MULTIFACTORIAL HORMESIS

MULTIFACTORIAL HORMESIS PART 2 - CONTINUED AN IMPORTANT CONTEXT FOR HEALTH AND LONGEVITY ON THE ROAD TO A GRAND UNIFIED THEORY (GUT) FOR BIOLOGY Investigating the Future of Medicine Symposium and Webcast Hawaii institute for Molecular Communications March 16, 2013 • Presentation by: • Vincent E. Giuliano, Ph.D. • Agingsciences.com

Hormesis pathwaysWell-studied ones include: • The adaptive immune system • ROS-mediated pathways • The heat shock response • The cold shock response • The unfolded protein response (UPR) • The mitochondrial UPR • The hypoxic response

The adaptive immune system • The system in vertebrates that confers immunity to a pathogen once you are exposed to it • The system that makes vaccines work A well-developed and well understood area of medical science Taught to every medical and nursing student 15,296 publications in PubMed since 1927 Image source

ROS-mediated hormesis • At high doses ROS create damage • At low dose levels, ROS act as signaling molecules triggering hormesis • Probably this is the main mechanism acting in radiation and many other forms of hormesis ROS have a dual character Image source

Nrf2 • A key pathway involved in ROS-mediated hormesis seems to be the keap1/NRF2 axis Nrf2 • When activated, Nrf2 turns on hundreds of natural antioxidant and other protective genes • Nrf2 seems to be strongly protective against many diseases and aging • Many phytochemicals as well as other substances turn on Nrf2 • Nrf2, a relative newcomer, is being intensely studied in the literature- 3038 Pubmed publications since 1994

Nrf2 – how it works • Nrf2 is a stress-sensing genetic transcription factor that is part of the cap’n’collar family. • Nrf2 appears to be a master regulator of cellular responses to oxidative damage and other stressful conditions. • Nrf2 is normally held in the cytosol by a protein called Keap1 which sends Nrf2 off for ubiquination and proteolysis breakdown • In the presence of stress signals including 0xidative stress, Keap1 releases Nrf2 into the cell nucleus where it activates multiple stress-responsive genes • Nrf2 activation is a basic mechanism of ROS-mediated hormesis

Nrf2 and antioxidant response elements (AREs) • Nrf2 and Nrf1 are transcription factors that bind to over 240 ARE genes and and activate them. • These genes produce enzymes that exercise anti-inflammatory, anti-stress, detoxifying and antioxidant effects. • involved is the Phase II detoxification of xenobiotics to reduce cellular stress include glutathione transferases, quinonereductase, epoxidehydrolase, hemeoxygenase, UDP-glucuronosyltransferases, and gamma-glutamylcysteine synthetase. • Expression of these genes protects cells from oxidative damage and can prevent mutagenesis and cancer.

There is a wild hormetic zoo of other reactive molecular species that trigger hormesis. E.g. • Nitric Oxide (NOS) – e.g. vasodialation, • Carbon monoxide (CO) – e.g. bronchodilation • Hydrogen sulfide (H2S)- e.g. regulation of cardiac injury responses • Superoxide (O2) - e.g. a growth signal via the ras-NAD(P)H oxidase-MAPK signaling pathway, All of these involve the Keap1/Nrf2 pathway among others These are important signaling molecules at low doses. And, they and can kill you at higher doses. Watson JD, 2012

Some of the pathways activated by reactive species Image source

Examples of reactive nitrogen species (RNS) hormesis pathwaysIschemic preconditioning • Seems to be mediated by reactive nitrogen species (RNS) via mitochondria Image source Image source

PATHWAYS OF HORMESIS The heat shock response • Don’t be confused: heat stress activates multiple pathways and the heat shock response is triggered by more than heat • “The cellular response to heat shock includes the transcriptional up-regulation of genes encoding heat shock proteins (HSPs) and chaperone proteins as part of the cell‘s internal repair mechanism.[1] “ • This is called The heat shock response • “ They are also called stress-proteins.[2] and respond to heat, cold and oxygen deprivation by activating several cascade pathways.”(ref) • HSPs have many biological effects. • E.g. they induce T-cell regulation of chronic inflammation HSP 90 Image source

Theheat shock response • Inducers of HSP proteins and hormesis Image source

The heat shock response (cont.) • “HSPs are 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] • HSPs help new or misfolded proteins to fold into their correct three-dimensional conformations, which is essential for their function.[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)” • Pathways can be complex. E.g. HSP hormesis can be induced by small doses of SIRT1acting through activation of HSP70. This keeps the protein HSF1 active via deacytelation. ”(ref)

