diseases. Salidroside, with its strong antioxidative and cytoprotective actions, is of particular interest in the development of antioxidative therapies for oxidative injury in cardiac diseases. We examined the pharmacological effects of salidroside on H9c2 rat cardiomyoblast cells under conditions of oxidative stress induced by hydrogen peroxide (H2O2) challenge. Salidroside attenuated H2O2-impaired cell viability in a concentration-dependent manner, and effectively inhibited cellular malondialdehyde production, lethal sarcolemmal disruption, cell necrosis, and apoptosis induced by H2O2 insult. Salidroside significantly augmented Akt phosphorylation at Serine 473 in the absence or presence of H2O2 stimulation; wortmannin, a specific inhibitor of PI3K, abrogated salidroside protection. Salidroside increased the intracellular mRNA expression and activities of catalase and Mn-superoxide dismutases in a PI3K-dependent manner. Our results indicated that salidroside protected cardiomyocytes against oxidative injury through activating the PI3K/Akt pathway and increasing the expression and activities of endogenous PI3K dependent antioxidant enzymes.”

Another study relating salidroside to cardiac cells is reported in the 2009 publication Salidroside protects cardiomyocyte against hypoxia-induced death: a HIF-1alpha-activated and VEGF-mediated pathway.  “Cardiomyocyte death (necrosis and apoptosis) plays a critical role in the progress of heart diseases. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L, has shown cardioprotective effects in vivo. However, whether salidroside has a protective effect against cardiomyocyte death is poorly understood. The present study was aimed to investigate the cardioprotective role of salidroside and the underlying mechanisms in hypoxia-induced cardiomyocyte death. Cardiomyocytes pretreated with or without salidroside for 24 h were exposed to hypoxic condition for 6 h and then cell viability, necrosis, apoptosis, the expressions of HIF-1alpha and VEGF were investigated. Pretreatment with salidroside markedly attenuated hypoxia-induced cell viability loss, cell necrosis and apoptosis in a dose-dependent manner. Mechanistically, pretreatment with salidroside up-regulated the HIF-1alpha protein expression and induced its translocation. Moreover, the level of VEGF, a downstream target of HIF, was significantly increased in parallel with the level of HIF-1alpha following pretreatment with salidroside. However, 2-methoxyestradiol (2-ME2), a HIF-1alpha inhibitor, attenuated the protection of salidroside and blocked the increase of HIF-1alpha and VEGF. These data indicated that salidroside has protective effect against hypoxia-induced cardiomyocytes necrosis and apoptosis by increasing HIF-1alpha expression and subsequently up-regulating VEGF levels.”

A number of cell-level studies look at the protective effects of salidroside of neural cells and the ability of salidroside to promote neurogenesis under various in-vitro conditions and speculate whether it might become the basis for treatment of Parkinson’s disease, Alzheimer’s disease and other neurodegenerative disorders.

The January 2012 publication Protective effects of a Rhodiola crenulata extract and salidroside on hippocampal neurogenesis against streptozotocin-induced neural injury in the rat is a case in point. “Previously we have demonstrated that a Rhodiola crenulata extract (RCE), containing a potent antioxidant salidroside, promotes neurogenesis in the hippocampus of depressive rats. The current study was designed to further investigate the protective effect of the RCE on neurogenesis in a rat model of Alzheimer’s disease (AD) induced by an intracerebroventricular injection of streptozotocin (STZ), and to determine whether this neuroprotective effect is induced by the antioxidative activity of salidroside. Our results showed that pretreatment with the RCE significantly improved the impaired neurogenesis and simultaneously reduced the oxidative stress in the hippocampus of AD rats. In vitro studies revealed that (1) exposure of neural stem cells (NSCs) from the hippocampus to STZ strikingly increased intracellular reactive oxygen species (ROS) levels, induced cell death and perturbed cell proliferation and differentiation, (2) hydrogen peroxide induced similar cellular activities as STZ, (3) pre-incubation of STZ-treated NSCs with catalase, an antioxidant, suppressed all these cellular activities induced by STZ, and (4) likewise, pre-incubation of STZ-treated NSCs with salidroside, also an antioxidant, suppressed all these activities as catalase: reduction of ROS levels and NSC death with simultaneous increases in proliferation and differentiation. Our findings indicated that the RCE improved the impaired hippocampal neurogenesis in the rat model of AD through protecting NSCs by its main ingredient salidroside which scavenged intracellular ROS.”

The 2009 publication Protective effect of salidroside against H2O2-induced cell apoptosis in primary culture of rat hippocampal neurons relates : “Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H(2)O(2))-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H(2)O(2)-stimulated apoptotic cell death in primary culture of hippocampal neurons in a concentration-dependent manner. The measurements of caspase-3 activity, nitric oxide (NO) production, and NO synthase (NOS) activity suggest that the protection of salidroside, shown in this study, might be mediated by inhibiting caspase-3 activity, and antagonizing NO production and NOS activity during H(2)O(2) stimulation. Perhaps, this study might contribute to the development of salidroside as a broad-spectrum agent for preventing and/or treating neuronal damage in neurodegenerative disorders.”

The 2010 publication Neuroprotective effects of salidroside against beta-amyloid-induced oxidative stress in SH-SY5Y human neuroblastoma cells reported: “Beta-amyloid (Abeta) peptide, the hallmark of Alzheimer’s disease (AD), invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. In this study, salidroside (Sald), an active compound isolated from a traditional Chinese medicinal plant, Rhodiola rosea L., was investigated to assess its protective effects and the underlying mechanisms against Abeta-induced oxidative stress in SH-SY5Y human neuroblastoma cells. Abeta(25-35)-induced neuronal toxicity was characterized by the decrease of cell viability, the release of lactate dehydrogenase (LDH), morphological alterations, neuronal DNA condensation, and the cleavage of poly(ADP-ribose) polymerase (PARP) by activated caspase-3. Pretreatment with salidroside markedly attenuated Abeta(25-35)-induced loss of cell viability and apoptosis in a dose-dependent manner. The mechanisms of salidroside protected neurons from oxidative stress included the induction of antioxidant enzymes, thioredoxin (Trx), heme oxygenase-1 (HO-1), and peroxiredoxin-I (PrxI); the downregulation of pro-apoptotic protein Bax and the upregulation of anti-apoptotic protein Bcl-X(L). Furthermore, salidroside dose-dependently restored Abeta(25-35)-induced loss of mitochondrial membrane potential (MMP) as well as suppressed the elevation of intracellular reactive oxygen species (ROS) level. It was also observed that Abeta(25-35) stimulated the phosphorylation of mitogen-activated protein (MAP) kinases, including c-Jun NH(2)-terminal kinase (JNK) and p38 MAP kinase, but not extracellular signal-regulated kinase1/2 (ERK1/2). Salidroside inhibited Abeta(25-35)-induced phosphorylation of JNK and p38 MAP kinase, but not ERK1/2. These results suggest that salidroside has protective effects against Abeta(25-35)-induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.”

Earlier publications on the effects of salidroside on neuroblastoma cells include the 2007 report Protective effects of salidroside on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells.

The neuroprotective effects of salidroside and the possibility of creating therapies for neurodegenerative diseases based on rhodiola compounds is far from new.  For example there is a 2003 publication Salidroside from Rhodiola sachalinensis protects neuronal PC12 cells against cytotoxicity induced by amyloid-beta.  “The amyloid beta-peptide (Abeta)-induced oxidative stress is a well-established pathway of neuronal cell death in Alzheimer’s disease (AD). Salidroside, one of the major compounds from the roots of Rhodiola species (Crassulaceae), was investigated in vitro for its cytoprotection against Abeta-induced toxicity on rat neuronal PCl2 cells. Salidroside significantly reduced Abeta-induced cytotoxicity in a dose-dependent manner. Salidroside also reduced Abeta-mediated intracellular accumulation of reactive oxygen species and malondialdehyde (MDA), a product of lipid peroxides, by preventing Abeta-induced decline of antioxidant enzyme activities. These results suggest that salidroside protects neuronal PC12 cells from Abeta-induced cytotoxicity via its antioxidant pathway.”

Fast-forwarding eight years to 2011, another report related to PC12 cells is Salidroside protects against MPP(+)-induced apoptosis in PC12 cells by inhibiting the NO pathway. “Oxidative stress plays an important role in Parkinson’s disease and other neurodegenerative disorders. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., has potent antioxidant properties. In the present study, we investigated the protective activity of salidroside against 1-methyl-4-phenylpyridinium (MPP(+))-induced apoptosis in PC12 cells. We found that incubation of PC12 cells with salidroside prior to MPP(+) exposure significantly reduced cell apoptosis and attenuated collapse of the mitochondrial membrane potential (MMP). Furthermore, salidroside inhibited the MPP(+)-induced nitric oxide (NO) increase and overexpression of nNOS and iNOS and suppressed accumulation of reactive oxygen species (ROS) and intracellular free Ca(2+). Our results show that the protective effects of salidroside on PC12 cells are mediated, at least in part, by inhibition of the NO pathway.”

Moving to right now, August 2012, we have the publication Salidroside protects PC12 cells from MPP(+)-induced apoptosis via activation of the PI3K/Akt pathway;  “Oxidative stress plays an important role in the pathogenesis of Parkinson’s disease (PD). Salidroside (SAL), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can exert potent antioxidant properties. In this study, we investigated the protective effects, and the possible mechanism of action, of SAL against 1-methyl-4-phenylpyridinium (MPP(+))-induced cell damage in rat adrenal pheochromocytoma PC12 cells. Pretreatment of PC12 cells with SAL significantly reduced the ability of MPP(+) to induce apoptosis in a dose and time-dependent manner. SAL significantly and dose-dependently inhibited MPP(+)-induced chromatin condensation and MPP(+)-induced release of lactate dehydrogenase by PC12 cells. SAL enhanced Akt phosphorylation in PC12 cells, and the protective effects of SAL against MPP(+)-induced apoptosis were abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. These findings suggest that SAL prevents MPP(+)-induced apoptosis in PC12 cells, at least in part through activation of the PI3K/Akt pathway.”

Different species of rhodiola may induce differing biological effects.

The 2006 publication Evaluation of Rhodiola crenulata and Rhodiola rosea for management of type II diabetes and hypertension reported: “In the current study, we investigated 2 species of the genus Rhodiola for the inhibition of alpha-amylase,alpha-glucosidase and angiotensin converting enzyme (ACE) inhibitory activity. Water extracts of Rhodiola crenulata had the highest alpha-amylase inhibitory activity (IC50,98.1 microg total phenolic /ml) followed by ethanol extract of R.crenulata (IC50, 120.9 microg total phenolic/ml) and ethanol extract of R.rosea (IC50, 173.4 microg total phenolic /ml). Ethanol R.rosea (IC50, 44.7 microg total phenolic/ml), water extract of R.rosea (IC50, 52.3 microg total phenolic/ml), water extract of R.crenulata (IC50, 60.3 microg total phenolic /ml) and ethanol extract of R.crenulata (IC50, 60.2 microg total phenolic/ml) also showed significant alpha-glucosidase inhibitory activity. The alpha-glucosidase inhibitory activity of the extracts was compared to standard tyrosol, which was significantly detected in the extracts using HPLC. Tyrosol had strong alpha-glucosidase inhibitory activity (IC50, 70.8 microg total phenolic/ml) but did not have any inhibitory effect on the alpha-amylase activity. Results suggested that alpha-glucosidase inhibitory activities of both Rhodiola extracts correlated to the phenolic content, antioxidant activity and phenolic profile of the extracts. The ability of the above Rhodiola extracts to inhibit rabbit lung angiotensin I-converting enzyme (ACE) was investigated. The ethanol extracts of R.rosea had the highest ACE inhibitory activity (38.5 %) followed by water extract of R.rosea (36.2 %) and R.crenulata (15.4 %).”

Rhodiola may be useful for treatment of metabolic syndrome or Type II diabetes.

Pursuing a theme of the 2006 publication just mentioned , a new (August 2012) publication Rhodiola crenulata root ameliorates derangements of glucose and lipid metabolism in a rat model of the metabolic syndrome and type 2 diabetes aeports: “AIM OF THE STUDY: To examine the effects of Rhodiola on glucose and lipid metabolism in the metabolic syndrome and type 2 diabetes.  MATERIALS AND METHODS: Zucker diabetic fatty (ZDF) rats were treated with Rhodiola crenulata root (RCR) powder (100 and 500mg/kg, by gavage, once daily for 4 weeks). In addition, the effects of RCR on sucrose-induced acute hyperglycemia in mice and olive oil-induced hypertriglyceridemia in rats were also examined. Biochemical variables were determined enzymatically or by ELISA.  RESULTS: In ZDF rats, RCR treatment decreased the increased plasma insulin and triglyceride concentrations at baseline, the index of the homeostasis model assessment of insulin resistance (HOMA-IR) and excessive hepatic triglyceride accumulation. This treatment also inhibited abnormal increases in plasma glucose and insulin concentrations during oral glucose tolerance test. Furthermore, RCR reversed the increased adipose insulin resistance index, and accelerated the decline of plasma concentrations of non-esterified fatty acids after exogenous glucose stimulation. However, RCR minimally affected sucrose-induced acute hyperglycemia in mice and olive oil-induced acute hypertriglyceridemia in rats.  CONCLUSIONS: The present results demonstrate that RCR treatment improves metabolic derangements in animal model of the metabolic syndrome and type 2 diabetes. Our findings may provide new pharmacological basis of therapeutics for the adaptogenic plants to treat metabolic derangements-associated disorders, such as asthenia.”

Some small clinical trials point to the safety and effectiveness of salidroside as a therapy or adjunct therapy for special disease conditions.

An example is discussed in the June 2012 publication Protective effects of salidroside on epirubicin-induced early left ventricular regional systolic dysfunction in patients with breast cancer.  “Background: Salidroside [2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside], one of the most potent ingredients extracted from the plant Rhodiola rosea L., has been shown to have a cardiovascular protective effect as an antioxidant, and early treatment of epirubicin-induced cardiotoxicity has been the focus of clinical chemotherapy in patients with breast cancer. However, the cardioprotective effects of salidroside on epirubicin-induced cardiotoxicity, especially early left ventricular regional systolic dysfunction, have to date been sparsely investigated. Objective: The aim of this study was to investigate the protective effects of salidroside in preventing early left ventricular regional systolic dysfunction induced by epirubicin. Methods: Sixty patients with histologically confirmed breast cancer were enrolled. Eligible patients were randomized to receive salidroside (600 mg/day; n = 30) or placebo (n = 30) starting 1 week before chemotherapy. Patients were investigated by means of echocardiography and strain rate (SR) imaging. We also measured plasma concentrations of reactive oxygen species (ROS). All parameters were assessed at baseline and 7 days after each new epirubicin dose of 100 mg/m2. Results: A decline of the SR peak was observed at an epirubicin dose of 200 mg/m2, with no significant differences between salidroside and placebo (1.35 ± 0.36 vs 1.42 ± 0.49/second). At growing cumulative doses of epirubicin, the SR normalized only with salidroside, showing a significant difference in comparison with placebo at epirubicin doses of 300 mg/m2 (1.67 ± 0.43 vs 1.32 ± 0.53/second, p < 0.05) and 400 mg/m2 (1.68 ± 0.29 vs 1.40 ± 0.23/second, p < 0.05). Moreover, a significant increase in plasma concentrations of ROS was found with placebo, but they remained unchanged with salidroside. Conclusion: Salidroside can provide a protective effect on epirubicin-induced early left ventricular regional systolic dysfunction in patients with breast cancer.”

Small-scale human trials as well as animal studies suggests that salidroside can enhance endurance exercise performance.

The May 2012 publication The Effects of an Acute Dose of Rhodiola Rosea on Endurance Exercise Performance reports: “The purpose of this study was to determine the effects of an acute oral dose of 3mg/kg of Rhodiola Rosea (Rr) on endurance exercise performance, perceived exertion, mood, and cognitive function. Subjects (n=18) ingested either Rr or a carbohydrate placebo 1 h prior to testing in a double blind, random crossover manner. Exercise testing consisted of a standardized 10 min warm-up, followed by a 6-mile time trial (TT) on a bicycle ergometer. Perceived exertion (RPE) was measured every 5 min during the TT using a BORG 10 pt scale. Blood lactate concentration, salivary cortisol and salivary alpha amylase were measured pre warm-up, 2 min post warm-up, and 2 min post TT (n=15). A Profile of Mood States (POMS) questionnaire and a Stroop’s Color Test were completed pre-warm up and post TT. Testing was repeated 2-7d later with the other condition. Rr ingestion significantly decreased heart rate during the standardized warm up (Rr=136+17bpm; Placebo=140+17bpm; mean+SD; p=0.001). Subjects completed the TT significantly faster following Rr ingestion (Rr=25.4+2.7 min; placebo=25.8+3.0 min; p=0.037). The mean RPE was lower in the Rr trial (Rr= 6.0+0.9; placebo= 6.6+1.0; p=0.04). This difference was even more pronounced when a ratio of the RPE relative to the workload was calculated (Rr= 0.048+0.01; placebo= 0.057+0.02; p=0.007). No other statistically significant differences were observed. Acute Rr ingestion decreases heart rate response to sub-maximal exercise, and appears to improve endurance exercise performance by decreasing the perception of effort.”

Biological impacts of salidroside under exercise conditions are examined in a mouse model in the January 2012 publication [Influence of salidroside from Rhodiola Sachalinensis A. Bor on some related indexes of free radical and energy metabolism after exercise in mice].  “OBJECTIVE: To study the anti-fatigue mechanism of salidroside from Rhodiola SachalinensisA.Bor (SRS)in anti-oxidation and energy metabolic systems in mice , some related indexes of free radical and energy metabolism after exercise were measured.  METHODS: Forty male mice were divided into four groups (n = 10): SRS sport group(SS), SRS quiet group(SQ),sport control group(SC), quiet control group (QC). The mice of SS and SQ groups received SRS solution of 150 ml/kg body weight per day for two weeks, while the mice of SC and QC groups received the same volume of distilled water. 30 min after the last treatment, the mice of SS and SC groups were forced to swim for 120 min without loads. then the biochemical parameters related to fatigue were determined.  RESULT: SRS could increase liver superoxide dismutase (SOD) , glutathione peroxidase (GSH-Px) activity of antioxidant enzymes and reduce the malondialdehyde (MDA) content, which might increase the body activity of antioxidant enzymes to play the role of anti-oxidation; SRS had some effect of stabilizing blood sugar, increasing liver glycogen and muscle glycogen reserves, preventing blood sugar, liver glycogen and muscle glycogen levels from reducing in long time exercise on mice; SRS could increase plasma total cholesterol (TC), triglyceride (TG) and free fatty acid (FFA) levels in exercise mice, and it had some effect on metabolism of fat under different conditions, and promoted the use of the role of fat.  CONCLUSION: Influence of SRS on some related indexes of free radical and energy metabolism is one of the mechanisms of anti-exercise-induced fatigue of SRS.”

A 2010 publication Effects of chronic Rhodiola Rosea supplementation on sport performance and antioxidant capacity in trained male: preliminary results relates: “AIM: Rhodiola Rosea, is an adaptogen plant which has been reported to promote fatty acids utilisation, to ameliorate antioxidant function, and to improve body resistance to physical strenuous efforts. The purpose of the present study was to investigate the effects on physical performance as well as on the redox status of a chronic Rhodiola Rosea supplementation in a group of competitive athletes during endurance exercise.  METHODS: Following a chronic supplementation with Rhodiola Rosea for 4 weeks, 14 trained male athletes underwent a cardio-pulmonary exhaustion test and blood samples to evaluate their antioxidant status and other biochemical parameters. These data were compared with those coming from the same athletes after an intake of placebo. RESULTS: The evaluation of physical performance parameters showed that HR Max, Borg Scale level, VO(2) max and duration of the test were essentially unaffected by Rhodiola Rosea assumption. On the contrary, Rhodiola Rosea intake reduced, in a statistically significative manner, plasma free fatty acids levels. No effect on blood glucose was found. Blood antioxidant status and inflammatory parameters resulted unaffected by Rhodiola Rosea supplementation. Blood lactate and plasma creatine kinase levels were found significantly lower (P<0.05) in Rhodiola Rosea treated subjects when compared to the placebo treated group. CONCLUSION: Chronic Rhodiola Rosea supplementation is able to reduce both lactate levels and parameters of skeletal muscle damage after an exhaustive exercise session. Moreover this supplementation seems to ameliorate fatty acid consumption. Taken together those observation confirm that Rhodiola Rosea may increase the adaptogen ability to physical exercise.”

Also relevant to the exercise issue is the 2009 study Attenuation of long-term Rhodiola rosea supplementation on exhaustive swimming-evoked oxidative stress in the rat.   “Rhodiola rosea improves exercise endurance and fatigue. We hypothesized that ingredients in Rhodiola rosea may increase antioxidant capability against swimming induced oxidative stress. In this study, we have identified the Rhodiola rosea ingredients, p-tyrosol, salidroside, rosin, rosavin and rosarin by high performance liquid chromatography-mass spectrometer and evaluated their O2(-)*, H2O2, and HOCl scavenging activities by a chemiluminescence analyzer. We next explored the effect and mechanism of Rhodiola rosea on 90-min swimming-induced oxidative stress in male Wistar rats fed with three doses of Rhodiola rosea extracts in drinking water (5, 25, 125 mg/day/rat) for 4 weeks. Our results showed that the 4 major ingredients (salidroside, rosin, rosavin and rosarin) from Rhodiola rosea extracts scavenged O2(-)*, H2O2, and HOCl activity in a dose-dependent manner. The ninety-min swimming exercise increased the O2(-)* production in the order: liver > skeletal muscle > blood, indicating that liver is the most sensitive target organ. The level of plasma malonedialdehyde, a lipid peroxidation product, was also increased after exercise. Treatment of 4 weeks of Rhodiola rosea extracts significantly inhibited swimming exercise-enhanced O2(-)* production in the blood, liver and skeletal muscle and plasma malonedialdehyde concentration. The expression in Mn-superoxide dismutase Cu/Zn-superoxide dismutase, and catalase in livers were all enhanced after 4 weeks of Rhodiola rosea supplementation especially at the dose of 125 mg/day/rat. Treatment of Rhodiola rosea extracts for 4 weeks significantly increased swimming performance. In conclusion, treatment of Rhodiola rosea extracts for 4 weeks could reduce swimming-enhanced oxidative stress possibly via the reactive oxygen species scavenging capability and the enhancement of the antioxidant defense mechanisms.”

Rhodiola might be useful for treating opioid addiction.

A May 2012 publication Effects of a Rhodiola rosea L. extract on the acquisition, expression, extinction, and reinstatement of morphine-induced conditioned place preference in mice reports: “RATIONALE: Opioid addiction is a chronic, recurrent brain disease that is characterised by compulsive drug seeking and a high rate of relapse even after long periods of abstinence. Prevention of relapse is the primary goal of addiction treatment and is still the major limitation in drug therapy.  OBJECTIVES: The present study investigated the effects of a Rhodiola rosea L. hydroalcoholic extract (RHO), a well-known traditional oriental medicine, on establishment and reinstatement of morphine-induced conditioned place preference (CPP) in mice.  METHODS: CPP was induced by intraperitoneal injection of morphine (10 mg/kg) as an 8-day conditioning schedule. The effects of RHO on the rewarding properties of morphine were tested in mice receiving oral administration of RHO (10, 15, and 20 mg/kg) 60 min prior to each morphine injection (acquisition) or prior to the CPP test on day 9 (expression). Once established, CPP was extinguished by repeated testing, during which conditioned mice were injected daily with different doses of RHO. Finally, the efficacy of RHO in blocking reinstatement of CPP provoked by priming injections and physical stress was also evaluated.  RESULTS: RHO administration showed dose dependency for prevention of establishment of CPP and was effective in facilitating extinction of morphine-induced CPP. RHO suppressed both priming- and stress-induced reinstatement of CPP in a dose-dependent manner.  CONCLUSIONS: In conclusion, as RHO was effective for reducing craving and vulnerability to relapse, it might be a very effective natural remedy for the treatment of opioid addiction.”

One of the traditional uses of rhodiola is for treatment of high-altitude mountain sickness.  The benefits are probably due to the presence of salidroside

The March 2012 publication Salidroside stimulates the accumulation of HIF-1α protein resulted in the induction of EPO expression: a signaling via blocking the degradation pathway in kidney and liver cells reports: “Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1α (HIF-1α) protein, but not HIF-2α protein: the salidroside-induced HIF-1α protein was via the reduction of HIF-1α degradation but not the mRNA induction. The increased HIF-1α could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside.”

Comments

I could go on with many more citations and observations but think what is included above is sufficient to convey a good idea of what rhodiola’s capabilities are.

Collectively, these citations lead me to general observations applicable not only to salidroside and rhodiola but to most other traditional folk remedies:  There have been more and more cell-level studies over the years establishing safety and efficacy of rhodiola and salidroside as an anti-oxidant and anti-apoptotic on the cell level. These cell-level studies are relatively easy and cheap to do and appear to me to be somewhat redundant over the years.  Many of the newer studies are also unveiling the molecular pathways through which rhodiola and its important constituent compounds such as salidroside work.  A smaller number of small-animal studies also establishing safety and efficacy for a number of health and medical conditions, and a yet-smaller collection of small-scale human trials have been held under various specialized conditions. 

But, in the West at least, mainline salidroside-based clinical therapies that have been subject to pharma-scale clinical trials are yet to emerge.  So salidroside and other traditional medical substances like andrographis, gambogic and gambogenic acids and icarin remain in the shadows.  They are unknown to most physicians and unused in Western clinical practice.  They may even be viewed as dangerous because of the possibility of their interactions with pharmaceutical drugs. 

It is difficult to establish whether this is due to the weaknesses of the substances as drug candidates or due to the economics of clinical drug development in the Western model of medical practice involving pharmaceutical companies with a strong profit motive and the FDA as a watchdog.  What drug company would want to spend a billion dollars and ten years to bring to market a natural substance that is not patentable, or an analog of such a natural substance that may be little or no better than the substance itself?  And how many doctors are willing to risk administering substance therapies that are not FDA sanctioned?

So, in the US and other advanced Western countries, herbal healing remains a splinter activity unconnected with mainline medical practice.  This may be slowly changing as 1.  More medical schools and health institutions are starting programs in complementary, alternative or integrative medicine, 2.  Big pharma and biotech companies turn more to traditional folk medicines in their searches for new drug candidates, and 3. There is increased public awareness among people that individuals need to take responsibility for their own health and longevity.

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FROM TIME TO TIME, THIS BLOG DISCUSSES DISEASE PROCESSES. THE INTENTION OF THOSE DISCUSSIONS IS TO CONVEY CURRENT RESEARCH FINDINGS AND OPINIONS, NOT TO GIVE MEDICAL ADVICE. THE INFORMATION IN POSTS IN THIS BLOG IS NOT A SUBSTITUTE FOR A LICENSED PHYSICIAN’S MEDICAL ADVICE. IF ANY ADVICE, OPINIONS, OR INSTRUCTIONS HEREIN CONFLICT WITH THAT OF A TREATING LICENSED PHYSICIAN, DEFER TO THE OPINION OF THE PHYSICIAN. THIS INFORMATION IS INTENDED FOR PEOPLE IN GOOD HEALTH. IT IS THE READER’S RESPONSIBILITY TO KNOW HIS OR HER MEDICAL HISTORY AND ENSURE THAT ACTIONS OR SUPPLEMENTS HE OR SHE TAKES DO NOT CREATE AN ADVERSE REACTION.

By Vince Giuliano

Small doses of radiation, such as from occasional X-rays or living at a high altitude, may actually be good for you according to the radiation hormesis hypothesis discussed here.  Although this hypothesis is about 30 years old now, its history has been controversial and related to political issues such as the safety of nuclear energy.  The hypothesis has not been embraced by those concerned with radiation safety in X-ray applications, nuclear plants and space travel.  Recent research lends strong support to the hypothesis however, and characterizes the biological pathways through which it works.  This blog entry briefly reviews the concept of hormesis and looks at a representative sample of hundreds of research articles related to radiation hormesis.

About hormesis

I introduced the concept of hormesis in this blog in November 2009.  My blog posting Hormesis and age retardation describes hormesis as a process of “challenging cells and body systems by mild stress resulting in them becoming stronger and resistant to aging(ref). The stress can be physical, chemical and even possibly psychological.” Exercise is an example. That blog entry reviews the science behind hormesis and some of its demonstrable anti-aging effects. Also, see my blog entry Stress and longevity for further discussion of how moderate stresses confer longevity.

From Wikipedia: “Hormesis (from Greek hórmēsis “rapid motion, eagerness,” from ancient Greek hormáein “to set in motion, impel, urge on”) is the term for generally favorable biological responses to low exposures to toxins and other stressors. A pollutant or toxin showing hormesis thus has the opposite effect in small doses as in large doses. — In toxicology, hormesis is a dose response phenomenon characterized by a low dose stimulation, high dose inhibition, resulting in either a J-shaped or an inverted U-shaped dose response.  – The hormesis model of dose response is vigorously debated.[1] The notion that hormesis is a widespread or important phenomenon in biological systems is not widely accepted.[2] — The biochemical mechanisms by which hormesis works are not well understood. It is conjectured that low doses of toxins or other stressors might activate the repair mechanisms of the body. The repair process fixes not only the damage caused by the toxin, but also other low-level damage that might have accumulated before without triggering the repair mechanism.” Image may be NSFW. Clik here to view.

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A good review of the current status of hormesis is provided in the 2011 e-publication Hormesis pervasiveness and its potential implications for pharmaceutical research and development. “ The volume of literature published in this area each year has grown 10-fold in large part due to single, original papers in a variety of high impact factor, scientific, peer-reviewed international journals, but also due to entire volumes of journals which have been devoted to hormesis. Some of the various areas include aging, benign prostate enlargement, biochemical and physiological cellular responses, caloric restriction, cardiovascular function, cancer and tumor development, chemo-sensitization, chemotherapy, dermatology, drug binding, hair growth, sexual dysfunction, ocular diseases, osteoporosis, oxidative stress, prion diseases and synaptic plasticity (Calabrese 2008a, Maynard et al. 2008). Four special volumes concentrated on neuroscience, including neuronal survival, neurite outgrowth, glial adaptive responses to neurotoxins, p-glycoprotein efflux transporter activity, anxiety and anxiolytic drugs, epilepsy, traumatic brain injury, stroke, addiction, memory and Alzheimer’s Disease (Calabrese 2008b).”

Historically, many scientists believed that hormesis, or “a little stress is a good thing,” was somewhat of a magical belief not fully to be trusted despite evidence supporting it.  As evidence for hormesis builds up, however, that skepticism may be slowly dissolving.  As pointed out below, I believe it is no longer true that “ The biochemical mechanisms by which hormesis works are not well understood.”

Radiation hormesis, though still controversial, is actually an old topic.

It has been studied in Japan since 1982 in multiple research centers.  The Nuclear Technology Research Center there has published a historical timetable of early events.

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Among the early studies from Japan, a 1993 publication  even went so far as to suggest that low dose radiation might be used as a protective measure to prevent lung cancer metastasis(ref).

Epidemiological evidence of various kinds exists for the radiation hormesis hypothesis.  For example, studies indicate that mortality of populations varies inversely with altitude and amount of natural background radiation.

The June 2012 publication Cancer mortality, state mean elevations, and other selected predictorsreports: “This ecological inquiry compares cancer mortality rates in the U.S. to the predictor of natural background radiation (via land elevation means) along with eight other predictors thought to be associated with cancer mortality. Age-adjusted cancer mortality in 2006 was compared to the predictors of mean land elevation, percent of smokers, educational attainment, percent of population without health insurance, income, obesity, health perception, physical activity, and diet. Among the six predictors considered appropriate for multiple linear regression, three were found to be statistically significant; from strongest to weakest, these three were: smoking, land elevation, and educational attainment. The predictors of smoking and educational attainment have long been considered associated with cancer mortality. The finding that the predictor of land elevation / natural background radiation is inversely related to cancer mortality is another piece of evidence supporting the theory of radiationhormesis. In this study, land elevation / natural background radiation ranked second in predictive strength regarding cancer mortality, behind smoking and ahead of educational attainment. Since this is an ecological inquiry, no causal inferences can be made.”

Other epidemiological studies related to radiation hormesis have looked at cancer risk.  For example, the December 2010 review publication [Cancer incidence and mortality after low-dose radiation exposure: epidemiological aspects]reports:“Current recommendations for limiting exposure to ionizing radiation are based on the linear-no-threshold (LNT) model for radiation carcinogenesis under which every dose, no matter how low, carries with it some cancer risk. In this review, epidemiological evidences are discussed that the LNT hypothesis is incorrect at low doses. A large set of data was accumulated that showed that cancer risk after ordinarily encountered radiation exposure (natural background radiation, medical X-rays, etc.) is much lower than projections based on the LNT model. The discovery of the low-level radiation hormesis (stimulating effect) implies a non-linear dose-response curve in the low-dose region. The further studies in this field will provide new insights about the mechanisms of radiation carcinogenesis.”

An epidemiological study looking at radon gas exposure risk also points to the presence of radiation hormesis.  The December 2010 publication Epidemiological Evidence for Possible RadiationHormesis from Radon Exposure: A Case-Control Study Conducted in Worcester, MA reported: “Data from a case-control study of lung cancer and residential radon exposure conducted in Worcester County, Massachusetts, are presented. Lung cancer risk was estimated using conditional logistic regression models that controlled for demographic, smoking, and occupational exposure covariates. Preliminary exploratory analyses using lowess smoothing revealed a non-linear association between exposure and the log odds of lung cancer. Radon exposure was considered by using linear spline terms in order to model this nonlinearity. The best fit of this linear spline model to these data predicted a shift from a positive to a negative slope in the log-odds of lung cancer at a radon concentration of 70 Bq m(-3). A statistically significant decrease in cancer risk with increased exposure was found for values ≤ 157 Bq m(-3) normalized to the reference exposure of 4.4 Bq m(-3), the lowest radon concentration measured(adjusted odds ratio (AOR) [95% CI] = 0.42 [0.180, 1.00], p = 0.049). This result is consistent with those reported elsewhere that considered radon exposure with cubic spline terms (Thompson, RE et al. 2008). Furthermore, this model predicts an AOR that is numerically less than 1.0 for radon exposures up to 545 Bq m(-3) versus the above baseline, reference exposure.”

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Graphic depiction of the stochastic hormetic relative risk model as given in Scott et al. (2009).

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Biological experiments also establish the existence of radiation hormesis.

For example, the January 2012 publication Low-dose-rate, low-dose irradiation delays neurodegeneration in a model of retinitis pigmentosa reports: “The existence of radiation hormesis is controversial. Several stimulatory effects of low-dose (LD) radiation have been reported to date; however, the effects on neural tissue or neurodegeneration remain unknown. Here, we show that LD radiation has a neuroprotective effect in mouse models of retinitis pigmentosa, a hereditary, progressive neurodegenerative disease that leads to blindness. Various LD radiation doses were administered to the eyes in a retinal degeneration mouse model, and their pathological and physiological effects were analyzed. LD gamma radiation in a low-dose-rate (LDR) condition rescues photoreceptor cell apoptosis both morphologically and functionally. The greatest effect was observed in a condition using 650 mGy irradiation and a 26 mGy/minute dose rate. Multiple rounds of irradiation strengthened this neuroprotective effect. A characteristic up-regulation (563%) of antioxidative gene peroxiredoxin-2 (Prdx2) in the LDR-LD-irradiated retina was observed compared to the sham-treated control retina. Silencing the Prdx2 using small-interfering RNA administration reduced the LDR-LD rescue effect on the photoreceptors. Our results demonstrate for the first time that LDR-LD irradiation has a biological effect in neural cells of living animals. The results support that radiation exhibits hormesis, and this effect may be applied as a novel therapeutic concept for retinitis pigmentosa and for other progressive neurodegenerative diseases regardless of the mechanism of degeneration involved.”

Another study showing a radiation hormesis effect on the cell level is reported in the 2011 publication The low-dose ionizing radiation stimulates cell proliferation via activation of the MAPK/ERK pathway in rat cultured mesenchymal stem cells: “Hormesis induced by low-dose ionizing radiation (LDIR) is often mirrored by its stimulation of cell proliferation. The mitogen-activated protein kinases (MAPK)/ extracellular-signal- regulated kinases (ERK) pathway is known to play important roles in cell growth. Therefore, this study was to examine the effects of LDIR on rat mesenchymal stem cell (MSC) proliferation and MAPK/ERK signaling pathway. Rat MSCs were isolated from the bone marrow from 6 to 8-week-old male Wistar rats and cultured in vitro. Exponentially growing cells within 4-5 passages were irradiated with low doses of X-rays at 20, 50, 75 and 100 mGy with a dose rate of 100 mGy/min. Cell proliferation was evaluated by counting total viable cell number with trypan-blue staining and MTT assay. Cell cycle changes were also evaluated by flow cytometry and the activation of MAPK/ERK signaling pathway was assayed by Western blotting. Results showed that LDIR at 50 and 75 mGy significantly stimulated the proliferation of rat MSCs with the most stimulating effect at 75 mGy. There was a significant increase in the proportion of S phase cells in MSCs in response to 75 mGy X-rays. Activation of several members in the MAPK/ERK signaling pathway, including c-Raf, MEK and ERK were observed in the cells exposed to 75 mGy X-rays. To define the role of ERK activation in LDIR-stimulated cell proliferation, LDIR-treated MSCs were pre-incubated with MEK specific inhibitor U0126, which completely abolished LDIR-increased phosphorylation of ERK and cell proliferation. These results suggest that LDIR stimulates MSC proliferations in the in vitro condition via the activation of MAPK/ERK pathway.”

