Histone modifying enzymes involved in metastasis.
\\n\\n
Dr. Pletser’s experience includes 30 years of working with the European Space Agency as a Senior Physicist/Engineer and coordinating their parabolic flight campaigns, and he is the Guinness World Record holder for the most number of aircraft flown (12) in parabolas, personally logging more than 7,300 parabolas.
\\n\\nSeeing the 5,000th book published makes us at the same time proud, happy, humble, and grateful. This is a great opportunity to stop and celebrate what we have done so far, but is also an opportunity to engage even more, grow, and succeed. It wouldn't be possible to get here without the synergy of team members’ hard work and authors and editors who devote time and their expertise into Open Access book publishing with us.
\\n\\nOver these years, we have gone from pioneering the scientific Open Access book publishing field to being the world’s largest Open Access book publisher. Nonetheless, our vision has remained the same: to meet the challenges of making relevant knowledge available to the worldwide community under the Open Access model.
\\n\\nWe are excited about the present, and we look forward to sharing many more successes in the future.
\\n\\nThank you all for being part of the journey. 5,000 times thank you!
\\n\\nNow with 5,000 titles available Open Access, which one will you read next?
\\n\\nRead, share and download for free: https://www.intechopen.com/books
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Preparation of Space Experiments edited by international leading expert Dr. Vladimir Pletser, Director of Space Training Operations at Blue Abyss is the 5,000th Open Access book published by IntechOpen and our milestone publication!
\n\n"This book presents some of the current trends in space microgravity research. The eleven chapters introduce various facets of space research in physical sciences, human physiology and technology developed using the microgravity environment not only to improve our fundamental understanding in these domains but also to adapt this new knowledge for application on earth." says the editor. Listen what else Dr. Pletser has to say...
\n\n\n\nDr. Pletser’s experience includes 30 years of working with the European Space Agency as a Senior Physicist/Engineer and coordinating their parabolic flight campaigns, and he is the Guinness World Record holder for the most number of aircraft flown (12) in parabolas, personally logging more than 7,300 parabolas.
\n\nSeeing the 5,000th book published makes us at the same time proud, happy, humble, and grateful. This is a great opportunity to stop and celebrate what we have done so far, but is also an opportunity to engage even more, grow, and succeed. It wouldn't be possible to get here without the synergy of team members’ hard work and authors and editors who devote time and their expertise into Open Access book publishing with us.
\n\nOver these years, we have gone from pioneering the scientific Open Access book publishing field to being the world’s largest Open Access book publisher. Nonetheless, our vision has remained the same: to meet the challenges of making relevant knowledge available to the worldwide community under the Open Access model.
\n\nWe are excited about the present, and we look forward to sharing many more successes in the future.
\n\nThank you all for being part of the journey. 5,000 times thank you!
\n\nNow with 5,000 titles available Open Access, which one will you read next?
\n\nRead, share and download for free: https://www.intechopen.com/books
\n\n\n\n
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\r\n\tNeoplasia is considered to be the result of aberrations in the homeostatic mechanisms regulating cell turnover. This may be due to a combination of genetic, epigenetic and stochastic factors. Drug resistance is one of the major reasons for treatment failure and tumor relapse. Hence, an improved understanding of the mechanisms of neoplastic growth is possible based on systematic cataloging of the druggable targets for each of the major hallmarks of cancer. This approach can also aid in the refinement and validation of the in vitro and in vivo models, as well as provide pointers for the development of novel systems. Genomic instability and mutation, as well as tumor-promoting inflammation should be included as underlying factors influencing the process of neoplasia along with factors dysregulating energetics. The focus of this book is to update the reader on an integrated perspective including epigenetics data and corroborative mechanistic evidence from multiple model systems including humans. This will enable the reader to better comprehend the current scenario in terms of the pharmacological aspects pertaining to cancer chemoresistance. Also, the challenges for the molecular oncology will be discussed as well as probable strategies and a road-map for cancer chemotherapeutic drug development.
