Invited ReviewNanoparticles in food. Epigenetic changes induced by nanomaterialsand possible impact on healthBozena Smolkovaa, Naouale El Yamanib,c, Andrew R. Collinsc, Arno C. Gutlebd,Maria Dusinskab,*aDepartment of Genetics, Cancer Research Institute of Slovak Academy of Sciences, Bratislava, SlovakiabHealth Effects Laboratory, Department of Environmental Chemistry (MILK), NILU- Norwegian Institute for Air Research, 2027 Kjeller, NorwaycDepartment of Nutrition, University of Oslo, Oslo, NorwaydCentre de Recherche Public Gabriel Lippmann, Luxembourg, LuxembourgAR T I C L E I NF OArticle history:Received 27 September 2014Accepted 18 December 2014Available onlineKeywords:Nanomaterials in foodNanoparticlesEpigenetic effectsImpact on healthRisk assessmentA B S T R AC TDisturbed epigenetic mechanisms, which developmentally regulate gene expression via modicationsto DNA, histone proteins, and chromatin, have been hypothesized to play a key role in many human dis-eases. Recently it was shown that engineered nanoparticles (NPs), that already have a wide range ofapplications in various elds including food production, could dramatically affect epigenetic processes,while their ability to induce diseases remains poorly understood. Besides the obvious benets of the newtechnologies, it is critical to assess their health effects before proceeding with industrial production. Inthis article, after surveying the applications of NPs in food technology, we review recent advances in theunderstanding of epigenetic pathological effects of NPs, and discuss their possible health impact withthe aim of avoiding potential health risks posed by the use of nanomaterials in foods and food-packaging. 2014 Published by Elsevier Ltd.1. IntroductionNanoparticles can be of natural origin or man-made and are ubiq-uitous in the environment (Buzea et al., 2007; Smita et al., 2012).Nowadays engineered nanoparticles (NPs) are already included invarious consumer products including food, feed, biocides and vet-erinary drugs, as well as in applications such as agriculture, waterpurication techniques and soil cleaning (Bradley et al., 2011; Li et al.,2008; Peters et al., 2014; Sekhon, 2010; Wei et al., 2007). Withinthe food sector they are used as supplements and additives thatprolong shelf-life for fresh and processed products. They may alsoserve as nano-sensors to provide information about the food itemstorage history. Dietary exposure to NPs is also happening throughfood packaging where NPs can minimize carbon dioxide leakage frombottles and inhibit bacterial growth (Neethirajan and Jayas, 2011;Silvestre et al., 2011). Nanotechnology is furthermore applied toimprove food colouring and avouring and to encapsulate and laterspecically release nutritional additives. Traces of NPs have beenfound in vegetables, derived from the use of agrochemicals and fer-tilizers (Bouwmeester et al., 2009). The majority of the NPs in foodproduction reported a fewyears ago were non-biodegradable metalsand metal oxides (EPA, 2007). However, the recent inventory ofnanomaterials in food, feed and agriculture performed under theEuropean Food Safety Authority (EFSA) shows a trend from inor-ganic materials (metals, metal oxides, clay and full carbon materials)to organic nanomaterials such as nano-encapsulates andnano-composites. Themostusednanomaterialsatpresentarenano-encapsulates, silver and titanium dioxide (TiO2) (Peters et al.,2014).Exposure of NPs to biological molecules results in the forma-tion of a bio-corona, and this is an important factor in subsequentinteractions with organelles and macromolecules that nally mayresult in negative effects on cells (Ahluwalia et al., 2013). Apart fromcytotoxicity, cell death, oxidativestress, immunotoxicityandgenotoxicity (Dusinska et al., 2013), NPs can also induce more subtleepigenetic processes associated with aberrant gene expression (Yaoand Costa, 2013) or induce changes in the proteome (personal com-munication). Epigenetics covers heritable changes in the functionsof genes that occur without direct alteration in the DNA sequenceitself (Egger et al., 2004). Epigenetic mechanisms include DNA meth-ylation at specic sites in regulatory regions; a complex set of histonemodications, including acetylation, methylation, phosphoryla-tion, ubiquitination and ATP-ribosylation that lead to chromatinremodelling; and post-transcriptional changes of gene expressionmediated by miRNAs. It was shown that epigenetic changes can betriggered by environmental and lifestyle factors (Alegra-Torres et al.,2011; Jaenisch and Bird, 2003). Epigenetic marks can in some cir-cumstances be transferred into the subsequent generation by the* Corresponding author. Health Effects Laboratory, Department of EnvironmentalChemistry (MILK), NILU- Norwegian Institute for Air Research, 2027 Kjeller, Norway.Tel.: +4763898157.E-mail address: mdu@nilu.no (M. Dusinska).http://dx.doi.org/10.1016/j.fct.2014.12.0150278-6915/ 2014 Published by Elsevier Ltd.Food and Chemical Toxicology (2015) ARTICLE IN PRESSPlease cite this article in press as: Bozena Smolkova, Naouale El Yamani, Andrew R. Collins, Arno C. Gutleb, Maria Dusinska, Nanoparticles in food. Epigenetic changes induced bynanomaterials and possible impact on health, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2014.12.015Contents lists available at ScienceDirectFood and Chemical Toxicologyj our nal homepage: www. el sevi er. com/ l ocat e/ f oodchemt oxQ2Q4Q11234567891011121314151617181920212223242526272829303132333435 3637383940414243444546474849505152535455565758596061626364656667686970717273747576777879808182838485868788899091process oftransgeneration inheritance (Jablonka and Raz, 2009;Skinner et al., 2013).A growing body of evidence suggests that epigenetic modica-tions have an important role in a subset of human diseases, includingcancer, neurological, autoimmune, cardiovascular and metabolic dis-eases (reviewed in Portela and Esteller, 2010; Nilsson and Skinner,2014). The observation that epigenetic changes are reversible makesthem an attractive target for disease prevention and treatment.2. Uses of NPs in the food industryNanotechnology has been used in the food industry for manyyears without considering it as nanotechnology an example beinglow-fat products such as