Of course, there are fruit fly studies showing radiation hormesis.  The 2009 publication [Low-dose rate irradiation induced hormesis, hypersensitivity and adaptive response in Drosophila melanogaster of radiosensitive strains] reports: “We have studied the adaptive response after chronic low dose irradiation (2.5 mGy/h) in wild type Drosophila melanogaster strains (Canton-S and Oregon-R), as well as mutant strains on DNA damage sensing (mei-41), DNA repair (mus209, mus210, mus309, rad54) and free radicals detoxification (sod). The effects of irradiation on the prolongation of the larval stage, pupa lethality, and imago whole body weight have been analyzed. The high dose irradiation (30 Gy, 0.05 Gy/s) induced prolongation of the prepupal period and lethality in all wild type and mutant strains under investigation. The chronic low dose irradiation resulted in shortening of the larval development period (hormetic effect) was observed in wild type (Oregon-R) and mutant (mus209) strains (absorbed dose was 20 cGy) of Drosophila. At the same time these strains demonstrated the hormetic effect after chronic low dose irradiation. The hypersensitivity effect was found in sod and rad54 larvae (20 cGy, prolongation of the prepural period), and rad54 and mei-41 pupa (40 cGy, increase of death rate). The larvae of hypersensitive strains and pupa of all strains under investigation did not have the adaptive response. The chronic irradiation in 6 and 60 cGy with the dose rate of 0.25 and 2.5 mGy/h induced the hormetic effect (imago whole body weight enhancement) Canton-S. The obtained results suggest the important role of free radical detoxification, of DNA damage sensing, and of DNA repair mechanisms in the whole organism radiation induced effects. The appearance of the adaptive response depends on the investigated effect and developmental stage of fly.”

The 2008 publication Evidence for radiation hormesis after in vitro exposure of human lymphocytes to low doses of ionizing radiation reports another of several cell-level studies.  “Previous research has demonstrated that adding a very small gamma-ray dose to a small alpha radiation dose can completely suppress lung cancer induction by alpha radiation (a gamma-ray hormetic effect). Here we investigated the possibility of gamma-ray hormesis during low-dose neutron irradiation, since a small contribution to the total radiation dose from neutrons involves gamma rays. Using binucleated cells with micronuclei (micronucleated cells) among in vitro monoenergetic-neutron-irradiated human lymphocytes as a measure of residual damage, we investigated the influence of the small gamma-ray contribution to the dose on suppressing residual damage. We used residual damage data from previous experiments that involved neutrons with five different energies (0.22-, 0.44-, 1.5-, 5.9-, and 13.7-million electron volts [MeV]). Corresponding gamma-ray contributions to the dose were approximately 1%, 1%, 2%, 6%, and 6%, respectively. Total absorbed radiation doses were 0, 10, 50, and 100 mGy for each neutron source. We demonstrate for the first time a protective effect (reduced residual damage) of the small gamma-ray contribution to the neutron dose. Using similar data for exposure to gamma rays only, we also demonstrate a protective effect of 10 mGy (but not 50 or 100 mGy) related to reducing the frequency of micronucleated cells to below the spontaneous level.”

Hormesis effects have been observed in a small study of interventional radiologists.

The February 2012 publication Cellular adaptive response to chronic radiation exposure in interventional cardiologists reports: “Aims: Invasive cardiologists are the most exposed to ionizing radiation among health professionals and show an increased rate of somatic DNA damage. To evaluate the effects of chronic low-dose exposure to ionizing radiation on redox state and apoptotic activation. Methods and results: We enrolled 10 healthy exposed professionals (all interventional cardiologists, Group II, exposed: age = 38 ± 5 years) and 10 age- and gender-matched unexposed controls (Group I, non-exposed). Exposed subjects had a median exposure of 4 mSv/year (range 1-8) by film badge dosimetry (below lead apron). We measured reduced glutathione (GSH, a marker of antioxidant response) in erythrocytes and plasma generation of hydrogen peroxide (a marker of oxyradical stress) by ferrous oxidation-xylenol orange assay in plasma. In both groups, lymphocytes were isolated and caspase-3 activity (a marker of apoptotic response) measured at baseline and following 2 Gy in vitro irradiation. Exposed subjects showed a three-fold increase in hydrogen peroxide (Group I = 2.21 ± 1.03 vs. II = 6.51 ± 1.55 μM H(2)O(2) equivalents) and a 1.7-fold increase in GSH (I = 12.37 ± 1.22 vs. II = 20.61 ± 2.16 mM). Exposed subjects also showed higher values of caspase-3 activity, both at baseline and-more strikingly-following high-dose radiation challenge. Conclusion: In interventional cardiologists, chronic exposure to low-dose radiation is associated with an altered redox balance mirrored by an increase in hydrogen peroxide and with two possibly adaptive cellular responses: (i) an enhanced antioxidant defence (increase in GSH, counteracting increased oxyradical stress) and (ii) an increased susceptibility to apoptotic induction which might efficiently remove genetically damaged cells.”

After 30 years the relevance and even existence of radiation hormesis remains controversial.  Some have argued that the statistical analyses establishing radiation hormesis are faulty.  Other publications suggest that not enough is known about it to justify changing current radiation safety standards..

The 2012 publication A meta-analysis of evidence for hormesis in animal radiation carcinogenesis, including a discussion of potential pitfalls in statistical analyses to detect hormesis is a case in point: “ A database containing 800 datasets on the incidence of specific tumor types from 262 radiation carcinogenicity experiments identified in a comprehensive literature search through September 2000 was analyzed for evidence of hormesis. This database includes lifetime studies of tumorigenic responses in mice, rats, and dogs to exposures to alpha, beta, gamma, neutron, or x-ray radiation. A J-shaped dose response, in the form of a significant decreased response at some low dose followed by a significant increased response at a higher dose, was found in only four datasets from three experiments. Three of these datasets involved the same control animals and two also shared dosed animals; the J shape in the fourth dataset appeared to be the result of an outlier within an otherwise monotonic dose response. A meta-analysis was conducted to determine whether there was an excess of dose groups with decreases in tumor response below that in controls at doses below no-observed-effect levels (NOELs) in individual datasets. Because the probability of a decreased response is generally not equal to the probability of an increased response even in the null case, the meta-analysis focused on comparing the number of statistically significant diminished responses to the number expected, assuming no dose effect below the NOEL. Only 54 dose groups out of the total of 2579 in the database had doses below the dataset-specific NOEL and that satisfied an a priori criterion for sufficient power to detect a reduced response. Among these 54, a liberal criterion for defining a significant decreases identified 15 such decreases, versus 54 × 0.2 = 10.8 expected. The excess in significant reductions was accounted for almost entirely by the excess from neutron experiments (10 observed, 6.2 expected). Nine of these 10 dose groups involved only 2 distinct control groups, and 2 pairs from the 10 even shared dosed animals. Given this high degree of overlap, this small excess did not appear remarkable, although the overlap prevented a formal statistical analysis. A comprehensive post hoc evaluation using a range of NOEL definitions and alternative ways of restricting the data entering the analysis did not produce materially different results. A second meta-analysis found that, in every possible low dose range ([0, d] for every dose, d) of each of the radiation types, the number of dose groups with significantly increased tumorigenic responses was either close to or exceeded the number showing significantly reduced responses. This meta-analysis was considered to be the more definitive one. Not only did it take dose into account by looking for consistent evidence of hormesis throughout defined low-dose ranges, it was also potentially less susceptible to limitations in experimental protocols that would cause individual animals to respond in a non-independent fashion. Overall, this study found little evidence in a comprehensive animal radiation database to support the hormesis hypothesis. However, the ability of the database to detect a hormetic effect was limited both by the small number of dose groups with doses below the range where positive effects have been found in epidemiological studies (≤ 0.1 Gy) and by the limited power of many of these dose groups for detecting a decrease in response.”

Another example, the June 2012 publication Radiationhormesis: Autophagy and other cellular mechanisms reports: “Purpose: To review the cellular mechanisms of hormetic effects induced by low dose and low dose rate ionising radiation in model systems, and to call attention to the possible role of autophagy in some hormetic effects. Results and conclusions: Very low radiation doses stimulate cell proliferation by changing the equilibrium between the phosphorylated and dephosphorylated forms of growth factor receptors. Radioadaptation is induced by various weak stress stimuli and depends on signalling events that ultimately decrease the molecular damage expression at the cellular level upon subsequent exposure to a moderate radiation dose. Ageing and cancer result from oxidative damage under oxidative stress conditions; nevertheless, ROS are also prominent inducers of autophagy, a cellular process that has been shown to be related both to ageing retardation and cancer prevention. A balance between the signalling functions and damaging effects of ROS seems to be the most important factor that decides the fate of the mammalian cell when under oxidative stress conditions, after exposure to ionising radiation. Not enough is yet known on the pre-requirements for maintaining such a balance. Given the present stage of investigation into radiationhormesis, the application of the conclusions from experiments on model systems to the radiation protection regulations would not be justified.”

There has been a Growing understanding of the epigenetic mechanisms through which radiation hormesis works.

An important contribution in this regard was the 2009 publication Radiation-Stimulated Epigenetic Reprogramming of Adaptive-Response Genes in the Lung: An Evolutionary Gift for Mounting Adaptive Protection Against Lung Cancer.  It establishes, among other things, that the hormetic protective effects of low-level gamma ray exposure can protect against lung cancers induced by alpha-ray exposure.  “Humans are continuously exposed to low-level ionizing radiation from natural sources. However, harsher radiation environments persisted during our planet’s early years and mammals survived via an evolutionary gift – a system of radiation-induced natural protective measures (adaptive protection). This system includes antioxidants, DNA repair, apoptosis of severely damaged cells, epigenetically regulated apoptosis (epiapoptosis) pathways that selectively remove precancerous and other aberrant cells, and immunity against cancer. We propose a novel model in which the protective system is regulated at least in part via radiation-stress-stimulated epigenetic reprogramming (epireprogramming) of adaptive-response genes. High-dose radiation can promote epigenetically silencing of adaptive-response genes (episilencing), for example via promoter-associated DNA and/or histone methylation and/or histone deacetylation. Evidence is provided for low linear-energy-transfer (LET) radiation-activated natural protection (ANP) against high-LET alpha-radiation-induced lung cancer in plutonium-239 exposed rats and radon-progeny-exposed humans. Using a revised hormetic relative risk model for cancer induction that accounts for both epigenetic activation (epiactivation) and episilencing of genes, we demonstrate that, on average, >80% of alpha-radiation-induced rat lung cancers were prevented by chronic, low-rate gamma-ray ANP. Interestingly, lifetime exposure to residential radon at the Environmental Protection Agency’s action level of 4 pCi L⁻¹ appears to be associated with on average a > 60% reduction in lung cancer cases, rather than an increase. We have used underlined italics to indicate newly introduced terminology.”

The single most-important biological pathway responsible for radiation hormesis is probably the activation of Nrf2 by radiation-created oxidative stress.

This observation became clear to me as an “of course” matter as soon as I begin digging into the area of radiation hormesis.  I have written extensively about Nrf2 in this blog.  Nrf2 is the master regulator of the body’s response to stresses of most kinds.  Activating hundreds of the body’s natural antioxidant and stress defense genes, the Nrf2/Keap1 pathway provides a central mechanism for most if not all forms of hormesis.  Specifically, you can see the blog entries The pivotal role of Nrf2. Part 1 – a new view on the control of oxidative damage and generation of hormetic effects, The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2 and The pivotal role of Nrf2. Part 3– Is promotion of Nrf2 expression a viable strategy for human human healthspan and lifespan extension?. 

Lending credence to this perception is the December 2011 publication Ionizing radiation activates the Nrf2 antioxidant response.  “The transcription factor NF-E2-related factor 2 (Nrf2) binds the antioxidant DNA response element (ARE) to activate important cellular cytoprotective defense systems. Recently several types of cancers have been shown to overexpress Nrf2, but its role in the cellular response to radiation therapy has yet to be fully determined. In this study, we report that single doses of ionizing radiation from 2 to 8 Gy activate ARE-dependent transcription in breast cancer cells in a dose-dependent manner, but only after a delay of five days. Clinically relevant daily dose fractions of radiation also increased ARE-dependent transcription, but again only after five days. Downstream activation of Nrf2-ARE-dependent gene and protein markers, such as heme oxygenase-1, occurred, whereas Nrf2-deficient fibroblasts were incapable of these responses. Compared with wild-type fibroblasts, Nrf2-deficient fibroblasts had relatively high basal levels of reactive oxygen species that increased greatly five days after radiation exposure. Further, in vitro clonogenic survival assays and in vivo sublethal whole body irradiation tests showed that Nrf2 deletion increased radiation sensitivity, whereas Nrf2-inducing drugs did not increase radioresistance. Our results indicate that the Nrf2-ARE pathway is important to maintain resistance to irradiation, but that it operates as a second-tier antioxidant adaptive response system activated by radiation only under specific circumstances, including those that may be highly relevant to tumor response during standard clinical dose-fractionated radiation therapy.”

As a matter if fact, radiation oncologists have been mainly concerned with Nrf2 and its hormetic effects because they could reduce the traditional cancer cell killing impacts of radiation cancer therapies.  Another relevant publication in this regard is the 2010 report Relationship between Radiosensitivity and Nrf2 Target Gene Expression in Human Hematopoietic Stem Cells.  “NFE2-related factor 2 (Nrf2), which belongs to the cap “n” collar family of basic region leucine zipper transcription factors, is a key protein in the coordinated transcriptional induction of expression of various antioxidant genes. The purpose of this study was to analyze the expression of Nrf2 target genes, such as heme oxygenase 1 (HO-1), ferritin heavy polypeptide 1 (FTH1), NAD(P)H dehydrogenase, quinone 1 (NQO1), glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, glutathione reductase (GSR) and thioredoxin reductase 1 (TXNRD1), after X irradiation of CD34⁺ cells that were prepared from human placental/umbilical cord blood hematopoietic stem cells (HSCs). We evaluated the relationship between radiosensitivity and expression of Nrf2 target genes in HSCs. The number of colony-forming cells derived from 2-Gy-irradiated HSCs decreased to approximately 20% of the nonirradiated control. At the same time, the mRNA expression of HO-1, FTH1, NQO1, GSR and TXNRD1 was significantly increased after X irradiation. A statistically significant negative correlation was observed between the surviving fraction of HSCs and the intrinsic NQO1 mRNA expression, indicating that HSCs in which NQO1 mRNA levels are low may also be radioresistant. The present results suggest that the antioxidant system associated with Nrf2 is involved in the radiosensitivity of HSCs.”

It is interesting though that in the publication of those directly concerned with radiation hormesis, I have yet to come across a mention of Nrf2.

Radiation hormesis is still not a mainline view

There is epidemiological, experimental and genetic pathway evidence for the existence of radiation hormesis.  However, given political and social situations, the radiation safety establishment has chosen to view even low-level radiation as unsafe, despite a growing body of scientific evidence contrary to this view.

This situation is characterized in the April 2012 publication Hormesis: A peep in to the human nature.  “Hormesis is a term to denote biphasic dose response to an agent which reveals a stimulatory or beneficial effects at low dose and an inhibitory or toxic consequence at a higher dose or concentration. Hormesis is a concept which is involved to biphasic dose response effects of environmental agents including ionizing radiation. Proponents of anti-nuclear lobby brand the idea of hormesis as the privilege of the cranks, and the ill informed. UNSECAR in 1958 and 1 CRP in 1959 adopted linear non threshold theory (LNT) in contrast to hormetic or biphasic relation. These organizations based their recommendation by extrapolation of the studies done on atomic bomb survivors who had received higher doses of radiation. According to LNT theory (a) The effect of low doses or radiation can be estimated by linear extrapolation from effects observed by high doses, and (b) There are not any safe doses as even very low doses of ionizing radiation produce some biological effect (Mortazan). — Science dislikes any vacuum of ideas, experiments, and evidence.  – It is generally assumed that science is based on specific results which is independent of personalities; in other words, the interpretation is secular. However, in reality, interpretation of results may to an extent, depend on the position of the interpreter.  The frenzied debates about hormesis in radiation or global warming attest to the above assertion. Jamea Miller observed mutation following radiation of Drosophila. He assumed a linear relation between radiation and mutation though his experiments did not include very low doses of radiation. Catastrophe following nuclear war on Hiroshima and Nagasaki has resulted in radiophobia and an aversion to the concept of hormesis. The evidence for hormesis is accumulating. According to UNSECAR (1994), amongst A-bomb survivors from Hiroshima and Nagasaki, who received doses below 200 mSv, there was no incidence of cancer deaths. And deaths due to leukemia in the sub group who received less than 100 mSv was less than age matched controls. Nambi and Soman in 1987 have demonstrated a significantly reduced death due to cancer in high back ground areas versus areas of low background radiation in Kerala. In a Canadian survey the mortality caused by cancer at nuclear plants was 58% lower than national average. (Abbat 1983). Thus, there is no evidence of harmful effects of radiation at lower doses. Yet, most people tend to go by LNT model including regulatory authorities. — There are instances in the history of medicine where intuitive decision have been disastrous. It took a while to establish a link between administration of saturated oxygen and retrolenticular fibroblasia in neonates. It was felt that life sustaining oxygen can never harm human life even at higher concentration. Thus, establishing a casual link between retrolenticular hyperplasia and saturated oxygen took considerable time by which time innumerable neonates were affected. The biphasic action of chlorpromazine as protector and senstiser at various concentrations is yet another instance of concentration dependent behavior. There are innumerable such examples of biphasic phenomenon. — Hormesis is integral to the normal physiological function of cells and organisms (Mark Mattson) Adaptive responses evolve due to biphasic response to many physical and chemical stimuli. Hormesis also has played a crucial role in the evolution of life. Life had to evolve despite the harsh environmental conditions, including higher cosmic radiation that is seen now. Cellular mechanism including signaling pathways responsible for adaptive pathways are emerging. Now, there is a mechanistic explanation for a possible hermetic response for a wide range of stimulants, including that for ionizing radiation. — Radiophobia has propelled many a nuclear activitists and scientists in to a narrow tunnel vision, obscuring the evidence, as well as disabling a congent thinking. Men are no Drosophila or cells in the Petri dish . Complex biological systems behave much differently than most people concede. Failure to acknowledge hormesis is also a failure to realize essential realities of biological complexity.”

There is a slow broadening of views in the area of radiobiology to where hormesis is being accepted.

The June 2012 publication Changing paradigms in radiobiologysummarizes the nature of this shift: “The last 25 years have seen a major shift in emphasis in the field of radiobiology from a DNA-centric view of how radiation damage occurs to a much more biological view that appreciates the importance of macro-and micro-environments, hierarchical organization, underlying genetics, evolution, adaptation and signaling at all levels from atoms to ecosystems. The new view incorporates concepts of hormesis, nonlinear systems, bioenergy field theory, uncertainty and homeodynamics. While the mechanisms underlying these effects and responses are still far from clear, it is very apparent that their implications are much wider than the field of radiobiology. This reflection discusses the changing views and considers how they are influencing thought in environmental and medical science and systems biology.”

For years, voices have been raised calling for acknowledgement of the existence of radiation hormesis and for revision of the LNT (linear no-threshold) standards for radiation protection which state that radiation exposure at any dose is biologically damaging.  These calls have not gotten anywhere so far. 

The 2008 publication It’s time for a new low-dose-radiation risk assessment paradigm–one that acknowledges hormesis is one of many over the years calling for revision of the LNT.  “The current system of radiation protection for humans is based on the linear-no-threshold (LNT) risk-assessment paradigm. Image may be NSFW.
Clik here to view.Perceived harm to irradiated nuclear workers and the public is mainly reflected through calculated hypothetical increased cancers. The LNT-based system of protection employs easy-to-implement measures of radiation exposure. Such measures include the equivalent dose (a biological-damage-potential-weighted measure) and the effective dose (equivalent dose multiplied by a tissue-specific relative sensitivity factor for stochastic effects). These weighted doses have special units such as the sievert (Sv) and millisievert (mSv, one thousandth of a sievert). Radiation-induced harm is controlled via enforcing exposure limits expressed as effective dose. Expected cancer cases can be easily computed based on the summed effective dose (person-sievert) for an irradiated group or population. Yet the current system of radiation protection needs revision because radiation-induced natural protection (hormesis) has been neglected. A novel, nonlinear, hormetic relative risk model for radiation-induced cancers is discussed in the context of establishing new radiation exposure limits for nuclear workers and the public.”

Given that individuals may vary significantly in their hormetic responses to radiation, establishing standards to replace LNT could be a daunting task.

Wrapping it up

It is unfortunate that radiation safety standards and therefore radiation hormesis seem to be so bound up with nuclear energy safety issues, issues of great public concern where most people want to be on the safe side.  It is thought by some that radiation hormesis is mainly an argument put forward by the nuclear industry to lessen the regulatory burden on it, and this may to some extent be so.  However, science is science.

My personal opinion is that radiation hormesis is real and important, first because of the collective research evidence to this effect, second because it is very well established that controlled oxidative stress induces hormesis, so it can be expected that free radicals produced by radiation would produce such stress and induce hormesis, and third, because the key biological pathway, the Nrf2/Keap1 pathway, is now understood. 

Radiation hormesis is ancient and evolutionary but good news, providing us with layers of protection we did not know about.

It also seems clear to me that the LNT standards for radiation safety should be revised given the presence of radiation hormesis, but how much so will require further concerted study and that in turn will require a new public safety initiative.  Fully acknowledging the effects of radiation hormesis conceivably could significantly reduce public concern for radiation safety from x-rays and radon as well as nuclear power plants.

This blog is about Mitohormesis, a different form of hormesis than that discussed in the previous blog entry Radiation Hormesis.  Mitohormesis has to do with cell metabolic pathways and oxidative stress, topics I have discussed in many previous blog entries.  The proper functioning of mitohormesis has to do with multiple aspects of health and with extended organism longevity in many species, quite probably including our own.  And the absence of proper mitohormesis functioning has been shown to be associated with certain disease processes and shortened lifespans in a range of animals including humans.  As is the case for radiation hormesis, the concept of mitohormesis has been around for some time but has not been universally accepted.  It is only recently being widely acknowledged as an important biological phenomenon.  And only relatively recently are its mechanisms of operation being unveiled.

I am grateful for an intensive round of communication with James P Watson, a follower of this blog and stem cell researcher, which helped me to improve this blog entry.  Also I feel free to selectively quote some of Watson’s comments sent me by e-mail while this blog entry was being researched.  This is not to say that Watson, clearly a very brilliant person, will necessarily agree with all I have to say.

In researching this blog entry I encountered a number of interesting recent research results having to do with topics I have discussed previously including mitochondrial metabolism, the OXPHOS system in general, mitochondrial biogenesis, frataxin, the key roles of SIRT3 in controlling ROS stress in mitochondria, and the health roles of PCG1alpha.  And I started to question whether it makes sense to supplement with PQQ.  And there are pathways which seem to produce health benefits independently of motohormesis, like inhibition of TOR.  Because of the great complexity involved I decided to cover most of those topics in subsequent blog entries – even though they all bear on mitohormesis.  So, my intent is to keep this present blog entry focused on mitohormesis itself, what turns it on and off, and the direct health implications of mitohormesis.  In particular, I deal with the important practical topic of mitohormesis and exercise, and how some antioxidants nullify the health benefits of regular exercise.

Hormesis in General

The first section of the previous blog entry Radiation Hormesis is a short introduction to the topic of hormesis in general, and I will not repeat that introduction here.

What is Mitohormesis?

A definition and summary introduction to mitohormesis is found in the abstract of the 2010 publication How increased oxidative stress promotes longevity and metabolic health: The concept of mitochondrial hormesis (mitohormesis):  “Recent evidence suggests that calorie restriction and specifically reduced glucose metabolism induces mitochondrial metabolism to extend life span in various model organisms, including Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans and possibly mice. In conflict with Harman’s free radical theory of aging (FRTA), these effects may be due to increased formation of reactive oxygen species (ROS) within the mitochondria causing an adaptive response that culminates in subsequently increased stress resistance assumed to ultimately cause a long-term reduction of oxidative stress. This type of retrograde response has been named mitochondrial hormesis or mitohormesis, and may in addition be applicable to the health-promoting effects of physical exercise in humans and, hypothetically, impaired insulin/IGF-1-signaling in model organisms. Consistently, abrogation of this mitochondrial ROS signal by antioxidants impairs the lifespan-extending and health-promoting capabilities of glucose restriction and physical exercise, respectively. In summary, the findings discussed in this review indicate that ROS are essential signaling molecules which are required to promote health and longevity. Hence, the concept of mitohormesis provides a common mechanistic denominator for the physiological effects of physical exercise, reduced calorie uptake, glucose restriction, and possibly beyond.”

The July 2011 review publication Extending life span by increasing oxidative stress also argues for the existence of mitohormesis across very diverse species: “Various nutritional, behavioral, and pharmacological interventions have been previously shown to extend life span in diverse model organisms, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, mice, and rats, as well as possibly monkeys and humans. This review aims to summarize published evidence that several longevity-promoting interventions may converge by causing an activation of mitochondrial oxygen consumption to promote increased formation of reactive oxygen species (ROS). These serve as molecular signals to exert downstream effects to ultimately induce endogenous defense mechanisms culminating in increased stress resistance and longevity, an adaptive response more specifically named mitochondrial hormesis or mitohormesis.  Consistently, we here summarize findings that antioxidant supplements that prevent these ROS signals interfere with the health-promoting and life-span-extending capabilities of calorie restriction and physical exercise. Taken together and consistent with ample published evidence, the findings summarized here question Harman’s Free Radical Theory of Aging and rather suggest that ROS act as essential signaling molecules to promote metabolic health and longevity.”

My regular readers will discover in these statements many themes discussed in previous blog entries: Reactive Oxygen Species (ROS) are important signaling molecules(ref),  increased ROS expression up to a certain point may be good instead of bad for you(ref)(ref), the stress induced by ROS leads (via the Nrf2 pathway) to activation of multiple endogenous antioxidant and other protective genes(ref)(ref).  And, in fact, suppression of ROS by taking antioxidants can be health-damaging and life-shortening(ref).  These points were also made in the publications related to radiation hormesis cited in the previous blog entry.  They are also articulated in several of the publications that will be cited here as relevant to mitohormesis.

Hormesis, Nrf2 and Antioxidant supplementation – a unifying framework

Before proceeding further with specific findings reported in the literature, I would like to outline a framework for viewing these findings, findings which otherwise sometimes seem inconsistent.

The major points of this framework are:

1. I hypothesize that the operation of the the Keap1-Nrf2 pathway is a fundamental mechanism of hormesis.  I do that because the operation of the Keap1-Nrf2 pathway is by itself sufficient to explain most if not all the observed phenomena of mitohormesis and hormesis in general. 

In reviewing hundreds of research articles relating  the Nrf2 pathway, I found hormesis mentioned only rarely.  And, in reviewing the literature of hormesis I likewise find that the keap1-Nrf2 pathway is mentioned only rarely.   Yet my research suggests that everything that is known to happen associated with hormesis is completely explainable by the operation of keap1-Nrf2 pathway, without reference to other pathways normally associated with hormesis such as the heat-shock response.  See the subsection entitled The single most-important biological pathway responsible for radiation hormesis is probably the activation of Nrf2 by radiation-created oxidative stress   in the blog entry Radiation Hormesis.   “I have written extensively about Nrf2 in this blog.  Nrf2 is the master regulator of the body’s response to stresses of most kinds.  Activating hundreds of the body’s natural antioxidant and stress defense genes, the Nrf2/Keap1 pathway provides a central mechanism for most if not all forms of hormesis.  Specifically, you can see the blog entries The pivotal role of Nrf2. Part 1 – a new view on the control of oxidative damage and generation of hormetic effects, The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2 and The pivotal role of Nrf2. Part 3– Is promotion of Nrf2 expression a viable strategy for human human healthspan and lifespan extension?.”

In stressing the role of the keap1-Nrf2 pathway in hormesis, I of course acknowledge that this is a simplification and far from tells the whole story.  Many other pathways downstream from keap1-Nrf2 are involved in a hormetic response , namely the ones activated by the AREs (antioxidant response genes) after Nrf2 translocates to the cell’s nucleus.  SIRT3and the heat shock proteins like HPS70 play important  roles in hormesis(ref)(ref), along with and possibly due to expression triggered by Nrf2 expression(ref)(ref)(ref).  L-proline catabolism can also be involved(ref).  HDAC inhibition and histone-dependent gene silencing can be involved.  And the whole subject is wrapped up in the vastly complex subjects of ROS dynamics and cell metabolism involving AMPK, PGC1-alpha, PPAR-gamma, SOD2, OXPHOS, P38, etc..  And of course there are other largely independent body stress-response pathways like the UPR (unfolded protein response)(ref) and the various kinds of DNA repair machinery.  But seeing the keap1-Nrf2 pathway as primary can be a very useful one, because research on Nrf2 has been yielding many insights relevant to understanding the scope and relevancy of hormesis  So, the hypothesis is an application of Occam’s Razor, where simplicity is paramount. 

2.     A key to understanding what is going on in hormesis is understanding how the hormetic dose-response curve works, and this can be formulated in terms of NRF2 expression.

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Graphic depiction of the stochastic hormetic relative risk model as given in Scott et al. (2009).

Understanding the typical dose-response curve associated with hormesis is critical for interpreting  seemingly contradictory research. In my interpretation, the horizontal axis depicts level of stress, say as driven by ROS load.  The vertical axis represents relative risk, level of probable pathological organismic response where normal level is 1.  To the left of the first axis crossing in the diagram (point D) the Keap1-Nrf2 pathway is progressively kicking in but not sufficiently so as to overcome the direct negative effects of ROS stress. So, in Transition zone A there is under-expression of the ARE genes and a negative health condition Between stress  levels b and D***  there is hormetic protection compared to what would be expected given a linear model of negative response to stress and better-than-expected health, due at least in part to activation of Nrf2 and the ARE genes.  The zone of maximum protection is between D* and D**.  Starting at D** to D*** the stress load begins to overwhelm the defensive activities of the ARE genes and the protection becomes less and less until at point D*** the hormetic response associated with ARE gene activation becomes negligible.  In the case of radiation damage at least, beyond point D*** the damage according to conventional wisdom is in linear proportion to the stressor, the amount of radiation.  Phantom risk is theoretical risk for low stress levels that would apply if the linear model were extrapolated for low stress dosages.

According to this model, supplementation with radical-scavenging supplements like vitamins C and E inhibits the ROS which triggers the release of Nrf2 which produces hormesis when the stress is in the hormesis range, i.e between b and D*** on the diagram.  The result of such supplementation is a lower health state.  That is, supplementation with such antioxidants when the stress is within the hormesis range of stress can turn off the stress that activates the keap1-Nrf2 pathway therefore turning off hormesis and can do more harm than good.  It appears, for example, that this is what happens with exercise where taking antioxidants eliminates the hormetic health benefits of the exercise.

On the other hand, taking radical-scavenging supplements can be very useful and even highly desirable when the stress at a level is to the right of point D*** The traditional Harman free radical theory aging and the invoking of traditional antioxidant protection is valid in the linear zone beyond point D*** or even possibly beyond point D**.  Some of the earlier research on free radicals and their damaging effects appears to be concerned with high dosages in this beyond-hormesis region.  I am thinking of publications like ß-Cell Glucose Toxicity, Lipotoxicity, and Chronic Oxidative Stress in Type 2 Diabetes,  Pancreatic islet β-cell and oxidative stress : The importance of glutathione peroxidase  (2007), and Feeding acetyl-L-carnitine and lipoic acid to old rats significantly improves metabolic function with decreasing oxidative stress (2002).  So these early anti-ROS and pro-antioxidant papers are valid in their own domains and not in fact contradicted by later papers that show positive health effects associated with lower levels of ROS stress.  I believe serious nuclear plant radiation overexposure and accidental ingestion of heavy metals are excellent reasons for taking heavy doses of radical-scavenging antioxidants.

Many phytochemical substances present in certain foods and sold as supplements are called “antioxidants,” but actually are not free radical scavengers.  They work by activating Nrf2 and triggering a hormesis response which lowers ROS and produces a wide range of positive health effects.  I discuss these and other substances which activate Nrf2 in the blog entry The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2.  I believe most of the phytosubstances mentioned there are useful to trigger hormesis or reinforce it within the hormesis stress range (up to point D*** in the diagram), and in no-way inhibit hormesis.  Of course, they can’t overcome massive doses of ROS which correspond to the region to the right of D***on the hormesis response curve.

Now, moving on to some results reported in the literature:

In some lower organisms, inducing enhanced ROS signaling can lead to mitohormesis and significantly extended lifespans.

The April 2012 publication Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal reports: “Impaired insulin and IGF-1 signaling (iIIS) in C. elegans daf-2 mutants extends life span more than 2-fold. Constitutively, iIIS increases mitochondrial activity and reduces reactive oxygen species (ROS) levels. By contrast, acute impairment of daf-2 in adult C. elegans reduces glucose uptake and transiently increases ROS. Consistent with the concept of mitohormesis, this ROS signal causes an adaptive response by inducing ROS defense enzymes (SOD, catalase), culminating in ultimately reduced ROS levels despite increased mitochondrial activity. Inhibition of this ROS signal by antioxidants reduces iIIS-mediated longevity by up to 60%. Induction of the ROS signal requires AAK-2 (AMPK), while PMK-1 (p38) and SKN-1 (NRF-2) are needed for the retrograde response. IIIS upregulates mitochondrial L-proline catabolism, and impairment of the latter impairs the life span-extending capacity of iIIS while L-proline supplementation extends C. elegans life span. Taken together, iIIS promotes L-proline metabolism to generate a ROS signal for the adaptive induction of endogenous stress defense to extend life span.”

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Image source

Many substances can promote mitohormesis at low doses, even poisons which are highly toxic at higher doses.

An example is the highly toxic pesticide rotenone.   The publicationEffects of Low Concentrations of Rotenone Upon Mitohormesis in SH-SY5Y Cells reports: “The mitochondrial toxin rotenone exerts cytotoxicity viaoverproduction of reactive oxygen species (ROS) and depolarization of the mitochondrial membrane. We investigated the effects of rotenone (12.5, 25, 50, 100 nmol/L) on mitochondrial biogenesis and the potential roles of ROS production in SH-SY5Y cells. Mitochondrial biogenesis was assessed by counting the number of mitochondria, determining protein expression of peroxisome proliferator-activated receptor γ coactivator α (PGC1-α) and its regulator, SIRT1, and oxygen consumption. ROS production and levels of reduced glutathione (GSH) and oxidized glutathione(GSSG) were also determined. Compared with controls, rotenone (12.5 nmol/L) significantly increased the quantity of mitochondria and amount of oxygen consumption, whereas rotenone at >12.5 nmol/L decreased the quantity of mitochondria and amount of oxygen consumption. GSH contents and GSH/GSSG were also significantly enhanced by rotenone at 12.5 nmol/L and decreased by rotenone at >12.5 nmol/L. Except for ROS production and SIRT1 protein expression, all concentration-response relationships showed a typical inverted-U shape. ROS production was continually increased in cells treated with rotenone. These data indicate that low concentrations of rotenone can induce mitohormesis, which may be attributed to ROS production.”

James P Watson comments about this finding: “This study will not be replicated in humans!  It does show that even mitochondrial poison pesticides can have a beneficial mitohormetic effect at low doses.  The “mitohormetic ROS dose” is 12.5-25 nmol/L whereas the mitochondrial toxic dose is > 25 nmol/L.  PGC-1alpha and SIRT1 increased with “mitohormetic doses” of rotenone, whereas they both decreased with higher doses.  Again, more proof of mitohormetic dose-response curve.”

One of several substances that promotes mitohormesis in nematodes is the phytochemical glaucarubinone.  The 2011 publication The phytochemical glaucarubinone promotes mitochondrial metabolism, reduces body fat, and extends lifespan of Caenorhabditis elegans reports: “Naturally occurring compounds that promote energy expenditure and delay aging in model organisms may be of significant interest, since these substances potentially provide pharmaceutical approaches to tackle obesity and promote healthy lifespan in humans. We aimed to test whether pharmaceutical concentrations of glaucarubinone, a cytotoxic and antimalarial quassinoid known from different species of the plant family Simaroubaceae, are capable of affecting metabolism and/or extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. — Adult C. elegans roundworms, maintained on agar plates, were fed with E. coli strain OP50 bacteria, and glaucarubinone was applied to the agar to test (i) whether it alters respiration rates and mitochondrial activity, (ii) whether it affects body fat content, and (iii) whether it may promote longevity by quantifying survival in the presence and absence of the compound. We have found that glaucarubinone induces oxygen consumption and reduces body fat content of C. elegans. Moreover and consistent with the concept of mitohormesis, glaucarubinone extends C. elegans lifespan when applied at a concentration of 1 or 10 nanomolar. Taken together, glaucarubinone is capable of reducing body fat and promoting longevity in C. elegans, tentatively suggesting that this compound may promote metabolic health and lifespan in mammals and possibly humans.”

Yet another substance that appears to induce a hormetic-like effect in muscle tissues is Stanozolol.  The 2011 publication Stanozolol treatment decreases the mitochondrial ROS generation and oxidative stress induced by acute exercise in rat skeletal musclereports: “Anabolic androgenic steroids are used in the sport context to enhance muscle mass and strength and to increase muscle fatigue resistance. Since muscle fatigue has been related to oxidative stress caused by an exercise-linked reactive oxygen species (ROS) production, we investigated the potential effects of a treatment with the anabolic androgenic steroid stanozolol against oxidative damage induced on rat skeletal muscle mitochondria by an acute bout of exhaustive exercise. Mitochondrial ROS generation with complex I- and complex II-linked substrates was increased in exercised control rats, whereas it remained unchanged in the steroid-treated animals. Stanozolol treatment markedly reduced the extent of exercise-induced oxidative damage to mitochondrial proteins, as indicated by the lower levels of the specific markers of protein oxidation, glycoxidation, and lipoxidation, and the preservation of the activity of the superoxide-sensitive enzyme aconitase. This effect was not due to an enhancement of antioxidant enzyme activities. Acute exercise provoked changes in mitochondrial membrane fatty acid composition characterized by an increased content in docosahexaenoic acid. In contrast, the postexercise mitochondrial fatty acid composition was not altered in stanozolol-treated rats. Our results suggest that stanozolol protects against acute exercise-induced oxidative stress by reducing mitochondrial ROS production, in association with a preservation of mitochondrial membrane properties.”

