Quality of our Food

19-03-18 Madl Madl 0

Is there any difference between a human being and an ecosystem? An ecosystem is a community of living things that interact with each other and with the abiotic entities around them. From that definition, the human body must likewise be regarded an ecosystem as it is composed of trillions of cells. The human ecosystem is similar to a forest or a tropical coral reef with symbiotic association among viruses, bacteria and bodily cells – both within but particularly at the interface to the outer world (what the membrane is to the cell is the skin to the organism – incl. respiratory-, excretory,- reproductive- and digestive system – the latter f.e. is in simple terms just a tube with two open endings where the environment is inversely folded in to nourish a microbial universe via a mucosal medium). So there are billions of them living on our skins and magnitudes more in our guts. Hence, all these associations represent communities known as the human microbiome. Because of this, each one of us is a unique ecosystem. – not even cloning would change that. However, as socio-cultural constraints, psychological stress momenta, eating habits, hygienic and medical standards etc. tend to equalize across the globe, our microbiomic diversity shifts towards a “standard uniformity” resulting in minor variations only. While this may seem less troublesome for some, it poses a serious threat in ecosystem stability, as new pathologies may easily kick this fragile balance out of its labile equilibrium resulting in propagation dynamics that are then difficult to come by with as in a homogenized ecosystem with little diversity left most members of such a society will be affected by it. This lecture shall shed light on the various aspects when assigning the human body an ecosystem-like status and what is needed to preserve, to take care and to properly nourish. It is the aim to increase microbiomic robustness and to maintain good health till old age. Contact details: Pierre MADL - Div. of Material Sciences, Dep. Physics & Biophysics, University of Salzburg (AT) / Edge-Institute, Golling (AT) [email protected] http://biophysics.sbg.ac.at/home.htm Intro Food Epigenome Method Implication

Intro

Points of Departure 1. Psychological and physical conditions; 2. Personal life attitude; 3. Personal life organization; 4. Epi-/Genetic predisposition; 5. Nutrition

19-03-18 Madl 1 p.21: »das ganze ist mehr als die summe seiner teile«. Kollath (1977) meint, dass nahrung mehr als nur das gemisch aus kohlehydraten, fetten, eiweissen, mineralsalzen, vitaminen, spurenelementen und ballaststoffen ist …. Legionen von forschern könnten jahrzehntelang mit dieser fragestellung sinnvoll beschäftigt werden; d.h.: - in wie weit biologische rhythmen die optimale auswahl der nahrung beeinflussen (wie es bsp-weise die mazdaznan-filosofie postuliert), - ob es zutrifft, dass das optimum der ernährung gleichbedeutend damit ist, dass möglichst wenig nahrung aufgenommen werden muss, - ob die optimale nahrung stets aus der jeweiligen lebensregion des verbrauchers stammen muss, wie es die makrobiotiker annehmen, - ob polaritätsdenken eine basis hat, bsp-weise ob die ernährungsfysiologische rolle der einzelkomponenten das säure-basen-gleichgewicht des bluts, des urins und des extrazellulär-raums eine übergeordnete funktion hat, - inwieweit den lebensmitteln vorwiegend eine evolutionsbiologische bedeutung zukommt, wie es in ansätzen der anthroposofen zum ausdruck kommt. Heute kennt man fast alle mangelerscheinungen, die beim ausfall »essentieller« substanzen in der nahrung auftreten. Weniger gut bekannt sind die langzeit-wirkungen von überschüssen …. In form von stärkungsmitteln, vitaminpräparaten oder »natürlichen« zusatzstoffen in der nahrung ….. Der gesunde appetit nimmt jedenfalls wenig rücksicht darauf. …. »Bei durstgefühl ist es mit hoher wahrscheinlichkeit günstig zu trinken.« fragt sich nur noch, was.

Source: Poop F.A. (2002). Die Botschaft der Nahrung, Zweitausendeins Verlag, FRA.a.Main - FRG Kollath W. (1997). Die Ordnung unserer Nahrung. 6th ed. K. F. Haug Verlag, Heidelberg – FRG. Intro Food Epigenome Method Implication

Threshold Levels

Threshold & Risks

• Threshold limits • No-effect level However: • The smaller the more toxic (Donaldson et al. 2000)

Riepe, 2003

Grenzwerte: sind gesetzlich verankerte (justiziable) grössen; eine überschreitung ist demnach klagbar; bei jeder messung treten allerdings messfehler auf (zufällige und systematische fehler); daher gilt das der grenzwert nur innerhalb eines bestimmten toleranzbereiches gültig ist; wird ein grenzwert gemessen, so lässt sich nicht genau sagen ob man jetzt schon darüber oder noch unterm grenzwert liegt! Vorsorge-, richt-, und orientierungs-werte sind nur empfehlungswerte und haben daher keine gesetzeskraft; In some fine powders, including ultrafine titanium dioxide, carbon blacks, and fumed silicas, specific surface areas in excess of 2·E5m2/kg [200m2/g] are achieved among particles with aerodynamic diameters less than 4 mm. In comparison, an aerosol of spherical particles 4mm in diameter and with unit density would have a specific surface area of 1.5 x 103m2/kg [1.5m2/g]. There is evidence that for some low- solubility materials toxic response may be associated with surface area or even particle number (Oberdorster et al., 1994; Lison et al., 1997; Donaldson et al., 1998).[1] Donaldson et al. (2000) documented that nano-particles have profound effects upon the recipient tissues.[2] They found that nano-particles are more inflammogenic than their coarser siblings made of the same material. The cut-off size for this increased toxicity was found among a gradient that showed the greatest effect at particles smaller 65 nm and gradually decreased and levelled off towards larger diameters around 200 nm. The properties that drive this toxicity are still largely unknown. Nonetheless, it was possible to relate oxidative stress responses of the target cells to particle number concentration and their associated surface area. In addition, while one kind of nano-particle exerted a moderate inflammogenic response, the effects increased or even became synergistic when otherwise harmless nano-aerosols were added to the mono-specific class of particles.

Legally binding threshold values and the corresponding alarm levels for authorities. General risk assessment chart, assigned to an exposed public (dots represent the individual members)

Source: [1] B.A.Baron & K.Willeke (2001). Aerosol Measurement Principles, Techniques and Applications, 2nd e.d; [1] p.781-782; [2] Donaldson K., Stone V., Gilmour P.S., Brown D. M., MacNee W. (2000). Ultrafine Particles: Mechanisms of Lung Injury. Philosophical Transactions: Mathematical, Physical and Engineering Sciences, Vol. 358, No. 1775, Ultrafine Particles in the Atmosphere, pp.2741-2749. Intro Food Epigenome Method Implication

Historic Review

19-03-18 Madl 3 Intro Food Epigenome Method Implication

History (1/6)

The Analyst

• Society for Analytical Chemistry (UK, 1874) published its first issue of • the Analyst in March 1876

Aim: …. the object of THE ANALYST is not only to present to its readers the latest and best authenticated processes of analysis …. but to publish all cases of prosecution for adulteration, and such parliamentary and other proceedings as may appear to touch the interests of Analysts in general. → a major step towards a fragmentated science; Analyst 1876, 1:1 19-03-18 Madl 4 p.23: die seinerzeitigen lebensmitt-elexperten witterten im erfindungsreichtum ihre chancen: sie vermischten besonders begehrte genussmittel mit minderwertigen produkten und liessen sich diese »streckung« der produkte so bezahlen, als ob sie nicht stattgefunden hätte …. Die erste wissenschaftliche zeitschrift der »society of public analysts« (1874): »the analyst« (1876) dienten dem erfahrungsaustausch mit dem ziel, den panschern auf die sprünge zu kommen …. p.25: Mit der zersplitterung des qualitätsbegriffs in einzelne, untergeordnete merkmale verliert sich die rechtfertigung, von qualität zu reden, zugunsten einer zweifelhaften angabe über die zweckmässigkeit …. Solange chemikalien der nahrung zugesetzt werden, erhöht sich das risiko verminderter qualität, während die verhinderung des zusatzes einzelner stoffe …. die höchstmengen-verordnung liefert deshalb unbedingt notwendige, aber keineswegs hinreichende qualitätsleitwerte …. Kurioserweise verdanken wir die »naturbelassenheit« der kost immer mehr der sorgfalt von lebensmittel-ingenieuren …. Die lebensmittelindustrie geht …. neuerdings der folgender strategie nach: die in der nahrung natürlich vorhandenen substanzen, die den genusswert erhöhen, werden angereichert, andere, die die frische günstig beeinflussen, zugefügt, wieder andere, die den verkauf der ware fördern, gezielt verstärkt oder beigemischt, jene aber, die nutzlos erscheinen, soweit es geht herausverdünnt …. (Kerbusk 1993). Dieses unter dem schlagwort »lebensmittel-funktionalitaet« betriebene vorgehen erinnert an optimierungsprozesse …. warum sollten lebensmittel-chemiker, mit höherem sachverstand ausgerüstet, diese optimierung nicht noch besser vornehmen können als eine erfahrene, aber rücksichtslose natur? …. p.xxi: Schauen sie sich doch mal die forschungslandschaft an. Wenn heute jemand irgendwo als forscher arbeitet, dann hat er ein spezielles gebiet .... Jeder hat so ein spezialgebiet und 99% der kollegen wären gar nicht in der lage die ganze problematik zu erfassen die ja von interdisziplinärer natur ist .... Man darf gar keine fachdisziplin in den vordergund stellen, sondern muss die fragestellung in den vordergrund rücken. Source: Kerbusk K.P.1993.Der Gen-Frass. Biotechniker bauen die Nahrung um. Der Spiegel 15, 202-218 http://en.wikipedia.org/wiki/The_Analyst_(chemistry_journal) http://www.rsc.org/Publishing/Journals/an/Article.asp?Type=CurrentIssue Intro Food Epigenome Method Implication

History (2/6)

Rene Descartes (1596 – 1650 )

• philosopher, mathematician, scientist, and writer; • scientific progress via technical means; • rational knowledge: “incapable of being destroyed”; search for truth in science;

• Discourse on the Method - accept only that which you are sure of; - divide into as small parts as necessary; - solve the simplest problems first; - make as complete list as possible; Wikipedia, 2009

19-03-18 Madl 5 p.33: …. Der französische mathematiker und filosof Rene Descartes lebte von 1596 bis 1650 …. Ähnlich widersprüchlich und deshalb vital - bei weitem nicht notwendig falsch - in der wissenschaft den fortschritt und die technischen mittel für arbeitserleichterung bis hin zur selbstverwirklichung des menschen zu erkennen. Von ihm geprägt, folgt die wissenschaft …. Noch heute einigen regeln seiner methode des richtigen vernunftgebrauchs: - vermeide alle vorurteile und erkenne nur das als wahr an, was sich klar und deutlich nachweisen lässt, - zerlege die probleme soweit als möglich in einzelteile, - schreite vom einfachsten objekt gleichsam stufenweise zum komplizierten voran, - sichere die vollständigkeit des systems durch aufzählung.

Source: http://en.wikipedia.org/wiki/Descartes http://en.wikipedia.org/wiki/Discourse_on_Method Intro Food Epigenome Method Implication

History (3/6)

Denis Papin (1647 - 1712)

Medical scholar who joined Christian Huygens, who was . working with air pumps …. Together they pioneered the a human invention of the steam digester, the forerunner of the w that e -engine … steam engine. kn steam Energetic requirements to power the human body: 1. energy supply for internal and external work, to ucible to maintain heartbeat or contraction of muscles; d Rene reDescartes 2. assurance of necessary materials for cell-growth, t t bu no 3. body heat regulation; is Wikipedia,…. 2009

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Begonnen aber hat es damit, dass beim abbau aller vorurteile und der beschränkung auf das, was klar und deutlich nachweisbar ist, der mensch sich scheinbar nicht mehr von einer maschine unterscheiden lässt. Descartes hätte hier freilich bereits protestiert, denn eine maschine kann nicht zweifeln …. p.34: Aber als man die parallelität des menschen mit der dampfmaschine erkannte - im übrigen lange zeit nach Descartes -, waren die selbstzweifel der wissenschaftler zugunsten der fortschritts-gläubigkeit ausgeräumt …. der treibstoff (nahrung) für den menschen dient nach diesem modell den folgenden drei funktionen: 1. der zufuhr notwendiger energie für innere und äussere arbeit, wie herzschlag beziehungsweise kontraktion der muskeln, 2. der sicherung notwendiger substanzen für das zellwachstum, 3. der wärmeregulation. p.35: …. Im menschen sterben etwa 1·E6 zellen pro sekunde ab - die sowohl im korrekten »timing« als auch mit submolekularer präzision genau ausgeglichen werden; und das trotz eines lückenhaften nahrungsangebots, mit nicht einmal annähernd passenden bausubstraten - wie proteine, lipide, enzyme, nukleinsäuren, vitamine - die nötig sind, um die gesamte substanz jeder fehlenden zelle zu nachzubilden …. p.36: Wäre die wachstumsrate der zellen z.b. des darms nur geringfuegig erhöht, stürbe der mensch innerhalb weniger tage an darmverschluss …. Source: www.kuhf.org/cdprojects/steam/track8.html http://www.nndb.com/people/558/000096270/ Intro Food Epigenome Method Implication

History (4/6)

Mismatch b/w Calories & Exercise

• Energy pathways (an/aerobic) • Energy contents of carbo-hydrates

sucrose C12H22O11 glucose C6H12O6 starch C6H10O5)n triglycerides (fat, RCOO-CH2CH(- OOCR')CH2-OOCR ) proteins (AA, H2NCHRCOOH ) • daily allowance: 2000 kCal

MedBio, 2008

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»Guter« treibstoff erfüllt diese aufgaben optimal, zeichnet sich dann durch eine hohe »ernährungsfysiologische« qualität aus. Um es klarzustellen: dieser standpunkt muss nicht falsch sein. Er könnte sich aber sehr wohl als unzureichend erweisen …. Wenn ein 75kg arbeiter einen 50kg zementsack 10m hochtragen will (PE [J] = mgh, m=75+30kg) …. dann muss er ca. 0.25 g fett verbrennen (1 kCal = 4187 J), oder zirka 1g proteine, oder ersatzweise 1g kohlehydrate. Die tägliche menge von zirka 2000 kCal, die der erwachsene benötigt, reicht immerhin aus, um …. vier klein-LKWs mit je rund 65 sack zement abzuladen und auf den 30m hohen turm zu tragen …. in einem tag (24h) hat er vergleichsweise nicht mehr geleistet als eine seiner 100W glühbirnen, die er möglicherweise abzuschalten vergessen hat (PE [J] = VIt, t=24h60min60s).

Source: www.kuhf.org/cdprojects/steam/track8.html http://www.nndb.com/people/558/000096270/ http://www.medbio.info/Horn/Time%206/muscle_metabolism_march_2007.htm Intro Food Epigenome Method Implication

History (5/6)

Mismatch b/w Calories & Exercise

• 5 kg fat deposits (= 45·E3 kCal !) • Loss is difficult to achieve via mechanical work only;

triathlete.com, 2008

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Physics alone can't supply the answer without biology. If we just consider the work done against gravity it would take a 200 pound bench press to burn 1 food calorie (4187J). From here the issue comes down to efficiency, how much effort is wasted? (A human can expend calories in muscle tension without doing any work, saying nothing of our core bio functions that run all the time). Using a cardio-machine to measure ones heart rate yields the wattage of ones physical effort. Once you know that heart rate x leads to wattage y, just multiply the y of your average heartrate by the length of your workout (measured in seconds) and by 1/4187 to get a vague idea of your expended calories.

Source: http://www.beginnertriathlete.com/ www.dna.caltech.edu/courses/cns187/references/brain.pdf Intro Food Epigenome Method Implication

History (6/6)

Mismatch b/w Calories & Exercise

• 5 kg fat deposits (= 45·E3 kCal !) • Loss is difficult to achieve via mechanical work only; • Brain uses 20% of total energy (20W) or 1kCal every 3.5 mins; • engaging in mental activity doubles / tripples energetic requirements!

triathlete.com, 2008

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The consensus is that a resting human brain uses energy at a rate of 20 Watts, that’s 20% of the overall energy budget of the human body. Hence, 20 W = 20 J/s = 1200 J/min = 28800 J/day, and with a kCal = 4187 J, one obtains: 4.8 Cal/s = 0.29kCal/min = 17.2 kCal/h = 412.7 kCal/day! Or in other words, the human brain uses 1 kCal of energy in about 3.5 min without performing any particular mental task!

…. One could find menu cards in restaurants indicating, in addition to the price, the energy content of every dish …. Taken literally, this is just absurd. For an adult organism the energy content is as stationary as the material content. Since any calorie is worth as much as any other calorie, one cannot see how a mere exchange could help. What then is that precious something contained in our food which keeps us from death? …. Everything that is going on in Nature means an increase of the entropy …. What an organism feeds upon is negative entropy. p.35: erste schlussfolgerung koennte lauten: - lohnt es sich überhaupt, sein übergewicht ehrlich abzuarbeiten? 5kg zuviel zwingen uns, 6100 sack zement (zirka 93 klein-LKWs) abzuladen und jeden einzelnen sack 30m hochzuschleppen. Dafür müsste man vier wochen schweren arbeitsurlaub nehmen. - sollen wir uns nicht lieber freuen darüber, welche enormen arbeitsreserven schon mit einer spärlichen mahlzeit aufgenommen werden? - konnte es der sinn der biologischen evolution sein, uns als energiemaschinen zu gestalten, die es dann noch nicht einmal mit einer einfachen glühbirne aufnehmen können?

Source: http://www.beginnertriathlete.com/ www.dna.caltech.edu/courses/cns187/references/brain.pdf Schrödinger E. (1948) What is Life? The Physical Aspect of the Living Cell. Cambridge: University Press. Intro Food Epigenome Method Implication

Brains (1a/6)

Gut-Brain Connection (our 3 Brains)

• Central- vs enteric-nervous system brain & gut connected by Nervus vagus;

→ Serotonin - common neurotransmitter of both brains (released mostly in the gut – reaches brain via circulatory system); → the gut shapes the brain & vice versa → gut in evolutionary terms is the 1st Brain (see invertebrates); Denjaeander, 2013 Nervus vagus Nervus

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Our two brains have much in common. the upper one is called the central-, the lower one the enteric-nervous system. But why was it necessary that evolution gave us two separate brains? Gershon Michael: "the outsourcing of the lower brain has a similar background to the PCs on our desks. By transferring it to the periphery (intestine), the upper brain works more effectively” .... In fact primitive multicellular organisms at the beginning consisted only of a digestive canal from which the enteric nervous system developed. Most likely did evolution create our upper brain to improve our nutrition (with the known adds-on such as eyes and ears), to facilitate the search for food. Without this division of labour we would still use all our energy today only for digestion .... One brain for thinking, the other one for digesting - a clear division of tasks, but as always reality is more complex, as (1) both nervous systems are in contact with each other by the Nervus vagus and are thus constantly in contact and (2) the both brains use the same neurotransmitters, e.g. one of them is serotonin. In the upper brain, serotonin means well-being (hypothalamus - control centre of emotions), whereas in the abdomen, it determines the rhythm of our intestinal activity and regulates our immune system. Not surprisingly, around 95 % of serotonin is generated in the gastrointestinal tract …. While the exchange of information via the Nervus vagus is well known, the role of serotonin which reaches the brain via the circulatory system has far reaching effects on our feelings. The fact that the belly is actually the shore of the brain is a source of surplus information, which actually has no place in the brain, leads to misunderstandings. it is known that feelings are mirrored in the abdomen, but via serotonin it has been shown that the abdomen can actually influence our feelings ! Thus, does our subconscious mind - an idea that already existed in ancient times and was rediscovered by Siegmund Freud - sit in our gut? Gershon Michael: "the enteric nervous system can influence our feelings by sending signals to the brain. these messages are not conscious to us, but can cause a changed perception of our environment. Our ability to think positively, to defend ourselves against depressions and negative feelings can be determined by signals that our intestines send to the brain, so it influences our subconsciousness.”

Source: Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/fr Intro Food Epigenome Method Implication

Brains (1b/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain: central- vs enteric-nervous system connected by Nervus vagus;

→ Serotonin - common neurotransmitter of both brains (released mostly in the gut – reaches brain via circulatory system); → Food poisoning induced by Bacillus cereus (& sepsis, pneumonia and meningitis); Cereulide (vomit-inducing toxin) binds to serotonin receptors in stomach and small bowel, stimulating Nervus vagus (controls muscle movement in the gut).

Mathur et al., 2018 Taylor, 2018 19-03-18 Madl 11

Host recognition of microbial components is essential in mediating an effective immune response. Cytosolic bacteria must secure entry into the host cytoplasm to facilitate replication and, in doing so, liberate microbial ligands that activate cytosolic innate immune sensors and the inflammasome activated by the enterotoxin, haemolysin BL (HBL). This toxin is highly conserved among the human pathogen Bacillus cereus. HBL binds to the cell membrane forming a lytic pore inducing activation of the NLRP3 inflammasome, secretion of interleukin-1β, -18, and pyroptosis. Furthermore, HBL- producing B.cereus induces rapid inflammasome-mediated mortality.[1] How does the body respond to Bacillus cereus, which can cause food poisoning and sometimes lead to serious infections elsewhere in the body, including sepsis, pneumonia and meningitis? The toxin secreted by the bacteria binds directly to cells in the human body and punches holes in the cells to kill them, triggering an immune response …. Even parts of our nervous system is targeted by the toxins produced by the bacteria .... Bacillus cereus produces more than 12 different toxins. One triggers vomiting and another diarrhea .... The vomit-inducing toxin, called cereulide, binds to serotonin receptors in the stomach and small bowel and stimulates the Nervus vagus, which controls muscle movement in the gut .... That signals back up to the vomiting centres of the brain …. It causes direct stimulation of the small bowel, triggering a reactive response of reflex mechanism called the gastro-colic reflex. The toxins also stimulate receptors in the gut lining ... triggering a lot more movement of gut and colon muscles.[2] Image: A secreted factor of B.cereus activates the NLRP3 inflammasome. Release of IL- 1β and IL-18 and death of bone-marrow-derived macrophages after stimulation with the supernatant of the corresponding bacteria.[1] Source: Mathur A, Feng S, Hayward JA, Ngo C, Fox D, Atmosukarto II, Price JD, Schauer K, Maertlbauer E, Robertson AAB, Burgio G, Fox EM, Leppla SH, Kaakoush NO, Man SM (2018) A multicomponent toxin from Bacillus cereus incites inflammation and shapes host outcome via the NLRP3 inflammasome. Nature Microbiology, 2058-5276; doi: 10.1038/s41564-018-0318-0 [2] Taylor T (2018) What's happening inside your body when you have food poisoning? ABC-Health https://www.abc.net.au/news/health/2018-12-11/food-poisoning-bacillus-cereus-what-happens-inside-your- body/10601390 Intro Food Epigenome Method Implication

Brains (2/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease Early symptoms: digestive disorders (e.g. constipation, reduced sense of smell);

→ digestive complaints appear up to 20 yrs before onset of first motor disorders; → tiny nerve sample (intestinal biopsy) is sufficient to deduce the neuronal state of the brain …. → Gut as 2nd brain ! Denjaeander, 2013 Lebouvier et al., 2010

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For a long time Parkinson's disease was regarded as a disease that exclusively affects a certain brain region (Substantia nigra), but then it was discovered that the disease was associated with severe digestive problems. This led to the conclusion that Parkinson's not only affects the head neurons but also those in the intestine. Damier Philippe: "in some patients, digestive disorders such as constipation or a reduction of the sense of smell can be detected at the beginning, even before the first motor symptoms appear. Since these signs occur very early, the hypothesis is put forward that the disease starts at the periphery of the body and then spreads via the nervous system up to the brain. there, the symptoms are much more diffuse before the patients mentally break down in the last phase and develop balance disorders” [1] …. A simple and harmless intestinal biopsy can therefore provide clarity .... 150 nerve cells are sufficient to be able to deduce the state of the neurons in the brain. The damage found in the intestinal neurons is an indication of the overall state of health among the nerves of the central nervous system .... Since the digestive complaints of 20 years before the first motor disorders allows an early diagnosis to occur an earlier and therefore more effective treatment of Parkinson's disease.[1] Four biopsies were taken from the ascending and descending colon during the course of a total colonoscopy. Immuno-histochemical analysis was performed using antibodies against phosphorylated alpha-synuclein, neurofilaments (NF 220 kDa) and tyrosine hydroxylase. The density of Lewy bodies and Lewy neurites (found in the enteric nervous system of Parkinson’s disease patients), labeled by anti-phosphorylated alpha-synuclein antibodies, was evaluated using a quantitative rating score. Lewy pathology was apparent in the colonic biopsies from 21 patients and in none of the controls. A decreased number of NF-immunoreactive neurons per ganglion was observed of PD patients compared to controls. The amount of Lewy neurites was inversely correlated with neuronal count and positively correlated with levodopa-unresponsive features and constipation.[2]

Source: [1] Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/fr [2] Lebouvier T, Neunlist M, des Varannes SB, Coron E, Drouard A, N’Guyen JM, Chaumette T, Tasselli M, Paillusson S, Flamand M, Galmiche JP, Damier P, Derkinderen P (2010) Colonic Biopsies to Assess the Neuropathology of Parkinson’s Disease and Its Relationship with Symptoms. PLOS-one, Vol.5(9): e127 28 Intro Food Epigenome Method Implication

Brains (3a/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain a recent (1972) extension of BAPs

→ direct influence on cognitive disturbance, depression & neurodegenerative disease can be treated

Denjaeander, 2013 Shipsey, 2015 Bo, 1972

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Chinese have been practicing acupuncture for 3000 years, for a long time without paying special attention to the abdomen, until Bo ZhiYun during a life lecture in 1972 discovered belly acupuncture - a completely new procedure in TCM. Bo ZhiYun: “I had 20 students who were trained in western medicine. One day an old worker was delivered in a pushcart. He complained heavily about back pain. I applied the classical acupuncture methods - nothing helped. The patient kept sweating a lot because of his pain and I also became nervous as the students looked over me trying to understand how I tackle this case .... Finally, I used two acupuncture points on the abdomen which are very deep on the Ren-meridian. The patient immediately stopped complaining and his face relaxed ... ” Yet, the position of the points however did not correspond with his symptoms, in order to understand that Bo ZhiYun started to investigate this more closely and was able to identify a highly sensitive area near the navel. Bo ZhiYun: "the umbilical cord is the center of human development and therefore plays a crucial role in the growth of the embryo. Even if it is cut, it remains an important coordinating center of the body .... the internal organs and viscera are directly connected to our feelings and our mental condition, which explains why there is a close relationship between the abdomen and the brain". Since this discovery, Bo ZhiYun has been able to treat numerous ailments that were previously considered difficult to deal with, ranging from depression to neurodegenerative diseases …. MRI images of the brain of patients before and after abdominal acupuncture treatment have shown that the number of activated brain areas has increased immediately after completion of the procedure.[1]

Image: Abdominal Acupuncture Wall chart. [1,2]

Source: [1] Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/fr [2] Shipsey D (2015) Mastering the Art of Abdominal Acupuncture – A concise guide to treating numerous painful conditions. Centerforce, Dublin (IRL) Intro Food Epigenome Method Implication

Brains (3b/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain a recent (1972) extension of BAPs

→ abdominal acupuncture improves allomeric function in brain cognition network of central nervous system !

Zhong et al., 2011

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Objective: to approach the central nervous mechanism of abdominal acupuncture by using resting- state functional magnetic resonance imaging (fMRI). Methods: twelve old healthy volunteers were treated by standardized abdominal acupuncture with invigorating the kidney and nourishing marrow method and ZhongWan (CV 12), XiaWan (CV 10), QiHai (CV 6), GuanYuan (CV 4), QiXue (KI 13), ShangQu (KI 17) and HuaRouMen (ST 24) were selected. They were examined by using resting-state fMRI before and after treatment of abdominal acupuncture and the brain functional connectivities were analyzed based on bilateral hippocampus chosen as seed points. Results: (1) Before acupuncture, functional connectivities were found in the bilateral middle frontal gyrus, bilateral superior temporal gyrus, bilateral middle temporal gyrus, bilateral thalamus, bilateral subiculum hippocampi, bilateral insula, the cupula and declivis of right cerebellum, left superior frontal gyrus, left head of caudate nucleus, left posterior cingulate gyrus and left inferior parietal lobule (all p <0.05). (2) After acupuncture, functional connectivities were found in the bilateral superior frontal gyrus, bilateral superior temporal gyrus, bilateral middle temporal gyrus, bilateral thalamus, bilateral insula, bilateral subiculum hippocampi, bilateral inferior parietal lobule, bilateral postcentral gyrus, bilateral cingulate gyrus, right head of caudate nucleus, right middle occipital gyrus and right posterior cingulate gyrus (all p <0.05). (3) After acupuncture, there were intensity functional connectivities greater than before acupuncture in the bilateral superior frontal gyrus, bilateral middle frontal gyrus, bilateral superior temporal gyrus, bilateral superior parietal lobule, right inferior frontal gyrus, right anterior cingulate gyrus, right fusiform gyrus, right precuneus and right postcentral gyrus, while the intensity of functional connectivities of left middle temporal gyrus, right lingual gyrus, left cuneus/precuneus and left inferior parietal lobule were attenuated. Conclusion: abdominal acupuncture with invigorating the kidney and nourishing marrow method can improve the allomeric function of the brain cognition network of central nervous system.

Image: A study on response of abdominal acupuncture by using resting state fMRI. [1]

Source: [1] Zhong ZP, Wu SS, Chen ZG, Bo L (2011) Study on response of resting-state functional magnetic resonance imaging induced by abdominal acupuncture with invigorating the kidney and nourishing marrow method. [Article in Chinese] ZhongGuo ZhenJiu. Vol.31(2):139-143 http://en.cnki.com.cn/Article_en/CJFDTotal-ZGZE201102016.htm Intro Food Epigenome Method Implication

Brains (4a/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain • entero-Microbiome – our 3rd Brain

→ faecal microbial transfusion among mice - renders calm ones (Balb/c) aggressive and vice versa (NIH swiss)

Collins et al., 2013 Denjaeander, 2013

der kluge bauch – swiss mice.mp4

19-03-18 Madl 15

Induction of adoptive transfer of behavioral phenotype exploited well-established differences in behavioral profiles and the microbiomes across commonly used strains of laboratory mice. At one end of the spectrum, Balb/c mice express more anxiety-like behavior and timidity than NIH-Swiss mice that are generally more gregarious and less timid. As shown above, this difference is reflected by more time spent in the light by NIH Swiss mice (left panel) and a shorter latency to step-down (right panel) …. In the first series of experiments, SPF NIH Swiss mice were colonized with NIH Swiss or Balb/c flora …. when NIH Swiss mice were colonized with Balb/c flora, the latency to step-down increased significantly from 20 s to 62 s …. This shift to more anxiety-like behavior in NIH Swiss mice receiving Balb/C flora was accompanied by a significant reduction in the level of Brain Derived Neurotropic Factor (BDNF) n the hippocampus evident at 1 week post colonization but not 3 weeks post-colonization …. Hippocampal BDNF is a therapeutic target for anxiolytic drugs and a reduction is associated with anxiety-like behavior …. Fecal content from NIH Swiss mice were transferred to germ free Balb/c mice, resulting in a significantly reduced latency to step down, indicating a less anxiety-like behavior. This change in behavior was accompanied by a significant increase in hippocampal BDNF.[1]

Image: Behavioral differences between Balb/c and NIH Swiss mice. The left panel shows the results of the light box-dark box test in Balb/c and NIH Swiss mice (n = 33 for each). The right panel shows the results of the step down test in Balb/c and NIH Swiss mice. Shown are the mean + 1 SEM. Data were analyzed by ANOVA and the Tukey test.[1]

Source: Collins SM, Kassam Z, Bercik P (2013) The adoptive transfer of behavioral phenotype via the intestinal microbiota: experimental evidence and clinical implications. Current Opinion in Microbiology, Vol.16(3): 240-245 [2] Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/ Intro Food Epigenome Method Implication

Brains (4b/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain • entero-Microbiome – our 3rd Brain

→ faecal transplantation in rhodents modify risk of obesity; i.e. from lean donors to obese recipients from obese donors to lean recipients

Ridaura et al., 2013

19-03-18 Madl 16

The role of specific gut microbes in shaping body composition remains unclear. We transplanted fecal microbiota from adult female twin pairs discordant for obesity into germ-free mice fed low-fat mouse chow, as well as diets representing different levels of saturated fat and fruit and vegetable consumption typical of the U.S. diet. Increased total body and fat mass, as well as obesity- associated metabolic phenotypes, were transmissible with uncultured fecal communities and with their corresponding fecal bacterial culture collections. Cohousing mice harboring an obese twin‘s microbiota (Ob) with mice containing the lean co-twin‘s microbiota (Ln) prevented the development of increased body mass and obesity-associated metabolic phenotypes in Ob cage mates. Rescue correlated with invasion of specific members of Bacteroidetes from the Ln microbiota into Ob microbiota and was diet-dependent. These findings reveal transmissible, rapid, and modifiable effects of diet-by-microbiota interactions

Image: Reliable replication of human donor microbiota in gnotobiotic mice. (A) Features of the four discordant twin pairs. (B) Assembly of bacterial communities in mice that had received intact and uncultured fecal microbiota transplants from the obese and lean co-twins in DZ pair 1. (C) Body composition, defined by QMR, was performed 1 day and 15 dpc of each mouse in each recipient group. Mean values (T SEM) are plotted for the percent increase in fat mass and lean body mass at 15 dpc for all recipient mice of each of the four obese co-twins‘ or lean co-twins‘ fecal microbiota, normalized to the initial body mass of each recipient mouse. Driven by a significant difference in adiposity and total body mass between mice colonized with a lean or obese co-twin donor‘s fecal microbiota. (D) Mean values (T SEM) are plotted for the percent change in fat mass at 15 dpc for all recipient mice of each of the four obese co-twins‘ or lean co-twins‘ fecal microbiota. (E) More prolonged time course study for recipients of fecal microbiota from co-twins in discordant DZ pair 1.

Source: Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, Muehlbauer MJ, Ilkayeva O, Semenkovich CF, Funai K, Hayashi DK, Lyle BJ, Martini MC, Ursell LK, Clemente JC, Van Treuren W, Walters WA, Knight R, Newgard CB, Heath AC, Gordon JI. (2013) Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science. Vol.341(6150): 1079-1089.

16 Intro Food Epigenome Method Implication

Brains (5a/6)

No-intervention group Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain rd • entero-Microbiome – our 3 Brain Probiotic enriched yoghurt

→ probiotic food alters brain activity (Danone co-funded “functional food” study)

Face-morphing.mp4

Tillisch et al., 2013 Fermented milk products w/ probiotic associated blood oxygenation level–dependent changes

19-03-18 Madl

Changes in gut microbiota have been reported to alter signaling mechanisms, emotional behavior, and visceral nociceptive reflexes in rodents. However, alteration of the intestinal microbiota with antibiotics or probiotics has not been shown to produce these changes in humans. We investigated whether consumption of a fermented milk product with probiotic (FMPP) for 4 weeks by healthy women altered brain intrinsic connectivity or responses to emotional attention tasks. Ingestion of FMPP is associated with altered reactivity of interoceptive and somatosensory regions to an emotional attention task: Supporting the findings from the connectivity analysis, region of interest, and whole-brain analyses identified FMPP- associated blood oxygenation level–dependent (BOLD) changes in the insular and somatosensory cortices. When pair-wise group differences in task response were assessed, the FMPP group showed a significant decrease in BOLD activity in the primary viscerosensory and somatosensory cortices (posterior and mid insula) compared with Control and No-Intervention groups …. At the whole-brain level, FMPP significantly decreased BOLD activity in the mid insula cortex and primary somatosensory cortex compared with the No-Intervention group.

Image: Regions showing reduced activity in response to an emotional faces attention task after FMPP intervention are shown, with significant regions demarcated.

Source: Tillisch K, Labus J, Kilpatrick L, Jiang ZG, Stains J, Ebrat B, Guyonnet D, Legrain-Raspaud S, Trontin B, Naliboff B, Mayer EA (2013) Consumption of fermented Milk Product with Probiotic modulates Brain Activity. Gastroenterology. Vol.144(7): 1394-401. doi:10.1053/j.gastro.2013.02.043. Intro Food Epigenome Method Implication

Brains (5b/6)

No-intervention group Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain rd • entero-Microbiome – our 3 Brain Probiotic enriched yoghurt

→ food and microbiome – improper nutrition Food alters the microbiome and thus our  Microbiome behavior !  Banjari et al., 2014 . Mood  Behavior

19-03-18 Madl

Studies have been showing the food we eat affects chemical composition of our brain and alters our mood. Nutrition affects cognitive possibilities, including alertness and the production or release of neurotransmitters, the chemical messengers that carry information from one nerve cell to another. Foods are made up of more than one nutrient, and their interaction is going to affect the production and release of neurotransmitters. Neural impulses are largely resulting from sodium-potassium exchange, but numerous others such as complex carbohydrates, amino acids (tryptophan and tyrosine), fatty acids, particularly omega-3 fatty acids, affect permeability of cell membrane, neurotransmitter metabolism and glial cells. The delicate brain chemical balance is somewhat controlled by the blood – brain barrier. Still, brain remains highly susceptible to changes in body chemistry resulting from nutrient intake and deficiency. The direct connection between nutrition, brain function and behaviour exists, without any doubt. It can be seen through brain’s capability of receiving, storing and integrating sensory information, while initiating and controlling motor responses. These functions correspond to mental activities and form the basis for our behaviour. Constant rise in number of evidence from epigenetic studies confirms that specific nutrients alter our brain development and susceptibility to diseases. Still, specific combination of foods can be extrapolated to a dietary regime, like the Mediterranean diet which has shown its positive impact on maintaining brain function and lower incidence of neurodegenerative diseases. This is of special importance since elderly population (people of 65 years and older) is on the rise all over the world, and the quality of life becomes a priority.

Source: Banjari I, Vukoje I, Mandic ML (2014) Brain food: how nutrition alters our mood and behaviour. Food in Health and Disease, Scientific journal of nutrition and dietetics. Vol. 3 (1):13-21 Intro Food Epigenome Method Implication

Brains (5c/6)

Hippocampus Gut-Brain Connection (our 3 Brains) A common phenomena among many parasites •1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain: • entero-Microbiome – our 3rd Brain

→ evidence #1: Toxoplasma gondii Amygdala T.gondii cyst. alter mouse-behavior and literally offers their host to cat predator, where it thrives better

Gatkowska et al., 2012

19-03-18 Madl 19

Toxoplasma gondii, a protozoan parasite, is capable of infecting a broad range of intermediate warm-blooded hosts including humans. The parasite undergoes sexual re- production resulting in genetic variability only in the intestine of the definitive host (a member of the cat family). The parasite seems to be capable of altering the natural behavior of the host to favor its transmission in the environment. The aim of this study was to evaluate the number of parasite cysts formed in the hippocampus and amygdala of experimentally infected mice as these regions are involved in defense behaviors control and emotion processing, and to assess the influence of the infection on mice behavior …. Infected mice showed significantly diminished exploratory activity described by climbing and rearing, smaller preference for the central, more exposed part of the open field arena and engaged in less grooming behavior compared to uninfected controls. Obtained results revealed that T.gondii invasion significantly influences the normal exploratory behavior of mice which was reflected by the statistically significant decrease in climbing during infection (ANOVA F(2,29)069.77, p<0.001) both in acute (LSD, p<0.001) and chronic (LSD, p<0.001) toxoplasmosis compared to uninfected controls.

Image: Light micrographs of H&E-stained brain sections from C57BL/ 6 mouse infected with ME49 T.gondii strain. The parasite cysts are present in hippocampus (top) and amygdala (bottom). Changes in the exploration parameters: climbing) of mice acutely and chronically infected with T.gondii in comparison to uninfected controls (time 0)

Source: Gatkowska J, Wieczorek M, Dziadek B, Dzitko K, Dlugonska H (2012) Behavioral changes in mice caused by Toxoplasma gondii invasion of brain. Parasitol Res. Vol.111(1): 53–58. Intro Food Epigenome Method Implication

Brains (6a/6)

Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain • entero-Microbiome – our 3rd Brain

→ one big ecosystem ….

Madl, 2018 Denjaeander, 2013

19-03-18 Madl 20

Gouyon Pierre-Henri: people perceive themselves strongly as individuals, whereas ecosystems are considered to be outsourced, in the form of tropical rainforests, coral reefs, etc., but reality is different in as such as we carry a bacterial biome inside us that also exists outside, so we bathe in a bacterial ecosystem that penetrates us and is a part of us. The separation between me and the outside world, between me and the other, no longer exists, rather should be aware that we are part of that amazing biological continuity. We are not individuals but ecosystems embedded in a wider ecosystem there is no boundary b/w the outer and the inner world, but an extraordinary bio-continuity that ties everything living together.

Image: the gut as a an external world popped inwards, fitted with two orifices - the mouth and the anus – connecting it to the outside world.[2]

Source: [1] Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/ Madl P (2018) Why QFT matters. A four-part lecture sieries on Biophotonics, Electromagnetism, Wate and Quantum-Field Theory in bioSciences. Available online: biophysics.sbg.ac.at Intro Food Epigenome Method Implication

Brains (6b/6) iome Microb t the ll abou not a t that’s …. ye Gut-Brain Connection (our 3 Brains)

•1st & 2nd Brain • the Evidence: Parkinson‘s Disease • abdominal Acupuncture & Brain • entero-Microbiome – our 3rd Brain

→ one big ecosystem …. …. connecting all & each of us → eversion of outer environment to a culture of micro-org’s under controlled conditions (T, rH, pH, pO2, pCO2, etc.) → coordinated microbial activity (homeostasis) → sustained by gut mucosa → a downscaled Ecosystem of Endosymbionts → exchange of energy, matter and information (!) Madl, 2018 18-Mar-19 Madl 21

The person is rooted in the planet. The relationship between the /and the Me is also the relationship between the conscious person and the planet: How-ever vast the quantity of information we receive through the outer surface of the body and its senses, it is as nothing compared to the enormous flow of information constantly being exchanged across the inner surface: the lungs and the gastrointestinal system. We breathe and eat and thus exchange enormous quantities of matter, energy, and information with the earth as a living system …. Originally, the evolution of life led to the formation of microorganisms such as bacteria, which developed properties that ensured their survival. At first the bacteria tried to eat or infect each other. But instead of one bacteria emerging victorious, they ended up cooperating. Together, two such organisms influenced each other so much that they developed a symbiotic relationship and could not survive without each other. Symbiotic relationships are common in nature, but what made Margulis's idea special was that they could exist within the cells— i.e., inside the living organisms themselves. An organism could thus consist of an internal cooperation: an endosymbiosis, with endo mean-ing "inside."

Image: The inner surface. A stream of matter flows through man from lips to the other end. A very big amount of information is exchanged across this inner surface to Gaia.

Source: Madl P (2018) Why QFT matters. A four-part lecture sieries on Biophotonics, Electromagnetism, Wate and Quantum-Field Theory in bioSciences. Available online: biophysics.sbg.ac.at

21 Intro Food Epigenome Method Implication

Microbiome (1/7)

our exoORGAN

• environmental influences

→ interface b/w environment & body → microbiome releases both beneficial a well as toxic products → selective pressure via dietary preferences, drugs, and pollutants Zhao, 2013

18-Mar-19 Madl 22

The gut microbiota has been linked with chronic diseases such as obesity in humans. However, the demonstration of causality between constituents of the microbiota and specific diseases remains an important challenge in the field. In this Opinion article, using Koch’s postulates as a conceptual framework, I explore the chain of causation from alterations in the gut microbiota, particularly of the endo- toxin- producing members, to the development of obesity in both rodents and humans. I then propose a strategy for identifying the causative agents of obesity in the human microbiota through a combination of microbiome- wide association studies, mechanistic analysis of host responses and the reproduction of diseases in gnotobiotic animals. Evans proposed a modified version as a unified concept for establishing causation of a putative cause (be it biotic or abiotic in nature) in infectious or non-infectious diseases. In this concept, three kinds of evidence are required to support causation: (i) an association between the disease phenotypes and the presence of the cells or genetic material of the putative cause, cross-sectionally and/or longitudinally; (ii) the reproduction or reduction of the disease phenotypes by experimental addition or removal of the putative cause in humans or animals; (iii) and the occurrence of host molecular responses that mechanistically connect the presence of the putative cause to the occurrence of the disease, for the whole concept to make biological or epidemiological sense. Image: Human health is influenced by interactions among the gut microbiota, the host and the environment. Humans are supraorganisms consisting of both human cells and microbial cells, particularly the gut microbiota. The gut microbiota interacts with host genetics and the environment (mainly diet) to influence the health of the human host. On the one hand, the gut microbiota releases toxins, such as lipopolysaccharides, and beneficial metabolites, such as vitamins and short-chain fatty acids, to damage or nourish humans, respectively. On the other hand, human genetics also imposes selective pressures on the gut microbiota through innate immunity or nutrient availability. The diet and particular drugs have a greater potential to shape the structure and function of the gut microbiota than host genetics, thus influencing the health state of the supraorganism.