",isbn:null,printIsbn:null,pdfIsbn:null,doi:null,price:0,priceEur:null,priceUsd:null,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"2a80fe34c552bb6ca76ef9cd8f21e377",bookSignature:"Dr. Suresh P.K.",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8640.jpg",keywords:"Molecular Mechanisms of Aberrations, Proliferation Mechanisms, Types of Cell Death, Signal Transduction Pathways, Angiogenesis in Cancer, Angiogenesis in Wound Healing, Molecular Mechanisms of Invasion, Molecular Mechanisms of Metastasis, Telomeres, Stem Cells, Delivery Challenges, Evasion Mechanisms",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"November 5th 2018",dateEndSecondStepPublish:"November 26th 2018",dateEndThirdStepPublish:"January 25th 2019",dateEndFourthStepPublish:"April 15th 2019",dateEndFifthStepPublish:"June 14th 2019",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"190244",title:"Dr.",name:"Suresh",middleName:null,surname:"P.K.",slug:"suresh-p.k.",fullName:"Suresh P.K.",profilePictureURL:"https://mts.intechopen.com/storage/users/190244/images/system/190244.jpeg",biography:"P.K. Suresh is a Professor at the School of Biosciences & Technology, VIT, Vellore (2009 to date). He has approximately 18.5 years of teaching, research and administrative experience in Biotechnology & Industrial Biotechnology and allied disciplines. He had also headed Biotechnology & Industrial Biotechnology departments and has handled a wide variety of theory papers. He has 45 research publications in SCOPUS-indexed journals and has completed 3 funded projects as the Principal Investigator. He has made several presentations at International conferences. He has guided 5 doctoral students (2 as co-guide) and is currently guiding 5 students in the doctoral program. He has organized and participated actively in several Faculty Development Programs in areas as diverse as Stem Cells, Bio-inspired Design and Pharmacokinetics and was also a resource person in these events.",institutionString:"School of Biosciences & Technology",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"Vellore Institute of Technology University",institutionURL:null,country:{name:"India"}}}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"6",title:"Biochemistry, Genetics and Molecular Biology",slug:"biochemistry-genetics-and-molecular-biology"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"177731",firstName:"Dajana",lastName:"Pemac",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/177731/images/4726_n.jpg",email:"dajana@intechopen.com",biography:"As a Commissioning Editor at IntechOpen, I work closely with our collaborators in the selection of book topics for the yearly publishing plan and in preparing new book catalogues for each season. This requires extensive analysis of developing trends in scientific research in order to offer our readers relevant content. Creating the book catalogue is also based on keeping track of the most read, downloaded and highly cited chapters and books and relaunching similar topics. I am also responsible for consulting with our Scientific Advisors on which book topics to add to our catalogue and sending possible book proposal topics to them for evaluation. Once the catalogue is complete, I contact leading researchers in their respective fields and ask them to become possible Academic Editors for each book project. Once an editor is appointed, I prepare all necessary information required for them to begin their work, as well as guide them through the editorship process. I also assist editors in inviting suitable authors to contribute to a specific book project and each year, I identify and invite exceptional editors to join IntechOpen as Scientific Advisors. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"61944",title:"The Landscape of Histone Modification in Cancer Metastasis",doi:"10.5772/intechopen.78383",slug:"the-landscape-of-histone-modification-in-cancer-metastasis",body:'Approximately 90% of cancer deaths are caused by metastasis [1]. Cancer metastasis is an exceedingly complex process involving tumor cell motility, intravasation, and circulation in the blood or lymph system, extravasation, and growth in new tissues and organs [2, 3]. During invasion, tumor cells lose cell–cell adhesion, gain mobility and leave the site of the primary tumor to invade adjacent tissues. In intravasation, tumor cells penetrate through the endothelial barrier and enter the systemic circulation through blood and lymphatic vessels. In extravasation, cells that survive the anchorage-independent growth conditions in the bloodstream attach to vessels at distant sites and leave the bloodstream. Finally, in metastatic colonization, tumor cells form macrometastases in the new host environment [2, 3]. All of these steps, from initial breakdown of tissue structure, through increased invasiveness, and ultimately distribution and colonization throughout the body, are developmental characteristics of the processes, epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET). EMT is a distinctive morphogenic process that occurs during embryonic development, chronic degeneration and fibrosis of organs, and tumor invasion and metastasis [4, 5, 6]. The similarity of genetic controls and biochemical mechanisms that underlie the acquisition of an invasive phenotype and the subsequent systemic spread of cancer cells highlights the concept that tumor cells usurp this developmental pathway for metastatic dissemination. In total, EMT provides tumor cells with the proclivity for early metastasis, renders them resistance to therapeutics and endows cells with cancer stem cell (CSC)-like traits [6].