The September 2011 publication Lonidamine extends lifespan of adult Caenorhabditis elegans by increasing the formation of mitochondrial reactive oxygen species reports: “Compounds that delay aging in model organisms may be of significant interest to antiaging medicine, since these substances potentially provide pharmaceutical approaches to promote healthy lifespan in humans. The aim of the study was to test whether pharmaceutical concentrations of the glycolytic inhibitor lonidamine are capable of extending lifespan in a nematodal model organism for aging processes, the roundworm Caenorhabditis elegans. Several hundreds of adult C. elegans roundworms were maintained on agar plates and fed E. coli strain OP50 bacteria. Lonidamine was applied to test whether it may promote longevity by quantifying survival in the presence and absence of the compound. In addition, several biochemical and metabolic assays were performed with nematodes exposed to lonidamine. — Lonidamine significantly extends both median and maximum lifespan of C. elegans when applied at a concentration of 5 micromolar by 8% each. Moreover, the compound increases paraquat stress resistance, and promotes mitochondrial respiration, culminating in increased formation of reactive oxygen species (ROS). Extension of lifespan requires activation of pmk-1, an orthologue of p38 MAP kinase, and is abolished by co-application of an antioxidant, indicating that increased ROS formation is required for the extension of lifespan by lonidamine. Consistent with the concept of mitohormesis, lonidamine is capable of promoting longevity in a pmk-1 sensitive manner by increasing formation of ROS.

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IMitohormesis appears to be a pathway response preserved by evolution over many species, ranging from yeast cells to nematode worms to humans.

An editorial article in the journal publishing the previous-cited report entitled Alternative mitochondrial fuel extends life span had to say: “In this issue of Cell Metabolism, Ristow and colleagues (Zarse et al., 2012) elucidate a conserved mechanism through which reduced insulin-IGF1 signaling activates an AMP-kinase-driven metabolic shift toward oxidative proline metabolism. This, in turn, produces an adaptive mitochondrial ROS signal that extends worm life span. These findings further bolster the concept of mitohormesis as a critical component of conserved aging and longevity pathways.”

The November 2011 publication Mitochondria, reactive oxygen species, and chronological aging: a message from yeast reports: “As a major intracellular source of reactive oxygen species (ROS), mitochondria are involved in aging and lifespan regulation. Using the yeast chronological aging model, researchers have identified conserved signaling pathways that affect lifespan by modulating mitochondrial functions. Caloric restriction and a genetic mimetic with reduced target of rapamycin signaling globally upregulate the mitochondrial proteome and respiratory functions. — Recent discoveries support the notion that an altered mitochondrial proteome induces mitohormesis. Mitohormesis involves a variety of ROS during several growth stages and extends lifespan in yeast and other organisms. Here we recap recent advances in understanding of ROS as signals that decelerate chronological aging in yeast. We also discuss parallels between yeast and worm hypoxic signaling. In sum, this mini-review covers mitochondrial regulation by nutrient-sensing pathways and the complex underlying interactions of ROS, metabolic pathways, and chronological aging.”

Exploitation of mitohormesis could become an important strategy for prevention and control of heart diseases.

The February 2012 publication Mitochondria as a source and target of lipid peroxidation products in healthy and diseased heart reports:  “The heart is a highly oxidativ1e organ in which cardiomyocyte turnover is virtually absent, making it particularly vulnerable to accumulation of lipid peroxidation products (LPP) formed as a result of oxidative damage. 2. Reactive oxygen and nitrogen species are the most common electrophiles formed during lipid peroxidation and lead to the formation of both stable and unstable LPP. Of the LPP formed, highly reactive aldehydes are a well-recognized causative factor in ageing and age-associated diseases, including cardiovascular disease and diabetes. 3. Recent studies have identified that the mitochondria are both a primary source and target of LPP, with specific emphasis on aldehydes in cardiomyocytes and how these affect the electron transport system and Ca(2+) balance. 4. Numerous studies have found that there are functional consequences in the heart following exposure to specific aldehydes (acrolein, trans-2-hexanal, 4-hydroxynonenal and acetaldehyde). Because these LPP are known to form in heart failure, cardiac ischaemia-reperfusion injury and diabetes, they may have an underappreciated role in the pathophysiology of these disease processes. 5. Lipid peroxidation products are involved in the transcriptional regulation of endogenous anti-oxidant systems.  Recent evidence demonstrates that transient increases in LPP may be beneficial in cardioprotection by contributing to mitohormesis (i.e. induction of anti-oxidant systems) in cardiomyocytes. Thus, exploitation of the cardioprotective actions of the LPP may represent a novel therapeutic strategy for future treatment of heart disease.”

The 2010 publication Activation of mitochondrial energy metabolism protects against cardiac failure spoke to the same basic points: “Cardiac failure is the most prevalent cause of death at higher age, and is commonly associated with impaired energy homeostasis in the heart. Mitochondrial metabolism appears critical to sustain cardiac function to counteract aging. In this study, we generated mice transgenically over-expressing the mitochondrial protein frataxin, which promotes mitochondrial energy conversion by controlling iron-sulfur-cluster biogenesis and hereby mitochondrial electron flux.   Hearts of transgenic mice displayed increased mitochondrial energy metabolism and induced stress defense mechanisms, while overall oxidative stress was decreased. Following standardized exposure to doxorubicin to induce experimental cardiomyopathy, cardiac function and survival was significantly improved in the transgenic mice. The insulin/IGF-1 signaling cascade is an important pathway that regulates survival following cytotoxic stress through the downstream targets protein kinase B, Akt, and glycogen synthase kinase 3. Activation of this cascade is markedly inhibited in the hearts of wild-type mice following induction of cardiomyopathy. By contrast, transgenic overexpression of frataxin rescues impaired insulin/IGF-1 signaling and provides a mechanism to explain enhanced cardiac stress resistance in transgenic mice.   Taken together, these findings suggest that increased mitochondrial metabolism elicits an adaptive response due to mildly increased oxidative stress as a consequence of increased oxidative energy conversion, previously named mitohormesis. This in turn activates protective mechanisms which counteract cardiotoxic stress and promote survival in states of experimental cardiomyopathy. Thus, induction of mitochondrial metabolism may be considered part of a generally protective mechanism to prevent cardiomyopathy and cardiac failure.”

A comment on this publication sent me by James P Watson is: “ This is a transgenic mouse model of chemotherapy-induced CHF. The chemotherapy drug is a common FDA approved drug that causes cardiomyopathy when patients are treated with too much of this drug.  The mechanism that causes the doxorubicin-induced cardiomyopathy is down-regulation at 3 points of the intra-cytoplasmic Insulin/IGF-1 pathway (Protein Kinase B, Akt, and glycogen synthetase kinase 3).   In this experiment, they created a transgenic mouse that over-expressed the mitochondrial protein frataxin, which increases expression of a critical rate-limiting complex in the OxPhos pathway (iron-sulphur-cluster, ISC) and in Kreb’s cycle (aconitase, which is also dependent on ISC).  If a human does not produce enough frataxin, this is called Fredrich’s ataxia and you will die in your 30s from heart failure (along with diabetes, muscle wasting, and ataxia).  In the frataxin-over expressed mice, there was an induction of mitochondrial metabolism and ROS defense.  When given doxorubicin, 85% of the frataxin mice survived instead of 55%  in the wild type mice.”

Statins induce mitohormesis, mitochondrial biogenesis and associated positive health effects in cardiac tissue but do not induce mitohormesis and instead induce excessive ROS stress and negative health effects in skeletal muscles.

This interesting observation is reported in the June 2012 publication Opposite effects of statins on mitochondria of cardiac and skeletal muscles: a ‘mitohormesis’ mechanism involving reactive oxygen species and PGC-1.  “AIMS: Statins protect against cardiovascular-related mortality but induce skeletal muscle toxicity. To investigate mechanisms of statins, we tested the hypothesis that statins optimized cardiac mitochondrial function but impaired vulnerable skeletal muscle by inducing different level of reactive oxygen species (ROS).  METHODS AND RESULTS: In atrium of patients treated with statins, ROS production was decreased and oxidative capacities were enhanced together with an extensive augmentation of mRNAs expression of peroxisome proliferator-activated receptor gamma co-activator (PGC-1) family. However, in deltoid biopsies from patients with statin-induced muscular myopathy, oxidative capacities were decreased together with ROS increase and a collapse of PGC-1 mRNA expression. Several animal and cell culture experiments were conducted and showed by using ROS scavengers that ROS production was the triggering factor responsible of atorvastatin-induced activation of mitochondrial biogenesis pathway and improvement of antioxidant capacities in heart. Conversely, in skeletal muscle, the large augmentation of ROS production following treatment induced mitochondrial impairments, and reduced mitochondrial biogenesis mechanisms. Quercetin, an antioxidant molecule, was able to counteract skeletal muscle deleterious effects of atorvastatin in rat.  CONCLUSION:  Our findings identify statins as a new activating factor of cardiac mitochondrial biogenesis and antioxidant capacities, and suggest the importance of ROS/PGC-1 signalling pathway as a key element in regulation of mitochondrial function in cardiac as well as skeletal muscles.” 

The report highlights the importance of dosage in determining whether ROS stress is in the range where the positive effects of hormesis are realized (where the hormetic response curve is above the line), or the stress is too great (where the hormetic response curve is below the line), and the net health result is negative.  The dose response curve must be scaled different for cardiac and muscle tissue.  Apparently, a dose of statins with a response above the line for cardiac tissue produces a result below the line for muscle tissue.

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A second point of the above publication is the importance of the ROS.PGC-1 signaling pathway in regulation of mitochondrial biogenesis and functions.

For background on PGC-1, see the blog entries PGC-1alpha and exercise, AMPK and longevity and PQQ – activator of PGC-1alpha, SIRT3 and mitochondrial biogenesis.  “PGC-1-alpha is a gene co-activator, necessary to turn on the PPARG gene and essential in the metabolic process. PGC-1-alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) “is a protein that in humans is encoded by the PPARGC1Agene.^[1] The protein encoded by this gene is a transcriptional coactivator that regulates the genes involved in energy metabolism. This protein interacts with the nuclear receptorPPAR-gamma, which permits the interaction of this protein with multiple transcription factors. This protein can interact with, and regulate the activities of, cAMP response element binding protein (CREB) and nuclear respiratory factors (NRFs). It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis, and is a major factor that regulates muscle fiber type determination. This protein may be also involved in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity(ref).^[2]”

Regarding this article, James P Watson had some very interesting comments which he communicated to me privately, as follow. 

“This is a very interesting article that basically say that statins are “good for your heart” and “bad for your skeletal muscles”.  They also clearly established  that this effect was mediated by PGC-1alpha and PGC-1beta in the nucleus and also affected mitochondrial gene expression.  IN BOTH NUCLEAR GENES AND MITOCHONDRIAL GENES,  THIS EFFECT WAS ABOLISHED BY ANTIOXIDATNS!  This is very convincing evidence of an ROS-mediated mitohormetic mechanism in the heart for statins and an opposite mitodestructive mechanism in skeletal muscles with statin therapy.

 If true, this is an example of “organ pleiotropy” – i.e. what is good for one organ is bad for another.  In medicine, we call this “drug side effects”.    In pharmacology, they call this  ”off target effects”.  The study included both an animal study and a human study of both cardiac and skeletal muscle  (well done) and also included a statin + quercetin arm to the study.  Here are the results summarized:

1. 1.     Animals: This was actually a mitochondrial biogenesis study

Nuclear co-activators and gene expression;

statin alone

=> reduced PGC-1alpha and PGC-1beta in skeletal muscle => reduced Cox1 and NRF1 gene expression

=> increased PGC-1beta in heart, no change in PGC-1alpha in the heart => increased Cox1 and NRF1 gene expression

statin + anti-oxidant (quercetin)

=> abolished all effects in both cardiac and skeletal muscle

mitochondrial gene expression

statin alone

=> reduced Cytochrome b and pyruvate kinase gene expression in skeletal muscle mitochondria

=> increased Cytochrome b and pyruvate kinase gene expression in cardiac muscle mitochondria

statin + quercetin (antioxidant)

=> abolished all effects in both cardiac and skeletal mitochondria

ROS production

statin alone

=> increased ROS in skeletal muscle

=> reduced ROS in cardiac muscle

statin + quercetin

=> abolished effect in skeletal muscle, no change in cardiac muscle

2,  Humans: 

This study did not include anti-oxidants.  It was just a skeletal muscle vs cardiac muscle biopsy study, done during open heart surgery.”

statins

=> reduced PGC-1alpha, PGC-1beta, SOD1, and SOD2 in skeletal muscle

=> increased PGC-1alpha, PGC-1beta, SOD1, and SOD2 in cardiac muscle

My comment (James P Watson, continuing) on this article and diagram:

This is the first article that I have seen that shows changes in both nuclear gene expression (of extrinsic mitochondrial genes) and intrinsic mitochondrial gene expression with statins and that this occurs via an ROS-dependent manner.  This study explains why patients on statins live longer than controls and that this may be a mitohormesis effect, and NOT a cholesterol lowering effect of statins.  It also is the first to show “organ pleiotropic” effects of a drug and explains  the known side effect of muscle pain due to statin therapy.  Taking anti-oxidants to prevent this side effect would abolish the beneficial effect of statins on the heart, so anti-oxidant supplementation would NOT be a good idea for patients on statins.

I really like this diagram, because it has one up arrow for ROS production in the heart and four up arrows for ROS production in skeletal muscle. This clearly shows that statins have a “hermetic ROS dose range” for the heart and a “toxic ROS dose range” for skeletal muscle.  This is “proof” that ROS dosing is the clear reason for the difference between hermetic doses and “Denham Harmon doses” of ROS.”

Another publication relating exercise, ROS and the impact of statins is the 2011 publication Atorvastatin treatment reduces exercise capacities in rats: involvement of mitochondrial impairments and oxidative stress.  “Physical exercise exacerbates the cytotoxic effects of statins in skeletal muscle. Mitochondrial impairments may play an important role in the development of muscular symptoms following statin treatment. Our objective was to characterize mitochondrial function and reactive oxygen species (ROS) production in skeletal muscle after exhaustive exercise in atorvastatin-treated rats. — Our results show that exhaustive exercise exacerbated metabolic perturbations and ROS production in skeletal muscle, which may reduce the exercise capacity and promote the muscular symptoms in sedentary atorvastatin-treated animals.” 

By blocking mitohormesis, regular supplementation with antioxidants may block the beneficial effects of regular exercise.

This is a sobering revelation for those who both regularly exercise and take antioxidant supplements, believing that these interventions work synergistically.  A 2009 publication Antioxidants prevent health-promoting effects of physical exercise in humans speaks as to how by blocking mitohormesis, supplementation with Vitamins C and E nullifies the health-producing effects of physical exercise.  “Exercise promotes longevity and ameliorates type 2 diabetes mellitus and insulin resistance. However, exercise also increases mitochondrial formation of presumably harmful reactive oxygen species (ROS). Antioxidants are widely used as supplements but whether they affect the health-promoting effects of exercise is unknown. We evaluated the effects of a combination of vitamin C (1000 mg/day) and vitamin E (400 IU/day) on insulin sensitivity as measured by glucose infusion rates (GIR) during a hyperinsulinemic, euglycemic clamp in previously untrained (n = 19) and pretrained (n = 20) healthy young men. Before and after a 4 week intervention of physical exercise, GIR was determined, and muscle biopsies for gene expression analyses as well as plasma samples were obtained to compare changes over baseline and potential influences of vitamins on exercise effects. Exercise increased parameters of insulin sensitivity (GIR and plasma adiponectin) only in the absence of antioxidants in both previously untrained (P < 0.001) and pretrained (P < 0.001) individuals. This was paralleled by increased expression of ROS-sensitive transcriptional regulators of insulin sensitivity and ROS defense capacity, peroxisome-proliferator-activated receptor gamma (PPARgamma), and PPARgamma coactivators PGC1alpha and PGC1beta only in the absence of antioxidants (P < 0.001 for all). Molecular mediators of endogenous ROS defense (superoxide dismutases 1 and 2; glutathione peroxidase) were also induced by exercise, and this effect too was blocked by antioxidant supplementation. Consistent with the concept of mitohormesis, exercise-induced oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous antioxidant defense capacity. Supplementation with antioxidants may preclude these health-promoting effects of exercise in humans.”

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Image source   “Mitohormesis links physical exercise and subsequent formation of reactive oxygen species to insulin sensitivity and antioxidant defense. Physical exercise exerts ameliorating effects on insulin resistance by increasing mitochondrial formation of reactive oxygen species in skeletal muscle to induce expression of PGC1α, PGC1β, and PPARγ as inducers of insulin sensitivity, as well as superoxide dismutases 1 and 2 and glutathione peroxidase 1, key enzymes of ROS defense. Notably, by blocking exercise-dependent formation of reactive oxygen species due to ingestion of antioxidant supplements, health promoting effects of physical exercise are abolished, and physical exercise fails to promote insulin sensitivity and antioxidant defense in the presence of vitamin C and vitamin E.”  I note that this finding seems to be relevant within the hormetic range of the ROS dose-response curve.

The patterns of coupling the taking of antioxidants along with regular physical exercise in the interest of general health is, for many people, deeply entrenched, encouraged by much advertising, and only recently being seriously questioned.  The August 2012 publication Does vitamin C and e supplementation impair the favorable adaptations of regular exercise? does not directly discuss mitohormesis but reports: “The detrimental outcomes associated with unregulated and excessive production of free radicals remains a physiological concern that has implications to health, medicine and performance. Available evidence suggests that physiological adaptations to exercise training can enhance the body’s ability to quench free radicals and circumstantial evidence exists to suggest that key vitamins and nutrients may provide additional support to mitigate the untoward effects associated with increased free radical production. However, controversy has risen regarding the potential outcomes associated with vitamins C and E, two popular antioxidant nutrients. Recent evidence has been put forth suggesting that exogenous administration of these antioxidants may be harmful to performance making interpretations regarding the efficacy of antioxidants challenging. The available studies that employed both animal and human models provided conflicting outcomes regarding the efficacy of vitamin C and E supplementation, at least partly due to methodological differences in assessing oxidative stress and training adaptations. Based on the contradictory evidence regarding the effects of higher intakes of vitamin C and/or E on exercise performance and redox homeostasis, a permanent intake of non-physiological dosages of vitamin C and/or E cannot be recommended to healthy, exercising individuals.” – “The discrepancy among studies described above regarding the influence of vitamin C and/or E supplementation on adaptations in redox homeostasis also applies to the effects of antioxidant supplementation on alterations in redox homeostasis in response to acute exercise. In fact, most of the relevant studies have reported that vitamin C and/or E supplementation does not affect redox status [14, 18, 36–40], less studies have reported that attenuates oxidative stress [41–45] and there are even reports indicating a pro-oxidant effect [46, 47]. We believe that this “agreement on disagreement” between responses and adaptations to exercise indicates the inherent complexity of redox biochemistry and signifies the difficulty in providing unidirectional predictions after supplementation with antioxidant agents.”  I speculate that these disagreements among studies can be explained by reference to the ROS dose-response curve for hormesis discussed above. .

Long-term antioxidant supplementation, specifically with Vitamin C and alpha-lipoic acid, reduces mitochondrial biogenesis in skeletal muscle tissue by interfering with ROS signaling that triggers mitohormesis.

Antioxidants produce a result similar to that produced by statins as described above, inhibiting mitohormesis,  The June 2012 publication Antioxidant supplementation reduces skeletal muscle mitochondrial biogenesisreports “PURPOSE: Exercise increases the production of reactive oxygen species (ROS) in skeletal muscle, and athletes often consume antioxidant supplements in the belief they will attenuate ROS-related muscle damage and fatigue during exercise. However, exercise-induced ROS may regulate beneficial skeletal muscle adaptations, such as increased mitochondrial biogenesis. We therefore investigated the effects of long-term antioxidant supplementation with vitamin E and α-lipoic acid on changes in markers of mitochondrial biogenesis in the skeletal muscle of exercise-trained and sedentary rats.  METHODS: Male Wistar rats were divided into four groups: 1) sedentary control diet, 2) sedentary antioxidant diet, 3) exercise control diet, and 4) exercise antioxidant diet. Animals ran on a treadmill 4 d · wk at ∼ 70%VO2max for up to 90 min · d for 14 wk.  RESULTS: Consistent with the augmentation of skeletal muscle mitochondrial biogenesis and antioxidant defenses, after training there were significant increases in peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) messenger RNA (mRNA) and protein, cytochrome C oxidase subunit IV (COX IV) and cytochrome C protein abundance, citrate synthase activity, Nfe2l2, and SOD2 protein (P < 0.05). Antioxidant supplementation reduced PGC-1α mRNA, PGC-1α and COX IV protein, and citrate synthase enzyme activity (P < 0.05) in both sedentary and exercise-trained rats.”

The March 2012 review study Antioxidants and Skeletal Muscle Performance: “Common Knowledge” vs. Experimental Evidence also shows absence of research support for the popular idea that antioxidant supplementation is a good thing to do associated with fitness exercising.  “Antioxidants are assumed to provide numerous benefits, including better health, a reduced rate of aging, and improved exercise performance. Specifically, antioxidants are commonly “prescribed” by the media, supplement industry, and “fitness experts” for individuals prior to training and performance, with assumed benefits of improved fatigue resistance and recovery. This has provoked expansion of the supplement industry which responded by creation of a plethora of products aimed at facilitating the needs of the active individual. However, what does the experimental evidence say about the efficacy of antioxidants on skeletal muscle function? Are antioxidants actually as beneficial as the general populous believes? Or, could they in fact lead to deleterious effects on skeletal muscle function and performance? This Mini Review addresses these questions with an unbiased look at what we know about antioxidant effects on skeletal muscle, and what we still need to know before conclusions can be made.” –  “Conclusion: Experimental evidence does not support the “common knowledge” that antioxidant treatment greatly improves exercise performance and recovery. On the contrary, studies with antioxidant supplementations generally show no effect on muscle function during and after exercise. The exception is NAC treatment, which has been found to improve performance during submaximal exercise. The limited effects of ROS/RNS and antioxidants during exercise are unexpected in that increases in ROS/RNS are likely to occur and these are potentially harmful. It appears that muscle fibers are in some way protected against deleterious effects of oxidants during exercise and fibers are generally much more sensitive to exposure to oxidants in the rested state than during fatigue.”  While mitohormesis is not mentioned, clearly that explains what is happening.

The March 2012 publication Rationale for antioxidant supplementation in sarcopenia sounds a similar note of caution regarding antioxidant supplementation: “Sarcopenia is an age-related clinical condition characterized by the progressive loss of motor units and wasting of muscle fibers resulting in decreased muscle function. The molecular mechanisms leading to sarcopenia are not completely identified, but the increased oxidative damage occurring in muscle cells during the course of aging represents one of the most accepted underlying pathways. In fact, skeletal muscle is a highly oxygenated tissue and the generation of reactive oxygen species is particularly enhanced in both contracting and at rest conditions. It has been suggested that oral antioxidant supplementation may contribute at reducing indices of oxidative stress both in animal and human models by reinforcing the natural endogenous defenses. Aim of the present paper is to discuss present evidence related to possible benefits of oral antioxidants in the prevention and treatment of sarcopenia. – Conclusion: In summary, there is some evidence that oral antioxidant supplementation may reduce muscle damage, but experimental results are largely preliminary and far to be clinically relevant, at least, as suggestive of positive benefits. In fact, a large body of evidence may indicate extreme cautiousness in taking antioxidant supplementation as preventive measures against aging process and age-related conditions. Further studies are needed to support the widespread practice of oral antioxidant supplementation and to determine appropriate recommendations in elderly. — ”  Although this article contains significant discussion on ROS during exercise and the body’s endogenous antioxidant defense system, no mention is made of the key mechanisms of mitohormesis and the actions of Nrf2.  Instead, seemingly puzzled, the authors suggest a need for more research to relate sarcopenia to antioxidant use.

In acknowledgement of the existence of mitohormesis, metabolic researchers are beginning to think in terms not only of oxidative stress but also in terms of antioxidant stress, especially associated with the consumption of antioxidants..

The May 2012 review publication The neglected significance of “antioxidative stress”summarizes the situation “Oxidative stress arises when there is a marked imbalance between the production and removal of reactive oxygen species (ROS) in favor of the prooxidant balance, leading to potential oxidative damage. ROSs were considered traditionally to be only a toxic byproduct of aerobic metabolism. However, recently, it has become apparent that ROS might control many different physiological processes such as induction of stress response, pathogen defense, and systemic signaling. Thus, the imbalance of the increased antioxidant potential, the so-called antioxidative stress, should be as dangerous as well. Here, we synthesize increasing evidence on “antioxidative stress-induced” beneficial versus harmful roles on health, disease, and aging processes. Oxidative stress is not necessarily an un-wanted situation, since its consequences may be beneficial for many physiological reactions in cells. On the other hand, there are potentially harmful effects of “antioxidative stress,” especially in the cases of overconsumption of synthetic antioxidants. Antioxidants can neutralize ROS and decrease oxidative stress; however, this is not always beneficial in regard to disease formation or progression (of, e.g., cancer) or for delaying aging.” 

Another publication making the same point is the February 2012 e-publication review Antioxidant-induced stress: “Antioxidants are among the most popular health-protecting products, sold worldwide without prescription. Indeed, there are many reports showing the benefits of antioxidants but only a few questioning the possible harmful effects of these “drugs”. The normal balance between antioxidants and free radicals in the body is offset when either of these forces prevails. The available evidence on the harmful effects of antioxidants is analyzed in this review. In summary, a hypothesis is presented that “antioxidant-induced stress” results when antioxidants overwhelm the body’s free radicals.”   Clearly, this applies only within the range of normal endogenous ROS loads.

Negative health effects due to inhibition of ROS and consequent inhibition of mitohormesis applies to classical antioxidants, substances like vitamins C and E that act as free radical scavengers.  On the other hand many phytosubstances commonly referred to also as “antioxidants” are not in fact free radical scavengers, do not inhibit mitohormesis and exercise positive health effects.  Instead such phytosubstances act via activating the body’s endogenous antioxidant defense and stress response system. 

Regarding this point, see the blog entry The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2.

Speaking also directly to this point is the March 2012 publication Oligomerized lychee fruit extract (OLFE) and a mixture of vitamin C and vitamin E for endurance capacity in a double blind randomized controlled trial.  “Antioxidant supplementations are commonly used as an ergogenic aid for physical exercise despite its limited evidence. The study aimed to investigate the effects of a polyphenol mixture and vitamins on exercise endurance capacity. Seventy regularly exercising male participants were randomly assigned to receive oligomerized lychee fruit extract, a mixture of vitamin C (800 mg) and E (320 IU), or a placebo for 30 consecutive days. The study results showed that oligomerized lychee fruit extract significantly elevated the submaximal running time (p = 0.01). The adjusted mean change was 3.87 min (95% CI: 1.29, 6.46) for oligomerized lychee fruit extract, 1.33 (-1.23, 3.89) for the vitamins, and 1.60 (-1.36, 4.56) for the placebo (p = 0.33 in between groups). Oligomerized lychee fruit extract significantly increased the anaerobic threshold by 7.4% (1.8, 13.0). On the other hand, vitamins significantly attenuated VO(2)max by -3.11 ml/kg/m (-5.35, -0.87). Their effects on plasma free radical amount, however, were similar. Our results suggest that a polyphenol-containing supplement and typical antioxidants may have different mechanisms of action and that the endurance-promoting effect of oligomerized lychee fruit extract may not directly come from the scavenging of free radicals but may be attributed to other non-antioxidant properties of polyphenols, which requires further investigation.”

Consistant with the above discussions regarding mitohormesis, researchers are continuing to discover additional important biological roles played by oxidative stress, even extending to sexual signaling.

The August 2012 e-publication Oxidative stress and condition-dependent sexual signals: more than just seeing red relates: “The links between fitness, health, sexual signals and mate choice are complex and subject to ongoing study. In 1999, von Schantz et al. made the valuable suggestion that oxidative stress may be an important missing piece of this complex puzzle. Their suggestion has been enthusiastically tested, with over 300 studies citing their paper, but most effort has concerned carotenoid-based (and to a lesser extent melanin-based) visual signals, predominantly in birds and fishes. Today, we know a great deal more about oxidative stress and related physiology, in both a pathological and regulatory sense, than we did in 1999. We revisit von Schantz et al.’s predictions and, more importantly, highlight novel mechanisms that could link oxidative stress with a range of energetically demanding signals, greatly increasing the scope from visual signalling systems that are usually discussed and nearly always tested. In particular, we argue that differences between individuals in their ability to regulate physiology related to oxidative stress may be an important factor influencing the production of sexual signals and the costs that are incurred from investment.”

Wrapping it up

Mitohormesis, like radiation hormesis exists and is important.  Here I have been able to discuss some key aspects of mitohormesis, but there is probably much more to come.  My experience in writing this blog entry was the usual one of more questions being raised than answered.  The leading edges of science are never neat and nicely wrapped up.  James P Watson has argued with me privately, for example, that most all scientifically proven lifespan extension phenomena are due to a mitohormetic principle.  And I hope that some combination of he and I will be able to lay that argument out in a blog posting to come.   

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Me, working up a little mitohormesis in the gym.  I also benefit from blogohormesis: Working to get my mind around the subject of this blog entry initially produced a similar experience of nearly-impossible weight and stress.  This was slowly replaced by a feeling of wellbeing, one that will be good hopefully until I get deeply into the next blog entry.

Vince

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 By Vince Giuliano

There appears to be a Great Divide in the world when it comes to health, manifest most clearly in Western Countries and in the US in particular.  The divide is between two major paradigms of thinking, philosophies, methods and institutions used to maintain health and treat diseases.  On one side of the divide we have what today is popularly called Health Care.  On the other side of the Divide we have the paradigm I will call Wellness - which is actually the current form of Folk Health. 

This blog entry updates and amplifies on the theme of my May 2011 blog entry Shift to the wellness-longevity paradigm. I reiterate some of the key points in that blog entry and focus particularly on the dynamics that keep the Great Divide in place.  I discuss how new scientific knowledge of certain plant-based substances can contribute to bridging the Great Divide.  And I express concern about how that can happen.

This is a long blog entry and if you want to go first to conclusions and suggestions you can scroll down to the final section entitled Wrapping it all up – bridging the Great Divide.

The two paradigms of Health Care and Wellness encompass behavior patterns, educational approaches, major institutions, government policies and agencies.  The divide is to some extent also between old and new, between East and West, and between how a person decides on what to do about his or her wellness and health.  It involves who people trust for health advice, and between what educated people think they are supposed to do and what they actually do. 

Though operating in very different spheres, there is some evidence that these two paradigms are increasingly overlapping and moving together.  I believe this is a very good thing.  In this blog entry I discuss the strengths and weaknesses of each paradigm.  I talk about how the two paradigms complement and require each other, and how the process of bringing them together might be accelerated.  My primary reference here is the situation in the US, although similar patterns exist in other advanced countries.

Health Care

First, let me outline the paradigm of Health Care as I see it.  Health Care is based mainly on Allopathic Medicine and goes on to encompass vast institutions.  Representing about 17% of the gross national product in the US, healthcare and its superstructure include doctors, nurses, dentists, hospitals, medical and nursing schools and clinics, pharmaceutical and biotech companies and institutions like the FDA and the NIH.  At the periphery lie health insurance companies, nursing homes and secondary care facilities, government bureaucracies and countless university and private laboratories and research institutions. 

The numbers involved in US health care are mind-numbing.  The latest CDC report Health United States 2011 lays out some of them.  Cost to the US economy of healthcare was $1.2 trillion in 2000 and $2.1 trillion in 2011 and rising at an unsustainable rate.  Direct cost of care was $4,122 per individual in 2000 and $6,797 in 2011.  And these costs do not include suffering from diseases, lost time from work for individuals and employers, loss of productivity, and care provided by unpaid caregivers.

The Health Care system facilitates progress in many important dimensions.  But the relationships among central players also limits progress in overall public health

What drives or limits progress in Health Care?  What is the source of innovation?  What inhibits innovation? The answers are complex and multi-facetted, but I think they lie mainly in the interaction among three central groups of players:  1.  MD doctors and the medical schools and boards that train and discipline them, 2.  The pharmaceutical, biotech and medical technology industries that convert research knowledge into practical drugs and educate doctors about these, and 3.  Government agencies, especially the FDA which sets the rules for how new drugs are developed and released to the public.  Only doctors and a few other highly licensed practitioners can prescribe legally-sanctioned drugs.  An individual can’t just go to the drugstore and buy them as in some countries.  The doctors and the public are educated about new drugs by advertising, promotional activities, and “detail men” paid for by the pharma companies – activities that may cost hundreds of million dollars for one drug.  Usually, more is spent on marketing and distribution than on the R&D required to develop a drug.   New drugs are developed typically through the course of an 8-15 years period of research and development, culminating with Phase 1, Phase2 and Phase 3 clinical trials.  These trials are overseen by the FDA and alone may cost from one to several hundred million dollars.  Between R&D and marketing the cost of researching, developing and getting out a new drug may exceed a billion dollars.  No wonder that big drug companies aim new drugs at massive markets and the business model is like that for blockbuster movies.  There are “fast track and accelerated” FDA approval provisions for “orphan drugs” aimed at rare diseases, but even getting one of these developed and to market may cost $100 million dollars and it may require over 5 years to do so.

Medical doctors are highly trained, regulated and respected in our society.  They function within practice frameworks prescribed by law and by rule-setting professional associations like the American College of Cardiology, the American Association for Thoracic Surgery, the American Academy of Pediatrics, etc.  Few doctors will administer or prescribe treatments that have not gone through the FDA approval process and that are not mainline in their professional subspecialty.  One reason is that straying beyond FDA and association-approved medications engenders a risk of ruinous lawsuits.  If a treatment prescribed is not an established standard of medical practice and someone claims to be harmed by that treatment, the doctor is at risk of ruin.  Use of standard and government approved drugs provides badly needed legal coverage in case of such commonly-occurring lawsuits.   Another reason, sadly, is that doctors, being extremely busy as they are, may simply not know about highly effective alternative treatments in Wellness which have been highly researched and found effective but which are not FDA sanctioned.  Physicians get much of their drug information not from reading research journals but from pharma company representatives that are constantly calling on them.   As a result, I believe there are numerous effective alternative treatments that are locked out of medical practice.  By-and-large, allopathic physicians do not know about them.  They live in limbo.

Health Care is basically a repair-oriented industry

The main context of Health Care has been repair rather than regular maintenance.  For the most part, patients invoke Health Care to fix a problem when it manifests itself.   You have a sore throat or a serious pain, you see a doctor. 

The most serious problem with the repair-oriented paradigm of Health Care is that by the time a serious degenerative disease problem shows up, it is frequently too late effectively to do anything about it.  Too many aging-related diseases like Alzheimer’s disease, Parkinson’s disease, ALS and many cancers are simply not curable.  And for them a diagnosis is like a death sentence.  Early-detection biomarkers for more and more of these diseases are being identified and there are a number of simple preventative maintenance interventions that can be applied to forestall actual diseases from emerging, even when there is a genetic risk factor for them.  However, the concept of Preventative Medicine is only now starting to receive semi-serious attentions.  For most of us, Preventative Medicine within the Health Care paradigm means relatively superficial checkups.   Blood pressure and pulse is checked at every office visit to a doctor.  An annual physical exam involves a few blood and urine tests, the doctor thumping us and looking in our orifices, asking us a few questions.  Once every few years there might be a cardiogram, chest x-ray, mammogram, colonoscopy or other diagnostic procedure.  For seemingly healthy people however, even aging ones, predictive biomarkers for specific deadly age-related diseases are not looked for.

In the blog entry Shift to the wellness-longevity paradigm, I contrast our Health Care  with healthcare for commercial airliners.  For airliners, healthcare is mainly about constant checkups and preventative maintenance, not about repair-after-breakdown.  An airliner must be inspected before each flight and is subject to a strictly enforced schedule of preventative maintenance required by law.  Imagine what it would be like if airliners were in a healthcare system like us humans are – where repairs are initiated only when a problem becomes clearly manifest.  For an airliner this could mean an engine stopping or catching fire in mid-air or a flat tire that is only discovered when taking off.  The idea is very scary and our airline accident death rate would be many times what it is now. 

In all fairness we can’t do preventative maintenance on the body like done on airliners because our system of biomarker indicators for health problems is too primitive, we can’t be taken apart, and we can’t readily have our parts swapped in and out.  However by moving in the direction of regular preventative maintenance we could do a lot better than we are doing now.

The term Health Care itself is misleading because the paradigm of Health Care actually cares a lot more about trying to cure problems of health than it cares about maintaining individuals or a population in a healthy state.

How well is Health Care doing? 

The answer depends on the perspective.  Health care is responsible for many achievements.  Sophisticated technologies have been brought to bear on a massive scale, such as the use of MRI machines for diagnosis and interventional radiology for performing surgeries without cutting.  A few formerly incurable diseases are now curable, although the treatments are sometimes draconian.   More cancers are being cured.  People who are walking around would have been dead without triple bypass surgeries or the use of special drugs that address certain deadly and rare diseases.  Orthopedic procedures such as knee, hip and shoulder replacements keep many people functional and moving, and there is progress in developing robotic appendages.  Measures of health such as longevity, diseases of all causes, infant mortality and disease incidences and cure rates have generally continued to improve year-to-year .  See ref for details. 