Source: Zhao L. (2013). The gut microbiota and obesity: from correlation to causality. Nat Rev Microbiol. Vol.11(9):639-647.

22 Intro Food Epigenome Method Implication

Microbiome (2a/7)

our exoORGAN

• environmental influences • chronic diseases

→ gut microbiome is altered in many chronic diseases; e.g. - obesity - asthma

Ehrlich, 2016 • 100 trillion microorganisms ; more cells than the human body; up to 2 kg of mass! • Interface between food and epithelium • In contact with the 1st pool of immune cells and the • 2nd pool of neural cells of the body

19-03-18 Madl 23

Chronic diseases potentially impacted by the gut microbiome Frailty in seniors Van Tongeren et al., 2005 Crohn’s disease Seksik et al., 2003; Sokol et al., 2006, 2008, 2009 Ulcerative colitis Sokol et al., 2008; Martinez et al., 2008 Rheumatoid arthritis Vaahtovuo et al., 2008; Scher et al., 2013 Obesity Ley et al., 2007; Kalliomäki et al., 2008 Type 2 diabetes Cani and Delzenne, 2009 Type 1 diabetes Dessein et al., 2009; Wen et al., 2008 Celiac disease Nadal et al., 2007; Collado et al., 2009 Allergy Kirjavainen et al., 2002; Björkstén, 2009 Autism Finegold et al., 2002; Paracho et al., 2005 Colorectal cancer Mai et al., 2007; Scanlan et al., 2008 Breast cancer Velicer et al., 2004 HIV Gori et al., 2008 Cirrhosis Gunnarsdottir et al. 2003 Atherosclerosis Wang et al. 2011

Source: Ehrlich DS (2017) The Human Microbiome in Medicine. IMI Stakeholders Forum, Brussels (BE) Ehrlich DS (2016) The intestinal microbiota and the link with diseases. Metagenopolis (mpgs.eu) 1st int. Conference on Clinical Metagenomics, Geneva (CH) Intro Food Epigenome Method Implication

Microbiome (2b/7)

our exoORGAN

• environmental influences • chronic diseases

→ obesity: Akkermansia muciniphila activates fat-metabolising genes; (inversely correlated w/ onset of inflammation, altered adipose tissue metabolism & metabolic disorders)

Schneeberger et al. 2015 Cani & deVos, 2017

19-03-18 Madl 24

Diet-induced obesity (DIO) has been associated to substantial changes in gut microbiota composition in rodent models. In the context of obesity, enhanced adiposity is accompanied by low-grade inflammation of this tissue but the exact link with gut microbial community remains unknown. In this report, we studied the consequences of high-fat diet (HFD) administration on metabolic parameters and gut microbiota composition over different periods of time .… Akkermansia muciniphila abundance was strongly and negatively affected by age and HFD feeding and to a lower extend Bilophila wadsworthia was the only taxa following an opposite trend. Different approaches, including multifactorial analysis, showed that these changes in A.muciniphila were robustly correlated with the expression of lipid metabolism and inflammation markers in adipose tissue, as well as several circulating parameters (i.e., glucose, insulin, triglycerides, leptin) from DIO mice. Thus, our data shows the existence of a link between gut A.muciniphila abundance and adipose tissue homeostasis on the onset of obesity, thus reinforcing the beneficial role of this bacterium on metabolism.[1]

Image: Effects of A.muciniphila and derived products on host metabolism. A.muciniphila has been found to be lower in several conditions such as during obesity, diabetes, intestinal inflammation, liver diseases, or chronic alcohol consumption. This is associated with an altered gut barrier function leading to an increased plasma LPS levels and eventually triggering low grade inflammation and metabolic disorders. A.muciniphila alive or pasteurized as well as Amuc_1100 has been shown to restore gut barrier function likely by acting on TLR2 and restoring appropriate tight junction expression. All these results are associated with an increased mucus later thickness and an improvement of metabolic disorders. It is worth noting that an exploratory human investigation has shown that A.muciniphila is apparently safe.[2]

Source: [1] Schneeberger M, Everard A, Gómez-Valadés AG, Matamoros S, Ramírez S, Delzenne NM, Gomis R, Claret M, Cani P (2015) Akkermansia muciniphila inversely correlates with the onset of inflammation, altered adipose tissue metabolism and metabolic disorders during obesity in mice. Scientific Reports Vol.5, Article number: 16643 [2] Cani PD, deVos WM (2017) Next-Generation Beneficial Microbes: The Case of Akkermansia muciniphila. Front. Microbiol., Vol.8 Article 1765 | https://doi.org/10.3389/fmicb.2017.01765 Intro Food Epigenome Method Implication

Microbiome (2c/7)

our exoORGAN

• environmental influences • chronic diseases

→ obesity: epigenetic activation SNPs (single nucleotide polymorphisms) morphologically affect FTO-gene (a non-protein coding intron) that controls IRX3-gene (active during embryo stage) …. deactivated reduced the body’s Ctrl: control (wild type) KO …. knock out ability to store fat (IRX3 KO) variant

Smeno et al., 2014

19-03-18 Madl 25

Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D)1–3. Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence bodymass and composition phenotypes4–6.However, no direct connection between the obesity-associated variants and FTO expression or function has been made7–9. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3.Consistentwith this, obesity-associated single nucleotide polymorphisms are associated with expression ofIRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3- deficientmice, primarily through the loss of fatmass and increase inb asal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficientmice. Our data suggest thatIRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition. Image: Figure 3 | Irx3-deficient mice are leaner and are protected against dietinduced obesity. Sections of inguinal white adipose tissue (IWAT; subcutaneous), perigonadal WAT (PWAT; visceral), brown adipose tissue (BAT), and liver from High Fat Diet mice.

Source: Smemo S, Tena JJ, Kim KH, Gamazon ER, Sakabe NJ, Gómez-Marín C, Aneas I, Credidio FL, Sobreira DR, Wasserman NF, Lee JH, Puviindran V, Tam D, Shen M, Son JE, Vakili NA, Sung HK, Naranjo S, Acemel RD, Manzanares M, Nagy A, Cox NJ, Hui CC, Gomez-Skarmeta JL, Nóbrega MA. (2014) Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature 507(7492): 371–375

25 Intro Food Epigenome Method Implication

Microbiome (2d/7)

our exoORGAN

• environmental influences • chronic diseases

→ obesity: epigenetic activation a missing protein (Toll-like receptor-5) – a component of the innate immune system – it induces:

highly irritated intestine obesity high blood pressure diabetes – type 2 ! Vijar-Kumar et al., 2010

19-03-18 Madl 26

Metabolic syndrome is a group of obesity-related metabolic abnormalities that increase an individual’s risk of developing type 2 diabetes and cardiovascular disease. Here, we show that mice genetically deficient in Toll-like receptor 5 (TLR5), a component of the innate immune system that is expressed in the gut mucosa and that helps defend against infection, exhibit hyperphagia and develop hallmark features of metabolic syndrome, including hyperlipidemia, hypertension, insulin resistance, and increased adiposity. These metabolic changes correlated with changes in the composition of the gut microbiota, and transfer of the gut microbiota from TLR5-deficient mice to wild-type germ-free mice conferred many features of metabolic syndrome to the recipients. Food restriction prevented obesity, but not insulin resistance, in the TLR5-deficient mice. These results support the emerging view that the gut microbiota contributes to metabolic disease and suggest that malfunction of the innate immune system may promote the development of metabolic syndrome

Image: T5KO mice develop obesity. T5KO mice and WT littermates were monitored for 20 weeks. Abdominal photograph of representative 20-week-old male mice.

Source: Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S, Sitaraman SV, Knight R, Ley RE, Gewirtz AT. (2010). Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science. Vol.328(5975):228-31.

26 Intro Food Epigenome Method Implication

Microbiome (2e/7)

our exoORGAN

• environmental influences • chronic diseases

→ obesity: epigenetic imprinting (paramutation) Mothers treated w/ gastro-intestinal bypass (to loose weight); 50 kids from 20 obese mothers; → kids of obese mothers tend to be likewise obese

some received gastro-intestinal bypass (GIB); these kids developed normally; approx. 5700 genes differently methylated (before & after mother underwent GIB) Guénardet al.,2012 Ng et al., 2010

19-03-18 Madl 27

Obesity and overnutrition during pregnancy affect fetal programming of adult disease. Children born after maternal bariatric gastrointestinal bypass surgery (AMS) are less obese and exhibit improved cardiometabolic risk profiles carried into adulthood compared with siblings born before maternal surgery (BMS). This study was designed to analyze the impact of maternal weight loss surgery on methylation levels of genes involved in cardiometabolic pathways in BMS and AMS offspring. Differential methylation analysis between a sibling cohort of 25 BMS and 25 AMS (2–25 y-old) offspring from 20 mothers was conducted to identify biological functions and pathways potentially involved in the improved cardiometabolic profile found in AMS compared with BMS offspring. A total of 5,698 genes were differentially methylated between BMS and AMS siblings, exhibiting a preponderance of glucoregulatory, inflammatory, and vascular disease genes. The global prevalence of obesity is increasing across most ages in both sexes. This is contributing to the early emergence of type 2 diabetes and its related epidemic1, 2. Having either parent obese is an independent risk factor for childhood obesity3. Although the detrimental impacts of diet-induced maternal obesity on adiposity and metabolism in offspring are well established4, the extent of any contribution of obese fathers is unclear, particularly the role of non-genetic factors in the causal pathway. Here we show that paternal high-fat-diet (HFD) exposure programs β-cell ‘dysfunction’ in rat F female offspring. Chronic HFD consumption in Sprague–Dawley fathers induced increased body weight, adiposity, impaired glucose tolerance and insulin sensitivity. Relative to controls, their female offspring had an early onset of impaired insulin secretion and glucose tolerance that worsened with time, and normal adiposity. Paternal HFD altered the expression of 642 pancreatic islet genes in adult female offspring; genes belonged to 13 functional clusters, including cation and ATP binding, cytoskeleton and intracellular transport. Broader pathway analysis of 2,492 genes differentially expressed demonstrated involvement of calcium-, MAPK- and Wnt-signalling pathways, apoptosis and the cell cycle. Hypomethylation of the Il13ra2 gene, which showed the highest fold difference in expression (1.76-fold increase), was demonstrated. This is the first report in mammals of non-genetic, intergenerational transmission of metabolic sequelae of a HFD from father to offspring.

Image: a, Body weight (control, HFD: n = 8 and 9, respectively). b, Specific growth rate (SGR, change in body weight/body weight; n = 8 and 9, respectively). c, Cumulative energy intake (n = 9 and 7, respectively). d–g Blood glucose (d) and plasma insulin (e) during a glucose tolerance test at 6 weeks (n = 8 and 8, respectively) and 12 weeks (f, g) (n = 5 and 7, respectively). h, Insulin resistance index (glucose (mM) × insulin (ng ml−1) × 0.0417/22.5) at 12 weeks. i, Blood glucose during an insulin tolerance test (0.5 U kg−1) at 11 weeks (n = 8 and 9, respectively). Source: Guénard F, Deshaiesb Y, Cianfloneb K, Krald JG, Marceauc P, Vohla MC (2012) Differential methylation in glucoregulatory genes of offspring born before vs. after maternal gastrointestinal bypass surgery. PNAS, vol.110(28) 11439 -11444. Ng SF, Ruby C, Lin Y, Laybutt DR, Barres R, Owens JA, Morris MJ (2010) Chronic high-fat diet in fathers programs β- cell dysfunction in female rat offspring. Nature Vol. 467: 963 -966

27 Intro Food Epigenome Method Implication

Microbiome (2d/7)

our exoORGAN

• environmental influences • chronic diseases

→ asthma suppression - fiber-rich diet boosts Bactero- idaceae & Bifidobacteria - yield short-fatty acids (sFA) migrate to bone marrow - over-expression of immune Remaining cells suppressed Other; Other; Other; Other Verrucomicrobia;Verrucomicrobiae;Verrucomicrobiales;VerrUcomicrobiaceae Firmicutes; Erysipelotrichi; Erysipelotrichales; Erysipelotrichaceae Firmicutes; Other; Other; Other Firmicutes; Clostridia; Clostridiales; Peptostreptococcaceae Firmicutes; Clostridia; Clostridiales; Ruminococcaceae Firmicutes; Clostridia; Clostridiales; Lachnospiraceae Firmicutes; Clostridia; Clostridiales; Other Firmicutes; Bacilli; Lactobacillales; Other Firmicutes; Clostridia; Clostridiales; Clostridiaceae Firmicutes; Bacilli; Bacillales; Staphylococcaceae Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae Trompete et al., 2014 . Bacteroidetes; Bacteroidia; Bacteroidales; Prevotellaceae Bacteroidetes; Bacteroidales; Rikenellaceae Bacteroidetes; Bacteroidia; Bacteroidales; Other Bacteroidetes; Bacteroidia; Bacteroidales;Prophyromonadaceae Actinobacteria; Actinobacteria; Coriobacteriales; Coriobacteriaceae Bacteroidetes; Bacteroidia; Bacteroidales;Bacteroidaceae Actinobacteria; Actinobacteria; Actinomycetales; CorynebacterIaceae Actinobacteria; Actinobacteria; Bifidobacteriales;Bifidobacteriaceae 19-03-18 Madl 28

Metabolites from intestinal microbiota are key determinants of host-microbe mutualism and, consequently, the health or disease of the intestinal tract. However, whether such host-microbe crosstalk influences inflammation in peripheral tissues, such as the lung, is poorly understood. We found that dietary fermentable fiber content changed the composition of the gut and lung microbiota, in particular by altering the ratio of Firmicutes to Bacteroidetes. The gut microbiota metabolized the fiber, consequently increasing the concentration of circulating short-chain fatty acids (SCFAs). Mice fed a high-fiber diet had increased circulating levels of SCFAs and were protected against allergic inflammation in the lung, whereas a low-fiber diet decreased levels of SCFAs and increased allergic airway disease. Treatment of mice with the SCFA propionate led to alterations in bone marrow hematopoiesis that were characterized by enhanced generation of macrophage and dendritic cell (DC) precursors and subsequent seeding of the lungs by DCs with high phagocytic capacity but an impaired ability to promote T helper type 2 (TH2) cell effector function. The effects of propionate on allergic inflammation were dependent on G protein-coupled receptor 41 (GPR41, also called free fatty acid receptor 3 or FFAR3), but not GPR43 (also called free fatty acid receptor 2 or FFAR2). Our results show that dietary fermentable fiber and SCFAs can shape the immunological environment in the lung and influence the severity of allergic inflammation.

Image: Image: Dietary fiber content alters the intestinal microbiota and both local and systemic levels of SCFAs. 454 pyrosequencing analysis of the fecal microbiota composition from mice fed the different fiber diets at the family level bacteria (c) and the proportions of the Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria phyla (not shown)

Source: Trompette A, Gollwitzer ES, Yadava K, Sichelstiel AK, Sprenger N, Ngom-Bru C, Blanchard C, Junt T, NicodTP, Harris NL, Marsland BJ (2014) Gut microbiota metabolism of dietary fiber influences allergic airway disease and hematopoiesis, Nature Med., Vol.20: 150-166 Huffnagle GB (2014) Increase in dietary fiber dampens allergic responses in the lung. Nature Med., Vol.20: 120–121 28 Intro Food Epigenome Method Implication

Microbiome (3/7)

our exoORGAN

• environmental influences • chronic diseases • diet & innate immunity

→ diet alters gut microbiom;

→ the signature of a ‘healthy gut‘ has direct implications on human health; i.e. favoring microbial composition improves human health.

Greiner & Bäckhed., 2011

19-03-18 Madl 29

The human gut is home to a vast number of bacteria, the microbiota, whose genomes complement our own set of genes. The gut microbiota functions at the intersection between host genotype and diet to modulate host physiology and metabolism, and recent data have revealed that the gut microbiota can affect obesity. The gut microbiota contributes to host metabolism by several mechanisms including increased energy harvest from the diet, modulation of lipid metabolism, altered endocrine function, and increased inflammatory tone. The gut microbiota could thus be considered to be an environ- mental factor that modulates obesity and other meta- bolic diseases.

Image: The gut microbiota suppresses enterocyte expression of Angptl4; this alleviates LPL inhibition and promotes LPL-mediated triglyceride storage in adipose tissue. In addition, reduced Angptl4 levels together with diminished activation of AMPK reduce fatty acid oxidation in skeletal muscle. The gut microbiota has also direct effects on enteroendocrine L-cells: microbially generated short-chain fatty acids (SCFAs) bind to the G-protein-coupled receptor (GPCR) Gpr41 which stimulates secretion of the gut hormone PYY. Secretion of PYY leads to reduced intestinal transit, increased energy harvest, and stimulates hepatic lipogenesis. The gut microbiota generates secondary bile acids that are the major ligands for the GPCR TGR5. Stimulation of TGR5 enhances GLP-1 secretion, and this promotes increased insulin secretion, satiety, and reduced gastric emptying.

Source: Greiner T, Bäckhed F. (2011) Effects of the gut microbiota on obesity and glucose homeostasis. Trends Endocrinol Metab. Vol. 22(4):117-123.

29 Intro Food Epigenome Method Implication

Microbiome (4/7)

our exoORGAN

• environmental influences • chronic diseases • diet & innate immunity • diet & acquired (gut)immunity

→ Bio-communication of bacteria via pilus exchange of drug- resistancy via plasmids (e.g. antibiotic resistance)

Kruse & Sorum., 1994

19-03-18 Madl 30

Image: Scienceprofonline.com/microbiology/bacterial-genetics-plasmid-dna- conjugation-gene-transfer.html

Source: Kruse H, Sorum H (1994). Transfer of Multiple FDrug Resistance Plasmids between Bacteria of Diverse Origins in Natural Microenvironments. Applied and Environ. Microbiology Vol.60(11): 4015-4021

30 Intro Food Epigenome Method Implication

Microbiome (5/7)

our exoORGAN

• environmental influences • chronic diseases • diet & innate immunity

Legend LF/PP: low-fat, plant polysaccharide-rich diet P: post-natal day

Turnbach et al., 2009 Graham, 2016 8 …. see next slide . :1 23 9– :5 21 o Formula fed.mp4 de vi 19-03-18 Madl 31

Diet and nutritional status are among themost importantmodifiable determinants of human health. The nutritional value of food is influenced in part by a person’s gut microbial community (microbiota) and its component genes (microbiome). Unraveling the interrelations among diet, the structure and operations of the gut microbiota, and nutrient and energy harvest is confounded by variations in human environmental exposures, microbial ecology, and genotype. To help overcome these problems, we created a well- defined, representative animal model of the human gut ecosystem by transplanting fresh or frozen adult human fecal microbial communities into germ-free C57BL/6J mice. Culture- independent metagenomic analysis of the temporal, spatial, and intergenerational patterns of bacterial colonization showed that these humanized mice were stably and heritably colonized and reproduced much of the bacterial diversity of the donor’smicrobiota. Switching from a low-fat, plant polysaccharide–rich diet to a high-fat, high-sugar “Western” diet shifted the structure of the microbiota within a single day, changed the representation of metabolic pathways in the microbiome, and altered microbiome gene expression. Reciprocal transplants involving various combinations of donor and recipient diets revealed that colonization history influences the initial structure of themicrobial community but that these effects can be rapidly altered by diet. Humanizedmice fed the Western diet have increased adiposity; this trait is transmissible via microbiota transplantation. Humanized gnotobioticmicewill be useful for conducting proof-of- principle “clinical trials” that test the effects of environmental and genetic factors on the gut microbiota and host physiology. Image: Postnatal assembly of the humanized gut microbiota. (A) Rarefaction curves measuring bacterial diversity in the fecal communities. (B) Taxonomic distribution [RDP level 3 (27)] of the gut microbiota sampled from mice from P14 to P85. Values rep- resent the average relative abun- dance across all samples within a given Source: Turnbaugh PJ, Ridaura VK, Faith JJ, Rey FE, Knight R, Gordon JI. (2009). The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci Transl Med. Vol.1(6):6ra14. Graham P (2016) Gut Reaction Pt-2, Catalyst, ABC – AUS; http://www.abc.net.au/catalyst/stories/4511643.htm

31 Intro Food Epigenome Method Implication

Microbiome (6/7) …. biochem. characterization only !

Water 1 4 1 2 1 Carbohydrates

our exoORGAN 2 Protein 1 6 Fats • environmental influences Formula Minerals • chronic diseases Vitamins 14 Enzyme 13 • diet & innate immunity Amino acid

Nucleotides → Breast milk as the ideal culture Soy Lecithin Water medium Legend Carbohydrates 121 5 34 Carboxylic acid 38 2 Proteins Herslett et al, 2007 9 Non‐protein N's Breast Fats Milk Minerals 49 Vitamins 59 Metal Growth Factors 1 14 18 Enzymes Antiproteases

19-03-18 Madl 32

LECHE MATERNA: Agua, Hidratos de carbono (fuente de energía), Lactosa, Oligosacáridos*, Ácidos carboxílicos, Alfa hidroxiácidos, Ácido láctico, Proteínas (para los músculos y los huesos), Lactoalbúmina, Alfalactoalbúmina, HAMLET (Alfa-Lactoalbúmina modificada para atacar células de tumores), Lactoferrina, Multitud de factores antimicrobianos, Caseína, Albúmina sérica, Nitrógenos no protéicos, Creatina, Creatinina Urea, Ácido úrico, Péptidos. Aminoácidos (los ladrillos que forman las proteínas): Ácido glutámico, Glutamina, Taurina, Alanina, Treonina, Serina, Glicina, Ácido aspártico, Leucina, Cistina, Valina, Lisina, Histidina, Fenilalanina, Tirosina, Arginina, Isoleucina, Ornitina, Metionina, Fosfoserina, Fosfoetanolaminaα-aminobutirato, Triptófano, Prolina, Carnitina (compuesto de aminoácidos necesarios para hacer uso de ácidos grasos como fuente de energía). Nucleótidos (las unidades estructurales del ADN y el ARN): 5- Adenosín monofosfato (5-AMP), 3:5-Adenosín monofosfato cíclico (3:5-cyclic AMP), 5-Citidina Monofosfato (5-CMP), Citidina Difosfato Colina (CDP colina), Guanosín difosfato (GDP), Guanosina difosfato Manosa, 3-Uridina monofosfato (3-UMP), 5 -Uridina monofosfato (5-UMP), Uridina difosfato (UDP), Uridina difosfato hexosa (UDPH), Uridina Difosfato N-Acetilglucosamina (UDPAH), Uridina Difosfato, Ácido Glucurónico (UDPGA) y otros muchos nucleótidos del tipo UDP recién descubiertos. Lípidos: Triglicéridos, Ácidos grasos poliinsaturados de cadena larga, Ácido docosahexaenoico (DHA importante para el desarrollo del cerebro), Ácido araquidónico (AA importante para el desarrollo del cerebro), Ácido linoleico (LA), Ácido alfa-linolénico (ALA), Ácido eicosapentaenoico (EPA), Ácido linoleico conjugado (ácido rumenico), Ácidos grasos libres, Ácidos grasos monoinsaturados, Ácido oléico, Ácido palmitoleico, Ácido heptadecanoico, Ácidos grasos saturados, Ácido esteárico, Ácido palmítico, Ácido laúrico, Ácido mirístico. Fosfolípidos: Fosfatidilcolina, Fosfatidiletanolamina, Fosfatidilinositol, Lisofosfatidilcolina, Lisofosfatidiletanolamina, Plasmalógenos. Esfingolípidos: Esfingomielinas, Gangliósidos GM1, GM2, GM3, Glucosilceramida, Glucoesfingolípidos, Galactosilceramida, Lactosilceramida, Globotriaosilceramida (GB3), Globósido (GB4). Esteroles: Escualeno, Lanosterol, Dimetilsterol, Metosterol, Latosterol, Desmosterol, Triacilglicerol, Colesterol 7-dehidrocolesterol, Estigmaesterol y campesterol 7-ketocolesterol, β-sitosterol, Latosterol, Metabolitos de la vitamina D, Hormonas esteroides. Vitaminas: Vitamina A, Beta caroteno, Vitamina B6, Vitamina B8 (Inositol), Vitamina B12, Vitamina C, Vitamina D, Vitamina E a-tocoferol, Vitamina K, Tiamina, Riboflavina, Niacina, Ácido fólico, Ácido pantoténico, Biotina. Minerales: Calcio, Sodio, Potasio, Hierro, Zinc, Cloruro, Fósforo, Magnesio, Cobre, Manganeso, Yoduro, Selenio, Colina, Azufre, Cromo, Cobalto, Flúor, Níquel. Metal: Molibdeno (elemento esencial en muchas enzimas), Factores de crecimiento (contribuyen a la maduración del recubrimiento intestinal), Citokinas interleukina-1β (IL-1β), IL-2, IL-4, IL-6, IL-8, IL-10, Factor estimulante de colonias de granulocitos (G-CSF), Factor estimulante de colonias de macrófagos (M-CSF), Factor de crecimiento derivado de las plaquetas (PDGF), Factor de crecimiento endotelial vascular (VEGF), Factor de crecimiento de los hepatocitos -α(hgf-α), HGF-β, Factor de necrosis tumoral-α Interferón-γ, Factor de crecimiento epitelial (EGF), Factor de crecimiento transformante-α (TGF-α), TGF β1, TGF-β2, Factor de crecimiento insulínico-i (IGF-Iconocido como somatomedina C), Factor de crecimiento insulínico- II, Factor de crecimiento nervioso (NGF), Eritropoyetina. ***Péptidos (combinaciones de aminoácidos): HMGF I, (Factor de crecimiento humano) HMGF II, HMGF III, Colecistoquinina (CCK) β-endorfinas, Hormona paratiroidea (PTH), Péptido relacionado con la hormona paratiroidea (PTHrP) β-defensinas-1, Calcitonina, Gastrina, Motilina, Bombesina (peptido liberador de gastrina, también conocido como neuromedina B), Neurotensina, Somatostatina. Hormonas (mensajeros químicos que mandan señales desde una célula, o grupo de células, a otras a través de la sangre): Cortisol, Tiroyodina (T3), Tiroxina (T4), Hormona estimulante de la Tiroides (TSH también conocida como Tirotropina), Hormona liberadora de Tirotropina (TRH), Prolactina, Oxitocina, Insulina, Corticosterona, Trombopoyetina, Hormona liberadora de Gonadotropina (GnRH GRH), Leptina (contribuye a la regulación de la ingesta de alimentos), Grelina (contribuye a la regulación de la ingesta de alimentos), Adiponectina, Factor inhibidor de la lactancia (FIL), Eicosanoides, Prostaglandinas (enzimáticamente derivadas de los ácidos grasos), PG-E1, PG-E2, PG-F2, Leucotrienos, Tromboxanos, Prostaciclinas. Enzimas (catalizadores que regulan las reacciones químicas en el cuerpo humano): Amilasa, Arisulfatasa, Catalasa, Histaminasa, Lipasa, Lisozima, PAF-acetilhidrolasa, Fosfatasa, Xantina oxidasa, Antiproteasas (se cree que se unen a las macromoléculas, como las enzimas, y, como consecuencia, previenen reacciones alérgicas y anafilácticas) a-1-antitripsina, a-1-antiquimotripsina. factores inmunológicos (utilizados por el sistema inmune para identificar y neutralizar objetos extraños, como bacterias o virus): Leucocitos (glóbulos blancos), Fagocitos, Basófilos, Neutrófilos, Eosinófilos, Macrófagos, Linfocitos, Linfocitos B (también conocidos como células B), Linfocitos T (también conocidos como células C) siga (Inmunoglobulina A Secretora factor inmunológico más importante), IgA2, IgG, IgD, IgM, IgE, Complemento C1, Complemento C2, Complemento C3, Complemento C4, Complemento C5, Complemento C6, Complemento C7, Complemento C8, Complemento C9, Glicoproteínas, Mucinas (atacan a las bacterias y los virus para evitar que se aferren a las mucosas), Lactadherina, Alpha-lactoglobulina, Alpha-2 macroglobulina, Antígenos Lewis, Ribonucleasa, Inhibidores de la hemaglutinina, Factor Bifidus (incrementa el crecimiento de Lactobacillus bifidus que son bacterias beneficiosas para el organismo), Lactoferrina (encierra el hierro para evitar que las bacterias lo utilicen para reproducirse y propagarse) Lactoperoxidasa (Proteína que aísla la vitamina B12 inhibe el crecimiento de microorganismos), Fibronectina (hace a los fagocitos más agresivos, minimiza la inflamación y repara el daño causado por la inflamación) *Oligosacáridos (más de 200 tipos diferentes).

FÓRMULA: Agua, Hidratos de carbono, Lactosa, Maltodextrina de maíz. Proteínas: Concentrado de lactoalbúmina parcialmente hidrolizado y con menos minerales (procedente de leche de vaca). Lípidos: Oleína de palma, Aceite de soja, Aceite de coco, Aceite de cártamo alto oleico o aceite de girasol, Aceite de M. alpina (DHA de hongos), Aceite de C.cohnii (AA de algas). Minerales: Citrato de potasio, Fosfato de potasio, Cloruro de calcio, Fosfato tricalcio, Citrato de sodio, Cloruro de magnesio, Sulfato ferroso, Sulfato de zinc, Cloruro de sodio, Sulfato de cobre, Yoduro de potasio, Sulfato de manganeso, Selenato de sodio. Vitaminas: Ascorbato de sodio, Inositol, Bitartrato de Colina, Alfa-tocoferol acetato, Niacinamida, Pantotenato de calcio, Riboflavina, Vitamina A acetato, Clorhidrato de piridoxina, Mononitrato de tiamina, Filoquinona, Biotina, Vitamina D3, Vitamina B12. Enzima: Tripsina. Aminoácidos: Taurina, L-Carnitina (una combinación de dos aminoácidos diferentes). Nucleótidos: Citidina 5, Monofosfato Uridina 5, Monofosfato Disódico Adenosín 5 Monofosfato, Guanosín disódico

Source: Herslett C, Hedberg S, Rumble H (2007) Did you ever wonder what's in …. Breastfeeding Course for Health Care Providers, Douglas College, New Westminster, BC (CDN); http://forms.lamaze.org/WhatsinBreastmilkPoster.pdf https://docplayer.es/4681697-La-alimentacion-industrializada-del-lactante-y-el-nino-pequeno.html#show_full_text http://www.fabriziodamiani.com/2017/08/03/latte-matterno-e-latte-artificiale-a-confronto/ Intro Food Epigenome Method Implication

Ecotone (1/5)

Ecotone of our exoORGAN

• rich bacterial species diversity (DNA extracted from fecal samples)

→ gut homeostasis: 100·E12 microbial cells diversity: >1·E3 different strains in mass: approx. 1-2 kg of body weight* → 99.1% of genes are of bacterial origin, (0.8% Achaea; 0.1% Eukaryotes & virus‘)

→ misbalances detune gut-homeostasis & facilitates proliferation of pathogens Qin et al., 2010 18-Mar-19 Madl 33

To understand the impact of gut microbes on human health and well-being it is crucial to assess their genetic potential. Here we describe the Illumina-based metagenomic sequencing, assembly and characterization of 3.3 million non-redundant microbial genes, derived from 576.7 gigabases of sequence, from faecal samples of 124 European individuals. The gene set, approx. 150 times larger than the human gene complement, contains an overwhelming majority of the prevalent (more frequent) microbial genes of the cohort and probably includes a large proportion of the prevalent human intestinal microbial genes. The genes are largely shared among individuals of the cohort. Over 99% of the genes are bacterial, indicating that the entire cohort harbours between 1,000 and 1,150 prevalent bacterial species and each individual at least 160 such species, which are also largely shared. We define and describe the minimal gut metagenome and the minimal gut bacterial genome in terms of functions present in all individuals and most bacteria, respectively.

Image: Relative abundance of 57 frequent microbial genomes among individuals of the cohort. See Fig. 2c for definition of box and whisker plot.

Source: NZZ – die Macht der Bakterien (13. Sept. 2012); (*) Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/ Qin J1, Li R, Raes J, Arumugam M, Burgdorf KS, Manichanh C, Nielsen T, Pons N, Levenez F, Yamada T, Mende DR, Li J, Xu J, Li S, Li D, Cao J, Wang B, Liang H, Zheng H, Xie Y, Tap J, Lepage P, Bertalan M, Batto JM, Hansen T, Le Paslier D, Linneberg A, Nielsen HB, Pelletier E, Renault P, Sicheritz-Ponten T, Turner K, Zhu H, Yu C, Li S, Jian M, Zhou Y, Li Y, Zhang X, Li S, Qin N, Yang H, Wang J, Brunak S, Doré J, Guarner F, Kristiansen K, Pedersen O, Parkhill J, Weissenbach J; MetaHIT Consortium, Bork P, Ehrlich SD, Wang J. (2010). A human gut microbial gene catalogue established by metagenomic sequencing. Nature, Vol.464(7285):59-65.

33 Intro Food Epigenome Method Implication

Ecotone (2/5)

Ecotone of our exoORGAN

• rich bacterial species diversity

• 4 blood-groups / 3 enterotypes Enrichment of strains among these 3 enterotypes: Bacteroides, Clostridiales & Parabacteroides (1) (EU-FP7 metaHIT) Prevotella & Streptococcus (2) Akkermansia, Methanobrevibacter & Ruminococcus (3) .... see above (obesity) → microbiome produces 30% of our calories); yet energy yield differs among these 3 types; so do vitamin concentrations → not nation/continent specific neither to sex & age All three enterotypes yield essential vitamins but do so in altering concentrations Arumugam et al., 2011

19-03-18 Madl 34

Combining 22 newly sequenced fecal metagenomes of individuals from 4 countries with previously published datasets, we identified three robust clusters (enterotypes hereafter) that are not nation or continent-specific …. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis for a community understanding. Enterotype 1, containing 8 samples, is enriched in Bacteroides (p<0.01), which co-occurs, for example, with Parabacteroides. The drivers of this enterotype seem to derive energy primarily from carbohydrates and proteins through fermentation, since these closely related genera have a very broad saccharolytic potential and since genes encoding enzymes involved in the degradation of these substrates (galactosidases, hexosaminidases, proteases) along with glycolysis and pentose phosphate pathways are enriched in this enterotype. Enterotype 2 contains 6 samples and is enriched in Prevotella (p<0.01) and the co-occurring Desulfovibrio, who can act in synergy to degrade mucin glycoproteins present in the mucosal layer of the gut: Prevotella is a known mucin-degrader and Desulfovibrio could enhance the rate- limiting mucin desulfation step by removing the sulfate. Enterotype 3 is the most frequent one and is enriched in Ruminococcus (p<0.01) as well as co- occurring Akkermansia, both known to comprise species able to degrade mucins. It is also enriched in membrane transporters, mostly of sugars, suggesting the efficient binding of mucin and its subsequent hydrolysis as well as uptake of the resulting simple sugars by these genera

Image: An Enterotye can be defined as a densely populated areas in a multi-dimensional space of community composition. Abundances of the main contributors of each enterotype from the Sanger metagenomes (top). Co-occurrence networks of the three enterotypes from the Sanger metagenomes (bottom); unclassified genera under a higher rank are marked by asterisks.

Source: Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, Bertalan M, Borruel N, Casellas F, Fernandez L, Gautier L, Hansen T, Hattori M, Hayashi T, Kleerebezem M, Kurokawa K, Leclerc M, Levenez F, Manichanh C, Nielsen HB, Nielsen T, Pons N, Poulain J, Qin J, Sicheritz-Ponten T, Guarner F, Pedersen O, de Vos WM, Brunak S, Doré J; MetaHIT Consortium, Antolín M, Tims S, Torrents D, Ugarte E, Zoetendal EG, Wang J, Artiguenave F, Blottiere HM, Almeida M, Brechot C, Cara C, Chervaux C, Cultrone A, Delorme C, Denariaz G, Dervyn R, Foerstner KU, Friss C, van de Guchte M, Guedon E, Haimet F, Huber W, van Hylckama-Vlieg J, Jamet A, Juste C, Kaci G, Knol J, Lakhdari O, Layec S, Le Roux K, Maguin E, Mérieux A, Melo Minardi R, M'rini C, Muller J, Oozeer R, Parkhill J, Renault P, Rescigno M, Sanchez N, Sunagawa S, Torrejon A, Turner K, Vandemeulebrouck G, Varela E, Winogradsky Y, Zeller G, Weissenbach J, Ehrlich SD, Bork P (2011) Enterotypes of the human gut microbiome. Nature. 2011 May 12;473(7346):174-80. doi: 10.1038/nature09944. http://enterotype.embl.de Intro Food Epigenome Method Implication

Ecotone (3/5)

Ecotone of our exoORGAN

• rich bacterial species diversity • 4 blood-groups / 3 enterotypes • mother-to-infant seeding

→ strategies to boost beneficial colonization (limited transmigration of maternal microbes through placenta & amniotic fluid); - vaginal delivery: substantial exposure; - breastfeeding: bacteria via skin & milk;

5 Rautava et al., 2012 :5 02 – 20 1: Birth microbiome.mp4 o de 19-03-18 Madlvi 35

Interaction with colonizing intestinal bacteria is essential for healthy intestinal and immunological development in infancy. Advances in understanding early host–microbe interactions indicate that this early microbial programming begins in utero and is substantially modulated by mode of birth, perinatal antibiotics and breastfeeding. Furthermore, it has become evident that this stepwise microbial colonization process, as well as immune and metabolic programming by the microbiota, might have a long-lasting influence on the risk of not only gastrointestinal disease, but also allergic, autoimmune and metabolic disease, in later life. Modulating early host–microbe interaction by maternal probiotic intervention during pregnancy and breastfeeding offers a promising novel tool to reduce the risk of disease. In this Review, we describe the current body of knowledge regarding perinatal microbial contact, initial intestinal colonization and its association with human disease, as well as means of modulating early host–microbe interaction to reduce the risk of disease in the child. The fetus comes into contact with microbes originating in the maternal gut through the placenta and amniotic fluid. A massive inoculum of maternal bacteria is encountered during vaginal delivery, which is also associated with increased maternal intestinal permeability and translocation of gut bacteria into breast milk. Breastfeeding not only modulates neonatal bacterial colonization and immune maturation, but is a direct source of maternal bacteria. After birth, skin-to-skin contact and nursing ensures direct transfer of maternal bacteria to the infant to enhance healthy immune and metabolic maturation.

Image: The gut microbiota programmes host health—from mother to infant.

Source: Rautava S, Luoto R, Salminen S, Isolauri E (2012). Microbial contact during pregnancy, intestinal colonization and human disease. Nature Reviews Gastroenterology and Hepatology Vol.9, 565-576 Graham P (2016) Gut Reaction Pt-1, Catalyst, ABC – AUS; http://www.abc.net.au/catalyst/stories/4506661.htm 35 Intro Food Epigenome Method Implication

Ecotone (4a/5)

Ecotone of our exoORGAN

• rich bacterial species diversity • 4 blood-groups / 3 enterotypes • mother-to-infant seeding

→ adverse microbiomic strategies (from mother to newborn): - medical treatment (antibiotics) - delivery mode (caesarean vs vaginal) - familial contamination (heavy metals) - breast or formula feeding

Cho & Blaser, 2012 . Denjaeander, 2013 . 19-03-18 Madl 36

We have about 100 times more bacteria in our gut than cells in our body …. Only a small part of us is the result of the fertilization of an egg by a sperm whereas the larger part consists of the microbiome, i.e. the bacteria that settle in and on it, as a result each on of us is an ecosystem. Collins Steppen M: "we are more bacterial than human beings because there are more bacteria in and on our bodies than we have human cells. So we have more bacterial DNA than human DNA …. each of us carries 1-2 kg of bacteria, which in turn produce about 30% of our calories”. Since we cannot store a large part of our food, the bacteria take over, in return for offering ideal growth conditions, human receive their energy. Bacteria help us to find out what is poisonous and what is beneficial for our body. Hence, without bacteria we could hardly survive. Collins Steppen M: "the largest immune system in our body is in our intestines and is trained by a multitude of bacteria that inform us about potential dangers and keep us on alert" .... after the relatively low-germ environment in the mother's womb the newborn is colonized by many billions of bacteria from the first day on. Those who manage to settle in the intestine select influence the successive colonization until the intestinal population stabilizes and forms a kind of personal signature .... the intake of antibiotics by mother or child, a caesarean section, bottle instant-milk instead of breastfeeding or excessive hygiene, reduces contact with beneficial bacteria and weakens the microbiome of the child.

Image: Acquisition of the microbiome in early life by vertical transmission, and factors modifying mother-to-child microbial transmission. Through live-birth, mammals have important opportunities for mother-to-child microbial transmission through direct surface contact. However, many modern practices can reduce organism and gene flow; several examples are illustrated. After initial introductions, there is strong selection by hosts for microbes with specific phenotypes, consistent with the extensive conservation. Acquisition is modified by differences in offspring genetics and epigenetics (with respect to both maternal and paternal genes) that inform the competition for host resources by the vertically transmitted or environmentally acquired microbes.[2]

Source: [1] Source: Denjeander C (2013) Le ventre, notre deuxième cerveau (der kluge bauch – unser zweites gehirn); arte.tv/ [2] Cho I, Blaser MJ (2012) The human microbiome: at the interface of health and disease. Nature Reviews Genetics, Vol.13: 260–270 Intro Food Epigenome Method Implication

Ecotone (4b/5)

Ecotone of our exoORGAN

• rich bacterial species diversity • 4 blood-groups / 3 enterotypes • mother-to-infant seeding

→ adverse microbiomic strategies (from mother to newborn): - medical treatment (antibiotics) - delivery mode (caesarean vs vaginal) - familial contamination - breast or formula feeding - intrauterine contamination Matamoros et al., 2013

19-03-18 Madl 37

Throughout the human lifetime, the intestinal microbiota performs vital functions, such as barrier function, metabolic reactions, trophic effects, and maturation of the host's innate and adaptive immune responses. Development of the intestinal microbiota in infants is characterized by rapid and large changes in microbial abundance, diversity, and composition. These changes are influenced by medical, cultural, and environmental factors such as mode of delivery, diet, familial environment, diseases, and therapies used. Thus, it is nearly impossible to define a universal standard for intestinal colonization and development of the intestinal microbiota. This review discusses recent data on the early colonization of the gut by microbial species, development of the intestinal microbiota, and its impact on health.

Image: Impact of external factors on the intestinal microbiota of the infant. Green arrows show beneficial modification; red arrows show modification considered negative for healthy development.

Source: Matamoros S, Gras-Leguen C, Le Vacon F, Potel G, de LaCochetiere MF. (2013) Development of intestinal microbiota in infants and its impact on health. Trends Microbiol. Vol.21(4): 167-173.

37 Intro Food Epigenome Method Implication

Ecotone (4c/5)

Ecotone of our exoORGAN

• rich bacterial species diversity • 4 blood-groups / 3 enterotypes • mother-to-infant seeding

→ additional maternal factors - lifestyle (obesity, smoking, alcohol, drugs) - enviro-exposure (food, water & air) - hygiene (lack / excessive) - stress (psychodynamics) 5 :0 10 – Pfeffer & Renz, 2014 25 8: o de vi Formula fed.mp4 19-03-18 Madl 38

More than 300 years after Antonie van Leeuwenhoek gave the first description of microbes that colonize human body surfaces, the re-discovery of this multifaceted microbial world within our bodies has challenged our principal view on microbes. Novel sequencing techniques provide a plethora of (meta)genomic data, which elucidate the unique properties of mircobiota in different subjects. Moreover, the variety of metabolic and immunologic interactions between the mircobiota and the host's epithelial surfaces has challenged the paradigm of a unidirectional interplay between a given pathogen and the host's immune defense. The newly discovered mechanisms that underlie the symbiosis between the host, specific colonizers, and the mircobiota as a whole indicate that this colonization is more than a friendly coexistence. In fact, it represents a complex ecosystem with implications for the human metabolic homeostasis and immune tolerance. The resilience of the mircobiota and the capability to maintain a well-established equilibrium between symbionts and potential pathogens seem to be determining factors in shaping health or disease. Maternal lifestyle factors such as dietary habits/obesity, smoking, a poor microbial environment (e.g. urban lifestyle), and chronic stress during pregnancy might lower the diversity of maternal mircobiota and decelerate the fetal immune development. Mode of delivery and immune maturation at birth might have an impact on the diversity and properties of the neo-/postnatal bacterial colonization. Neo-/postnatal nutrition, early chronic stressful events, and low microbial diversity of the living environment (e.g. exaggerated hygiene, frequent and early use of antibiotics) might hamper the development of a diverse and adaptive mircobiota, which favors a non-enriched mircobiota accompanied by pro-inflammatory conditions at the mucosal interfaces, a metabolic imbalance, and the loss of immune tolerance. This might result in a higher risk for chronic inflammatory and metabolic diseases in later life. Image: Pre- and postnatal environmental factors pave the way for later chronic inflammatory disease through the microbiome.