Heat shock responsepathways Image source Image source

Thecold shock response • 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. • The cold shock hormetic response has been recognized as something going on in lower organisms for some time. It is now known to be evolutionarily conserved in humans. 1042 Pubmed publications since 1955

Cold shock and related hormesis pathways • For cold shock, hypoxia, UV Image source

The hypoxic stress response • The hypoxic stress response pathway. This is the pathway activated when there is insufficient oxygen, such as at high altitudes. • “The hypoxia response leads to the activation of multiple cellular signaling pathways involved in regulation of respiration, metabolism, cell survival, etc. • 16313 PubMed.org publications since 1946 The Hypoxia-Inducible-Factor (HIF) pathway plays a central role during the hypoxia response Its expression and activity are regulated in an oxygen-dependent manner and it also regulates the expression of multiple hypoxia responsive genes.

Among benefits of hypoxia hormesis • Inhibition of cancers • Protects against cerebral ischemia and neurological disorders • Important for survival plants, e.g citrus crops • Response potentiated by acute anxiety • Repeated immersion in cold water leads to habituation • This habituation can be fairly permanent, lasting 7-14 months in humans Image source

Hypoxia pathways Image source Image source

Unfolded protein response (UPR) hormesis pathways • Heat, ROS, RNS, RSS, 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. • TheUPRssense and deal with accumulation of unfolded proteins, protecting the cell and blocking apoptosis. Image source

Unfolded protein response (UPR) hormesis pathways • 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. • One of the UPR pathways involved in hormesis is the heat shock response pathway (HSP) mentioned above. • There are two other UPR pathways to be considered: • the mitochondrial UPR and the • endoplasmic reticulum UPR. • All these pathways have been extensively researched

Unfolded protein response (UPR) hormesis pathways

Cytoplasmic and endoplasmic reticulum UPR pathways Cytoplasm E.R. Image source

The mitochondrial UPR • The mtUPR occurs through both JNK2 and AKT signaling Image source Image source Both the cell nucleus and mitochondria are involved as a result mtUPR signaling

There are many interesting additional stress-activated hormetic pathways • Example, formulation of stress granules (SGs) • “Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. • Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. • -- cold shock is a novel trigger of SG assembly in yeast and mammals. • Whereas cold shock-induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures.” (ref)

Literature reports of exotic forms of hormesis are increasing, e.g., Thursday • A publication related to wound healing. Of particular interest were the observations “that wound healing could be accelerated in the 30-50% range with the dose response displaying biphasic characteristics consistent with the hormesis dose-response model. Such findings set the stage for the hormetic dose-response revolution that is occurring within the biological and biomedical sciences, including wound healing,” (ref) • A publication relating to infantile hemangiomashowing that “short exposure of the endothelial cells to hypoxia resulted in increased cell survival and proliferation, accompanied by occurrence of autophagy. Prolonged hypoxia-induced autophagy, correlating with increased cell death, (ref) • A publication Xenohormetic and anti-aging activity of secoiridoidpolyphenols present in extra virgin olive oil: A new family of gerosuppressant agents. “the "xenohormesis hypothesis," which states that stress-induced synthesis of plant polyphenols and many other phytochemicals provides an environmental chemical signature that upregulates stress-resistance pathways in plant consumers, can be explained in terms of the reactivity of the AMPK/mTOR-axis to so-called xenohormetins. “

Mitochondrially-mediated hormesis Kolb et all, 2012

IMPORTANT GENERAL PROPERTIES OF HORMESIS • Dose response is critical – the general curve seems to always apply though its quantitative parameters will vary widely • Timing and duration of stressor is critical. • Cannot be for too long. • Works best when synchronized with circadian time windows • For hormetic behavior, stressors must be pulsed; cannot be constant. Periods of rest and recuperation essential. • Stress responses are limited by stress pathways activated; • Multiple stressors may produce no better responses than single ones • Repeated cycles of hormetic stress changes parameters of stress response curve and makes organism capable of handling higher and higher levels of stress • E.g. exercise training, hypoxic accommodation

IMPORTANT GENERAL PROPERTIES OF HORMESIS 6. Hormesis applies to hundreds or thousands of situations • Via a large multiplicity of pathways • In some cases, dominant pathway shifts at hormetic curve crossover point 7. Hormetic affect can be highly dependent on the state of the system effected • E.g. state of inflammation in an organ 8. Hormetic effects can be seen across the entire spectrum of biology Applies on multiple levels to multiple systems • Ranging from molecular pathway effects to whole-organism behavior and social behavior 9. Yet, despite its universality, hormesis is not generally embraced by many in the biological community • It is known mainly by a number of different names for different applications

GENERAL PROPERTIES OF HORMESIS HORMETIC ACCOMODATION IS KEY TO EVOLUTION AND POSSIBLY IS A CENTRAL DRIVER OF EVOLUTION • If an environment gradually shifts the pattern of stresses normally encountered by the organism (e.g. climate change or change in predators), the organism evolves through repeated hormetic exposure to accommodate to the new pattern of stresses. • Therefore I speculate that hormesis applies to all commonly occurring stress pathways in any successful species.