Yet, the US with by far the highest per-capita expenditure for health care, trails many other countries in terms of most key health indicators  The WHO Statistics document tells the story,  Given that we are the bastion of Health Care and spend so much on it, Why does the US rank  as the 49^th country in the world in terms of infant mortality(ref)?  Why have we dropped from being ranked 23th in 1960?  Why are we not even in the top 20 countries when it comes to life expectancy when we ranked 7^th. in 1960?  And the same pattern exists for most other health statistics related to specific diseases and conditions.  Our performance ranks down with that of third-world countries while, all at the same time that we spend at least twice as much per healthcare per capita than in other countries. 

Despite hundreds of billions spent by the Health Care establishment on research  and ineffective drugs over the years, most of the degenerative diseases of old age like Alzheimer’s Disease, Parkinson’s Disease, solid tumor cancers and diabetes and other metabolic diseases remain mainly incurable.  And as the population ages and lifespans become longer, these diseases become more and more important.

Health Care is not the major factor in determining health of a population.  Wellness appears to be more important.   

The conclusion seems inescapable.   There is great concern today in improving the efficiency of health care, controlling health care costs, speeding up the FDA approval process, eliminating unnecessary medical procedures, developing better medical records, etc..  These could be very good things to do.  However, improving the efficiency of Health Care is not going to be enough.  By itself the Health Care paradigm is incomplete, broken and in key respects obsolete.  Unless other initiatives are taken, health care costs are likely to continue to spiral upwards and our relative health performance as a country is likely to spiral even further downwards. .  We have to do something else, and I believe the place to look for that something else is in the Wellness paradigm.

Wellness – Folk Health

Wellness, Folk Health, has deep historical roots and includes but is not confined to Folk Medicine.  Wellness is not focused on sickness but rather on maintenance of health and longevity. It is comparable to preventive maintenance where steps are taken to prevent debility or sickness sufficiently in advance so that actual debility or sickness becomes relatively scarce. While important aspects of wellness are captured in the traditional Health Care idea of preventative medicine, wellness-longevity goes much further in expectations for ever-enhanced longevity, personal productivity and transformed lifestyles.

As I see it, Wellness encompasses all health-oriented activities that take place outside of the health care (allopathic medicine) establishment.  Wellness includes all the things people do as individuals to keep themselves healthy including eating healthy food, fastening seat belts, watching their weight, exercising regularly, using air and water purifiers in their homes and consuming dietary supplements.  And Wellness includes public health initiatives.

Historically, the most striking gains in health and population longevity have been due to public health initiatives.  In recent history for example, automobile injury deaths and rates of certain cancers have declined due to safer cars and anti-smoking campaigns.  AIDS educations has been another important health-promoting factor, as is the use of mosquito netting in the third world to control malaria.   Over the last 200 years water treatment and sewage systems have been major contributors to longevity.  Some public health gains have been the results of other technological developments.  The replacement of horses with automobiles eliminated fecal materials in the streets and reduced bacterial contamination in cities, and modern oil and gas furnaces eliminates the cancerous clouds of smoke from wood burning fireplaces that once hung over our cities.  Catalytic converters and other emission-control measures are reducing smog in cities that contributes to pulmonary problems.    And today anti-obesity campaigns and public awareness programs about diabetes are kicking in. 

Folk Medicine

Folk medicine, a subset of Wellness, is still the mainly practiced form of medicine in many parts of the world, and is surprisingly vital and growing today, even in the US.  “The term folk medicine refers to healing practices and ideas of body physiology and health preservation known to a limited segment of the population in a culture, transmitted informally as general knowledge, and practiced or applied by anyone in the culture having prior experience.^{[1] —} Folk medicine may also be referred to as Traditional medicine, Alternative medicine, Indigenous medicine, Complementary medicine, and Natural medicine(ref).”  It is often called holistic medicine.  Practiced in almost every country in the world, Folk Medicine has very deep roots in ChineseMedicine and Indian Aryuvedic Medicine.  Each of these is thousands of years old and has involved the passage of knowledge from generation to generation primarily by oral means.  These and other forms of folk Medicine tend to look at diseases more in terms of perturbations of whole-body systems than in Health Care which is more reductionist.  Most forms of Folk Medicine practiced throughout the world are heavily focused on herbal treatments.  Folk Medicine is usually passed on by an oral tradition and its practitioners may be trained or be self-declared.  They can be known by such names as healers, shamans, bush doctors and curanderos. 

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Image source.   “Dhanvantari (धन्वंतरी), known as an avatar of Vishnu is the Hindu god associated with Ayurveda”Image may be NSFW.
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Image source.   Contemporary Chinese pharmacy

Folk Health is mainly focused on disease prevention, staying healthy and longevity, and only partially on disease repair.  Folk Medicine tends to be concerned with both health maintenance and treating diseases, in most cases viewing the two as intrinsically related.  Often, Folk Medicine as well as other practices of Folk Health have strong spiritual and mental wellbeing components.  In the US for example, many practitioners of yoga and meditation view themselves as being important contributors to Wellness.

Folk health today – called “wellness”

I believe Wellness is alive and thriving in the US and Western countries with ever-new forms of expression.  Like traditional Folk Health it is largely separate and independent from Health Care.  Like traditional Folk Health the major focus is on wellbeing, general health and longevity. 

One major trend in wellness is that individuals increasingly are taking personal responsibility for their own health.  There is no one strong group of respected healers, curanderos or bush doctors in our US society.  But there are many groups with their individual wellness-related specialties like athletic trainers, specialized non-medical therapists of all kinds, dietary advisors and TV Wellness gurus, practitioners of homeopathy and naturopathy, and teachers of yoga, tai chi, massage, meditation and acupuncture.  Signs of the Wellness paradigm are everywhere in our culture.  For one thing, the value of regular exercise is being widely acknowledged:  exercise and health clubs are thriving, more and more people are regularly “working out,” there are dozens of new forms of exercise and yoga for everyone to participate in. 

Manifestations of the Wellness trend seem to be everywhere.  Public policy and institutions are continuing to evolve to better support the health and Wellness trend.  Cigarette smoking has already been banned in most public places, cigarettes are highly taxed and their use in the first-world is on the decline.  Recently, and New York City has banned large-size sugary drinks in fast-food restaurants.  Bike paths are augmenting roads in cities and old railroad lines are becoming hike-and-bike trails.  There are hundreds of health and wellness apps available at little or no cost for smart cell phones and computer tablets.  New condo buildings are constructed with built-in health clubs. Michelle Obama has launched a crusade against childhood obesity, “junk food” chains like McDonalds have been induced to offer healthier alternatives, and there is a focus on healthier school lunches.  These Wellness activities are all supported  by the Health Care establishment.

Dietary Supplements in Wellness

Another trend in Wellness is broader recognition of the importance of dietary intake for health and longevity.  Supermarkets are offering organic and other forms of “healthier” foods and chains that offer premium healthier foods are thriving. 

Dietary health-producing supplements is a large and growing business.  “The global nutraceuticals (health-producing dietary supplements) market is projected to exceed US$243 billion by 2015, owing to consumer desire for leading a healthy life and increasing scientific evidence supporting health foods(ref).” According to a CDC report, at least 54% of Americans consume dietary supplements in 2006 (the last year available)(ref).  There is belief in the industry that the number is now hovering around 70%.  And I suspect that more often than not, this consumption of pills is without the consent or even knowledge of individual’s medical doctors.  “The US continues to be the largest market for nutrients & supplements in the world in terms of retail sales. In 2010, the US nutrition and dietary supplements market reached USD 28.04 billion compared to USD 25.00 billion of 2009(ref).”

Herbal substances and Wellness

Although Folk Medicine can be practiced in hundreds of different forms and traditions, a common denominator appears to be usages of plant-based substances for healing and health maintenance.  And I believe it is exactly these plant-based substances that can provides a needed bridge between Wellness and  Health Care.   

Remarkably, many of the most important medicinal herbs are found in many very different traditions of Folk Medicine.  They have passed not clinical trials but instead multiple trials-by-culture over thousands of years.   And for many categories of these Phyto (plant-based) substances, science has recently been discovering how and why they work in terms of gene activation. 

These substance do not exist by accident.  They have been carefully crafted by millions of years of plant evolution.

They are signaling molecules that govern multiple survival pathways in plants, regulating responses to light and dark, temperature, moisture, resistance to insects, etc.  .  In many cases the same molecular pathways are evolutionarily conserved in humans.  So the substances can produce important gene-activation signaling and health benefits in humans too.  Finally, many of the most important health-producing phytosubstances are readily available in foods we can opt to eat and in dietary supplements we can purchase.

One example of a universal health-producing phytosubstances is Bitter Melon.  See the blog entry Focus on Bitter melon.  This plant (Momordica charantia) “is known as bitter melon, bitter gourd or bitter squash in English, and  has many other local names. (including “which include kugua (Chinese: 苦瓜, pinyin: kǔguā, “bitter gourd”); parya (Ilokano), pare or pare ayam (Javanese and Indonesian), pavayka or kayppayka (Malayalam:പാവയ്ക്ക, കയ്പ്പക്ക ), goya (Okinawan: ゴーヤー) or nigauri (Japanese: 苦瓜; although the Okinawan word goya is also used in Japanese), paakharkaai (Tamil: பாகற்காய்), hāgalakāyi (Kannada: ಹಾಗಲಕಾಯಿ), ma’reah (Khmer: ម្រះ), kaakarakaya(Telugu: కాకరకాయ), করলা (korola) (Bengali), ampalaya (Tagalog), muop dang (Vietnamese: mướp đắng) or kho qua (Vietnamese: khổ qua). It is also known as caraille or carilley on Trinidad and Tobago, carilla in Guyana, cundeamor is a small variety very common in Puerto Rico (actually is the Momordica balsamina), “asorosi” or assorosie” in Haiti, and cerasee or cerasse in Jamaica and elsewhere in the Caribbean, including parts of South America (although is known in Portuguese as melão de São Caetano – and Spanish-speaking areas, however is known by the Okinawan or Japanese names in others regions). It is karela in Hindi – and Urdu-speaking areas, कारले (karle) in Marathi. It is known as तीतेकरेला (tite karela) in Nepali. In Suriname, it is known as sopropo. The fruit is called kudhreth narhy (kudret narı) in Turkey, faaga in Maldives, and karavila in Sri Lankan(Sinhalese). Additional local names include hagala kayi(ಹಾಗಲಕಾಯಿ) in Kannada, karla in Bengali and Marathi, kakarakaya in Telugu, and paakal-kaai (பாகற்க்காய்) in Tamil.)(ref).    . 

How could the same plant show up in so many different traditions of health and medicine ?.  World commerce?  To some extent perhaps.  But, I think mainly not so since usages of the herb appear to predates significant international commerce.  And the cultural forms of Folk Health that use the same herb can be very different.  I think bitter melon and many other traditional herbs were discovered and incorporated into multiple folk traditions of health and medicine for one simple reason, they actually work to produce health.  The most popular medicinal herbs happened to grow wild in a particular area, were consumed randomly by people who lived in that area, they were found to have particular health benefits, and this knowledge was passed down orally from generation to generation.

“Good” supplements and “bad” ones: Not all dietary supplements are equal, either according to research studies or large population tests.  This leads some in the Health Care paradigm mistakenly to paint them all as bad.

Although in the public’s mind antioxidants, vitamins and plant and herbal substances may be part of the same mix of little green and brown bottles on the shelf of a drugstore or health food store, studies are showing that they are very different. 

• Entries in this blog have gone into detail as to why indiscriminant consumption of classical antioxidants and vitamins like vitamins C and E, beta-carotene and vitamin A and folic acid may do nothing or even have negative health effects – they can interfere with operation of the body’s natural anti-oxidant defense system.  And the free radicals that classical antioxidants go after are not necessarily bad; they are essential biological signaling molecules(ref)(ref).  While at the same time a great many plant-based substances sold as supplements like curcumin and resveratrol have a hormesis-producing health effect due to their activation of Nrf2 which turns on hundred of natural anti-oxidant and health promoting genes(ref)(ref)(ref)(ref) (ref).
• Large-scale population research supports the same conclusion.  But some scientists in the Health Care establishment fail to recognize the critical distinctions between types of supplements and paint them all as bad.  For example, a October 2012 article in Gastroenterology and Endoscopy News reported: Use of Dietary Supplements May Raise Cancer Risk: by George Ochoa    “Dietary supplements have little to no effect in preventing cancer and may actually increase cancer risk, according to a review published in the Journal of the National Cancer Institute (Martínez ME et al. 2012;104:732-739).  Restricting their review to supplements that have been researched in sufficiently powered clinical trials or large observational studies, the authors focused on antioxidants, folate and folic acid, vitamin D and calcium.  – Despite early evidence suggesting an anticancer benefit from antioxidants, clinical studies have not borne out that promise, the authors said. For example, b-carotene does not prevent recurrence of non-melanoma skin cancer (Greenberg ER et al. N Engl J Med 1990;323:789-795); b-carotene and vitamin A do not protect against lung cancer (Omenn GS et al. N Engl J Med 1996;334:1150-1155); vitamins C and E do not protect against total cancer incidence (Gaziano JM et al. JAMA 2009;301:52-62); and a-tocopherol, vitamin C, and b-carotene do not protect against total cancer or cancer mortality (Lin J et al. J Natl Cancer Inst 2009;101:14-23). — However, several trials have shown evidence of an increased cancer risk from antioxidants, the review authors, led by María Elena Martínez, PhD, at the University of California, San Diego, reported. One such study, conducted in a population at high risk for lung cancer, found a 39% increase in lung cancer incidence in the b-carotene arm compared with the placebo arm (Omenn GS et al. N Engl J Med 1996;334:1150-1155). — Similarly, the authors did not find evidence that folic acid and folate protect against cancer, whereas they did note evidence of increased risk for cancer from long-term folic acid supplementation. They found insufficient evidence to draw conclusions about vitamin D, and “diverse results” regarding calcium. — Many expert groups have reached a “general consensus” that “nutritional supplements have little to no benefit in preventing cancer,” the authors wrote.”  

The quoted “general consensus” statement paints all nutritional substances as equal and is not at all justified by the research cited.  This statement can be viewed as propaganda in a war the pharma industry is waging against the dietary supplement industry, a war I describe below.  The public could benefit considerably from better and more authoritative information highlighting the distinctions among dietary supplements and their health benefits based on current research.  Unfortunately, large numbers of Internet sites concerned with dietary supplements, particularly sites selling popular health books or supplements, completely blur those distinctions and promulgate now-obsolete research results as the truth.  They irresponsibly continue to try to scare people about free radicals and taut conventional antioxidants as well as phytosubstances. 

Much additional research is needed about dietary supplements

A great deal of knowledge now exists as to how a number of dietary substances work, what they do in terms of gene activation, and what their health benefits are.  We now know, for example, that broad classes of polyphenols work via hormesis, activate the Nrf2 pathway which activates hundreds of anti-oxidant and other protective genes, and turn off NF-kappaB and its pro-inflammatory activities.  See my blog entries cited below.  But, unlike many proprietary drugs, plant extracts usually consist of many compounds which activate multiple biological channels.  We are missing reliable systematic information as to which polyphenols are superior to others, which are short acting and which are long-acting, and what their hormetic range of dosage is, the range where health effects are maximized.  We know that many such substances have poor bioavailability and we don’t really have good dose-response curves for humans. 

When the question is asked “What dietary supplements should I take when; what is the best combination?” the answers can at best be roughly estimated.  Finally, there is a serious issue of herb-drug interactions(ref) which also can benefit from additional research.  So, much additional research on dietary supplements is required.

The dietary supplement industry can’t pick up the slack and do the required research on its products.  By itself, it cannot develop and bring to market new innovative Wellness products based on research it has done on plant substances.  It must depend on research of others.

The companies in this industry are not well-positioned to do the required research.  It is mainly a low-margin consumer commodity product industry comprising tens of thousands of manufacturers marketing more than 29,000 products.  Companies are mainly focused on manufacturing,  marketing and distribution of  me-too products.  They mostly cannot afford and do not do any scientific research and only little product-oriented research.  The idea of such a company spending $300,000 to a million dollars for a clinical trial of a single product is unthinkable. 

Further, a supplement company cannot obtain major venture funding to bring a new plant-based product to market.  Because it cannot patent such a product, it can’t protect itself from competitors sweeping in and selling the same product cheaper.  What a few supplement companies have done, however, is develop proprietary counterparts of commodity phytosubstances like resveratrol and curcumin that are –or at least claimed to be –more bioactive.  And they often package several substances together which they market under such names as “Men’s Formula,” “Power Weight Loss Formula,” or “Immune Strengthener.” 

Natural substances, the mainstays of Wellness, cannot make the transition into becoming medicines in Health Care because they can’t be patented and owned by any company. 

Therefore, no pharma company will spend the hundreds of million dollars required to put such substances through clinical trials.  It’s that simple.  And not being FDA-approved medicines, most doctor’s shy away from them

Supplement manufacturer trade associations have focused primarily on regulatory issues and legislation, though they are aware of and are interested in addressing some of the key issues facing this industry.

The IADSA (International Alliance of Dietary/Food Supplement Associations) is an umbrella association of food supplement associations with member associations in six continents.  IADSA is structured to play an integrative role in a widely dispersed industry.  “IADSA is the leading international expert association regarding the globalisation of food supplement markets and increasing regulatory challenges. Bringing together food supplement associations from 6 continents, IADSA aims to build an international platform for debate and a sound legislative and political environment for the development of the food supplement sector worldwide.” –“IADSA is primarily composed of member associations in the food supplement sector representing over 20,000 companies worldwide. As a member of one of these associations you become part of the global network, receiving information and documentation developed by IADSA and able to play a part in shaping future global regulation(ref).”  Besides legislative activities, IDSA sponsors research and publications on major issues confronting the industry 

For example, regarding the issue of arriving at standards for dosage, IADSA sponsored a 2011 publication Bioactive Food Components: Changing the Scientific Basis for Intake Recommendations. October 2011:  “This publication proposes a new framework for recommended intake, enabling the incorporation of aspects of basic, pre-clinical and clinical research – including the Evidence Based Medicine approach of Randomized Controlled Trials (RCT) – but allowing for decision-making based not primarily on RCT but on the totality of the evidence. It suggests the need for human intervention studies of a smaller scale than those used to evaluate drug efficacy and safety, arguing that RCT used to establish the safety and efficacy of drugs is, alone, not an appropriate method for establishing recommended intakes for nutrients and other bioactive substances.

Peace and War between Health Care and Wellness

There is peace and co-operation

In important respects Health Care and Folk Health (Wellness) are at peace and cooperate with each other.  In other respects they are at war.  Example of the former are schools of public health and growing  emphasis in medical schools and some clinics on preventative medicine, holistic medicine and complementary medicine..More Doctors are advising exercise and good diets, though many do not know what exactly to recommend.  And some doctors are at the forefronts of public health initiatives. Finally, some insurance companies pay for and some HMOs offer access to “complementary medicine” treatments by non-MD practitioners.

It is slowly dawning on the US government and insurance companies that one dollar spent on wellness education may pay off ten-to-one or better in terms of reduced medical costs.  An intestinal transplant is likely to cost $1,121,800, a heart transplant $787,000, and a bone morrow transplant $676,800(ref).  And the figure for open heart surgery is $324,000.  And these figures do not include costs of personal suffering or lost time, or costs to family members.  These amounts, just for one procedure on one individual, can buy substantial programs of Wellness education that affect large numbers of people.

And then, there is war

An example of ongoing war between Medical Care and Wellness/Folk Medicine is the Pharma industry’s efforts to see laws enacted that establish much tighter FDA regulation over the dietary supplement industry – even going so far as to having many familiar herbal substances reclassified as drugs that would require clinical trials before they are made available to the public.  I agree that the supplement industry in the US is currently under-regulated and that indiscriminant use of the substances it provides can lead to negative health results..  There is currently no requirement that effectiveness be established for any natural-product dietary supplement.  Labeling requirements for dietary supplements are quite lax, and people often do not know what they are getting.  Nobody checks.  Supplement dosages or ingredients may be incorrectly labeled and not reveal contaminants such as heavy metals. 

Further, certain supplements relating to athletic performance and weight loss have been shown to be outright dangerous.  Scarcely a day passes without some news item about negative effects of dietary supplements.

The Durbin Bill

However, in my opinion some of the efforts to regulate the supplement industry like the Durbin Bill (the “Dietary Supplement Labeling Act of 2011)  have been over-reaching.  They are threatening to put the supplement industry squarely under the FDA’s heavy thumb so this industry becomes tamed and reshaped as yet-another appendage of Health Care.  “Currently, there is no requirement for pre-market testing for safety and efficacy for dietary supplements, and dietary supplement manufacturers are not required to register with the FDA or to report adverse events from product usage.[10] —  that may change if Senator Durbin’s proposed bill becomes law(ref)” The FDA already has some authority to assure product safety.  The bill led to concern that some familiar substances like curcumin could be reclassified as drugs, handed to pharma companies for clinical trials, eventually packaged in proprietary formulations owned by drug companies, and then sold to the public by prescription for 20-50  times the current prices.  All this would be enforced by law.  This possible scenario is frightening to the supplement industry and also frightening of takers of dietary supplements who cannot afford to pay pharmaceutical-level prices for them.  Is this scenario a serious possibility?  I think it possibly is. In Germany, France, Sweden and Australia, herbs are classified and regulated as medicines, while in the US these herbs are almost all unregulated and freely available to the public(ref).  So, there has been controversy over whether the Durbin Bill was a serious attempt to protect consumers or whether it is a power grab by the pharma industry(ref).  Possibly it is both.  All the natural product dietary supplement trade associations rallied against the bill and critical amendment to the bill was defeated May 24, 2012.  Yet the legislative struggle between the pharma and dietary supplement industries goes on and is not likely to abate.    

Wrapping it all up – Bridging the Great Divide

In summary, several forces are working to bridge the practical divide between Health Care and Folk Health/Wellness.  I see the most important ones as:

• Wellness is in the air and manifesting itself everywhere, part of the evolution of our social fabric of our time. 
• Elements of Wellness are slowly transforming Health Care.  And there is a clear economic incentive for this to happen
• People who also rely on doctors are increasingly taking responsibility for their own wellness – be this via some form of exercise, diet, mental discipline, etc  The Internet makes a wealth of wellness and medical information available.  Individuals are taking all kinds of Wellness initiatives independently of their doctors.
• The unquestionable authority of medical doctors is increasingly being challenged.  Lay people are learning more and more about wellness.  Lay people are starting to research their own illnesses on Internet.  They use social media to learn about the experiences of others.  They sometimes look to exercise trainers and others more than to their doctors for wellness information.  And they are likely to barrage their doctor’s with hard questions or confront them with information that the doctor may not know about.  Asking for second opinions is increasingly common.
• This blog, focused on health and wellness-research results that have largely not been embraced yet by Health Care, is another manifestation of the trend.
• Big pharma is increasingly turning to plant-based products for discovering chemicals that can be the basis of new drugs.   
• Health Care used to have the advantage of pointing out that it had science on its side and that Traditional Folk Health had only folk tales and anecdotal evidence that could not be really trusted.  That is no longer the case as shown by a great many entries in this blog which focus on the science behind herbal substances.  It is quite the opposite.  It appears that most practitioners of Health Care either do not know about or ignore in practice the science behind Health and Wellness.

For me personally, the most exciting shift relates to an important theme of this blog – hard scientific evidence that elements of Folk Health, particularly the use of certain foods and dietary supplements are supported by a wealth of current scientific research.  Many traditional herbs have been extensively studied using the latest knowledge and tools of science.  We now know not only that many substances work as shown by the test of time in multiple countries and social contexts, but we are clarifying how and why they work in terms of molecular biology and genetics.  I have written blog entries reviewing a number of lesser known traditional herbal substances like curcumin, resveratrol, ginger, cat’s claw (ucaria tomentosa or Uncaria guianensis), bitter melon (Momordica charantia), epimedium and icarin, andrographis, rhodiola,  dashen root (salvia miltiorrhiza bunge), fucoidan, cordyceps militaris, rosmarinic acid, caffeic acid, gambogenic and gambogic acid and grape seed extract. I have written a number of blog entries explaining how these substances work in terms of hormesis and Nrf2 activation(ref)(ref)(ref)(ref) (ref).  I have written extensively about the advantages of phytosubstances(ref)(ref)(ref).  And I have written about healthy foods like extra-virgin olive oil, blueberries, and dark chocolate.

There are many challenges still

• While Health Care is well organized and tightly regulated, Wellness is wild and open to everybody, including snake oil salesmen and practitioners that offer quack cures.  And today the quest for Wellness sometimes leads people to trust offshore clinics that offer dangerous stem cell “therapies” as cures for just about everything.  And, unregulated purveyors of dietary supplements often make misleading claims that certain products have research-proven health benefits when the opposite may be the case.  
• Many available herbal substances may offer significant health benefits, but they are eschewed by Health Care and live in a limbo state as part of Wellness.  As far as their use for our personal Wellness goes, we continue to be largely on our own.
• The general public is quite ignorant about the distinctions between classical vitamins and antioxidants, and plant-based substances.  And this critical distinction remains blurred for commercial reasons and because of ignorance.  So the public is barraged and confused by news reports and infomercials that say supplements are good for you and ones that say that supplements are bad for you.
• Much further research is needed to verify safety and efficacy and proper dosages of certain herbal substances, ones which clearly promote wellness and may have specific therapeutic values.
• Governments and foundations must pick up the tab for the needed research on phytosubstances; the supplement industry can’t do it and the pharma industry won’t.  At present, there is no clear indication that that is going to happen on the needed scale. 
• The public can benefit from additional forms of regulation of the supplement industry, probably along the lines suggested by the IADSA.
• Most people respect their medical doctors and look to them as a source of wellness advice.  Doctors need to be better prepared and supported to fulfill this role by moving more heavily into Wellness.  This process has been started but needs to be carried much further.
• It would be good if Health Care seriously embraced herbal medicine as a specialty.  Elements of herbal medicine should to be taught in medical and nursing schools.   Family doctors should be made familiar with a small subset of herbal substances for which research establishes safety and efficacy

Health Care and Wellness are both needed and can work better together

In a nutshell, Wellness is needed to insure general health and longevity; this cannot be achieved by Health Care alone.  Health Care is needed to treat diseases and ailments and provide care for the sick; tasks far beyond the scope of Wellness.  It is ridiculous for the two paradigms to be at war with one another and balances need to be established between them.  Health care insurance companies, government and businesses that bear the costs of health care have enormous economic stakes in further integration of Health Care and Wellness.  So do we as individuals.  Let’s do it!

I am grateful to Melody Winnig for significant help in assembling the ideas for this document and creating the document itself.

By James P Watson

There are 2 “Mantras” Here in the Zoo – Hormesis and Nrf2 inducible Isoforms

Preface – By Vince Giuliano

I want to introduce and welcome James P Watson as a senior researcher and writer for this blog.  James, a surgeon and stem cell researcher, has been responsible for contributing very astute comments in this blog    James (Jim) and I have been in intensive interaction by e-mail and phone over the past few of weeks on a number of interesting topics relating to longevity science, including epigenetics, stem cell senescence, hormesis and the roles of Nrf2.  I have been learning a lot in the course of this dialog and have come to deeply respect James depth in molecular biology as well as his breadth of his knowledge relative to the multiple scientific disciplines bearing on aging.  I am honored that Dr. Watson  has decided to join me as a researcher and writer for this blog.

This blog entry is a natural extension to the blog entries on hormesis and those on Nrf2 expression(ref)(ref)(ref)(ref) (ref).  It is based on an e-mail sent me by Jim and our phone interactions.  I have edited it a bit and added a few remarks  for context.  And I added the pictures.

Introduction

I have discovered a zoo in our cells!  The “animals” are gases (CO, NO, H2S) and non- gases (HCN, O2-, H2O2, etc.), reactive species which are signaling molecules that act as 2nd messengers at hormetic doses.  (“Second messengers are molecules that relay signals from receptors on the cell surface to target molecules inside the cell, in the cytoplasm or nucleus. They relay the signals of hormones like epinephrine (adrenaline), growth factors, and others, and cause some kind of change in the activity of the cell. They greatly amplify the strength of the signal.^[1][2] Secondary messengers are a component of signal transduction cascades(ref).”)    The zoo animals are definitely wild and some like carbon monoxide and hydrogen cyanide have very nasty reputations.  Although they are  deadly poisons, they are NOT toxins at hormetic doses!  They are ACTIVELY produced and destroyed by enzymes in our bodies.  Nrf2 controls the production of the enzymes that create and destroy these 2nd messengers, at least in most known cases.  The regulation of these 2nd messengers is very tightly controlled by both synthesis and degradation.  I suspect that these zoo animals most have half-lives of less than a second

Nitric Oxide (NOS)      Image may be NSFW.
Clik here to view.

• This is a small gaseous molecule enzymatically produced physiologically in minute quantities. 
• At small concentrations it has major beneficial physiologic effects such as vasodilatation – ie. controlling blood flow.
• At higher concentrations, it has major detrimental effects, such as death!  “It is the proximate cause of septic shock and may play a role in many diseases with an autoimmune etiology(ref)”
• NOS is being intensively studied as a signaling molecule and plays key roles in neuroinflammation and neurogenesis(ref)
• NOS is produced by nitric oxide synthethases (NOS) – “(NOSs) are a family of enzymes that catalyze the production of nitric oxide (NO) from L-arginine(ref).”
• Isoforms of NOS (forms of the protein) are regulated differently:

1. Nrf2 inducible isoform – NOS-2 – regulated by cellular stress

2. non-inducible isoform – eNOS – controlled by endothelial cell stress, blood flow turbulence, etc.

• Transcription factor controlling NOS-2: Nrf2
• how to non-invasively measure NO: expired air (i.e. in your breath!): machine: multichannel gas analyzer: Logan 2500, Logan Research Limited, Kent, UK.  reproducible? – yes. sensitivity – 0.1%.  time taken to measure this – 6-13 minutes

The wild animal is NO and the zoo keeper keeping it under control is NOS-2, via a NRF2 mechanisms.

Carbon monoxide (CO)      Image may be NSFW.
Clik here to view.

• This is a small gaseous molecule enzymatically produced for signaling purposes in physiologically minute quantities
• It has major beneficial physiologic effects such as bronchodilation.  
• At higher concentrations, it has major detrimental effects, such as competitive binding with oxygen on hemoglobin, which results in death!
• CO is produced by heme oxygenases (HO) – which again exist in different isoforms which are regulated differently.

1. NRF2 Inducible isoform – HO-1 – regulated by cellular stress

2. non-inducible isoform – HO – I don’t know what triggers this form

• Transcription factor controlling HO-1:  NRF2
• how to non-invasively measure CO:  expired air (i.e. in your breath!): machine: multichannel gas analyzer: Logan 2500, Logan Research Limited, Kent, UK.  reproducible? – yes.  Time taken to measure this – 6-13 min

The wild animal is CO and the zoo keeper keeping it under control is HO-1, via NRF2 mechanisms

Reference: Ryter SW, Otterbein LE, Morse D, Choi AM  Heme oxygenase/carbon monoxide signaling pathways: regulation and functional significance  Mol Cell Biochem. 2002 May-Jun;234-235(1-2):249-63. 

See also Rochette L, Cottin Y, Zeller M, Vergely C.  Carbon monoxide: Mechanisms of action and potential clinical implications.  Pharmacol Ther. 2012 Sep 29. pii: S0163-7258(12)00209-4. doi: 10.1016/j.pharmthera.2012.09.007. [Epub ahead of print]

Hydrogen sulfide (H2S)-   Image may be NSFW.
Clik here to view.

• H2S is again a small gaseous molecule, enzymatically produced in physiologic minute quantities
• It has major beneficial physiologic effects such as vasodilation – i.e. controlling blood vessel caliber and flow and involved in inflammation and in the regulation of cardiac response to ischemia and reperfusion injury.
• The nice thing about H2S is that it does not have the capacity to form a toxic metabolite (i.e. it is different in this regard from CO and NO), even though it smells very bad.
• H2S is produced by cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS) (ref)(ref), and probably by other naturally occurring enzymes.

1. Nrf2 Inducible isoform: CSE

2. non-inducible isoform -

• Transcription factor controlling CSE: Nrf2
• How to non-invasively measure H2S:  no one has described this yet – we could win the Nobel prize for this! 

The wild animal is H2S and the zoo keeper keeping it under control is CSE, via NRF2 mechanisms

Reference: Ling L, Rose P, Moore P, Hydrogen Sulfide and Cell Signaling, Annual Review of Pharmacology and Toxicology, Vol 51, pp 169-187, February, 2011

Hydrogen Cyanide (HCN)  –   Image may be NSFW.
Clik here to view.

• HCN is a small molecule , that is enzymatically produced in physiologic minute quantities.
• HCN has major beneficial effects. (I am not sure what they all are yet). 
• HCN signaling appears to mainly have been studied in plants.  We know that HCN plays a signaling role in the germination of and development apple seedlings(ref)(ref)
• I also don’t know if HCN is under the control of Nrf2.
• What I do know is that at higher concentrations, HCN has major detrimental effects such as death!  

Superoxide (O2)      Image may be NSFW.
Clik here to view.

• O2 is a  small molecule (NOT a gas), that is enzymatically produced in physiologic minute quantities.
• O2  tat has major beneficial physiologic effects as a growth signal via the ras-NAD(P)H oxidase-MAPK signaling pathway,

- peroxisome proliferator-activator-activated receptor gamma (PPAR-gamma) pathway (involved with PGC-1alpha), etc.

• At higher concentrations, superoxide has major detrimental effects, such as death!
• Production: There are several inducible systems and several non-inducible systems

1. NRF2 Inducible O2- systems: NAD(P)H oxidase

2. Non-inducible O2- systems: mitochondria

• Scavengers:SOD1 (cytoplasm), SOD2 (mitochondria), SOD3 (plasma, extracellular space)
• Transcription factor controlling SODs: Nrf2
• How to non-invasively measure O2?  No one has described this yet!  We could win our 2nd Nobel prize for this

The wild animal is O2- and here the zoo keeper controls both the production of the wild animal (NADPH oxidase) and the destruction of the wild animal (SOD), via Nrf2

Reference: Buetler T, Krauskopf A, Ruegg U, Role of Superoxide as a Signaling Molecule, Physiology,Vol 19(3), pp 120-123, June 1, 2004

Hydrogen Peroxide (H2O2)   Image may be NSFW.
Clik here to view.

• H202 is a small molecule (NOT a gas), that is enzymatically produced in physiologic minute quantities.
• H202  has major beneficial physiologic effects as a cell signaling molecule.  
• It does this via interaction with redox sensitive proteins with a cystein-sulfhydryl group on them with a low pKa at their active sites.
• Here is a list of them (i.e. redox sensitive cell signaling molecules:

Cysteine Residue proteins:

- Thioredoxin

- Disulfide isomerase

- Cysteine proteases – Ex: caspaces

- peroxiredoxin -

- serine/threonine kinases – cAMP-dependent kinase, S6Kinase, cGMP-dependent kinase, etc.

• What molecules trigger H2O2 as a signaling molecule?   PDGF 
• At high levels, H2O2 can have detrimental effects such as death!
• Scavenger for H2O2:catalase, GST, etc.
• Transcription factor controlling scavenger system: Nrf2
• How to non-invasively measure H2O2?  No one has described this. We could win our 3rd Nobel prize!
• In this case I don’t know if the zookeeper controls the production of the wild animal

The wild animal is H2O2 and here the zookeeper Nrf2 controls the destruction of the wild animal (catalase).

Reference: Rhee SG, Redox signaling: hydrogen peroxide as intracellular messenger, Experimental and Molecular Medicine, 

Vol 31(2), pp 53-59, June, 1999

 Summary:  All of these small molecules function as intracellular 2nd messengers in some aspect, but do not work via the classic lock-and-key, enzyme pocket interactions with proteins.  They are way too “reactive” to communicate this way!  They are wild animals!  They attack!  They don’t shake hands and sing Kumbaya!.  The way they communicate as 2nd messengers is to react with cysteine sulfhydryl groups on their targets which affects the protein they interact with, via some conformational change in shape of the protein.  And they don’t live very long at all.

It was once thought that in cells these zoo animals were by-products of metabolism.  No more.  They are messengers which can bend proteins out of shape.

There are probably many more “wild animals in the zoo.  Here are the ones I have not studied: peroxynitrate (ONOO-), thiyl peroxyl radical (RSOO), ferry radical (FeO2++), and the hydroxyl radical (OH).  These may all interact with cystein residues with pKa of < 7.0.   I think we have quite a zoo here!

One implication of all this is that we must bury the FRTA (Free radical theory of aging) as soon as possible!  Free radicals and stress are essential in the fabric of life (ref)(ref)(ref).  Another is that we have to stop thinking of substances as good and bad.  It all depends on the dose and context and the very worst stuff like ccarbon monoxide gas – well – it is made by us, used by us and we need it.  Hormesis rules the day! (ref)(ref)(ref).  And oh yes, we recall that the master zookeeper Nrf2 is regulated by Keap1, which is the zoo keaper.  A final thought relative to taking conventional antioxidants which kill free radicals:  Don’t shoot the 2^nd messengers.

Certain plant polyphenols not only exercise general positive health effects but also inhibit oncogenic transformation or the proliferation of cancer cells.  Or, they outright kill cancer cells.  Included are a substantial number of cancer-fighting plant polyphenols, some of which are found in familiar substances like green tea, turmeric, ginger, garlic, broccoli and bitter melon.  This blog entry is about the biological pathways and epigenetic transformations through which these cancer-fighting effects take place.  I start with short descriptions of six semi-independent modes of epigenetic action through which certain polyphenols attack  cancer cells – the six knockout punches delivered by these polyphenols.  Then I cite a selection of research publications supporting this general picture and filling in the details.

Image may be NSFW.
Clik here to view.        Image may be NSFW.
Clik here to view.

Picture source                     Image source
Cancer-fighting polyphenols (CFPs) exert their actions through at least six mechanisms:

First knockout punch mechanism – downregulation of expression of NF-kappaB

CFPs downregulate the expression of the pro-inflammatory intermediary NF-kappaB.  Cancer cells proliferate most readily in a pro-inflammatory environment and find anti-inflammatory environments hostile.