Source: Pfefferle PI, Renz H. (2014). The mucosal microbiome in shaping health and disease. F1000Prime Rep. Vol.6: 11-20 Graham P (2016) Gut Reaction Pt-1, Catalyst, ABC – AUS; www.abc.net.au/catalyst/stories/4506661.htm

38 Intro Food Epigenome Method Implication

Ecotone (5a/5)

Ecotone of our exoORGAN

• rich bacterial species diversity bacterial community • 4 blood-groups / 3 enterotypes characterization via 16S • mother-to-infant seeding rRNA gene pyrosequencing

→ modulating infant mibrobiome & determines future health • communities in vaginal borns resemble vaginal microbiota (Lactobacillus, Prevotella, or Sneathia spp. etc,), • communities among C-section babies resemble maternal skin surface (Staphylococcus, Coryne-bacterium, Propionibact. spp. etc.) Dominguez-Bello et al., 2010

19-03-18 Madl 39

Upon delivery, the neonate is exposed for the first time to a wide array of microbes from a variety of sources, including maternal bacteria. Although prior studies have suggested that delivery mode shapes the microbiota's establishment and, subsequently, its role in child health, most researchers have focused on specific bacterial taxa or on a single body habitat, the gut. Thus, the initiation stage of human microbiome development remains obscure. The goal of the present study was to obtain a community-wide perspective on the influence of delivery mode and body habitat on the neonate's first microbiota. We used multiplexed 16S rRNA gene pyrosequencing to characterize bacterial communities from mothers and their newborn babies, four born vaginally and six born via Cesarean section. Mothers’ skin, oral mucosa, and vagina were sampled 1 h before delivery, and neonates’ skin, oral mucosa, and nasopharyngeal aspirate were sampled <5 min, and meconium <24 h, after delivery. We found that in direct contrast to the highly differentiated communities of their mothers, neonates harbored bacterial communities that were undifferentiated across multiple body habitats, regardless of delivery mode. Our results also show that vaginally delivered infants acquired bacterial communities resembling their own mother's vaginal microbiota, dominated by Lactobacillus, Prevotella, or Sneathia spp., and C-section infants harbored bacterial communities similar to those found on the skin surface, dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp. These findings establish an important baseline for studies tracking the human microbiome's successional development in different body habitats following different delivery modes, and their associated effects on infant health. Image: Bacterial 16S rRNA gene surveys reveal that the first microbiotas of human newborns are primarily structured by delivery mode. (A) Communities clustered using principal coordinates analysis of the unweighted UniFrac distance matrix. PC1 and PC2 are plotted on x-and y-axes. Each point corresponds to a community colored according to the mother's body habitat or the newborn's delivery mode. All newborn body habitats are shown. The percentage of variation explained by the plotted principal coordinates is indicated on the axes. The white arrow indicates a pair of superimposed points. Vaginal samples were not obtained from two of the mothers who delivered by C-section. (B) Average relative abundances of the dominant taxa found in this study in aggregated samples. (C) Relative abundances of the 20 most abundant taxa in mothers’ vaginal communities and in the babies they delivered vaginally. Sequences were classified to highest taxonomic level to which they could be confidently assigned. Source: Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R. (2010) Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. PNAS Vol.107(26):11971-11975.

39 Intro Food Epigenome Method Implication

Ecotone (5b/5)

Ecotone of our exoORGAN

• rich bacterial species diversity • 4 blood-groups / 3 enterotypes • mother-to-infant seeding D DV h- → modulating infant mibrobiome irt ob & determines future health icr M • communities in vaginal borns resemble vaginal microbiota (Lactobacillus, Prevotella, or Sneathia spp. etc,), • communities among C-section babies resemble maternal skin surface (Staphylococcus, Coryne-bacterium, Propionibact. spp. etc.) Dominguez-Bello et al., 2010

19-03-18 Madl 40

Upon delivery, the neonate is exposed for the first time to a wide array of microbes from a variety of sources, including maternal bacteria. Although prior studies have suggested that delivery mode shapes the microbiota's establishment and, subsequently, its role in child health, most researchers have focused on specific bacterial taxa or on a single body habitat, the gut. Thus, the initiation stage of human microbiome development remains obscure. The goal of the present study was to obtain a community-wide perspective on the influence of delivery mode and body habitat on the neonate's first microbiota. We used multiplexed 16S rRNA gene pyrosequencing to characterize bacterial communities from mothers and their newborn babies, four born vaginally and six born via Cesarean section. Mothers’ skin, oral mucosa, and vagina were sampled 1 h before delivery, and neonates’ skin, oral mucosa, and nasopharyngeal aspirate were sampled <5 min, and meconium <24 h, after delivery. We found that in direct contrast to the highly differentiated communities of their mothers, neonates harbored bacterial communities that were undifferentiated across multiple body habitats, regardless of delivery mode. Our results also show that vaginally delivered infants acquired bacterial communities resembling their own mother's vaginal microbiota, dominated by Lactobacillus, Prevotella, or Sneathia spp., and C-section infants harbored bacterial communities similar to those found on the skin surface, dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp. These findings establish an important baseline for studies tracking the human microbiome's successional development in different body habitats following different delivery modes, and their associated effects on infant health.

Image: The effect of delivery mode on the direct transmission of bacteria from mother to newborn. Average (±SEM) weighted UniFrac distance for pairwise comparisons between (A) vaginal microbiota of mothers who delivered vaginally, or (B) the skin microbiota of mothers who delivered via C-section and the microbiotas of the newborn babies. The maternal microbiota (vagina or skin) was compared with the microbiota of her own baby, or babies of the same or different delivery mode. Baby samples were from the skin (arms and forehead), oral mucosa, nasopharyngeal aspirate, and meconium. The mothers’ skin samples were from both arms.

Source: Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R. (2010) Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. PNAS Vol.107(26):11971-11975.

40 IntroFood Epigenome Method Implication

Food

19-03-18 Madl 41 IntroFood Epigenome Method Implication

Production (1/7)

Communicative Dynamics: Systems relies on matter, energy and information:

EQM = h·ν0 2 ERT = m 0c dE + dm + dI = 0 (P.Manzelli, 2006)

Energy: the potential for causing change. Information: a »bit« of information is definable as a difference that makes a difference (Bateson) - the content about Form & Gestalt in a message. Matter: the physical & objectifyable world.

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Information-Energy-Matter Triad: With the quantum potential, its effect on a particle depends on its form rather than its magnitude. The effect is the same regardless of the strength of the wave. The wave may have larger effects even at long distances, for the wave does not carry energy; it is an information wave with mental & physical properties (see ship travelling on auto-pilot controlled by satellite: the information contained within the radio waves actually guides the enormous energy possessed by the ship). Matter: is the substance of which physical objects are composed. It constitutes the observable (objectifyable) universe. According to the theory of relativity there is no distinction between matter and energy, because matter can be converted to energy, and vice versa.

Source: Madl P., Yip M.; 2007; The Light of Life – Biophotonics; Proceedings of the 6th Gathering in Biosemiotics 2006, Umweb - Helsinki; Manzelli P. 2006. Evolution in "Bio–Quantum Physics“; General Science Journal; IntroFood Epigenome Method Implication

Production (2a/7)

• Agro Revolution (~8kyrs ago) • Sessile Lifestyle & Population Growth (current)

modified after Cunningham et al,. 2003; AAAS 2000;

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Human populations began to increase rapidly after about A.D. 1600. Many factors contributed to this rapid growth. Increased sailing and navigating skills stimulated commerce and communication among nations. Agricultural developments, better sources of power, and improved health care and hygiene also played a role. We are now in an exponential, or J curve, pattern of growth …. It took all of human history to reach 1 billion people in 1800 but only 156 more years to get to 3 billion in 1960. It took us about 12 years to add the seventh billion. Another way to look at population growth is that the number of living humans tripled during the 20th century. Will it do so again in the twenty-first century? If it does, will we overshoot our environment’s carrying capacity and experience a catastrophic dieback …. As you will see later in this chapter, there is some evidence that population growth already is slowing, but whether we will reach equilibrium soon enough and at a size that can be sustained over the long term remains a difficult but vital question.[1] The explosive burst of highly developed urban civilizations - after millions of years without a face – and which spread over the whole world (so far known to them; e.g. Egypt, Sumer & Akkad in the Hindu valey, Xia dynasty at the HuangHo, the indigene cultures on the american continent among others) is remarkable. It must have been this development in which humans finally have left the line of animal ancestors. Image: Human population levels throughout history. It is clear from the J-shaped growth curve that human population is growing exponentially. When will the growth curve assume an S shape and population growth level off? Many factors influence ideal family sizes. Insert: Exponential growth rises in a J-shaped curve. In contrast, logistic growth rates from an S-shaped curve as carrying capacity slows or stops population growth.

Source: [1] Cunningham WP, Cunningham MA (2013) Principles of Environmental Science: Inquiry & Applications, 7th ed. McGrawHill Publ. USA; AAAS (2000) Overview – The Scale of our Presence. American Atlas of Population & Environment. University of California Press, USA; http://atlas.aaas.org/index.php?part=1&sec=scale IntroFood Epigenome Method Implication

Production (2b/7)

• Agro Revolution (~8kyrs ago) • Sessile Lifestyle & Population Growth (current)

modified after Cunningham et al,. 2003; AAAS 2000; Bryant et al., 1998;

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The agro-revolution made us sessile, thereby enabling communities to establish villages, cities, and ultimately larger urban aggregations IntroFood Epigenome Method Implication

Production (4/7)

Food & Environment: •Excessive Fertilization – excess nitrogen & phosphate content in water & foods since the 1950s; •since 1840: Agro-Industrial eutrophication / Liebig‘s law

Galloway et al., 2004 Reid et al., 2005

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Since the discovery of Liebig’s law (1850), agricultural productivity skyrocketed – so did the collateral effects of airborne relocation of essential minerals such as phosphorous and nitrogen. In 1840 Justus von Liebig proposed that the single factor in shortest supply relative to demand is the critical determinant in the distribution of that species (be it bacteria, plant, animal, fungi). Nitrogen Cycle: The total amount of reactive, or biologically available, nitrogen created by human activities increased ninefold between 1890 and 1990, with most of that increase taking place in the second half of the century in association with increased use of fertilizers. A recent study of global human contributions to reactive nitrogen flows projected that flows will increase from approximately 165 Tg of reactive nitrogen in 1999 to 270 Tg in 2050, an increase of 64%. More than half of all the synthetic nitrogen fertilizer (which was first produced in 1913) ever used on the planet has been used since 1985. Human activities have now roughly doubled the rate of creation of reactive nitrogen on the land surfaces of Earth. The flux of reactive nitrogen to the oceans increased by nearly 80% from 1860 to 1990, from roughly 27 Tg of nitrogen per year to 48 Tg in 1990. (This change is not uniform over Earth, however, and while some regions such as Labrador and Hudson’s Bay in Canada have seen little if any change, the fluxes from more developed regions such as the northeastern United States, the watersheds of the North Sea in Europe, and the Yellow River basin in China have increased 10- to 15-fold.[2]

Image: Estimated Total Reactive Nitrogen Deposition from the Atmosphere (Wet and Dry) in 1860, Early 1990s, and Projected for 2050. [1] Atmospheric deposition currently accounts for roughly 12% of the reactive nitrogen entering terrestrial and coastal marine ecosystems globally, although in some regions, atmospheric deposition accounts for a higher percentage (about 33% in the United States). (Note: the projection was included in the original study and is not based on MA scenarios.) [2]

Source: [1] Galloway JP, Dentener FJ, Capone G, Boyer EW, Howarth RW, Seitzinger SP, Asner GP, Cleveland CC Green PA, Holland AE, Karl DM, Michaels AF, Porter JH, Townsend AR, Vöosmarty CJ (2004), Nitrogen cycles: past, present, and future. Biogeochemistry, Vol.70: 153–226 [2] Reid WR, Mooney HA, Cropper A, Capistrano D, Carpenter SR, Chopra K, Dasgupta P, Dietz T, Duraiappah AK, Hassan R, Kasperson R, Leemans R, May RM, McMichael T, Pingali P, Samper C, Scholes R, Watson RT, Zakri AH, Zhao SD, Ash NJ, Bennett E, Kumar P, Lee MJ, Raudsepp-Hearne C, Simons H, Thonell J, Zurek MB (2005) Ecosystem and Human Well-Being, Synthesis Ch.5. Millennium Ecosystem Assessment, Ecosystems and Human Well-being: Synthesis. Island Press, Washington, DC - USA IntroFood Epigenome Method Implication

Production (4/7)

Food & Environment: •Excessive Fertilization – excess nitrogen & phosphate content in water & foods since the 1950s; •Persistent Organic Pollutants (POP) after WWII (e.g. DDT); •industry helped to cope with famine …. But at what price? •environmental movement kicked off in the early 1960s;

CERPA 2009

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In the 1950s .... the issue of "chemicals in food" played no or only a marginally role .... p.13: Today, however, there is hardly a foodstuff that has not already been warned about, such as fish, meat, intestinal organs, even bread, vegetables, fruit, milk, noodles, rice, spices, eggs and so forth .... The key topic in fact is the quality of the food .... excessive nitrogen and phosphate content in the top soil layer, in drinking water, food contaminated with pesticides, preservatives, residues of veterinary drugs, substitution and booster chemicals, etc intended to increase the quantity rather than the quality of the food.... p.15: "subsidized destruction of nature" (1990): "our tables are amply loaded .... but the price we have to pay is high, and tha tab we habe to deal with includes increasing destruction of the environment .... This is all the more serious in the long term, especially since it is still not taken seriously and is even strongly promoted by agricultural policy measures .... p.17: The displacement of wild plants and animal species (competing with cultivated plants) is not an industrial accident, but the declared goal of agricultural land use .... Foreign substances and residues determine the health and quality of the food far less than its nutrients, which means in plain language that “it is still cheaper to take in toxic substances with the food than to starve” .... p.123: the current situation: .... Modern industry has eliminated famine .... but are we paying for today's abundance with widely unknown risks. Chemicals that pushed up yields in agricultural practices, leave behind the “silence of a cemetary, desolation, and with the destroction of the fields not only the so called "pests" are gone but also the insects and with them the birds feeding on them. threaten our own health with latent long-term damage .... At first glance it seems elegant to bring harmless and natural substances into the food chain or "genetically" manipulate them .... but food is more than just the sum of its parts ....

Source; http://en.wikipedia.org/wiki/Silent_Spring Carson R (1962) Silent Spring Houghton Mifflin, USA IntroFood Epigenome Method Implication

Production (5/7)

Food & Environment: •Excessive Fertilization – excess nitrogen & phosphate content in water & foods since the 1950s; •Persistent Organic Pollutants (POP) after WWII (e.g. DDT); enter food chain - often associated with endocrine disrupting properties; Florida alligators: pesticide spill in Lake Apopka in 1980 w elo •Micro-contaminants (adsorb • ‘feminized’ males; er b urth EDC on micro-sized particles • birth rates crashed; – f tox (Trojan-horse effect – are not icro • population declined; e M cleansed in waste-water treatment plants) . se Keith, 1997 … 19-03-18 Madl 47

Endocrine Disruptors (ECDs): The physiological processes of the endocrine (hormonal) system can be disrupted by a number of artificially and naturally occurring chemicals. These compounds may potentially cause adverse effects on human health and wildlife by interfering with the function of endogenous hormones in the body (Kim et al.). The endocrine system include the pituitary, thyroid, adrenal glands, and the male and female reproductive systems, all of which release hormones into the bloodstream. In particular the sex hormones include estrogens in females and androgens in males. EDCs consist of synthetic and natural chemicals (Persistant Organic Pollutants, POP) that affect the balance of normal hormonal functions in animals. Depending on their activity they may be characterized as estrogen modulators or androgen modulators. They may mimic the sex hormones estrogen or androgen, thereby producing similar responses to them or they may block the activities of estrogen or androgen; i.e. anti-estrogens or anti-androgens (Keith, 1997).

Source: Keith L.H. (1997). Environmental endocrine disruptors – A handbook of properties, John Wiley & Sons, Inc., New York - USA. Kim T.UK., Lee H.J., Lee S.Y., Cho J.W., Moon S.H. 2005. A Study on Analysis of EDCs by Mass Spectrometry and Removal of EDCs by Membranes. A UNU Pilot Programme on Science and Technology for Sustainability. http://en.wikipedia.org/wiki/Lake_Apopka http://www.epa.gov/endocrine/ IntroFood Epigenome Method Implication

Production (6a/7)

Food & Environment: • Excessive Fertilization • Persistent Organic Pollutants (POP) biocides used in modern-style agro-industry: - Inorg. / organochlor. insecticides, - Herbicides, - Fungicides, etc. Uptake via - skin, (dermal applications) - lungs (inhalation) - intestinal tract (ingestion) Kick-Raack., 2004

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Modern food production employs large scale use of biocides that are introduced into the food chain. Areas of skin have different rates of absorption. Note that area around head absorbs well. Studies with farm applicators have shown absorption on back of neck. Certainly do not want pesticide contaminated hat etc to remain on head. Scrotum area is most sensitive. While palm and forearm not most sensitive, they are still where most exposure occurs for mixers/loaders and often applicators. (Lawn care applicators have exsposure on legs because the walk into their application. So potential for harm depends on sensitivity of the route of exposure, where exposure actually occurs, how long the exposure occurs, and the toxicity of the product. Acute Effects: Immediate “poisoning” or harm and are based on LD50 values. Most sensitive route determines Signal Word. Chronic Effects: Long-term effects from small doses over time. Not necessarily on label. Some information on MSDS (carcinogenicity, mutagenicity, teratogenicity = birth defects, oncogenicity, liver damage, Reproductive disorders-sperm count, sterility, miscarriage, nerve damage, allergenic sensitization). Source: Joanne Kick-Raack, 2004. Pesticide Poisoning Symptoms & Other Health Issues. Pesticide Education Programs, Ohio State Univ. The EXtension TOXicology NETwork: http://extoxnet.orst.edu/ http://hhs.sbo.hampton.k12.va.us/IB%20AP%20PowerPoints/APES%20pesticides.ppt Pesticides: •Algicides: Control algae in lakes, canals, swimming pools, water tanks, and other sites. •Antifouling agents: Kill or repel organisms that attach to underwater surfaces, such as boat bottoms. •Antimicrobials Antimicrobials: Kill microorganisms (such as bacteria and viruses). •Attractants: Attract pests (for example, to lure an insect or rodent to a trap). (However, food is not considered a pesticide when used as an attractant.) •Biopesticides: Biopesticides are certain types of pesticides derived from such natural materials as animals, plants, bacteria, and certain minerals. •Biocides: Kill microorganisms. •Disinfectants and sanitizers: Kill or inactivate disease-producing microorganisms on inanimate objects. •Fungicides: Kill fungi (including blights, mildews, molds, and rusts). •Fumigants: Produce gas or vapor intended to destroy pests in buildings or soil. •Biocides: Kill microorganisms. •Disinfectants and sanitizers: Kill or inactivate disease-producing microorganisms on inanimate objects. •Fungicides: Kill fungi (including blights, mildews, molds, and rusts). •Fumigants: Produce gas or vapor intended to destroy pests in buildings or soil. •Herbicides: Kill weeds and other plants that grow where they are not wanted. •Insecticides: Kill insects and other arthropods. •Miticides (also called acaricides): Kill mites that feed on plants and animals. •Microbial pesticides: Microorgs that kill, inhibit, or out compete pests, including insects or other microorgs. •Molluscicides: Kill snails and slugs. •Nematicides: Kill nematodes (microscopic, worm-like organisms that feed on plant roots). •Ovicides: Kill eggs of insects and mites. •Pheromones: Biochemicals used to disrupt the mating behavior of insects. •Repellents: Repel pests, including insects (such as mosquitoes) and birds. •Rodenticides: Control mice and other rodents. IntroFood Epigenome Method Implication

Production (6b/7)

Food & Environment: • Excessive Fertilization • Persistent Organic Pollutants (POP) - introduction of toxins into the food chain after WWII (e.g. DDT); - often associated with endocrine disrupting properties;

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It is 1948 Paul Muller receives a Nobel Prize for his discovery of the insecticidal properties of DDT (dichloro-dipheynl-tricholoethane) was first synthesized 1873. But at that time, it was just another molecule, with no notable qualities. In 1935, Muller, a researcher at Geigy (Basle, Switzerland), began looking for chemicals that might help control Colorado potato beetles. In 1939, he announced finding the insecticidal properties of DDT. Research on pesticides -- against typhus louse and mosquitoes -- had been underway since 1941 (by the medical branch of OSRD). Disease was no peripheral military issue: 2.5 million had died of typhus in World War I. Malaria was now a concern especially in the Pacific theater. The National Research Council had, since 1940, been organizing research among industries and foundations for an effective mosquito repellant. DDT research in focused on "military" effectiveness. By Feb 1943, DDT was shown to be one hundred times more toxic to mosquito larvae than any alternative. "In October of 1943 a heavy outbreak of typhus occurred in Naples and the customary relief measures proved totally inadequate. General Fox thereupon introduced DDT treatment with total exclusion of the old, slow methods of treatment. As a result, 1,300,000 people were treated in January 1944 and in a period of three weeks the typhus epidemic was completely mastered …. DDT … proved to be extremely valuable in the “fight” against malaria, this the most widespread of all contagious diseases which yearly affects about 300,000,000 people with a death rate of 1%.

Source: http://www.tc.umn.edu/~allch001/1815/pestcide/sim/background.htm http://www.fws.gov/Pacific/ecoservices/envicon/pim/reports/Olympia/HoodCanalEagle.htm IntroFood Epigenome Method Implication

Production (6c/7)

Food & Environment: • Excessive Fertilization • Persistent Organic Pollutants (POP)

Salmon farming.mp4 often found in farmed produce; POPs do bio-accumulate and are toxic;

Bartle A, 2013 . Ibrahim et al., 2011 . Biddinger & Kahn, 2006 .

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Background: Dietary interventions are critical in the prevention of metabolic diseases. Yet, the effects of fatty fish consumption on type 2 diabetes remain unclear. The aim of this study was to investigate whether a diet containing farmed salmon prevents or contributes to insulin resistance in mice …. Our data indicate that intake of farmed salmon fillet contributes to several metabolic disorders linked to type 2 diabetes and obesity, and suggest a role of POPs in these deleterious effects. In addition it is shown that the presence of environmental contaminants, like persistent organic pollutants (POPs), in fish oil may counteract the benefits of LC n-3 PUFA. Overall, these findings may participate to improve nutritional strategies for the prevention and therapy of insulin resistance.[1]

Image: Salmon vaccination (and use of antibiotics) to increase population densities within aquacultural plant.[4] https://www.youtube.com/watch?v=RYYf8cLUV5E

Source: [1] Ibrahim MM, Fjære E, Lock JE, Naville D, Amlund D, Meugnier E, Battistoni BL, Frøyland L, Madsen L, Jessen N, Lund S, Vidal H, Ruzzin J (2011) Chronic Consumption of Farmed Salmon Containing Persistent Organic Pollutants Causes Insulin Resistance and Obesity in Mice. PLoS ONE, Vol.6(9: e25170 Bartle A (2013) What - eating salmon may not be good for me? BioNews Highlights, EnviroTox, University Bergen (http://www.uib.no/filearchive/final.pdf) [3] Biddinger SB, Kahn CR (2006) From Mice to Men: Insights into the Insulin Resistance Syndromes. Annual Review of Physiology, Vol. 68:123-158 [4] Bruehlwiller T (2017) Chilenian Salmon industrie suffers persistant decline after the booming years (In Chiles Lachsindustrie herrscht Katerstimmung nach dem Boom), Fokus on Economy, NZZ. CH http://static1.squarespace.com/static/5608b122e4b013120ad28e89/t/560c10b4e4b05486deaee59c/14436 31289824/ IntroFood Food Method Implication

Production (7b/7)

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Unwanted Medications: • Don’t flush down toilets / drains • Properly dispose / recycle • Prudent usage

Source: http://www.epa.gov/nerlesd1/chemistry/pharma/image/drawing.pdf Daughton C. 2004. Pharmaceuticals and Personal Care Products (PPCPs) as Environmental Pollutants. Environmental Sciences Division. National Exposure Research Laboratory. U.S. Environmental Protection Agency. IntroFood Epigenome Method Implication

Production (7c/7)

Food & Environment: • Excessive Fertilization • Persistent Organic Pollutants Glyphosphate [N-(phosphono-methyl)-glycine] • Glyphosphate / Nonylphenol an organo-phosphorus compound

a LC50 or EC50 [mg/L] (95% confidence limits) …. hazardous to resident fauna, particularly if Organism Temp 24 h 48 h 96 h the water temperatures are elevated or pH >7.5. [°C]

Daphnids 22 3.0 (2.6-3.4) …. avoided reapplication for at least 7 days to Scuds 12 ≈ 100 62 (40-98) prevent bio-accumulation to toxic levels. Midge larvae 22 18 (9.4-32) 43 (28-66) Rainbow trout 12 8.3 (7.0-9.9) 8.3 (7.0-9.9) Fathead minnows 22 2.4 (2.0-2.9) 2.3 (1.9-2.8) Folmar et al. 1979

Channel catfish 22 13 (11-16) 13 (11-16) Bluegills 22 6.4 (4.8-8.6) 5.0 (3.8-6.6)

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Studies were initiated to determine the acute toxicity of technical grade glyphosate (MON0573), the iso-propyl-amine salt of glyphosate (MON0139), the formulated herbicide Roundup (MON02139), and the Roundup surfactant (MON0818) to four aquatic invertebrates and four fishes: daphnids (Daphnia magna), scuds (Gammarus pseudolimnaeus), midge larvae (Chironomous phcmosus), mayfly nymphs (Ephemerella walkeri), Rainbow trout (Salmo gairdneri), fathead minnows (Pimephales promelas), channel catfish (Ictalurus punctatus), and bluegills (Lepomis macrochirus). Acute toxicities for Roundup ranged from 2.3 mg/L (96h LC50, fathead minnow) to 43 mg/L (48h EC50, mature scuds). Toxicities of the surfactant were similar to those of the Roundup formulation. Technical glyphosate was considerably less toxic than Roundup or the surfactant; for midge larvae, the 48h EC50 was 55 mg/L and for rainbow trout, the 96h LC50 was 140 mg/L. Roundup was more toxic to rainbow trout and bluegills at the higher test temperatures, and at pH 7.5 than at pH 6.5. Toxicity did not increase at pH 8.5 or 9.5. Eyed eggs were the least sensitive life stage, but toxicity increased markedly as the fish entered the sac fry and early swim-up stages. No changes in fecundity or gonadosomatic index were observed in adult rainbow trout treated with the isopropylamine salt or Roundup up to 2.0 mg/L. The aging of Roundup test solutions for seven days did not reduce toxicity to midge larvae, rainbow trout or bluegills. In avoidance studies, rainbow trout did not avoid concentrations of the isopropylamine salt up to 10.0 mg/L; mayfly nymphs avoided 10.0 mg/L of Roundup, but not 1.0 mg/L. In a simulated field application, midge larvae avoided 2.0 mg/L of Roundup.

Image: Glyphosate (N-(phosphono-methyl)-glycine) is a broad-spectrum systemic herbicide and crop desiccant. It is an organophosphorus compound, specifically a phosphonate. It is used to kill weeds, especially annual broadleaf weeds and grasses that compete with crops. It was discovered to be an herbicide by Monsanto chemist John E. Franz in 1970.[3] Monsanto brought it to market in 1974 under the trade name Roundup.

Source: Folmar LC, Sanders HI, Julin AM (1979) Toxicity of the herbicide glyphosate and several of its formulations to fish and aquatic invertebrates. Arch. Environm. Contain. Toxicol. 8, 269-278 (1979) Archives of Environmental Contamination and Toxicology IntroFood Epigenome Method Implication

Production (7d/7)

Food & Environment: • Excessive Fertilization • Persistant Organic Pollutants IS, injected side. • Glyphosphate / Nonylphenol NIS, noninjected side. Gly-inj, embryo injected with glyphosate. Cephalic malformations & abnormal development of the craniofacial skeleton: A-D: diluted glyphosate-based herbicides treatment (C-D, stained). E-I: 360 pg direct glyphosate injection @ 2-cell stage. J: 100 µM Cyclopamine (Hedgehog pathway inhibitor - leads to malformations) Paganelli et al. 2010 Ho, 2010

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Xenopus laevis embryos were incubated with 1/5000 dilutions of a commercial glyphosatebased herbicides (GBH). The treated embryos were highly abnormal with marked alterations in cephalic and neural crest development and shortening of the anterior-posterior axis. Alterations on neural crest markers were later correlated with deformities in the cranial cartilages at tadpole stages. Embryos injected with pure glyphosate showed very similar phenotypes. Moreover, GBH produced similar effects in chicken embryos, showing a gradual loss of rhombomere domains, reduction of the optic vesicles, and microcephaly. This suggests that glyphosate itself was responsible for the phenotypes observed, rather than a surfactant or other component of the commercial formulation. A reporter gene assay revealed that GBH treatment increased endogenous retinoic acid (RA) activity in Xenopus embryos and co-treatment with a RA antagonist rescued the teratogenic effects of the GBH. Therefore, we conclude that the phenotypes produced by GBH are mainly a consequence of the increase of endogenous retinoid activity.

Image: A-D: 1/5000 GBH-treated embryos analyzed at stage 45-47. A: Control embryo; eyes (arrows);. B: Embryo exposed to GBH showing reduced eyes (arrows) and head structures (89%, n=38) compare yellow bar spacing in A & B. C-D: Embryos stained with Alcian blue. C: Control embryo, showing facial cartilages: Meckel (Me), ceratohyal (Ce), infrarrostral (I), quadrato (Qu), and branchial (Br). D: Reduction of Me and Qu cartilages in GBH-exposed embryos (77%, n=39). E-I: Embryos injected with 360 pg of glyphosate per cell in one or both cells at the 2-cell stage and analyzed at stage 47 by Alcian blue staining ‘E-F’ or gross morphology ‘H-I’, which was compared with that of sibling controls ‘G’. E-F: Unilaterally injected embryos showing reduced Alcian blue staining and smaller Qu and Me cartilages on the IS (56%, n=16). G: Control embryo. Arrows indicate the position of the eyes. H: Notice the reduction of the eye in the IS (arrow) (54%, n=13). I: Bilaterally injected embryo exhibiting cyclopia (arrow) (38%, n=8). J: Cyclopamine-treated embryo - observe the proximity of both eyes (arrows), due to midline defects - compare w/ control embryo in ‘G’.

Source: Paganelli A, Gnazzo V, Acosta H (2010) Glyphosate-Based Herbicides Produce Teratogenic Effects on Vertebrates by Impairing Retinoic Acid Signaling. Chem. Res. Toxicol. 2010, 23, 1586–1595 Ho MW (2010) Lab Study Establishes Glyphosate Link to Birth Defects. I-SIS. http://www.i-sis.org.uk/glyphosateCausesBirthDefects.php IntroFood Epigenome Method Implication

Production (7e/7)

Food & Environment: • Excessive Fertilization • Persistant Organic Pollutants • Glyphosphate / Nonylphenol

Excess of Retinoic acid as a result of low dose Glyphosphate exposure induces birth defects in verterbrate animals Teglia et al., 2015

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Retinoids are known to regulate important processes such as differentiation, development, and embryogenesis of vertebrates: Alteration in endogenous retinoids concentration is linked with teratogenic effects. Retinol (ROH), retinoid acid (RA), and isoform 13-Cis-retinoic acid (13-Cis-RA), in plasma of a native adults frog, Leptodactylus chaquensis from a rice field (RF) and a forest (reference site; RS) were measured. ROH did not vary between treatment sites. RA and 13-Cis-RA activities were higher (93.7 8.6 µg·mL-1 and 131.7 11.4 µg·mL-1, respectively) in individuals collected from RF than in those from RS (65.5 8.6 µg·mL-1 and 92.2 10.2 µg·mL-1, respectively). The ratios retinoic acid-retinol (RA/ROH) and 13-Cis-RA/ROH revealed significantly higher values in RF than in RS. RA and 13-Cis-RA concentrations in plasma on wild amphibian’s species such as L.chaquensis would be suitable biomarkers of pesticide exposure in field monitoring. Finally, the mechanism of alteration in retinoid metabolites alteration should be further explored both in larvae and adult, considering that the potential exposition and uptake contaminants vary between the double lives of these vertebrates.

Image: Chromatograms corresponding to a standard solution of 0.100 µg·mL-1 and plasma samples of L.chaquensis from the reference site (RS) and plasma sample of L.chaquensis from a rice field (RF) - top. Comparative values of retinoic acid (RA) in individuals of L.chaquensis. Bars mean SEM. *p <0.05 compared with RS - bottom.

Source: Teglia CM, Attademo AM, Peltzer PM, Goicoechea HC, Lajmanovich RC (2015) Plasma retinoids concentration in Leptodactylus chaquensis (Amphibia: Leptodactylidae) from rice agroecosystems, Santa Fe province, Argentina. Chemosphere, Vol.135: 24-30 IntroFood Epigenome Method Implication

Production (7f/7)

Food & Environment: • Excessive Fertilization • Persistant Organic Pollutants • Glyphosphate / Nonylphenol

Excess of Retinoic acid as a result of low dose Glyphosphate exposure induces birth defects in verterbrate animals Peltzer et al. 2010 Mariel et al, 2014

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We present the first compilation and analysis of cases of morphologically abnormal anurans from the mideastern region of Argentina (Córdoba, Santa Fe, and Entre Ríos Provinces). We sampled for abnormal individuals at 51 sites in agricultural, suburban, and forest settings between January 2000 and December 2009. We recorded 71 abnormal individuals, including 16 types of abnormalities in 15 anuran species. In agricultural sites, we found 12 types of abnormalities, with ectromelia being the most abundant …. This study is the first catalog of anurans with morphological abnormalities in Argentina, thus expanding the geographic range of observed abnormalities in amphibians and illustrating the ubiquity of this phenomenon.[1] The toxicity of Nonylphenol, an emerging pollutant, on the common South American toad Rhinella arenarum was stage and time dependent, thus Median Lethal Concentrations (LC50) for acute (96 h), short-term chronic (168 h) and chronic exposure (336 h) were 1.06; 0.96 and0.17 mgNP/L from embryonic period, whereas for exposure from larvae, LC50 remained constant at 0.37 mgNP/L from 96 h to 168 h, decreasing to 0.11 mgNP/L at 336 h. NOEC-168 h for exposure from embryos was 0.025 mgNP/L. The Teratogenic Potential (NOEC-lethality/NOEC-sublethal effects) was 23 times higher than the threshold value, indicating a high risk for embryos to be malformed in absence of significant lethality and representing a threat for the species conservation. By comparing with other amphibians, the early development of R.arenarum was very sensitive to NP. The results highlight the relevance of extending the exposure time and look for the most sensitive stage in order to perform the bioassays for conservation purposes[2] Image: Types of abnormalities recorded in the mid-eastern region of Argentina …. Hatched chick kept in close proximity to glyohosphpate-base herbicide treated farmland …. ectromelia of the femur in L. atrans …. forked tail in S.nasicus tadpole …. forelimb remains under skin in L. latrans.[1] Panoramic views of malformed larvae of Rhinella arenarum as a result of Nonylphenol exposure Control (top) vs 0.45 mg NP/L (bottom): reduced body size, axial flexures, microcephaly, gut miscoiling, underdeveloped gills, abdominal and generalized edema as well as extrusion of the fin axis.[2]

Source: [1] Peltzer PM, Lajmanovich RC, Sanchez LC, Attademo AM, Junges CM, Bionda CL, Martino AL, Basso A (2010) Morphological abnormalities in amphibian populations from the mid-eastern region of Argentina. Herpetological Conservation and Biology 6(3):432–442. [2] Mariel AC, Alejandra BP, Silvia PCC (2014) Developmental toxicity and risk assessment ofnonylphenol to the South American toad,Rhinella arenarum . Environm. Tox. Pharm., Vol. 38; 634– 642 IntroFood Epigenome Method Implication

Production (7g/7)

Food & Environment: • Excessive Fertilization • Persistant Organic Pollutants • Glyphosphate / Nonylphenol

Excess of Retinoic acid as a result of low dose Glyphosphate exposure induces birth defects in verterbrate animals Pederson, 2016 Krueger et al, 2014

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A feed-change from GM-soya to non-GM-soya deliveredresulted in remarkable differences. Diarrhoea in the herd disappeared, medicine usage fell to a third of the level prescribed when GMsoya was being fed to the animals and at the same time, illnesses such as, stomach ulcers and bloat in the sows disappeared. This had been one of the main causes of death amongst sows when consuming GM-feed time. A recorded number of 36 sows had died due to stomach issues in the last 2 years on GM-soya. At least one died a monthdue to ulcers or bloat. Two sows died due to lack of appetite in the last year before the changeover. Another sow had kidney damage, both kidneys transformed to huge balloon-like structures; one was entirely transformed to a structure, containing about 50 L of fluid, while the other kidney had started the same transformation, having a “balloon” on each end, containing about 0.5 and 2 L respectively, and only about 5 cm of the kidney in the middle looked like a normal kidney. The sow was in good health, having just farrowed, and had just been in heat again. In some areas people are experiencing more kidney problems due to Roundup. This is the reason that Sri Lanka has prohibited the use of Roundup. On Non-GM feed the sows became calmer and they produced more milk. Each sow, on average, weaned two extra piglets, meaning that each sow was able to look after two piglets more than before, so reducing the need for “nursing sows”. We saw litter size increase, and fertility improve in times of “low” glyphosate levels in feed and Non-GM soya fed to the sows. We noticed that many old sows that had had 6 layers of piglets and that had never weaned more than 10 to 11 pigs/L, could suddenly wean 14 piglets, on Non-GMO soya. It is good, but not normal for sows to be most productive that late in life. In the weaning barn, health improved notably: the manure got drier; the faeces got more solid and we did not need to use medicine, just after changing to feed containing soya, (at this “change” to a diet containing soya, most Medicine is used in weaners today at farms feeding GM-soya). Efficiency went up and the overall economy of the herd improved.[1] Glyphosate residues in different organs and tissues as lungs, liver, kidney, brain, gut wall and heart of malformed euthanized one-day-old Danish piglets (N= 38) were tested using ELISA. All organs or tissues had glyphosate in different concentrations. The highest concentrations were seen in the lungs (Range 0.4-80 μg/ml) and hearts (Range 0.15-80 μg/ml). The lowest concentrations were detected in muscles (4.4-6.4 μg/g). The detection of such glyphosate concentrations in these malformed piglets could be an allusion to the cause of these congenital anomalies. Further investigations are urgently needed to prove or exclude the role of glyphosate in malformations in piglets and other animals.[2] Image: Selected pictures of malformed Danish piglets. Source: [1] Pedersen IB (2016) A farm study showing health issues, deformities, abortion and fertility problems in pigs linked to GM soya and roundup. Intl Monsanto Tribunal. Amsterdam (NL) http://www.monsanto-tribunal.org/upload/asset_cache/223719479.pdf [2] Krüger M, Schrödl W, Pedersen Ib, Shehata AA (2014) Detection of Glyphosate in Malformed Piglets. J Environ Anal Toxicol 4(5): 230. doi: 10.4172/2161-0525.1000230 IntroFood Epigenome Method Implication

Production (7h/7)

Food & Environment: • Excessive Fertilization • Persistant Organic Pollutants • Glyphosphate / Nonylphenol

Excess of Retinoic acid as a result of low dose Glyphosphate exposure induces birth defects in verterbrate animals and humans Robin, 2017 Peru 21

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Fabian Tomasi, was never trained to handle pesticides properly. His job was just spraying the liquid on the crops tank after tank. Today, at 47, he is a living skeleton. Aixa Cano, is only 5 years old and her body is full of hairy moles. However, even though it is almost impossible to prove it, doctors relate it to agrochemicals. Camila Veron, 2 years old, was born with multiple organ problems, serious innate organic disorders and disability. Doctors point out that agrochemicals are the culprits. Ezequiel Moreno was born with hydrocephalus. Her mother, Silvia Alvarez, blames continued exposure to agrochemicals for two miscarriages and her son's health problems. Dr. Lescano points at the effects of a 12yr female that has ran across a Glyphosphate treated soy-field.

Source: Robin MM (2017) Le Roundup – face a ses juges. Arte France https://peru21.pe/mundo/fotos-efectos-agroquimicos-poblacion-argentina-129616?foto=1 IntroFood Epigenome Method Implication

Xenobiotica (1/2)

Food & Environment: Estimated Chemicals in use (1990)

• chemicals in commerce: 100·E3 • industrial chemicals 72·E3 • annual increase (new): 2·E3 • pesticides: 600 • food additives: 8.7·E3 • cosmetic ingredients.: 7.5·E3 • human pharmaceuticals: 3.3·E3

Keith, 1997

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Environmental toxicology: science that examines the effects of poisonous chemicals and other agents on human and wildlife health. Toxicology is increasingly important: * production have expanded the number of new chemicals; * concentration of chemical contaminants in the environment have increased; but artificially produced chemicals give us the standard of living we enjoy today; * industrial agriculture; * medical advances; * modern materials and conveniences; Thousands of synthetic chemicals are toxicants that affect the health of wildlife and people; they are ubiquitous in the environment and can end up far from their source. Pesticides used to kill insects and weeds are some of the most widespread and toxic synthetic chemicals (e.g. source: lawns, farmlands, golf courses). Increased concern about environmental quality; deal specifically with those toxicants that come from or are discharged into the environment; e.g. Tyrone Hayes of University of California; illustrate battles where stakes are high, both in economics and human health; wasn't until 1960s that people began to learn about the risks of exposure to many synthetic pesticides.

The key event was the publication of Rachel Carson's 1962 book, Silent Spring (DDT)

Source: http://web.bryant.edu/~dlm1/sc372/readings/toxicology/toxicology.htm IntroFood Epigenome Method Implication

Xenobiotica (2/2)

Food & Environment: Estimated Chemicals in use [Mt] (1990) • chemicals in commerce: 100·E3 • industrial chemicals 72·E3 • annual increase (new): 2·E3 • pesticides: 600 • food additives: 8.7·E3 • cosmetic ingredients.: 7.5·E3 • human pharmaceuticals: 3.3·E3 e.g. Hg-exposure Stenstrup , 2003

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Mercury, due to its toxic properties has been classified among the most dangerous substances to human health. There are three groups of sources of mercury release into the environment: natural, anthropogenic and remission. Coal mining, its processing and use in the energy sector has the greatest relevance regarding the pollution of the environment with mercury compounds in Poland. A review of reference literature shows that the average content of mercury in Polish bituminous coal varies within a wide range of 41–399 ppb, which is conditional on the origin, age and type of coal. The production of coal has led to a number of facilities in the form of structurally and age-varied landfills, heaps and mining waste dumps. The content of mercury in post-mining waste is in the range from approximately 55 to 380 ppb. The problem of environmental contamination with mercury has attracted considerable interest due to the effects that its concentration have in the biosphere. On the basis of the existing data it has been found that the content of mercury in soils in areas degraded by mining and processing of coal is even 10–16 times higher, compared to the geochemical background. It is necessary to conduct research in this area due to the limited results of research on mercury content in deposited waste from the preparation and flotation of Polish bituminous coals and the potential harmful effect of mercury on the environment. The paper is dedicated to the mercury content in waste from the extraction and processing of bituminous coal. Image: schematic drawing of mercury cycling in an aquatic ecosystem. With the exception of isolated cases of known point sources, the source of most mercury to most aquatic ecosystems is deposition from the atmosphere, primarily associated with rainfall.