The hallmark of EMT is the loss of E-cadherin expression, an important caretaker of the epithelial phenotype. Loss of E-cadherin expression is often correlated with the tumor grade and stage because it results in the disruption of the cell–cell adhesion and an increase in the nuclear β-catenin. Several transcription factors have been implicated in the regulation of EMT, including the zinc finger proteins of the SNAIL family (SNAIL1/2/3), the basic helix–loop–helix (HLH) factor TWIST (TWIST1/2, E12/E47), and two double zinc finger and homeodomain ZEB family (ZEB1/ZEB2). These factors act as a molecular switch for the EMT program by repressing a subset of common genes that encode cadherins, claudins, integrins, mucins, plakophilin, occludin and ZO1, and thereby induce EMT.
EMT is a dynamic process that preserves plasticity [6]. In this instance, the reprogramming of gene expression provides a rapid and dynamic regulatory mechanism to switch between the epithelial and mesenchymal conditions during cancer progression. Consistent with this, these EMT-activating transcriptional factors (EMT-TFs) are liable proteins that turn over rapidly and do not have long residence times at their binding sites. Interestingly, disseminating cells orchestrate a metastatic cascade without a concomitant need for genomic mutations, which indicates that this dissemination is epigenetically templated. Both EMT and epigenetic modification (DNA methylation and histone modifications) are dynamic and efficient processes during development, differentiation and carcinogenesis. These studies indicate that the epigenetic mechanism plays an important role in modulating the induction of EMT and tumor metastasis.
The term “epigenetics” was first coined by Conrad H. Waddington in his Principles of Embryology textbook in 1942 to designate a process in which gene regulation modulated development. The final definition of epigenetics was confirmed in the Epigenetic Meeting held by the Banbury Conference Center and Cold Spring Harbor Laboratory in 2008 as “a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.” In general, epigenetic regulation includes changes that impact histone modification, DNA methylation, histone variants, chromatin looping, noncoding RNAs and nucleosomal occupancy and remodeling.
Genomic DNA is tightly packaged in chromatin by both histone and nonhistone proteins in the nucleus of eukaryotic cells. The basic chromatin subunits, nucleosomes, are formed by wrapping 146 base pairs (bp) of DNA around an octamer of four core histones: H2A, H2B, H3, and H4. Whereas the nucleosomal core is compact, eight flexible lysine-rich histone tails protrude from the nucleosome that modulate internucleosomal contacts and provide binding sites for nonhistone proteins. From the perspective of gene transcription, chromatin structure can be divided into two distinct categories: euchromatin and heterochromatin. “Euchromatin” is an open chromatin structure that affords accessibility of transcription factors to DNA, resulting in gene activation. In contrast, “heterochromatin” is a closed chromatin structure with a low interaction between transcription factors and the genome, leading to gene repression.
The histone code hypothesis was first proposed by Strahl and Allis in 2000. They suggested that “multiple histone modifications, acting in a combinatorial or sequential fashion on one or multiple histone tails, specify unique downstream functions” [7]. The histone “language,” based on this “histone code,” is encoded in these modifications and read by chromatin-associated proteins. So far, several histone post-translational modifications (PTMs) have been identified, including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP ribosylation, proline isomerization, biotinylation, citrullination and their various combinations [8]. These modifications constitute a unique “code” to regulate histone interactions with other proteins and thereby allow modification (either overcoming or solidifying) of the intrinsic histone barrier to transcription. Accordingly, with these modifications, the various proteins that add, recognize and remove these PTMs, termed writers, readers and erasers, respectively, have been identified and structurally characterized. While “writer” and “eraser” enzymes modify histones by catalyzing the addition and removal of histone PTMs, respectively; “reader” proteins recognize these modified histones and ‘translate’ the PTMs by executing distinct cellular programs. In addition, numerous core histone chaperones also facilitate core histone deposition or removal from chromatin. Histone modifications control dynamic transitions between transcriptionally active or silent chromatin states, and regulate the transcription of genetic information encoded in DNA (the “genetic code”) [9]. Analyses of genome-wide profiles of histone modifications and gene expression identified three distinct types of configurations: repressed, active and bivalent. First, the closed chromatin configuration is linked with suppression of gene transcription, the repressed state. Second, an open chromatin configuration is associated with active gene transcription, the active state. Third, bivalent chromatin consists of domains that have both repressive and active histone markers, predominately on developmental genes, which allows phenotypic plasticity before committing to a specific cell fate.
During EMT, histone modifications provide a regulatory platform to orchestrate the repression or activation between epithelial and mesenchymal genes. Here, we only focus on the well-studied histone acetylation and methylation, and discuss their diverse regulation and role in transcriptional reprogramming of tumor metastasis (Table 1).
Histone modifying enzymes involved in metastasis.