Second knockout punch mechanism – CFPs creates stress and stress signaling in cells

In normal cells, the stress generated by normal dietary intake of CFPs is within the hormetic range of response.  Nrf2 translocates into the nucleus, ARE genes are activated including anti-oxidant and Phase 2 detoxifying genes.  The net result is health-producing for the cell.  The same is true when the stress is due to normal doses of CFP supplements.   In cancer cells which are relatively deregulated to start with, the stress generated by CFP intake can be beyond the hormetic range, with the result being damage or death to the cancer cell.

Third knockout punch mechanism – HDAC inhibition which re-activates tumor suppressor genes.

CFPs act as HDAC inhibitors or HATs (histone deacetylase inhibitors or histone acetyl transferases).  Expression of protective pro-apoptotic genes like P53 and P21 is turned on in acetylated cancer cells while it is usually turned off in deacetylated cancer cells.  So, sensing self-damage, acetylated cancer cells are more likely to terminate themselves via apoptotic death.

Fourth knockout punch mechanism – impact on DNA repair machinery

For the same reasons related to both non-hormetic oxidative stress and histone acetylation, cancer cells in an acetylated state are more likely to experience damage to their DNA repair machinery.

Fifth knockout punch – impact of CFPs on mRNAs

CFPs act on mRNAs so as to induce tumor suppressor mRNAs and block oncogenic mRNAs

Sixth knockout punch mechanism – – CFPs inhibit DNA methyltransferases (DNMT) in cancer cells, thus blocking the methylation and inactivation of tumor suppressor genes in cancers.

These mechanisms are further elucidated in the research publications cited below.

What are the major CFPs?

A listing of many of them is found in the abstract for the publication Molecular targets of dietary agents for prevention and therapy of cancer, although in 2006, the time of that publication, the epigenetic mechanisms were only partially understood.  “While fruits and vegetables are recommended for prevention of cancer and other diseases, their active ingredients (at the molecular level) and their mechanisms of action less well understood. Extensive research during the last half century has identified various molecular targets that can potentially be used not only for the prevention of cancer but also for treatment. However, lack of success with targeted monotherapy resulting from bypass mechanisms has forced researchers to employ either combination therapy or agents that interfere with multiple cell-signaling pathways. In this review, we present evidence that numerous agents identified from fruits and vegetables can interfere with several cell-signaling pathways. The agents include curcumin (turmeric), resveratrol (red grapes, peanuts and berries), genistein (soybean), diallyl sulfide (allium), S-allyl cysteine (allium), allicin (garlic), lycopene (tomato), capsaicin (red chilli), diosgenin (fenugreek), 6-gingerol (ginger), ellagic acid (pomegranate), ursolic acid (apple, pears, prunes), silymarin (milk thistle), anethol (anise, camphor, and fennel), catechins (green tea), eugenol (cloves), indole-3-carbinol (cruciferous vegetables), limonene (citrus fruits), beta carotene (carrots), and dietary fiber. For instance, the cell-signaling pathways inhibited by curcumin alone include NF-kappaB, AP-1, STAT3, Akt, Bcl-2, Bcl-X(L), caspases, PARP, IKK, EGFR, HER2, JNK, MAPK, COX2, and 5-LOX. The active principle identified in fruit and vegetables and the molecular targets modulated may be the basis for how these dietary agents not only prevent but also treat cancer and other diseases. This work reaffirms what Hippocrates said 25 centuries ago, let food be thy medicine and medicine be thy food.”

The overall epigenetic actions of CFPs on regulating cancers is summarized in the Sept 2012 publiction Cancer Chemoprevention and Nutri-Epigenetics: State of the Art and Future Challenges: “The term “epigenetics” refers to modifications in gene expression caused by heritable, but potentially reversible, changes in DNA methylation and chromatin structure. Epigenetic alterations have been identified as promising new targets for cancer prevention strategies as they occur early during carcinogenesis and represent potentially initiating events for cancer development. Over the past few years, nutri-epigenetics – the influence of dietary components on mechanisms influencing the epigenome – has emerged as an exciting new field in current epigenetic research. During carcinogenesis, major cellular functions and pathways, including drug metabolism, cell cycle regulation, potential to repair DNA damage or to induce apoptosis, response to inflammatory stimuli, cell signalling, and cell growth control and differentiation become deregulated. Recent evidence now indicates that epigenetic alterations contribute to these cellular defects, for example epigenetic silencing of detoxifying enzymes, tumor suppressor genes, cell cycle regulators, apoptosis-inducing and DNA repair genes, nuclear receptors, signal transducers and transcription factors by promoter methylation, and modifications of histones and non-histone proteins such as p53, NF-κB, and the chaperone HSP90 by acetylation or methylation.The present review will summarize the potential of natural chemopreventive agents to counteract these cancer-related epigenetic alterations by influencing the activity or expression of DNA methyltransferases and histone modifying enzymes. Chemopreventive agents that target the epigenome include micronutrients (folate, retinoic acid, and selenium compounds), butyrate, polyphenols from green tea, apples, coffee, black raspberries, and other dietary sources, genistein and soy isoflavones, curcumin, resveratrol, dihydrocoumarin, nordihydroguaiaretic acid (NDGA), lycopene, anacardic acid, garcinol, constituents of Allium species and cruciferous vegetables, including indol-3-carbinol (I3C), diindolylmethane (DIM), sulforaphane, phenylethyl isothiocyanate (PEITC), phenylhexyl isothiocyanate (PHI), diallyldisulfide (DADS) and its metabolite allyl mercaptan (AM), cambinol, and relatively unexplored modulators of histone lysine methylation (chaetocin, polyamine analogs). So far, data are still mainly derived from in vitro investigations, and results of animal models or human intervention studies are limited that demonstrate the functional relevance of epigenetic mechanisms for health promoting or cancer preventive efficacy of natural products. Also, most studies have focused on single candidate genes or mechanisms research has the potential to explore nutri-epigenomics at a genome-wide level to understand better the importance of epigenetic mechanisms for gene regulation in cancer chemoprevention.”. With the emergence of novel technologies such as next-generation sequencing, future

Background for first knockout punch mechanism – downregulation of expression of NF-kappaB

Regarding cancer-fighting polyphenols: I have discussed the anti-cancer activities of CFPs in a number of blog entries.  In the blog entry New, emerging and potential treatments for cancers: Part 3 – selected less-known phytochemicals that have long been used in traditional Chinese medicine – focus on gambogic and gambogenic acids I related “ Many traditional Chinese medicines have been extensively studied in China during the last 10-20 years using the current tools and intellectual frameworks of modern Western science. These medicines have been looked at in terms of their detailed chemical structures, their proteomic properties, the molecular biological pathways through which they work, their gene activation and epigenetic properties, their pharmacological properties, etc, This work has generally been of high quality and has resulted in thousands or tens of thousands of research reports, many of them published in highly respected Western journals. Abstracts to these publications can be found in the definitive US National Library of Medicine database pubmed.org.  – A good place to start is with the July 2011 e-publication Anti-cancer natural products isolated from chinese medicinal herbs, as to be expected written by a team of Chinese researchers. “In recent years, a number of natural products isolated from Chinese herbs have been found to inhibit proliferation, induce apoptosis, suppress angiogenesis, retard metastasis and enhance chemotherapy, exhibiting anti-cancer potential both in vitro and in vivo. This article summarizes recent advances in in vitro and in vivo research on the anti-cancer effects and related mechanisms of some promising natural products. These natural products are also reviewed for their therapeutic potentials, including flavonoids (gambogic acid, curcumin, wogonin and silibinin), alkaloids (berberine), terpenes (artemisinin, β-elemene, oridonin, triptolide, and ursolic acid), quinones (shikonin and emodin) and saponins (ginsenoside Rg₃), which are isolated from Chinese medicinal herbs. In particular, the discovery of the new use of artemisinin derivatives as excellent anti-cancer drugs is also reviewed.”

Regarding expression of NF-kappaB: From my Treatise: “An important line of epigenomic research relates to NF-kappaB signaling. NF-kappaB is a nuclear transcription factor involved in cell signaling, i.e. a protein that binds to a specific sequence of DNA. It is present in a latent (non-activated) form in many cell types. On the one hand, expression of NF-kB appears to be one of the body’s regulatory means for handling situations of stress, cancer, damage or disease. In eukaryotic cells NF-kB is an important regulator of genes that control cell proliferation and cell survival. NF-kB regulates anti-apoptotic genes that protect healthy cells from cell death and activates the expression of genes that keep cells proliferating. On the other hand, activated NF-kB binding to genes has long been known to play a central role in promoting runaway inflammation and inflammation’s negative consequences. — These consequences include promotion of angiogenesis, proliferation, metastasis and invasiveness in cancer tumors, autoimmune diseases, neurodegenerative diseases and contributing to the activation of human immunodeficiency virus (HIV) leading to AIDS.  – There appears to be increasing evidence that inhibition of expression of NF-kB could be a key approach for fighting cancers, controlling inflammatory diseases, AIDS, neurodegenerative conditions like Parkinson’s Disease and a number of other significant age-related maladies. — Recent studies position NF-kB even more centrally with respect to longevity. It is likely that NF-kB expression is central to a programmed set of changes which we call aging. One study(ref1) confirms that that in multiple mammalian tissues (including skin fibroblasts, kidney, cortex, kidney medulla, abdominal muscle, skeletal muscle, and brain), aging involves continuing changes in expression of hundreds of genes. And, further, NF-kB signaling appears to be a major regulator of gene expression related to the aging progress. In fact, by inhibiting NF-kB cell signaling the researchers were able to cause the epidermal tissue of old mice to revert to the state of very young mouse tissue, both in observable characteristics and in genetic expression profile. The authors show that NF-kB cell signaling is a meta-factor for determining aging of nine other key cell types as well, and they argue that the results should apply equally to humans and other mammals.”  The relationship of NF-kappaB expression to oncogenic processes.  cancer proliferation and metastasis runs deep and wide.  See for example The activated NF-kappaB-Snail-RKIP circuitry in cancer regulates both the metastatic cascade and resistance to apoptosis by cytotoxic drugs.

 Regarding CFPs and the expression on NF-kappaB:   Discussion in my treatise as well as many blog entries point to how CFPs inhibit the expression of NF-kappaB.

“Regarding proinflammatory environments and cancer susceptibility:  It has long been known that a pro-inflammatory environment can significantly enhance cancer susceptibilities.  The 2002 publication Inflammation and cancer pointed out: “Recent data have expanded the concept that inflammation is a critical component of tumour progression. Many cancers arise from sites of infection, chronic irritation and inflammation. It is now becoming clear that the tumour microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. In addition, tumour cells have co-opted some of the signalling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. These insights are fostering new anti-inflammatory therapeutic approaches to cancer development.  — The functional relationship between inflammation and cancer is not new. In 1863, Virchow hypothesized that the origin of cancer was at sites of chronic inflammation, in part based on his hypothesis that some classes of irritants, together with the tissue injury and ensuing inflammation they cause, enhance cell proliferation¹. Although it is now clear that proliferation of cells alone does not cause cancer, sustained cell proliferation in an environment rich in inflammatory cells, growth factors, activated stroma, and DNA-damage-promoting agents, certainly potentiates and/or promotes neoplastic risk.”  Inflammation ,au contribute to cancers via engendering higher rate of DNA mutations.  An author of a 2011 study reported “”Our study shows that miR-155 is upregulated by inflammatory stimuli and that overexpression of miR-155 increases the spontaneous mutation rate, which can contribute to tumorigenesis.”

Together, points a. – d. above characterize the first mechanism through which  CFPs combat cancers: CFPs downregulate the expression of the pro-inflammatory intermediary NF-kappaB.  Cancer cells proliferate most readily in a pro-inflammatory environment and find anti-inflammatory environments hostile.

Background for the second knockout punch mechanism – CFPs creates stress and stress signaling in cells

In normal cells, the stress associated with CFP intake is within the hormetic range of response, Nrf2 translocates into the nucleus, ARE genes are activated including anti-oxidant and Phase 2 detoxifying genes.  The net result is health-producing for the cell.  See the blog entries Nrf2: Part 1 – a new view on the control of oxidative damage and generation of hormetic effects.  The pivotal role of Nrf2. Part 3 – Part 3 – Is promotion of Nrf2 expression a viable strategy for human human healthspan and lifespan extension?, and Part 2 – foods, phyto-substances and other substances that turn on Nrf2.

However, in cancer cells which are relatively deregulated to start with, the stress created by ingestion of CFPs can be beyond the hormetic range, with the result being damage or death to the cancer cell. See the blog entries Mitohormesis and Radiation Hormesis for discussion of hormetic range.  For example, for human head and neck squamous cell carcinoma cells, DNA damage has been shown to be caused by resveratrol(ref).  Grape seed extract likewise causes DNA damage to squamous cell carcinoma cells (ref).  Grape seed extract leads to apoptotic death of human prostate carcinoma DU145 cells via caspases activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release(ref).  Sulphoraphane, a naturally occurring isothiocyanate induces apoptosis in breast cancer cells by targeting heat shock proteins(ref).

Background for the third knockout punch mechanism – HDAC inhibition which re-activates tumor suppressor genes

Cancer cells tend to be epigenetically dysregulated.   This can be seen in several epigenetic dimensions, for example that of gene methylation profiles.  The just-published (October 2012) publication  LRpath analysis reveals common pathways dysregulated via DNA methylation across cancer types  reports: “BACKGROUND: The relative contribution of epigenetic mechanisms to carcinogenesis is not well understood, including the extent to which epigenetic dysregulation and somatic mutations target similar genes and pathways. We hypothesize that during carcinogenesis, certain pathways or biological gene sets are commonly dysregulated via DNA methylation across cancer types. The ability of our logistic regression-based gene set enrichment method to implicate important biological pathways in high-throughput data is well established. RESULTS: We developed a web-based gene set enrichment application called LRpath with clustering functionality that allows for identification and comparison of pathway signatures across multiple studies. Here, we employed LRpath analysis to unravel the commonly altered pathways and other gene sets across ten cancer studies employing DNA methylation data profiled with the Illumina HumanMethylation27 BeadChip. We observed a surprising level of concordance in differential methylation across multiple cancer types. For example, among commonly hypomethylated groups, we identified immune-related functions, peptidase activity, and epidermis/keratinocyte development and differentiation. Commonly hypermethylated groups included homeobox and other DNA-binding genes, nervous system and embryonic development, and voltage-gated potassium channels. For many gene sets, we observed significant overlap in the specific subset of differentially methylated genes. Interestingly, fewer DNA repair genes were differentially methylated than expected by chance.  CONCLUSIONS: Clustering analysis performed with LRpath revealed tightly clustered concepts enriched for differential methylation. Several well-known cancer-related pathways were significantly affected, while others were depleted in differential methylation. We conclude that DNA methylation changes in cancer tend to target a subset of the known cancer pathways affected by genetic aberrations.”

Histone deacetylase inhibition (HDACi) is an important emerging approach to cancer therapy.

I have made this point before(ref).  HDAC inhibitors are of interest for treating many medical conditions besides cancers including targeting Alzheimer’s disease(ref), other neurodegenerative conditions(ref), and diabetes(ref). While many substances are HDAC inhibitors including a number of CFPs, the drug panobinostat is being particularly studied and experimentally used in the cancer research community. “Panobinostat (LBH-589) is an experimental drug developed by Novartis for the treatment of various cancers. It is a hydroxamic acid^[1] and acts as a non-selective histone deacetylase inhibitor (HDAC inhibitor) — Panobinostat inhibits multiple histone deacetylase enzymes, a mechanism leading to apoptosis of malignant cells via multiple pathways.^[1] (ref).^[2]”

Going back to a 2004 publication Role of histone deacetylase inhibitors in the treatment of cancer (Review):  “Acetylation and deacetylation of nucleosomal histones play an important role in the modulation of chromatin structure, chromatin function and in the regulation of gene expression. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) are two opposing classes of enzymes, which tightly control the equilibrium of histone acetylation. An imbalance in the equilibrium of histone acetylation has been associated with carcinogenesis and cancer progression. So far, a number of structurally distinct classes of compounds have been identified as HDAC inhibitors including the short-chain fatty acids, hydroxamates, cyclic tetrapeptides and benzamides. These compounds lead to an accumulation of acetylated histone proteins both in tumor cells and in normal tissues. HDAC inhibitors are able to activate differentiation, to arrest the cell cycle in G1 and/or G2, and to induce apoptosis in transformed or cancer cells. Attention is currently being drawn to molecular mechanisms involving histone deacetylases. An induction of p21(WAF/CIP1) and a suppression of angiogenic stimulating factors have been observed in tumor cells following exposure to HDAC inhibitors. In xenograft models, several HDAC inhibitors have demonstrated antitumor activity with only few side effects. Several clinical trials showed that HDAC inhibitors in well tolerated doses have significant antitumoral activities. A combination of HDAC inhibitors with differentiation-inducing agents and cytotoxic drugs is an innovative therapeutic strategy that carries the potential for significant improvements in the treatment of cancer.”

The 2010 publication Histone deacetylase inhibitors: mechanisms and clinical significance in cancer: HDAC inhibitor-induced apoptosis tells a part of the story.  “Epigenic modifications, mainly DNA methylation and acetylation, are recognized as the main mechanisms contributing to the malignant phenotype. Acetylation and deacetylation are catalyzed by specific enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. While histones represent a primary target for the physiological function of HDACs, the antitumor effect of HDAC inhibitors might also be attributed to transcription-independent mechanisms by modulating the acetylation status of a series of non-histone proteins. HDAC inhibitors may act through the transcriptional reactivation of dormant tumor suppressor genes. They also modulate expression of several other genes related to cell cycle, apoptosis, and angiogenesis. Several HDAC inhibitors are currently in clinical trials both for solid and hematologic malignancies. Thus, HDAC inhibitors, in combination with DNA-demethylating agents, chemopreventive, or classical chemotherapeutic drugs, could be promising candidates for cancer therapy.”

HDAC-inhibiting drugs have been approved for cancer indications by the FDA and are in clinical use.  A list of HDAC-inhibiting drugs for cancers and other disease indications in clinical trials in 2010 can be found here.   As reported in the 2011 publication Rational therapeutic combinations with histone deacetylase inhibitors for the treatment of cancer, HDAC-inhibiting drugs have been approved by the FDA and are used generally in combination with other drugs.  ”Histone deacetylases (HDACs) regulate the acetylation of a variety of histone and nonhistone proteins, controlling the transcription and regulation of genes involved in cell cycle control, proliferation, survival, DNA repair and differentiation. Unsurprisingly, HDAC expression is frequently altered in hematologic and solid tumor malignancies. Two HDAC inhibitors (vorinostat and romidepsin) have been approved by the US FDA for the treatment of cutaneous T-cell lymphoma. As single agents, treatment with HDAC inhibitors has demonstrated limited clinical benefit for patients with solid tumors, prompting the investigation of novel treatment combinations with other cancer therapeutics. In this article, the rationales and clinical progress of several combinations with HDAC inhibitors are presented, including DNA-damaging chemotherapeutic agents, radiotherapy, hormonal therapies, DNA methyltransferase inhibitors and various small-molecule inhibitors. The future application of HDAC inhibitors as a treatment for cancer is discussed, examining current hurdles to overcome before realizing the potential of this new approach.”

Going beyond the above, it is possible now to surface deeper mechanisms through which HDAC inhibitors fight cancers

There are at least two mechanisms through which HDAC inhibitors fight cancers: 1. turning on pro-apoptotic tumor suppresssor genes like P53 and P21 in cancer cells and 2. downregulates the cancer cell’s DNA repair machinery.

It has long been known that histone deacytlases negatively regulate tumor suppressor genes thus promoting carcinogenesis(ref).  Similarly HDAC inhibitors can cause re-expression of tumor suppressor genes like P53 which are turned off in cancer cells.  For example, benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis.  Its mode of operation appears to involve upregulation of P53(ref), likely to be due to HDAC inhibitory activity.

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Image source  “Effects of dietary polyphenols on the DNA methylation and histone modifications  -  Simplified scheme demonstrates a number of epigenetic changes that occur during carcinogenesis. In cancers, tumor suppressor genes become “inactivated” (shown as red circles) while oncogenes are “activated” (green circles). Epigenetic gene expression regulation is a complex process and several key enzymes play crucial roles. DNA methyltransferase (DNMT) is responsible for transfer of methyl group to 5′-cytosine. Histone acetylases (HAT) and histone deacetylases (HDAC) are responsible for the acetylation and de-acetylation of lysine residues within histone tails, respectively. Because of these histone modifications, conformational changes in chromatin structure lead to changes in DNA accessibility for transcription regulators and polymerases. Polyphenols can impact these enzymes in specific ways induces reversibility of epigenetic dysregulation in cancer cells(ref).”

The 2010 publication Downregulation of Homologous Recombination DNA Repair Genes by HDAC Inhibition in Prostate Cancer Is Mediated through the E2F1 Transcription Factor discusses the second mechanism in the case of prostate cancer: “Background Histone deacetylase inhibitors (HDACis) re-express silenced tumor suppressor genes and are currently undergoing clinical trials. Although HDACis have been known to induce gene expression, an equal number of genes are downregulated upon HDAC inhibition. The mechanism behind this downregulation remains unclear. Here we provide evidence that several DNA repair genes are downregulated by HDAC inhibition and provide a mechanism involving the E2F1 transcription factor in the process. Methodology/Principal Findings Applying Analysis of Functional Annotation (AFA) on microarray data of prostate cancer cells treated with HDACis, we found a number of genes of the DNA damage response and repair pathways are downregulated by HDACis. AFA revealed enrichment of homologous recombination (HR) DNA repair genes of the BRCA1 pathway, as well as genes regulated by the E2F1 transcription factor. Prostate cancer cells demonstrated a decreased DNA repair capacity and an increased sensitization to chemical- and radio-DNA damaging agents upon HDAC inhibition. Recruitment of key HR repair proteins to the site of DNA damage, as well as HR repair capacity was compromised upon HDACi treatment. Based on our AFA data, we hypothesized that the E2F transcription factors may play a role in the downregulation of key repair genes upon HDAC inhibition in prostate cancer cells. ChIP analysis and luciferase assays reveal that the downregulation of key repair genes is mediated through decreased recruitment of the E2F1 transcription factor and not through active repression by repressive E2Fs. Conclusions/Significance Our study indicates that several genes in the DNA repair pathway are affected upon HDAC inhibition. Downregulation of the repair genes is on account of a decrease in amount and promoter recruitment of the E2F1 transcription factor. Since HDAC inhibition affects several pathways that could potentially have an impact on DNA repair, compromised DNA repair upon HDAC inhibition could also be attributed to several other pathways besides the ones investigated in this study. However, our study does provide insights into the mechanism that governs downregulation of HR DNA repair genes upon HDAC inhibition, which can lead to rationale usage of HDACis in the clinics.

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Clik here to view.By Vince Giuliano  

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The popular “life extension” blogs have been lit up recently with exchanges related to a recent publication that reports that a homogenized solution of olive oil and C60 carbon buckyballs fed to middle age rats extends their lifespans by an average of 90%.  If this result stands up it is truly amazing.   Compared with other longevity interventions such as rapamycin feeding or calorie restriction which at best extend lifespans by 15-20%, the 90% figure is off the scale.  So I decided to delve into the research literature to clarify what is known and what is not known about C60 carbon fullerenes as related to biological impacts and health.  I report on this expedition here.  I also chime in with my own hypotheses about the mechanisms through which the C60-olive oil cocktail extends rats lifespans, assuming it really does.  The main points I will be documenting are:

1. There is a substantial research literature related to C60 buckyballs and their health-producing biological impacts.  The research goes back some 30 years and Pubmed.org shows some 1089 publications related to C60 fullerenes.
2. The recent rat study stands on the shoulders of many prior research results and, like all good science, is carefully documented though of too-small a scale to lend certitude that the longevity results will stand up on a much larger scale. 
3. Remarkably, of the super-aged rats that had consumed the C60-olive oil mixture and experienced the 90% life extension effect in this study, none contracted a cancer.
4. An earlier study also indicates that C60 extends the life of mice.
5. For it to be biologically active and not toxic, the form of administration of C60 is critical.  It tends to clump and dissolves only very poorly in water.  Consumed orally in water or olive-oil solutions, C60 appears to be nontoxic in rats and is excreted by the body within days.
6. C60 being a nanomaterial readily penetrates the blood-brain barrier and into cell interiors, mitochondria and the nucleus.  It is believed to exercise major effects in mitochondria.
7. C60 can be very biologically active and a body of responsible research indicates it exercises powerful antioxidant, anti-cancer, immunomodulatory, neuromodulatory, antiamyloid, and other health-producing effects.  It is being actively investigated as an anti-cancer agent.
8. C60 can have multiple effects in cells: it binds to DNA and can affect gene expression; it can affect protein shapes and cell morphology and geometry; it binds to microtubules and can affect their forms and multiple functions including tubulin polymerization, and nuclear envelope shape.
9. Fullerenes can be employed as targeted drug and gene delivery vehicles.
10. C60 buckyballs are small enough to fit within a physical cavity in the HIV-1 virus.  And once inside that cavity the C60 blocks replication of the virus.  Thus, C60 is seen as an important potential weapon in the battle against HIV and AIDS.
11. Among the other interesting biological properties of fullerenes is inhibiting the allergic response,  affecting cells involved on phagocytosis, affecting platelet aggregation, affecting the native structure of DNA, impacting gene expression, affecting microtubules in cells, influencing cell mitosis and of course the really important one – potentiating  hair growth.  Mechanisms of action are generally poorly understood or not understood at all, although antioxidant action is given the biggest credit.
12. The major context for studying C60 has been the development of new structural or semiconductor materials with extraordinary properties.  For this reason, C60 is being manufactured in ever-increasing quantities.  And there is concern that it is being released into the environment with unknown consequences.
13. These factors have led many scientists to caution about possible biological impacts of C60.  One known negative impact is that C60 cannot be used as a basis of a skin cream because when it is photo-excited it produces destructive singlet oxygen radicals. In general, there seems to be mixed evidence and little agreement about overall negative effects of C60. 
14. The C60-olive oil cocktail used in the rat longevity study is available commercially from several sources.  It appears from popular blog comments that there is an emerging cohort of people who are buying this cocktail and using themselves as human lab rats.  We don’t know whether these people will in the future be viewed as as long-lived personal health and longevity innovators or as reckless risk-takers who paid heavily for their self-experimentation.   
15. Most research publications attribute the health and longevity producing effects of C60 to its powerful antioxidant and membrane penetration qualities.  I disagree.  I doubt that antioxidant protection alone could lend 90% life extension in rats.  Further, given that the super-aged rats lived long beyond the period of months when they were treated with the C60-olive oil cocktail and that C60 rapidly clears from the body, some long-lasting shifts in cell and organs must have been created.  I hypothesize that other more-fundamental mechanisms are critically involved, such as C60 impacts on strengthening DNA, effects on microtubules or in the mitochondria and epigenetic impacts.  These may well involve quantum-level phenomena largely ignored in biology up to this point. 
16. I believe we are so far fairly ignorant of how C60 exercises its health and longevity effects.  If this is so, we may be on the verge of surfacing new biological mechanisms critical to health and longevity – ones unknown and unimagined until now.

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C60 buckyball Image source      Buckminister Fuller

 What is a C60 fullerene?

From wickipedia: “A fullerene is any molecule composed entirely of carbon, in the form of a hollow sphere, ellipsoid or tube. Spherical fullerenes are also called buckyballs, and they resemble the balls used in football (association football). Cylindrical ones are called carbon nanotubes or buckytubes. Fullerenes are similar in structure to graphite, which is composed of stacked graphene sheets of linked hexagonal rings; but they may also contain pentagonal (or sometimes heptagonal) rings.^{[1] –}  – The first fullerene molecule to be discovered, and the family’s namesake, buckminsterfullerene (C₆₀), was prepared in 1985 by Richard Smalley, Robert Curl, James Heath, Sean O’Brien, and Harold Kroto at Rice University. The name was an homage to Buckminster Fuller, whose geodesic domes it resembles. The structure was also identified some five years earlier by Sumio Iijima, from an electron microscope image, where it formed the core of a “bucky onion.”^[2] Fullerenes have since been found to occur in nature.^[3] More recently, fullerenes have been detected in outer space.^[4] According to astronomer Letizia Stanghellini, “It’s possible that buckyballs from outer space provided seeds for life on Earth.”^[5]

C60 buckyballs and longevity of rats, mice and other lower species

I start out with the publication that has initiated the current buz, The prolongation of the lifespan of rats by repeated oral administration of [60] fullerene published in August 2011. The study reported was designed to assess the toxicity of C60 in an olive oil suspension, not to assess impact on lifespans of rats.  The result that not only was the suspension not toxic but radically increased the livespans of the rats was a surprise to the researchers. “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 subacute 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.”

A nice thing about this publication is that it describes the experimental conditions in meticulous detail.  For example, getting a good solvent vector for administration of C60 to animals has been a serious problem.  Unlike many other studies which employed water-based solutions of C60 with poor or uncertain bioavailability and toxic effects, this study used an olive oil brew.  “Fifty mg of C60 were dissolved in 10 ml of olive oil by stirring for 2 weeks at was increased to 60% for 10 min and then hold constant for the remaining 7 min of ambient temperature in the dark. The resulting mixturewas centrifugedat 5.000gfor each sample run. At least 10 column volumes of the initial composition were flushed 1 h and the supernatant was filtered through a Millipore filter with 0.25 mmporosity.”

There were several sub-studies reported in this paper.  In the chronic toxicity and longevity sub-study, only 18 rats middle-aged were involved divided into three cohorts of six rats each: a) a control cohort fed normal rat chow, b) a cohort fed food plus olive oil by gavage, and c) a cohort feed the C60-olive oil brew by gavage (forced feeding).  “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(10 months old,  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.”  All rats in cohort (a) were alive until week 18 of the experiment and all were dead by week 38.  All rats in cohort (b). were alive until week 36 and all were dead by week 58.  In cohort (c). all rats were alive until week 60 and all dead by week 66 (the last one being sacrificed at week 66).  Between weeks 38 and 60 all the control rats were dead and all the C60-fed rats were alive and well.  Olive oil alone produced a weighted average of 18% life extension while the weighted average for the C60-olive oil brew was 90%.  Remarkably, no rats in cohort (c) contracted cancers.

In the sub-study of oxidative stress, the C60-olive oil mix almost completely protected against carbon tetrachloride oxidative liver damage.  “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 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.”  The animals were subsequently sacrificed and their livers examined. “– the liver sections of GA and GC animals co-treated with water and CCl4 or with olive respectively,  showed important damage including many inflammatory areas as well as large necrotic areas with ballooning necrotic cells associated with an important steatosis (Fig. 4). In contrast, microscopic examination of the liver sections of GE animals co-treated with C60-olive oil and CCl4, revealed few necrotic areas with some ballooning cells without apoptosis limited to some cords of hepatocytes (Fig. 4).”

The study also investigated the pharmacodynamics and pharmacokinetics of C60 administration.   “The results of this pharmacokinetic study show for the first time that C60 is absorbed by the gastro-intestinal tract (Fig. 1). — In the case of highly hydrophobic drugs (Log P > 5) it is well known that the absorption of the molecules by the gastro-intestinal tract occurs via the mesenteric lymphatic system after association with developing lipoproteins in the enterocytes rather than via the portal blood [40]. Therefore, as the octanol/water partition coefficient of C60 is estimated to be 6.67 [41], the absorption of C60 occursvia the mesenteric lymphatic system rather than via the portal blood.” – “The elimination half-lives indicate that C60 is completely eliminated from blood 97 h after administration irrespective of the route of administration.” – “The elimination process follows a non-urinary route because unmodified C60 was not detected in urine samples taken up 48 h after administration.  Previous investigations showed that C60 is mainly eliminated through the bile ducts [21] –. 

“Conclusion:  The effect of pristine C60 on lifespan emphasizes 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 optimize 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”

A 2008 publication also indicated that a fullerene is capable of extending the lifespans of mice: A carboxyfullerene SOD mimetic improves cognition and extends the lifespan of mice. “In lower organisms, such as Caenorhabditis elegans and Drosophila, many genes identified as key regulators of aging are involved in either detoxification of reactive oxygen species or the cellular response to oxidatively-damaged macromolecules. Transgenic mice have been generated to study these genes in mammalian aging, but have not in general exhibited the expected lifespan extension or beneficial behavioral effects, possibly reflecting compensatory changes during development. We administered a small-molecule synthetic enzyme superoxide dismutase (SOD) mimetic to wild-type (i.e. non-transgenic, non-senescence accelerated) mice starting at middle age. Chronic treatment not only reduced age-associated oxidative stress and mitochondrial radical production, but significantly extended lifespan. Treated mice also exhibited improved performance on the Morris water maze learning and memory task. This is to our knowledge the first demonstration that an administered antioxidant with mitochondrial activity and nervous system penetration not only increases lifespan, but rescues age-related cognitive impairment in mammals. SOD mimetics with such characteristics may provide unique complements to genetic strategies to study the contribution of oxidative processes to nervous system aging.”

Another 2011 publication Polyhydroxy Fullerenes (Fullerols or Fullerenols): Beneficial Effects on Growth and Lifespan in Diverse Biological Models indicates that fullerenes can extend the lifespans of certain more primitive organisms.  The publication reports “Recent toxicological studies on carbon nanomaterials, including fullerenes, have led to concerns about their safety. Functionalized fullerenes, such as polyhydroxy fullerenes (PHF, fullerols, or fullerenols), have attracted particular attention due to their water solubility and toxicity. Here, we report surprisingly beneficial and/or specific effects of PHF on model organisms representing four kingdoms, including the green algae Pseudokirchneriella subcapitata, the plant Arabidopsis thaliana, the fungus Aspergillus niger, and the invertebrate Ceriodaphnia dubia. The results showed that PHF had no acute or chronic negative effects on the freshwater organisms. Conversely, PHF could surprisingly increase the algal culture density over controls at higher concentrations (i.e., 72% increase by 1 and 5 mg/L of PHF) and extend the lifespan and stimulate the reproduction of Daphnia (e.g. about 38% by 20 mg/L of PHF). We also show that at certain PHF concentrations fungal growth can be enhanced and Arabidopsis thaliana seedlings exhibit longer hypocotyls, while other complex physiological processes remain unaffected. These findings may open new research fields in the potential applications of PHF, e.g., in biofuel production and aquaculture. These results will form the basis of further research into the mechanisms of growth stimulation and life extension by PHF.”

C60 is a powerful antioxidant

This point is long known and confirmed in a number of studies.  From (2007) Medicinal applications of fullerenes: “Results published in 1999 have shown that fullerenes have a potential as biological antioxidants. The antioxidant property is based on the fact that fullerenes possess large amount of conjugated double bonds and low lying lowest unoccupied molecular orbital (LUMO) which can easily take up an electron, making an attack of radical species highly possible. It has been reported that up to 34 methyl radicals have been added onto a single C60 molecule. This quenching process appears to be catalytic. In other words the fullerene can react with many superoxides without being consumed. Due to this feature fullerenes are considered to be the world’s most efficient radical scavenger and are described as radical sponges (Krusic et al 1991). The major advantage of using fullerenes as medical antioxidant is their ability to localize within the cell to mitochondria and other cell compartment sites, where in diseased states, the production of free radicals takes place. — Experiments on rats done by Najla Gharbi and coworkers proved this remarkable trait. They showed that aqueous C60 suspensions prepared without using any polar organic solvent, not only have no acute or sub acute toxicity in rodents, but also protect their livers against free-radical damage (Gharbi et al 2005).”

The 2005 publication [60]fullerene is a powerful antioxidant in vivo with no acute or subacute toxicity confirms both this and another point made in the recent rat study: C60 does not engender toxicity in rodents.  “In the present work, we report the effects of C(60)-pretreatments on acute carbon tetrachloride intoxication in rats, a classical model for studying free-radical-mediated liver injury. Our results show that aqueous C(60) suspensions prepared without using any polar organic solvent not only have no acute or subacute toxicity in rodents but they also protect their livers in a dose-dependent manner against free-radical damage. To be sure, according to histopathological examinations and biological tests, pristine C(60) can be considered as a powerful liver-protective agent.”

The 2011 report Antioxidant activity of fullerene C60 against OH free radicals: A Quantum Chemistry and Computational Kinetics Studyreports “Fullerenes are considered to be the world’s most efficient radical scavenger, and represents an attractive tool for biological applications. Indeed, it have been demonstrated in vivo and in vitro, that fullerenes and related structures reduce the toxicity of free radical assault on neuronal tissue, reacting readily and at a high rate with free radicals, which are often the cause of cell damage or death. Although there is strong evidence that antioxidant activity is an intrinsec property of fullerenes, the mechanism of radical scavenging and neuroprotection are still unclear. In this work, we have studied the reaction between fullerene C60 and hydroxyl radicals, using high level quantum chemistry and computational kinetics methods. Energy profiles are calculated using different basis sets, and reaction rate constant are reported for the first time. The presence of nonpolar environments seems to enhance the reactivity of fullerene molecule toward OH radicals, compared to the gas phase. Energetic considerations show that, once a first radical is attached to the fullerene cage, further additions are increasingly feasible, suggesting that fullerene can act as OH radical sponges.  They also protect their livers in a dose-dependent manner against free-radical damage. To be sure, according to histopathological examinations and biological tests, pristine C(60) can be considered as a powerful liver-protective agent.”

C60 has low toxicity and can cross the blood-brain barrier and may lead to many medical applications.