Source: Antoszczyszyn T, Michalska A (2016) The potential risk of environmental contamination by mercury contained in Polish coal mining waste. Journal of Sustainable Mining, Vol.15(4): 191-196 http://www.coastalreview.org/2012/05/state-tries-to-grapple-with-mercury/ http://www.theworldcounts.com/stories/Chemicals-Used-in-Daily-Life IntroFood Epigenome Method Implication

Distribution (1/1)

Food Distribution - Globalization

• globalization boosted focus on monetary value of production; • is reflected in the shift from bulk commodities towards processed foods; • encompasses organic foods as well;

Regmi & Gehlhar, 2001, 2006

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Area devoted to pear growing areas has increased sharply in Chile in the last 12 years, reaching in 1995 about 17.500 hectares (compared to 6.600 ha in 1985). Cultivation of this crop in Chile extends between the latitude 32° and 35° South, corresponding to Metropolitana, VIth and VIIth Regions. The main cultivated varieties are Packham's Triumph (37.4%), Beurré Bosc (23.7%), Anjou (4.9%) and Red Sensation (11.4%). The main problems in this specie are: inadequate zonification of varieties and lack of precocity in most of them. The profitability of the cultivation has been, however, seriously impacted by the increase of the volume produced and low fruit sales prices, obtaining returns between US$ 2.8– 3.0 per box for the grower (this mean 17% of the final sales price at the port). The export volumes have surpassed 9 million boxes. The main Chilean pears importers are North America (25.0%) and Europe (25.5%). 20 yrs ago, bulk commodities that consisted primarily of grains and oilseeds accounted for most agricultural trade; however, in recent years processed and semi-processed products have jointly accounted for 2/3 of total agricultural trade. Highvalue food products are nonbulk commodities that either require special handling, such as fresh produce, or are processed, which adds substantial value beyond the farm level. Processed foods are edible foodstuffs that have been transformed from their original post-harvest states to either semi-processed products (flour and meal) or final products (bread and breakfast cereal).

Image: U.S. Bulk Commodities Export Share Dropped About 30 Percent During 1980-98

Source: Regmi A., Gehlhar M., 2001. Global Food Trade - Consumer Preferences and Concerns. Food Review. Vol. 24 (3): 2-8; Yuri, J.A. and Torres, C. 1998. PEAR PRODUCTION IN CHILE: GROWING AREAS, CULTIVARS, EXPORTS AND PROFITABILITY. Acta Hort. (ISHS); 475:27-34 (http://www.actahort.org/books/475/475_1.htm) IntroFood Epigenome Method Implication

Processing (1/9)

Food Additives - E-numbers

• number codes for food additives; • on food labels throughout the EU;

Two examples i) Conservants i) Packaging i) Radiation i) GMO

INDIAWO, 2006

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Pleasure without quality: for over 2000 years, the philosophers have been arguing about whether true enjoyment consists by exhausting it to the full (hedonism) or of doing without it altogether (asceticism). Until today, this oscillation between roast pork and fasting has been maintained, even developed into a new ingenious variation, whereby the taste of the food has been "perfected" or replaced by reinforcing substances, aromatic additives, colouring and consistency agents and even includes psycho-pharmaceutical medication. Consumers no longer know whether they have fallen into crapulence or whether they have already slipped into ascetism. The agro-industry very much so considers the needs of customers, as their major goals are: - to preserve the "natural" freshness as much as possible, - to imitate or even surpass the "natural" taste in a deceptively similar way, - to maintain the "natural" odour even if the "food" system had long since rotted under natural conditions and been affected by decay, -to make the appearance as appetizing as possible, so that you would like to bite immediately and it can hardly wait .... - and most importantly to increase their profit margins

Source: www.ksoe.ch/lernangebot/indiawo06/ IntroFood Epigenome Method Implication

Processing (2/9)

Food Additives – Conserving Life:

Consequences of improper processing, “western” diet & medical overdose: …we …. • consume over 3kg of chemicals/year; • live longer; • die slower; • become older – the older we are, the more “conserved” we become;

Engel, 2004 & wikipedia, 2009,

19-03-18 Madl 62 p.xl: unfortunately, contemporary western diet tends to be lethal. Although we are living longer lives we do so in a very „conserved“ manner and as such we are “dying” at a slower pace (implying extended physical suffering with a concomitant loss in the quality of our lifes) .... p.79: As if this is not bad enough, further strain is placed on the body by the assortment of chemicals needed to preserve colour, dry, flavour and tenderise processed food, in order to get people to eat it. By this stage, it is not really food but more a composition of reconstituted, chemical-laden organic matter. A recent study has shown that the average person now consumes about three kilograms of chemicals per year, in the form of food additives. The human body was never designed to be so constantly abused. It becomes overloaded and develops allergies to all the unnatural rubbish with which it has been constantly fed. It is again a matter of degree. A little processed food, now and again, is no problem for a healthy body. But, when it is eaten on a daily basis, year after year, as the main ingredients of the diet, the body will lose its capacity to cope, and an insidious form of ill health will result.

Source: http://en.wikipedia.org/wiki/Parkinson%27s_disease Engel C.C. 2004. Stepwise Care for The Recently Deployed and Their Families - A Population- based Model of Care. Uniformed Services University, Walter Reed Army Medical Center. Griffiths S. 1996. Allergy overload. HarperCollins (Australia) IntroFood Epigenome Method Implication

Processing (3a/9)

Food Additives - E-numbers

• number codes for food additives; • on food labels throughout the EU;

E100–E199 (colours) E200–E299 (preservatives) E300–E399 (antioxidants, acidity regulators) E400–E499 (thickeners, stabilizers, emulsifiers) E500–E599 (acidity regs, anti-caking agents) E600–E699 (flavour enhancers) E700–E799 (antibiotics) E900–E999 (miscellaneous) E1000–E1999 (additional chemicals)

zusatzstoffe-online.de, 2009 19-03-18 Madl 63

Preservatives are additives that prolong the shelf-life of foods by “shielding” them from the harmful effects of microorganisms. They have to be approved and may only be used for certain foods and in limited doses; e.g. the shells of oranges are often treated with wax, to which (except in organic farming) preservatives are usually added (generally questionable as 12mg/kg body weight of these substances must not be exceeded at all): i) thia-bendazole (E 233), (i) ortho-phenyl-phenol (E 231), (i) sodium ortho-phenyl-phenol (E 232), (i) biphenyl (=diphenyl) (E 230) and (i) imazalile (0,03 mg/kg/day)

Due to their fungicidal effect, they are approved exclusively for surface treatment of citrus fruits. The preservatives are either applied directly to the shell of the fruit or, in the case of biphenyl (E 230), indirectly via the wrapping paper, cardboard or inlays. The fungicide thia-bendazole (E 233) is also used for the preservation of citrus fruits.

Source: http://www.landwirtschaft-mlr.baden- wuerttemberg.de/servlet/PB/menu/1104695_l1/index.html IntroFood Epigenome Method Implication

Processing (3b/9)

Food Additives - E-numbers

• number codes for food additives; • on food labels throughout the EU;

E100–E199 (colours) E200–E299 (preservatives) E300–E399 (antioxidants, acidity regulators) E400–E499 (thickeners, stabilizers, emulsifiers) E500–E599 (acidity regs, anti-caking agents) E600–E699 (flavour enhancers) E700–E799 (antibiotics) E900–E999 (miscellaneous) E1000–E1999 (additional chemicals)

zusatzstoffe-online.de, 2009 19-03-18 Madl 64 p.31: It seems unfair that normal consumers today are more and more forced to shop with a biochemistry lexicon or a short-list in their hands in order to avoid exposing and altering their chromosomes if they intend to eat healthy and sustainably .... Indeed, we must make it clear what we actually want. That is why it is necessary to find objective and holistic food quality standards that can explain to us the following: - wherein lies the difference between a naturally fresh salad and the artificially freshly maintained one? - what are the consequences of the large-scale manipulation of natural durability, natural appearance, colour and sensory characteristics (such as smell and taste) and what does it mean to our microbiome once it is consumed?

In its essence we need to ask ourselves this question: not only do we need to know what food is, but above all what "life" itself means.

Source: http://www.zusatzstoffe-online.de/home/ http://www.minutella.ch/e/index.htm IntroFood Epigenome Method Implication

Processing (3c/9)

Trend Intake P None Low Medium High Children's hot dog consumption Adjusted ORa 1.0 0.6 1.3 9.5 Long-Term Effects: 0.01 (CI)b -- (0.3-1.5) (0.6-3.0) (1.6-57.6) Case/Control 43/47c 38/55 44/34 14/3 Fathers' hot dog consumption Adjusted ORd 1.0 0.9 3.1 11.0 Mutagenic effects of E250 (Sodium Nitrite) in 0.01 (CI)b -- (0.5-1.7) (1.2-8.0) (1.2-98.7) curd meat products Case/Control 45/57 57/61 30/18 7/3 Children's coke or cola consumption Adjusted ORe 1.0 1.5 0.5 1.3 0.8 (CI)b -- (0.5-4.1) (0.2-1.1) (0.5-3.4) • conserving agent Case/Control 53/62 19/15 23/28 44/34 Children's ham/bacon/sausage • has bacteriocidal properties consumption 1.0 0.8 0.7 1.3 Adjusted ORe -- (0.4-1.7) (0.3-1.7) (0.4-4.5) 1.0 • meat maintains it “appealing” pinkish coulor (CI)b 47/50 33/33 33/43 24/11 Case/Control even after cooking Children's lunch meat consumption Adjusted ORe 1.0 0.6 1.0 1.4 • sausages & hams attain their characteristic taste 0.4 (CI)b -- (0.3-1.5) (0.4-2.5) (0.6-3.4) Case/Control 51/59 17/28 34/28 37/24 Children's hamburger consumption Adjusted ORe 1.0 0.7 0.6 1.0 0.5 (CI)b -- (0.3-1.7) (0.2-1.4) (0.3-3.8) Case/Control 46/49 33/33 49/48 10/8

DeKok & vanMaanen, 2000 Peters et al., 1994

19-03-18E250 Comet assay.mp4 Madl 65

In this review, we focus on exposure of the large bowel to five different classes of fecal mutagens that have previously been related to colorectal cancer risk …. Including N-nitroso compounds (NOC) …. The combined results from in vitro and in vivo research unequivocally demonstrate that these classes of compounds comprise potent mutagens that induce many different forms of genetic damage and that particularly bile acids and fecapentaenes may also affect the carcinogenic process by epigenetic mechanisms.[1] The relation between the intake of certain food items thought to be precursors or inhibitors of NOC and risk of leukemia was investigated in a case-control study among children from birth to age 10 yrs. [2] Two mechanisms of endogenous nitrosation have been identified. The first is a chemical reaction at the low pH of the stomach between amine and amide precursors and nitrite generated from nitrate. About 5% of exogenous nitrate is reduced in the oral cavity by bacterial nitrate reductase to nitrite after gastrointestinal recirculation; this nitrite can react in the stomach to form NOC …. The concentrations of non-volatile NOC in cured meats, including N-nitroso-proline, N-nitroso-hydroxy-proline and N-nitroso-2-hydroxy-methyl.- thiazolidine-4-carboxylic acid can be as high as 100–1000 µg/kg …. The intake of preformed non-volatile NOC from the diet is about 10–100 times higher, up to 1 µmol/day.[1] Table: Multiply-adjusted odds ratios (OR) between childhood leukemia risk & intake of various foods. a) Adjusted for self-reported use of indoor pesticides, children's hair dryers, fathers' occupational spray paint, fathers' other occupational chemicals, fathers' hot dog consumption, wiring configurations, and nursing. b CI = 95% confidence interval. c) Numbers are smaller than in Table 1 due to missing data on individual confounding factors. d) Adjusted for self-reported use of indoor pesticides, children's hair dryers, father's occupational spray paint, father's other occupational chemicals and child's hot dog consumption, wiring configurations, and nursing. e) Adjusted for self-reported use of indoor pesticides, children's hair dryers, fathers' occupational exposure to spray paint, other occupational chemical exposure, fathers' and children's hot dog consumption, wiring configurations, and nursing.

Source: [1] deKok TMCM, vanMaanen JMS (2000) Evaluation of fecal mutagenicity and colorectal cancer risk. Mutation Research Vol.463: 53–101 [2] Peters JM, Preston-Martin S, London SJ, Bowman JD, Buckley JD, Thomas DC (1994). Processed meats and risks of childhood leukemia (California, USA). Cancer Causes and Control 1994, 5, 195 - 202 http://ukfoodguide.net/e250.htm IntroFood Epigenome Method Implication

Processing (3d/9)

Long-Term Effects:

Mutagenic effects of E250 (Sodium Nitrite) in curd meat products

• conserving agent • has bacteriocidal properties • meat maintains it “appealing” pinkish coulor even after cooking • sausages & hams attain their characteristic taste

Hainz, 2015

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Some organic butcher have succeeded in substituting nitrite salt when treating their hams and sausages by it with an herbal extract (e.g. Herba-Pure). Nitrates have only been in use since around 1920, to maintain the fresh red colour, its aroma and at the same time extend shelf life. While it is possible to obtain an excellent product while maintaining colouring it takes more time and, above all, is a very sensitive procedure to perform.

Source: Bio-Hofmetzgerei Hainz Stierlingwaldstrasse 12, A-5111 Bürmoos / e-post: [email protected] IntroFood Epigenome Method Implication

Processing (5a/9)

Food Additives - Packaging

• Aluminium i) Al as wrappers & containers

i) Al-chlorohydrate: AlnCl(3n-m)(OH)m used in deodorants & antiperspirants i) as coagulant in water purification. i) Al-hydroxide, Al(OH) to induce 3 u inflammation in vaccines

• see slide on Al - further below – 00:32:10) e Akte Al Di Wikipedia,(00:31:05 2009

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•Al-chlorohydrate-salt: AlnCl(3n-m)(OH)m is used in deodorants & antiperspirants and as a coagulant in water purification. − •Al-hydroxide, Al(OH)3, (hydroxoaluminate: Al(OH)4 anion @ pH>8 of Al(OH)3

Timelog: dieaktealuminium.files.wordpress.com/2013/04/pressemappe.pdf ‎ 19:04: Philippa Dabre - investigations of toxic potential of minute traces of aluminium in antitranspirant comparative studies with women who suffer breast cancer and those who do not, higher traces of Al have been found in breast-liquids women who had breast cancer 21:05: breast cells degenerating into tumor cells when in contact w/ Al http://www.sciencedirect.com/science/article/pii/S0162013405001613 Darbre PD. (2005). Aluminium, antiperspirants and breast cancer. J.o.Inorg Biochem. Vol.99(9):1912-1919. 21:35: Al promotes transmigration across cells & formation of metastasis 23:27: 2/3 of all anti-transpirants contain Al-compounds - Al reacts w/ skins cells & blocks cysts - sweat doesn‘t form 23:50: pure Al does not exist .... Al occurs only in its oxidated form; e.g. bauxite @ Porta Trompetas 27:25: bauxite results from the weathering of Al-rich ores. 250 soccer fields/year of pristine tropical rainforest sacrifieced 31:05: commercial to promote the use of Al-products 32:15: University of Vienna - allergy trigger (asthma, seasonal rhinitis, neuro-dermitis, etc.) Isabella Pali-Schoell & Erika Jensen Jarolim: Al-hydroxide (AlOH) used to induce allergic reaction to boost immunisation response of vaccines; AlOH used to moderate pH during heart burn (sodbrennen); inducing allergic response in mice by feeding apples together with Al-hydroxide 34:30 kids are more likely to become allergic when mothers took Al-containig medicine during pregnancy (epigenetics) e.g. heart burn medication during pregnancy 35:40: Herwig Holzer - neurological disorders, speech problems, stroke-like symptoms in young individuals, Alzheimer-like symptoms have been observed in patients that underwent dialysis, these patients have been given Al-containing medications …. See Wawschinek O., Petek W., Lang J, Pogglitsch H, Holzer H (1982) The determination of aluminium in human plasma. Microchim. Acta, Vol.77(5-6): 335-339 http://link.springer.com/article/10.1007/BF01197113# 42.10: Al is the only compound in nature that is not used in organismic metabolism or may serve for a specific functions Christoopher Exley http://www.keele.ac.uk/aluminium/groupmembers/chrisexley/ Darbre PD, Mannello F and Exley C (2013) Aluminium and breast cancer: Sources of exposure, tissue measurements and mechanisms of toxicological actions on breast biology. Journal of Inorganic Biochemistry 128, 257-261. http://www.sciencedirect.com/science/article/pii/S0162013413001608 Minshall C, Nadal J and Exley C (2014) Aluminium in human sweat. Journal of Trace Elements in Medicine and Biology 28, 87-88. http://www.sciencedirect.com/science/article/pii/S0946672X13001612 Exley (2013). Human exposure to aluminium. Environ.Scie.Proc.& Impacts.15:1807-1816.http://pubs.rsc.org/en/Content/ArticleLanding/2013/EM/c3em00374d#!divAbstract House E, Polwart A, Darbre P, Barr L, Metaxas G, Exley C (2013) The aluminium content of breast tissue taken from women with breast cancer. Journal of Trace Elements in Medicine and Biology. 27, 257-266. http://www.sciencedirect.com/science/article/pii/S0946672X13000576 Chuchu N, Patel B, Sebastian B, Exley C (2013). The aluminium content of infant formulas remains too high. BMC Pediatrics. 13:162 http://www.biomedcentral.com/1471-2431/13/162 48:00 reddish NaOH-enriched sludge as far as the eye can see 48:30 http://en.wikipedia.org/wiki/Aluminium_hydroxide Al(OH)3 for the cosmetic & pharmaceutical industry, too boost immunological reaction 51:50 workers affected by Al-exposure .... brittle bones, sleeplessness, headaches, acid-burnt skin, 56:20 locals using the water on a daily basis .... skin ruptures, skin itches, water burns like pepper ..... dam ruptured and flooded land where locals live, dust contaminated their fields 59:10: http://wwf.panda.org/wwf_news/press_releases/special_coverage/hungary_mud_sludge_toxic_red/ 1:05:00: molten Al-meets water from the sprinkler during the collapse of the twin-tower in NY; Boing: 82,3t (Al-share: 30t) Christian Simensen:

Al+H2O at 800C -> Al2 (OH) 3 + H2 http://www.sintef.no/home/Press-Room/Research-News/New-theory-explains-collapse-of-Twin-Towers/ 1:07.00 solid rocket boosters use Al-compounds as rocket fuel 1:10:00 Camelford (UK): using Al-compounds to cleanse water - dead fish, bluish water, odd taste .... flushing the drinking water pipeline. Al-sulfate has been erroneously into the wrong pipe people suffered from rheumatic, 10 out of 40 sheep who drank of it died, neurologial disfunction among several members of the village. Exley found traces of Al in deceased 1.2ug/g brain mass in most adults in Alzheimer patients even 4-6ug/g and 23ug/g in deceased victims of Camelford 1:19:00 Ruhrgebiert uses AlCl3 to cleanse water 1:20:20: Paris likewise .... there remains always a rest of Al-traces in the water; Andre Picot induces shift to Fe-oxide powder 1:22:30 Alzheimer patients 1:24:00 Exley claims that Al is a neurotoxin 1:28:26 Romain Gherardi macrophages filled with Al-particles introduced into vaccines redistributed throughout the body, i/o lymphatic system and into the CNS Al-hydroxide is used in vaccines since 1927; in some individuals it is never excreted; Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, Pellissier JF, Chariot P, Authier FJ. Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle. Brain. 2001 Sep;124(Pt 9):1821-31. http://brain.oxfordjournals.org/content/124/9/1821.long 1:31.30: 8th int. congress on autoimmunity 2012, Granada Spain). Yehuda Shoenfeld on the topic of Al-hydroxide and Mg-carbonate .... Al-particles can penetrate i/o the brain congress conclusion: pharmaceutical companies are urged to invest in Al-free vaccines 1:33:00 EU does not see the potential danger of it

Shaw CA, Tomljenovic L. (2013) Aluminum in the central nervous system (CNS): Toxicity in humans and animals, vaccine adjuvants, and autoimmunity. Immunol Res. 2013 Jul;56(2-3):304-16. doi: 10.1007/s12026-013-8403-1. http://link.springer.com/article/10.1007%2Fs12026-013-8403-1 Tomljenovic L. (2011). Aluminum and Alzheimer's disease: after a century of controversy, is there a plausible link? J Alzheimers Dis. 2011;23(4):567-98. doi: 10.3233/JAD-2010- 101494. http://iospress.metapress.com/content/vq1p78553222661m/?genre=article&issn=1387-2877&volume=23&issue=4&spage=567 Source: http://en.wikipedia.org/wiki/ IntroFood Epigenome Method Implication

Processing (5b/9)

Food Additives - Packaging

• Aluminium i) Al as wrappers & containers

i) Al-chlorohydrate: AlnCl(3n-m)(OH)m in deodorants & antiperspirants i) as coagulant in water purification.

i) Al-hydroxide, Al(OH)3 to induce inflammation in vaccines • see slide 43 ff

19-03-18 Madl 68

The only two measures which, according to the conditions tested here, prevent an increase in the natural low aluminium (Al) content in lye-treated b bread-ware during production are the use of stainless steel sheets and baking sheeting. None of the coatings tested here for Al-baking trays (whether damaged or not) as well as the mesh fabric used as a protective coating could reliably prevent Al from being detached by the washing soda under certain conditions and thus allowing a certain amount of Al to pass into the dough. The concentration of the pretzel also influences an Al-transition in such a way that the Al-content in such breads also increases with increasing pH - which can be observed in all types of baking trays and baking layers, with the exception of stainless steel trays and baking sheeting, which remain unaffected by this.

Image: various foodstuffs in contact with Al - Prezel or lye rolls in contact with Al-trays during preparation adsorb a significant amount of Al (Al-quick-test), whereas warm foods covered with Al-foil become easily stained, therefore indicating that a substantial amount of Al-ions leacked into the food (shown is the foil of marinated chicken wings).

Source: LGL (2015) Aluminium in Laugengebäck – Untersuchungsergebnisse 2014 (Al- concentration in Lye-Breads – Research Report 2014) https://www.lgl.bayern.de/lebensmittel/warengruppen/wc_17_brote_kleingebaecke/ue_2014_laugengebaec k_aluminium.htm IntroFood Epigenome Method Implication

Processing (6a/9)

Food Additives - Packaging

• PolyEthylene Terephthalate;

…. Acetaldehyde-formation

CH3CHO …. a question of dosage 0 –1:03:23) (determines toxicity) 01:0 Plastic(1: Era

Wikipedia,Antimon 2009

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PolyEthylene Terephthalate (PET, PETE, or the obsolete PETP or PET-P), is a thermoplastic polymer of the polyester family and is used in synthetic fibers; beverage, food and other liquid containers. PET consists of polymerized units of the monomer ethylene terephthalate, with repeating C10H8O4 units. It contains the chemical elements carbon, hydrogen, and oxygen. If fully burned, it produces only carbon dioxide (CO2) and water (H2O). PET is commonly recycled, and has the number "1" as its recycling symbol. PET degradation: formation of acetaldehyde …. It is a colorless liquid with a fruity smell. When acetaldehyde is produced, some of it remains dissolved in the walls of a container and then diffuses into the product stored inside, altering the taste and aroma. This is not such a problem for non- consumables such as shampoo, for fruit juices, which already contain acetaldehyde or for strong- tasting drinks, such as soft drinks. For bottled water, low acetaldehyde content is quite important, because if nothing masks the aroma, even extremely low concentrations can be tasted (10-20 parts per billion parts of resin, by weight).

Acetaldehyde (ethanal) is an organic chemical compound with the formula CH3CHO or MeCHO. Acetaldehyde occurs naturally in ripe fruit, coffee, and fresh bread, and is produced by plants as part of their normal metabolism. It is popularly known as a chemical that causes hangovers …. Most people of East Asian descent have a “mutation” in their alcohol dehydrogenase gene that makes this enzyme unusually effective at converting ethanol to acetaldehyde, and about half of such people also have a form of acetaldehyde dehydrogenase that is less effective at converting it to acetic acid. This combination causes them to suffer from the alcohol flush reaction, in which acetaldehyde accumulates after drinking, leading to severe and immediate hangover symptoms. Acetaldehyde is toxic when applied externally for prolonged periods, an irritant, and a probable carcinogen. In addition, acetaldehyde is damaging to DNA and causes abnormal muscle development as it binds to proteins. Source: http://en.wikipedia.org/wiki/PET_bottle http://en.wikipedia.org/wiki/Acetaldehyde IntroFood Epigenome Method Implication

Processing (6b/9)

Food Additives - Packaging quotidien • Bisphenol A (BPA); …. Endocrine disrupting activity

C15H16O2 Wikipedia, 2009 eo: Notre poison d Ana Soto (1:28:00 –1:32:23) Vi

Mammary gland of unborn mouse: untreated vs. 250ng/kg treated Sonnenschein & Soto, 2010

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There are seven classes of plastics used in packaging applications. Type 7 is the catch-all "other" class, and some type 7 plastics, such as polycarbonate (sometimes identified with the letters "PC" near the recycling symbol) and epoxy resins, are made from BPA monomer. When such plastics are exposed to hot liquids, BPA leaches out 55 times faster than it does under normal conditions, at up to 32 ng/hour (boiling for an hour: 29 parts per trillion) Type 3 (PVC) can also contain BPA as antioxidant in plasticizers. Types 1 (PET), 2 (HDPE), 4 (LDPE), 5 (polypropylene), and 6 (polystyrene) do not use BPA.[1]

Bisphenol A (BPA), is an organic compound with two phenol functional groups. It is an important plastics and plastic additives. BPA has low acute toxicity, with an oral LD50 of 3250 mg/kg in rats, but it is an endocrine disruptor. Low doses of BPA can mimic the body's own hormones, possibly causing negative health effects. There is thus concern that long term low dose exposure to BPA may induce chronic toxicity in humans. 0.025 "Permanent changes to genital tract” 0.025 "Changes in breast tissue that predispose cells to hormones and carcinogens” 2 "increased prostate weight 30%” 2 “< bodyweight, >anogenital distance in both genders, early puberty and longer estrus.” 2.4 "Decline in testicular testosterone” 2.5 "Breast cells predisposed to cancer” 10 "Prostate cells more sensitive to hormones and cancer” 10 "Decreased maternal behaviors” 30 "Reversed the normal sex differences in brain structure and behavior” 50 U.S. human exposure limit (not a result from an animal study, but a guideline set by EPA); Lang and colleagues found that high BPA levels were significantly associated with heart disease, diabetes, and abnormally high levels of certain liver enzymes. [2] Perinatal exposure to low-doses of bisphenol-A (BPA) results in alterations in the ovary, uterus, and mammary glands and in a sexually dimorphic region of the brain known to be important for estrous cyclicity.[3]

Source: http://en.wikipedia.org/wiki/Acetaldehyde [1] Saal F.S., Myers J.P., 2008. Bisphenol A and Risk of Metabolic Disorders. JAMA. 2008;300(11):1353-1355. Lang I.A., Galloway T.S., Scarlett A., Henley W.E., Depledge M., Wallace R.B., Melzer D. 2008. Association of Urinary [2]Bisphenol A Concentration With Medical Disorders and Laboratory Abnormalities in Adults. JAMA. 2008;300(11):1303-1310 [3] Cabaton N.J., Wadia P.R., Rubin B.S., Zalko D., Schaeberle C.M., Askenase M.H., Gadbois J.N., Tharp A.P., Whitt G.S., Sonnenschein C., Soto A.M. 2010. Perinatal Exposure to Environmentally Relevant Levels of Bisphenol-A Decreases Fertility and Fecundity in CD-1 Mice. Environmental Health Perspectives. doi: 10.1289/ehp.1002559 IntroFood Epigenome Method Implication

Processing (6c/8)

Food Additives - Packaging • Bisphenol A (BPA); …. Endocrine disrupting activity

C15H16O2 Wikipedia, 2009

3 :3 24 – Horstman, 2015 0 :5 21 o de BP-A & epigenetics.mp4 vi

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Even though chemicals like BP-A only stay in our bodies for a few hours, can exposure in children affect their obesity risk for the rest of their lives? The level of BP-A in the thermal paper of shop receipts is more than 100,000 times higher than in canned foods. It's still too early in this study to link the variability of growth rates in babies with early childhood chemical exposure, but there's alarming evidence from elsewhere about the effects of BPA exposure in school-age children …. Those children with the highest levels of BP-A exposure in the first 3 month of their life have a 35%-40% increased risk of being obese compared to those children with the lowest level of exposure. The likely explanation is that BPA is an endocrine disrupter that interferes with the hormone system. But could obesogens be reshaping our genetic destiny as well? …. There's this other science called epigenetics where the environment changes, not the structure of the gene, but the function of the gene. This epigenetics is a normal part of our human development. That's the way babies grow. Organs develop and change through these epigenetic processes. It's when they get hijacked by environmental toxicants that things go wrong.

Source: Horstman M (2015) Why am I still fat?. Catalyst, ABC (AUS) http://www.abc.net.au/catalyst/stories/4327346.htm IntroFood Epigenome Method Implication

Processing (6d/8)

Food Additives - Packaging of blood bags • Phthalate Esters (PE); …. Endocrine disrupting activity pro-inflammatory cytokine booster

Lozano, 2013 Rael et al., 2009 Salamin et al, 2016

DEHP & MEHP detected in day 42 supernatants e.g. autologous & homologous blood doping 19-03-18 Madl 72

It was found that the plasticizer di-2-ethylhexyl-phthalate (DEHP) used in combination with polyvinylchloride (PVC), leached to the blood component contained in the plastic bag and that in the case of the red blood cells, stabilized the membrane allowing the extension of storage up to 49 days, depending on the additive solution used. This extraction of the plasticizer to the components provokes that not only the recipients of transfusions are exposed to significant levels of DEHP but also plasma and platelet apheresis donors are exposed.[1] Phthalate esters (PEs) are a group of plasticizers produced commercially to impart flexibility to an otherwise rigid polyvinylchloride (PVC). PVC and phthalates are used extensively in the production of blood collection bags and other medical devices. PE's are the most abundant man- made pollutants and increase the risk of developing an allergic respiratory disease or a malignancy …. The cumulative chronic exposure to all phthalates and possible synergistic endocrine effects need to be investigated …. The development of some allergic respiratory diseases has been attributed to DEHP and MEHP with the former causing an increase in IgG and the latter promoting increased levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-8 (IL-8).[2] This approach allows for the detection of both autologous and homologous blood transfusion and represents an advantage of being independent from the doping technique or the marketing of new pharmaceutical drugs.[3]

Image: Mass chromatograms measuring DEHP & MEHP detected in day 42 supernatants collected from stored packed red blood cell (PRBC) units (N = 10). DEHP (RT = 10.5 min, [M + H+] = 391.28; MEHP (RT = 7.4 min, [M + H+] = 279.16) was detected using prepared standards (1–1,000 µM DEHP & 1–100 µM MEHP). For each run, 10 µL of sample was injected onto a YMC-Pack Protein-RP HPLC column utilized with a flow rate of 1 mL/min.

Source: [1] Lozano M (2013) DEHP plasticizer and blood bags: challenges ahead. ISBT Science Series Vol.8: 127–130 [2] Rael LT, Bar-Or R, Ambruso DR, Mains CW, Slone DS, Craun ML, Bar-Or D (2009) Phthalate esters used as plasticizers in packed red blood cell storage bags may lead to progressive toxin exposure and the release of pro-inflammatory cytokines. Oxid Med Cell Longev. Vol.2(3): 166–171. [3] Salamin O, DeAngelis S, Tissot JD, Saugy M, Leuenberger N (2016) Autologous blood transfusion in sports: emerging biomarkers. Transfusion Medicine Reviews, Vol.30(3): 109-115 IntroFood Epigenome Method Implication

Processing (7a/9)

Food Additives - Radiation

• Radura-logo …. indicates that a food has been treated with ionizing radiation;

Food irradiation can - mask spoiled food, - discourage to good manufacturing practices, Wikipedia, 2009 - kill 'good‘ / encourage growth of 'bad' bacteria, - devitalise and denature irradiated food, - impair the flavour, - not destroy bacterial toxins already present, - cause harmful chemical changes, - unnecessary in today's food system. 19-03-18 Madl 73

Food irradiation pursues a series of objectives that are achieved by irradiation doses of varying levels: a) inhibition of germination and maturation; b) control of insects and parasites; c) increase of shelf life; d) elimination of microorganisms; e) sterilization (!); The effect of irradiation is based on the destruction of the genome and thus the reproductive and survival capacity of the irradiated organisms. Due to its size, DNA is much more sensitive to ionizing radiation than smaller molecules. Electron radiation and X-ray radiation from particle accelerators as well as gamma rays from 60Cobalt or 137Cesium are used. The photon radiation energy of 60Co (1.12 & 1.33 MeV) and 137Cs (0.66 MeV) is too low to trigger nuclear activation (radioactive decay). International bodies (FAO, IAEA and WHO) have confirmed that food of any kind can be irradiated without hesitation up to an 'average threshold value of 10 kGy (Gray: is the quotient of the absorbed energy and the mass of the body. 1 Gy = 1 J/kg = 100 rd). Low Dose applications (up to 1 kGy) * sprout inhibition in bulbs and tubers 0.03-0.15 kGy * delay in fruit ripening 0.25-0.75 kGy * insect disinfestations (quarantine) elimination of food borne parasites 0.07-1.00 kGy Medium Dose applications (1 kGy to 10 kGy) * reduction of spoilage microbes, prolonging shelf-life (meat, poultry & frozen seafoods 1.50- 3.00 kGy * reduction of pathogenic microbes in fresh and frozen meat, poultry & seafoods 3.00-7.00 kGy * reducing the number of microorganisms in spices to improve hygienic quality 10.00 kGy High Dose applications (above 10 kGy) * sterilisation of packaged meat, poultry &their products which are shelf stable w/o refrigeration. 25.00-70.00 kGy * sterilization of hospital diets 25.00-70.00 kGy * product improvement as increased juice yield or improved re-hydration These doses are above those currently permitted for these food items by the US-FDA and other regulators around the world. The Codex Alimentary Standard on Irradiated Food does not specify any upper dose limit. NASA is authorized to sterilize frozen meat for astronauts at doses of 44 kGy as a necessary exception.

Source: http://en.wikipedia.org/wiki/Food_irradiation http://de.wikipedia.org/wiki/Lebensmittelbestrahlung IntroFood Epigenome Method Implication

Processing (7b/9)

Community Science Action Guides, 2009

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Food Irradiation: After the World War II, scientists looked for new ways to improve life utilizing applications of nuclear energy. One of these applications is that of food irradiation. This process is used to kill dangerous diseases associated with foods. The first use of food irradiation was used during the early 60's, this technique was applied to foods such as wheat, flour, and potatoes. As population grew, the need for new solutions for "cleaning food" also grew. So in the early 80's people started using food irradiation on spices, seasonings, fruits, vegetables and for meat such as pork to prevent trichinosis. Irradiation has become very popular among the growing community today. Today, over 40 nations have accepted the use of food irradiation. These countries include China, France, Germany, Great Britain, Israel, Japan, the Netherlands, South Africa, and the United States among others.

Process: The process of food irradiation is to exposes food to gamma rays from radioactive 60Co. Food is passed through a small machine where 60Co is located. The food is exposed to the radioactive material for approximate 15 to 45 minutes depending on the type of food. The food is packaged before being introduced to the irradiator so the food does not contact other types of bacteria after being irradiated. When the process is completed, the rods of 60Co are retracted into a pool of water, which acts as a radiation barrier. This process has been used not only for food but also to sterilize medical devices, bandages, condoms, tampons, contact lens solution, and food for astronauts. Irradiation is used to extend the life period of food. This is advantageous in areas where food refrigeration is not available. The purpose of food irradiation is to clean food in the very best way possible. The radiation penetrates all parts of the food, killing harmful bacteria that cause diseases, such as salmonella in seafood, trichinosis in pork, and cholera from fish. Source: fi.edu/guide/wester/applications.html IntroFood Epigenome Method Implication

Processing (8a/9)

Food Alteration - GMO:

Genetically Modified Organisms (GMO) …. • to generate pest-resistant strains (gene-gun); • acc. to Epigenetics, genes play a subordinate, role, hence gene manipulation is hit-or-miss production of potential monstrosities (environmental incompatibility); • Many GM-DNAs are combinations of genes and genetic material that have never existed …. risk unknown …. • but are released into our environment and into the food chain and may be more invasive to natural habitats than their conventional counterparts; • GMO-foods: corn, tomato, potato, cotton, squash, papaya, rice, canola, sugar beet, flax, soy, radish, cantaloupe, bananas, etc.; Walker 2005 19-03-18 Madl 75

Advocates of genetic engineering claim that it is no different from what evolution has done, and that it is in fact a new form of evolution. But genetically engineered crops are not analogous to products of normal evolution. If epigenetic causation is the motor of evolution as proposed, and genes play a subordinate, consolidating role, then going at the properties of an organism by manipulating its genes is not even really “engineering.” It is the hit-or-miss production of potentially useful monstrosities. This sharing of genetic information is not an accident. It is nature's method of enhancing the survival of the biosphere. As discussed earlier, genes are physical memories of an organism’s learned experiences. The recently recognized exchange of genes among individuals disperses those memories, thereby influencing the survival of all organisms that make up the community of life (GMO - shifting the nodes w/n the web of life).

Source: Walker C., 2005: Epigenetics vs. Genetic Determinism - An Interview with Stuart Newman; Wild Duck Review, Vol.V, (2) Lipton B. (2005). Biology of Belief. Elite Books p.45 http://en.wikipedia.org/wiki/GMO_food IntroFood Epigenome Method Implication

Processing (8b/9)

Food Alteration - GMO:

Genetically Modified Organisms (GMO) …. • are illegitimate recombinations (DNA-scrambling): unspecific DNA-insertion (distinguishes GM from conventional methods) - not analogous to products of normal evolution; • horizontal gene transfer: GM-DNA fragments cross species boundaries; • ‘Growthfactor-like’ effects in the stomach and intestinal lining of young rats fed on GM-potato for just 10 days; • Horizontal transfer of GM-DNA to bacteria in the soil and into human gut has been found; • GM DNA of Lactobacillus in heat-treated fermented sausage could still be detected after 9 months of storage; I-SIS 2003 19-03-18 Madl 76

Making a GM plant or animal involves breaking and joining the DNA of the host genome at many unspecifiable locations, a process referred to as "illegitimate recombination", which distinguishes GM from all conventional methods. This process leads to substantial scrambling of both foreign and host DNA at the sites of integration. …. Topping the list of the direct evidence of hazard inherent to the technology is the study of Pusztai and co‐workers, who found dramatic ‘growthfactor-like’ effects in the stomach and intestinal lining of young rats fed GM potato for just 10 days, which were not present either in rats fed non‐GM potatoes or in rats fed non‐GM potatoes spiked with the transgene product.

Source: http://www.i-sis.org.uk/GMSRDF.php THE GM SCIENCE REVIEW PANEL (2003) FIRST REPORT. An open review of the science relevant to GM crops and food based on interests and concerns of the public (very controversial). Pusztai A, Bardocz S, Ewen SWB (2003) Genetically Mofified Foods: Potential Human Health Effects. In: d’Mello JPF, (ed) Food Safety: Contaminants and Toxins. CAB International Straub AJ, Hertel C, Hammes P W (1999). The fate of recommbinat DNA in thermally treated fermented sausages. Eur. Food research.Techno.210: 62-67 IntroFood Epigenome Method Implication

Processing (8d/9)

–1:33:55) 00 32: –1:40:25) (1:08:38 –1:09:20)0 n :0 GMO-paper -Monsanto (1:06:38 –1:08:40)38 :35 –1:47:00) Mais (1: 45 1: Journal icks (1: GMO-invasio Transgenicdirty tr Promo-clip (1:40:57 –1:41:36) undup-toxicity ( o R

Heritage, 2004

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For example, tinkering with the genes of a tomato may not stop at that tomato, but could alter the entire biosphere in ways that we cannot foresee. Already there is a study that shows that when humans digest genetically modified foods, the artificially created genes transfer into and alter the character of the beneficial bacteria in the intestine [Heritage 2004; Netherwood, et al, 2004]. Similarly, gene transfer among genetically engineered agricultural crops and surrounding native species has given rise to highly resistant species deemed superweeds (for epigenetic modifications see Caulerpa taxifolia) [Milius 2003; Haygood, et al, 2003; Desplanque, et al, 2002; Spencer and Snow 2001]

Image: possible route for transfer of DNA from plant cells in the human diet to intestinal bacteria. Some DNA in food is degraded during cooking and processing, but the remainder is ingested intact. Consumed DNA is largely hydrolyzed during digestion. Netherwood et al. provide evidence that intact transgenic DNA can be recovered in the human ileum and taken up by bacteria in this environment.

Source: Lipton B. (2005). Biology of Belief. Elite Books p.45 Heritage, J. (2004). The fate of transgenes in the human gut. Nature Biotechnology 22, 170-172. Netherwood T., Martín-OrÚe S.M., O'Donnell A.G., Gockling S., Graham J., Mathers J.C., Gilbert H.J. (2004) Assessing the survival of transgenic plant DNA in the human gastrointestinal tract. Nature Biotechnology 22, 204-209. IntroFood Epigenome Method Implication

Processing (8e/9)

Recommendations for the FDA. 1)delay implementation of decision to permit use of GM-herbicides. 2)urgently assess toxicology of glyphosate (formulated & mixtures) and other herbicides. 3)label GM foods (GM crops are agro-products most heavily treated with herbicides some of the latter pose serious cancer-inducing risks). Landrigan & Benbrook, 2015 19-03-18 Madl 78

We offer two recommendations. First, we believe the EPA should delay implementation of its decision to permit use of Enlist Duo. This decision was made in haste. It was based on poorly designed and outdated studies and on an incomplete assessment of human exposure and environmental effects. It would have benefited from deeper consideration of independently funded studies published in the peer-reviewed literature. And it preceded the recent IARC determinations on glyphosate and 2,4-D. Second, the National Toxicology Program should urgently assess the toxicology of pure glyphosate, formulated glyphosate, and mixtures of glyphosate and other herbicides. Finally, we believe the time has come to revisit the United States’ reluctance to label GM foods. Labeling will deliver multiple benefits. It is essential for tracking emergence of novel food allergies and assessing effects of chemical herbicides applied to GM crops. It would respect the wishes of a growing number of consumers who insist they have a right to know what foods they are buying and how they were produced. And the argument that there is nothing new about genetic rearrangement misses the point that GM crops are now the agricultural products most heavily treated with herbicides and that two of these herbicides may pose risks of cancer. We hope, in light of this new information, that the FDA will reconsider labeling of GM foods and couple it with adequately funded, long-term post-marketing surveillance.

Source: Landrigan PJ, Benbrook C (2015) GMOs, Herbicides, and Public Health. N Eengl J Med Vol.373: 693-695 IntroFood Epigenome Method Implication

Processing (9/9)

Ultra-processed food & Cancer risk:

• Cat-I processed: single additives (sugar or salt, etc.) • Cat-II ultra-processed: beverages (-”-, colorants, texturizing agents), canned food (seasoned, marinated, flavoured, hydrogenated, modified starches, etc.)

Cat-II-foods: increased cancer risk, especially • breast, by ? a la • prostate, & b l or mu fo r • colorectal cancer; s fo on t ti an • 10 % share of Cat-II foods increases risk by 10% ! ca st li in mp - Imp ds Thibault et al., 2018 oo 19-03-18 Madl f 79

This study assesses the prospective associations between consumption of ultra-processed food and risk of cancer based on a cohort study. Some 104 980 participants aged at least 18 years (median age 42.8 years) from the French NutriNet-Santé cohort (2009-17). Dietary intakes were collected using repeated 24 hour dietary records, designed to register participants’ usual consumption for 3300 different food items. These were categorised according to their degree of processing by the NOVA classification. Associations between ultra-processed food intake and risk of overall, breast, prostate, and colorectal cancer assessed by multivariable Cox proportional hazard models adjusted for known risk factors. The results showed that ultra-processed food intake was associated with higher overall cancer risk (n=2228 cases; hazard ratio for a 10% increment in the proportion of ultra-processed food in the diet 1.12 (95% confidence interval 1.06 to 1.18); P for trend <0.001) and breast cancer risk (n=739 cases; hazard ratio 1.11 (1.02 to 1.22); P for trend = 0.02). These results remained statistically significant after adjustment for several markers of the nutritional quality of the diet (lipid, sodium, and carbohydrate intakes and/or a Western pattern derived by principal component analysis). In this large prospective study, a 10% increase in the proportion of ultra-processed foods in the diet was associated with a significant increase of greater than 10% in risks of overall and breast cancer. Rapidly increasing consumption of ultra-processed foods may drive an increasing burden of cancer and other non-communicable diseases. Thus, policy actions targeting product reformulation, taxation, and marketing restrictions on ultra-processed products and promotion of fresh or minimally processed foods may contribute to primary cancer prevention.