mayonnaise in which emulsied fat drop-letsarediluted bytheinclusioninthemofnano-sizedwaterdroplets with no effect on taste, when compared with the high-fatversionoftheproduct. NPscangivebenetssuchasbetterdispersibility of water-insoluble additives in food products withoutthe use of additional fat or surfactants. Nano-texturing of food-stuffstargetsthedevelopmentof nano-structuresandstableemulsions to improve consistency, taste, avour and texture attri-butes. Major applications of NPs can be found in the health-foodsector, where nano-sized supplements and nutraceuticals have beendeveloped to improve health and well-being. The focus in Asiancountries is on development of micronized starch, cellulose, wheatand rice our, and a range of spices and herbs for herbal medicineand food applications (Tsukamoto et al., 2010). Recent research foundthat foods with caramelized sugar, including bread and corn akes,contain carbon NPs (Sk et al., 2012).Uses of nanomaterials in the food industry include food addi-tives, food ingredients, food contact materials, novel food, avourings,enzymes, and supplements (Table 1). An extensive literature searchand inventory of EFSA resulted in the selection of 779 relevant ref-erences to nanomaterials used in the food, agriculture and feedsectors. Of these, 633 nanotechnology applications described physico-chemical characteristics of nanomaterials, reporting product namesand related suppliers; and 55 types of nanomaterials in 12 differ-ent applications were identied with the most common applicationas food additives and food contact materials (Peters et al., 2014).The EU FP7 project ObservatoryNANO (http://www.observatorynano.eu) collected information about existing nano-patents on food; 35 patents were found worldwide. Nanotechnologyhas a wide range of potential applications in the food processing,fromdelivery and formulation to nano-sensors and nano-tracers forfood safety (Prakash et al., 2013). Given the fact that the consum-er is so intimately exposed to food, the food industry faces manychallengesinintroducingthistechnologyintotheirrepertoire.However, for some applications, such as protection of certain labilecompounds in food or the addition of compounds with generallyunacceptable taste into relative stable lipid materials, nanotech-nology may be a way to proceed (Souto and Severino, 2013).In the food industry, synthetic amorphous silica (E551) has beenused for many years to clear beers and wines, as an anti-caking agentto maintain the ow properties of powder products, and to thickenpastes. The conventional bulk form of TiO2 is approved as an ad-ditive for food use (Mura et al., 2013). However, EUFDA, US-FDA,and FAO/WHO regulations do not require manufacturers to specifythe particle size on the label of consumer products (Dekkers et al.,2010; Peters et al., 2012; Tran and Chaudhry, 2010) and it is likelythat in some cases such food grade additives may contain nanosizedparticles whose properties differ from those ofthe conventionalchemical form.TiO2, silicon dioxide (SiO2) and magnesium oxide NPs are usedin food to preserve colour or durability and to improve handling.TiO2, aluminum silicate and SiO2 NPs, for example, are added asanticakingagentstopowderedproducts(invendingmachine Table1Overviewofnanoparticlesandnanomaterialspresentinfoodandfoodpackagingintentionallyandunintentionally.NanoparticleapplicationsinfoodNanoparticlesappliedinfoodpackagingNanoparticlesunintentionallypresentinfoodFoodadditives/ingredientsNovelfoodFlavouringsFoodenzymesFoodsupplementContactmaterialsNanocapsulestoimprovedispersion,bioavailabilityandabsorptionofnutrientsNanomaterialsascolourenhancersNano-structuresinstableemulsionstoimproveconsistency,taste,avourandtextureattributesNanoparticlesforselectivebindingandremovalofchemicalsandpathogensfromfoodNanomaterialsasanti-cakingagents,ortothickenpastesNanoparticlesinpackagingmaterialsNano-sizednutraceuticalstoimprovehealthandwell-beingMicronizedstarch,cellulose,wheatandriceour,andarangeofspicesandherbsforherbalmedicineandfoodapplicationsNano-encapsulatedavourenhancersCarriersforenzymeimmobilizationandbasedproteinseparation-purication.(e.g.allowingenzymestobeeasilyreusedmultipletimesforthesamereactionwithlongerhalf-livesandlessdegradation)Stableenzymebasedcoatingorspraysinfoodsupplyinfrastructure(e.g.enzyme-basedlistericidalnanocompositecreatingaspecialcoatingtoselectivelykillharmfulbacteria)Nano-encapsulationfortargeteddeliveryofnutraceuticalsNano-sensorsforthefooditemstoragehistoryorasoxygensensorsNano-tracersforfoodsafetySolidlipidNPsincombinationwithfunctionalfoodtoincreasestabilityandprotectionfromdegradationinthegastricsystemCalciumandmagnesiumsaltsNPs,ironbasedNPs,ashealthsupplementsNanoparticlesuspensionsasantimicrobialsNanomaterialsimmersedingelsasediblelmstocoatfoodNanoparticlestodetectchemicalsoffoodbornepathogensBiodegradablenanosensorsfortemperatureandmoisturemonitoringNanoclaysandnanolmsasbarriermaterialstopreventspoilageandoxygenabsorptionNanoparticlesforantimicrobialandantifungalsurfacecoatingsNanomaterialsfromagricultureuse(Pesticides,fertilizerssuchasbiosolids)Nanomaterialsasfeedadditives,supplementsandfeedenzymesVeterinarydrugsBiocidesFoodcontaminantsfromothersources(NMsinwater,soil,foodprocessing,air,etc.)ARTICLE IN PRESSPlease cite this article in press as: Bozena Smolkova, Naouale El Yamani, Andrew R. Collins, Arno C. Gutleb, Maria Dusinska, Nanoparticles in food. Epigenetic changes induced bynanomaterials and possible impact on health, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2014.12.0152 B. Smolkova et al./Food and Chemical Toxicology (2015) Q51234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071powders, milk and cream powder substitutes, cheese and sugar).Variousmaterialssuchasnanoclays, cellulosicnanomaterials,electrospun nanobres and nanocapsules, carbon-basednanomaterials, NPs of metal and metal oxides, and NP-containingcarriers all nd application in food packaging (Ahmadi et al., 2009;Monteiro-Riviere et al., 2011; Nemmar et al., 2008; Roblegg et al.,2012).TiO2 nanocomposites act as oxygen sensors and could be usedas packaging lms for a variety of oxygen-sensitive food products.Their major drawback is the requirement of UVA light for their ac-tivation (Azeredo, 2009). Another example of the