GENERAL PROPERTIES OF HORMESIS Many if not most hormetic effects are conserved across species, from yeast to us. E.g. calorie restriction hormesis for lifespan extension Image source

Hormesis has a dark side • Hormesis can also lead to negative results, e.g. the stress survival of cancer cells • E.g. via the Nrf2 pathway • Therefore a number of cancer researchers are looking for ways to turn hormesis stress survival pathways in cancer cells off • Pathogens adopt to the stresses of antibiotics and become antibiotic resistant – a major problem

The non-linear nature of the hormesis dose-response curve can help sort out paradoxes.Different scientists who look at research related to different segments of the dose-response curve come up with opposite conclusions. • Taking antioxidants can be very important for control of ROS and inflammation and are very important for health • Antioxidants can inhibit the body’s endogenous antioxidant defense system and can be unhealthful • IGF1is a pro-aging, pro-inflammatory pathway (Most scientists studying aging) • IGF1is essential. It induces neurogenesis and reduces brain and muscle deterioriation and combats aging (Some endocrinology researchers)

Different scientists who look at research related to different segments of the dose-response curve come up with opposite conclusions. More examples • All radiation exposure is dangerous, creating damage proportional to dose (official radiology establishment view) • Radiation exposure can produce health effects (multiple studies of radiation hormesis) • Carbon monoxide is a dangerous poison absolutely to be avoided (everybody knows that) • Carbon monoxide is an important signaling molecule (691 research publications in PubMed)

Different scientists who look at research related to different segments of the dose-response curve come up with opposite conclusions. Another example • Beta-amloid protein is a toxic substance responsible for the onset of Alzheimer’s disease • Billions of dollars have been spent on developing drugs that clear out beta-amloid to treat AD, and they have not worked • Beta-amloid is an essential signaling molecule in a hormetic feedback pathway that controls cellular cholesterol levels* • When the chosterol-related stress in that pathway is greater than in the hormetic range, excess beta-amloid is produced that can lead to AD. • Getting rid of all beta-amloid is NOT a good idea * 2013 publicationRational heterodoxy: Cholesterol reformation of the amyloid doctrine

It is time to stop thinking of stressors like ROS, heat, cold, various hormones and pathways as “good” and “bad,” and instead to start thinking of how they work in terms of the hormetic dose-response curve.

HORMESIS AND AGING The hormesis pathways have a lot to do with longevity of individuals and of species Experiments with lower species indicate that lifespans of lower species can be enhanced via inducing hormesis, e.g. • Multiple stressors in Caenorhabditiselegans . Up to 40% life extension due to heat shock • Multiple species via calorie restriction • Multiple species via radiation • Drosophila via repeated application of mild heat stress Image source

HORMESIS AND AGING • A large number of other stimuli can promote hormetic pathways and longevity in lower species • E.g. a current studies shows that feeding C-elegans a bacteria that produces nitric oxide promotes hsf-1 and daf-16, resulting in a high resistance to stress and a 15% longer life, Image source

HORMESIS AND AGING • Some very-long lived species are exposed to repeated hormetic shocks and have markedly upgraded stress-response pathways, e.g. Bristle cone pines is the world’s longest-lived non-clonal species, with livespans of up to 5,000 years • Grow on mountains between 5,600 and 11,200 feet, White Mountains, California • Subject to strong and highly variable stresses: cold temperatures, dry soils, high winds, short growing seasons, and constant hypoxia • Water/moisture stress a major factor • Very disease resistant • Needles alone may last 40 years

HORMESIS AND AGING • More on long-lived pines • At least four stress-response pathways are involved in (hormetic) pre-conditioning to drought in the Loblolly Pine. • one cycle of mild stress followed by two cycles of severe stress creates a "photosynthetic acclimation to water deficit". • Consecutive cycles of severe drought up-regulates mitochondrial heat shock proteins. Gene networks involved in drought stress response and tolerance Image source