Image: Relative contribution of each food group to ultra-processed food consumption in diet

Source: Thibault F, Srour B, Sellem L, Kesse-Guyot E, Allès B, Méjean C, Deschasaux M, Fassier P, Latino-Martel P, Beslay M, Hercberg S, Lavalette C, Monteiro CA, Julia C, Touvier M (2018) Consumption of ultra-processed foods and cancer risk: results from NutriNet-Santé prospective cohort BMJ 2018; 360 :k322; doi: https://doi.org/10.1136/bmj.k322 https://soundcloud.com/bmjpodcasts/ultraprocessed-food-and-increased-cancer-risk weekly science column, arte-journal from 17.02.2018 IntroFood Epigenome Method Implication

Aluminium (1/10)

21st century - the Aluminium Age:

• was biologically non-available to the biochemical evolution until 20th century • Al enters geochemical cycling - is deliberately introduced into biopshere • Al is not required for cellular metabolism • Al is toxic to life Exley, 2013

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Human activities have circumvented the efficient geochemical cycling of aluminium within the lithosphere and therewith opened a door, which was previously only ajar, onto the biotic cycle to instigate and promote the accumulation of aluminium in biota and especially humans. Neither these relatively recent activities nor the entry of aluminium into the living cycle are showing any signs of abating and it is thus now imperative that we understand as fully as possible how humans are exposed to aluminium and the future consequences of a burgeoning exposure and body burden. The aluminium age is upon us and there is now an urgent need to understand how to live safely and effectively with aluminium.

Image: Aluminium's exposome. A schematic which explores relationships between exposure, immediate targets mediating exposure, sinks and sources of biologically available aluminium with putative mechanisms of action and finally excretion of aluminium.

Source: Exley C. (2013). Human exposure to aluminium. Environ. Sci. Processes Impacts, Vol.15:1807-1816.

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Aluminium (2/10)

Al-compounds as food supplements: Walton, 2012 • 300 BCE, romans coloured wine with it (!) • Nowadays Al-sulfates used to teat water (> levels of monomeric Al3+) • Basic variants form precipitates; deprotonated (

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Aluminum (Al) is a recognized neurotoxin causally linked to several neurodegenerative diseases with a dementia component. Al has long had a GRAS (Generally Recognized As Safe) rating by the US FDA, that allows Al salts to be used in food manufacture and clarification of urban drinking water supplies. Routine ingestion of Al salts throughout life causes bioavailable Al to accumulate in the brain where it specifically deposits in regions most vulnerable in Alzheimer’s disease (AD). The main source of Al exposure for humans is their total dietary Al intake from foods, water, other beverages, and Al additives. Animal studies provide a parallel in that their feed and water intakes can be rigorously controlled throughout the life span. Such a study revealed that a significant proportion of rats, particularly those that consumed Al in an equivalent amount to the high end of the human total dietary Al range, progressively accumulated Al in their brain, accompanied by AD-related neuropathology and cognitive deterioration in old age.

Inlet: The concentrations of soluble, mononuclear Al ion species, present in aqueous solutions, change as the pH is raised or lowered. Ionic or free Al3+ is an inorganic monomer and is regarded as the most bioavailable and toxic Al-species.

Source: Walton JR (2012). Evidence that Ingested Aluminum Additives Contained in Processed Foods and Alum-Treated Drinking Water are a Major Risk Factor for Alzheimer’s Disease. Current Inorganic Chemistry, Vol. 2: 000-000

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Aluminium (2/10)

Al-compounds as food supplements: Walton, 2012 • 300 BCE, romans coloured wine with it (!) • Nowadays Al-sulfates used to teat water (> levels of monomeric Al3+) • Basic variants form precipitates; deprotonated (

19-03-18 Madl 82

Aluminum (Al) is a recognized neurotoxin causally linked to several neurodegenerative diseases with a dementia component. Al has long had a GRAS (Generally Recognized As Safe) rating by the US FDA, that allows Al salts to be used in food manufacture and clarification of urban drinking water supplies. Routine ingestion of Al salts throughout life causes bioavailable Al to accumulate in the brain where it specifically deposits in regions most vulnerable in Alzheimer’s disease (AD). The main source of Al exposure for humans is their total dietary Al intake from foods, water, other beverages, and Al additives. Animal studies provide a parallel in that their feed and water intakes can be rigorously controlled throughout the life span. Such a study revealed that a significant proportion of rats, particularly those that consumed Al in an equivalent amount to the high end of the human total dietary Al range, progressively accumulated Al in their brain, accompanied by AD- related neuropathology and cognitive deterioration in old age.

• Aluminium chlorohydrate-salt: AlnCl(3n-m)(OH)m is used in deodorants and antiperspirants and as a coagulant in water purification.

• Aluminium hydroxide, Al(OH)3, − • Hydroxoaluminate: Al(OH)4 anion is known in high pH solutions of Al(OH)3 Image:Use of Al-salts in processed foods and drinking water; Other uses: to strengthen dough; swell pastas; bleach flour and cheeses. Al is also included as an ingredient in reconstituted vegetable protein, as a stabilizer, thickener, acidifying agent, buffer, neutralizer and texturizer [15]. Calcium supplements, especially those based on oyster shells, provide about 12 mgAl/day .

Source: Walton JR (2012). Evidence that Ingested Aluminum Additives Contained in Processed Foods and Alum-Treated Drinking Water are a Major Risk Factor for Alzheimer’s Disease. Current Inorganic Chemistry, Vol. 2: 000-000

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Aluminium (3/10)

Epigenetics of Al-exposure : • Heterochromatin: genetically inactive • Al inhibits eukaryotic gene transcription • Al acts as transcriptional repressor (of A+T-rich templates) • Found across species AT, Arabidopsis thaliana (cress); LU, Linum usitatissimum (flax); CE, Caenorhabditis elegans (nematode); MS, Mus musculus (mouse); HS, Homo sapiens (human); (L) liver; (B) brain. Lukiw, 2010

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Averaging 8.1% (w/w) of the earth's crust, aluminum is the most highly abundant metal in our biosphere, yet has long been thought to serve no essential biological function. In aqueous solutions, aluminum salts and hydroxides are exceptionally potent aggregators of biological molecules, often coalescing molecular species to the point that they precipitate out of solution. A biological function for aluminum is proposed in which this abundant, high charge density metal cation has a significant role in biomolecular compaction. Sometimes, molecules ectopically aggregated by aluminum are associated with pathological conditions. The data further suggests that a specific consequence of ‚aluminum biocompaction‘ may be particularly important in the condensation of A + T-rich chromatin domains, and in silencing the expression of specific kinds of genetic information.

Image: Aluminum content and genetic activity are inversely correlated. Correlation of interphase heterochromatin status and aluminum content of chromatin in select eukaryotic nuclei. Eukaryotic nuclei contain complex arrangements of heterochromatic and euchromatic domains: ‘inactive, constitutive‘ heterochromatin is associated with the absence of gene expression. Mean constitutive heterochromatin was assayed for A+T-rich heterochromatic regions, and heterochromatin was expressed as a percent of total nuclear area; aluminum content, expressed as μg/g total chromatin.

Source: Lukiw WJ (2010). Evidence supporting a biological role for aluminum in chromatin compaction and epigenetics. Journal of Inorganic Biochemistry Vol.104: 1010-1012.

83 IntroFood Epigenome Method Implication

Aluminium (4a/10)

Average personal Al-exposure :

pregnant women suffer often from gastroesophageal reflux: • Al-hydroxide is also often used as antiacids (heartburn, gastro-esophageal reflux disease) • Al-hydroxide is a known neurotoxin & contraindicated in pregnancy

Wikipedia 2014

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Antacids either directly neutralize acidity, increasing the pH, or reversibly reduce or block the secretion of acid by gastric cells to reduce acidity in the stomach. When gastric hydrochloric acid reaches the nerves in the gastrointestinal mucosa, they signal pain to the central nervous system. This happens when these nerves are exposed. In addition to the reduction of gastric acidity, antacids also alter the profile of prostaglandins produced by gastroduodenal mucosa and this may promote mucosal healing and be related to its therapeutic effect Antacids are taken by mouth to relieve heartburn, the major symptom of gastroesophageal reflux disease, or acid indigestion. Treatment with antacids alone is symptomatic and only justified for minor symptoms. The treatment of ulcers resulting from excessive acidity may require H2-receptor antagonists or proton pump inhibitors, and eradication of H. pylori. Aluminum hydroxide: may lead to the formation of insoluble aluminium- phosphate-complexes, with a risk for hypophosphatemia and osteomalacia. Although aluminium has a low gastrointestinal absorption, accumulation may occur mainly in the presence of renal insufficiency. Aluminium-containing drugs often cause constipation and are neurotoxic. Aluminium-containing drugs are contraindicated in pregnancy.

Image: Wyeth amphojel tablets of aluminum hydroxide.

Source: http://en.wikipedia.org/wiki/Antacid

84 IntroFood Epigenome Method Implication

Aluminium (4b/10)

Average personal Al-exposure :

• Inhalation: 1.4 µg/day • Ingestion: 1-20 mg/day • Epidermal: 2 g/day Franz-cell exposure system to test transmigration of Aluminium chlorohydrate (ACH) via dermal layer

Pineau et al., 2012

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Aluminum salts such as aluminum chlorohydrate (ACH) are known for use as an active antiperspirant agent that blocks the secretion of sweat. A local case report of hyperaluminemia in a woman using an aluminum-containing antiperspirant for 4 years raises the problem of transdermal absorption of aluminum (Al). Only a very limited number of studies have shown that the skin is an effective barrier to transdermal uptake of Al. In accordance with our analytical procedure, the aim of this study with an in vitro Franz™ diffusion cell was to measure aluminum uptake from three cosmetic formulations of antiperspirant: the base for an "aerosol" (38.5% of ACH), a "roll-on" emulsion (14.5% ACH), and a "stick" (21.2%), by samples of intact and stripped human skin (5 donors). The Al assays were performed by Zeeman Electrothermal Atomic Absorption Spectrophotometry (ZEAAS). Following contacts lasting 6, 12 and 24h, the Al assays showed only insignificant transdermal absorption of Al (≤0.07% of the quantity of Al deposited) and particularly low cutaneous quantities that varied according to the formulations (1.8 μg/cm² for "aerosol base" and "stick" - 0.5 μg/cm² for the "roll-on"). On stripped skin, for which only the "stick" formulation was tested, the measured uptake was significantly higher (11.50 μg/cm² versus 1.81 μg/cm² for normal skin). These results offer reassurance as regards to the use of antiperspirants for topical application of ACH- containing cosmetic formulations on healthy skin over a limited time span (24h). On the other hand, high transdermal Al uptake on stripped skin should compel antiperspirant manufacturers to proceed with the utmost caution. Image: Franz™ diffusion cell (static type) to test transmigration of Aluminium chlorohydrate-salt:

AlnCl(3n-m)(OH)m across skin.

Source: Pineau A, Guillard O, Favreau F, Marrauld A, Fauconneau B. (2012) In vitro study of percutaneous absorption of aluminum from antiperspirants through human skin in the Franz™ diffusion cell. J. o. Inorg. Biochem., Vol.110: 21-26.

85 IntroFood Epigenome Method Implication

Aluminium (4c/10)

Average personal Al-exposure :

• Inhalation: 1.4 µg/day • Ingestion: 1-20 mg/day • Epidermal: 2 g/day

Exley, 2013

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While the evidence to-date is that only a very small proportion of aluminium in topically-applied antiperspirant enters the bloodstream to ultimately be excreted via the kidney this observation does not preclude the persistence of such aluminium within the structures of the skin and neither does it preclude the entry of this aluminium into the lymphatic system. The nature of the aluminium compounds which are present in topical applications, the amount of aluminium which is often applied and the regularity of many such applications must mean that skin is a significant sink for aluminium and a persistent source of biologically available aluminium both locally and systemically.

Image: The skin is a sink for topically applied aluminium and will act as a source of biologically reactive aluminium both to structures within the skin and to the systemic circulation.

Source: Exley C. (2013). Human exposure to aluminium. Environ. Sci. Processes Impacts, Vol.15:1807-1816.

86 IntroFood Epigenome Method Implication

Aluminium (5/10)

Hormone Mimicry induced by Xenobiotics via: • high concentrations of a chemical w/ low binding affinity to oestrogen receptors (OR) • Cocktail of several compounds w/ low affinity to OR - acting synergistically!

Dabre & Fernandez, 2013

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Although diet, alcohol, radiation and inherited loss of BRCA1/2 genes have all been associated with increased incidence, the main identified risk factors are life exposure to hormones including physiological variations associated with puberty/pregnancy/menopause, personal choice of use of hormonal contraceptives and/or hormone replacement therapy. On this basis, exposure of the human breast to the many environmental pollutant chemicals capable of mimicking or interfering with oestrogen action should also be of concern. Hundreds of such environmental chemicals have now been measured in human breast tissue from a range of dietary and domestic exposure sources including persistent organo- chlorine pollutants (POPs), polybromi- nated diphenylethers and polybromobiphenyls, polychlorinated biphenyls, dioxins, alkyl phenols, bisphenol- A and chlorinated derivatives, as well as other less lipophilic compounds such as parabens (alkyl esters of p-hydroxybenzoic acid), but studies investigating any association between raised levels of such compounds and the development of breast cancer remain inconclusive.

Image: The many ways in which different environmental chemicals may induce an oestrogenic stimulus to the human breast through a receptor-mediated mechanism. Colour gradient from black to grey to white indicates a decreasing contribution to oestrogenic load. Compounds with higher relative binding affinity (RBA) for the oestrogen receptor only need to be present at lower concentrations.

Source: Darbre PD, Fernandez MF (2013). Environmental oestrogens and breast cancer: long-term low-dose effects of mixtures of various chemical combinations. J Epidemiol Community Health March Vol.67(3): 203-205.

87 IntroFood Epigenome Method Implication

Aluminium (6/10)

Hormone Mimicry induced by Xenobiotics via: • high concentrations of a chemical w/ low binding affinity to oestrogen receptors (OR) • Cocktail of several compounds w/ low affinity to OR - acting synergistically!

Dabre, 2011

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Although epidemiological studies conclude that the majority of breast cancers are environmental in origin, the main underlying environmental causes remain to be identified. Loss of function of the deoxyribonucleic acid (DNA) repair tumor suppressor genes breast cancer susceptibility gene 1/2 (BRCA1/2), exposure to excess prolactin, smoking, diet, alcohol, and radiation have all been identified as risk factors, but lifetime exposure to estrogen remains the main influence in the development of breast cancer. Epidemiological studies have linked estrogen exposure to breast cancer incidence through studies of the effects of age of me- narche/menopause, nulliparity, age of first pregnancy, practices of breastfeeding, and use of the oral contra- ceptive pill or hormone replacement therapy (HRT). However, the role of estrogen is also well documented in studies of the growth of tumors in animals and of human breast cancer cells in culture, and the established in- volvement of estrogen in clinical studies has formed the basis for the successful use of endocrine therapy as a targeted approach to treatment for breast cancer.

Image: Chemical components of personal care products that have been shown to possess estrogenic activity using assay systems in vitro and in vivo. Estrogenic chemicals are grouped according to their function in the product. Insert: The molecular basis of estrogen action. Physiological estrogen or estrogen- mimicking compound (E) enters a cell and binds to an intracellular receptor protein (R), resulting in the release of receptor-interacting proteins (RIP). The estrogen-receptor complexes dimerize and bind to specific nucleotide sequences in the DNA in the vicinity of estrogen-regulated genes. RNA polymerase binds to the DNA (POL), as do several other coactivators of transcription (CO), and this results in regulation of gene expression.

Source: Darbre PD (2011). Personal Care Products and Breast Cancer. Elsevier Science, UK

88 IntroFood Epigenome Method Implication

Aluminium (7a/10)

Al-compounds in nipple aspirate found to induce breast tumors, as Al is: • is a metalloestrogen (enabling oestrogenic effects) • transmigrates across cells • known to interfere with DNA (micronucleus asssays) • dermal absorption (e.g. via anti-transpirants); •Ac • cumulates in biological fluid (nipple aspirate)

Darbre et al. 2011

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The human breast is exposed to aluminium from many sources including diet and personal care products, but dermal application of aluminium-based antiperspirant salts provides a local long-term source of exposure. Recent measurements have shown that aluminium is present in both tissue and fat of the human breast but at levels which vary both between breasts and between tissue samples from the same breast. We have recently found increased levels of aluminium in noninvasively collected nipple aspirate fluids taken from breast cancer patients (mean 268 +/-28 μg/l) compared with control healthy subjects (mean 131 +/-10 μg/l) providing evidence of raised aluminium levels in the breast microenvironment when cancer is present. The measurement of higher levels of aluminium in type I human breast cyst fluids (median 150 μg/l) compared with human serum (median 6 μg/l) or human milk (median 25 μg/l) warrants further investigation into any possible role of aluminium in development of this benign breast disease. Emerging evidence for aluminium in several breast structures now requires biomarkers of aluminium action in order to ascertain whether the presence of aluminium has any biological impact. To this end, we report raised levels of proteins that modulate iron homeostasis (ferritin, transferrin) in parallel with raised aluminium in nipple aspirate fluids in vivo, and we report overexpression of mRNA for several S100 calcium binding proteins following long-term exposure of MCF-7 human breast cancer cells in vitro to aluminium chlorhydrate. It is possible that if levels of iron binding proteins are found to correlate with raised aluminium in larger repeat studies, that measurement of ferritin and/or transferrin might provide useful biomarkers in human breast tissue for the existing link between aluminium and iron suggested as pathogenetically impor- tant for human neurodegenerative disorders. Image: Mean levels of aluminium, ferritin and transferrin in nipple aspirate fluid samples obtained from women with (Cancer) or without breast cancer and as compared to levels in blood serum or breast milk. Source: Darbre PD, Pugazhendhi D, Mannello F. (2011) Aluminium and human breast diseases. Journal of Inorganic Biochemistry Vol.105:1484-1488

89 IntroFood Epigenome Method Implication

Aluminium (7b/10)

approx. 10 μL Al-compounds in nipple aspirate found to induce breast tumors, as Al is: • is a metalloestrogen (enabling oestrogenic effects) • transmigrates across cells • known to interfere with DNA (micronucleus asssays) • dermal absorption (e.g. via anti-transpirants); • accumulates in biological fluid (nipple aspirate)

Egartner et al. 2013

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Source: Ehgartner B, Roth C., Ripper T., Freingruber A., Hohl T. (2013) – die Akte Aluminium. ARTE, ORF, SRF

90 IntroFood Epigenome Method Implication

Aluminium (7c/10)

Al-compounds in nipple aspirate found to induce GCBD: • GCBD: Gross cystic breast disease - most common benign breast disorder, (7% of Western women) • antiperspirant formulations are designed to block apocrine sweat ducts of the axilla

Fluid obtained from human sweat glands • apocrine type I • transudative type II

Manello et al. 2009

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Gross cystic breast disease (GCBD) is the most common benign breast disorder, but the molecular basis of cyst formation remains to be identified. If the use of aluminium-based antiperspirant salts is involved in the etiology of gross breast cyst formation, it might be expected that aluminium would be at elevated levels in human breast cyst fluid (BCF). Aluminium was measured by ICP-MS in 48 samples of BCF, 30 samples of human blood serum and 45 samples of human breast milk at different stages of lactation (colostrum, intermediate, mature). The median level of aluminium in apocrine type I BCF (n = 27, 150 microg l(-1)) was significantly higher than in transudative type II BCF (n = 21, 32 microg l(-1); P < 0.0001). By comparison, aluminium measurements gave a median concentration of 6 microg l(-1) in human serum and 25 microg l(-1) in human breast milk, with no difference between colostrum, intermediate and mature milk. Levels of aluminium were significantly higher in both types of BCF than in human serum (P < 0.0001). However when compared with human breast milk, aluminium levels were only significantly higher in apocrine type I BCF (P < 0.0001) and not in transudative type II BCF (P = 0.152). It remains to be identified why such high levels of aluminium were found in the apocrine type I BCF and from where the aluminium originated. However, if aluminium-based antiperspirants are found to be the source and to play any causal role in development of breast cysts, then it might become possible to prevent this common breast disorder.

Image: Median levels of aluminium in human breast gross cyst fluids according to breast gross cyst subtypes (*P < 0.0001).

Source: Mannello F, Tonti GA, Darbre PD. (2009). Concentration of aluminium in breast cyst fluids collected from women affected by gross cystic breast disease. J Appl Toxicol. Vol.29(1):1-6.

91 IntroFood Epigenome Method Implication

Aluminium (8/10)

Al-induced Neurodegenerative Diseases:

Al & Blood-Brain-Barrier (BBB) • Dialysis Encephalopathy Syndrome (DES) • Amyotrophic Lateral Sclerosis (ALS) • Parkinsonism Dementia (PD) • Alzheimer‘s Disease (AD)

Exley & House, 2011 Szutowicz A, 2002

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The brain must expend energy in its ‘unconscious‘ response to an exposure to biologically available aluminium. There are many examples where ‚biological effect‘ has resulted in aluminium-induced neurotoxicity and most potently in conditions that have resulted in an aluminium-associated encephalopathy. However, since aluminium is non-essential and not required by the brain, its biological availability will only rarely achieve such levels of acuity, and it is more pertinent to consider and investigate the brain‘s response to much lower though sustained levels of biologically reactive aluminium. This is the level of exposure that defines the putative role of aluminium in chronic neurodegenerative disease and, though thoroughly investigated in numerous animal models, the chronic toxicity of aluminium has yet to be addressed experimentally in humans. The simplest way reduce the Al-burden is by facilitating the urinary excretion of aluminium through the regular drinking of a silicic acid-rich mineral water over an extended time period. This will lower the body and brain burden of aluminium, and by doing so will test whether brain aluminium contributes significantly to chronic neurodegenerative diseases such as Alzheimer‘s and Parkinson‘s. AD: The main clinical feature of AD is a slowly-developing, relentless dementia characterized by memory deficit, confusion, inability to focus attention, incontinence, and per- severative behaviors. Neuropathological hallmarks of AD include cerebral NFTs, amyloid plaques, and granulo- vacuolar degeneration. Many studies have shown that NFTs correlate better with disease severity than amyloid plaques. DES: Al increase in neural cells stimulates Ca2+ uptake, leading to seizures and death ALS: is characterized by progressive motor weakness affecting the limbs and premature death. PD: dementia is a severe and progressive dementia accompanied by extrapyramidal disease. ALS and parkinsonism dementia often occur together in the same individuals of these populations. Spinal neurons of ALS/PD-affected Guamanians contain NFTs rich in Al, silicon and Ca2+. Granulovacuolar degeneration (GVD) occurs in the ALS/PD cortex, substantia nigra, and other subcortical nuclear. There is evidence that Al induces both NFT formation and GVD, which are also prominent in AD. Image: A schematic of the possible distribution of aluminium in plasma, the blood- brain barrier (BBB), the cerebrospinal fluid (CSF), the brain interstitial fluid (BIF), and the cellular and pathological compartments of the human brain. Source: Exley C, House ER (2011). Aluminium in the human brain. Monatsh Chem Vol.142: 357-363 Walton JR (2012). Evidence that Ingested Aluminum Additives Contained in Processed Foods and Alum- Treated Drinking Water are a Major Risk Factor for Alzheimer’s Disease. Current Inorganic Chemistry, Vol. 2: 000-000 Szutowics A (2002) Aluminium Neurotoxicity. In: Masaro EJ (ed) Handbook of Neurotoxicology, Humana Press, Totowa (NJ), USA

92 IntroFood Epigenome Method Implication

Aluminium (9/10)

Al-compounds & Alzheimer's Disease (AD): Walton, 2012 • dietary patterns are akin to a grand-scale experiment whereby some individuals are consuming large quantities of Al • selective accumulation over lifetime selective w/n neurons of the brain • Al shows affinity to specific neuronal transporter proteins • small amounts needed to promote AD

Tomljenovic, 2011

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The brain is a highly compartmentalized organ exceptionally susceptible to accumulation of metabolic errors. Alzheimer disease (AD) is the most prevalent neurodegenerative disease of the elderly and is characterized by regional specificity of neural aberrations associated with higher cognitive functions. Aluminum (Al) is the most abundant neurotoxic metal on earth, widely bioavailable to humans and repeatedly shown to accumulate in AD-susceptible neuronal foci. In spite of this, the role of Al in AD has been heavily disputed based on the following claims: 1) bioavailable Al cannot enter the brain in sufficient amounts to cause damage, 2) excess Al is efficiently excreted from the body, and 3) Al accumulation in neurons is a consequence rather than a cause of neuronal loss. Research, however, reveals that: 1) very small amounts of Al are needed to produce neurotoxicity and this criterion is satisfied through dietary Al intake, 2) Al sequesters different transport mechanisms to actively traverse brain barriers, 3) incremental acquisition of small amounts of Al over a lifetime favors its selective accumulation in brain tissues, and 4) since 1911, experimental evidence has repeatedly demonstrated that chronic Al intoxication reproduces neuropathological hallmarks of AD. Misconceptions about Al bioavailability may have misled scientists regarding the significance of Al in the pathogenesis of AD. The hypothesis that Al significantly contributes to AD is built upon very solid experimental evidence and should not be dismissed. Immediate steps should be taken to lessen human exposure to Al, which may be the single most aggravating and avoidable factor related to AD. Image: Major routes of Al transport in and out of the brain: from the blood, Al enters the brain extracellular fluid (ECF) primarily through the blood brain barrier (BBB) via transferrin-mediated uptake. Some Al in the brain is rapidly effluxed as Al-citrate by the MCT-transporter. A significant portion of Al is retained in the cellular compartments (nucleus, ER, bound to ATP or membrane phospholipids). Inlet: Staging of aged rat (upper row) and aged human (lower row) of hippocampal CA1neurons stained; Stage 0: the entire cell appears Al-negative and has normal morphology; Stage I: magentanucleolus, no other staining for Al. Stage II: magenta nucleolus in pink nucleoplasm with visible chromatin; the cytoplasm is blue. Stage III: magenta nucleolus in an elongated or irregularly shaped purple nucleus.The cytoplasm is blue. Many apical dendrites from this stage onwards have a serpentine appearance. Stage IV: the magenta staining appears in the elongated nucleus which now shows less structural detail; the shrunken cytoplasm is still blue. Stage V: purple to magenta staining appears throughout the nucleus and cytoplasm. Cell shape is distorted and the axon and dendrites are disrupted. Source: Tomljenovic L. (2011). Aluminum and Alzheimer‘s Disease: After a Century of Controversy, Is there a Plausible Link? Journal of Alzheimer‘s Disease Vol.23: 567-598. Walton JR (2012). Cognitive Deterioration and Associated Pathology Induced by Chronic Low-Level Aluminum Ingestion in a Translational Rat Model Provides an Explanation of Alzheimer‘s Disease, Tests for Susceptibility and Avenues for Treatment. Journal of Alzheimer‘s Disease Vol.194947: 1-17. Walton JR (2006). Aluminum in hippocampal neurons from humans with Alzheimer“s disease. NeuroToxicology Vol.27:385-394 93 IntroFood Epigenome Method Implication

Aluminium (10a/10)

Vaccine Production Indication Al content Producer (trade marks) technique declared Berna Inflexal V inactivated virus anti-flu No Biotech inactivated, split Sanofi Pasteur Vaxigrip anti-flu No Al-compounds in Vaccines virion MSD Novartis contains Anatetall anti-tetanus Yes Vaccines and anatoxin Diagnostics Srl recombinant Baxter AG Tetabulin anti-tetanus No subunits diphtheria, GSK • Al(OH)3 used to induce Infanrix contains tetanus, Spa (DTPa) Yes immune response anatoxin acellular paediatric pertussis, Stamaril attenuated 17 D GSK • present in most vaccines yellow fever No Pasteur strain Spa Vi GSK such as Engerix, Hevac, Thypherix Salmonella typhi No polysaccharide Spa Tetavax, Tetracoq, Havrix, measles, GSK Priorix attenuated mumps, rubella, No Spa Twinrix, etc. varicella recombinant GSK Engerix-B subunits hepatitis B Yes Spa Gatti & Montanari, 2015 GSK Varilrix attenuated varicella No Spa Novartis contains diphtheria, Dif-Tet-All Yes Vaccines and anatoxin tetanus Diagnostics Srl Novartis meningococcal Menjugate kit conjugate Yes Vaccines and group C Diagnostics Srl Novartis Focetria inactivated H1N1-flu No Vaccines and Diagnostics Srl Merck & Co. Gardasil recombinant cervical cancer Yes 18-Mar-19 Madl 94Inc.

Few drugs are the object of controversies and disputes as much as vaccines, including the actual validity of the premarket trials that particularly the new ones undergo; the safety of the chemicals added; the appropriateness of their qualitatively-different, concentrated administration to newborn babies and to soldiers; their real efficacy; their side effects; the information released by producers and those printed in the leaflets enclosed in the package; and the reliability of the medical literature in most cases sponsored by producers are just a few of the points generally discussed and, unfortunately, unscientific opinions and lobby interests too often overwhelm objectivity …. It is our duty to declare that the first 19 of a total of 27 …. vaccines were analyzed …. All 27 presented some anomalies in their composition, as we found particulate matter that should not be present in a drug destined to be injected in the human body and definitely not in a newborn baby. The quantity of each vaccine we analyzed was 20 µL deposited on a cellulose filter …. If, given the presence of non- biodegradable particles, the formation of a common local reaction (e.g., a granuloma) at the vaccine inoculation site, a reaction often seen in animals, can be understandable, but correlating the administration with systemic adverse effects including neurological ones may need some not-yet- available or, at least, not-scientifically indisputable explanations.

Image: List of some of the vaccines analyzed with their indications (right). Infanrix (a) shows the presence of debris of (b) titanium-silicon-sulfur immersed in an environment of aluminium diluted in saline solution (sodium-chlorine). Not shown samples: Anatetall show 2-micron-sized debris of aluminium-silicon-sulfur immersed in a saline solution (sodium-chlorine) and iron-chromium-nickel- silicon sulfur with aluminium in saline. Inflexal shows a 4-micron-sized particle of copper-lead-zinc- tin-phosphorus identified in the Inflexal vaccine. Stamaril of a 1.8-micron-sized debris. It contains chromium-silicon-copper-iron-magnesium-sulfur-potassium-calcium. Thypherix shows spherical nanoparticles of tungsten-calcium in a saline solution. Gardasil reveals micro-sized particulate matter composed of aluminium-copper-iron and nanosized ones composed of lead-bismuth.

Source: Gatti AM, Montanari S (2015) Food, Drugs and Nanoparticles, Ch.8. In: Gatti & Montanari (eds) Case Studies in Nanotoxicology and Particle Toxicology. Academic Press/Elsevier, London, UK

94 IntroFood Epigenome Method Implication

Aluminium (10b/10)

Al-compounds & Macrophagic Myofascitis (MMF):

• Al(OH)3 used to induce immune response • Present in most vaccines such as Engerix, Hevac, Tetavax, Tetracoq, Havrix, Twinrix, etc.

Gherardi er al., 2001

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Macrophagic myofasciitis (MMF) is an emerging condition of unknown cause, detected in patients with diffuse arthromyalgias and fatigue, and characterized by muscle infiltration by granular periodic acid-Schiff's reagent-positive macrophages and lymphocytes. Intracytoplasmic inclusions have been observed in macrophages of some patients. To assess their significance, electron microscopy was performed in 40 consecutive cases and chemical analysis was done by microanalysis and atomic absorption spectrometry. Inclusions were constantly detected and corresponded to aluminium hydroxide, an immunostimulatory compound frequently used as a vaccine adjuvant. A lymphocytic component was constantly observed in MMF lesions. Serological tests were compatible with exposure to aluminium hydroxide-containing vaccines.

Image: Deltoid muscle biopsy (A) tighly packed macrophages intermingled with lymphocytes (x100); (B) adjacent section of the same showing immuno-localization of macrophage marker CD68 (x100); (C) adjacent section of the same showing immuno- localization of T-cell marker CD3 (x100); (D) nodular aggregation of lymphocytes with microvascular neo-angio-genesis (x70); (E) HLA antigen expression in macrophages & muscle fibers; (F) lzmphocytes stuck to a granular macrophage (x600). Insert: Microanalytic Spectral Analysis showing an abnormal presence of Al in muscle tissue strictly restricted to the area of macrophage infiltration (53 mg Al/g MF)

Source: Gherardi RK, Coquet M, Cherin P, Belec L, Moretto P, Dreyfus PA, Pellissier JF, Chariot P, Authier FJ.(2001). Macrophagic myofasciitis lesions assess long-term persistence of vaccine-derived aluminium hydroxide in muscle. Brain, Vol.124(Pt 9): 1821-1831.

95 IntroFood Epigenome Method Implication

Aluminium (10c/10)

Vaccine Adjuvant Hepatitis-B (HepB) booster Al-salt + thiomersal Diphtheria-Tetanus-Pertussis (DTaP) Al-salt + thiomersal Al-compounds & Autoimmune- DTaP-booster Al-salt + thiomersal + formol DTaP/HepB/Hib (Haemophil Flu-B) Al-salt + phenoxyethanol Inflammatory Syndrome (ASIA): Influenza vaccine Thiomersal + NaCl + KCl + MgCl Influenza H1N1 Squalene Influenza H1N1 + H3N2 Thiomersal + NaCl + KCl + MgCl • Al(OH)3 used to induce Inactivated Polio vaccine (IPV) MgCl Pneumococ. Conjug. vac. (PCV) S23 Thiomersal + phenol immune response Measles-Mumps-Rubella (MMR) vac. Neomycin + sorbitol + albumin Measles/Rubella (MR) booster Neomycin + sorbitol + albumin • present in most vaccines Bacillus Calmette-Guérin (BCG) ?

such as Engerix, Hevac, Thiomersal organo-Hg); Neomycin (broadband antibiotic)

Tetavax, Tetracoq, Havrix, Vaccine AEFI Moderate Severe P Twinrix, etc. HepB booster 23 14 9 1.00 DTaP 1 1 0 1.00 DTaP booster 4 1 1 0.14 • triggering ASIA DTaP + IPV + HepB 8 7 1 0.11 Flu vaccine 5 4 1 0.64 Flu H1N1 7 4 3 1.00 Cerpa-Cruz et al., 2013 Flu H1N1 + H3N2 12 7 5 0.49 IPV vaccine 6 3 3 0.66 PCV S23 vaccine 3 1 2 0.55 MMR 2 0 2 0.13 MR booster 30 19 11 0.59 BCG 6 5 1 0.39 Data pool: 120 patients subject to AEFI (adverse event following immunization); 18-Mar-19 Madl 96

A traditional infectious disease vaccine is a preparation of live attenuated, inactivated or killed pathogen that stimulates immunity. Vaccine immunologic adjuvants are compounds incorporated into vaccines to enhance immunogenicity. Adjuvants have recently been implicated in the new syndrome named ASIA autoimmune/inflammatory syndrome induced by adjuvants. The objective describes the frequencies of post-vaccination clinical syndrome induced by adjuvants. We performed a cross-sectional study; adverse event following immunization was defined as any untoward medical occurrence that follows immunization 54 days prior to the event. Data on vaccinations and other risk factors were obtained from daily epidemiologic surveillance. Descriptive statistics were done using means and standard deviation, and odds ratio adjusted for potential confounding variables was calculated with SPSS 17 software. Forty-three out of 120 patients with moderate or severe manifestations following immunization were hospitalized from 2008 to 2011. All patients fulfilled at least 2 major and 1 minor criteria suggested by Shoenfeld and Agmon– Levin for ASIA diagnosis. The most frequent clinical findings were pyrexia 68 %, arthralgias 47 %, cutaneous disorders 33 %, muscle weakness 16 % and myalgias 14 %. Three patients had diagnosis of Guillain–Barre syndrome, one patient had Adult-Still’s disease 3 days after vaccination. A total of 76 % of the events occurred in the first 3 days post-vaccination. Two patients with previous autoimmune disease showed severe adverse reactions with the reactivation of their illness. Minor local reactions were present in 49 % of patients. Vaccines containing adjuvants may be associated with an increased risk of autoimmune/inflammatory adverse events following immunization

Tables: Vaccines and adjuvants (top), Vaccines and severity of AEFI (bottom)

Source: Cerpa-Cruz S, Paredes-Casillas P, Navarro EL, Bernard-Medina AG, Martinez-Bonilla G, Gutierrez- Urena S (2013) Adverse events following immunization with vaccines containing adjuvants. Immunol Res (2013) 56:299–303

96 IntroFood Epigenome Method Implication

Aluminium (10d/10)

Effectiveness of Vaccination

• Evidence is weak 1853 smallpox vaccination act 1867 stringent laws passed

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The Evidence is weak that Vaccines have saved more lives than they have cost

Image: Insert: Inverse Relation between the Incidence of Prototypical Infectious Diseases (Panel A) and the Incidence of Immune Disorders (Panel B) from 1950 to 2000.[1]

Source: UK-Statistics (1997) Record of mortality in England and Wales for 95 years. Office of National Statistics, UK Cornewall Bart GCL (1860) Her Majesty’s Principle Secretary of State of the Home Department Pearce CT (1899) Essay on Vaccination. Parliament Paper of the 62nd Return of the Registrar General 181-1989 Member of the Royal Collage of Surgeons of England [2] UK Public Health (2017) Historical vaccine development and introduction of routine vaccine programmes in the UK; https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/650485/Vaccinatio n_timeline.pdf http://truthsift.com/graph_view?gid=520 http://www.dissolvingillusions.com/graphs/ https://korenwellness.com/blog/never-vaccinate-child/

97 IntroFood Epigenome Method Implication

Aluminium (10e/10)

Effectiveness of Vaccination

• Evidence is weak i) aging society i) shift in disease weighting - prevalence towards chronic diseases

…. for which satisfying treatments are not yet available ….

Jones et al., 2012 Bach, 2002

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At first glance, the inaugural 1812 issue of the New England Journal of Medicine and Surgery, and the Collateral Branches of Science seems reassuringly familiar: a review of angina pectoris, articles on infant diarrhea and burns. The apparent similarity to today’s Journal, however, obscures a fundamental discontinuity. Disease has changed since 1812. People have different diseases, doctors hold different ideas about those diseases, and diseases carry different meanings in society. To understand the material and conceptual transformations of disease over the past 200 years, one must explore the incontrovertibly social nature of disease .... Just as organisms evolve to keep up with changing environmental conditions (the “Red Queen Effect”), medicine struggles to keep up with the changing burden of disease. Since therapeutic innovation takes time, the burden shifts even as solutions appear. By the time antibiotics and vaccines began combating infectious diseases, mortality had shifted toward heart disease, cancer, and stroke. Great progress has been made to meet these challenges, but the burden of disease will surely shift again. We already face an increasing burden of neuropsychiatric disease for which satisfying treatments do not yet exist.[1]

Image: Top 10 Causes of Death: 1900 vs. 2010. Data are from the Centers for Disease Control and Prevention.[1] / Insert: Inverse Relation between the Incidence of Prototypical Insert: Infectious Diseases (Panel A) and the Incidence of Immune Disorders (Panel B) from 1950 to 2000.[2]

Source: [1] Jones DS, Podolsky SH, Greene JA (2012) The Burden of Disease and the Changing Task of Medicine. N. Engl J Med Vol.366: 2333-2338 [2] Bach JF (2002) The Effect of Infections on Susceptibility to Autoimmune and Allergic Diseases. N Engl J Med, Vol.347(12): 911-920

98 IntroFood Epigenome Method Implication

Aluminium (10f/10)

Effectiveness of Vaccination

• Evidence is weak i) aging society i) shift in disease weighting - prevalence towards chronic diseases • surge in antimicrobial resistance

• …. for which satisfying treatments are not yet available …. Cassini et al., 2018

18-Mar-19 Madl 99

Infections due to antibiotic-resistant bacteria are threatening modern health care. However, estimating their incidence, complications, and attributable mortality is challenging. We aimed to estimate the burden of infections caused by antibiotic- resistant bacteria of public health concern in countries of the EU and European Economic Area (EEA) in 2015, measured in number of cases, attributable deaths, and disability-adjusted life-years (DALYs). From EARS-Net data [2] collected between Jan 1, 2015, and Dec 31, 2015, we estimated 671 689 (95% uncertainty interval [UI] 583148–763966) infections with antibiotic-resistant bacteria, of which 63.5% (426277 of 671689) were associated with health care. These infections accounted for an estimated 33110 (28480–38430) attributable deaths and 874541 (768837– 989068) DALYs. The burden for the EU and EEA was highest in infants (aged <1 year) and people aged 65 years or older, had increased since 2007, and was highest in Italy and Greece.

Our results present the health burden of five types of infection with antibiotic-resistant bacteria expressed, for the first time, in DALYs. The estimated burden of infections with antibiotic-resistant bacteria in the EU and EEA is substantial compared with that of other infectious diseases, and has increased since 2007. Our burden estimates provide useful information for public health decision-makers prioritising interventions for infectious diseases.

Image: Burden of infections with antibiotic-resistant bacteria in DALYs, EU and European Economic Area, 2015 Error bars are 95% uncertainty intervals. Greece did not report data on S pneumoniae isolates to the European Antimicrobial Resistance Surveillance Network in 2015. DALY rates are age-standardised to limit the effect of demographic differences across countries; numbers of cases and deaths are not age-standardised. DALYs=disability-adjusted life-years. *Excludes those resistant to carbapenem or colistin. †In 2015, most of the third-generation cephalosporin-resistant E coli (88·6%) and K pneumoniae (85·3%) isolates reported to the European Antimicrobial Resistance Surveillance Network produced an extended-spectrum β-lactamase. Table: Table 2: Estimated annual burden of infections with selected antibiotic-resistant bacteria of public health importance, age-group standardised, EU and European Economic Area, 2007–15; Data are median (95% uncertainty interval) and are age-standardised. Note that only bacteria under surveillance in both 2007 and 2015 are included in this analysis. *Excluding isolates resistant to colistin or carbapenems. †In 2015, most of the third- generation cephalosporin-resistant E coli (88·6%) and K pneumoniae (85·3%) isolates reported to EARS-Net produced an extended-spectrum β-lactamase.9 ‡Resistance to three or more antibiotic groups as marker of multidrug resistance. §Excluding isolates resistant to macrolides. ¶Excluding isolates resistant to penicillins, but not to macrolides.

Source: [1] Cassini A, Högberg LD, Plachouras D, Quattrocchi A, Hoxha A, Simonsen GS, Colomb-Cotinat M, Kretzschmar ME, Devleesschauwer B, Cecchini M, Ouakrim DA, Oliveira TC, Struelens MJ, Suetens MJ, Monnet DL, AMR (2018) Attributable deaths and disability-adjusted life-years caused by infections with antibiotic-resistant bacteria in the EU and the European Economic Area in 2015: a population-level modelling analysis. The Lancet Infectious Diseases, Vol.0(0): xxx; DOI: 10.1016/S1473-3099(18)30605-4 [2] https://ecdc.europa.eu/en/about-us/networks/disease-networks-and-laboratory-networks/ears-net-data

99 IntroFood Epigenome Method Implication

Microtox (1a/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc.

Immediate effects of ocean plastics dumping:

– serve as rafts for species over long distances (introduction of invasive species into new biomes)

– marine life starved or strangled to death (ingested plastics mistaken for eatable invertebrates)

– polymers degrade into monomers (release of toxic additives, e.g. plasticizers) Winston et al, 1997

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As unlikely as it may seem, transport of terrestrial organisms (plants, invertebrates, and vertebrates) possibly has a greater probability of significant biological impact …. Insects, snails, isopods, millipedes, and plants can survive transport, for example, on rafts of vegetation or logs or both …. Because of its smooth, nonporous surface, plastic is not as well suited to their survival as natural vegetation, but the report of viable seeds being transported in a plastic toy boat …. strongly suggests the possibility that insects or other hard-shelled invertebrates could be passively carried in the same manner …. Rafting long distances may be extremely unlikely for vertebrates (e.g., lizards, rats) because the very large masses of trash noted in some urban harbors are unlikely to stay together in stormy seas. The greatest potential for impact is local, Le., the introduction of pests like rats from populated coasts to nearby islands where attempts are being made to preserve an endangered native biota …. It is conceivable that islands of high conservation value and meriting heritage or scientific reserve status, with biotas supposedly protected from disturbance, could unknowingly be placed at risk. Events of this kind could lead to the introduction of exotic and aggressive alien taxa, thus eroding or rapidly destroying years of environmental effort and progress. It is a danger of which those who manage or have stewardship over protected coastal commons should remain cognizant.