application of TiO2is in polypropylene lms to inhibit the growth of microorganisms.As an additive, TiO2is labelled as E171 but it is not specied whetheror not it is nanosized. In fact, when E171 was analysed, a consid-erable fraction was found to have dimensions below100 nm. As TiO2is used as a white colourant, typical uses of TiO2 are in confection-ery, white-coloured sauces and dressings, and non-dairy creamers(Lomer et al., 2002).Nanotechnology offers new possibilities to develop food-baseddelivery systems for a range of labile or sensitive compounds rangingfrom bioactive natural compounds to pharmaceuticals, thereby po-tentially improving health as such systems may result in increasedbioavailability of the active compounds (McClements, 2013). Suchfunctional compounds can be incorporated into solid lipid NPs re-sulting in increased stability and protection fromdegradation in thegastrointestinal tract (GIT) (Weiss et al., 2008). Overall the high po-tential of such technologies has not yet been fully exploited.It has been estimated that TiO2 NPs were produced at a level of5000 t/year in 2010. Altogether 58 000 tons of NPs are expected tobe produced by 2020. Unfortunately, it is very dicult to estimatethe real production volumes of NPs. Apart fromEFSA external report(Peters et al., 2014) only scientic reports containing admittedly wildguesses are available (Landsiedel et al., 2010).US Patent (US5741505) describes the potential application of na-noscale inorganic coatings directly on food surfaces to provide abarrier to moisture and oxygen, and thus to improve shelf life and/or the avour impact of foods (Mura et al., 2013). The materials usedfor the nanocoatings, intended to be applied in a continuous processas a thin amorphous lm of50 nm or less, include TiO2, silicondioxide and magnesium oxide (Ma et al., 2010).AluminiumNPsareusedtoimprovethefunctioningofalu-miniumfoil. It acts as an ecient barrier for gases (CO2and oxygen),and has a UV screening effect. It is also used as an anti-adhesivecoating to prevent sticking and for black coating of baking foil sothat it does not reect heat in the oven. Also in nanopackaging, com-positelayerscontainingnanoplateletsof clayminerals, e.g.,montmorillonite, have been used since 1986 when the process wasinvented by Toyota Corporation. Different layers in nanopackagingslow down the passage of gases. Such polymers have been appliedalso to produce plastic bottles (Miller Brewing Company) that areimpermeable to O2 and CO2 (Duncan, 2011).Another important application is related to the fact that bioactivecompounds with low resorption rates may be more bioavailablewhen present in a nanosized form (Oehlke et al., 2014; Rao et al.,2013). Selenium NPs are being marketed as an additive to a greentea product, with a number of (proclaimed) health benets result-ing from enhanced uptake of selenium (Mura et al., 2013). Calciumsalts NPs are the subject of patent applications (Sustech GMBH &Co, Dsseldorf, Germany; http://www.nanomat.de) for intended usein chewing gums. Calcium and magnesium salts NPs, as well as ironbased NPs, are also available as health supplements (Mura et al.,2013).While food grade nanomaterials have a high likelihood to enterwaste (water) streams following digestion and excretion (Yang et al.,2014), at the same time TiO2 NPs are used as a photocatalysts inwater treatment applications, especially to oxidize heavy metals andorganic pollutants, and to kill microbial pathogens (Liu et al., 2014).Iron-based NPs are also used in the treatment of contaminated water,where they are claimed to decontaminate water by breaking downorganic pollutants and killing microbial pathogens (Scherer et al.,2000).Silver NPs (Ag NPs) are used as an antibacterial agent, or as e-noses to detect food degradation while not being in direct contactwith the foodstuffs (Tung et al., 2014). Ag NPs release toxic ions,which could contaminate food. It is even likely that we regularlyingest Ag NPs that are shed fromour forks, spoons and knives (Gloveret al., 2011).Ag NPs immersed in gels (e.g., alginate) so called edible lms have been used to coat food products such as carrots, asparagus,etc. Such treated food has less water loss and higher consumer ac-ceptability. Ag NPs immersed in cellulose pads placed in contact withbeef were shown to reduce the microbial loads in meat extracts.Copper and zinc oxides are still mostly used in scientic studies,but are currently being targeted also at food applications.Another aspect of engineered NPs in food that needs to be men-tioned is the fact that the detection of NPs is still not straightforward.No validated methods or suitable reference standards are avail-able for detecting, identifying and quantifying nanomaterials incomplex matrixes such as food. Food structure is highly complexand variable and many types of NPs exist. No single, universally ap-plicable method is expected to be developed. The methods to choosefrominclude but are not limited to Inductively Coupled Plasma MassSpectrometry (ICP-MS), High Performance Liquid Chromatogra-phy (HPLC), Field FlowFractionation (FFF), Atomic Force Microscopy(AFM), Transmission Electron Microscopy (TEM) etc. The fact thatphysical, chemical and biological transformations may alter surfaceand other properties of the nanomaterials, only further increasesthe challenge for analytical determinations of nanomaterials in food(Szakal et al., 2014).There are also NPs that are not intended to enter the food supply,but because of their minuscule size they can slip through waste-water treatment and take up residence in the biosolids created atthe end of the wastewater treatment process. These biosolids arelater applied to elds as fertilizer for their nitrogen and phospho-rus content, and so food may also contain traces of NPs (Buzea et al.,2007).There is concern about possible release of NPs from packagingmaterial. A study by Avella et al. (2005) analysed vegetables thatwere in contact with nanopolymer packaging: no increase in mineralcontent of the vegetables was observed. On the contrary,nanopolymer packaging lm was effective in decreasing the mi-gration of optical whitener 2,5-thiophenediylbis(5-tert-butyl-1,3-benzoxazole) into food products. In another study, Simon et al. (2008)reported that only