Evidence has established that histone acetylation is associated with gene activation. A genome-wide study demonstrated that all forms of histone acetylation are positively correlated with gene expression [10]. Histones contain amino acids with basic side chains that are positively charged and attracted to the negatively charged genomic DNA. Ultimately, histone acetylation reduces the positive charge on histones and decreases the interaction between nucleosomes and DNA. Generally, histone acetylation is greater in the promoters of active genes and influences both the initiation and elongation of gene transcription. Histone acetylation also stabilizes the binding of chromatin remodeling factors at promoter regions and induces nucleosomes unfolding as well as reduces nucleosome occupancy. The acetylation state of a chromatin leads to the structural modification of the nucleosome. Acetylated (or hyperacetylated) chromatin is in a relaxed confirmation and associated with active transcription. In contrast, deacetylated (or hypoacetylated) chromatin is condensed and supercoiled, and is associated with transcriptional silencing (and, in the context of cancer, the inhibition of tumor suppressor genes).
Histone acetylation is a rapid and reversible process controlled by histone acetyltransferases (HATs) and histone deacetylases (HDAC)s. The HATs transfer acetyl groups from acetyl-coenzyme A (CoA) to the ε-amino groups of lysine residues in histone tails, which results in gene activation. HATs contain a bromodomain that recognizes and binds to acetylated histones, categorized into three major families, GNAT (GCN5 and PCAF), MYST (Tip60 and MOF), and CBP/p300. The HDACs remove acetyl groups from lysine residues, leading to gene silencing. Sequence homology, subcellular location, and the features of the catalytic site have been used to classify the 18 members of the human HDAC family into 4 groups: class I (HDACs 1, 2, 3, and 8), class II (HDACs 4, 5, 6, 7, 9, and 10), class III (SIRT1, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, and SIRT7), and class IV (HDAC11) [11]. Class I HDACs have sequence homology to class II HDACs and class IV HDACs but not class III HDACs. Class I, II, and IV HDACs are zinc-dependent, whereas class III HDACs are nicotinamide adenine dinucleotide (NAD)+-dependent. Genome-wide mapping of the binding of HATs and HDACs to the human genome demonstrate that these enzymes regulate the activation and repression of transcription, respectively. The dysfunctional balance between acetylation and deacetylation is clearly associated with human disease and tumorigenesis.
p300 cooperates in an epigenetic manner with a DOT1L-c-Myc complex to induce EMT in breast metastasis [12]. The elevated level of p300-DOT1L-c-Myc is associated with the acquisition of CSC-like properties during breast carcinogenesis, which implies that p300 functions as a potential oncogene to influence the clinical outcome of breast cancer. In addition, transforming growth factor-beta (TGF-β) and WNT co-operated to mediate EMT. TGF-β induces the translocation of β-catenin to the nucleus where it binds to T- cell Factor (TCF); this complex recruits p300/CBP to assemble a transcriptional complex on target gene promoters that promotes EMT signaling. Intriguingly, over-expression of SNAIL/SLUG up-regulates TGF-β-receptor 2 (TGFBR2) expression with an increase of H3K9 acetylation on TGFBR2 promoter to increase TGF-β signaling [13]. In contrast, however, p300 was reportedly recruited by the hepatocyte nuclear factor (HNF) 3 to the E-cadherin promoter, increasing expression, and thus reducing the metastatic potential of breast cancer cells [14]. Similarly, the p300-CBP-associated factor (PCAF) has functions that can differ among cancer types. PCAF is an anti-oncogene and its expression is down-regulated and negatively correlated with tumor metastasis in hepatocellular carcinoma (HCC) [15]. This complex plays an important role in suppressing EMT and HCC metastasis and by targeting Gli1 [16]. However, it was also reported that PCAF acetylates the enhancer of zeste homolog 2 (EZH2) at K348 to augment EZH2 stability, and thus promotes lung cancer cell migration and invasion [17]. These reports indicate that the role of PCAF is context-dependent. In several breast cancer cell lines, hMOF catalyzes promoter H4K16 acetylation, which is critical to maintain expression of EMT-related tumor suppressor genes [18]. Consistent with this, MOF also acetylates the histone demethylase lysine-specific histone demethylase 1 (LSD1), to suppress EMT, indicating that MOF is a critical suppressor of EMT and tumor progression [19]. Recently, we found that Tip60 appears to be an important regulator of TWIST activity by acetylating at H3K73 and H3K76 of the GK-X-GK motif, resulting in an interaction between BRD4 and TWIST, hence promoting the aggressiveness of basal-like breast cancer (BLBC) [20].