Image: Artist's conception of rafts of plastic debris as a dispersal agent for marine and terrestrial organisms.

Source: Winston JE, Gregory MR, Stevens LM (1997) Encrusters, epibionts, and other biota associated with pelagic plastics: a review of biogeographical, environmental, and conservation issues. In: Coe JM, Rogers DB (eds) Ch.7. Marine debris: sources, impacts, and solution. Springer (NY), USA

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Microtox (4/6)

Microplasts in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc.

both of global …. • plastics introduced i/o oceans (5 garbage patches) • runoff via ricers i/o oceans • storm-water drains routed i/o seas

…. & much more of local concern • 48 kt (FRG) 6.8 kt (AT) annually rubber wear & tear

• polyacrylate as H2O-buffer in agriculture • polystyren in gardening to increase aeration Plastic bags mistaken for • sewage used as fertilizer containing MPs fom jelly fish in turtle stomach cosmetic care products (1.5 kt) Sea of Shame.mp4 Neubauer, 2018

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There are increasing signs that our soils are even more polluted than the seas. How these tiny plastic particles affect terrestrial ecosystems has yet to be investigated …. When washing synthetic clothing, synthetic fibres dislodge into the wastewater. Clothing abrasion as well as plastics from peelings and shower gels collect in the sewage sludge and end up in the fields. Traffic also produces a lot of microplastic; the Austrian Federal Environment Agency reports the annual tire abrasion in AT of almost 6.8 kt tons, around 40% of which is plastic. In Germany, rubber abrasion is estimated at over 43 kt/year.... Some plastics are even deliberately introduced into the soil: swellable plastic granules of polyacrylates - the same material that binds moisture in diapers - are mixed into soils in hot regions as water storage. Similar plastics that absorb a lot of moisture are added to sewage sludge and fermentation residues to drain them. This makes them easier to handle as fertilizer. Styrofoam balls are a popular loosening agent for soil, especially for greenhouses, indoor and pot plants. They also released into the environment when they blow away or are disposed of withered potted plants in compost or in the garden. Now plastics are not toxic per se. However, many contain harmful additives such as plasticizers, flame retardants and heavy metals. In addition, plastic remains in the environment for decades, if not centuries. Even some of the plastics described as degradable often only decompose into smaller fragments instead of rotting completely. However, such fragmentation cannot be equated with biological degradation.[1] Image: Through the Gyre - It’s more than plastic bags and empty bottles.[3] Widespread contamination of plastic debris reported in the scientific literature. The current and growing scientific evidence reports widespread contamination of plastic debris among many aquatic habitats and animals, including 50% of all species of seabirds, 66% of all species of marine mammals, and 100% of all species of sea turtles.[4] Source: [1] Neubauer U (2018) Auch unsere Böden sind voller Mikroplastic; Wissenschaft & Forschung, NZZ; https://www.nzz.ch/wissenschaft/auch-unsere-boeden-sind-voller-mikroplastic-ld.1380551 [2] Nine Network 60 Minutes, Sea of Shame; https://www.youtube.com/watch?v=cxzhrZ4JkDY [3] ec.europa.eu/environment/marine/pdf/through_gyre.pdf [4] Rochman CM, Cook AM, Koelmans AA (2016) Plastic debris and policy: Using current scientific understanding to invoke positive change Environ Tox & Chem, Vol.35(7): 1617-1626 101 IntroFood Epigenome Method Implication

Microtox (1b/6)

Microplasts (MP) in our Environment as fibers, fragments & microbeads Composed of poly-ethylene, -propylene, - Microplastics: <5 mm styrene, -ester, -acrylates, nylon, etc. Nanoplastics: <0.1 μm

Size Description Primary MP: intentionally made at micro-level 5 mm Upper size limit of microplastics 2.5 mm Size of a flea – Microbeads in personal care products 330 μm Lower size limit of neuston nets – Media for air-blasting machinery or boat hulls 200 μm Microplastic fragments in a facial scrub – Pre-production plastics (nurdles) 100 μm Thickness of a sheet of paper 7 μm Diameter of red blood cells Secondary MP: degradation of polymers via 5 μm Microplastic particles in toothpaste – Photolytic 1 μm Width of anthrax bacterium Into monomers + release of – Mechanical 100 nm Upper size limit of nanoplastics toxic additives 20 nm Diameter of small viruses – Biological 2 nm Diameter of DNA Clothes washing: shedding of synthetic fibers 1 nm Diameter of carbon nanotube (single-walled)

Pachkowski, 2016

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Microbeads as a Source of Microplastics (MPs) • Used in personal care products (e.g., facial cleansers, soaps, toothpaste) as exfoliants or for aesthetics; typically polyethylene; • Enter wastewater treatment plants and either retained in sewage sludge or introduced into environment via effluent; • Estimated 19 tons of microbeads enter wastewater treatment plants in the state of New York each year; [2] • Estimated 8 billion microbeads enter aquatic environments each day in US; [3]

Source: [1] Pachkowski B (2016) Microplastics as Contaminants of Emerging Concern. New Jersey Department of Environmental Protection Division of Science, Research and Environmental Health [2] Report from NY State Attorney General’s Office (Nalbone, 2014); [3] Rochman CM, Kross SM, Armstrong JB, Bogan MT, Darling ES, Green SJ, Smyth AR, Veríssimo D (2015). Scientific evidence supports a ban on microbeads. Environ. Sci. Technol. Vol.49(29), 10759–10761.

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Microtox (2/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc.

Cosmetics & detergents …. • MPs in personal care products (filling materials & coating agents to protect against heat stress of hair drier, producing a "silky" skin effect) • MPs in detergants and washing powders (delayed release of contained perfumes)

Lahrtz, 2019

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One shower with some shower gel and a hair wash with a bit of shampoo releases some 100·E3 tiny plastic particles flow into the drain. On the other hand, the load of MPs sticking to the skin is carried around all day long (So far, nothing like this has been found in certified natural cosmetics). Standard shower gels and body care creams form a very thin film rendering the skin pleasantly silky. The effect is owed to the acrylate co- or cross-polymers. In such a shampoo, the plastic film wraps the individual hair as a film and thus makes combing easier. The plastic film also protects against the heat of the hairdryer. Other plastics such as polyethylene or nylon polymers increase the viscosity in lipsticks or mascara. Even most detergents contain MPs, as mini containers for fragrances, as fillers to make gels supple, or to achieve an emery effect. The MPs released from cosmetics float via wastewater into the sewage treatment plant. There - at least in most European plants - up to 98 % of the MPs are retained in the sewage sludge. Unlike in Switzerland, however, in many countries, including Germany, the sludge (and with it some 50 % of the retained MPs) is reintroduced as fertilizer or used as a building material into the environment. In this way, more than 50 % of sewage sludge throughout Europe ends up in the environment.

Source: Lahrtz S (2019) Viele Pflegeprodukte enthalten Mikroplastik – doch das sollte vermieden werden. NZZ https://www.bund.net/fileadmin/user_upload_bund/publikationen/meere/meere_mikroplastik_einkaufsfuehrer.p df https://www.umsicht.fraunhofer.de/content/dam/umsicht/de/dokumente/publikationen/2018/kunststoffe-id- umwelt-konsortialstudie-mikroplastik.pdf https://blog.energiedienst.de/mikroplastik/

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Microtox (3a/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc.

Global MP (<5 mm) concentrations reported by aquatic and sedimentary environment.

Hurley et al, 2018 Tekman et al., 2019

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Global- and catchment-scale patterns of MP-contamination. By compiling the first global MP-map and ranking for marine and freshwater environments, we show that the concentration of microplastics in river bed sediments of the Mersey catchment is higher than in any other environment - either deposited or in suspension - so far reported. The most contaminated site (~517k particles m−2) is on the River Tame (146 km2), a few kilometres upstream of its confluence with the Etherow, which is 50% higher than the maximum value for the next ranked site of the Incheon-Kyeonggi beach sediments in South Korea. The maximum value for the Irwell, on the River Tonge (96.5 km2), is 191k particles m−2.[1]

Image: Global MP (<5 mm) concentrations reported by aquatic and sedimentary environment. MP’s are mapped as maximum concentrations (top) and ranked by average concentration (bottom). Note that in the absence of standardized reporting of MP-data, this compilation includes data from studies employing a number of methods. Concentration refers to both the actual and surface concentration. Interactive platform: Litter and microplastic distribution taken from publications with most commonly used units [items/km²; items/km; items/m³] can be used to filter data. If possible, other dimensions were standardised to these units to allow comparison. [2]

Source: [1] Hurley R, Woodward J, Rothwell JJ. (2018) Microplastic contamination of river beds significantly reduced by catchment-wide flooding. Nature Geoscience Vol.11: 251-257 [2] Tekman MB, Gutow L, Macario A, Haas A, Walter A, Bergmann M (2019): Alfred Wegener Institute Helmholtz Centre for Polar and Marine Research - https://litterbase.awi.de/litter

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Microtox (3a/6)

Microplasts (MP) in our Environment Concentration factor estimated to be: ~1·E5 to 1·E6 Composed of poly-ethylene, -propylene, - 16 styrene, -ester, -acrylates, nylon, etc.

Concentration of beached plastic resin pellet of Persistent Organic Pollutants (POPs) •Polycyclic aromatic Hydrocarbons (PAHs) •Polychlorinated biphenyl (PCBs) •Bisphenol-A (BPA) •Dichlor-diphenyl-trichlorethan (DDT) •Hopanes

Kwan & Takada, 2019 Yamashita et al., 2019

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Additives and monomers can leach out from plastics (polymers). BPA, NP, and PBDEs are widely detected in landfill leachates. Although the concentrations in the leachates may be considered low as they are usually in the range of less than 1 mg/L, the concentration levels sometimes exceed water quality standards, e.g., for surface waters that have been set for the protection and preservation of aquatic life …. Plastic products contain various additives to maintain their performances …. and include plasticizers, UV absorbers, antioxidants, anti-static agents, flame retardants and so on. One example regards the concentrations of NP in caps of mineral water and beverage polyethylene tere-phthalate (PET) bottles (which does not contain NPs). NP concentrations were up to 600 ng/g. NP or its derivatives are thought to be compounded as antioxidants and/or anti-static agents.

Image: Under the influence of abiotic stress exposure (UV-radiation, saltwater, wave action, microbial mobilisation, etc). contained POPs are leak out into the environment. In the case of ocean water these rather hydrophobic chemicals sooner or later adsorb on surfaces likewise hydrophobic such as MPs. The observed magnification factor of POPs on MPs can be as high as 1·E6-fold !

Source: [1] Kwan CS, Takada H (2019) Release of Additives and Monomers from Plastic Wastes. https://meetings.pices.int/publications/presentations/PICES-2014/2014-S8/S8-1115-Takada.pdf [2] Yamashita R, Tanaka K, Yeo BG, Takada H, van Franeker JA, Dalton M, Dale E (2019) Hazardous Chemicals in Plastics in Marine Environments: International Pellet Watch. In: Takada H, Karapanagioti HK (eds) Hazardous Chemicals Associated with Plastics in the Marine Environment. The Handbook of Environmental Chemistry, Springer, Chan (CH).

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Microtox (3a/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc.

Concentration of beached plastic resin pellet of Persistent Organic Pollutants (POPs) •Polycyclic aromatic Hydrocarbons (PAHs) •Polychlorinated biphenyl (PCBs) •Bisphenol A (BPA) •Dichlor-diphenyl-trichlorethan (DDT) •Hopanes

Yamashita et al., 2019 Takada et al., 2019

Plastik- der Fluch der Meere.mp4

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Plastics carry various hazardous chemicals in marine environments …. The chemicals include polychlorinated biphenyls (PCBs), dichloro-diphenyl- trichloro-ethane and its metabolites (DDTs), polycyclic aromatic hydrocarbons (PAHs), poly-brominated di-phenyl ethers (PBDEs), nonyl-phenols (NP), octyl- phenol (OP) and bis-phenol-A (BPA). Their concentrations ranged from 1 to 10,000 ng/g. They are derived from two origins. One group of chemicals consists of additives such as BPA that were originally compounded into plastic products. Another group of chemicals consists of hydrophobic compounds such as PCBs and DDTs that were sorbed from surrounding seawater. Some chemicals such as PBDEs and NP come from both origins..

Image: Median concentrations of PCBs, PHAs, DDTs, HCHs and Hopanes in plastic resin pellets found on beaches across the globe.[1]

Source: [1] Takada H () Global Monitoring of Persistent Organic Pollutants (POPs) using Beached Plastic Resin Pellets: http://www.pelletwatch.org/ [2] Yamashita R, Tanaka K, Yeo BG, Takada H, van Franeker JA, Dalton M, Dale E (2019) Hazardous Chemicals in Plastics in Marine Environments: International Pellet Watch. Both in: Takada H, Karapanagioti HK (eds) Hazardous Chemicals Associated with Plastics in the Marine Environment. The Handbook of Environmental Chemistry, Springer, Chan (CH). Moench M, Nottenbacher F (2012) Plastik - Fluch der Mere. Arte.TV: 23:30-27:35

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Microtox (3a/6)

Microplasts (MP) in our Environment rafting taxa (incl. <5% of global plastic enter microbes) Composed of poly-ethylene, -propylene, - ocean waters annually styrene, -ester, -acrylates, nylon, etc.

• microbes-accelerated weathering (monomers + Degredation of MPs into monomers & additives); • influencing zoo-plankton- toxic additives and biomagnification fish interaction; • MPs adsorb of toxins • Elution / transfer of Mincer et al., 2019 sorbed toxins to macro- MPs grazers altering gut mediated microbiome; exposure • Microbial coating - altered gravitational settling; • Abyssal MPs enriched Biomagnified not Biomagnified sediments, influencing e.g. psammon community

PCBs vs. PBDEs

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Microbial inhabitants on plastic marine debris (PMD) are now known to fluctuate depending upon season, geographic location, substrate, and age. Cursory calculations estimate a range of 1-15 kt of microbial biomass harbored on PMD …. Drifter buoys and physical oceanographic models have shown that plastic can migrate over 1 Mm, from the nutrient rich coastal waters of the Eastern Seaboard of the USA to the oligotrophic interior of the North Atlantic Subtropical Gyre (NASG), in less than 60 days and a bit longer for the North Pacific Subtropical Gyre (NPSG) …. It is currently unclear if Plastisphere microbes directly degrade plastic resins or sorbed POPs; however, it is clear that heterotrophic bacteria and fungi and autotrophic microalgae in the marine environment have the ability to generate highly reactive oxygen species known to oxidize refractive carbon compounds.[1] Plastic-mediated exposure could be insignificant. In remote areas where background pollution is low, plastics bring higher concentrations of chemicals, plastic-mediated exposure could be significant. Also, plastics could be a significant pathway of additives that are not biomagnified in higher-trophic-level organisms. The lipophilic nature of digestive fluid is key to facilitate the leaching and, consequently, the transfer of hydrophobic chemicals from ingested plastics to the tissues of the organisms.[2]

Image: Plastic resin and its multiple fates in the marine environment: significant plastic debris inputs from land can be transported directly out to open ocean gyres where they could persist for decades. Microbial biofilms rapidly attach to plastic debris and possibly influence weathering of PMD, sorptive properties, and its interactions with indigenous biota.[1] Models of chemical exposure in seabirds via ingested plastics and food web.[2]

Source: [1] Mincer TJ, Zettler ER, Amaral-Zettler LA (2019) Biofilms on Plastic Debris and their Influence on Marine Nutrient Cycling, Productivity, and Hazardous Chemical Mobility. Tanaka K, Yamashita R, Takada H (2019) Transfer of Hazardous Chemicals from Ingested Plastics to Higher-Trophic-Level Organisms. Both in: Takada H, Karapanagioti HK (eds) Hazardous Chemicals Associated with Plastics in the Marine Environment. The Handbook of Environmental Chemistry, Springer, Chan (CH). 107 IntroFood Epigenome Method Implication

Microtox (5a/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc. μm-sized MPs in 20 μm polystyrene MPs Bio-accumulation & -magnification supernatant of marine salt in copepod – MP: S:V-ratio serve as vectors for chemicals – release of PCBs, PAH, PBA, pesticides, heavy metals etc.; biohazard: microbial & viral associations found on MPs – via ocean currents MPs could serve as vehicles 20 μm MPs in Pachkowski, 2016 Rat liver Rat kidney .

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MPs have been found throughout the world, primarily in aquatic environments. Because of their size and ability to adsorb and release chemicals, MPs may have ecological impacts. MPs have been found in species (e.g., shellfish) consumed by humans raising the possibility for human exposure and health effects. Oral exposure is likely to be the main route of human exposure …. Bivalves (found in farm-raised mussels and oysters; potential human exposure to 11·E3 MPs pieces per year; Finfish (found in gastrointestinal tracts; possibility for translocation across the gut, into circulation, and embedded into edible tissue; Lab experiments demonstrate MPs in liver; Table salt (sea salt harvesting) [1] Image top: Particles in the supernatant of marine salt solutions.[4] Center: 20 μm diameter fluorescent polystyrene MPs internalized by the pigmented calanoid copepod Temora longicornis; Insert: following enzymatic digestion the biological tissue was mineralized, releasing the fluorescent MPs , which were then retained on a glass fibre filter.[2] Bottom: Polarized-light microscopy images (x100) showing the presence of plastic particles in liver of exposed fishes. Black arrow indicate MP particles inside the liver tissue. Particles in the supernatant of marine salt solutions.[3] Accumulation of different sizes of MPs in mice tissues after exposure for 28 days.[5]

Source: [1] Pachkowski B (2016) Microplastics as Contaminants of Emerging Concern. New Jersey Department of Environmental Protection Division of Science, Research and Environmental Health [2] Cole M, Webb H, Lindeque PK, Fileman ES, Halsband C, Galloway TS (2014) Isolation of microplastics in biota-rich seawater samples and marine organisms. Scientific Reports 4:4528 [3] Avio CG, Gorbi S, Regoli F (2015) Experimental development of a new protocol for extraction and characterization of microplastics in fish tissues: First observations in commercial species from Adriatic Sea. Marine Environmental Research, Vol.111: 18-26. [4] Yang et al. (2015) Microplastic Pollution in Table Salts rom China. Environ Sci Technol, Vol.49(22):13622-13627, [5] Deng YF, Zhang Y, Lemos B, Ren HQ (2017) Tissue accumulation of microplastics in mice and biomarker responses suggest widespread health risks of exposure. Scientific Reports Vol.7: 46687

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Microtox (6a/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc. Stool sample analysed w/ FTIR micro-spectroscopy

Schwabl et al., 2018

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Methods (exclusion criteria): Medically set on diet; Diarrhoea or obstipation; Antibiotics taken within the last 2 weeks; Drugs affecting stool frequency and consistency (e.g. loperamide); Drugs affecting resorption (e.g. activated charcoal, cholestyramine); Diagnosed gastrointestinal disease (e.g. Ulcerative colitis, Crohn's disease); Invasive or abrasive dental treatment within the last 2 weeks Data collection: Food protocol 6-7 days prior to stool sampling; Brand name of tooth paste and cosmetic products; Information about chewing gum and alcohol intake; Information about drinking habits from PET bottles; Plastic-free stool sampling and shipping to Vienna;

Image: Stool sample filtrate and placed in petri dish; the FTIR-spectrogram of POM is shown along with a POM-sample obtained from a comercial dealer (insert highlights an areal section of the stool sample with the MP-particle from which the FTIR-scan was taken);

Source: Schwabl P, Liebmann B, Köppel S, Reiberger T, et. al. (2018) Assessment of microplastic concentrations in human stool. UEG-week

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Microtox (6b/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc. Stool sample analysed w/ FTIR micro-spectroscopy

Schwabl et al., 2018

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Descriptive Statistics: 8 participants (3 males, 5 females); aged 33-65 years; no vegetarian; 2 of them daily chewing-gum users; 6 ingested sea-food during the observation period; all of them had contact to plastic-wrapped food; on average, 750 ml/day were drunk from PET bottles Results: Stool weight 34 [8-39] g; all samples tested positive for MP-particles (20 [18-172] particles / 10g stool sample); MP-particle size ranged from 50-500 μm; Plastic types detected: 9 out of 10 types) How representative are these results? Consultation with our Department of Medical Statistics (Prof. Daniela Dunkler): in this study MPs were present in all stool samples (100%). The confidence interval of this finding is 68-100%, when applying it to larger cohorts. Hence, more than 50% of the world population might have MPS in their stool. However, sample size was rather low, thus only larger studies will be able to confirm this assumption. The source terms of these MPs most likely are attributable to food itself Ingestion of sea-food correlated with MP-content (R=0.648; p=0.089) and Food in contact with plastics during Packaging and processing. How representative are these results? MP-translocates from the intestine and particles with sizes up to 130 μm have been detected in the blood stream, lymphatic vessels and the liver of fish and various mammals. MPs may harm via bioaccumulation (causing local immunoreactions) or can serve as a vector for other chemicals. In birds and fish oral plastic caused remodeling of the intestinal villi, distortion of iron absorption and hepatic stress. Especially patients with inflammatory bowel diseases might be vulnerable to MPs.

Image: from the 10 synthetic polymers selected, four are regularly used as wrappers/packaging material in the food industry, whereas the others most likely originate from translocation of degrading materials (wear & tear) that somehow found their way into the food chain. Thus the four main components as used in the food-industry can be found again in the human gut and amout to >95 % if the MP-particles identified in human stool samples.

Source: Schwabl P, Liebmann B, Köppel S, Reiberger T, et. al. (2018) Assessment of microplastic concentrations in human stool. UEG-week 110 IntroFood Epigenome Method Implication

Microtox (6b/6)

Microplasts (MP) in our Environment Composed of poly-ethylene, -propylene, - styrene, -ester, -acrylates, nylon, etc.

Neuro-toxicological effects of Oxasulfuron ECD’s (incl.BPA, PCB‘s Molinate Quinoclamine flame retardants) Cymoxanil Fenpropidin → interfere w/ TSH-receptors Tri-allate → de-myelinization of neurons Mancozeb tau-Fluvalinate Pirimicarb EFSA, 2013 Isoxaflutole Mestari, 2015 Pyridate Jansen et al, 2015

Das Raetsel unserer Intelligenz.mp4 Ferraro, 1937 20:00-30:00

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Endocrine disrupting chemicals affect the production of thyroid hormone, which is needed for the development of the brain (mediates myelinizatio of neurons) – both t young and advanced age. Newborns in particular are affected as they have a very low level of myelinization – formation of the myelin sheets starts only at after few weeks post-partum. The myelin sheet surrounds the axons and ensures that information is passed through the brain faster and more efficiently (coherence).[1,2] EFSA has identified over 100 pesticides that affect hormone production in the thyroid gland, particularly due to BP-A, PCB‘s and flame retardants.[2]. These endocrine disrupting substances block the effectiveness of thyroid hormone in our brain with serious consequences; i.e. exposed mothers give birth to children with a lowered level of intelligence (by 5-6 points) compared to non-exposed mothers and their children.[1] The hypothalamic-pituitarythyroid (HPT) axis maintains circulating levels of thyroid stimulating hormone (TSH) and thyroid hormone (TH) in an inverse relationship. Greater longevity has been associated with higher TSH and lower TH levels, but mechanisms underlying TSH/TH differences and longevity remain unknown. The HPT axis plays a pivotal role in growth, development and energy metabolism …. TSH-receptors are also expressed on bone cells, and other cells, including adipocytes, hepatocytes, skeletal muscle cells, neuronal cells, astrocytes and mesenchymal stem cells.[3]

Image: Macroscopic aspect of an area of concentric demyelinization, shown (a) with low and (b) with high magnification. The center of the focus was an elliptic area, 3 by 1.5 mm. Around this concentric rings, composed of alternating gray and white lamellae, were present. Near the center the rings were about 1 mm. thick, while the peripheral rings were wider.

Source: [1] Mestari A (2015) Notre intelligcne devoilee (Das Raetsel unserer Intelligenz), Arte (Fra). [2] EFSA (2013) Scientific Opinion on the identification of pesticides to be included in cumulative assessment groups on the basis of their toxicological profile, EFSA Journal, Vol.11(7):3293, [3] Jansen SW, Akintola AA, Roelfsema F, van der Spoel E, Cobbaert CM, Ballieux BE, Egri P, Kvarta-Papp Z, Gereben B, Fekete C, Slagboom PE, van der Grond J, Demeneix BA, Pijl H, Westendorp RGJ, van Heemst D (2015) Human longevity is characterised by high thyroid stimulating hormone secretion without altered energy metabolism. Scientific Reports Vol.5, Article number: 11525. [4] Ferraro A (1937) Primary Demyelinating Process of the CNS. Archives of Neurology & Psychiatry, Vol.37(5): 1100-1160

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Nanotox (1/4)

NPs in our Environment The smaller the more toxic

• exposure relevant issued during (i) production & manufacturing (i) during wear & tear (i) after usage, disposal & degradation

• Unique physical and chemical properties; • Interaction with biomolecules; • Contamination can occur during the life cycle;

RSE, (2004)

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Nanoscience and nanotechnologies are widely seen as having huge potential to bring benefits to many areas of research and application …. In June 2003 the UK Government therefore commissioned the Royal Society and the Royal Academy of Engineering to carry out this independent study into current and future developments in nano-science and nanotechnologies and their impacts. The remit of the study was to: · define what is meant by nanoscience and nanotechnologies; · summarise the current state of scientific knowledge about nanotechnologies; · identify the specific applications of the new technologies, in particular where nanotechnologies are already in use; · carry out a forward look to see how the technologies might be used in future, where possible estimating the likely timescales in which the most far-reaching applications of the technologies might become reality; · identify what health and safety, environmental, ethical and societal implications or uncertainties may arise from the use of the technologies, both current and future; and · identify areas where additional regulation needs to be considered.

Image: Some possible exposure routes for nanoparticles and nanotubes based on current and potential future applications. Very little is known about exposure routes for nanoparticles and nanotubes and this figure should be considered with this in mind.

Source: RSE, (2004) Nanoscience and nanotechnologies: opportunities and uncertainties. Nanoscience and nanotechnologies. Royal Society of Engineering, London http://www.nanotec.org.uk/report/Nano%20report%202004%20fin.pdf

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Nanotox (2/4)

NPs in our Environment

By now, exposure to NP is a fact

• in different fields of applications; • variopus types of nanoparticles are used in the different nanoproducts;

e.g. micro-nano agglomerates of Ag- particles in toothpaste or shampoo.

Gatti & Montanari, 2015

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The amount of nanoproducts available on the market is already enormous, and increases every day. It is not always clear in what countries each product is available. Some producers deliberately omit information about the presence of nanoparticles in their products, as there is no worldwide specific regulation requiring the obligatory notification of all nanomaterials intentionally added to a product …. Some companies claim the presence of non-existent nanoparticles, while others are shy about it, fearing a lack of acceptance by customers uncertain about the safety of nanoparticles. In any case, even when nanoparticles are reported correctly in the product’s label, no quantities, size ranges and chemical final formulations in the products are declared …. What is obvious is that if bio-persistent particles enter into the food chain, animals and humans have a high probability to ingest them and their permanence in the body can trigger adverse biological reactions. With increasing awareness regarding the dangers of eating contaminated food, organic food is growing more and more popular. The few organic food samples we analyzed with the aim of searching for particulate contamination did not show any significant differences compared to nonorganic food. We can say the same thing regarding biodynamics. In both cases, the problems involving particulate contamination are ignored either by the law and by the producers. Organic and biodynamic productions are limited to the obligation to observe the steps of certain processes but the controls of the results are not particularly stringent. At least not as far as particulate contamination is concerned …. Once inside the body, due to their tiny size, they crossed the digestive barrier and entered the blood flow …. And the brain is one of the possible targets of those particles.

Image: Nanoproducts identified per product-category (left). Chemical composition of nanoparticles in nanoproducts (right).

Source: Gatti AM, Montanari S (2015) Food, Drugs and Nanoparticles, Ch.8. In: Gatti & Montanari (eds) Case Studies in Nanotoxicology and Particle Toxicology. Academic Press/Elsevier, London, UK

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Nanotox (3/4)

NPs in our Environment

By now, exposure to NP is a fact

• in different fields of applications; • variopus types of nanoparticles are used in the different nanoproducts; e.g. micro-nano agglomerates of Ag- particles in toothpaste or shampoo.

Gatti & Montanari, 2015

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Just as a couple of examples: a NanoUP Toothpaste Au-Ag where gold nano- particles have not been found and a large amount of titanium-rich nano- contaminations has been identified in the NanoCare NanoSilver Beauty Soap, where no silver nano-particles have been found (no shown). Additionally, in most of the samples, the nano-particles intentionally introduced in the nano-products are hardly submicrometric or are even micrometric entities and are not truly nano-sized materials, as nano is bureaucratically understood to refer to size. The above images show the presence of nanosized additives in products. Of course, there is the possibility that some nano-particles were not seen by the FEG-ESEM especially those with a size below 10 nm and if present in isolated form. In many cases we identified them gathered together as aggregates. Small aggregates of 100 nm silver nano-particles have been found in the NanoCare NanoSilver Shampoo. No significant research has ever been carried out regarding the effects of silver nano-particles in repeated and very often long-lasting contacts with the skin.

Image: Low-magnification photograph of NanoCare NanoSilver Toothpaste (a) and EDS spectrum (b): silver micrometric debris have been detected (top). Drop of NanoCare shampoo (c) and EDS spectrum (d) of aggregated silver nanoparticles can be clearly seen (bottom).

Source: Gatti AM, Montanari S (2015) Food, Drugs and Nanoparticles, Ch.8. In: Gatti & Montanari (eds) Case Studies in Nanotoxicology and Particle Toxicology. Academic Press/Elsevier, London, UK

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Nanotox (4/4)

NPs in our Environment

By now, exposure to NP is a fact

• in many processed foods; • nano-Ag to unfold antibacterial properties for water filters, air conditioners, washing machines, paints, toothbrushes; e.g. micro-nano agglomerates of Ag- particles in hamburgers

Gatti & Montanari, 2015

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Nano-silver is more and more frequently used in many products mainly because of its well-known antibacterial properties: garments, air conditioners, washing machines, paints, toothbrushes and in the tap-water filters or the barrier pitchers to kill bacteria in drinking water. Silver has a bacteriostatic and, maybe, even a bactericidal action, but is also a well-known pesticide and so has been investigated by the EPA in the US: “Silver, a naturally-occurring element, is registered for use in water filters to inhibit the growth of bacteria within the filter unit of water filter systems designed to remove objectionable taste, odors, and color from municipally-treated tap water; these bacteriostatic water filters account for over 90% of its pesticidal use”, and ingesting a pesticide is something hard to justify. At present farmers can find commercially available nano-pesticides whose biopersistence in the environment and in agricultural products is mostly unknown, with all the consequences linked, for instance, to microflora and microfauna. Among others, we tested one of those commercially available filters (Brita, US) and found that silver nano-particles had migrated into the water used for drinking.

Image: Image (a) shows a silver aggregate (b) found in an industrial hamburger.

Source: Gatti AM, Montanari S (2015) Food, Drugs and Nanoparticles, Ch.8. In: Gatti & Montanari (eds) Case Studies in Nanotoxicology and Particle Toxicology. Academic Press/Elsevier, London, UK

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Epigenome - Epigenetics

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Epigenetics (1/7)

Memory in planarian flatworms

Thompson & McConnell:

• flatworms conditioned to light;

• using electroshocks to induce learning & • feeding samples of parental flatworm to offspring.

Block & McConnell, 1967

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In 1955, Thompson & McConnell conditioned flatworms by pairing a bright light with an electric shock. After repeating this several times they took away the electric shock, and only exposed them to the bright light. The flatworms would react to the bright light as if they had been shocked (conditioning). They found that if they cut the worm in two, and allowed both worms to regenerate each half would develop the light-shock reaction. In 1962, McConnell extended the experiment by feeding the trained and ground flatworms to other flatworms. Incredibly these flatworms learned to associate the bright light with a shock much faster than flatworms who had not been fed trained worms (epigenetics).[3] p.54: Upon repeating McConnell's experiment it was observed that the reaction to light is significantly faster than other worms fed with particles of non-conditioned flatworms. A clear connection between food and learning (epigenetics) .... With some African tribes the belief is still prevalent today that you have the power of a lion when you eat lion meat .... In this regard Ostertag proposed to regard "living macromolecules" (DNA) as elixirs of life. Complex biopolymers have an extremely high information density (1·E9 times higher than the technical food) - this could be the secret of the "informative" function of food per se .... [1]

Regeneration capabilities of flatworms: Planarian has very simple organ systems. It has small brain and perceptual organs and digestive system which consists of a mouth, pharynx, and an intestine. However, its morphogenesis is somewhat complicated and mysterious. Planaria has highly robust regeneration system. When its part of body is cut, each piece of the part regenerates itself.

Source: [1] Ungar G. (1974): Der molekulare Code des Gedächtnisses. In: Mannheimer Forum. Studienreihe Boehringer Mannheim; 141-192, p.163 [2] Ostertag W. (1981): Lebende Makromoleküle als Lebens-Elixier. Humata Verlag Harold S. Blume, Bern. [3] Block R.A. McConnell J.V. (1967): Classically Conditioned Discrimination in the Planarian, Dugesia dorotocephala. Nature 215, 1465-1466; http://en.wikipedia.org/wiki/Planaria Intro FoodEpigenome Method Implication

Epigenetics (2/7)

A phenotypic variation (paramutation):

• same parents; • same age; • no mutation in pigmentation;

Image: Jirtle, 2007

…. all mice are genetically IDENTICAL ….

…. but they are epigenetically different !

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A study found that an enriched environment can even override genetic mutations in mice[1]. The scientists looked at the effect of dietary supplements on pregnant mice with the abnormal "agouti" gene. Agouti mice have yellow coats and are extremely obese, which predisposes them to cardiovascular disease, diabetes and cancer …. Methyl groups also inactivate remnants of past viral infections, called transposons. 45% of the human genome is made up of parasitic transposons. Methylation is nature's way of allowing environmental factors to tweak gene expression without making permanent mutations, Dr. Jirtle said. Methyl groups are entirely derived from the foods people eat. And the effect may be good or bad. Maternal diet during pregnancy is consequently very important, but in ways that are not yet fully understood.[2] For his experiment Jirtle chose a mouse that happens to have a transposon right next to the gene that codes for coat color. The transposon induces the gene to overproduce a protein that turns the mice pure yellow or mottled yellow and brown. The protein also blocks a feeding control center in the brain. Yellow mice therefore overeat and tend to develop diabetes and cancer. To see if extra methylation would affect the mice, the researchers fed the animals a rich supply of methyl groups in supplements of vitamin B12, folic acid, choline and betaine from sugar beets just before they got pregnant and through the time of weaning their pups. The methyl groups silenced the transposon, which in turn affected the adjacent coat color gene. The babies, born a normal brownish color, had an inherited predisposition to obesity, diabetes and cancer negated by maternal diet.

Source: http://blog.plantpoisonsandrottenstuff.info/category/vitamins/ [1] Waterland R.A. & Jirtle R.L.(2003). "Transposable Elements: Targets for Early Nutritional Effects on Epigenetic Gene Regulation." Molectilar and Cell Biology 23(15): 5293-5300. [2] Randy L. Jirtle & Michael K. Skinner (2007) Environmental epigenomics and disease susceptibility. Nature Reviews Genetics 8, 253-262 Intro FoodEpigenome Method Implication

Epigenetics (3/7)

These transgenic Mice: • yellow pups develop obese, cancer & diabetes; • brown pups are healthy;

Wong, et al. Hum Mol Genet 14:R11, 2005 IJirtle R. 2007

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Gene expression is conditioned by environment: developmental interactions occur from conception till death - Depends on what environments and what sequence the organism encounters them. Epigentic signals can be transmitted to the next generation i.e. display meiotic stability. Not all epigenetic signals are erased and reprogrammed during gametogenesis. This partly explains incomplete penetrance and variable expressivity – not all offsprings are affected (see phenotypic expression of agouti mice mutants).

Example: mouse agouti locus Isogenic Avy/a mice range in colour from yellow to black (pseudoagouti) Darkness proportional to amount of DNA methylation in agouti gene (complete methylation  black). Transplants’ colour influenced by genetic mother not surrogate.

Source: http://www.pbs.org/wgbh/nova/sciencenow/3411/02.html Waterland R.A. & Jirtle R.L.(2003). "Transposable Elements: Targets for Early Nutritional Effects on Epigenetic Gene Regulation." Molectilar and Cell Biology 23(15): 5293-5300. Albert H.C. Wong, Irving I. Gottesman and Arturas Petronis (2005) Phenotypic differences in genetically identical organisms: the epigenetic perspective. Human Molecular Genetics 14:R11- R18 Intro FoodEpigenome Method Implication

Epigenetics (4/7)

These transgenic Mice: • yellow pups develop obese, cancer & diabetes; • brown pups are healthy; • 05:00)

(03:26- eo NOVAd

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Epigenet Jirtle R, 2007

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Agouti mothers received methyl-group-rich supplements …. When methyl groups attach to a gene's DNA, it changes the binding characteristics of regulatory chromosomal proteins. If the proteins bind too tightly to the gene, the protein sleeve cannot be removed and the gene cannot be read. Methylating DNA can silence or modify gene activity …. The mothers who got the methyl group supplements produced standard, lean, brown mice, even though their offspring had the same agouti gene as their mothers. The agouti mothers who didn't get the supplements produced yellow pups, which ate much more than the brown pups. The yellow pups wound up weighing almost twice as much as their lean, "pseudo-agouti" counterparts ….

Source: Lipton B. (2005) Biology of Belief. Elite Books, p.70-71 Waterland R.A. & Jirtle R.L.(2003). "Transposable Elements: Targets for Early Nutritional Effects on Epigenetic Gene Regulation." Molectilar and Cell Biology 23(15): 5293-5300. Video: http://www.pbs.org/wgbh/nova/sciencenow/3411/02.html Intro FoodEpigenome Method Implication

Epigenetics (5/7)

These transgenic Mice: • yellow pups develop obese, cancer & diabetes • brown pups are healthy • cause: maternal diet (!) 06:45)

(05:00- eo NOVAd

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Epigenet Jirtle R, 2007

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Feeding the mother with methyl-enriched food supplements …. one can see that how dramatically the mice shifted the coat color and we get many, many more brown animals And that matters because your coat color is a tracer, is an indicator…. of the fact that you have turned off that gene. This epigenetic fix was also inherited by the next generation of mice, regardless of what their mothers ate. And when an environmental toxin was added to the diet instead of nutrients, more yellow babies were born, doomed to grow fat and sick like their mothers. Basically, what we eat (our mother / our grandparents ate) affects/ed our gene expression!

Source: Video: http://www.pbs.org/wgbh/nova/sciencenow/3411/02.html Waterland R.A. & Jirtle R.L.(2003). "Transposable Elements: Targets for Early Nutritional Effects on Epigenetic Gene Regulation." Molectilar and Cell Biology 23(15): 5293-5300. Intro FoodEpigenome Method Implication

Epigenetics (6/7)

Rats & ECD (Paramutation):

• Epigenetic transgenerational actions of endocrine disruptors through the male germ line;

• Reduced sperm motility – up to the 4th generation (F4);

Skinner, 2006

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Michael Skinner (Washington State University, USA), exposed pregnant rats to toxins that upset their sex hormone balance. Upon investigating how these toxins affected germ cells of male offspring he noted altered DNA methylation in various genes and reduced sperm buoyancy. If the male offspring of these mothers were subsequently crossed with untreated females, the researchers were surprised to discover that both the methylation profile and the reduced sperm mobility were passed on to the fourth generation. The conditions in the womb of the first generation thus shaped the offspring over several generations. As several studies by Emma Whitelaw (University of Sydney, AUS) have shown, epigenetic inheritance is not only restricted to male germ cells, but can also take place via oocytes. Endocrine disruptors have recently been shown to promote an epigenetic transgenerational phenotype involving a number of disease states (e.g. male infertility). The anti-androgenic fungicide vinclozolin was found to act transiently at the time of embryonic sex determination to promote in the F1 generation a spermatogenic cell defect and subfertility in the male. When the animals were allowed to age up to 1 yr, a number of other disease states developed. This phenotype was transferred through the male germ line to all subsequent generations analyzed (F1–F4). The ability of an environmental factor (i.e. endocrine disruptor) to promote an epigenetic transgenerational phenotype impacts the potential hazards of environmental toxins, mechanisms of disease etiology, and evolutionary biology. The biological importance of the epigenetic actions of environmental agents is reviewed in the context of the primordial germ cell and development of epigenetic transgenerational phenotype

Source: Anway D.M. & Skinner M.K. (2006) Epigenetic Transgenerational Actions of Endocrine Disruptors. Endocrinology Vol. 147, No. 6 s43-s49 Intro FoodEpigenome Method Implication

Epigenetics (7a/7)

Emotion – the limbic cell (Bookmarking)…. quality of life in womb bookmarks our susceptibility to diseases later in life; • newborns “download” huge volumes of information for their personal development; •information programmed into the subconscious mind is defined as "truth“ (incl. abuses); •social world hugely influencing expression of genes (determines neuronal connections);

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p.156: The fetal and infant nervous system has vast sensory and learning capabilities and a kind of memory that neuroscientists call implicit memory …. Small creatures with unexpectedly large thoughts" [Chamberlain 1998] …. p.158: "For the growing brain the social world supplies the most important experiences influencing the expression of genes, which determines how neurons connect to one another, which give rise to mental activity" [Siegel 1999] …. p.163: The newborn faces a huge challenge – a new environment to cope with, an extremely stressy situation. Hence, b/w birth and age 6, children “download" an incredible amounts of information they need to thrive in their environment, thereby establishing the neuronal connections b/w the cortex and the limbic system ….They do so by carefully observing their environment and download the worldly wisdom offered by parents directly into their subconscious memory. As a result, their parents' behavior and beliefs become their own. p.164: Once programmed i/o the subconscious mind it becomes defined as "truths" (incl. eating habits, verbal abuses, traumas) that unconsciously shape the behavior and potential of the child through life (reprogramming via hypnotic state?) …. As we get older, we become less susceptible to outside programming (however, under extremely emotional events and in an effort to cope with that challenge, one can relapse into this stage thereby establishing the required connections (emotional) b/w the limbic system & the cortex, see Sacks p.26-27) …. While most of our senses, such as eyes, ears and nose, observe the outer world, consciousness resembles a "sense organ" that behaves like a mirror reflecting back the inner workings of the body's own cellular community; it is an awareness of "self". p.166: The subconscious mind is like a programmable "hard drive" into which our life experiences are downloaded and become hardwired stimulus-response behaviors …. When a stimulus is perceived, it will automatically engage the behavioral response that was learned when the signal was first experienced (later on our sensors are needed only to evoke an emotional response to environmental stimuli). In fact, people who realize the automated nature of this playback response frequently admit to the fact that their "buttons have been pushed" …. Source: Lipton B. (2005) Biology of Belief. Elite Books Sacks O. (2008) Musicophilia – Tales of Music and the Brain, 5th ed.. A.A.Knopf, Toronto - CDN Siegel, D. J. (1999). The Developing Mind: How Relationships and the Brain Interact to Shape Mo We Are. New York, Guilford - USA Chamberlain, D. (1998). The Mind of Your Newborn Baby. N.Atlantic Books. Berkeley – USA Intro FoodEpigenome Method Implication

Epigenetics (7b/7)

Emotion – the limbic cell (Bookmarking) •newborns “download” huge volumes of information for their personal development •information programmed into the subconscious mind is defined as "truth“ (incl. abuses); •social world hugely influencing expression of genes (determines neuronal connections); •generates emotions – via controlled release of regulatory signals (nervous-, endocrine system); •conscious mind reads the flow of cellular signals (which comprises the mind of the body); 19-03-18 Madl 124 p.131: The evolution of the limbic system …. converted the chemical communication signals into sensations that could be experienced by all of the cells in the community. Our conscious mind experiences these signals as emotions. The conscious mind not only "reads" the flow of the cellular coordinating signals that comprise the body's "mind", it can also generate emotions, which are manifest through the controlled release of regulatory signals by the nervous system (unfolding EXformation)…. p.132: Candace Pert …. established that the "mind" was not focused in the head, but was distributed via signal molecules to the whole body (s. also M.W.Ho) …. While proper use of consciousness can bring health to an ailing body, inappropriate unconscious control of emotions can easily make a healthy body diseased …. [Pert 1997] …. p.135: Our responses to environmental stimuli are indeed controlled by perceptions, but not all of our learned perceptions are accurate. Not all snakes are dangerous! Yes, perception "controls" biology, but …. perceptions can be true or false. Therefore, it is more accurate to refer to these perceptions as beliefs. Beliefs control biology p.167: e.g., if a ball flies t/w your eye …. the subconscious mind, which processes some 20·E6 environmental stimuli per second vs. 40 environmental stimuli interpreted by the conscious mind in the same second, will cause the eye to blink [Norretranders 1998] …. Reflex behaviors …. e.g. driving a car at 65km/h on a highway while your conscious mind is fully engaged in conversation with a passenger …. Through the conditioned learning process, neural pathways between eliciting stimuli and behavioral responses become hardwired to ensure a repetitive pattern ("habits") …. Image: Overview of the information flow through a human being. A so-called organogram. shows that more information goes in and out of humans than consciousness perceives.