NPs of 1 nm are able to diffuse from packagingmaterial to food, while bigger particles do not migrate. Ag or ZnOantimicrobials were found to enter orange juice from antimicro-bialpackagingmaterial(Emamifar, 2011). AgNPsinpackagingmaterial may release Ag ions into the exudates of meat containedin it (Fernndez et al., 2010).3. Food as vehicle for NPsThe main physiological routes of NP uptake are inhalation, dermalabsorption or ingestion; NPs can be distributed to the same organby several routes of exposure. The extent of NP exposure is affect-edbytheircharacteristics, inuencingabsorption, metabolism,distribution and excretion (reviewed in Hagens et al., 2007). NPsentering the GIT are separated fromblood by epithelial barriers. Theycan reach the capillaries in the connective tissue under the epithe-lial layer covering the intestines, which can lead to translocationinto secondary organs. Uptake of NPs by healthy orogastrointestinalmucosa appears to be low but absorption differs between variousARTICLE IN PRESSPlease cite this article in press as: Bozena Smolkova, Naouale El Yamani, Andrew R. Collins, Arno C. Gutleb, Maria Dusinska, Nanoparticles in food. Epigenetic changes induced bynanomaterials and possible impact on health, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2014.12.0153 B. Smolkova et al./Food and Chemical Toxicology (2015) Q6123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132types of GIT cells; for instance it is higher in the small intestine thanin the oral cavity (Roblegg et al., 2012).Previous investigations have found that TiO2 accumulates in theintestine in rats (Jani et al., 1994) and sh (Zhang et al., 2007) andmigrates to other organs. Accumulation of TiO2 inside the intesti-nal cells, especially in lymphoid-rich areas (Peyers patches), mightlead to damaging outcomes such as inammation and could be in-volved in the pathogenesis of inammatory bowel disease (Evanset al., 2002; Lomer et al., 2002).Lesions and inammation may lead to increased uptake of NPsfromtheGIT. Alsowithoutpenetrationintothebody, NPsinintraepithelial compartments can trigger inammatory responsesandcompetitionbetweenfoodNPswithnormal uptake(re-viewed by Frhlich and Roblegg, 2012).For the estimation of a health risk of ingested NPs we have totake into account that they are suspended in biological uids suchas the mucus of saliva and they are affected by pH, ionic strength,protein, sugar and lipid content that can change their surface prop-erties and size (Frhlich and Roblegg, 2012). When NPs reach thesystemic circulation they can interact with plasmaproteins, coag-ulation factors, platelets and red and white blood cells as well aswith organelles and DNA of the cells.Although penetration of NPs into the human body appears tobe low, long-term exposure can cause accumulation of NPs due totheir limited excretion rate. Commonly used toxicity screening isbased on testing rather high-dose short exposures. In the humanbody more often long-term low-dose exposures occur, and so ac-cumulation of minor changes rather than acute toxicity is probable.Currently we lack knowledge about the biological effects of com-bined, long-term NP exposure.4. Epigenetic processes affected by NP exposureEpigenetic regulation involves complex interacting structures thatalong with genetic information in DNA allow sophisticated, time-and tissue-specic control of gene expression, with major conse-quencesforcellfatedecision, inbothnormalandpathologicaldevelopment (Jenuwein and Allis, 2001). Although it is widely ac-ceptedthatepigeneticmechanismsarekeyregulatorsofgenetranscription, withabilitytorespondtoenvironmentalsignals(Jaenisch and Bird, 2003), there are still many gaps in our under-standing ofthe role ofindividual epigenetic processes and theirinteractions in health and disease.4.1. DNA methylationDNA methylation is a postreplication modication that predomi-nantly involves the covalent addition of a methyl (CH3) group, inmammalsalmostexclusivelyfoundonthe5 positionsofcy-tosines in the dinucleotide sequence CpG. CpG dinucleotides in thegenome are generally methylated except for CpG clusters locatedin the promoter region and rst exon of many genes. CpG islandshave been found in all known housekeeping genes and some tissue-specic genes (Larsen et al., 1992). Methylation/demethylation ofCpG islands is important for maintenance of cell- or tissue-specicgene expression; as a general rule, methylation in promoters sup-pressestranscription. Methylationpatternsarecell-ortissue-specicandarereversible. DNAmethylationplaysaroleinembryogenesis, imprinting, X chromosome inactivation and main-taining genome integrity (Holliday and Pugh, 1975; Reik, 2007; Riggs,1975). Global hypomethylationhasbeenassociatedwithin-creased chromosome instabilities and oncogenesis and is commonlyaccompanied by gene-specic hypermethylation of CpG islands. Epi-genetic inactivation of tumour suppressors or activation of oncogenesleads to unregulated cell growth and carcinogenesis. Recently re-portedgenebodymethylationor so-calledintragenic DNAmethylation that is not located in the promoter region of a gene (re-viewed in Shenker and Flanagan, 2012) might stimulate transcriptionelongation and might have an impact on splicing. The role of DNAmethylation in altering the activities of enhancers, insulators andotherregulatoryelementsisonlyjustbeginningtobeunder-stood. Patterns of DNA methylation are generated duringdevelopment involving de novo methylation and demethylation ac-tivities. DNAmethyltransferases(DNMTs)areresponsibleformaintaining methylation status in the genome, and catalyse thetransfer of a methyl group from S-adenosyl-l-methionine (SAM) tothe cytosine of a CpG dinucleotide. A family of DNMTs is involvedindenovoDNAmethylationandits maintenance. Recently5-hydroxymethyl-2-deoxycytidine (hmdC) has been discovered asa marker of methylation in the brain (Kriaucionis and Heintz, 2009)and is suggested to play a role in the epigenetic regulation of neu-ronal function. 