HORMESIS AND AGING • Some very-long lived species are exposed to repeated hormetic shocks and have markedly upgraded stress-response pathways, e.g. The naked mole rat lives eight times as long as comparable-size mice and can live up to 30 years • Lives in conditions of hypoxia, high carbon dioxide, acidification, excrements, urine, wars between colonies • Shows little decline due to aging, maintains high activity, bone health, reproductive capacity, and cognitive ability throughout its lifetime. • Never get cancers, the main killer of mice • Has incredible hypoxic response. Can withstand brain oxygen deprivation for more than 30 minutes • Exhibits mutation in the specificity of HIF1a • Has six times the amount of endogenous expression of the hormetic anti-oxidant defense protein Nrf2. • Has high endogenous levels of NRG-1, P16

How about bats? • Compared with other mammals of similar size, bats live a long time, with lifespans of between 20 and 40 years. Rats live between 2 and 3 years, on average • Despite living in dank, dark, dung-filled caves, bats only rarely get sick or are debilitated by infections • Out of more than 5,000 types of mammals on the planet, bats are the only one capable of sustained flight and some species can fly more than 1,000 km in a single night. • Such exertion entails great free radical stress • Bats have evolved to have powerful free radical defenses and DNA repair systems

These and many other long-lived species experience repeated hormetic stresses and have remarkable stress-resisting defenses. • Raises the question: • What role do repeated hormetic stress experiences play in the longevity of very long-lived species? Or, lack of stress?

Then, there is the whole area of practical personal stress management to keep it hormetic Image source Stress management techniques known to be effective

In the course of a typical day, we have numerous minor hormetic experiences, e.g. me Feb 13, 2013 • Cold semi-naked in bedroom and bathroom morning – cold shock pathway • Blueberries, walnuts and hytosubstances supplements for breakfast– Nrf2 pathway • Frustration stress related to a computer help line conversation – cortisol, multiple pathways • Minor fight with my wife - same pathways • Exercising/shoveling snow/treadmilling – PGC1 pathway

In the course of a typical day, we have numerous minor hormetic experiences, e.g. me Feb 13, 2013 (continued) • Chain sawing, fumes, gas spilled on my hand – Nrf2 pathway - phase II detoxifying enzyme genes via AREs. • Pepperoni snack containing nitrites – same pathway • More plant-based supplements during day • Supper included salmon 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 – Nrf2 path again • Bedrom/bathroom icy cold again at night – cold shock again

HORMESIS AND DIETARY SUBSTANCES From the Hormesis project, the National Institute of Aging Shows the impacts of broccoli, garlic, hot peppers, turmeric, grape skins and numerous other phyto-substances on the Nrf2, Sirt1, FOXO3 and NF-kappaB pathways to create positive hormetic reactions in the nucleus of cells. • Pathological stress and over-expression of the stress hormone cortisol can block the good benefits from happening.

HORMESIS AND SYSTEMS DYNAMICS HORMESIS IS A NECESSARY PROPERTY DICTATED BY THE DYNAMIC SYSTEMS NATURE OF BIOLOGICAL PHEMENONA • Dynamic systems, no matter how simple, need feedback systems to keep them in normal safe ranges of operation – e.g. a boiler requires a pressure release valve to blow off steam if the pressure gets too high • Biological systems consist of complex networks of interacting dynamic sub systems embodying multiple points of positive or negative reinforcement. • Biological systems are subject to constantly changing external stresses as well as ones due to normal operation, stresses that can easily drive the operation of one or more subsystems out of its safe normal operating range • So, for example, is a nuclear power plant, with its complexity of feedwater, boiler, heat and pressure management, radiation control, reactor and electrical grid subsystems.

A few of many nuclear plant subsystems Containment spray Servicewater Emergency feedwater Safety injection

HORMESIS AND SYSTEMS DYNAMICS HORMESIS IS A NECESSARY PROPERTY DICTATED BY THE DYNAMIC SYSTEMS NATURE OF BIOLOGICAL PHEMENONA • If a critical subsystem in either a human body or nuclear power plant starts operating out of its normal and safe operating range, disaster can quickly ensue • So, the system must employ rapid feedback control mechanisms to ensure that operation of each critical subsystem is always within acceptable ranges of stress. • Further, there must be learning and adaption from each experienced stress event to make sure the system can better handle the event next time. • In biology, this systems management is called Hormesis • In nuclear plants, chemical plants, refineries and other complex industrial situations it is called Dynamic Systems Control

MULTIFACTORIAL HORMESIS