The 2012 publication C60 fullerene derivatized nanoparticles and their application to therapeutics reports “Fullerenes can be formed into many new materials and devices. They have a wide range of applications in medicine, electronics, biomaterials, and energy production. An overview of the nanostructure and the physical and chemical characteristics of fullerene-drug derivatives is given. The biological behavior of fullerene derivatives shows their potential to medical application fields because C(60) is rapidly absorbed by tissues and is excreted through urinary tract and enterons, which reveals low toxicity in vitro and in vivo studies. Nanomedicine has become one of the most promising areas of nanotechnology, while many have claimed its therapeutic use against cancer, human immunodeficiency virus (HIV), and neurodegenerative disorders. Water-soluble C(60) fullerene derivatives that come from chemical modification largely enhance the biological efficacy. The blood-brain barrier (BBB) is a physical barrier composed of endothelial tight junctions that restrict the paracellular permeability. A major challenge facing neuropharmacology is to find compounds that can be delivered into the brain through the bloodstream. Fullerene C(60) was demonstratively able to cross the BBB by hybridizing a biologically active moiety dyad, which provides a promising clue as a pharmacological therapy of neural disorders.”

Fullerene C60 is neuroprotective

The 2001 publication Fullerene-based antioxidants and neurodegenerative disorders reports: “Water-soluble derivatives of buckminsterfullerene (C60) derivatives are a unique class of compounds with potent antioxidant properties.  Studies on one class of these compounds, the malonic acid C60 derivatives (carboxyfullerenes), indicated that they are capable of eliminating both superoxide anion and H2O2, and were effective inhibitors of lipid peroxidation, as well. Carboxyfullerenes demonstrated robust neuroprotection against excitotoxic, apoptotic and metabolic insults in cortical cell cultures. They were also capable of rescuing mesence-phalic dopaminergic neurons from both MPP1 and 6-hydroxydopamine-induced degeneration. Although there is limited in vivo data on these compounds to date, we have previously reported that systemic administration of the C3 carboxyfullerene isomer delayed motor deterioration and death in a mouse model of familial amyotrophic lateral sclerosis (FALS). Ongoing studies in other animal models of CNS disease states suggest that these novel antioxidants are potential neuroprotective agents for other neurodegenerative disorders, including Parkinson’s disease.”

C60 derivative and hybrid structure compounds are also being studied for their neurprotective as well as other medical properties.  See for example [Study of the neuroprotective action of hybrid structures based on fullerene C60]. “The neuroprotective action of hybrid structures based on fullerene C60 with attached proline amino acid has been studied. Hybrid structures contained natural antioxidant carnosine or addends with one or two nitrate groups. It has been shown that all studied compounds had antioxidant activity and decreased the concentration of malondialdehyde in homogenates of the rat brain.”

Fullerene C60 might be useful for the treatment of Alzheimer’s disease.

The 2012 publication Fullerene C60 prevents neurotoxicity induced by intrahippocampal microinjection of amyloid-beta peptide reports: “The dynamics of the state of hippocampal pyramidal neurons after intrahippocampal microinjections of (1) amyloid-beta25-35 (1.6 nmol/1 microl), (2) an aqueous molecule-colloidal solution of C60 (0.46 nmol/1 microl) and (3) an aqueous molecule-colloidal solution of C60 before amyloid-beta25-35 administration were analysed in rats. This model allowed us to study the role of amyloid-beta25-35 in the pathogenesis of Alzheimer’s disease and to test anti-amyloid substances. Methods of fluorescent (acridine orange) and brightfield (cresyl violet and immunohistochemistry) microscopy were used. Acridine orange staining indicated changes in protein synthesis intensity due to alterations in the rRNA state of neuron ribosomes. One day after administration of amyloid-beta25-35, the intensity of protein synthesis in the population of morphologically intact cells decreased by 45%. By day 14, degeneration occurred in the majority of pyramidal cells, and amyloid-beta25-35 deposits were observed in the neuronal cytoplasm. In necrotic cells, acridine orange staining of the cytoplasm was drastically increased as a result of RNA degradation rather than due to an increase in protein synthesis. Because amyloid-beta25-35 administration provoked oxidative stress, we assumed that an aqueous molecule-colloidal solution of C60 administered before amyloid-beta25-35 prevented protein synthesis changes on day 1, while acting as an antioxidant, and by day 14 it inhibited neurodegeneration and amyloid-beta25-35 accumulation. Based on the data that an aqueous molecule-colloidal solution of C60 prevented amyloid-beta25-35 aggregation in in vitro experiments and based on our present evidence on the antitoxicity of an aqueous molecule-colloidal solution of C60, we suggest that functionalised C60 prevents/diminishes amyloid-beta25-35 aggregation in vivo as well. Thus, an aqueous molecule-colloidal solution of C60 administered at a low concentration before amyloid-beta2-35, prevented disturbances in protein synthesis, neurodegeneration and formation amyloid-beta25-35 deposits in hippocampal pyramidal neurons in vivo. This evidence gives promise that functionalised C60 can be used to develop anti-amyloid drugs combining antioxidant and anti-aggregative properties.”

The 2012 publication [Antiamyloid properties of fullerene C60 derivatives]reports “A comparative estimation of the ability of complexes of fullerene C60 with polyvinylpyrrolidone and fullerene C60 derivatives (the sodium salt of the polycarboxylic derivative of fullerene C60, sodium fullerenolate), has been carried out. The fullerenes destroyed amyloid fibrils of the Abeta(1-42) peptide of the brain and the muscle X-protein. A study of the effect of fullerenes on muscle actin showed that complexes of fullerene C60 with polyvinylpyrrolidone and sodium fullerenolate did not prevent the filament formation of actin, nor did they destroy its filaments in vitro. Conversely, sodium salt of the polycarboxylic derivative of fullerene C60 destroyed actin filaments and prevented their formation. It was concluded that sodium fullerenolate and complexes of fullerene C60 with polyvinylpyrrolidone are the most effective antiamyloid compounds among the fullerenes examined.”

Fullerenes may enable new anticancer therapies via various mechanisms: one is as a carrier for conventional anticancer drugs; another is enhancing cytotoxic effects of chemotherapy drugs; another yet is based on the anti-cancer activities of the fullerene molecules themselves.

With respect to the first role, as a potential carrier of conventional anti-cancer drugs, the new (November 2012) publication Water-Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo- and Photodynamic Therapeutic Actions reports” “Prodrug therapy is one strategy to deliver anticancer drugs in a less reactive manner to reduce nonspecific cytotoxicity. A new multifunctional anticancer prodrug system based on water-dispersible fullerene (C60) aggregates is introduced; this prodrug system demonstrates active targeting, pH-responsive chemotherapy, and photodynamic therapeutic (PDT) properties. Incorporating (via a cleavable bond) an anticancer drug, which is doxorubicin (DOX) in this study, and a targeting ligand (folic acid) onto fullerene while maintaining an overall size of approximately 135 nm produces a more specific anticancer prodrug. This prodrug can enter folate receptor (FR)-positive cancer cells and kill the cells via intracellular release of the active drug form. Moreover, the fullerene aggregate carrier exhibits PDT action; the cytotoxicity of the system towards FR-positive cancer cells is increased in response to light irradiation. As the DOX drug molecules are conjugated onto fullerene, the DOX fluorescence is significantly quenched by the strong electron-accepting capability of fullerene. The fluorescence restores upon release from fullerene, so this fluorescence quenching-restoring feature can be used to track intracellular DOX release. The combined effect of chemotherapy and PDT increases the therapeutic efficacy of the DOX-fullerene aggregate prodrug. This study provides useful insights into designing and improving the applicability of fullerene for other targeted cancer prodrug systems.” 

Another publication, dated 2013, related to use of fullerenes for anti-cancer drug delivery is PEI-derivatized fullerene drug delivery using folate as a homing device targeting to tumor.You can also see (1007) Nanotubes, Nanorods, Nanofibers and Fullerenes for Nanoscale Drug Delivery.

C60 compounds are also promising as delivery vehicles for drugs.

For example, related to myocardial treatments the 2010 publicationThe C60-fullerene porphyrin adducts for prevention of the doxorubicin-induced acute cardiotoxicity in rat myocardial cellsreports:  “This is a fullerene-based low toxic nanocationite designed for targeted delivery of the paramagnetic stable isotope of magnesium to the doxorubicin (DXR)-induced damaged heart muscle providing a prominent effect close to about 80% recovery of the tissue hypoxia symptoms in less than 24 hrs after a single injection (0.03 – 0.1 LD50). Magnesium magnetic isotope effect selectively stimulates the ATP formation in the oxygen-depleted cells due to a creatine kinase (CK) and mitochondrial respiratory chain-focusing “attack” of 25Mg2+ released by nanoparticles. These “smart nanoparticles” with membranotropic properties release the overactivating cations only in response to the intracellular acidosis. The resulting positive changes in the energy metabolism of heart cell may help to prevent local myocardial hypoxic (ischemic) disorders and, hence, to protect the heart muscle from a serious damage in a vast variety of the hypoxia-induced clinical situations including DXR side effects.”

C60 can enhance the cytotoxic action of chemotherapeutic agents against cancer through autophagy.

The 2009 publication Autophagy-mediated chemosensitization in cancer cells by fullerene C60 nanocrystalreports: “Autophagy may represent a common cellular response to nanomaterials, and modulation of autophagy holds great promise for improving the efficacy of cancer therapy. Fullerene C60 possesses potent anti-cancer activities, but its considerable toxicity towards normal cells may hinder its practical applications. It has been reported that fullerene C60 induces certain hallmarks of autophagy in cancer cells. Here we show that the water-dispersed nanocrystal of underivatized fullerene C60 (Nano-C60) at noncytotoxic concentrations caused authentic autophagy and sensitized chemotherapeutic killing of both normal and drug-resistant cancer cells in a reactive oxygen species (ROS)-dependent and photo-enhanced fashion. We further demonstrated that the chemosensitization effect of Nano-C60 was autophagy-mediated and required a functional Atg5, a key gene in the autophagy signaling pathway. Our results revealed a novel biological function for Nano-C60 in enhancing the cytotoxic action of chemotherapeutic agents through autophagy modulation and may point to the potential application of Nano-C60 in adjunct chemotherapy.”

With respect to the direct anticancer activities of C60 molecules, you can check our the 2011 publicationPristine C 60  Fullerenes Inhibit The Rate Of Tumor Growth And Metastasis.  “AIM: To estimate the impact of C(60) fullerene aqueous solution (C(60)FAS) on the rate of transplanted malignant tumor growth and metastasis.  METHODS: Lewis lung carcinoma was transplanted into С57Bl/6J male mice. Conventional methods for the evaluation of antitumor and antimetastatic effects have been used. RESULTS: The C(60)FAS at low single therapeutic dose of 5 mg/kg inhibited the growth of transplanted malignant tumor (antitumor effect) and metastasis (antimetastatic effect): the maximum therapeutic effect was found to be of 76.5% for the tumor growth inhibition; the increase of animal life span by 22% was found; the metastasis inhibition index was estimated as 48%.  CONCLUSION: It was found that water-soluble pristine С(60) fullerenes efficiently inhibit the transplanted malignant tumor growth and metastasis.”

C60 protects against radiation-induced cell damage

The 2010 publication Dendro[C(60)]fullerene DF-1 provides radioprotection to radiosensitive mammalian cells reports: “In this study, the ability of the C(60) fullerene derivative DF-1 to protect radiosensitive cells from the effects of high doses of gamma irradiation was examined. Earlier reports of DF-1′s lack of toxicity in these cells were confirmed, and DF-1 was also observed to protect both human lymphocytes and rat intestinal crypt cells against radiation-induced cell death. We determined that DF-1 protected both cell types against radiation-induced DNA damage, as measured by inhibition of micronucleus formation. DF-1 also reduced the levels of reactive oxygen species in the crypt cells, a unique capability of fullerenes because of their enhanced reactivity toward electron-rich species. The ability of DF-1 to protect against the cytotoxic effects of radiation was comparable to that of amifostine, another ROS-scavenging radioprotector. Interestingly, localization of fluorescently labeled DF-1 in fibroblast was observed throughout the cell. Taken together, these results suggest that DF-1 provides powerful protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, and cell death.”

See also the 2010 publication The polyhydroxylated fullerene derivative C60(OH)24 protects mice from ionizing-radiation-induced immune and mitochondrial dysfunction.

C60 fullerenes have anti-viral properties and might be useful for preventing or delaying the onset of AIDS.

From (2007) Medicinal applications of fullerenes:“Compounds with antiviral activity are generally of great medical interest and different modes of pharmaceutical actions have been described. Replication of the human immunodeficiency virus (HIV) can be suppressed by several antiviral compounds, which are effective in preventing or delaying the onset of acquired immunodeficiency syndrome (AIDS). Fullerenes (C60) and their derivatives have potential antiviral activity, which has strong implications on the treatment of HIV-infection. The antiviral activity of fullerene derivatives is based on several biological properties including their unique molecular architecture and antioxidant activity. It has been shown that fullerenes derivatives can inhibit and make complex with HIV protease (HIV-P) (Friedman et al 1993; Sijbesma et al 1993). Dendrofullerene 1 (Figure 1) has shown the highest anti-protease activity (Brettreich and Hirsch 1998; Schuster et al 2000). Derivative 2, the trans-2 isomer (Figure 1), is a strong inhibitor of HIV-1 replication. The study suggests that relative position (trans-2) of substituents on fullerenes and positive charges near to fullerenes cage provide an antiviral structural activity.”  Also “Amino acid derivatives of fullerene C60 (ADF) are found to inhibit HIV and human cytomegalovirus replication (Kotelnikova et al 2003).”

Fullerenes inhibit the allergic response

The 2007 publication Fullerene nanomaterials inhibit the allergic response reports “Fullerenes are a class of novel carbon allotropes that may have practical applications in biotechnology and medicine. Human mast cells (MC) and peripheral blood basophils are critical cells involved in the initiation and propagation of several inflammatory conditions, mainly type I hypersensitivity. We report an unanticipated role of fullerenes as a negative regulator of allergic mediator release that suppresses Ag-driven type I hypersensitivity. Human MC and peripheral blood basophils exhibited a significant inhibition of IgE dependent mediator release when preincubated with C(60) fullerenes. Protein microarray demonstrated that inhibition of mediator release involves profound reductions in the activation of signaling molecules involved in mediator release and oxidative stress. Follow-up studies demonstrated that the tyrosine phosphorylation of Syk was dramatically inhibited in Ag-challenged cells first incubated with fullerenes. In addition, fullerene preincubation significantly inhibited IgE-induced elevation in cytoplasmic reactive oxygen species levels. Furthermore, fullerenes prevented the in vivo release of histamine and drop in core body temperature in vivo using a MC-dependent model of anaphylaxis. These findings identify a new biological function for fullerenes and may represent a novel way to control MC-dependent diseases including asthma, inflammatory arthritis, heart disease, and multiple sclerosis.”

C60 fullerenes exercise immunomodulary effects.

The 2012 publication [The condition of lipid peroxidation in mice and the effect of fullerene C60 during immune response] reports: “The aim of this study was to assess the influence of fullerene C60 on lipid peroxidation (POL) and antioxidant protection during the induction of the immune response to heteroantigen. Balb/c mice were immunized intraperitoneal (i.p.) with sheep erythrocytes for the primary immunization. Water dispersion of fullerene C60 was injected i.p. once at the dose 50 ng to mice on first, third and sixth days after immunization. During immune response, the increment ofmalonic dialdehide (MDA) was enhanced in liver, kidneys and heart tissues. Fullerene C60 induced POL during the latent phase of immune response, but inhibited this process during progression of immune response. Activities of superoxide dismutase (SOD) and catalase in liver and spleen tissues were induced after injection of fullerene C60 to intact mice. After immunization, high level of activity of antioxidant enzymes and low level of organs mass factor were determined. Injection of fullerene C60 reduced the activities of SOD and catalase in spleen tissues. The results of our study indicate that fullerene C60 can display positive effect on POL processes and antioxidant enzymes activity which is probably due to membrane’s stabilization action or the ability of fullerene C60 to bind free radicals independently.”

Another 2012 publication that demonstrates anti-arthritis immunomodulatory activity in rats is [Fullerene C60 exhibits immunomodulatory activity during adjuvant-induced arthritis in rats].“The effect of fullerene C60 (FC60) on the immune processes during experimental adjuvant-induced arthritis (AA) in rats has been studied. The results indicate the inhibitory action of FN60 during AA on cellular splenocyte proliferation, neutrophil phagocytic and oxygen-stimulatory activities in the NBT test, and humoral immune mechanisms involved in the production of antinuclear antibodies, formation of circulating immune complexes, and restoration of morphological structure of spleen. Taken together, these results allow FC60 to be considered as a new potential pharmacological agent that can realize its effects mainly through anti-inflammatory and immunomodulatory activity.”

C60 fullerenes appear to affect the innate immune system

An august 2012 publication Effect of buckminsterfullerenes o cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cellsreported: “C60 nanoparticles, the so-called buckminsterfullerenes, have attracted great attention for medical applications as carriers, enzyme inhibitors or radical scavengers. However, publications evaluating their immunological mechanisms are still rather limited. Therefore, we aimed to analyze systematically the in vitro influence of polyhydroxy-C60 (poly-C60) and N-ethyl-polyamino-C60 (nepo-C60) on peripheral blood mononuclear cells (PBMC) from healthy individuals, angling their effect on proliferation, expression of surface markers, and cytokine production. We isolated PBMC from 20 healthy subjects and incubated them in a first step only with poly-C60 or nepo-C60, and in a second step together with recall antigens (purified protein derivative, tetanus toxoid, bacillus Calmette-Guérin). Proliferation was determined by (3)H-thymidine incorporation, activation of PBMC-subpopulations by flow cytometry by measurement of the activation marker CD69, and secretion of T helper cell type 1 (TH1)- (interferon-gamma [IFN-γ], tumor necrosis factor beta [TNF-β]), TH2- (interleukin-5 [IL-5], -13, -10) and macrophage/monocyte-related cytokines (IL-1, IL-6, TNF-α) into the supernatants by enzyme-linked immunosorbent assay. Both fullerenes did not influence T cell reactivity, with no enhanced expression of CD69 and production of T cell cytokines observed, the CD4/CD8 ratio remaining unaffected. In contrast, they significantly enhanced the release of IL-6 and CD69-expression by CD56 positive natural killer cells. PBMC, which had been cultured together with the three recall antigens were not affected by both fullerenes at all. These data indicate that fullerenes do not interact with T cell reactivity but may activate cells of the innate immune system. Furthermore, they seem to act only on ‘naïve’ cells, which have not been prestimulated with recall antigens, there are however, large inter individual differences.”

C60 may affect platelet aggregation

A 2012 Russian publicationEffects of fullerene C60 nanocomposites on human platelet aggregationREPORTS: “The effects of fullerene C(60) nanocomposites on human platelet aggregation induced by ADP, ristocetin, and collagen were studied. The nanocomposite containing fullerene C(60) in polyvinyl pyrrolidone solution did not change platelet aggregation, while fullerene C(60) in crown ether and Twin-80 solutions inhibited ADP-induced platelet aggregation by 20 and 30%, respectively.”  I do not know if the study was controlled to take account the effects of the solvents used. 

Fullerenes can potentiate hair growth

The 2009 publicationFullerene nanomaterials potentiate hair growthreports “Hair loss is a common symptom resulting from a wide range of disease processes and can lead to stress in affected individuals. The purpose of this study was to examine the effect of fullerene nanomaterials on hair growth. We used shaved mice as well as SKH-1 “bald” mice to determine if fullerene-based compounds could affect hair growth and hair follicle numbers. In shaved mice, fullerenes increase the rate of hair growth as compared with mice receiving vehicle only. In SKH-1 hairless mice fullerene derivatives given topically or subdermally markedly increased hair growth. This was paralleled by a significant increase in the number of hair follicles in fullerene-treated mice as compared with those mice treated with vehicle only. The fullerenes also increased hair growth in human skin sections maintained in culture. These studies have wide-ranging implications for those conditions leading to hair loss, including alopecia, chemotherapy, and reactions to various chemicals.”

Less perspective be lost, it is important to keep in mind that the major interests in C60 relate to developing new structural materials and electronic applications.

For these reasons C60 is currently being manufactured in industrial quantities measured in tons and there has been considerable concern about the biological impact of C60 and other fullerenes being released into the environment.

Literature related to the toxicity of C60 comes to mixed conclusions.  One the one hand, there has been much general concern about toxicities and long-term biological impacts of fullerenes.  And theoretical studies strongly suggest toxic actions of C60 against DNA and other cell components.  On the other hand, specific studies of C60 show few or no toxic effects on whole animals.  Researchers caution against possible yet-unobserved long-term effects.

The rat longevity study mentioned earlier was basically conducted to measure C60 toxicity, and found little or none.   Another 2012 study Sub-acute oral toxicity study with fullerene C60 in ratsreports: “To obtain initial information on the possible repeated-dose oral toxicity of fullerene C60, Crl:CD(SD) rats were administered fullerene C60 by gavage once daily at 0 (vehicle: corn oil), 1, 10, 100, or 1,000 mg/kg/day for 29 days, followed by a 14-day recovery period. No deaths occurred in any groups, and there were no changes from controls in detailed clinical observations, body weights, and food consumption in any treatment groups. Moreover, no treatment-related histopathological changes were found in any organs examined at the end of the administration period and at the end of the recovery period. Blackish feces and black contents of the stomach and large intestine were observed in males and females at 1,000 mg/kg/day in the treatment group. There were no changes from controls in the liver and spleen weights at the end of the administration period, but those weights in males in the 1,000 mg/kg/day group increased at the end of the recovery period. Using liquid chromatography-tandem mass spectrometry, fullerene C60 were not detected in the liver, spleen or kidney at the end of the administration period and also at the end of the recovery period. In conclusion, the present study revealed no toxicological effects of fullerene C60; however, the slight increases in liver and spleen weights after the 14-day recovery period may be because of the influence of fullerene C60 oral administration. In the future, it will be necessary to conduct a long-term examination because the effects of fullerene C60 cannot be ruled out.” 

More on the cautious side is the 2009 book chapter Cytotoxicity and Genotoxicity of Carbon Nanomaterials:  “With the recent development in nanoscience and nanotechnology, there is a pressing demand for assessment of the potential hazards of carbon nanomaterials to humans and other biological systems. This chapter summarizes our recent in vitro cytotoxicity and genotoxicity studies on carbon nanomaterials with an emphasis on carbon nanotubes and nanodiamonds. The studies summarized in this chapter demonstrate that carbon nanomaterials exhibit material-specific and cell-specific cytotoxicity with the general trend for biocompatibility: nanodiamonds > carbon black powders > multiwalled carbon nanotubes > single-walled carbon nanotubes, with macrophages being much more sensitive to the cytotoxicity of these carbon nanomaterials than neuroblastoma cells. However, the cytotoxicity to carbon nanomaterials could be tuned by functionalizing the nanomaterials with different surface groups. Multiwalled carbon nanotubes and nanodiamonds, albeit to a less extend, can accumulate in mouse embryonic stem (ES) cells to cause DNA damage through reactive oxygen species (ROS) generation and to increase the mutation frequency in mouse ES cells. These results point out the great need for careful scrutiny of the toxicity of nanomaterials at the molecular level, or genotoxicity, even for those materials like multiwalled carbon nanotubes and nanodiamonds that have been demonstrated to cause limited or no toxicity at the cellular level.”

Despite its apparent benevolence when ingested by rats, C60 and its derivatives solutions when photo-activated can produce singlet oxygen radicals which are biologically damaging.

For example, see Photo-Induced Damages of Cytoplasmic and Mitochondrial Membranes by a [C₆₀]Fullerene Malonic Acid Derivative.  On the one hand, the photo-activation properties of C60 appear to make it toxic and dangerous for some aquatic species(ref)(ref)(ref).  So, there is serious concern about release of manufactured C60 into natural aquatic environments.  On the other hand, there has been thought of exploiting these properties in photo-based anticancer therapies(ref). “–fullerenes can effectively photoinactivate either or both pathogenic microbial cells and malignant cancer cells. The mechanism appears to involve superoxide anion as well as singlet oxygen, and under the right conditions fullerenes may have advantages over clinically applied photosensitizers for mediating photodynamic therapy of certain diseases(ref).”  Photo-responsiveness of cells exposed to C60 can be fairly complex(ref).    

I strongly suspect that a deeper biological mechanism is involved in the health and longevity-producing effects of C60 despite the prevailing wisdom.  As I see it the candidates for these deeper effects of C60 are (1) effects exercised on DNA including impacts on structural configuration, epigenetic gene activation effects, histones and nuclear envelope shape, (2) effects exercised on microtubule structures in cells, (3) effects on mitochondria, and (4) epigenetic impacts such as on histones and DNA methylation.

I cannot prove this suspicion; that will require further research.  However I can cite arguments that tend to confirm my suspicion.

(1)  C60 is known to bind to and have impact on DNA.  While the results of modeling studies indicate toxic effects on DNA, certain effects could possibly be beneficial.

That C60 binds to and deforms DNA has been known for some time.  A 2005 publication C60 binds to and deforms nucleotides reported: “Atomistic molecular dynamics simulations are performed for up to 20 ns to monitor the formation and the stability of complexes composed of single- or double-strand DNA molecules and C60 in aqueous solution. Despite the hydrophobic nature of C60, our results show that fullerenes strongly bind to nucleotides. The binding energies are in the range -27 to -42 kcal/mol; by contrast, the binding energy of two fullerenes in aqueous solution is only -7.5 kcal/mol. We observe the displacement of water molecules from the region between the nucleotides and the fullerenes and we attribute the large favorable interaction energies to hydrophobic interactions. The features of the DNA-C60 complexes depend on the nature of the nucleotides: C60 binds to double-strand DNA, either at the hydrophobic ends or at the minor groove of the nucleotide. C60 binds to single-strand DNA and deforms the nucleotides significantly. Unexpectedly, when the double-strand DNA is in the A-form, fullerenes penetrate into the double helix from the end, form stable hybrids, and frustrate the hydrogen bonds between end-group basepairs in the nucleotide. When the DNA molecule is damaged (specifically, a gap was created by removing a piece of the nucleotide from one helix), fullerenes can stably occupy the damaged site. We speculate that this strong association may negatively impact the self-repairing process of the double-strand DNA. Our results clearly indicate that the association between C60 and DNA is stronger and more favorable than that between two C60 molecules in water. Therefore, our simulation results suggest that C60 molecules have potentially negative impact on the structure, stability, and biological functions of DNA molecules.”

The recent 2012 publicationA large-scale association study for nanoparticle C60 uncovers mechanisms of nanotoxicity disrupting the native conformations of DNA/RNA,a modeling study, reports: “Nano-scale particles have attracted a lot of attention for its potential use in medical studies, in particular for the diagnostic and therapeutic purposes. However, the toxicity and other side effects caused by the undesired interaction between nanoparticles and DNA/RNA are not clear. To address this problem, a model to evaluate the general rules governing how nanoparticles interact with DNA/RNA is demanded. Here by, use of an examination of 2254 native nucleotides with molecular dynamics simulation and thermodynamic analysis, we demonstrate how the DNA/RNA native structures are disrupted by the fullerene (C60) in a physiological condition. The nanoparticle was found to bind with the minor grooves of double-stranded DNA and trigger unwinding and disrupting of the DNA helix, which indicates C60 can potentially inhibit the DNA replication and induce potential side effects. In contrast to that of DNA, C60 only binds to the major grooves of RNA helix, which stabilizes the RNA structure or transforms the configuration from stretch to curl. This finding sheds new light on how C60 inhibits reverse transcription as HIV replicates. In addition, the binding of C60 stabilizes the structures of RNA riboswitch, indicating that C60 might regulate the gene expression. The binding energies of C60 with different genomic fragments varies in the range of -56 to -10 kcal mol(-1), which further verifies the role of nanoparticle in DNA/RNA damage. Our findings reveal a general mode by which C60 causes DNA/RNA damage or other toxic effects at a systematic level, suggesting it should be cautious to handle these nanomaterials in various medical applications.”

A 2011 publication DNA Exposure to Buckminsterfullerene (C₆₀): Toward DNA Stability, Reactivity, and Replicationconveys a somewhat different story, indicating that fullernols not only have major impacts on the structures and biological properties of DNA, but also that they can contribute remarkably to DNA stability against thermal degredation.

“Buckminsterfullerene (C₆₀) has received great research interest due to its extraordinary properties and increasing applications in manufacturing industry and biomedical technology. We recently reported C₆₀ could enter bacterial cells and bind to DNA molecules. This study was to further determine how the DNA–C₆₀ binding affected the thermal stability and enzymatic digestion of DNA molecules, and DNA mutations. Nano-C₆₀ aggregates and water-soluble fullerenols were synthesized and their impact on DNA biochemical and microbial activity was investigated. Our results revealed that water-soluble fullerenols could bind to lambda DNA and improve DNA stability remarkably against thermal degradation at 70–85 °C in a dose-dependent manner. DNase I and HindIII restriction endonuclease activities were inhibited after interacting with fullerenols at a high dose. Experimental results also showed the different influence of fullerenol and nano-C₆₀ on their antibacterial mechanisms, where fullerenols contributed considerable impact on cell damage and mutation rate. This preliminary study indicated that the application of fullerenols results in significant changes in the physical structures and biochemical functions of DNA molecules.”

The general topic of nanopartucles binding is covered in a 2012 review publication Prospects of nanoparticle–DNA binding and its implications in medical biotechnology. This remains a very new and immature area of research. 

Right now it seems fair to conclude that C60 is very likely to bind to and interact with DNA/RNA, but the macroscopic outcomes of such interactions are unknown.  There does seem to be contradictions between rodent studies that suggest no overall toxic effects of C60 and the molecular-chemical studies which suggest that C60 could play havoc with DNA.

(2)  C60 is known to affect the formation and durability of microtubules.

First of all, a little on microtubules for those not familiar with them.  Although almost never mentioned in the longevity literature they are critical to health and longevity.  According to Wikipedia, “Microtubules are a component of the cytoskeleton. These cylindrical polymers of tubulin can grow as long as 25 micrometers and are highly dynamic. The outer diameter of microtubule is about 25 nm. Microtubules are important for maintaining cell structure, providing platforms for intracellular transport, forming the mitotic spindle, as well as other cellular processes.^[1] There are many proteins that bind to microtubules, including motor proteins such as kinesin and dynein, severing proteins like katanin, and other proteins important for regulating microtubule dynamics — Microtubules are part of a structural network (the cytoskeleton) within the cell’s cytoplasm. The primary role of the microtubule cytoskeleton is mechanical. However, in addition to structural support, microtubules also take part in many other processes. A microtubule is capable of growing and shrinking in order to generate force, and there are also motor proteins that allow organelles and other cellular factors to be carried along a microtubule. This combination of roles makes microtubules important for organising cell layout. — A notable structure involving microtubules is the mitotic spindle used by most eukaryotic cells to segregate their chromosomes correctly during cell division. — The process of mitosis is facilitated by a subgroup of microtubules known as astral microtubules, defined as a microtubule originating from the centrosome that does not connect to a kinetochore. Astral microtubules develop in the actin skeleton and interact with the cell cortex to aid in spindle orientation. They are organized into radial arrays around the centrosomes. The turn-over rate of this population of microtubules is higher than that of any other population. Astral microtubules function in concert with specialized dynein motors, which are oriented with the light chain portion attached to the cell membrane and the dynamic portion attached to the microtubule. This allows for dynein contraction to pull the centrosome toward the cell membrane, thus assisting in cytokinesis. — Astral microtubules are not required for the progression of mitosis, but they are required to ensure the fidelity of the process; they are required for the correct positioning and orientation of the mitotic spindle apparatus. They are also involved in determination of cell division site based on the geometry and polarity of the cells (ref).^[2][3] ”

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Microtubules and microfiliments

I first discussed microtubules in my blog entry Quantum Biology.  There I pointed out how some quantum biologists argue that there is yet-another role for microtubules – they are quantum computers possibly exercising command and control functions for cell processes.  In fact it is known that microtubules are semiconductors as are certain arrays of fullerenes.  However, the quantum computer role for microtubules remains controversial.  For now, it is enough to know that microtubules are important for cell structure and are main rail lines for transport of molecules within cells.

The 2004 publication In Vitro and In Vivo Investigation of Collagen – C60(OH)24 Interactionargues that fullerole affects intermolecular communications from collegen fibers through integrines and microtubules to cell nucleus. 

A 2011 publication In vitro polymerization of microtubules with a fullerene derivative reports that a fullerene C60 derivative inhibits the polymerization of tubulin and therefore inhibits the formation of new microtubules.  “Fullerene derivative C(60)(OH)(20) inhibited microtubule polymerization at low micromolar concentrations. The inhibition was mainly attributed to the formation of hydrogen bonding between the nanoparticle and the tubulin heterodimer, the building block of the microtubule, as evidenced by docking and molecular dynamics simulations. Our circular dichroism spectroscopy measurement indicated changes in the tubulin secondary structures, while our guanosine-5′-triphosphate hydrolysis assay showed hindered release of inorganic phosphate by the nanoparticle. Isothermal titration calorimetry revealed that C(60)(OH)(20) binds to tubulin at a molar ratio of 9:1 and with a binding constant of 1.3 ± 0.16 × 10(6) M(-1), which was substantiated by the binding site and binding energy analysis using docking and molecular dynamics simulations. Our simulations further suggested that occupancy by the nanoparticles at the longitudinal contacts between tubulin dimers within a protofilament or at the lateral contacts of the M-loop and H5 and H12 helices of neighboring tubulins could also influence the polymerization process. This study offered a new molecular-level insight on how nanoparticles may reshape the assembly of cytoskeletal proteins, a topic of essential importance for illuminating cell response to engineered nanoparticles and for the advancement of nanomedicine.”  An in-vitro result, it suggests the opposite of a health-producing effect of C60 on microtubules.

Again, the interactions of C60 with cell microtubules and their creation and destruction appear to be not well understood.  It seems such interactions do exist.  Although modeling studies suggest that the macroscopic results of such interactions may be toxic rather than health-producing, we just don’t know for sure.

“When fullerene is derivatized with polar groups, as in case of polyhydroxylated fullerenes (fullerenol) and C60 tris(malonic)acid, they become water soluble enabling them to cross the cell membrane and localize preferentially to mitochondria (Foley et al 2002; Youle and Karbowski 2005), which generate great masses of cellular oxygen free radicals. This phenomenon makes them useful for a variety of medical applications (Tsai et al 1997; Lotharius et al 1999; Bisaglia et al 2000). These radical scavengers have shown to protect cell growth from various toxins that can induce apoptotic injuries in vitro (Lin et al 1999; Lin et al 2002; Chen et al 2004) in different cell types such as neuronal cells (Dugan et al 1997; Bisaglia et al 2000), hepatoma cells (Huang et al 1998), or epithelial cells (Straface et al 1999).(ref)” 

Does C60 do more in the microchondria than act as a super anti-oxidant?  Or does the super antioxidant power of C60 create permanent changes in the mitochondria?  If the research literature is indicative, no one has so far grappled with these questions or even asked them for that matter.

Final comments

I could quote and discuss here only a small but hopefully representative sample of the unfolding literature related to C60 and its biological impacts.  The rodent longevity studies are tantalizing but tiny and hopefully will be soon followed by much larger ones.  There appear to be some basic contradictions and many more basic questions are raised than those answered.  For rodents at least, far from being toxic pure C60 appears to be not only benevolent but life-extending.  On the other hand, mostly-theoretical studies of the likely impacts of C60 on DNA and on microtubules and cell morphology suggest that C60 may generate all kinds of havoc on the cell level.  Without question C60 is a powerful antioxidant.  However it tends to generate permanent longevity-enhancing changes and it is not at all clear how an antioxidant could do that?  How does it work to so grossly extend longevity?  Are there other means through which C60 works its health and longevity benefits, and if so, what are they? 

The literature references I have been able to surface seemed to focus on the lipid membrane and antioxidant and other chemical properties of C60 – mostly 1990s ways of looking at biological mechanisms which are valid but limited.  The research literature so far seems to be remarkably silent on certain issues that could turn out to be key:  C60 and DNA methylation, impacts of C60 on histones, C60 and the DNA repair machinery, C60 as related to stem cells, C60 and siRNAs, and C60 as related to key known aging pathways.   It the longevity impacts of C60 hold up, there are important layers of knowledge here yet to be revealed.  If this were an archeological dig, we have so far only gone down a foot or two.

Written by Vince Giuliano, with major contributions by James P Watson

This is the first of a series of blog entries examining the roles of mitochondria in key biological activities.  The goal of the series is to shed light on whether focusing on mitochondria can lead to new approaches for averting or curing neurological, cardiac and other diseases, and for practical interventions that extend lifespans.  In my treatise ANTI-AGING FIREWALLS – THE SCIENCE AND TECHNOLOGY OF LONGEVITY originally drafted in May 2008, I discussed Mitochondrial Damage as the third-most important of some 18 theories of aging.  I have also written blog entries related to mitochondrial biogenesis(ref)(ref) and to miotohormesis(ref).  These have all remained highly relevant.

My recent refocusing on mitochondria is due to two factors: 1. the incredible life-extension results in rats reported in the previous blog entry Buckyballs, health and longevity – state of knowledge thought to be due to a mitochondrial antioxidant intervention, and 2. an incredibly creative series of phone and e-mail exchanges over the ten days with James P Watson (Jim).  Many of the bolder ideas that will put forward in this series are Jim’s. And a number of the diagrams that appear in this series were put together by Jim.  When I use “we” here, I think I am speaking for both Jim and I.