5-hydroxymethylcytosine(5hmC)isthemostabundant of 5-methylcytosine (5mC) oxidation products. The bio-logical role of 5hmC is still unclear. Current models propose that5hmCis anintermediatebaseinanactiveor passiveDNAdemethylation process that operates during important reprogram-ming phases of mammalian development. Tumour tissues have beenshown to have strongly depleted levels of 5hmC (Pfeifer et al., 2013).Even though the role of aberrant DNA methylation in rare andcomplex disorders is well documented, still many key questionsremain unanswered. One of the most intriguing is the interactionbetween the various epigenetic mechanisms (Portela and Esteller,2010).4.2. Histone modicationsIn the nucleus, DNA is associated with histone proteins in thecomplex that constitutes chromatin. The nucleosome is 147 bp ofDNA wrapped around an octameric core of histone proteins con-sisting of two H3-H4 histone dimers surrounded by two H2A-H2Bdimers. The N-terminal histone tail protrudes fromnucleosomes intothe nuclear lumen. Nucleosome spacing determines chromatin struc-ture, broadly described as hetero- and eu-chromatin. Histones canbe post-transcriptionally modied to restructure chromatin in manyways, including acetylation, methylation, phosphorylation,ubiquitination, sumoylation and ATP-ribosylation (Bannister andKouzarides, 2011; Suganuma and Workman, 2008). The histone codehypothesis suggests that different combinations of histone modi-cations may regulate chromatin structure and transcriptional status(Jenuwein and Allis, 2001). Post-translational modications of his-tones lead to changes in chromatin structure that can inuencetranscription. The most common modications are acetylation andmethylation on histone lysine residues. Increased acetylation resultsin open chromatin conguration and transcription activation, whilemethylation of histones can result either in repression or activa-tion of transcription (Rice and Allis, 2001). Histone modicationsare extremely dynamic and highly regulated by a complex of histone-modifying enzymes including histone acetyltransferases (HATs),deacetylases (HDACs), histone methyltransferases (HMTs) and histonedemethylases (HDMs). Their aberrant functions cause misregulationof chromatin structure and activity with impact on access of tran-scription factors to DNA and gene transcription.4.3. RNA interferenceRNA interference is a process of posttranscriptional control ofprotein expression. One type of molecule involved in this processis microRNA (miRNA) whose role is to regulate gene expression byinterfering with mRNA processes, affecting mRNA stability, target-ing mRNA for degradation or both; or in rare cases it can also increasegene transcription (Mathers et al., 2010). miRNAs can also be in-volved in establishing DNA methylation and may inuence chromatinARTICLE IN PRESSPlease cite this article in press as: Bozena Smolkova, Naouale El Yamani, Andrew R. Collins, Arno C. Gutleb, Maria Dusinska, Nanoparticles in food. Epigenetic changes induced bynanomaterials and possible impact on health, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2014.12.0154 B. Smolkova et al./Food and Chemical Toxicology (2015) 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132structure by regulating histone modiers. Eukaryotic cells use RNAinterference to silence transposable elements after transcription, pro-tecting the cell against genomic damage. More than 1050 miRNAhave been identied up to nowin human cells with impact on almostall genetic pathways, through their effects on transcription factors,receptorsandtransporters(Esquela-KerscherandSlack, 2006;Griths-Jones et al., 2008). The aberrant expression of miRNAs hasbeen linked to various human diseases, including cancer, Alzheim-ers disease and heart disease (Montgomery and van Rooij, 2010;Olive et al., 2010; Provost, 2010).5. Epigenetic effects of NPs in vitro and in vivoAlthough emerging evidence indicates that environmental factorsaffect epigenetic patterns and disease risk (reviewed by Ho et al.,2012), epigenetic effects of NPs have been examined only rarely (re-viewed by Stoccoro et al., 2013; Mytych and Wnuk, 2013). Initiallythe focus of research was on the abilities of studied NPs to changegene expression, without analysing the relationship with epigen-etic modications. Gene expression studies were performed witha wide range of NPs showing that certain NPs can impair the ex-pression of genes involved in DNA methylation reactions leadingto changes in global as well as gene specic DNA methylation. Up-and down-regulation of DNA damage response genes, cell cycle pro-gression, DNArepair, metalmetabolism, proteinfoldinggenes,inammatory markers and many other effects were shown (Asharaniet al., 2012; Cui et al., 2012; Dawson and Kouzarides, 2012; Gao et al.,2012; Gojova et al., 2009; Gui et al., 2013; Moos et al., 2011; Parket al., 2008). Elevated oxidative stress, lipid peroxidation, mem-brane damage and reduced antioxidant levels were also associatedwith exposure to NPs (Li et al., 2011a; Lin et al., 2006).Generally, there are two main mechanisms through which en-vironmental factors including NPs can affect DNA methylation. Firstis a decreased availability of methyl donors and second is an alteredactivity of the DNMT enzymes. Histone marks can be affected byaltered abundance and/or ecacy of the enzymes responsible fortheir modication and changed availability ofthe enzyme sub-strate (McKay and Mathers, 2011).Stoccoro and colleagues summarized the epigenetic effects of NPs.They showed the potential of NPs to induce global DNA methyla-tionchanges, aswellaschangesofgene-specicmethylationpatterns, including tumour suppressor genes (APC, p16, RASSF1A, p53),inammatory genes (iNOS, IFNG, IL4), DNA repair genes (PARP-1),and impaired expression of genes involved in DNA methylation re-actions (DNMT1, DNMT3A, DNMT3B, MBD2), all potentially relevantto cancer development. These authors also showed that exposureto nanomaterials induced changes in the acetylation and methyla-tion ofthe histone tails and global or gene-specic alteration ofmiRNA expression (Stoccoro et al., 2013).An altered global DNA methylation pattern was associated withpro-oxidative properties of NPs. Oxidative damage to DNA may affectthe ability of DNMTs to interact with DNA (Simk et al., 2011). More-over, reactive oxygen species (ROS) can alter the expression of genesthat are regulated by DNA methylation (Fratelli et al., 2005).SiO2 NPs are used as anticaking agents in food or packaging ma-terials where their direct contact with food allows