Source: Lipton B. (2005) Biology of Belief. Elite Books Pert, Candace (1997). Molecules of Emotion: The Science Behind Mind-Body Medicine, New York Norretranders T. (1998) The User Illusion Cutting Consciousness Down to Size. Penguin Books, NY – USA S. Lehrl and B. Fischer (1985). "Der maximale zentrale Informationsfluss" Grundlagenstudien aus Kybernetik und Geistenswissenschaji/Humankybernetik 26: 147-154 Intro FoodEpigenome Method Implication

Epigenetics (7c/7)

Emotion – the language of the cell • the “upper-I” (regime of morality); • the “I” (regime of reality);

• the “IT” (regime of lust); :45) -06 Liu, 2009

SHINE

chology (05:00 y Ps

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Engaging the subconscious in battle is as pointless as kicking the jukebox in the hope that it will reprogram its play list …. p.171: Tensions between conscious will power and subconscious programs can result in serious neurological disorders (movie “Shine”: why we should not challenge the subconscious) …. Most of us engage in less dramatic battles with our subconscious mind as we try to undo the programming we received as children …. Decision making: i) every decision is made within an emotional framework, i.e. every decision must be emotionally tolerable - only on this basis can the intellect be used; i) major changes (patterns of thinking and behaviour) must develop on an emotional basis; i) dramatic changes - deep involvement - means fundamental behavioural change; i) intellectual impulse (e.g. you should not smoke) is not enough; only by addressing the emotional, deeper realms can a change be induced. In Freud's model of 3 instances, the superego can be seen simplified as the moral instance (conscience) and represents the antagonist for the elementary pleasure instincts of the ES. It is formed in early childhood (up to the year 6) and contains the (moral) norms and internalized values of the cultural environment in which the individual grows up (especially those of the parents !). The super-ego is created by adapting one's own person to others with whom this person identifies (introjection through the wider community). The process of a person consciously engaged in thinking happens under the influence of the superego, and the fundamental values contained therein. In the human psyche, the superego functions as a controlling authority whose goal is to bring one's own behaviour into accordance with the ideal image by self-observation. With deviations from this ideal due to ES, the superego has an effect on the human being in the form of feeling of guilt.

Source: Klussmann R. (2000) Psychotherapie, 3te Auflage. Springer, p.5 & 20-28 Source: Lipton B. (2005) Biology of Belief. Elite Books http://en.wikipedia.org/wiki/Id,_ego,_and_super-ego Intro FoodEpigenome Method Implication

Diet (1a/10) Eastern versus Western Diet • Local eating habits evolved over centuries / fast food over one decade; Oldways, 2008

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Biophycist F.A.Popp who lived some time in China made the following observation:: Having lived there like "God in France“, nonetheless he has lost 5kg during that time .... How is that possible? Because back then the food was healthy and knowing what is good or not good for him helped as well .... p.xl: The opposite happens when someone travels to the USA with an almost inexistent eating culture .... eating habits that literally kill. Americans may live longer, but basically do so in a conserved state, implying that the decay much slower along with pathologies that become chronic .... p.14: Americans live almost exclusively on processed (canned) food; Chinese have not yet been able to afford it. they depend on fresh food .... It seems as if preservatives not only conserve the food but also the consumer .... on average, a chinese is about the same age as an american. nevertheless the chinese have a much lower risk of suffering from "civilization diseases" such as circulatory failure, cancer and rheumatism after the age of 50 than their US-counterparts of the same age .... p.52: as impractical as this way of judging food quality may seem .... One can deduct that "subjective" components play a decisive role. It would probably be better to feed the chinese in general with rice, the european with wheat, than vice versa in an effort to undertake missionary attempts with fatal consequences.

Source: http://www.oldwayspt.org/store/index.php/cPath/5 www.momskitchen.in/articles.php http://www.isrealli.org/another-reason-to-trash-junk-food/ Intro FoodEpigenome Method Implication

Diet (1b/10) Eastern versus Western Diet • Local eating habits evolved over centuries / fast food over one decade; Oldways, 2008 Yet still …. USDA, 2005 …. the currently accepted recommendations pose serious health related consequences .... So, what‘s wrong with it?

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The MyPyramid was released in April 2005 and replaced the Food Guide Pyramid (1992). In 2011, the MyPlate replaced MyPyramid and represents the current USDA guidance.

Source USDA, 2005 https://www.cnpp.usda.gov/mypyramid-graphics Intro FoodEpigenome Method Implication

Diet (1c/10) Eastern versus Western Diet • dietary protein >10% is a risk factor for cancer; • tumor growth in rats greatly enhanced by diets containing ≈10% animal protein (casein); • tumor growth repressed with either 5% animal or ≈20% plant protein; • tumor development, both early and late stages of development can be turn on / off by modulating animal protein intake;

Excess intake of animal proteins depress activity of an enzyme complex responsible for carcinogen activation, subsequent, promotion of preneoplastic clones & their sequelae and life-long development of full-blown tumors. This effect also existed both for chemically and virally induced cancers. Campbell, 2007

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We showed that tumor growth in rats was greatly enhanced by diets containing ≈10% animal protein (casein) and was completely repressed with either 5% animal protein or ≈20% plant protein .... This protein effect depressed the activity of the major enzyme complex responsible for carcinogen activation .... and for the subsequent and dominant promotion of preneoplastic clones and their sequelae ....the life-long development of full-blown tumors ....This effect also existed both for chemically and virally induced cancers and was explained, .... by accelerated cell replication as well as by a variety of cellular and enzymatic mechanisms. We could turn on and off tumor development, both in its early .... and late .... stages of development. Shortly after the identification of the IGFs, we observed in hepatitis B virus–transfected mice that the tumor-enhancing effects of dietary protein were closely associated with a greater hepatic synthetic synthesis of IGF-2 but not of IGF-1.... For example, it is not only the adverse effects of animal protein, per se, but the combined biologically integrated and consistent effects of animal protein with its dietary covariates that are far more significant ....

Source: Campbell TC (2007) Dietary protein, growth factors, and cancer. The American Journal of Clinical Nutrition, Vol.85(6): 1667; doi: 10.1093/ajcn/85.6.1667 Intro FoodEpigenome Method Implication

Diet (1d/10) Eastern versus Western Diet Combined stress exposure of

• Aflotoxin-B1 (AFB1)& • casein (diary protein) mimicking real-life multi-factorial exposure conditions

Youngman et al., 1992

Dietary Tumor V Tumor Time AFB,- Tumor No of tumor casein incidence occupied mass [wks] treated per animal2 [%] [%] [%] index3 Tumor study design – The initiation (dosing) 0 + 0/20 (0) - - - period lasted 3 weeks and included 15 doses of 6 + 0/15 (0)a - - - 40 AFB, and 22% casein diet feeding .... Numbers 14 + 0/15 (0)a - - - 22 + 2/15 (13) a - - - of animals killed at each time point are shown 0 + 0/9 (0) - - - in parentheses. 6 + 0/15 (0)a - - - 58 14 + 3/13 (0)b - - - 22 + 7/15 (0)c - - - 0 + 0/26 (0) - - - 6 + 12/60 (20)a 12 ±5a 1.9 ±0.2a 240 100 14 + 32/38 (84)b 28 ±9a,b 3.5 ±0.4b 2350 22 + 52/58 (90)b 27 ±8b 3.5 ±0.3b 3330 19-03-18 Madl 129

Previous studies in this laboratory with young Fischer 344 male rats have shown that the post-initiation + development of aflatoxin-B1 (AFB1)-induced gamma-glutamyl-transpeptidase-positive (GGT ) hepatic foci was markedly inhibited by low protein feeding, even though the energy intake was greater. This dietary effect, however, did not necessarily apply to hepatic tumor development. Thus, the present investigation was undertaken to examine this dietary effect upon the development of hepatic tumors and, in so doing, to

determine the correlation of foci development with tumor development. Following AFB1 dosing (15 daily doses of 0.3 mg/kg each), animals were fed diets containing 6, 14 or 22% casern (5.2, 12.2, 19.1% protein) for 6, 12, 40, 58 and 100 weeks. Foci at 12 weeks and tumors at 40, 58 and 100 weeks developed dose- dependently to protein intake. Foci development, tumor incidence, tumor size and the number of tumors per animal were markedly reduced while the tune to tumor emergence was increased with low protein feeding. Non-hepatic tumor incidence also was lower in the animals fed the lowest protein diet. Foci development indices (foci number, per cent liver volume occupied) were highly correlated with tumor incidence at 58 and 100 weeks (r = 0.90–1.00). Tumor and foci inhibition occurred in spite of the greater energy intake.

Image: Tumor study design. The initiation (dosing) period lasted 3 weeks and included 15 doses of AFB1, and 22% casein diet feeding. After 1 week of no dosing to allow for the clearance of AFB, and its metabolites, dietary treatment commenced. Kill times for subsets of animals were at 6, 12, 40, 58 and 100 weeks. At the beginning of the experiment, 110, 80 and 110 animals comprised the 6, 14, 22% AFB|-treated groups, respectively. Numbers of animals killed at each time point are shown in parentheses. Table: Liver tumor development in AFB,-dosed rats at 40, 58 and 100 weeks post-initiation1 Means ±SEM. Statistical analyses are restricted to comparisons within columns and within the same time period. Different lettered superscripts are statistically different at P <0.05. 1 None of the control (i.e. tricaprylin-dosed) animals developed liver tumors: 0/20 at 40 weeks, 0/9 at 58 weeks and 0/26 at 100 weeks. 2 Number of liver tumors per tumor-bearing animal. 3 Product of per cent incidence x mean per cent liver volume occupied by tumors.

Source: Youngman LD, Campbell TC (1992) Inhibition of aflatoxin B1-induced gamma- glutamyltranspeptidase positive (GGT+) hepatic preneoplastic foci and tumors by low protein diets: evidence that altered GGT+ foci indicate neoplastic potential. Carcinogenesis, Vol.13(9): 1607–1613, doi: 10.1093/carcin/13.9.1607 Intro FoodEpigenome Method Implication

Diet (1e/10) Eastern versus Western Diet Combined stress exposure of

• Aflotoxin-B1 (AFB1)& • casein (diary protein) mimicking real-life multi-factorial exposure conditions

comparative effects of dietary protein feeding and AFB1-dose on liver volume occupied by foci.  positive dose-response relationship w/ increasing AFB1 at constant dietary casein at 20%.  negative the dose-response curve with increasing AFB1 at decreasing dietary casein intake. Pre-neo-plastic lesions in rat liver – indicated by Dunaif & Campbell, 1987 y-glutamyl-transferase-positive (GGT+) foci

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Male weanling F344 rats were orally gavaged with aflatoxin B1 (AFB1) in daily doses of 200, 235, 270, 300, and 350 micrograms/kg/day for a total of 10 doses over a 12-day period, and then 1 week after the last dose they were fed diets of varying protein (casein) content to compare the contribution of AFB1 dose and dietary protein level on the development of presumptive preneoplastic gamma-glutamyltransferase-positive (GGT+) foci in rat liver. All animals were fed the same 20% dietary casein level during the dosing period. One week after the end of the dosing period, one-half of the animals in each dose group were then continued on the 20% casein diet for the entire 12-week foci-development period; the remaining half in each dose group were fed lower levels of dietary casein during the foci-development period for the increasing AFB1 dose groups (20, 16, 12, 8, and 4% casein for the 235-, 250-, 270-, 300-, and 350-μg /kg/day groups, respectively). The AFB1 dose groups used were determined in a preliminary experiment. In this previous experiment, a clearly discernible threshold dose at about 100-150 μgAFB1/kg/day (below which no GGT+ foci were observed) and a steep slope between 150 and 400 μg/kg/day were produced. In the second experiment, while the expected positive slope of (AFB1) dose versus (GGT+ foci) response relationship was found for animals fed the 20% casein diet, the dose response for the animals fed the lower levels of casein was eliminated, providing evidence that nutrient intake during the postdosing foci development is more rate limiting toward the development of these preneoplastic lesions than is the carcinogen dose.

Image: Comparative effects of AFBI dose and postdose dietary protein on GGT+ foci response.... Data points are means ±SE. Numbers in parentheses indicate numbers of animals with evidence of GGT+ foci. Means are compared between corresponding values for upper and lower curves.

Source: Dunaif GE, Campbell TC (1987) Relative contribution of dietary protein level and aflatoxin B1 dose in generation of presumptive preneoplastic foci in rat liver. J Natl Cancer Inst. Vol.78(2): 365-369. Intro FoodEpigenome Method Implication

Diet (1f/10) Eastern versus Western Diet Combined stress exposure of

• Aflotoxin-B1 (AFB1)& • casein (diary protein) mimicking real-life multi-factorial exposure conditions

Alternating high (20%) & low (5%) casein diets fed to growing rats during the dosing

and postdosing periods of ATB1.  dose-related induction of hepatic pre- Treatment No of No of loci/mL VTumor Diameter of neoplastic lesion in the liver animals of liver occupied loci [μm] [%] 55 12 9.4 ±4.1 1.2 ±0.77 77 ±17 Appleton & Campbell, 1983 5 20 11 53.9 ±17.8 3.0 ±0.91 67 ±13 20 5 12 15.9 ±7.3 0.19 ±0.06 28 ±3 20 20 12 96.5 ±26.3 1.96 ±0.40 35 ±2

China Study.mp4 12:38 – 14:30

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Aflatoxin B1-induced liver lesion development is readily modified by dietary protein intake. Earlier work had shown that low-protein diets enhanced the acutely toxic lesion but depressed the carcinogenic lesion. This study examined the emergence of these lesions as a function of dietary protein intake, particularly with respect to whether the protein modification occurred during or after the aflatoxin B12 dosing period. High (20%) and low (5%) casein diets were fed to growing Fischer 344 rats during the dosing and postdosing periods of aflatoxin B2-induced hepatic preneoplastic lesion development. Focal areas of hepatocellular alteration were identified and quantitated by staining sections of liver for gamma-glutamyl transferase (GGT). Animals fed low casein diets during the dosing period displayed a characteristic spectrum of lesions including hepatomegaly, severe bile duct proliferation, cholangiofibrosis, and a tendency for developing large remodeling GGT- positive foci. These lesions were regarded as symptomatic of acute hepatoxicity. Animals fed high-protein diets during the dosing period had small, densely stained, GGT-positive foci, with only mild bile duct proliferation and no cholangiofibrosis, hepatomegaly, or large, remodeling GGT-positive foci. During the postdosing period, protein modulation markedly influenced the total number of foci. Animals fed high casein diets during this period exhibited an approximate 6-fold increase in the number of foci, regardless of the level of protein fed during the earlier dosing period. The marked increase in foci number (as well as area of liver occupied) in high casein diet animals during the postdosing period is regarded as an increased tendency for tumor development.

Image: Temporal relationships between AFB, dosing and diet treatments. Table: Effect of different dietary treatments on GGT+ foci development in AFB,-treated rat. Statistical significance between values is noted by footnotes b and d. Means which are different at p <0.05 have different superscripts.

Source: Appleton BS, Campbell TC (1983) Effect of high and low dietary protein on the dosing and postdosing periods of aflatoxin B1-induced hepatic preneoplastic lesion development in the rat. Cancer Res. Vol.43(5): 2150-2154. Intro FoodEpigenome Method Implication

Diet (1g/10) Eastern versus Western Diet Inducing havoc to endothelial lining of arteries

Disruption of endothelial lining  Coronary Heart Disease  reversal of effect when switching to plant- based diet Esselstyn, 1983 Esselstyn et al., 2014

Nitric oxides (NO) produced by endothelial cells lubricates & dilates arteries

NO – forks over knives.mp4

27th Nov. 1996 22nd Jul. 1999 19-03-18 Madl 132

The world's advanced countries have easy access to plentiful high-fat food; ironically, it is this rich diet that produces atherosclerosis. In the world's poorer nations, many people subsist on a primarily plant-based diet, which is far healthier, especially in terms of heart disease. To treat coronary heart disease, a century of scientific investigation has produced a device-driven, risk factor-oriented strategy. Nevertheless, many patients treated with this approach experience progressive disability and death. This strategy is a rear-guard defensive one. In contrast, compelling data from nutritional studies, population surveys, and interventional studies support the effectiveness of a plant-based diet and aggressive lipid lowering to arrest, prevent, and selectively reverse heart disease. In essence, this is an offensive strategy. The single biggest step toward adopting this strategy would be to have United States dietary guidelines support a plant-based diet. An expert committee purged of industrial and political influence is required to assure that science is the basis for dietary recommendations. Image: Coronary angiography reveals a diseased distal left anterior descending artery (left). Following 32 months of a plant- based nutritional intervention without cholesterol- lowering medication, the artery regained its normal conguration (right). Positron emission tomography performed on a patient with coronary artery disease shows an area of myocardium with insufficient blood flow (top). Following only 3 weeks of plant- based nutritional intervention, normal blood flow was restored (bottom).

Source: Esselstyb CB (2001) Resolving the Coronary Artery Disease Epidemic Through Plant-Based Nutrition. Prev cardiol, Vol.4(4):171-177. Esselstyn CB, Gendy G, Doyle J, Golubic M, Roizen MF (2014) A way to reverse CAD? J. Fam. Practice, Vol.63(7): 356-364. Endothelial Cells to the Rescue!: https://www.drcarney.com/blog/entry/endothelial-cells-to-the-rescue / https://www.youtube.com/watch?v=9jkRaxMRS_U Intro FoodEpigenome Method Implication

Diet (1h/10) Wheat flour intake [g/day] Eastern versus Western Diet Meat – a tumor promoting food

Plant-based diet (very low content of animal protein) significantly reduces  tumors  coronary heart disease  stroke & heart attacks Rice intake [g/day] a plant-based diet promotes health !

JunShi et al., 2006

China Study.mp4 15:24 – 14:30 China Study – Campbell-Collin.mp4 1:24 – 3:21 19-03-18 Madl 133

A mortality study throughout China involving about 800 million people in 1973- 75 is summarised, followed by a detailed study in 65 counties ranging from plasma, red cells, and urine measurements to food samples, dietary surveys, and a lifestyle questionnaire. The basic presentation comprises 733 A4 pages of data including maps and correlations. Two plotted correlations display something of the breadth of the data - mortality rate from stomach cancer for each county against the percentage of individual plasma samples positive for Campylobacter pylori, IgG antibody, and mortality rate for colorectal cancer against mortality from schistosomiasis.

Image: Wheat is grown and eaten predominantly in the north (and rice in the south), hence there are correlations with many other ‘north-south’ variables. Dietary intakes are standardized per “reference man”, defined as an adult male 19-59 yrs of age, 65 kg of body weight and undertaking very light physical work. Top: in areas where wheat is eaten, the mean county intake varies from 0,1-0,7 kg/day, but there are many southern counties where wheat intake is negligibly small. Bottom: In areas where rice is eaten the men county intake varies from 0,3-0,8 kg/day.

Source: JunShi C, Peto R, Pan WH, Liu BQ Campbell TC, Boreham J, Parpia B, Cassano P, Chen ZM, Feng ZL, Gelband H, Li JY, Pan HC, Root M, Wu YP, Zoungman L (2006) Diet, life- style and mortality in China – Geographic study of the characteristics of 69 counties in mainland china and 16 areas in Taiwan. Oxford University Press, Oxford (UK) ISBN019-2618431 Intro FoodEpigenome Method Implication

Diet (1h/10) Endocrine-nutrit.& metabol. Disease age 35-69 [1/100k] Eastern versus Western Diet Meat – a tumor promoting food

Plant-based diet (very low content of animal protein) significantly reduces  tumors  coronary heart disease  stroke & heart attacks Diabetes age 35-69 a plant-based diet promotes health ! [1/100k]

JunShi et al., 2006

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Image-top: This category is dominated by deaths from diabetes (78 % male, 77 % female); Diabetes mortality is greater in urban than in rural China. Bottom: Risk of dying from diabetes at ages 35-69 in rural China (male 0.2 %; female 0,3 %) with rates lower in rural China than in urban China. The low rates may be due to the low prevalence of obesity in rural China, but compositions with other counties and possibly with urban China or Taiwan are not easily interpretable, because what is certified as death from diabetes varies considerably; e/g/ deaths of diabetes in the USA from vascular disease may not be coded as diabetes.

Source: JunShi C, Peto R, Pan WH, Liu BQ Campbell TC, Boreham J, Parpia B, Cassano P, Chen ZM, Feng ZL, Gelband H, Li JY, Pan HC, Root M, Wu YP, Zoungman L (2006) Diet, life- style and mortality in China – Geographic study of the characteristics of 69 counties in mainland china and 16 areas in Taiwan. Oxford University Press, Oxford (UK) ISBN019-2618431 Intro FoodEpigenome Method Implication

Diet (1h/10) Plant food intake [g/day] Eastern versus Western Diet Meat – a tumor promoting food

Plant-based diet (very low content of animal protein) significantly reduces  tumors  coronary heart disease  stroke & heart attacks Animal food intake [g/day] a plant-based diet promotes health !

JunShi et al., 2006

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Image: Dietary intakes are standardized per “reference man”, defined as an adult male 19-59 yrs of age, 65 kg of body weight and undertaking very light physical work. Intake of plant foods in negatively correlated with intake of animal foods and the two have opposite correlations with plasma lipids. Top: four variables should be considered together, i.e. mean 0.1 kg/day, range 0.0-0.4 kg/day, percentage of plant food intake by weight of total diet (mean 30 %) and percentage animal food intake by weight of total diet (mean 7%, range 0-30 %). Bottom: Very low levels in most counties but higher in more prosperous coastal areas and in northern herding areas. Positively correlated with blood lipids 64 %, tot. cholesterol 48 % non-HDL 46 %. Four variables should also be considered together like plant food intake (1,1 kg/day range 0.7-1.6 kg/day), animal food intake (mean 0.1 kg /day, range 0.0-0.4 kg/day), percentage of plant food intake by weight of total diet (mean 93 %) and percentage of animal food intake by weight (7 %, range 0-30 %).

Source: JunShi C, Peto R, Pan WH, Liu BQ Campbell TC, Boreham J, Parpia B, Cassano P, Chen ZM, Feng ZL, Gelband H, Li JY, Pan HC, Root M, Wu YP, Zoungman L (2006) Diet, life-style and mortality in China – Geographic study of the characteristics of 69 counties in mainland china and 16 areas in Taiwan. Oxford University Press, Oxford (UK) ISBN019-2618431 Intro FoodEpigenome Method Implication

Diet (1h/10) Oesophageal cancer age 35-69 [1/100k] Eastern versus Western Diet Meat – a tumor promoting food

Plant-based diet (very low content of animal protein) significantly reduces  tumors  coronary heart disease  stroke & heart attacks Malignant neoplasms age 0-34 a plant-based diet promotes health ! [1/100k]

JunShi et al., 2006

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Image-top: Risk of dying from oesophageal cancer at ages 35-69 in rural China (male 4.2 %, female 2.3 %). In the 11 counties with the highest rates (black dots), oesophageal cancer is a major cause of death (12 % males, 7 % females, but ranging up to 14 % male and 10 % female in worst affected county, i.e. CiXian). Little change over time with stable rates in most counties. Strong geographic correlations (52 % death rate) with stomach cancer, with below-average rates for both cancers in 40 counties; there are counties where oesophageal cancer is common and stomach cancer is not and others where the revers is true. Lack of correlations with alcohol and smoking variables indicate that other factors cause most of the geographic variations. Oesophageal cancer is however more common among smokers than among non-smokers and smoking is a cause of about 28 % of male deaths from oesophageal cancer in China at ages 35-69. Bottom: Risk of dying from oesophageal cancer at ages 0-34 in rural China (male 0,6 %, female 0,4 %). Category includes deaths from all malignant neoplasms. Mean rates increased by 1/3 b/w 1974-1987 mainly reflecting the apparent increase in liver cancer deaths among young adults. High rates in specific areas are chiefly due to liver cancer mortality in young men, especially GuangXi, JaingSu and FuJian province. The high rate recorded for females in 1987 in ErChang county involves small numbers of deaths from several different types of cancer and may be chiefly by chance.

Source: JunShi C, Peto R, Pan WH, Liu BQ Campbell TC, Boreham J, Parpia B, Cassano P, Chen ZM, Feng ZL, Gelband H, Li JY, Pan HC, Root M, Wu YP, Zoungman L (2006) Diet, life- style and mortality in China – Geographic study of the characteristics of 69 counties in mainland china and 16 areas in Taiwan. Oxford University Press, Oxford (UK) ISBN019-2618431 Intro FoodEpigenome Method Implication

Diet (1i/10) Eastern versus Western Diet Cow milk – bad for the bones

Cow milk as the biggest dietary cause of osteoporosis  the higher the milk consumption, the higher the rate of hip fractures  low-fat milk has an elevated protein content  metabolic acidosis depletes finite reserves of osteoblasts

Hegsted , 1986

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The currently available data, although limited, suggest that osteoporosis, like several other major chronic diseases, is largely a disease of affluent, western cultures. Diet must be suspected as a factor, but the causal agent is not known. Much more data from populations where calcium intakes are known to differ substantially using a variety of techniques now available are urgently needed. Since the susceptible populations apparently consume more, rather than less calcium, the epidemiological data do not implicate calcium deficiency as a cause. It seems quite clear that we do not understand the etiology of osteoporosis; the epidemiological data need an explanation, and something is wrong when current explanations are inconsistent with general experience. It is dangerous to ignore the epidemiological data. The first rule in formulating public health policy should be the assurance that the recommendations are not detrimental. It will be embarrassing enough if the cur rent calcium hype is simply useless; it will be immeasurably worse if the recommendations are actually detrimental to health.

Image: Available calcium in the food supply compared with the incidence of hip fractures in females of several nations.

Source: Hegsted DM (1986) Calcium and Osteoporosis. J Nutr Vol.986;116(11): 2316-2319 Intro FoodEpigenome Method Implication

Diet (1i/10) Eastern versus Western Diet Cow milk – bad for the bones

Cow milk as the biggest dietary cause of osteoporosis  the higher the milk consumption, the higher the rate of hip fractures  low-fat milk has an elevated protein content  metabolic acidosis depletes finite reserves of osteoblasts

Hall, 2011

China Study.mp4 15:24 – 14:30 19-03-18 Madl 138

Metabolic acidosis is a condition that occurs when the body produces excessive quantities of acid or when the kidneys are not removing enough acid from the body. If unchecked, metabolic acidosis leads to acidemia, i.e., blood-pH is less than 7.35 due to increased production of protons by the body or the inability of the body to − form bicarbonate (HCO3 ) in the kidney. Its causes are diverse, and its consequences can be serious, including coma and ultimately if untreated death. Together with respiratory acidosis, it is one of the two general causes of acidemia.[2] In some instances, acid-base disorders are not accompanied by appropriate compensatory responses. When this situation occurs, the abnormality is referred to as a mixed acid-base disorder, which means that there are two or more underlying causes for the acid-base disturbance. For example, a patient with low pH would be categorized as acidotic. If the disorder was metabolically mediated, this would also be accompanied by a low − plasma HCO3 concentration and, after appropriate respiratory compensation, a low pCO2. However, if the − low plasma pH and low HCO3 concentration are associated with elevated pCO2, one would suspect a respiratory component to the acidosis, as well as a metabolic component. Therefore, this disorder would be − categorized as a mixed acidosis. This disorder could occur, for example, in a patient with acute HCO3 loss from the gastrointestinal tract because of diarrhea (metabolic acidosis) and emphysema (respiratory acidosis). A convenient way to diagnose acid-base disorders is to use an acid-base nomogram. This diagram can be used − to determine the type of acidosis or alkalosis, as well as its severity. In this acid-base diagram, pH, HCO3 concentration, and pCO2 values intersect according to the Henderson-Hasselbalch equation. The central open circle shows normal values and the deviations that can still be considered within the normal range. The shaded areas of the diagram show the 95 percent confidence limits for the normal compensations to simple metabolic and respiratory disorders. [1]

- Image: Acid-base monogram showing arterial blood-pH, arterial plasma HCO3 and PCO2 values The central area shows the approximate limits for acid-base status in normal people. The shaded areas in the nomogram show the approximate limits for the nor al compensations caused by simple metabolic and respiratory disorders. For values lying outside the shaded areas one should suspect a mixed acid-base disorder. [1]

Source: [1] Hall, JE (2011) Guyton and Hall’s Textbook of Medical Physiology, 13th ed. Elsevier, Philadelphia (PA), USA [2] https://en.wikipedia.org/wiki/Metabolic_acidosis Intro FoodEpigenome Method Implication

Diet (1g/10)

Western Diet – Olestra • inhibits absorption of some vitamins (A, D, E, & K); • may cause abdominal cramping; • sometimes causes underwear staining associated with ll clip) u "anal leakage“; lestra CSPI, 2009, Wikipedia, 2009 O

p Line (f el H

Personal enquiry confirmed that Olestra is currently being used in deep fried products in a FFR (Salzburg, Getreidegasse

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Olestra (brand name Olean) is a fat substitute that adds no fat, calories, or cholesterol to products; used in the preparation of high-fat foods such as potato chips, thereby lowering or eliminating their fat content. The irony is that it encourage consumers to eat more of the "top of the pyramid" foods due to the perception of it being more healthy, a paradox resulting in over-consumption thinking that Olestra allows that without consequence. The FDA was particularly hesitant to approve the product due to the side effects such as diarrhea and concern for the loss of fat-soluble vitamins.

Chemistry: Normal fats consist of a glycerol molecule with three fatty acid tails attached. Olestra is synthesized using a sucrose molecule, which can support from six to eight fatty acid chains arranged radially like an octopus, and is too large to move through the intestinal wall and be absorbed. Olestra has the same taste and mouthfeel as fat, but since it does not contain glycerol and the fatty acid tails can not be removed from the sucrose molecule for digestion, it passes through the digestive system without being absorbed and adds no calories or nutritive value to the diet. Since it contains fatty acid functional groups, Olestra is able to dissolve lipid-soluble vitamins such as vitamin D, E, K, & A, along with carotenoids. Fat soluble nutrients consumed along with Olestra products are excreted along with the undigested Olestra molecules. To counteract this loss of nutrient, products made with Olestra are fortified with oil soluble vitamins to compensate for this loss to fecal matter. In 1999, researchers discovered that Olestra helps facilitate the removal of dioxins from the body, as it apparently binds to dioxins in a manner similar to that of normal fats. This unexpected side effect may make the substance useful in treating victims of dioxin poisoning.

Source: http://www.cspinet.org/olestra/11cons.html & http://en.wikipedia.org/wiki/Olestra Virginia M. (1993) Food – A Source Book, 1st ed. OSU Department of Chemistry College of New Rochelle New Rochelle, NY – USA JA Weststrate , KH van het Hof (1994). Sucrose polyester and plasma carotenoid concentrations in healthy subjects. Am J Clin Nutr 1995 62: 591-597 Seddon J.M.; Ajani U.A.; Sperduto R.D.; Hiller R.; Blair N.; Burton T.C.; Farber M.D.; Gragoudas E.S.; Haller E.; Miller D.T. (1994). Dietary carotenoids, vitamins A, C, and E, and advanced age-related macular degeneration. Eye Disease Case-Control Study Group. JAMA 272:1413-1420. Intro FoodEpigenome Method Implication

Diet (1h/10)

Western Diet - SCFA • short-chain fatty acids prevalent in high-fiber diet • Lowerd risk of Asthma Thorburn et al., 2015 Graham, 2016 0 :1 13 1– :3 12 o de vi

Dietic asthma .mp4

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Asthma is prevalent in Western countries, and recent explanations have evoked the actions of the gut microbiota. Here we show that feeding mice a high-fibre diet yields a distinctive gut microbiota, which increases the levels of the short-chain fatty acid, acetate. High-fibre or acetate-feeding led to marked suppression of allergic airways disease (AAD, a model for human asthma), by enhancing T-regulatory cell numbers and function. Acetate increases acetylation at the Foxp3 promoter, likely through HDAC9 inhibition. Epigenetic effects of fibre/acetate in adult mice led us to examine the influence of maternal intake of fibre/acetate. High-fibre/acetate feeding of pregnant mice imparts on their adult offspring an inability to develop robust AAD. High fibre/acetate suppresses expression of certain genes in the mouse fetal lung linked to both human asthma and mouse AAD. Thus, diet acting on the gut microbiota profoundly influences airway responses, and may represent an approach to prevent asthma, including during pregnancy.

Image: Adult (6 week old female C57Bl6) mice (obtained from the same facility and cohoused for 3 weeks) were provided different diets for 3 weeks then faeces and blood were collected. SCFA levels in faeces and serum measured by 1H-NMR spectroscopy. Data represent mean+sem., n=5. Significance is represented by *P<0.05, **P<0.01, Student’s t-test (above). The effect of high-fibre diet and acetate on the development of

AAD in adult mice. Airway hyper-responsiveness in terms of airway resistance (RL) and dynamic compliance (Cdyn). Data represent mean+s.e.m., n=8. Significance is represented by *P<0.05, **P<0.01, ***P<0.001, Student’s t-test (below).

Source: Thorburn AN, McKenzie Ci, Shen S, Stanley D, Macia L, Mason LJ, Roberts LK, Wong CHY, Shim R, Robert R, Chevalier N, Tan JK, Mariño E, Moore RJ, Wong L, McConville MJ, Tull DL, Wood LG, Murphy VE, Mattes J, Gibson PG, Mackay CR (2015) Evidence that asthma is a developmental origin disease influenced by maternal diet and bacterial metabolites. Nature Comm., Vol.6, Article number: 7320 (2015)doi:10.1038/ncomms8320 Intro FoodEpigenome Method Implication

Diet (1i/10)

Western Diet – Nutrasweet (Aspartame)

•C14H18N2O5 • health hazard to those born w/ phenylketonuria (PKU; • acute & chronic toxicity studies in mice, rats, hamsters & dogs have found no adverse up to at least 4000 mg/kg BW/day; • synergistic effects? • long-term studies? Hodgson, 2001

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In 1985, Monsanto bought G.D. Searle and the aspartame business became a separate Monsanto subsidiary, the NutraSweet Company.

Chemistry: Aspartame is the methyl ester of the dipeptide of the natural amino acids L-aspartic acid and L-phenylalanine. Under strongly acidic or alkaline conditions, aspartame may generate methanol by hydrolysis. Under more severe conditions, the peptide bonds are also hydrolyzed, resulting in the free amino acids. In certain markets aspartame is manufactured using a genetically modified variation of E. coli. Upon ingestion, aspartame breaks down into natural residual components, including aspartic acid, phenylalanine, methanol, and further breakdown products including formaldehyde, formic acid, and a diketopiperazine. High levels of the naturally-occurring essential amino acid phenylalanine are a health hazard to those born with phenylketonuria (PKU), a rare inherited disease that prevents phenylalanine from being properly metabolized.

Source: http://en.wikipedia.org/wiki/Aspartame Hodgson A.S. (2001). Falsifications and Facts about Aspartame. Cooperative Extension Service, CTAHR, Hawaii – USA. Magnuson BA, Burdock GA, Doull J, et al (2007). "Aspartame: a safety evaluation based on current use levels, regulations, and toxicological and epidemiological studies". Crit. Rev. Toxicol. 37 (8): 629–727 Intro FoodEpigenome Method Implication

Diet (2/10)

Bioaccumulation in the Food Chain

Western Diet vs Eastern Diet The so-called, four basic foods are • Grains and cereals; • Milk and dairy products; • Meat, fish and poultry; • Fruit and vegetables;

An industry-oriented approach!

Example: Hg (s.a. slide 20)

19-03-18 Madl 142 i) 'Feed the man meat!' (ecologically unsustainable) i) 'Milk is good for you!' (overload of protein) i) 'Bread is the stuff of life!' (is not a vegetable, just a filler) p.146: We are encapsulated by the 'four-foods philosophy'. Yet, many people are allergic to at least two of these four foods (e.g. milk, dairy products, & grains, cereals). Intolerance and allergy comes about because of the body's inability to process certain foods properly .... Undigested food particles enter the bloodstream and wreak havoc throughout the body. These toxins continue to accumulate in the body, lowering tolerance, wrecking the immune system and finally evolving into specific disease. p.148: Grains and cereals …. Symptoms can range from overweight, tiredness, mental illness, to chronic disease …. Grains are acid-forming in the body. Even when cooked, they are, comparatively, difficult to digest and can produce abdominal swelling and flatulence. Because they are a seed, they contain an enzyme inhibitor, which is not easily broken down, even by cooking …. Wheat in particular, cause deposits of calcium salts in the tissues, leading to degeneration and hardening of the arteries …. White flour …. can't keep laboratory animals alive …. p.151: Milk and dairy products …. Pasteurization destroys the natural enzymes present in raw milk. Without them milk cannot be properly digested …. Effects to infants (<12 months of age): gastrointestinal blood loss due to the baby's underdeveloped GIT. Cow's milk is too high in protein, Na, and K for a baby's immature kidneys - places them under great strain. Anaemia may develop …. Cow's milk, fed too early …. can damage their digestive systems and render them allergic, or sensitive, to milk for life …. p.154: Meat, fish and poultry …. A lot of meat in the diet is particularly bad for allergy sufferers because it places a greater strain on bodily functions, already overstressed by constant allergic reactions (bioaccumulation of environmental toxins) …. p.157: Fruit and vegetables …. the ultimate diet for the onward maintenance of good health, is one that consists mainly of fresh fruit and vegetables …. For food/chemical allergy sufferers, a largely vegetarian diet is often the best answer, perhaps supplemented, if tolerance allows, by a small, irregular quantity of the other basic foods.

Source: Griffiths S. 1996. Allergy overload. HarperCollins (Australia) Intro FoodEpigenome Method Implication

Diet (3/10)

Bookmarking & sudden change of diet during the Bengali famine 1943: • emergency procedures to feed the needy; • 110·E6 free meals; • intolerance to novel food (e.g. wheat)intestinal microbiomic collapse; • → still 3·E6 people starved to death; Wikepedia, 2009

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In 1943 three million people died in Bengal because they had tried to replace their usual but scarce rice supplies with wheat .... Their intestinal microbiome was not able to adapt quick enough to maintain survivability of the host organism. The consumer, too, by no means is seeking a uniform diet .... it is diversity that one needs – similar to a concert experience where one would get bored when served with a constant repetition of a single tune ....

Source; http://en.wikipedia.org/wiki/Bengal_famine_of_1943 http://farm4.static.flickr.com/3022/2599462069_3983e36cfd.jpg?v=0 Intro FoodEpigenome Method Implication

Diet (4/10)

Paramutation - Oeverkalix study

•exposure to cigarette smoke can lead to a trans-generational effect but is restricted to boys; •poor food supply of paternal grandparents was followed by reduced mortality in grandchildren; •rich food supply of paternal grandparents was followed by increased mortality in grandchildren; •effects last over 2 generations; AW-EPIGENETIK.mp4 Bygren et al., 2001 Nutrition (07:24 – 09:07) Kaati et al, 2002 Pembrey et al., 2006 19-03-18 Madl 144

Oeverkalix study (unconventional epigenetic) is a long-term study based on life habits and causes of death of the grandparent generation available at local municipal archive; this material contains also information about 14·E3 children from birth onwards till reaching puberty wherbe the following aspects could be investigated: i) influence of diet on body weight and health of offspring; i) Gross oversupply of food available to grandparents drastically shortened the grandchildren's life expectancy (increased risk of diabetes). i) Shortfall of food supply among fathers drastically reduced the risk of cardiovascular disease and diabetes among offspring; i) epigenetic imprints (paramutation) last at least for 2 generations if not more;

Source: Bygren, Lars Olov; Kaati, Gunnar; Edvinsson, Sören (2001) Longevity Determined by Paternal Ancestors' Nutrition during Their Slow Growth Period. Acta Biotheoretica, 49:53-59. Kaati G, Bygren LO, Edvinsson S (2002) Cardiovascular and diabetes mortality determined by nutrition during parents and grandparents slow growth period. Eur J Hum Genet. Vol.10: 682- 688 Pembrey ME, Bygren LO, Kaati G, Edvinsson S, Northstone K, Sjöström M, Golding J, ALSPAC Study Team (2006) Sex-specific, male-line transgenerational responses in humans. Eur J Hum Genet 14: 159-166 Intro FoodEpigenome Method Implication

Diet (5/10)

Bookmarking – Fetal diet & adult health:

• Rich fetal diet: i) adjusted to a high level of nutrition in adult life develop a large phenotype – better adult conditions = better health (except at very high levels of nutrition). • Poor fetal diet: i) adjusted to poor adult nutrition in adult life develop a small phenotype i) in very poor adult conditions they are expected to be healthier than those with large phenotypes. Bateson, et al., 2004 19-03-18 Madl 145

The adult health and likelihood of survival of two groups of individuals with extreme phenotypes are given for a variety of nutritional environments. For individuals whose early environment has predicted a high level of nutrition in adult life and who consequently develop a large phenotype, the better the conditions, the better will be their health except perhaps at very high levels of nutrition. For individuals whose conditions in fetal life predicted poor adult nutrition and who develop a small phenotype, the expected outcome is less clear. In very poor conditions they are expected to be healthier than those with large phenotypes. It seems plausible that their health would be benefited by some improvements in their nutritional environment in later life, but these improvements would diminish with further increases in the nutritional environment. Whether such individuals would be worse off in absolute terms than in a low nutritional environment, and the slope of the graph eventually becomes negative, is difficult to assess, but relative to the large phenotype individuals they are expected to be much less healthy.

Image: The hypothetical relationship between adult health and nutritional level during later development for two extreme human phenotypes that were initiated by cues received by the fetus.

Source: Bateson P., Barker D., Clutton-Brock T., Deb D., D’Udine B., Foley R.A, Gluckman P., Godfrey K., Kirkwood T., Lahr M.M., McNamara J., Metcalfe N.B., Monaghan P., Spencer H.G. & Sul1 S.E. 2004. Developmental plasticity and human health. Nature 430: 420-421. Intro FoodEpigenome Method Implication

Diet (6/10)

Paramutation - Maternal under- nutrition & low birth weight:

• increased risk of obesity; • increased risk of diabetes; • increased risk of cardiovascular disease during adult life; • increased risk of colon cancer;

Jimenez–Chillaron et al.,2008

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"The quality of life in the womb, our temporary home before we were born, programs our susceptibility to coronary artery disease, stroke, diabetes, obesity and a multitude of other conditions in later life," …. [Nathanielsz 1999] …. An expectant mother might well logically reason that what she eats will affect her unborn child. But the evidence is mounting that not only her children, but her grandchildren and subsequent generations will be affected by her nutrition. What she eats may not only affect her descendants as they develop, but potentially throughout their adult lives. A study published Jimenez– Chillaron et al., indicate that low birth weight is associated with increased risk of obesity, diabetes and cardiovascular disease during adult life, the team wanted to know whether such disease risks might be passed on to future generations. Maternal exposure to glucocorticoids in pregnancy induces hypertension and/or evidence of insulin resistance, obesity and altered muscle mass as well as alterations in the hypothalamic–pituitary– adrenal axis in the adult progeny of several experimental species. When pregnant rats are given restricted diets, their offspring are smaller at birth; but when the offspring are subsequently given plenty of food they become more obese than the offspring of mothers given an unrestricted diet; moreover, for mice that were malnourished in utero, the richer the post-natal diet, the shorter is the lifespan. The modern western diet, so full of fats and sugars, could be exerting epigenetic effects on future generations, positive or negative. Abnormal methylation patterns are a hallmark of most cancers, including colon, lung, prostate and breast cancer …. The anticancer properties attributed to many foods can be linked …. to the distinct methylation patterns of people who eat those foods.