Our purpose in this and following blog entries in this series is to delve more deeply into exactly what is known and unknown and see what answers can be found to such questions as:

• Just how important is mitochondrial signaling in driving disease and aging processes that may play out and manifest through DNA genetic and epigenetic processes?  Have we been looking in the right places in our search for basic causes of neurological diseases?
• In our focus on stem cells, regenerative medicine, iPSCs and cellular senescence, have we paid enough attention to mitochondrial DNA?  In ignoring mitochondrial DNA senescence, are we perhaps missing where basic action is?
• Is there a solid theoretical basis for seeing a mitochondrial origin to neurological diseases like Parkinson and Alzheimer’s diseases and ALS?  If so, what needs to be done to further explore new therapeutic possibilities?  Further, what practical interventions might be available right now?
• Is there a solid theoretical basis for believing that mitochondrial interventions, antioxidant or otherwise, might lead to extension of lifespans?  If so, what is that evidence?
• To what extent has lifespan extension through mitochondrial interventions in lifeforms actually been demonstrated?  What about experiments in yeast, nematodes, mice and rats?  How does this possibility as applied to humans require further testing?
• Are mitochondria subject to replicative or stress-related senescence?  If so what are the key implications of that?  Can such senescence be reversed?
• What roles do mitochondria play in cell senescence; can a mitochondrial intervention affect the senescence process or lead to apoptosis in senescent cells?
• What about the roles mitochondria play in various key processes and pathways such as the unfolded protein response, apoptosis in normal and cancer cells, autophagy, etc.?
• What about human antioxidant supplementation?  Does it make sense given what is known about mitohormesis?  When might it be particularly useful or harmful?   What are its limits likely to be?  Does the body change and adapt to increasing doses of antioxidants/
• Could strategies for reducing mitochondrial electron chain leakage be better ones for health and longevity than focusing on mitochondrial-active antioxidants?  Do buckyballs affect electron chain leakage?
• What experimental work is needed to resolve outstanding questions?  What new biomarkers are needed?

To answer these and related questions, we draw on research from many different perspectives, in many cases on work done by researchers who do not appear to be in communication with each other.  These blog entries will blog will summarize information from several independent streams of publications, collectively a large number of them.   Entire schools of researchers are studying closely related processes but seem to either not know of or to ignore each other’s work.  Trying to put together the pieces of the jigsaw puzzle of health, longevity and aging, sometimes we wonder if all the pieces are from the same puzzle.  We nonetheless try to fit those pieces together, and in doing so hope that in doing that we will be able to surface new important insights and perspectives.  In some cases we speculate on how results from different streams are related, our own efforts to contribute.  It is not at all an easy process and some of our conjectures could be off-base, but the process is a very exciting one for us.

This Part 1 blog reviews basic facts about mitochondria, discusses the electron transport chain and electron leakage in mitochondria.  We discuss the sroles of mitochondria in aging and  genetic mutational diseases for important insights they might lead us to.  And we review and expand upon some of the material covered in previous blogs related to mitochondrial biogenesis and mitohormesis.  Some of this material is basic and well known and some is derived from fairly new research.  The next blog entry in this series, Mitochondria Part 2: mitochondrial pathways, diseases and aging will dig deeper and examine underlying pathway mechanisms for observed disease and aging effects.  We will touch upon mitochondrial signaling and the mitochondrial UPR (unfolded protein response), into the roles of mitochondria in UPR in the endoplasmic reticulum, some of the pathways between mitochondria and the nucleus, and into signaling transport mechanisms such as microtubules and motor proteins.  And we will speculate on a possible underlying mitochondrial cause common to aging and age-related neurological, cardiac and other diseases.  As I now see it, a Part 3 blog entry will focus on practical interventions.  It will review what is known about the impact of administration of key mitochondrial antioxidants, alpha-lipoic acid, acetyl-l-carnitine, melatonin and buckyballs in particular. And it will speculate on how research needs to be directed to answer critical outstanding questions.

Basics about Mitochondria

Those of you who already know about mitochondria might want to skip this section.  First, as to the relevance of mitochondria from my treatise: “Mitochondria are tiny organelles within cells responsible for producing the cell’s source of chemical energy known as adenosine triphosphate (ATP). Many researchers believe that decline in the integrity of mitochondrial DNA is a root cause of aging and that maintaining mitochondrial health is central for longevity. Mitochondria, have their own DNA which is extremely subject to mutation such as resulting from attack by ROS. At least 40 diseases have been identified that involve pathology of mitochondrial functioning. They can affect brain and other nerve cells, kidneys, eyes, ears, the pancreas, and the liver. Also, damage to mitochondrial DNA can come about through insufficiency of certain micronutrients like zink and copper, and deficiencies of Vitamin D, C, E, B12, B6, niacin, and folic acid(ref). See also the candidate theory of aging based on micronutrient triage. — Cells have mechanisms for repair of damaged DNA and mitochondrial DNA, but these have their own side effects. There is a DNA repair enzyme known as NADH. However, repair results in depletion of NADH and of ATP. ATP depletion is believed to be one of the most critical factors leading to necrosis or cell apoptosis. –Mitochondria play additional critical roles in the cell life and reproductive cycle including roles involving cell signaling, growth, differentiation and death. They can be the sources of many important signals relating to cancers, neurological diseases such as Parkinson’s, cardiovascular diseases, type 2 diabetes, and optical neurological problems. Presence of oxidative conditions in a cell can signal the mitochondria to generate signals to other parts of the cell leading to cell apoptosis. It is thought that selective inhibition of this chain could be one means of retarding aging. The signaling relationships between mitochondrial DNA and regular cellular DNA is one example of the systems relationships between the various theories of aging.  And it points out how addressing one “cause” of aging can sometimes also mitigate several others as well. The blog entry Mitohormesis discusses a number of processes that go on in mitochondria and the complex pathways through which they play their key role in energy metabolism.”

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Mitochondria are like simple separate biological organisms, and are thought to have possibly evolved from such very long ago.  In animal cells, they are in a complex set of symbiotic relationships with nuclear and other elements of the cells, relationships critical to the survival of mitochondria, the cells, the organs concerned,  and the animals themselves.  We believe that exploring the nature of those relationships may lead us to new insights related to diseases and aging – and may also suggest basic new health and longevity-promoting interventions.

Another good place to look for basics is of course Wikipedia: “In cell biology, a mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells.^[1] These organelles range from 0.5 to 1.0 micrometer (μm) in diameter. Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy.^[2] In addition to supplying cellular energy, mitochondria are involved in other tasks such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth.^[3] Mitochondria have been implicated in several human diseases, including mitochondrial disorders^[4] and cardiac dysfunction,^[5] and may play a role in the aging process. The word mitochondrion comes from the Greek μίτος mitos, thread, + χονδρίον chondrion, granule. — Several characteristics make mitochondria unique. The number of mitochondria in a cell varies widely by organism and tissue type. Many cells have only a single mitochondrion, whereas others can contain several thousand mitochondria.^[6][7] The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria,^[8] whereas in Murinae (rats), 940 proteins encoded by distinct genes have been reported.^[9] The mitochondrial proteome is thought to be dynamically regulated.^[10] Although most of a cell’s DNA is contained in the cell nucleus, the mitochondrion has its own independent genome. Further, its DNA shows substantial similarity to bacterial genomes.^[11] ”

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About mitochondrial DNA

Mitochondrial DNA is very different than nuclear DNA: it is much simpler, it is organized differently, it generates far fewer proteins, and it is passed on from generation to generation differently.   But like nuclear DNA it is subject to damage and mutations,  Again quoting selectively from Wikipedia, I have highlighted some key points in bold italics: “Mitochondrial DNA (mtDNA or mDNA^[2]) is the DNA located in organelles called mitochondria, — Mitochondrial DNA can be regarded as the smallest chromosome. Human mitochondrial DNA was the first significant part of the human genome to be sequenced. In most species, including humans, mtDNA is inherited solely from the mother^[3]. — Nuclear and mitochondrial DNA are thought to be of separate evolutionary origin, with the mtDNA being derived from the circular genomes of the bacteria that were engulfed by the early ancestors of today’s eukaryotic cells. This theory is called the endosymbiotic theory. Each mitochondrion is estimated to contain 2-10 mtDNA copies.^[4] In the cells of extant organisms, the vast majority of the proteins present in the mitochondria (numbering approximately 1500 different types in mammals) are coded for by nuclear DNA, but the genes for some of them, if not most, are thought to have originally been of bacterial origin, having since been transferred to the eukaryotic nucleus during evolution. — In most multicellular organisms, mtDNA is inherited from the mother (maternally inherited). Mechanisms for this include simple dilution (an egg contains 100,000 to 1,000,000 mtDNA molecules, whereas a sperm contains only 100 to 1000), degradation of sperm mtDNA in the fertilized egg, and, at least in a few organisms, failure of sperm mtDNA to enter the egg. Whatever the mechanism, this single parent (uniparental) pattern of mtDNA inheritance is found in most animals, most plants and in fungi as well. – “

“In sexual reproduction, mitochondria are normally inherited exclusively from the mother. The mitochondria in mammalian sperm are usually destroyed by the egg cell after fertilization. Also, most mitochondria are present at the base of the sperm’s tail, which is used for propelling the sperm cells. Sometimes the tail is lost during fertilization. In 1999 it was reported that paternal sperm mitochondria (containing mtDNA) are marked with ubiquitin to select them for later destruction inside the embryo.^[5] Some in vitro fertilization techniques, particularly injecting a sperm into an oocyte, may interfere with this.”– Because mtDNA is not highly conserved and has a rapid mutation rate, it is useful for studying the evolutionary relationships – phylogeny – of organisms. Biologists can determine and then compare mtDNA sequences among different species and use the comparisons to build an evolutionary tree for the species examined(ref).”

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Mitochondrial DNA – circular and closed in shape

A few key points are worth emphasis here, points we will be coming back to these later(ref):

• Any mtDNA an individual has is passed on only from his or her mother.
• mtDNA as in does not undergo recombination as in sexual reproduction.
• Changes to mtDNA come only from mutations, ones occuring during cell division or resulting from ROS damage.
• Mutations that occur in the mtDNA control region tend not to be repaired, since that region does not code for any specific product .  Such mtDNA mutations may accumulate with aging.
• Thus, only mutations that occur in female germline cells (those that become eggs) are passed on to offspring.

Mitochondrial electron transport chain, electron leakage and free radical production

In mitochondria, energy production depends on a well-known electron transport chain.   As described in Wikipedia:  “Most eukaryotic cells have mitochondria, which produce ATP from products of the citric acid cycle, fatty acid oxidation, and amino acid oxidation. At the mitochondrial inner membrane, electrons from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. The electron transport chain comprises an enzymatic series of electron donors and acceptors. Each electron donor passes electrons to a more electronegative acceptor, which in turn donates these electrons to another acceptor, a process that continues down the series until electrons are passed to oxygen, the most electronegative and terminal electron acceptor in the chain. Passage of electrons between donor and acceptor releases energy, which is used to generate a proton gradient across the mitochondrial membrane by actively “pumping” protons into the intermembrane space, producing a thermodynamic state that has the potential to do work. The entire process is called oxidative phosphorylation, since ADP is phosphorylated to ATP using the energy of hydrogen oxidation in many steps”

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About mitochondrial uncoupling proteins

Mitochondrial uncoupling proteins play an important role in the electron transport chain.  From The Biology of Mitochondrial Uncoupling Proteins (2004):  “Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They are found in all mammals and in plants. They belong to the family of anion mitochondrial carriers including adenine nucleotide transporters. The term “uncoupling protein” was originally used for UCP1, which is uniquely present in mitochondria of brown adipocytes, the thermogenic cells that maintain body temperature in small rodents. In these cells, UCP1 acts as a proton carrier activated by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP synthase. Activation of UCP1 enhances respiration, and the uncoupling process results in a futile cycle and dissipation of oxidation energy as heat. UCP2 is ubiquitous and highly expressed in the lymphoid system, macrophages, and pancreatic islets. UCP3 is mainly expressed in skeletal muscles. In comparison to the established uncoupling and thermogenic activities of UCP1, UCP2 and UCP3 appear to be involved in the limitation of free radical levels in cells rather than in physiological uncoupling and thermogenesis. Moreover, UCP2 is a regulator of insulin secretion and UCP3 is involved in fatty acid metabolism.”

Electron leakage from the electron transport chain in mitochondria normally produces a low level of superoxide free radicals.  These are useful signaling molecules performing useful positive effects.

“A small percentage of electrons do not complete the whole series and instead directly leak to oxygen, resulting in the formation of the free-radical superoxide, a highly reactive molecule that contributes to oxidative stress and has been implicated in a number of diseases and aging(ref).”  Mitochondrial electron leakage has been studied for a long time.  You can go back to the 1993 publication Electron leakage from the mitochondrial NADPH-adrenodoxin reductase-adrenodoxin-P450scc (cholesterol side chain cleavage) system.  You can also see (2010) Mitochondrial proton and electron leaks.

The normal “electron leak rate” (ELR). from healthy mitochondria is 2%. These electrons that leak out function to create hormetic dose signaling ROS and RNS that have many healthy effects, including the altering of redox sensitive signaling proteins such as Keap-1 and many others. ROS and RNS sensitive “redox proteins” allow for many gene pathways to be upregulated for xenobiotic enzymes and phase II enzymes that protect us from cellular stress.  These pathways work well for coping with bursts of increases free radicals and peroxynitrates that occur with acute stresses such as infection, trauma, exercise, etc.  See the blog entry Mitohormesis for details on this.

Regarding mitochondrial ROS generation control

It is a genetic/epigenetically controlled phenomenon, depicted out in this diagram:

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Image source:  James P Watson.  As Jim explains it: “Representation of the major signalling pathways regulating mitochondrial ROS. p53, FOXOs and c-Myc orchestrate the transcription of genes encoding effector molecules (i.e. p66shc and MnSOD) which directly regulate mitochondrial ROS level in response to several intra- and extra-cellular stimuli. Note that p66shc acts both as a transducer and as an effector for mitochondrial oxidative stress.”  From the 2009 publication The p53–p66shc–Manganese Superoxide Dismutase (MnSOD) network: A mitochondrial intrigue to generate reactive oxygen species: “Once considered as a mere by-product of respiration, mitochondrial generation of reactive oxygen species (ROS) has recently emerged as a genetically controlled phenomenon, involved in complex intracellular signal transduction cascades that directly regulate cell survival and death in responses to environmental stressors. These cascades are involved in the pathogenesis of several major age-related diseases, such as cancer and neurodegeneration, and also appear to somehow regulate the “normal” ageing process. — The present short review summarizes recent discoveries on mitochondrial reactive oxygen species regulation by p53, a tumor suppressor protein and p66shc, a protein implicated in the life-span determination. It also outlines the emerging network whereby these molecules cross-talk with each other and with the mitochondrial antioxidant system, namely MnSOD (SOD2), another life-span determining protein, to regulate oxidative stress in the organelle. This molecular circuit, which comprises two genetic determinants of longevity and a major tumor suppressor gene, also provides a theoretical framework connecting senescence and cancer.”

Extrinsic vs Instrinsic Control of ROS Production

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Source: James P Watson.  As Jim explains it: “Caloric restriction, the IGF-1/insulin pathway, and oxidative stress are known inducers of mitochondrial ROS production. The downstream signaling pathways that mediate this cellular response are closely interrelated. Indeed, SIRT1, activated by caloric restriction, deacetylates and thus activates the FoxO transcription factors, whose phosphorylation is controlled by the IGF-1 pathway. Furthermore, the adaptor protein p66^shc, which directly regulates mitochondrial ROS production, inhibits FoxO activation.”  Reference: Mitochondrial longevity pathways, Biochimica et Biophysica Acta (BBA) – Molecular Cell Research, Volume 1813, Issue 1, January 2011, Pages 260–268Interplay between the signaling pathways controlling mitochondrial ROS production.”

Now, with this base of information we go on to effects of aging and certain diseases on mitochondria, electron leakage, and ROS production.

There seems to be evidence that lifespans of species are correlated with lower rates of leakage in electron transport chains.  Cutting down on mitochondrial electron leakage might be a better strategy for creating mitochondrial health than focusing on mitochondrial antioxidants.

The 2006 publication Plugging the Mitochondrial Leak reports: “– While an inverse correlation between resting metabolic rate and longevity in animals generally holds true, there are some exceptions to the rule. Birds, bats, and humans live several times longer than their metabolic rates would suggest. The reason lies in the rate at which reactive oxygen species (ROS) leak out of the mitochondrial respiratory chain, the succession of membrane-bound proteins that passes electrons from NADH to oxygen. According to Gustavo Barja at the Complutense University in Madrid, pigeons leak barely a tenth the ROS of rats, and live nearly ten times longer, yet their resting metabolic rates are similar. “ROS leakage is so low in pigeons that they can afford to have much lower antioxidant levels than rats, and still live longer,” says Barja. “The question is, why are pigeon mitochondria so leak-proof?” — The answer could have profound implications. According to Alan Wright at Edinburgh University, the cellular threshold for apoptosis is calibrated by the rate of ROS leakage: “Species that leak ROS slowly have a lower rate of apoptotic cell loss in degenerative conditions, including those that apparently have nothing to do with oxidative or nitrosative stress.” Analyzing single mutations in 10 different degenerative conditions across five species, Wright and collaborators found that age of onset and severity of disease correlates closely with the rate of ROS leakage. “If we could slow ROS leakage, there’s a prospect we could delay the onset of a wide spectrum of degenerative diseases,” he says. — In June 2005, Douglas Wallace’s group at the University of California, Irvine, showed that the approach could work in mammals.¹ They generated transgenic mice that overexpress the antioxidant enzyme catalase in mitochondria (to break down hydrogen peroxide). Not only are average and maximal lifespans increased by about five months, but also degenerative conditions such as cardiac pathology and cataract formation are delayed.  Other work suggests that antioxidants targeted to the mitochondria, such as mitoQ, concentrate 1000-fold in the mitochondrial matrix, where they inhibit apoptosis. But antioxidants have the potential to interfere with ROS signaling, which plays a major role in the physiology of the cell. Birds solve the problem by cutting leakage from complex I, not by raising intramitochondrial antioxidant levels. — “The critical factor determining ROS leakage is not antioxidant status but the redox state of complex I, which is the major source of ROS,” says Martin Brand at the MRC Dunn Unit in Cambridge, UK. “Redox state is dependent on numerous factors like substrate supply, ATP use, uncoupling, amount of complexes, and allosteric influences, such as Ca2+ activation or NO inhibition of cytochrome oxidase. So predicting the outcome depends on knowing the state of all these variables.”

The writer goes on: “–Such variables explain conundrums such as the exercise paradox-why physically active people don’t die early. During exercise, the flow of electrons down the respiratory chain quickens, as does oxygen consumption. The overall effect is greater oxidation of complex I, and lower leakage. — A fall in the reduction state of complex I explains other apparent anomalies, such as the long lifespan of mice with high resting metabolic rates. Brand, working with John Speakman and colleagues at the University of Aberdeen, showed that these mice had more uncoupling proteins in their mitochondria, enabling electron flow to be uncoupled from ATP production, dissipating energy as heat. Uncoupling meant they consumed more oxygen at rest, yet they lived longer than other mice.²  — Uncoupling may be important in people, too. Mitochondrial DNA haplotypes vary geographically, with some types predominant in tropical regions, others in colder climes. The pattern might reflect differing degrees of uncoupling, restricting internal heat generation in hot climates, and vice versa. A consequence might be a higher rate of ROS leak in tropical peoples, and a correspondingly higher susceptibility to degenerative conditions such as heart disease. — Intervention might be possible. Vladimir Skulachev at Moscow State University points to recent work showing that the reduction state of complex I depends strongly on the NAD+ and NADH levels. “Perhaps we could lower ROS leakage, and correspondingly apoptosis, by maintaining a tighter control over the NADH pool.”

The mitochondrial electron transport chain works less well with aging, and this is associated with increased oxidative damage, in all probability due to increased electron leakage.  Further, increasing the efficiency of the electron transport chain can extend lifespansof mice.

Several publications discuss this point including the 2003 publication Mitochondrial electron transport chain complexes in aging rat brain and lymphocytes, the 2007 publication The mitochondrial energy transduction system and the aging process and the 2010 publication Mitochondrial electron transport chain functions in long-lived Ames dwarf mice.  Also relevant is the recent 2012 publication RhTFAM treatment stimulates mitochondrial oxidative metabolism and improves memory in aged mice.  Free radicals associated with increasing age-related increases in electron leakage seem to be the culprits associated with age-related decline in neural functionality.  “Free radical-mediated oxidations are determining factors of mitochondrial dysfunction and turnover, cell apoptosis, tissue function, and lifespan. Inner membrane enzyme activities, such as those of complexes I and IV and mitochondrial nitric oxide synthase, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests(ref).”

Since mtDNA spin off so much ROS and are damage-prone and have limited self-repair capability, it seems logical to surmise that what is going on with aging is increasing amounts of mutationally damaged mtDNA and that this is correlated with greatly increased susceptibility to age-related neurological and other diseases.  This is actually a quite established view of aging, articulated the years ago in the publication Oxidative Stress, Mitochondrial DNA Mutation, and Impairment of Antioxidant Enzymes in Aging¹.  “Mitochondria do not only produce less ATP, but they also increase the production of reactive oxygen species (ROS) as by-products of aerobic metabolism in the aging tissues of the human and animals. It is now generally accepted that aging-associated respiratory function decline can result in enhanced production of ROS in mitochondria. Moreover, the activities of free radical-scavenging enzymes are altered in the aging process. The concurrent age-related changes of these two systems result in the elevation of oxidative stress in aging tissues. Within a certain concentration range, ROS may induce stress response of the cells by altering expression of respiratory genes to uphold the energy metabolism to rescue the cell. However, beyond the threshold, ROS may cause a wide spectrum of oxidative damage to various cellular components to result in cell death or elicit apoptosis by induction of mitochondrial membrane permeability transition and release of apoptogenic factors such as cytochrome c. Moreover, oxidative damage and large-scale deletion and duplication of mitochondrial DNA (mtDNA) have been found to increase with age in various tissues of the human. Mitochondria act like a biosensor of oxidative stress and they enable cell to undergo changes in aging and age-related diseases. On the other hand, it has recently been demonstrated that impairment in mitochondrial respiration and oxidative phosphorylation elicits an increase in oxidative stress and causes a host of mtDNA rearrangements and deletions. Here, we review work done in the past few years to support our view that oxidative stress and oxidative damage are a result of concurrent accumulation of mtDNA mutations and defective antioxidant enzymes in human aging.”

Earlier publications relating lifespans of rodents to mitochondrial coupling and electron leakage include: Extension of murine life span by overexpression of catalase targeted to mitochondria (2005) and Uncoupled and surviving: Individual mice with high metabolism have greater mitochondrial uncoupling and live longer (2004).

Mitochondrial mutations seem highly associated with exceptional longevity across a number of species including our own. 

Certain mitochondrial mutation polymorphisms appear to be associated with exceptional longevity in human subpopulations.  And mitochondrial antioxidant interventions extend the lives of mice and other lower species.  Among the publications relating mitochondrial mutation polymorphisms to longevity and disease processes are:

Association of mitochondrial DNA haplogroups with exceptional longevity in a Chinese population.

Mitochondrial DNA haplogroups in a Chinese Uygur population and their potential association with longevity.

Mitochondrial DNA polymorphisms associated with longevity in a Finnish population.

A combination of three common inherited mitochondrial DNA polymorphisms promotes longevity in Finnish and Japanese subjects.

Absence of association between mitochondrial DNA C150T polymorphism and longevity in a Han Chinese population.

Mitochondrial DNA haplogroup D4a is a marker for extreme longevity in Japan.

Association of mtDNA haplogroup F with healthy longevity in the female Chuang population, China.

Mitochondrial DNA haplogroups associated with age-related macular degeneration.

Association of the mitochondrial DNA haplogroup J with longevity is population specific.

Mitochondrial DNA haplogroups in Spanish patients with hypertrophic cardiomyopathy.

Are mitochondrial haplogroups associated with extreme longevity? A study on a Spanish cohort.

Mitochondrial DNA haplogroups: role in the prevalence and severity of knee osteoarthritis.

Mitochondrial DNA haplogroups J and K are not protective for Parkinson’s disease in the Australian community.

Some variations in the mitochondrial genome are correlated with disease susceptibilities and aging.

The 2006 publication The role of mitochondria in ageing and carcinogenesis reported: “Mitochondria can perform multiple cellular functions including energy production, cell proliferation and apoptosis. These organelles contain their own genetic material, mitochondrial DNA (mtDNA), which is maternally inherited. Although much smaller than the nuclear genome, mtDNA is equally important, as it has been hypothesized to play a crucial role in ageing and carcinogenesis. This is partly due to the fact that mitochondria represent the major site for the generation of cellular oxidative stress and play a key role in mediating programmed cell death (apoptosis). Damage to mtDNA is therefore an important contributor to human ageing, cancer and neurodegenerative diseases. The most relevant footprints of mtDNA damage are point mutations of single bases, or deletions of the 16.5-kb mitochondrial genome. This review will focus on the key roles of mitochondrial function and mtDNA in oxidative stress production and as a mediator of apoptosis, and on the use of mtDNA as a biomarker of sun exposure. This will be related to the contribution of mitochondria and mtDNA in the ageing process and cancer, with a specific focus on human skin. In conclusion, it is likely that the interplay between nuclear and mitochondrial genes may hold the final understanding of the mitochondrial role in these disease processes.”

The 2011 publication The role of the mitochondrial genome in ageing and carcinogenesis relates: “Mitochondrial DNA mutations and polymorphisms have been the focus of intensive investigations for well over a decade in an attempt to understand how they affect fundamental processes such as cancer and aging. Initial interest in mutations occurring in mitochondrial DNA of cancer cells diminished when most were found to be the same mutations which occurred during the evolution of human mitochondrial haplogroups. However, increasingly correlations are being found between various mitochondrial haplogroups and susceptibility to cancer or diseases in some cases and successful aging in others.”

Other publications discussing the roles of mitochondria in disease processes and aging include:

Mitochondrial haplogroups, control region polymorphisms and malignant melanoma: a study in middle European Caucasians.

Mitochondrial DNA content varies with pathological characteristics of breast cancer.

Identification of an mtDNA mutation hot spot in UV-induced mouse skin tumors producing altered cellular biochemistry.

Mitochondrial dysfunction mediates aldosterone-induced podocyte damage: a therapeutic target of PPARγ.

Estrogen receptor mediates a distinct mitochondrial unfolded protein response.

Oxygen tension changes the rate of migration of human skin keratinocytes in an age-related manner.

The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens.

Potential therapeutic benefits of strategies directed to mitochondria.

Spectrum of mitochondrial DNA deletions within the common deletion region induced by low levels of UVB irradiation of human keratinocytes in vitro.

Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer.

Mitochondrial mutation diseases provide other models of how dysfunctional mitochondria lead to pathologies.

There are perhaps 50 such diseases, mostly rare ones that show up in infants and children.  There is a substantial number of research publications about them, most of which are how about a certain mutation correlates with its pathological manifestations.  The diseases of these mitochondrial DNA mutations appear to lead to all kinds of disease manifestations: neurological, immunological, cardiovascular, etc.  Examples of these publications are:

Mitochondrial tRNA(Phe) mutation as a cause of end-stage renal disease in childhood, A novel mutation in the mitochondrial tRNA(Ser(AGY)) gene associated with mitochondrial myopathy, encephalopathy, and complex I deficiency.

Mutations in the mitochondrial seryl-tRNA synthetase cause hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis, HUPRA syndrome,

Mutation in the mitochondrial tRNA(Val) causes mitochondrial encephalopathy, lactic acidosis and stroke-like episodes,

Infantile presentation of the mtDNA A3243G tRNA(Leu (UUR)) mutation.

An autopsy case of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) with intestinal bleeding in chronic renal failure.

Novel homoplasmic mutation in the mitochondrial tRNATyr gene associated with atypical mitochondrial cytopathy presenting with focal segmental glomerulosclerosis.

Reversible infantile respiratory chain deficiency is a unique, genetically heterogenous mitochondrial disease.

Mutation of the mitochondrial tyrosyl-tRNA synthetase gene, YARS2, causes myopathy, lactic acidosis, and sideroblastic anemia–MLASA syndrome.

Novel mitochondrial DNA mutations associated with myopathy, cardiomyopathy, renal failure, and deafness.

Studies of such rare diseases may provide clues as to what is going on in major neurological diseases such as ALS, Huntington’s disease (HD), Multiple Scleroris (MS), Parkinson’s Disease and Alzheimer’s Disease – and even autism.  The root cause is thought to be mitochondrial dysfunction resulting from mtDNA damage and leading to excess levels of reactive oxygen and nitrogen species.

This point is discussed in the publication  Oxidative stress, mitochondrial dysfunction and cellular stress response in Friedreich’s ataxia.  “There is significant evidence that the pathogenesis of several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, Friedreich’s ataxia (FRDA), multiple sclerosis and amyotrophic lateral sclerosis, may involve the generation of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) associated with mitochondrial dysfunction. The mitochondrial genome may play an essential role in the pathogenesis of these diseases, and evidence for mitochondria being a site of damage in neurodegenerative disorders is based in part on observed decreases in the respiratory chain complex activities in Parkinson’s, Alzheimer’s, and Huntington’s disease. Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant imbalance, are thought to underlie defects in energy metabolism and induce cellular degeneration. The precise sequence of events in FRDA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain, associated with increased free radical generation and oxidative damage, may be considered possible mechanisms that compromise cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, that decreased expression of frataxin protein is associated with FRDA. Many approaches have been undertaken to understand FRDA, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to FRDA pathogenesis. Brains of FRDA patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat shock response, which is one form of stress response. Heat shock proteins are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress. In the central nervous system, heat shock protein (HSP) synthesis is induced not only after hyperthermia, but also following alterations in the intracellular redox environment. The major neurodegenerative diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington’s disease (HD) and FRDA are all associated with the presence of abnormal proteins. Among the various HSPs, HSP32, also known as heme oxygenase I (HO-1), has received considerable attention, as it has been recently demonstrated that HO-1 induction, by generating the vasoactive molecule carbon monoxide and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. Given the broad cytoprotective properties of the heat shock response there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat shock response. This may open up new perspectives in medicine, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. In particular, manipulation of endogenous cellular defense mechanisms, such as the heat shock response, through nutritional antioxidants, pharmacological compounds or gene transduction, may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration.”  Italics emphasis on the presence of abnormal proteins is our own since it a theme we will pick up later in this discussion.

“Mitochondrial disease can look like any number of better known diseases, including: Autism, Parkinson’s disease, Alzheimer’s disease, Lou Gehrig’s disease, muscular dystrophy and chronic fatigue, among others. Adults and children with it can have features similar to other disorders like: Epilepsy, Myopathy, Developmental Delay, learning disabilities and, Fibromyalgia –  Research shows that mitochondrial dysfunction is often a central element of these more commonly recognized diseases. Studies and reports indicate the “orange” ones are more influenced. A cure for mitochondrial disease could impact cures for Autism, Parkinson’s, Alzheimer’s and Muscular Dystrophy(ref).”

With mitochondrial mutation diseases, there is an electron leak rate as high as 40% producing much larger volumes of free radicals outside of the hormetic range.

Again, it seems that Mitochondrial  electron leakage is a smoking gun for ROS production that is highly correlated with disease processes and longevity.  In this series of blog posts, we explore the nature of some of those correlations.  And we discuss practical intervention strategies.

Certain autoimmune and neurological diseases are also characterized by deficiency in electron chain operation, more electron leakage and excessive ROS production.

Here the literature suggesting the problem goes back a bit.  The 2006 publication Mitochondria, apoptosis and autoimmunity reports:  “– Several mitochondrial proteins have been implicated as regulators of apoptosis in the immune system that are required for prevention of autoimmunity. Recent discoveries have shed light on mitochondrial functions as they relate to cell death, including caspase-dependent and -independent apoptosis, mitochondrial death substrates and events that disable mitochondrial functions during apoptosis. These discoveries, taken with reports that the specific manner by which a cell dies greatly impacts on the nature of subsequent immune responses, highlight an exciting era of research on mitochondrial function and its role in apoptosis and the effects on immune responses.”

The just-published report Sjøgren’s syndrome-associated oxidative stress and mitochondrial dysfunction: Prospects for chemoprevention trials relates: “An involvement of oxidative stress (OS) was found in recent studies of Sjøgren’s syndrome (SS) that reported significant changes in protein oxidation, myeloperoxidase activity, TNF-α, nitrotyrosine and GSH levels in plasma from SS patients. Excess levels of OS markers, as oxidative DNA damage and propanoyl-lysine were reported in saliva from SS patients. Previous reports concurred with a role of OS in SS pathogenesis, by showing a decreased expression of antioxidant activities in conjunctival epithelial cells of SS patients and in parotid gland tissue samples from SS patients. A link between OS and mitochondrial dysfunction (MDF) is recognized both on the grounds of the established role of mitochondria in reactive oxygen species (ROS) formation and by the occurrence of MDF in a set of OS-related disorders. Early studies detected mitochondrial alterations in cells from SS patients, related to the action of antimitochondrial autoantibodies, and affecting specific mitochondrial activities. Thus, a link between MDF and OS may be postulated in SS, prompting studies aimed at elucidating SS pathogenesis and in the prospect of chemoprevention trials in SS clinical management.”

It appears that in certain disease processes, induced oxidative stress precipitates a long-lasting chain of stress response events in mitochondria

The just-published (November 2012 document Self-evolving oxidative stress with identifiable pre- and postmitochondrial phases in PC12 cells reports: “During the neurodegenerative process in several brain diseases, oxidative stress is known to play important roles in disease severity and evolution. Although early events of stress, such as increased lipid peroxidation and decreased superoxide dismutase, are known to characterize early onsets of these diseases, little is known about the events that participate in maintaining the chronic evolving phase influencing the disease progression in neurons. Here, we used differentiated PC12 cells to identify premitochondrial and postmitochondrial events occurring during the oxidative stress cascade leading to apoptosis. Our data indicate that an acute and strong oxidative impulse (500 μM H(2) O(2) , 30 min) can induce, in this model, a 24-hr self-evolving stress, which advances from a premitochondrial phase characterized by lysosomes and cathepsin B and D translocations to cytosol and early mitochondrial membrane hyperpolarization. This phase lasts for about 5 hr and is followed by a postmitochondrial phase distinguished by mitochondrial membrane depolarization, reactive oxygen species increase, caspase-9 and caspase-3 activations, and apoptosis. Inhibition of cathepsins B and D suggests that cells can be protected at the premitochondrial phase of stress evolution and that new cathepsins regulators, such as glycosaminoglycans mimetics, can be considered as new therapeutic prototypes for neurodegeneration. Insofar as early oxidative stress markers have been related to the early onset of neurodegeneration, strategies protecting cells at the premitochondrial phase of oxidative stress may have important therapeutic applications.

Since mitochondria are short-lived and mtDNA is so subject to mutational damage, mitochondrial biogenesis is an important consideration.

Since I have discussed mitochondrial biogenesis before I will treat it only in a summary fashion here.  From Wikipedia: “Mitochondrial biogenesis is the process by which new mitochondria are formed in the cell. Mitochondrial biogenesis is activated by numerous different signals during times of cellular stress or in response to environmental stimuli. — It is reckoned that higher mitochodrial copy number (or higher mitochondrial mass) is protective for the cell. — Mitochondria are produced from the transcription and translation of genes both in the nuclear genome and in the mitochondrial genome. The majority of mitochondrial protein comes from the nuclear genome, while the mitochondrial genome encodes most parts of the electron transport chain along with mitochondrial rRNA and tRNA. A major adaptation to mitochondrial biogenesis results in more mitochondrial tissues which increases metabolic enzymes for glycolysis, oxidative phosphorylation and ultimately a greater mitochondrial metabolic capacity.[Peter D. Wagner Summer 2011 Lecture]– The master regulators of mitochondrial biogenesis appear to be the peroxisome proliferator-activated receptor gamma (PGC) family of transcriptional coactivators, including PGC-1α, PGC-1β, and the PGC-related coactivator, PRC. PGC-1α, in particular, is thought to be a master regulator. It is known to co-activate nuclear respiratory factor 2 (NRF2/GABPA), and together with NRF-2 coactivates nuclear respiratory factor 1 (NRF1). The NRFs, in turn, activate the mitochondrial transcription factor A (tfam), which is directly responsible for transcribing nuclear-encoded mitochondrial proteins. This includes both structural mitochondrial proteins as well as those involved in mtDNA transcription, translation, and repair.”

I have discussed PGC-1α and Nrf2 in a number of blog entries related to mitochondrial biogenesis. In a June 2010 blog entry AMPK and longevity, I discussed how exercise activates the AMPK pathway and the role of PGC-1alpha (peroxisome-proliferator-activated receptor gamma co-activator-1alpha) as a co-transcriptional regulation factor that induces mitochondrial biogenesis by activating transcription factors.  The August 2010 blog entry PGC-1alpha and exercise provides a further and more general introduction to PGC1alpha.  I said “You can probably expect to hear a lot about PGC-1alpha as time goes on because this remarkable substance is turning out to have a lot to do with health and longevity. It appears to be the mediator of the health benefits produced by exercise. This blog post is about PGC-1alpha, about its relationship to exercise, and about efforts to stimulate it with various substances, in essence seeing if it is possible to provide “exercise in a pill.”  And in the blog entry PQQ – activator of PGC-1alpha, SIRT3 and mitochondrial biogenesis I discuss how Pyrroloquinoline quinone (PQQ), a redox cofactor available as a dietary supplement, appears to have at least three central biological effects with powerful downstream health and longevity consequences: it stimulates the generation of PGC1-alpha, results in expression of SIRT3, and induces mitochondrial biogenesis. If there is such a thing as exercise in a pill, it is probably PQQ.

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Image source:  James P Watson.  Since the image source raises certain critical questions, Jim is not sure it is helpful.  The questions Jim identified are:

• What part is important?
• What changes with aging?
• Can we affect mitochondrial ROS and health?
• What can you do about it?

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Image source: James P Watson.  Jim comments: “Mitochondrial Biogenesis and Reactive Oxygen SpeciesShown is mitochondrial biogenesis during calorie restriction versus ad libitum feeding in mice and its proposed effects on reactive oxygen species (ROS). In the ad libitum case, the number of electron transport chains is low, and if the rate of entry of electrons (red e⁻) exceeds the slowest step of flow through the chain, stalling of electrons at mitochondrial complexes I and III (blue e⁻) and production of ROS will be favored. During calorie restriction, mitochondrial biogenesis increases the number of electron transport chains, thereby reducing the rate of electron entry per electron transport chain. Calorie restriction may also increase the fraction of electrons that bypass complex I by entering the electron transport chain via the electron transfer flavoprotein dehydrogenase (ETF). These effects may reduce the production of ROS during calorie restriction and hence mitigate cellular damage, aging, and disease.  Reference: Leonard Guarante, Mitochondria- A Nexus for Aging, Calorie Restriction, and Sirtuins?, Cell, Vol 132(2), pp171-176, January 25, 2008”

Mitohormesis is another important concept relating to oxidative stress, health of mitochondria and probably organism longevity.