penetration intothe human body. SiO2 NPs were reported to show size- and dose-dependent cytotoxicity, cellular uptake, to increase ROS levels andto be pro-inammatory (Napierska et al., 2010). In vivo studies dem-onstrate largely reversible lung inammation, granuloma formationandfocal emphysema. SiO2NPswerefoundtoinduceglobalhypomethylation in the immortalized epithelial HaCaT cell line inassociation with a dose-dependent decrease in DNMT1, DNMT3a andmethyl-CpG binding protein 2 (MBD2) gene and protein expres-sions (Gong et al., 2010). The DNA-binding domain protein familythatincludesalsoMBD2, bindstomethylatedDNAandsup-presses transcription from a methylated target gene, promotinginteractions with other proteins (Bogdanovic and Veenstra, 2009).SiO2 NP treatment induced also global hypoacetylation implying aglobal epigenomic response (Gong et al., 2010). A decrease in themRNAexpressionofpoly(ADP-ribose)polymerase-1(PARP-1), apivotal repair enzyme, was associated with PARP-1 genehypermethylation (Gong et al., 2012) following SiO2 NP exposure.PARP-1 modies various nuclear proteins including histones bypoly(ADP-ribosyl)ation, thus inducing local relaxation of the chro-matin and facilitating the access of repair proteins to damaged DNA.PARP1 detects and relocates to single strand breaks or nicks in chro-mosomal DNA playing an important role in the initiation of the DNArepair pathway. PARP-1, was found in a complex with DNMT1, thehistone H3K9 methyltransferase G9a and the histone ubiquitin ligaseNp95, indicative of a link between poly(ADP-ribosylation) and theepigenome (Shall and de Murcia, 2000). Low expression of PARP-1is thought to be involved in carcinogenesis. High levels of activa-tion are associated with increased apoptosis in response to genotoxicstress (Kauppinen and Swanson, 2007).Gold NPs (Au NPs), frequently used in food packaging, bever-ages, toothpaste and Au NPs based biomedical products, are beingdeveloped for drug delivery, cancer therapy, diagnostic devices, andbiosensing. Au NPs were found to penetrate the nucleus and mod-ulate heterochromatin connections with lamin proteins and corehistones (Mazumder and Shivashankar, 2007). Chromatin conden-sation and reorganization was also observed in the nucleus of humanfoetal lung broblasts exposed to Au NPs (Ng et al., 2011). In thesame study Au NPs exposure signicantly altered the expression of19 genes; among themup-regulation of miR-155 was observed con-comitant with down-regulation of the PROS1 gene with no changesin methylation. PROS1 codes for S protein that acts as a cofactor inprocesses controlling blood clotting. Au NPs were also found to de-crease HDAC activity by binding to sulfhydryl groups on the surfaceof HDAC8 (Sule et al., 2008). Balansky and colleagues showed thatAu NPs produced signicant changes in microRNA expression andtransplacental size-dependent clastogenic and epigenetic effects inthe mouse foetus. The authors reported dys-regulated expressionof 28 out of 1281 miRNAs in prenatally exposed mouse foetal lungand 5 up-regulated miRNAs in foetal liver. Let-7a and miR-183 weresignicantly up-regulated in both organs (Balansky et al., 2013). Up-regulation of miR-183 expression was recently correlated with lungcancer incidence (Vsa et al., 2013).TiO2 NPs are one of the most commonly used NPs in food in-dustry as anticaking agents or for antimicrobial plastic packaging.The majority of in vitro studies with TiO2 NPs show increased levelsof DNAbreaks and insome studies oxidized DNAlesions(Magdolenova et al., 2014). However, the induction of DNA damagedepends on the conditions in which NPs are dispersed and the sec-ondary physical-chemical properties of TiO2NPs. Magdolenova et al.(2012) tested the same TiO2NPs in two different agglomeration stateson TK6 human lymphoblastoid cells, EUE human embryonic epi-thelial cells and Cos-1 monkey kidney broblasts, using the cometassay. Depending on the state of agglomeration different genotoxicresponses were obtained. The TiO2 NPs, in dispersion with large ag-glomerates, induced DNA damage in all three cell lines, while theTiO2NPs dispersed with agglomerates less than 200 nmhad no effecton genotoxicity (Magdolenova et al., 2012).Treatment of HaCaT cells with TiO2 NPs caused dysfunction ofthe methylation cycle and methionine deciency (Tucci et al., 2013).As the methionine cycle has a crucial role in the availability of methylgroups for methylation processes in living cells, deregulation of thecyclecanhaveadramaticimpactonglobal aswell asgene-specic DNA methylation particularly during embryogenesis. GlobalDNA hypomethylation and hypermethylation of tumour suppres-sorpromotershavebeenconsistentlyassociatedwithcancerinitiation and progression. Several miRNA were signicantly alteredARTICLE IN PRESSPlease cite this article in press as: Bozena Smolkova, Naouale El Yamani, Andrew R. Collins, Arno C. Gutleb, Maria Dusinska, Nanoparticles in food. Epigenetic changes induced bynanomaterials and possible impact on health, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2014.12.0155 B. Smolkova et al./Food and Chemical Toxicology (2015) 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132by TiO2NP exposure (miR-449, miR-1 and miR-135b) (Halappanavaret al., 2011).The main ndings of in vitro studies analysing epigenetic effectof NP exposure on various cell types are summarized in Table 2.An increasing number of studies reporting NP-induced epigen-etic alterations call for implementation of new testing methods toallow their safety assessment. To achieve this, there is a need forbetterunderstandingoftheiradverseepigeneticeffectsandtoimprove currently available technologies to study DNA methyla-tion, histone modications and non-coding RNAs. There are severallarge-scale initiatives to study epigenomic organization (Greally andJacobs, 2013). We hope these studies will provide insights into howepigenomes are organized and regulated, and how the three majormechanisms of epigenetic regulation interact to inuence gene ex-pression. Among them, DNA methylation has been the most widelystudiedtodate andthere are a number of techniques for5-methylcytosinequantication. Methodsbasedonsodiumbisulphite sequencing, pyrosequencing or quantitative real time PCR(qPCR)arecommonlyusedbutmajorlimitationsoftheseap-proaches