Source: Nathanielsz 1999, Life in the Womb: The Origin of Health and Disease. Ithaca, NY http://epigenome.eu/en/1,63,0 & http://blog.plantpoisonsandrottenstuff.info/category/vitamins/ Josep C. Jimenez-Chillaron, Elvira Isganaitis, Marika Charalambous, Stephane Gesta, Thais Pentinat-Pelegrin, Ryan R. Faucette, Jessica P. Otis, Alice Chow, Ruben Diaz, Anne Ferguson- Smith & Mary-Elizabeth Patti (2008) Intergenerational Transmission of Glucose Intolerance and Obesity by In Utero Undernutrition in Mice. Diabetes 58:460-468. Melissa Woo1 and Mary-Elizabeth Patti (2008). Diabetes Risk Begins In Utero. Cell Metabolism, Volume 8, Issue 1: 5-7. Intro FoodEpigenome Method Implication

Diet (7/10)

Paramutation - Obese Babies:

• obese mothers give birth to obese babies; • 20 genes seem to be involved; • overstimulation of fetal insulin production; • however, only 1-3% is of genetic origin the remaining 97-99% acquired (!); • metabolic related diseases in later life; e.g.: hormonal disbalance of leptin, neuropeptides (orexin, etc).

Lahrts, 2009 Jimenez–Chillaron et al.,2008

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Recent studies investigating metabolic disorders of the mother (e.g. diabetes), her weight before and during pregnancy play an important role. Obese mothers therefore have fat children much more often than women of normal weight .... Different factors are decisive for the overweight of the mothers. There are about 20 genes, some of which are susceptible to overweight. But at most only 1-3% of all obese people are of genetic origin .... For overweight mothers with diabetes significantly more sugar gets into the bloodstream of the unborn child via the umbilical cord. This constantly stimulates the fetal pancreas to produce insulin. This insulin is transported with the blood into the child's brain. There it works via special receptors, especially in the hypothalamus. Among other things, this receptor is also responsible for regulating the intake of food. If too much insulin constantly arrives in the developing hypothalamus, the saturation setpoint is set higher – and this occurs already in the womb. Together with the growth-promoting effect of insulin, this leads to many fat deposits in the fetus of an overweight woman. In addition, the hormone leptin is produced in fat cells. This normally signals to the brain when there are enough depots. However, if a fetus already has too many fat cells, it produces more leptin than slender unborn babies. Thus, the developing brain has both too much insulin and leptin. Animal experiments have indicated that overweight fetuses and newborns have a certain leptin insensitivity. In later life, this leads to leptin signals being less well perceived and noticed by the brain. This could cause more unnecessary fat deposits to accumulate. And since insulin and leptin, together with other hormones such as orexin or neuropeptide Y, regulate the intake and utilization of food, an oversupply of the first two hormones would lead to an imbalance in the complex regulation system.

Source: Lahrtz SA (2009) übergewichtige babys – mit schwerer hypothek ins leben NZZ 70: 25.3.09 Intro FoodEpigenome Method Implication

Diet (8/10)

Paramutation - Starvation in Holland 1944 (compare obesity in Africans)

People exposed to severe People conceived in famine had …. undernutrition in utero – • F1-babies w / normal birth weights; F2- 2nd gen. offspring had …. babies weighed 200-300g less; • more diabetes; • higher LDL/HDL cholesterol ratios; • 3-fold increase in coronary heart; • more likely to be obese; disease prevalence; • suffer from altered coagulation; • more pulmonary disease; • consider th/s psychologically less fit; • increased levels of micro- • aggerated response to stress - higher albuminuria; blood pressure rise under stress; • increased risk of stroke;

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The Dutch famine near the end of World War II led to an increased incidence of schizophrenia in adults who had been food-deprived during the first trimester of their mothers' pregnancy. Malnourishment among pregnant women in the South during the Civil War and the Depression has been proposed as an explanation for the high incidence of stroke among subsequent generations. Microa-lbuminuria occurs when the kidney leaks small amounts of albumin into the urine. In other words, when there is an abnormally high permeability for albumin in the renal glomerulus. More examples of epigenetic effects: i) hunger-winter in Holland 1944 (due to NS-embargo): mothers with deficient nutrition (400-800 kcal daily intake) gave birth to children with low birth weight, these children in turn - despite normal nutrition - give birth to underweight children again; i) at the time of the examination (xxxx) suffered twice as often from cardiovascular diseases as their peers; i) frequent breast cancer and obesity

Source: http://www.hongerwinter.nl/index.php?lang=english RC Painter, TJ Roseboom, OP Bleker . Prenatal Exposure to the Dutch Famine and disease in later life: an overview. Reprod Toxicol. 2005 Sep-Oct; 20 (3): 345-352. http://linkinghub.elsevier.com/retrieve/pii/S0890623805000882 Intro FoodEpigenome Method Implication

Diet (9/10)

Bookmarking – BSE & CJD:

• BSE (bovine spongiform encephalopathy; • CJD (Creutzfeldt-Jakob disease); • Scrapie (sheep);

All prions-transmissible infectious protein complexes and induce brain cell degeneration introduced via food uptake (epigenetic bookmarking);

Aguzzi et al, 2007

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Epigenetic Epidemiology: There is an association of CJD (Creutzfeldt-Jakob disease) and BSE (bovine spongiform encephalopathy), as well as scrapie in sheep. All are caused by prions- transmissible infectious protein complexes, whose reproduction and reconstruction involve some type of three-dimensional structural templating.,30 It is thought that BSE is a result of cannibalism in which faulty industrial practices produced prion-contaminated feed for cattle. There is now considerable concern that bovine prions may have been passed to humans, resulting in a new form of CJD. 29 Dementia: It’s an umbrella term for diseases typified by brain cell degeneration beyond what is expected for a certain age group, due to damage or disease. Dementias that affect the cortex region of the brain – the outer region that is densely packed with nerve cells, or neurons – include Alzheimer’s, Parkinson’s, Huntingdon’s and Creutzfeldt-Jakob Disease …. Memory loss and learning difficulties are the major consequences of these diseases, characterized by abnormal protein deposits, brain cells dying, and a reduction in new cell growth, which all contribute to brain circuit damage. Parkinson’s and Huntingdon’s disease affect the regions of the brain just below the outer cortex, and cause motor symptoms such as involuntary movements.

Image: Reported bovine spongiform encephalopathy (BSE) cases in the UK (blue) and in countries excluding the UK (orange). Non-UK BSE cases include cases from countries of the European Union (EU) and outside the EU (CDN, Israel, Liechtenstein, JPN, CH & USA). b) Reported cases of variant Creutzfeldt–Jakob disease (vCJD) in the UK (blue) and in countries outside the UK (orange). Non-UK vCJD cases include cases reported in FRA, IRL, ITA, USA, CDN, Saudi Arabia, JPN, NL, P and ESP. (see the National Creutzfeldt–Jakob Disease Surveillance Unit web site for vCJD data).

Source: http://epigenome.eu/en/2,58,1112 http://www.cjd.ed.ac.uk/vcjdworld.htm http://www.rdg.ac.uk/progspecs/pdf06/PFTFTQASSM06.pdf Jablonka E., Lamb M.J. 2002. The Changing Concept of Epigenetics. Ann. N.Y. Acad. Sci. 981: 82–96 29 Prusiner, S.B. 1998. Prions. Proc. Natl. Acad. Sci. USA 95: 13363–13383. 30 Chernoff, Y.O. 2001. Mutation processes at the protein level: is Lamarckback? Mutat. Res. 488: 39–64. Intro FoodEpigenome Method Implication

Diet (10a/10)

Bookmarking – Food intolerance:

• DNA-methyl. & histone modifs.; • Hypothalamus-Pituitary-Adrenal- Axis (HPA-axis); • Conditioning;

Garcia et al., 2007 19-03-18 Madl 150

At present, research focuses on the modulation of epigenetic factors regulating the immune system conditioning…. Such modulators are antisense oligonucleotides, ribozymes and siRNA. Johansen conducted a study on mice to determine the long-term effects of antihistamines …. 50 mice have been injected with bee venom, a substance to which almost all organisms develop an allergy upon exposure. Half of the mice were also given 100 micrograms of the antihistamine Clemastine just before they were given venom, and 100μg on each of the two days afterwards. After six weeks, the researchers injected the mice with another dosage of bee venom, and monitored the allergic reactions. They found that mice given antihistamines reacted more violently to the second venom injection. The findings suggest that the mice on allergy medication had not developed tolerance to the allergen. Effects of Conditioning: p.100: By repeatedly injecting rats with cyclo-phosphamide (a drug that induces nausea & suppressed the immune system) whenever they drank sweetened water, Ader and Cohen trained rats to avoid sweet water …. The rats responded by suppressing their immune systems themselves when faced with what they thought was the same situation again (sweet water & injection, cyclo-phosphamide had been replaced by a saline solution) …. p.102: …. All sorts of immune activity can be conditioned, including activation of the inflammatory response (placebo &) …. (& conditioning) Since the phenomenon of immune conditioning is already well defined, while the placebo response is not, it would seem preferable to keep the two terms separate, rather than having two words for the same thing …. Guinea pigs could be conditioned to associate gentle scratching and inflammation …. Inflammation is …. a symptom of immune activation, so it seems that immune enhancement can be conditioned as well as immune suppression. p.107: Some biologists have suggested that the immune system also functions as a sensory organ in higher animals, including humans.10 Just as the eyes allow us to detect light waves, so the immune system, like a sixth sense, enables us to detect the presence of the tiny invaders that constantly assail us from both within and without.

Source: Isidoro-García M, Dávila-González I, Pascual de Pedro M, Sanz-Lozano C, Lorente-Toledano F (2007): Interactions between genes and the environment. Epigenetics in allergy. Allergol Immunopathol (Madr). 35(6):254-8 Evans D. (2004). PLACEBO - Mind Over Matter in Modern Medicine; Harper Collins Publishers; London - UK Intro FoodEpigenome Method Implication

Diet (10b/10)

Bookmarking – Food intolerance:

• DNA-methylation & histone modifications; •HPA-axis;

Griffiths, 1996 Epigenetic conditioning

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The alarming number of people who are now suffering from this problem, in varying degrees, indicates that it is widespread due to the following reasons (incomplete list): i) Conserved foods: The excessive consumption of refined, pre-packed, tinned, frozen and artificially-preserved foods, in our Western civilization. The over- consumption of refined carbohydrates and the almost daily intake of 'fast foods', are aggravating factors. i) Toxins: Increasing pollution of the environment with chemicals such as fertilizers, insecticides and hydrocarbons. i) Overmedication: The vast array of drugs and medication being prescribed daily by doctors for every conceivable complaint.

The study of ecological illness in the United Kingdom, Europe and the United States, has shown that food and chemical sensitivities are often multiple. This can make diagnosis complex and difficult. Most doctors are not generally conversant with the concept that ecological illness can produce allergies with non-specific symptoms. A wide range of recurring symptoms, resulting in serious general malaise is often misunderstood.

Source: Griffiths S. 1996. Allergy overload. HarperCollins (Australia) Intro FoodEpigenome Method Implication

Diet (10b/10)

Bookmarking – Food intolerance:

• DNA-methylation & histone modifications; •HPA-axis;

…. for details see Epigenetics

Griffiths, 1996 Epigenetic conditioning

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For an in-depth role of blood in transbiosis see Epigenetics Lecture – The Ghost in the Gene ….. Intro FoodEpigenome Method Implication

Weightloss (1/12)

Yo-Yo-effect diet-induced weight loss and rebounce effect • there are alterations in the postprandial release of amylin and pancreatic polypeptide and, • changes in levels of leptin, ghrelin, peptide YY, gastric inhibitory polypeptide, pancreatic poly-peptide, amylin, and cholecystokinin, Mechanistic solution: • appetite-regulating medications will probably be required. Sumithran et al., 2011

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After weight loss, changes in the circulating levels of several peripheral hormones involved in the homeostatic regulation of body weight occur. Whether these changes are transient or persist over time may be important for an understanding of the reasons behind the high rate of weight regain after diet-induced weight loss. One year after initial weight reduction, levels of the circulating mediators of appetite that encourage weight regain after diet-induced weight loss do not revert to the levels recorded before weight loss. Long-term strategies to counteract this change may be needed to prevent obesity relapse. A multitude of hormones, pep- tides, and nutrients are involved in the homeostatic regulation of body weight, many of which are perturbed after weight loss. Leptin, an adipocyte hormone, is an indicator of energy stores and acts in the hypothalamus to reduce food intake and increase energy expenditure Ghrelin, peptide YY, gastric inhibitory polypeptide, GLP-1, cholecystokinin, pancreatic polypeptide, and amylin are released from the gastrointestinal tract and pancreas in response to nutrient intake; all but two inhibit intake. The exceptions are ghrelin, which stimulates hunger, and gastric inhibitory polypeptide, which may promote energy storage. Our study shows that after diet-induced weight loss, there are alterations in the postprandial release of amylin and pancreatic polypeptide and, more important, that changes in levels of leptin, ghrelin, peptide YY, gastric inhibitory polypeptide, pancreatic poly-peptide, amylin, and cholecystokinin, Given the number of alterations in appetite-regulating mechanisms that have been described so far, a combination of medications will probably be required. Image: Mean (+/-SE) Changes in Weight from Baseline to Week 62. The weight-loss program was started at week 0 and completed at week 10. ITT denotes intention to treat.

Source: Sumithran P, Prendergast LA, Delbridge E, Purcell K, Shulkes A, Kriketos A, Proietto J. (2011). Long-term persistence of hormonal adaptations to weight loss. N Engl J Med. Vol.365(17):1597-1604.

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Weightloss (2/12)

Yo-Yo-effect: Obesity trends • Body Mass Index (BMI) of 30 or higher, • obeise – but more so extremely obeise – seem to be carriers of Adenovisur 36

CDC, 2017

!

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The data shown in these maps were collected through CDC’s Behavioral Risk Factor Surveillance System (BRFSS). Each year, state health departments use standard procedures to collect data through a series of telephone interviews with U.S. adults. Height and weight data are self-reported. Prevalence estimates generated for the maps may vary slightly from those generated for the states by BRFSS as slightly different analytic methods are used.[1] In 1990, among states participating in the Behavioral Risk Factor Surveillance System, 10 states had a prevalence of obesity less than 10% and no state had prevalence equal to or greater than 15%. By 2000, no state had a prevalence of obesity less than 10%, 23 states had a prevalence between 20–24%, and no state had prevalence equal to or greater than 25%. In 2010, no state had a prevalence of obesity less than 20%. Thirty- six states had a prevalence equal to or greater than 25%; 12 of these states (Alabama, Arkansas, Kentucky, Louisiana, Michigan, Mississippi, Missouri, Oklahoma, South Carolina, Tennessee, Texas, and West Virginia) had a prevalence equal to or greater than 30%. [2] Image: Obesity Trends among US-adults between 1985 and 2010. (Body Mass Index, BMI: A measure of an adult’s weight in relation to his or her height, specifically the adult’s weight in kilograms divided by the square of his or her height in meters). The animation refers to BMI ≥30, or ~13kg overweight for a 1,6255 m tall person.

Source: [2] CDC (2006,‘08,‘10) State-specific prevalence of obesity among adults - United States, 2005,’07,‘10. MMWR Vol.55(36): 985–988. MMWR Vol. Vol.57(28): 765–768. MMWR Vol.59: 1–5. [1] (http://aps.nccd.cdc.gov/brfss) BMI-calculator: https://www.heartfoundation.org.au/your-heart/know-your-risks/healthy-weight/bmi- calculator

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Weightloss (3/12)

Yo-Yo-effect: Control Ad-36 CELO Experiment 1 (chickens) 23.07 69.23* 46.15 Obesity trends Experiment 2 (chickens) 18.1 63.6* • Body Mass Index (BMI) of 30 or higher, Experiment 3 (chickens) 12.5 70.0* • obese – but more so extremely obese – are Experiment 4 (mice) 22.22 60.0** likely to carry Adenovisur 36 *P<0.02; **P<0.05. Obesity was defined as greater than the 85th percentile of adiposity of the control group. Dhurandhar et al., 2000 Control Ad-36 CELO Number 13 13 13 Body weight [g] 502 18.2 538 18.2 527 16,0 Visceral fat [%] 0.27 0.05 0.54 0.07* 0.37 0.04 Total body fat [%] 5.80 0.60 6.90 0.10* 5.90 0.60 Cholesterol [mmol/L] 7.14 0.57 4.53 0.31* 5.58 0.61 Triglycerides [mmol/L] 0.84 0.05 0.69 0.03** 0.68 0.02* Mean sem.; *P_0.02; **P<0.05 compared to control.

Dhurandhar et al., 2002

Salehian et al., 2010

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Animals inoculated with Ad-36 developed a syndrome of increased adipose tissue and paradoxically low levels of serum cholesterol and triglycerides. This syndrome was not seen in chickens inoculated with CELO virus. Sections of the brain and hypothalamus of Ad-36 inoculated animals did not show any overt histopathological changes. Ad-36 DNA could be detected in adipose tissue, but not skeletal muscles of randomly selected animals for as long as 16 weeks after Ad-36 inoculation. Data from these animal models suggest that the role of viral disease in the etiology of human obesity must be considered.[1] Image: Experiment 1: infection of chickens with human adenovirus Ad-36 and Prevalence of obesity.[1] Cumulative body weight gains in marmoset monkeys after inoculation with human adenovirus Ad-36. Values are means SD, n=3 per group. *Different from control, P <0.05.[2] Tomography scans of a Ad36- infected patient, showing marked visceral adipose tissue in the abdomen (A) and thorax (B). Diffuse intrabadominal, retroperitoneal lipomatosis, and herniation of the mediastinum can be seen through the esophageal hiatus. Intrapericardial adipose infi ltration and adipose tissue bilaterally are seen within the pleura.[3]

Source: [1] Dhurandhar NV, Israel BA, Kolesar JM, Mayhew GF, Cook ME, Atkinson RL (2000) Increased adiposity in animals due to a human virus. Int J.Obesity Vol.24: 989-996 [2] Dhurandhar NV, Whigham LD, Abbott DH, Schultz-Darken NJ, Israel BA, Bradley SM, Kemnitz JW, Allison DB, Atkinson RL (2002) Human Adenovirus Ad-36 Promotes Weight Gain in Male Rhesus and Marmoset Monkeys. J Nutr., Vol.132: 3155-3160 [3] Salehian B, Forman SJ, Kandeel FR, Bruner DE, He J, Atkinson RL (2010) Adenovirus 36 DANN in Adipose Tissue of Patient with Unusual Visceral Obesity. Emerging Infectious Diseases Vol.16(5): 850-852

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Weightloss (4/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet • consists of i) low protein, i) low essential amino acid, i) low sugar diet, • Observed benefits: i) no loss of muscle mass i) visceral fat loss i)

…. can be induced with NR- supplements as well Canto et al, 2012 Schematics of the different actions of nicotinamide riboside in metabolic homeostasis

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As NAD+ is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38 - both NAD+ consumers - increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age- related disorders characterized by defective mitochondrial function …. Altogether, these results suggest that NR feeding increases mitochondrial biogenesis in a tissue-specific manner, consistent with the tissue-specific nature of the increases in NAD+ and sirtuin activity observed in NR-fed mice. The higher number of mitochondria, together with the different morphological mitochondrial profiles found in NR-fed mice would contribute to explain the higher oxidative profile, energy expenditure, and protection against metabolic damage observed upon NR feeding.

Image: Hypothesis by which NR supplementation would increase NAD+ content in key metabolic tissues, leading to SIRT1 and SIRT3 activation and the deacetylation and modulation of the activity of key metabolic regulators.

Source: Cantó C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y, Fernandez- Marcos PJ, Yamamoto H, Andreux PA, Cettour-Rose P, Gademann K, Rinsch K, Schoonjans K, Sauve AA, Auwerx J (2012) The NAD+ Precursor Nicotinamide Riboside Enhances Oxidative Metabolism and Protects against High-Fat Diet-Induced Obesity. Cell Metabolism, Vol.15(6): 838-847

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Weightloss (5/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet • consists of i) low protein, i) low essential amino acid, i) low sugar diet, • Observed benefits: i) no loss of muscle mass i) visceral fat loss i) no loss of muscle mass i) visceral fat loss. i) significant reduction in WBC i) increased mental “sharpness” i) learning & memory improves. i) slows aging i) Tayler, 2016; Choi et al., 2016

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Image: that cycles of a fasting mimicking diet (FMD) ameliorate disease severity by suppressing autoimmunity and stimulating remyelination via oligodendrocyte regeneration in multiple sclerosis (MS) mouse models. They also show that a similarFMDis a safe, feasible, and possibly a potentially effective treatment for patients with relapsingremitting MS.[1]

Source: [1] Choi Y, Piccio L, Childress P, Bollman B, Ghosh A, Brandhorst S, Suarez J, Michalsen A, Cross AH, Morgan TE, Wie M, Paul F, Bock M, Longo VD (2016) A Diet Mimicking Fasting Promotes Regeneration and Reduces Autoimmunity and Multiple Sclerosis Symptoms. Cell Reports , Volume 15 , Issue 10 , 2136 - 2146 [2] Taylor A (2016) Live Long Die Young. Catalyst, ABC (AUS) http://www.abc.net.au/catalyst/stories/4485468.htm

157 Intro FoodEpigenome Method Implication

Weightloss (6/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet • consists of i) low protein, i) low essential amino acid, i) low sugar diet, • Observed benefits: i) no loss of muscle mass 6 :4 i) visceral fat loss 13 0– i) no loss of muscle mass :5 19 i) visceral fat loss. o de i) significant reduction in WBCvi i) increased mental “sharpness” Tayler, 2016 i) learning & memory improves. Lee et al., 2012 i) slows aging i) Inflammation rates  & dermatitis  fasting mimicking diet.mp4 i) tumour incidents  (50% lower), or postponement & switching to more benign type 18-Mar-19 Madl 158

Thanks to everyone for your interest in the program. The fasting mimicking diet trialled in Prof Valter Longo's labs is produced by a private company called Prolon (Prof Longo does not receive profit from this company). Prolon does not distribute the product to Australia and there isn't a fresh food version released by Prof Longo's labs. However, the description of the fasting mimicking diet in their published journal paper lists a composition of "at least 9-10% proteins, 34-47% carbohydrates (plant based) and 44-56% fats (no animal fats). The amount of calories in total was around 700 - 800 per day. Unlimited herbal teas - no dairy, advised to drink lots of water. Each daily packet during my five day diet contained a nut bar for breakfast, a vegetable soup for lunch, a vegetable soup for dinner and afternoon snack consisted of 7 olives or some kale crackers. I am obviously not qualified to give medical or dietary advice and suggest you consult your doctor before undertaking any substantial change to your diet. This is a relatively strong intervention …. We saw a reduction in inflammation, dermatitis, we saw a major reduction in tumours, about 50% lower, but also a postponement and also the switching of the two modes from the more malignant type to more benign type. So it's really a triple effect. Then we also looked at cognitive function and they were improved in every measure that we looked at and they made less mistakes, so they were a lot sharper, they could learn and remember better. We also saw visceral fat loss. Another thing that was remarkable - there was no loss of muscle mass.[2] Image: A model for fasting-dependent sensitization of tumor cells to chemotherapy. In response to fasting, glucose, IGF-1, and other pro-growth proteins/factors (including oncogenes) are reduced in the serum. Malignant cells respond to this reduction by activating Akt/S6K. Notably, S6K can also be activated independently of Akt via energy-sensing pathways such as AMPK-mTORC1. These changes lead to an increase in oxidative stress, an increase in DNA damage, activation of caspase-3, and eventually cell death, particularly in the presence of chemotherapy/[1]

Source: [1] Lee C, Raffaghello L, Brandhorst S, Safdie FM, Bianchi G, Martin-Montalvo A, Longo VD (2012). Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy. Science Trans. Med., 4(124), 124ra27. http://doi.org/10.1126/scitranslmed.3003293 [2] Taylor A (2016) Live Long Die Young. Catalyst, ABC (AUS) http://www.abc.net.au/catalyst/stories/4485468.htm

158 Intro FoodEpigenome Method Implication

Weightloss (7/12) g un tj lu 0 Yo-Yo-effect B :5 & 07 Obesity trends t – al 0– r :2 Fasting Mimicking Diet U 06 • consists of i) low protein, i) low essential amino acid, i) low sugar diet, • Observed benefits: Mitochondrial dysfunction & Metformin Δ = 11% treating Type 2 Diabetes (T2D) 78241 patients treated w/ metformin 12222 patients treated w/ sulphonyl-urea 90463 matched non-diabetic controls T2D-patients treated w/ metformin had significantly longer survival rates ! Bannister et al., 2016

NAD+ NR(Vit B3) metformin 18-Mar-19 Madl 159

Clinical and observational studies have shown an increased risk of cardiovascular events and death associated with sulphonylureas versus metformin …. The objective of this study was therefore to compare all-cause mortality in diabetic patients treated first-line with either sulphonylurea or metformin monotherapy with that in matched individuals without diabetes …. We identified 78241 subjects treated with metformin, 12222 treated with sulphonylurea, and 90463 matched subjects without diabetes. This resulted in a total, censored follow-up period of 503384 years. There were 7498 deaths in total, representing unadjusted mortality rates of 14.4 and 15.2, and 50.9 and 28.7 deaths per 1000 person-years for metformin monotherapy and their matched controls, and sulphonylurea monotherapy and their matched controls, respectively …. Patients with type 2 diabetes initiated with metformin monotherapy had longer survival than did matched, non-diabetic controls. Those treated with sulphonylurea had markedly reduced survival compared with both matched controls and those receiving metformin monotherapy.[1]

Image: The Kaplan–Meier curves comparing metformin monotherapy with their matched control group without diabetes, sulphonylurea monotherapy with their matched control group without diabetes and patients aged 71–75 years at baseline for all four cohorts. Favouring metformin, these survival curves show that overall there was a small yet statistically significant difference between metformin cases and their non- diabetic controls (p=0.037). However those treated with sulphonylureas had markedly reduced sur- vival (p < 0.001) compared with their controls. Nicotinamide Adenine Dinucleotide (NAD)+ vs Nicotinamide Riboside (NR) vs metformin.[1] We report that per-oral NR, a vitamin B3 form and NAD+ precursor, effectively delayed mouse myopathy progression (MM). NR robustly induced mitochondrial mass and function and cured structural abnormalities of mitochondria, as well as delayed accumulation of mitochondrial DNA mutations. We show here that MM pseudo-starvation response is linked with a protective stress response, mitochondrial unfolded protein response, with induction of fasting cytokine FGF21.[2]

Source: [1] Bannister CA, Holden SE., Jenkins-Jones S, Morgan CL, Halcox JP, Schernthaner G, Mukherjee J Currie CJ (2014), Can people with type 2 diabetes live longer than those without? A comparison of mortality in people initiated with metformin or sulphonylurea monotherapy and matched, non-diabetic controls. Diabetes Obes Metab, Vol.16: 1165–1173. [2] Khan NA, Auranen M, Paetau I, Pirinen E, Euro L, Forsström S, Pasila L, Velagapudi V, Carroll CJ, Auwerx J, Suomalainen A (2014) Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3. EMBO Mol Med. Vol.6(6): 721–731.

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Weightloss (8/12)

Top: egg cell of a young ♀ mouse Yo-Yo-effect Obesity trends Bottom left: “post-menopausal” egg cell Fasting Mimicking Diet • consists of Bottom right: same but given Metformin as a supplement in i) low protein, drinking water i) low essential amino acid, i) low sugar diet, • Observed benefits: Mitochondrial dysfunction & aging "One week of NAD+ booster is sufficient to restore the mitochondrial homeostasis and key biochemical markers of muscle health in 22-month-old mice to levels similar to 6-month-old mice" 6 :4 Taylor, 201613 Massudiet al., 20120– :3 07 o de vi Longevity drug .mp4

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Image: NAD+ concentrations decline with age in males (blue) decrease significantly in males aged between 0–77 years (line a; p = 0.0007; n = 27). Pearson's correlation coefficient for a normally distributed population, r = −0.76’ and females likewiese decrease significantly with age (36–76) in post-pubescent females (p = 0.01; n = 22). Pearson's correlation coefficient for a normally distributed population,r = −0.537. An exponential (first-order) least squares fit was used to generate the nonlinear trend line.[1] Most of our experiments were done on male mice. They just happen to be biochemically simpler than females. But when studying female mice one notices that the old mice that were given metformin as a supplement were able to reproduce for longer. Here is an egg cell from a young mouse (top). It's the equivalent of a 20-year-old human female. What you see here in the blue is DNA which is wrapped up in these chromosomes and these chromosomes have to divide evenly between two cells. What happens during old age, the strings which attach normally to the chromosomes, which are in blue, are completely dysfunctional and disorganized (bottom left). And as a result, the offspring will either not have the correct number of chromosomes or won't even develop or become fertilised in the first place. And what we found is that by taking an old mouse and then feeding it with a supplement, we're able to completely normalise that (bottom right).[2]

Source: [1] Massudi H, Grant R, Braidy N, Guest J, Farnsworth B, Guillemin GJ (2012) Age- Associated Changes In Oxidative Stress and NAD+ Metabolism In Human Tissue. PLoS One, Vol.7(7): e42357. [2] Taylor A (2016) Live Long Die Young. Catalyst, ABC (AUS) http://www.abc.net.au/catalyst/stories/4485468.htm

160 Intro FoodEpigenome Method Implication

Weightloss (9/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet • consists of i) low protein, i) low essential amino acid, i) low sugar diet, • Observed benefits: s al Mitochondrial dysfunction & aging du vi + di n "One week of NAD booster is sufficient in a e th to restore the mitochondrial homeostasis es lly b ca and key biochemical markers of muscle o bi in ro lls e . health in 22-month-old mice to levels e na … , c a rg similar to 6-month-old mice" od ze u fo li rb f bo a u ta W Cancer is a shift away ssGomese et al., 2013→ a m ly from OXPHOS toward anaerobic glycolysis that provides cells with pl o al ur r t ic sufficient substrates for biomass (Warburg effect) s ie b to s ro e ea e u it a : d d 18-Mar-19Q fin Madl 161

Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1- dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear- mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.[1]

Image: A hallmark of cancer is a shift away from OXPHOS toward anaerobic glycolysis that provides cells with sufficient substrates for biomass. This metabolic reprogramming, known as the Warburg effect, is driven by several different pathways, including mTOR, c-Myc, and hypoxia- inducible factor 1 (HIF-1α). Interestingly, SIRT1 increases HIF-1α transcriptional activity, SIRT3 destabilizes HIF-1α protein, and SIRT6 functions as a HIF-1α corepressor, raising the possibility that HIF-1α may also be relevant to aging. Consistent with this, in C.elegans, Hif-1 regulates lifespan and the response to CR. A role for HIF-1α in mammalian aging, however, has not been explored yet.

Source: [1] Gomes AP, Price NL, Ling AJY, Moslehi JJ, Montgomery MK, Rajman L, White JP, Teodoro JS, Wrann CD, Hubbard BP, Mercken EM, Palmeira CM, deCabo R, Rolo AP, Turner N, Bell EL, Sinclair DA (2013) Declining NAD+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging. Cell. Vol.155(7): 1624–1638.

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Weightloss (10/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet • consists of i) low protein, i) low essential amino acid, i) low sugar diet, • Observed benefits: Mitochondrial dysfunction & gametes Infertility “when biochemical pathway becomes upset" 46 3: Imai & Guarente, 2014 1 0– Agarwal et al., 2012 :3 07 o de vi

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Hypoxia drives cellular metabolism towards glycolysis, which results in excessive production of lactate and pyruvate. However, conflicting results have been obtained by different investigators …. These biochemical changes can be explained by several mechanisms, for example, a reduced availability of glucose under anoxic conditions. Alternatively, toxic levels of NO can suppress the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, causing a subsequent reduction in the ratio of NADH to NAD+, which contributes to intracellular acidification. Finally, hypoxic suppression of the expression of aquaporin-9 (the major lactate influx channel between Sertoli cells and developing germ cells) might also account for the biochemical changes observed.[2] Image: Varicocele-induced sperm biochemical pathways of reactive oxygen species (ROS) generation. In the mitochondria, heat and hypoxic stress can directly activate complex III of the electron transport chain to release ROS. NO generated from testicular and endothelial cells in the testis with varicocele, can nitrosylate complexes-I and IV to promote excessive release of ROS by complex-III. In the sperm tail, where glycolytic units are present, NO can nitrosylate glyceraldehyde-3-phosphate dehydrogenase, contributing to intracellular acidification through reducing the ratio of NADH to NAD+ and reducing the production of lactate. SOD, superoxide dismutase.[2] Electron transport via NADH generates NAD+ in mitochondria and may decline with age. In young mitochondria, NADH, made by the citric acid cycle, readily donates its electrons to complex-I of the electron transport chain (ETC) and thereby generates NAD+. During aging, DNA damage accumulates in the nucleus, causing poly-ADP-ribose polymerase activation and NAD+ reduction. Consequently, SIRT1 activity is reduced, resulting in increased PGC-1α acetylation and decreased TFAM levels. These nuclear events reduce mitochondrial function in old mitochondria by affecting mitochondrial complex-I and other mitochondrial components, or blocking the entry of electrons from NADH into the ETC, thereby creating an NAD deficiency.[1]

Source: [1] Imai SI, Guarente L (2014) NAD+ and Sirtuins in Aging and Disease. Trends Cell Biol. Vol.24(8): 464–471. [2] Agarwal A, Hamada A, Esteves SC (2012) Insight into oxidative stress in varicocele-associated male infertility: part 1. Nature Reviews Urology Vol.9, 678–690 162 Intro FoodEpigenome Method Implication

Weightloss (11/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet Young blood • Heterochronic Parabiosis Joining an elderly with a young organism to induce “rejuvenation”; i.e: → regeneration of muscle tissue → hepatic steatosis reversed w/n days → reacquisition of vitality g un tj 6 cons: huge burden for the young lu :2 -B 19 → “aged” blood tends to be toxic lt – ra 5 → loss of intellectual skills U :0 16

Conboy et al., 2005, 2013

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The decline of tissue regenerative potential is a hallmark of ageing and may be due to age-related changes in tissue-specific stem cells. A decline in skeletal muscle stem cell (satellite cell) activity due to a loss of Notch signalling results in impaired regeneration of aged muscle. The decline in hepatic progenitor cell proliferation owing to the formation of a complex involving cEBP-alpha and the chromatin remodelling factor brahma (Brm) inhibits the regenerative capacity of aged liver. To examine the influence of systemic factors on aged progenitor cells from these tissues, we established parabiotic pairings (that is, a shared circulatory system) between young and old mice (heterochronic parabioses), exposing old mice to factors present in young serum. Notably, heterochronic parabiosis restored the activation of Notch signalling as well as the proliferation and regenerative capacity of aged satellite cells. The exposure of satellite cells from old mice to young serum enhanced the expression of the Notch ligand (Delta), increased Notch activation, and enhanced proliferation in vitro. Furthermore, heterochronic parabiosis increased aged hepatocyte proliferation and restored the cEBP-alpha complex to levels seen in young animals. These results suggest that the age-related decline of progenitor cell activity can be modulated by systemic factors that change with age.[2] In this review, we focus on the history of heterochronic parabiosis, methodological considerations and caveats, and the major advances that have emerged from those studies, including recent advances in our understanding of stem cell aging. Unlike transbiosis, there are no formal donor and host in parabiosis as each animal can be viewed as an equal partner in the pairing, each influencing the other parabiont.[1]

Image: Heterochronic parabiosis restores muscle regeneration and muscle stem cell activation in aged animals. a , Five days after injury, muscles from parabiotic mice were analysed for indices of regeneration and nascent myotube formation by conventional histological analysis (top panels: haematoxylin and eosin staining) and by immunostaining for eMHC (bottom panels: eMHC (red);

Source: [1] Conboy M, Conboy IM, Rando TA (2013) Heterochronic parabiosis: historical perspective and methodological considerations for studies of aging and longevity. Aging Cell, Vol.12: 525–530. [2] Conboy M, Conboy IM, Wagers AJ, Girma ER, Weissmann IL, Rando TA (2005) Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature Vol.433(7027): 760-764. http://conboylab.berkeley.edu/

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Weightloss (12/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet Young blood • Heterochronic Parabiosis • Transbiosis transfusion of key chemokines extracted from blood of young Young Elderly mice

g un tj 0 lu :0 -B 26 lt – ra 7 U :3 21

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In the central nervous system, ageing results in a precipitous decline in adult neural stem/progenitor cells and neurogenesis, with concomitant impairments in cognitive functions1. Interestingly, such impairments can be ameliorated through systemic perturbations such as exercise1. Here, using heterochronic parabiosis we show that blood-borne factors present in the systemic milieu can inhibit or promote adult neurogenesis in an age-dependent fashion in mice …. Accordingly, exposing a young mouse to an old systemic environment or to plasma from old mice decreased synaptic plasticity, and impaired contextual fear conditioning and spatial learning and memory. We identify chemokines—including CCL11 (also known as eotaxin)—the plasma levels of which correlate with reduced neurogenesis in heterochronic parabionts and aged mice, and the levels of which are increased in the plasma and cerebrospinal fluid of healthy ageing humans. Lastly, increasing peripheral CCL11 chemokine levels in vivo in young mice decreased adult neurogenesis and impaired learning and memory. Together our data indicate that the decline in neurogenesis and cognitive impairments observed during ageing can be in part attributed to changes in blood-borne factors.[1] Image: Systemic chemokine levels increase during ageing and heterochronic parabiosis, and correlate with decreased neurogenesis. a, Venn diagram of results from ageing and parabiosis proteomic screens. In grey are shown the seventeen age-related plasma factors that correlated most strongly with decreased neurogenesis, in red are shown the fifteen plasma factors that increased between young isochronic and young heterochronic parabionts, and in the brown intersection are the six factors elevated in both screens. Data from 5–6 mice per age group. b, c, Changes in plasma concentrations ofCCL11 with age (b) and young heterochronic parabionts pre- and post- parabiotic pairing (c). d, e, Changes in plasma (d; r50.40; P55.631027; 95% confidence interval 50.26–0.53) and CSF (e) concentrations of CCL11 with age in healthy human subjects. All data represented as dot plots with mean; *P,0.05; **P,0.01; ***P,0.001, t-test (c, e), ANOVA, Tukey’s post-hoc test (a, b), and Mann–Whitney U Test (d). Source: [1] Villeda SA, Luo J, Mosher KI, et al. (2011) The ageing systemic milieu negatively regulates neurogenesis and cognitive function. Nature 477:90-94. http://www.alkahest.com/about/management-team/ Brack AS, Conboy MJ, Roy S, et al. (2007) Increased Wnt signaling during aging alters muscle stem cell fate and increases fibrosis. Science. 317:807-810. Smith LK, He Y, Park JS, et al. (2015) β2-microglobulin is a systemic pro-aging factor that impairs cognitive function and neurogenesis. Nature Medicine. Villeda SA, Plambeck KE, Middeldorp J, et al. (2014) Young blood reverses age-related impairments in cognitive function and synaptic plasticity in mice. Nature Medicine 20:659-663. 164 Intro FoodEpigenome Method Implication

Weightloss (12/12)

Yo-Yo-effect Obesity trends Fasting Mimicking Diet Young blood • Heterochronic Parabiosis • Transbiosis transfusion of key chemokines extracted from blood of young Young Elderly mice

…. for details see g Modules un tj 0 BP & EMF lu :0 -B 26 lt – ra 7 U :3 21

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For an in-depth role of blood in transbiosis see …. i) Module-I, Biophotonics & Life i) Module-III, Electromagnetism in Biology

165 Intro Food Epigenome Method Implication

Preliminary Conclusion (1/2)

Biodynamic sustainable farming to promote good health: •agro-practices that respect biodiversity (practice w/o agro-chemical additives); •minimize consumption of processed food (incl. produce from intensive agro-farming practice); • grow & use seasonal / local produce only (get to know your grower); •reduce / eliminate animal proteins (eco-footprint ↓↓); •reduce waste - don’t wrap produce unless necessary, - avoid food contamination (leaking toxins f/ wrapper); •join the common-good-economy movement; Edaphon - 25 t biomass/ha Remiarz, 2017 Neuper & Pietsch, 2008 19-03-18 Madl 166

This presentation focusses on the enterprise potential of multi-species, multi-layer polycultures. Using examples from Britain, Europe and North America it explores how the food forest/forest garden concept can be adapted to commercial situations. This includes discussion of layout, plant selection, crop succession, management patterns, yield potential and marketing options. The presentation also highlights challenges that are commonly encountered by practitioners, ways that these challenges can be met and next steps in research and development.

Successful commercial polycultures in e.g. Austria: Langerhorst Mischkultur-Hof, Upper Austria - This family business has been cultivating their 2 ha (5 acres) smallholding since the late 1970s. The current pattern was established in 1989 and has not been greatly modified since then. One half of it is broadleaved woodland, the other half an intensive mixed polyculture of annuals and perennials. They have modified the linear pattern into a grid, with 20 x 20 metre “bays” around a central access path. Each bay is surrounded by a spine of fruit trees, with fruit bushes either side and herbs/strawberries below them along the outer edge. Inside the perennial belt, a clover path surround a 10 x 10 metre vegetable rotation. This layout provides a very sheltered spaces for annuals in the potentially harsh condition of the Austrian uplands. The market garden has largely been cultivated by hand since the beginning. Produce is consumed by the family, and the surplus is marketed via mail order to people across Austria who buy weekly or monthly boxes between 2 and 30kg, with an annual turnover of £20,000

Image: A healthy Edaphon - 25 t biomass per ha within 0,3m depth horizon (10 t bacteria & actinomycetes, 10 t fungi, 4 t earthworms, 1 t other soil dwelling organisms.

Source: Remiarz T (2017) Successful commercial polycultures. Conference: Real Farming Conference 2017, Oxford (UK); https://www.researchgate.net/publication/313038213_Successful_commercial_polycultures Neuper W, Pietsch H (2008) Lehr -& Foraschungsgemeinschaft fuer biodynamische Lebensfelder, Judenburg (AT) Biolandbau-Betrieb in Mischkultur - Margarete Langerhorst, Gugerling 5, A-4730 Waizenkirchen, Tel.: 0676 4604127 https://www.hope-theproject.com/the-film/ Intro Food Epigenome Method Implication

Preliminary Conclusion (2/2)

Eating habits to promote good health: (Slow food principles) • eat only when hungry; end)

• eat slowly and relax; –till 40 • don’t eat too many foods at once; 9: in TCM1 i (1: • do not mix raw fruits and vegetables; Q • always eat protein foods last;

• undereat whenever possible; Asian Food • don’t drink with meals; • don’t distract yourself while eating with other activities;

Griffiths, 1996

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Eating habits to promote good health Eat only when hungry: Hunger should be a comfortable feeling, resulting from the body needing food for energy, rather than from the ravenous cravings, caused by food allergy. Eat slowly and relax: Remember, saliva contains digestive enzymes. Thorough chewing considerably aids digestion and assimilation of food. Food broken down into fine particles by plenty of chewing, places much less strain on the digestive system. Eat small meals: It is better to eat five, or six, small meals per day, than three larger ones. If you eat smaller meals, your digestive system is not overloaded and, thus, operates more efficiently, nutrient absorption is much better and fat assimilation is less. Do not eat too many foods at once The fewer foods you eat at one meal, the better. Digestion and nutrient absorption is far more efficient. Do not mix raw fruits and vegetables: Both raw fruits and raw vegetables should be eaten separately. The reason is that totally different enzymes are needed to digest each one. Always eat protein foods last: Proteins require a generous amount of hydrochloric acid, in the stomach, to enable the enzymes to break them down effectively. This process takes about twice as long as that of carbohydrates. When these are eaten, little hydrochloric acid is secreted, as carbohydrates do not need much for digestion. Accordingly, they tend to pass much quicker into the duodenum. If carbohydrates are eaten after protein, they are held back in the stomach, by the protein, and fermentation commences. Undereat whenever possible: Systematic undereating is the greatest single aid to good health. Even the best possible diet, eaten to excess, will lead to food intolerances and degenerative disease. Over indulgence in high protein foods is especially harmful. Light meals lead to better digestion, greater utilisation of nutrients and, believe it or not, fewer hunger pangs. Do not drink with meals Although a glass of wine occasionally is harmless, generally, drinking with meals, or immediately before or after meals, should be avoided. The dilution of essential hydrochloric acid in the stomach, can have a detrimental effect on efficient digestion and absorption.

Source: Griffiths S. 1996. Allergy overload. HarperCollins (Australia)