I have devoted a recent blog entry to this topic Mitohormesis, so will devote only a few words to it here.  The central concept is that energy metabolism in mitochondria produce reactive oxygen species ROS, due to electron leakage and other factors.  At a low level these ROS perform essential cell signaling functions, including triggering of NRF2 signaling which activates the body’s endogenous antioxidant defense system.  Numerous health-producing effects result as well as demonstrated longevity-related impacts in lower species.  This signaling is a phenomenon evolutionarily conserved across a variety of species.  Consuming antioxidant supplements can sometimes interfere with such signaling and have negative biological impacts.  However, if the oxidative stress is to great, such as might be the case due to excessive electron leakage in the mitochondrial electron transport chain due to age-related mitochondrial gene mutations, in that case the benefits of the signaling might be outweighed by the destructive effects of the ROS.  In such a case exogenous antioxidant administration might be a good thing.

A key to understanding what is going on in hormesis is understanding how the hormetic dose-response curve works, and this can be formulated in terms of NRF2 expression.

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Graphic depiction of the stochastic hormetic relative risk model as given in Scott et al. (2009).

Understanding the typical dose-response curve associated with hormesis is critical for interpreting  seemingly contradictory research. In my interpretation, the horizontal axis depicts level of stress, say as driven by ROS load.  The vertical axis represents relative risk, level of probable pathological organismic response where normal level is 1.  To the left of the first axis crossing in the diagram (point D) the Keap1-Nrf2 pathway is progressively kicking in but not sufficiently so as to overcome the direct negative effects of ROS stress. So, in Transition zone A there is under-expression of the ARE genes and a negative health condition Between stress  levels b and D***  there is hormetic protection compared to what would be expected given a linear model of negative response to stress and better-than-expected health, due at least in part to activation of Nrf2 and the ARE genes.  The zone of maximum protection is between D* and D**.  Starting at D** to D*** the stress load begins to overwhelm the defensive activities of the ARE genes and the protection becomes less and less until at point D*** the hormetic response associated with ARE gene activation becomes negligible.  In the case of radiation damage at least, beyond point D*** the damage according to conventional wisdom is in linear proportion to the stressor, the amount of radiation.  Phantom risk is theoretical risk for low stress levels that would apply if the linear model were extrapolated for low stress dosages.

For further discussion of this curve and aspects of Mitohormesis, please see the Mitohormesis blog entry.

Moving forward

This blog entry summarizes some basic information about mitochondria, some found in textbooks and some not. By now it should be clear that mitochondria play central roles related to biology in general, health and longevity.   This blog entry is only the appetizer with the main course to follow. As I now see it, the next blog entry in this series, Mitochondria Part 2: mitochondrial pathways, diseases and aging will dig deeper and examine underlying pathway mechanisms for observed disease and aging effects.  We will touch upon the sources and natures of mitochondrial stress.  We go some into mitochondrial-nuclear signaling and the mitochondrial UPR (unfolded protein response), the roles of mitochondria in UPR in the endoplasmic reticulum, and into signaling transport mechanisms such as microtubules and motor proteins.  And we will speculate on a possible underlying mitochondrial cause common to aging and age-related neurological, cardiac and other diseases.  As I see it now, a Part 3 blog entry will focus on practical interventions.  It will review what is known about the impact of administration of key mitochondrial antioxidants, alpha-lipoic acid, acetyl-l-carnitine, melatonin and buckyballs. And we will speculate on how further research needs to be directed to answer critical outstanding questions.

• By Vince Giuliano and James P Watson

This blog entry generalizes on the concept of hormesis, discusses the multiple pathways through which hormesis takes place, and suggests a myriad of ways that ordinary people can take advantage of hormesis to maintain their health and possibly extend their lifespans.  Some of the ideas laid out here emerged from a series of e-mail and phone exchanges between Jim Watson and myself although I (Vince) am the primary writer of this blog.  We believe that hormesis is a fundamental process of human biology widely applicable in both a theoretical and practical sense across a very wide variety of health and aging issues.  It is time that this concept, so long in the closet, be accorded front-stage status.

Background

Hormesis is a process through which moderate stress induces a body response that is protective against insults, confers health and possibly even longevity benefits.  It is a process much mentioned in previous entries in this blog. I introduced the concept of hormesis without naming it in a July 2009 entry Stress and longevity.  “Longevity is correlated with having and meeting a healthy level of challenge – not too little and not too much stress.” A later 2009 blog entry Hormesis and age retardation started out by saying “An important approach to retarding aging that I have not discussed explicitly so far is hormesis, challenging cells and body systems by mild stress resulting in them becoming stronger and resistant to aging(ref).  The stress can be physical, chemical and even possibly psychological.”  The definition applies to the maintenance of health as well as to slowing aging.  That entry was the first to discuss the roles of heat shock and chaperone proteins in hormesis.  One important heat shock protein is discussed further in the blog entry HSP70 to the rescue  The relatively recent blog entries Mitohormesis and Radiation hormesis define hormesis in more detail and discuss it as it relates to mitochondrial oxidative stress and stress induced by radiation.  Unlike the conventional wisdom, there is much evidence that small doses of X or gamma rays are health producing.  Jim Watson’s recent post The Hormetic Wild Animal “Zoo” and Their “Zookeepers” discusses some familiar gasses like CO, NO, H2S and non- gases like HCN, O2-, and H2O2.  These are Dr. Jekyll and Mr. Hyde substances.   They serve as signaling molecules that act as 2nd messengers at very low doses triggering healthful body reactions in animals. Yet, at higher doses they are very toxic and even deadly.

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Two sides of a familiar hormetic character

On hormesis and homeostasis

The basic purpose of hormesis is maintaining a health condition of homeostasis or better in a cell, organ or entire organism.  That is, it is to keep conditions wihthin a normal functioning range and also improve that functioning.  “All organisms live in a world of changing conditions. But, to remain alive, the conditions inside of every organism need to remain fairly constant.  An organism must have ways to keep the conditions inside of itself from changing (too much, that is) as its external environment changes. One of the most important characteristics of all living things is the ability to maintain a (reasonably) constant internal environment. This ability is known as homeostasis(ref).”  Homeostasis is not a static state; rather it is a dynamic process of constant changes and adjustments.  A hormetic response to a stress not only maintains a functional internal environment but also improves it.  It can be thought of as a tune-up on homeostasis.

The basic hormesis response curve

Fundamental to understanding hormesis is a dose response-curve which I have discussed in previous blog entries and included yet-again here.  Forgive me, I would rather err here by redundancy rather than by incompleteness.

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Understanding the typical dose-response curve associated with hormesis is critical for interpreting seemingly contradictory research. I explain the curve somewhat more generally here than in previous blog entries.  In my interpretation, the horizontal axis depicts level of applied stress, say as driven by ROS load in a cell.  On a more macroscopic level it could represent the amount of whole-body radiation received due to an exposure event or to the amount of exposure to carbon monoxide or to stress due to being at high altitude (hypoxia).  The vertical axis represents relative risk, level of probable pathological organism response where normal level is 1.  Below 1, there is a “health reserve,” such as an enhanced ability to do exercise or enhanced resistance to disease.  Above 1 there is a less than normal “health reserve,” such as a lessened ability to resist disease or to do additional exercise.  To the left of the first axis crossing in the diagram (point b), positive body reactions to the stress situation is progressively kicking but not sufficiently so as to overcome the direct negative effects of the stress.  In the case discussed in other blog entries of ROS stress in cells, the Keap1-Nrf2 pathway is progressively becoming activated but in Transition zone A to the left of point b there is under-expression of the ARE genes and a negative health reserve.   Between stress levels b and D***  there is hormetic protection compared to what would be expected given a linear model of negative response to stress.  In the case of oxidative stress in cells, this is due at least in part to activation of Nrf2 and the ARE genes.  The zone of maximum protection is between D* and D**.  Starting at D** to D*** the stress load begins to overwhelm the hormetic defensive activities and the hormetic protection becomes less and less until at point D*** it vanishes.   In the case of oxidative stress in cells, at that point the hormetic response associated with ARE gene activation becomes negligible.  In the case of radiation damage at least, beyond point D*** the damage according to conventional wisdom is in linear proportion to the stressor, the amount of radiation.  Phantom risk is theoretical risk for low stress levels that would apply if the linear model were extrapolated for low stress dosages.

We conjecture that the hormesis curve applies to all forms of stress, physical or psychological.  However the scaling will vary widely from individual to individual and from one stress event to another for a given individual.  We explore how some of those variances work in this blog entry.

History of hormesis – The establishment of hormesis as a sound biological principle has been a slow process and until recently many scientists did not recognize its legitimacy. 

From the 2010 publication Cellular Stress Responses, The Hormesis Paradigm, and Vitagenes: Novel Targets for Therapeutic Intervention in Neurodegenerative Disorders: “Hormesis is a dose–response phenomenon  characterized by a low-dose stimulation and a highdose inhibition (Fig. 1). It may be graphically represented by either an inverted U-shaped dose response or by a J- or U-shaped dose response. The term hormesis was first presented in the published literature in 1943 by Southam and Ehrlich, who reported that low doses of extracts from the red cider tree enhanced the proliferation of fungi with the overall shape of the dose response being biphasic. However, credit for experimentally demonstrating the occurrence of hormesis goes to Hugo Schulz (396), who reported biphasic dose responses in yeast after exposure to a large number of toxic agents. The work of Schulz inspired a large number of investigators in diverse fields to assess whether such low-dose effects may be a general feature of biological systems. In fact, similar types of dose–response observations were subsequently reported by numerous researchers assessing chemicals (49) and radiation (41, 50–53, 246, 307, 313, 367, 381, 397, 431,432) with investigators adopting different names such as the Arndt-Schulz Law, Huppe’s Rule, and other terms to describe these similar dose–response phenomena (368). Despite the rather substantial historical literature concerning hormetic dose responses, this concept had a difficult time being incorporated into routine safety assessment and pharmacological investigations, principally because it (a) required more rigorous evaluation in the low-dose zone, (b) failure of investigators to understand its clinical significance, (c) failure to appreciate the quantitative features of the hormetic dose response, (d) failure to understand the limitations of its implications for commercial applications in agriculture as well as medicine, (e) because of the predominant interest in responses at relatively high doses during most of the 20^th century, and (f ) the continuing, yet inappropriate, tendency to associate the concept of hormesis with the medical practice of homeopathy (64, 89, 91). However, from the late 1970s (423, 433) there has been a growing interest in hormetic-like biphasic dose responses across the broad spectrum of biomedical sciences.  This resurgence of interest resulted from a variety of factors, including the capacity to measure progressively lower doses of drugs and chemicals, the adoption of cell culture methods, which has permitted more efficient testing of numerous doses and the need to reexamine the validity of linearity at low-dose modeling of cancer risks due to their enormous cost implications for regulations (379), as well the astute observations of independent investigators and their capacity to generalize their findings across biological systems (267, 423).”

Hormesis is a remarkably general phenomenon, producing the same results across a wide variety of stimuli.  Yet, it has taken a long time for its importance to be recognized.

“What has emerged from these research initiatives from highly diverse biomedical areas is the recognition that hormetic dose responses were common and highly generalizable, being independent of biological model, endpoints measured, and chemical class and/or physical agent studied (50–54, 68, 306, 448). This was an unexpected finding as hormetic responses were often considered by many in the so-called mainstream branches of toxicology and pharmacology to be paradoxical, not commonly expected and being of questionable reliability with a lack of capacity for replication. The casual dismissal of the hormesis concept during the mid decades of the last century is reflected in the general absence of the hormesis concept from the leading toxicological and biomedical textbooks. This situation has radically changed such that hormesis is now incorporated into all leading textbooks of toxicology (e.g., ref. 167) encyclopedias (89, 94) and other leading monographs. In fact, while the terms hormetic and hormesis were cited only about 160 times during the entire decade of the 1980s within the Web of Science database, in 2008 alone these terms were cited nearly 2300 times(ref).”

“Analysis of nearly 8,000 dose responses within the hormesis database indicates that quantitative features of phenotypic plasticity are highly generalizable, being independent of biological model, endpoint measured and chemical/physical stress inducing agent(ref).”

Hormesis played a key role in evolution.

The 2010 book The Fundamental Role of Hormesis in Evolution  relates: “Hormesis can be considered a major mechanism underlying Darwin’s and Wallace’s theory of evolution by natural selection. The ability of organisms to respond adaptively to low levels of exposure to environmental hazards in a manner that increases their resistance to more severe similar or different hazards is fundamental to the evolutionary process. The organisms that survive and reproduce are those best able to tolerate or avoid environmental hazards while competing successfully for limited energy (food) resources. Therefore many of the genes selected for their survival value encode proteins that protect cells against stress (heat-shock proteins, antioxidant enzymes, antiapoptotic proteins, etc.) or that mediate behavioral responses to environmental stressors (neurotransmitters, hormones, muscle cell growth factors, etc.). Examples of environmental conditions that can, at subtoxic levels, activate hormetic responses and examples of the genes and cellular and molecular pathways that mediate such adaptive stress responses are provided to illustrate how hormesis mediates natural selection.”

So, from the viewpoint of evolution, we would expect that adaptive mechanisms would have evolved for all the important forms of stress that might be encountered by an organism.  This appears to be the case.  As organisms become more sophisticated the exact pathways used to produce a given stress response also evolve.  For example, this diagram summarizes the hormetic effects of calorie restriction for yeast, nematodes, mice and us.Image may be NSFW.
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Calorie Restriction Pathways in Different Species  Image source: Leonard Guarante, Mitochondria- A Nexus for Aging, Calorie Restriction, and Sirtuins?.

“In yeast, SIR2 has been implicated downstream of mitochondrial changes in response to calorie restriction (CR), whereas in mammals the SIR2 ortholog SIRT1 has been implicated upstream of mitochondrial changes. In C. elegans, sirtuins have not been implicated in dietary restriction to date. The pathway in mice shows that the increase in mitochondrial number and activity may work via the mitochondrial sirtuins SIRT3, 4, and 5 or by reducing reactive oxygen species (ROS). The drug Resveratrol and CR may increase SIRT1 activity, which is part of an autoregulatory feedback loop that includes the enzyme endothelial nitric oxide synthase (eNOS).”  SIRT1 is the epicenter for linking the CR Mechanisms found in lower life forms to human CR.

A.    The personal practice of hormesis

This short section of this blog entry relates to the personal practice of hormesis, Section B below will further discuss the science of hormesis.

What do you mean, “take it easy?” 

Well-meaning friends often tell me that at my age I should take it easy and avoid stress.  I tell them that they are 100% wrong.  I thrive on stress because that is what keeps me young and going.  I tell them that some stress is not only a good thing.  It is absolutely necessary for health and longevity.

Hormesis is a commonly-occurring every-day phenomenon.  Hormesis is also multi-factorial because multiple stimuli can activate multiple pathways producing multiple results with multiple time and response characteristics.

Let me illustrate these points with a down-home highly personal example, based on yesterday which is like most days:

A day of hormesis and me

• When I got out of bed, I woke up in a cold (60 degree F) bedroom and immediately experienced feeling cold.  Though I turned up the heat, with no clothes on I was cold for 10-20 minutes while washing up and shaving until I warmly dressed.  I was not cold enough to shiver or suffer but definitely cold enough to experience a body reaction.  In doing this I triggered the cold shock hormetic pathway to start the morning.
• Before breakfast, I took some phytosubstance supplement pills including curcumin and ashwagandha which triggering the Nrf2 hormesis pathway.  Breakfast included blueberries and walnuts, also substances triggering this pathway.  The Nrf2 activation happens very quickly.  It not only promotes the immediate activation of my ARE genes but also, in ways not completely known, creates a permanent recoding of parts of my epigenome.
• After breakfast coming up to the computer I found some critical software was crashed.  I experienced a frustrating time wading through endless phone menus and trying to reason with a customer care representative in India whose mastery of US English was poor.  Finally the problem was resolved.  There was an amount of emotional/neurological stress.  Heart pounding a bit, I probably triggered several hormetic stress pathways.
• Other events producing emotional/neurological stress during the day was learning that an uncle of my wife had died, a small fight with my wife about not emptying the dishwasher, and, especially, trying to get my mind around all the research involved in this hormesis issue (a repeated event).  I like to think that each such event was a hormetic one.
• My daily 45 minutes on the treadmill produced exercise-related hormesis, probably via a combination of the Nrf2, heat shock and HIF pathways.  I end up breathing hard and sweating.  The exercise effect is not only short-term.  It affects my daily circadian rhythm clock, my weight, metabolism, muscles, hunger response and critical components of epigenetic encoding.
• Chain-sawing some trees fallen by our last hurricane, I spilled a couple of drops of gasoline on my hand and breathed chainsaw fumes for a short time.  This no-doubt activated the ARE detoxifying enzymes.  Hopefully the toxic stresses where in the hormetic range.
• During the day I take additional phytosubstance supplement pills and drink a little green tea, again all Nrf2 hormesis activators.
• For lunch I opened a can of Italian tuna fish, a favorite of mine.  This exposed me to both mercury and BHP, both toxins.  Hopefully the Phase 2 detoxifying enzymes kicked in and the total effect was hormetic.  To prevent chronic mercury exposure, I take some dietary supplements in my daily regimen which are heavy-metal chelators.
• I ate a couple of small packaged snacks during the day, ones probably containing traces of cancer-producing chemicals and pesticides.  These may help too.  Yes, at very low doses they too induce hormesis.
• Supper included salmon seasoned with olive oil, garlic, ginger, oregano and a touch of pepper sauce, broccoli and mixed greens – After supper, for desert I munched on 80% coco chocolate.  All these are Nrf2 hormesis-promoting phytosubstances.  I am also working on low-density lipoprotein-induced hormesis.  No steak for several weeks now.
• In the process of going to bed I again exposed myself to a cold bedroom with no clothes on, like in the morning.  Again, in doing this, I triggered the cold shock hormetic pathway.

These are events that I suspect produced hormetic results in me.  There probably were many other hormetic events in me in the course of the day that I don’t know about.

Once hormesis was mainly regarded as a curious laboratory phenomenon that happened when you exposed pseudomonas aeruginosa to a polychlorinated dibenzodioxin in a Petri dish.  Not just so!  It can go off in us multiple times every day.

B.   More on the science of hormesis

Mechanisms of hormesis

There are several different pathways through which hormesis can take place, including;

1. The keap1/Nrf2 pathway.  In simplistic terms, ROS stress in cells causes the protein keap1 to release Nrf2 which is resident in the cytoplasm whereupon Nrf2 translocates into the nucleus and activates at least 242 health-producing genes called antioxidant response elements (AREs)(ref).  The same holds in the case of electrophilic stress.  The basic operation and utility of this pathway is detailed in a trio of blog entries The pivotal role of Nrf2. Part 1 – a new view on the control of oxidative damage and generation of hormetic effects, The pivotal role of Nrf2. Part 2 – foods, phyto-substances and other substances that turn on Nrf2 and The pivotal role of Nrf2. Part 3– Is promotion of Nrf2 expression a viable strategy for human human healthspan and lifespan extension?.”  This pathway is also discussed in the Mitohormesis blog entry.  There is much more to what Nrf2 does than I have been able to cover so far.  It appears for example that Nrf2 impacts on the Notch1 pathway.
2. The heat shock response pathway, involving the actions of heat shock and chaperone proteins.  “The cellular response to heat shock includes the transcriptional up-regulation of genes encoding heat shock proteins (HSPs) as part of the cell‘s internal repair mechanism.^[1] They are also called stress-proteins.^[2] and respond to heat, cold and oxygen deprivation by activating several cascade pathways. HSPs are also present in cells under perfectly normal conditions.^[2] Some HSPs, called chaperones, ensure that the cell’s proteins are in the right shape and in the right place at the right time.^[1][2] For example, HSPs help new or misfolded proteins to fold into their correct three-dimensional conformations, which is essential for their function.^[2] They also shuttle proteins from one compartment to another inside the cell, and target old or terminally misfolded proteins to proteases for degradation.^[2] Heat shock proteins are also believed to play a role in the presentation of pieces of proteins (or peptides) on the cell surface to help the immune system recognize diseased cells.^[3] — The up-regulation of HSPs during heat shock is generally controlled by a single transcription factor; in eukaryotes this regulation is performed by heat shock factor (HSF) –(ref)”  The blog entry the HSP70 to the rescue offers an introductory discussion of HSPs, their role in the unfolded protein response (UPR), how HSPs play their roles in multiple species, and how HSP70 plays a role in the hormesis process.   Induction of hormesis by small doses of SIRT1acting through activation of HSP70 which keeps HSF1 active via deacytelation is also discussed in the blog entry SIRT1, the hypoxic response, autophagy and hormesis
3. The cold shock response pathway.  Yes, cold shock such as from taking a cold shower can also induce a hormetic response.  This pathway involves the cold-inducible RNA binding proteins: CIRP & RBM3.    Although the molecular dynamics of the cold shock response has been less-understood than the heat shock response, the cold shock hormetic response has been recognized as something going on in lower organisms for some time(ref).  It is now known to be evolutionarily conserved in humans and a considerable number of publications have been devoted to this subject(ref). From Cold-shock response and cold-shock proteins (1999) “Both prokaryotes and eukaryotes exhibit a cold-shock response upon an abrupt temperature downshift. Cold-shock proteins are synthesized to overcome the deleterious effects of cold shock. CspA, the major cold-shock protein of Escherichia coli, has recently been studied with respect to its structure, funtion and regulation at the level of transcription, translation and mRNA stability. Homologues of CspA are present in a number of bacteria. Widespread distribution, ancient origin, involvement in the protein translational machinery of the cell and the existence of multiple families in many organisms suggest that these proteins are indispensable for survival during cold-shock acclimation and that they are probably also important for growth under optimal conditions.”
4. The hypoxic stress response training pathway. This is the pathway activated when there is insufficient oxygen, such as at high altitudes.   I briefly discussed the hypoxic response and its relationship to hormesis in the 2010 blog entry SIRT1, the hypoxic response, autophagy and hormesis. The key stress-responsive transcription factor involved in the hypoxic stress response is HIF-1. From Wikipedia: “Hypoxia-inducible factors (HIFs) are transcription factors that respond to changes in available oxygen in the cellular environment, specifically, to decreases in oxygen, or hypoxia.^[1] The HIF signaling cascade mediates the effects of hypoxia, the state of low oxygen concentration, on the cell. Hypoxia often keeps cells from differentiating. However, hypoxia promotes the formation of blood vessels, and is important for the formation of a vascular system in embryos, and cancer tumors. The hypoxia in wounds also promotes the migration of keratinocytes and the restoration of the epithelium.^[9]– In general, HIFs are vital to development. In mammals, deletion of the HIF-1 genes results in perinatal death. HIF-1 has been shown to be vital to chondrocyte survival, allowing the cells to adapt to low-oxygen conditions within the growth plates of bones. HIF plays a central role in the regulation of human metabolism.^[10](Wiki – HIF-1).”
5. UPR hormesis pathways.  Heat, ROS, RNS, RSS, XRT, UV, and aging alter proteostasis resulting in the accumulation of unfolded or misfolded proteins.  These stressors can up-regulate stress coping mechanisms or induce cellular damage and apoptosis.  Unfolded Protein Responses (UPRs) sense and deal with accumulation of unfolded proteins, protecting the cell and blocking apoptosis.  One of the UPR pathways involved in hormesis is the heat shock response pathway mentioned above.  There are two other UPR pathways to be considered: the mitochondrial UPR and the endoplasmic reticulum UPR.  Diagrams of how these three UPR pathways work are thought to work can be found below. The UPR plays a role in a number of diseases of aging including cancer, heart disease, cerebrovascular disease, arthritis, osteoporosis, neurodegenerative disease like Alzheimer’s and Parkinson’s and Type II diabetes.
6. Cell-membrane mediated hormesis.  From the 2010 publication Cellular Stress Responses, The Hormesis Paradigm, and Vitagenes: Novel Targets for Therapeutic Intervention in Neurodegenerative Disorders “Evidence is emerging to support hormetic roles for low and transient increases in membrane oxidative stress. Levels of membrane lipid peroxidation are relatively low under most normal conditions. However, in some types of cells, lipid peroxidation increases considerably during periods of increased energy demand. For example, during vigorous physical exercise there is a marked increase in production of superoxide and hydrogen peroxide, hydroxyl radical, peroxynitrite, and lipid peroxidation (377). Evidence suggests that free radicals and products of lipid peroxidation generated during moderate exercise play important roles in hormetic effects of exercise on muscles, including changes in energy metabolism pathways, mitochondrial biogenesis, and upregulation of protein chaperones and antioxidant systems (377). Benefits of exercise on the cardiovascular system may also involve membrane oxidative stress-related mechanisms. Thus, it was reported that HNE activates nuclear factor erythroid 2-related factor 2 (Nrf2) and antioxidant gene expression in vascular cells (412). HNE may also activate other adaptive stress response pathways that promote the survival and plasticity of cells (349).  Ceramide is also believed to mediate hormetic effects of moderate/transient increases in membrane-associated oxidative stress. For example, pretreatment of neurons with subtoxic concentrations of ceramide results in increased resistance of the neurons to subsequent high levels of oxidative stress (191). Other studies have provided evidence for a pivotal role for ceramide in the cardioprotective effect of preconditioning ischemia in animal models of myocardial infarction (16, 149). Preconditioning ischemia is a classic example of hormesis, wherein exposure of cells to a moderate transient stress protects them against more severe stresses. Changes in the PMRS in response to stress may also allow cells to adapt to potentially damaging conditions. A dramatic example comes from a study in which the mitochondria of cellswere rendered dysfunctional, and the cells were able to survive because of a compensatory upregulation of PMRS enzyme activities (212).”

Hormesis operates through multiple channels for cell survival, dependent on the stimulation.

Illustrating a few of many possible situations, the following diagram illustrates the signaling operations for hormesis originated by three toxic gasses, hydrogen sulfide, carbon monoxide and nitric oxide:

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Regarding these noxious gasses and hormesis, see Jim Watson’s recent blog entry The Hormetic Wild Animal “Zoo” and Their “Zookeepers.”

Hormesis, specifically the scaling of the hormesis curve, depends on several critical factors:

1. Hormetic dose response is a critical consideration encountered in every situation of hormesis.  The hormetic dose response curve is central to the process. Not enough or too much of a given stress can be harmful.  A tiny whiff of carbon monoxide may be good for you but too much will kill you.  Avoiding all stress in your life could be a good approach to early death.
2. The time duration of the hormesis-generating stress is critical.  If a hormetic level of stress is maintained too long, the result could be non-hormetic and dangerous.  An example could be exercising to utter exhaustion; another would be breathing a very low level of carbon monoxide for too long.
3. To be effective, the stress must be pulsed; it cannot be chronic.  Chronic exposure to even very low levels of carcinogens, like second-hand tobacco smoke, could result in negative effects outside the hormesis zone.  Another example is that radiation hormesis cannot be expected from a chronic stress source like radiation from Strontium70 absorbed in bones.  You might be able to experience hormesis from cold shock by taking a cold shower, but if you stay in the shower too long your immune system might not be able to protect you from viral infections like common colds.
4. Multiple stressors that address a certain pathway response may produce no better hormetic benefits than single ones.  From Hormesis provides a generalized quantitative estimate of biological plasticity:  “These quantitative features of the hormetic dose response have important medical implications.  Most significantly, the hormetic dose response imposes constraints upon the magnitude of a drug to induce a desired effect. For example, if a drug increased cognitive performance in an elderly patient by approximately 25%–30%, the hormetic model suggests that this level of performance could not be further increased using a new drug combination. This concept has been supported in a variety of studies on hormesis and drug interaction. Flood (173–176) has demonstrated that the hormetic response for memory was bounded by the 30%–60% increase even when several drugs were used in combination that were designed to maximize memory outcome. This response magnitude constraint has been reported for immune stimulation, bacterial growth, increases in hair growth, plant growth, decrease in anxiety, decreases in tumor incidence, and numerous other endpoints (73).”  The limits of hormetic responses are set by the nature of the hormetic response machinery that is involved.  For example, consuming a phytosubstance like green tea may cause release of Nrf2 into the nucleus of cells activating ARE genes and creating a healthful hormetic response.  But there is only so much Nrf2 sitting around in the cytoplasm of cells at any given time.  So consuming green tea, curcumin, resveratrol, and ginger pills in the same gulp of pills may release no more Nrf2 or produce no better results – and might even exceed the hormetic dose.
5. Periods of rest are required between stress impulses.  For example, we all know that periods of rest are required between bouts of exercise.  We conjecture that the cells require time to replenish stocks of Nrf2 in the cytoplasm after it is suddenly released into the nucleus.  After oxidative stress the body requires time to clear out oxidation byproducts and otherwise re-establish homeostasis.
6. Hormesis is most effective when synchronized with circadian time frame windows.  For example, getting up from sleep and exercising at 3AM is not a good idea.  There are likely to be a number of other windows-of-best-opportunity for other circadian clocks.  This is an area requiring further research.
8. The body can increase its stress tolerance and affect the height and breadth of the hormetic response curve to a stressor by repeated hormetic exposures to the stressor.
10. A familiar example is exercise endurance training, where a runner may gradually increase his running time and speed to levels unthinkable for an ordinary individual.  Another example relates to pot, yes, marijuana.  THC, the active ingredient in marijuana, appears to be a hormetic molecule capable of promoting neurogenesis at low doses (probably smaller doses than ones from puffing on a marijuana cigarette)(ref).  I know a couple of individuals who are regular marijuana smokers and appear consistently to be functional and mentally present.  They tell me they smoke the substance ever day or so and that it leads them to clearer thinking.  I conjecture that they have increased their stress tolerance to THC.  I also know, based on personal experimentation during my hippy days 45 years ago or so, that just a few puffs of the same weed these friends are smoking would stop me from thinking clearly for a week or more.  Another good example of increasing stress tolerance through repetition discussed below is myocardial ischemic preconditioning.

Jim Watson summarized these hormesis factors to me in a phone conversation.  Though they are “of course” common sense observations, I have never seen them written down in one place.

The impacts of simple hormetic events on health and longevity might be profound and long-lasting.

Take for example, simple everyday events which activate Nrf2.  I am talking about eating some broccoli, drinking some green tea, swallowing a curcumin or resveratrol capsule, and many other sucheveryday actions.  We know Nrf2 activates the body’s endogenous antioxidant defense system.  But there is solid research that says it does much more including.

1. Affecting the differentiation and apoptosis of stem cell populations so as to affect organ regeneration and lifespan.
2. Affecting the repair of damaged DNA and tissues.  Many believe accumulated DNA damage is a fundamental driver of aging.
3. Responding to electrophilic stress.  It is possible that electrophilic stress is a more important cause of cellular aging than oxidative stress.
4. Creating permanent changes to one’s epigenome.

Although we do not know the magnitude or relative importance of these responses, I briefly cite evidence for each one.

Hormetic expression of Nrf2 can affect the differentiation and apoptosis of stem cell populations so as to affect organ regeneration and lifespan.

The 2010 publication Regulation of Notch1 Signaling by Nrf2: Implications for Tissue Regeneration makes the case in point. “–Through transcriptional analyses in Keap1- or Nrf2-disrupted mice, we identified interactions with the Notch1 signaling pathway. We found a functional antioxidant response element (ARE) recognized by Nrf2 in the promoter of Notch1. Notch1 regulates processes such as proliferation and cell fate decisions. We report a functional role for this cross talk between the two pathways and show that disruption of Nrf2 impeded liver regeneration following partial hepatectomy and was rescued by re-establishment of Notch1 signaling. — The Notch family of transmembrane receptors participates in a signaling pathway controlling a broad spectrum of metazoan cell fates and developmental processes through local cell-cell interactions (21). Alteration of signaling through the Notch family of receptors can markedly affect differentiation, proliferation, and apoptotic events. Genetic ablation studies indicate that Notch1 is crucial for early development and re-growth of several tissues (22, 23). Activation of the Notch pathway inhibits differentiation in different developmental contexts and has been associated with the amplification of some somatic stem cells— not only the neural (24) and hematopoietic stem cells (25), but also hepatocyte (26, 27) and intestinal epithelial stem cells (28, 29). Considering the importance of the Notch1 signal cascade in developmental biology, the microarray observations indicated the possibility that Nrf2 could be a key molecule affecting both embryonic and adult tissue stem cell renewal as well as cell fates. This study characterizes the effects of Nrf2 genotype on the expression of Notch1 and its effector genes and the importance of Nrf2-Notch1 crosstalk in liver regeneration.”

Hormetic expression of Nrf2, among other impacts, can affect the repair of damaged DNA and tissues.

This point is illustrated in the following graphic from the publication When NRF2 Talks, Who’s Listening?

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Possible means for regulation of cell survival and other cell-fate responses through interactions of NRF2 with additional cell-signaling pathways, including AhR, NF-κB, p53, and Notch1.

“Nonetheless, it is clear that the protective effects of upregulation of NRF2 signaling can take several forms. Protection can be immediate, reflecting induction of genes directly regulated through NRF2 binding to AREs in target genes (e.g., the innate immune response and elevated cytoprotective responses to blunt cytokine surges or detoxify reactive intermediates, respectively) (73). The protective effects can be secondary through induction of macromolecular damage repair/removal systems (proteasome, DNA repair) (84, 106). Last, the protective effects can be tertiary through activation of tissue repair/regeneration pathways. In these latter cases, involvement in cross talk with additional pathways affecting cell survival and other aspects of cell fate most certainly play important collaborating roles.”

Like oxidative stress, Nrf2 hormetically responds to electorphilic stress via the Keap1 pathway.  This phenomenon may be highly relevant to aging.

Electrophilic stress is stress induced by the stealing of pairs of electrons from compounds in cells by electrophiles, creating new and sometimes radical molecular structures.  “In general, electrophiles are positively charged species that are attracted to an electron rich centre. In chemistry, an electrophile (literally electron-lover) is a reagent attracted to electrons that participates in a chemical reaction by accepting an electron pair in order to bond to a nucleophile.(ref)”  Electrophilic stress is often discussed along with oxidative stress for both appear to activate Nrf2 via a common process.

As stated in Relationship of electrophilic stress to aging:  “In the present review, I will discuss the nature of electrophilic stress and its role in aging. I hope to present compelling evidence that electrophiles are, in fact, a long-neglected causal contributor to aging, and that electrophilic stress, while initiated by an oxidative event, is distinct, and can be functionally decoupled, from oxidative stress. — “This review begins with the premise that an organism’s life span is determined by the balance between two countervailing forces: (i) the sum of destabilizing effects and (ii) the sum of protective longevity-assurance processes. Against this backdrop, the role of electrophiles is discussed, both as destabilizing factors and as signals that induce protective responses. Because most biological macromolecules contain nucleophilic centers, electrophiles are particularly reactive and toxic in a biological context. The majority of cellular electrophiles are generated from polyunsaturated fatty acids by a peroxidation chain reaction that is readily triggered by oxygen-centered radicals, but propagates without further input of reactive oxygen species (ROS). Thus, the formation of lipid-derived electrophiles such as 4-hydroxynon-2-enal (4-HNE) is proposed to be relatively insensitive to the level of initiating ROS, but to depend mainly on the availability of peroxidation-susceptible fatty acids. This is consistent with numerous observations that life span is inversely correlated to membrane peroxidizability, and with the hypothesis that 4-HNE may constitute the mechanistic link between high susceptibility of membrane lipids to peroxidation and shortened life span.” – “Where do biologically relevant electrophiles come from? There are two major sources of such compounds. The first is external. Xenobiotics can be present in food, especially of plant origin, can be inhaled, or can be administered on purpose, e.g., as pharmacological agents. Many xenobiotics are directly electrophilic or can be metabolically converted to electrophiles [activation of toxins or drugs; see ref. 8 for a review]. The other source of electrophiles is the cell’s own metabolism. Certain intermediary metabolites are electrophilic.”  “The –  paradigm I am proposing, (is) namely that electrophiles such as 4-HNE are relevant to aging, and that the formation of 4-HNE is largely decoupled from the initiating oxidative stress but is a function of membrane peroxidizability.”

Electrophile Response System for Cellular Stress Tolerance

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Clik here to view. Image source   Overview of Activation and Inactivation of Nrf2 Signaling by electrophiles or ROS.

“Normally, INrf2 directs the degradation of Nrf2 by recruiting Cul3/Rbx1. After cells are exposed to stressors such as reactive oxygen species, INrf2 undergoes cysteine modification and Nrf2 is phosphorylated by PKC, resulting in dissociation of the INrf2:Nrf2 heterodimers. Phosphorylated Nrf2 translocates to the nucleus and binds the Antioxidant Response Element (ARE) with either small MAF or Jun. This leads to antioxidant gene expression that protects the cell, (A). The pathway is subsequently inactivated by two separate mechanisms. First, Maf homodimers and Bach1:Maf heterodimers compete with Nrf2 for ARE binding, resulting in diminished antioxidant gene expression. Second, GSK3b phosphorylates Fyn which leads to the Fyn translocating to the nucleus. Fyn subsequently phosphorylates Nrf2 at Tyr568. Nrf2 then is exported from the nucleus, binds to INrf2 and is subsequently degraded, (B).”

Hormetic expression of Nrf2 may create permanent health-inducing modifications to one’s epigenome.

The idea of permanently changing one’s epigenome in a positive manner by eating a bit of broccoli or kale or exercising a little may not be completely preposterous.  Consider the following three graphics from the publication Epigenetic impact of dietary polyphenols in cancer chemoprevention: Lifelong remodeling of our epigenomes

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