when used for screening purposes are that they are labour-intensive and low-throughput. Large-scale studies of the epigenome,based on microarray technology or massive parallel sequencing(MPS), on the other hand, allow acquisition of a huge amount ofdata;buttherearestillunsolvedtechnicalandnancialprob-lems. The analysis challenge is also substantial and newbioinformatics tools for data analysis and integration, using high-performance computing resources with cloud computing will becrucial. However, the trend of MPS is towards rapidly growing ca-pacity with decreasing costs which makes it a promising new toolfor future epigenomic studies. Regulation of chromatin organiza-tion is part of the mechanism for rapid activation or silencing ofgene expression. Several methods have been developed to analysethe state of histone modications, including chromatin immuno-precipitation (ChIP), ChIP coupled with microarray hybridization(ChIP-chip) and ChIP coupled with next generation sequencing (ChIP-seq). ChIP-based assays are largely qualitative with more complexsample preparation which is the major reason for limited use of thismethodcomparedwithDNAmethylationanalyses. miRNAre-search techniques include three major methods; miRNA microarrayplatforms, qPCR and MPS. Use of these tools has strengths and limi-tations (infrastructure, processing time, costs). Although there arecertain methods available to measure toxicant-induced changes inmiRNA, our current understanding of these processes is limited. Agoal of many ongoing studies is to be able to dene optimal methodsfor integrating different types of genome-wide data in order to beable to understand epigenomic and transcriptional regulation andto consider these endpoints in hazard identication and productsafety management (Rasoulpour et al., 2011).6. Implications for human healthThe recent ndings that common genetic variants alone tend notto identify causal loci of complex diseases or predict individual sus-ceptibility opens the possibility that epigenetic factors as functionalmodiers of the genome are key determinant of disease risk (Gibson,2012; Heyn and Esteller, 2012; Petronis, 2010). During early de-velopment epigenetics plays a key role in embryogenesis controllingcell differentiation while later in development it regulates tissue-specic protein expression. Epigenetic patterns not only vary fromtissue to tissue but alter with advancing age and are sensitive toenvironmental exposures. They are mitotically inheritable and there-fore can be long-lasting or passed to the next generation. The conceptof continued editing of early-life epigenetic markings or memo-ries has been proposed by Tang et al. (2008). According to thishypothesis epigenetic marks gained during the prenatal period areedited over the life course and may modify disease susceptibility.The reversible character of epigenetic patterns and their respon-sivenesstonutritionalandenvironmentalfactorsthusgiveusopportunities for prevention, delay or treatment of complex dis-eases (Hou et al., 2012).The role of aberrant epigenetic processes has been well estab-lished in several developmental and neurobehavioural disorders. Therst group comprises diseases whose aetiologies include deregu-lationofoneormoreimprintedgenes;theyincludeAlbrightshereditary osteodystrophy, Angelman, BeckwithWiedemann, andPraderWilli syndromes (Nicholls, 2000). The second group of dis-eases are those whose aetiologies include aberrant methylation ofrepeated sequences or gene-specic sequences such as ATR-X, FragileX and ICF syndromes. Syndromes whose aetiology involves muta-tions in the genes related to epigenetic regulation, such as DNAmethyltransferase 3b (DNMT3b) mutations in ICF syndrome, mu-tations of CpG binding protein 2 (MeCP2) gene in Rett syndrome,Table 2Main ndings of in vitro studies analysing epigenetic effects of nanoparticle (NP) exposure on various cell types.NP Cells Effect of NPs exposure ReferenceSiO2 Immortalized epithelial HaCaT cell line Global hypomethylation; dose-dependent decrease in DNMT1,DNMT3a and methyl-CpG binding protein 2 (MBD2) gene andprotein expressionsGong et al., 2010HaCaT cell line PARP-1 hypermethylation and repression of gene expression Gong et al., 2012CdTe QDs THP1 human monocytic cell line Binding to core histones and stimulation of aggregate formation Conroy et al., 2008MCF-7 human breast adenocarcinoma cells Histone 3 hypoacetylation and chromatin decondensation leadingto reduction in global gene transcription especially for anti-apoptotic genes; increase in p53 protein level by its activation viaphosphorylation, nuclear and mitochondrial translocationChoi et al., 2008NIH/3T3 mouse embryonic broblast line Global alteration of miRNAs expression patterns resulting in theapoptosis-like cell deathLi et al., 2011bAu HeLa cervical cancer cell line Modulation of heterochromatin connections with lamin proteinsand core histonesMazumder andShivashankar, 2007Enzymatic method Decrease of histone deacetylase activity by NPs binding tosulfhydryl groups on the surface of HDAC8Sule et al., 2008MRC5 lung broblast line Upregulation of miR-155 with downregulation of PROS1 gene,chromatin condensationNg et al., 2011TiO2 HaCaT cells Dysfunction of methylation cycle and methionine deciency Tucci et al., 2013MWCNTs NIH/3T3 cell line Changes of various miRNAs expression Li et al., 2011aSuper paramagnetic Fe Human prostate cancer cells PC-3 and humanbreast cancer cells Sk-Br-3K-182 HDACI-coated cationic NPs resulted in an increase in geneexpression and core histone hyperacetylationIshii et al., 2009Different cancer cell lines(U87, U373, Lipari, DF)Cholesterylbutyrate solid lipid NPs releasing butyric acid havebeen shown to act as histone deacetylase inhibitorsBrioschi et al., 2008ARTICLE IN PRESSPlease cite this article in press as: Bozena Smolkova, Naouale El Yamani, Andrew R. Collins, Arno C. Gutleb, Maria Dusinska, Nanoparticles in food. Epigenetic changes induced bynanomaterials and possible impact on health, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2014.12.0156 B. Smolkova et al./Food and Chemical Toxicology (2015) 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109mutationsinCREBBPgene(histoneacetylation)inRubinsteinTaybi syndrome and mutations in RSK-2 gene (histonephosphorylation) in ConLowry syndrome, constitute the lastgroup of inherited disorders with epigenetic aetiology. Most ofthese disorders are associated with mental retardation.Despite the large body of evidence for an association betweenenvironmental exposure and epigenetic alterations, data support-ing the link between epigenetic variation and common complexdisease phenotypes, with the exception of cancer, are still missing.Cancer was the rst human disease linked to epigenetics (Feinbergand Vogelstein, 1983). Since then several recent discoveries pointto a genome-scale disruption of the epigenome that involves largeblocks of DNA hypomethylation, mutations of epigenetic modiergenes and alterations of heterochromatin. Epigenome-modifyinggene mutations include histone variants; DNMTs; HATs; HDACs;HMTs; HDMs; and chromatin remodelling factors, which can inducechanges in euchromatin and heterochromatin. Surprisingly, the mu-tation frequencies of epigenome modiers in cancer are quite highin haematopoietic malignancies and rare solid tumours and epi-genetic mutations also frequently occur in cancers that relapse orthat are otherwise resistant to therapy (Timp and Feinberg, 2013).Teschendorff and colleagues recently showed that the variability ofDNA methylation was more predictive of cancer progression thanwere mean changes in DNA methylation, suggesting a fundamen-tal disruption in the mechanismfor maintaining epigenetic integrityin cancer cells (Teschendorff et al., 2012). This has important im-plications for cancer diagnosis and therapy.The role of DNA methylation in the function of the CNS (Guo et al.,2011) and ageing-related cognitive decline (Chouliaras et al., 2013;Oliveira et al., 2012; Sanchez-Mut et al., 2013) has recently beenhighlighted. Aberrant DNA methylation was also associated with os-teoarthritis (Fernndez-Tajes et al., 2014) and cardiovascular disease(Kim et al., 2013), pre-eclampsia (Anderson et al., 2013), rheuma-toid arthritis (Liu et al., 2013) and metabolic disorders (Bruce andCagampang, 2011; Kirchner et al., 2013). Epigenomic alteration inimmune function is associated with systemic lupus erythemato-sus, with T lymphocyte DNA demethylation contributing to lupusare severity (Sawalha et al, 2012).Studying the role of deregulated epigenetic mechanisms causedby NP exposure in disease pathogenesis, we have to challenge manyobstacles. First of all epigenetic alterations are cell type- and tissue-specic. DNA methylation patterns were shown to reect cell lineagesrather than inter-individual differences (Reinius et al., 2012). We haveto consider the impact oflong-term exposures and their conse-quences for patterns of gene expression, cell function and death.Induction of oxidative stress, inammation and inhibition of anti-oxidant defence mechanisms could be induced by NP exposure. Theterm exposome refers to the summation of all exposures expe-rienced by an individual over a lifetime. Another issue is instabilityand changes in behaviour ofNPs in the heterogeneous environ-ment ofbiological systems including food. Nevertheless, ndingepigenetic biomarkers may allow for estimation of past exposureand may have application in identifying high-risk individuals ordiagnostics.7. Risk assessment of nanomaterial in foodLegislation and regulation of nanomaterials in food in Europe-an Union (EU) and non-EU countries was reviewed on the basis ofa literature research and a questionnaire by Peters et al. (2014). Alegal basis for regulating nanomaterials and the application of nano-technologyinthefoodindustryhasbeensetup. In2011theEuropean Commission published a Recommendation on the de-nitionofananomaterial(2011/696/EU(EuropeanCommission,2011)) and recently also a draft law on novel food (COM/2013/0435 (European Commission, 2013)) was released. Additionally theEFSA Scientic Committee published guidance on the risk assess-ment of the application of nanoscience and nanotechnologies in thefood and feed chain (EFSA Scientic Committee, 2011). These docu-ments provide a framework for regulation of nanomaterials in thefood sector. In a similar way as for chemical safety, the strategy forhazard assessment of nanomaterials is based on using standard testsrecommended by Organisation of Economic Co-operation and De-velopment (OECD) guidelines.The current absence of standardized assays and analytical ap-proaches to test epigenetic effects of NPs is a reason for concern.There are many obstacles in the implementation and validation ofthe tests and there remains a need for further fundamental re-searchtoallowamorerobustbasisforOECDTestGuideline(OECD TG) recommendations. At the beginning there is a need toimprove our knowledge on the links between modulation of theepigenome by chemical compounds, including nanomaterials, andthe phenotype.8. ConclusionNotwithstanding the potential advantages of new technologiesutilizing nano-sized materials, NPs may cause undesirable hazard-ousinteractionswithbiologicalsystems. Theconsequencesofepigenetic changes induced by exposure to NPs and their causal re-lationship with complex diseases are still poorly understood. Inparticular, the possible epigenetic effects of NPs present in food haveas yet hardly been investigated. Development of new test guide-line recommendations for hazard identication and product safetymanagement, including epigenetic endpoints, will be possible onlyafter a further increase in our understanding of the normal vari-abilityoftheepigeneticlandscapeandcausalitybetweenNPexposure-induced epigenetic alterations and their consequences forthe phenotype.Conict of interestThe authors declare that there are no conicts of interest.Transparency documentThe Transparency document associated with this article can befound in the online version.AcknowledgementsThis work was supported by the European Commission FP7 proj-ects NANoREG, [NMP.2012.1.3-3], Contract no. 310584andQualityNano [INFRA-2010-1.131], Contract no: 214547-2.ReferencesAhluwalia, A., Boraschi, D., Byrne, H.J., Fadeel, B., Gehr, P., Gutleb, A.C., et al., 2013.The bio-nano-interface as a basis for predicting nanoparticle fate and behaviourin living organisms: towards grouping and categorising of nanomaterials andnanosafety by design. 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