Dysfunctional class I HDAC expression and activity is associated with cancer metastasis. HDAC1 regulates invasiveness by increasing matrix metalloproteinase (MMP) expression. Furthermore, HIF-2α is a transcriptional regulator of the HDAC1 gene, and hypoxia increase HIF-2α and HDAC1 expression [21]. TGF-β-driven E-cadherin silencing and EMT in human pancreatic cancer cells also depend on HDAC activity [22]. HDAC1 and HDAC2 form a transcriptional repressor complex with SNAIL to downregulate the E-cadherin expression of pancreatic cancer cells during metastasis. Intriguingly, SNAIL also recruits the HDAC1/2-containing SIN3A complex to deacetylate histones on the E-cadherin promoter for gene silencing [23]. In a similar manner, SLUG recruits the HDAC1-containing CtBP complex to silence genes by binding to the E-box on the BRCA2 promoter [24]. ZEB1/2 also recruits the CtBP/HDAC1 complex to the E-cadherin promoter, while ZEB1 recruits SIRT1, a class III HDAC, to silence E-cadherin and promote EMT and metastasis in prostate cancer cells [25, 26]. Moreover, ZEB1-induced EMT is accomplished by repression of other epithelial genes, including EPCAM, ESRP1 and RAB25, and is accomplished by a reduced acetylation of H3K9 and H3K27 at their promoters. In fact, evidence suggests that H3K27 deacetylation is a key epigenetic event in ZEB1-induced transcriptional reprogramming [27]. In addition to the EMT transcriptional factors, HDAC1 co-purifies with TCF12 and promotes migration and invasion; elevated expression of TCF12 and HDAC1 correlate with a poor prognosis in gallbladder cancer. These findings suggest that this HLH transcription factor TCF12 could also target HDACs for epithelial gene silencing during EMT [28]. The expression of the HDAC1 and HDAC3 correlates with nuclear receptor (NR) (i.e., ER and PR) status. HDAC assembles a complex with ERα that binds to the SLUG promoter to repress expression [29]. Interestingly, HDAC6 and SIRT1 counteract the p300-catalyzed acetylation on Cortactin, which enhances its F-actin binding ability to facilitate EMT and tumor progression [30]. Conversely, HDAC6 and ERα are co-localized in the cytoplasm of renal cell carcinoma (RCC) cells and HDAC6 enhances cell motility by decreasing acetylated α-tubulin expression [21]. Loss of α-Tubulin acetylation by HDAC6 is also associated with TGF-β-induced EMT [31].
Recently, the clinical relevance of HDACs and the therapeutic potential of HDAC inhibitors (HDACi) have been reported. HDACi can generally be classified into hydroximates, cyclic peptides, aliphatic acids, and benzamides [32], and grouped according to their specificity. Thus far three HDACi: vorinostat (SAHA), romidepsin (Istodax) and PTCL (Belinostat or Beleodaq) are approved by the FDA for some T-cell lymphomas [33]. However, these molecules have not produced favorable and expected outcomes in solid tumors. Currently, a number of small molecules HDACi were investigated in clinical trials with variety of solid neoplasms, including breast cancer, either alone or in combination with hormonal treatments. Entinostat (MS-275), a benzamide with high specificity for the class I HDACs, is currently in a phase II/III trial for advanced ER+ breast cancer [34, 35]. Vorinostat exerts EMT reversal effects by restoring the expression of E-cadherin. An expanded screen on 41 HDACi further identified 28 HDACi compounds, such as the class I-specific inhibitors Mocetinosat, Entinostat and CI994, that restore E-cadherin and ErbB3 expressions in ovarian, pancreatic and bladder carcinoma cells [36]. Mocetinostat, but not other HDACi, specifically interferes with ZEB1 function, restores miR-203 expression, represses stemness properties, and induces sensitivity against chemotherapy by restoring histone acetylation on the E-cadherin promoter [37]. Given that persistent genes activation may require targeting of multiple epigenetic silencing machineries, a combination of HDACi with anticancer drugs and/or radiotherapy demonstrate synergistic or additive effects in clinical trials. For example, HDACi have been utilized in combination with 5 Aza-dC as a synergistic strategy [38]. However, recent reports also found that HDACi could promote EMT in prostate and nasopharyngeal cancer cells [39, 40], indicating the application of HDACi in anti-cancer therapy is cancer-context dependent and may limit application.
Histone methylation occurs at specific lysine or arginine residues on the histone tails. This modification is associated with either transcriptional activation or repression. Histone methylation does not change the electrostatic charge of histones or affect the chromatin structure. The functional effects of histone methylation are affected by both the position of the modified residues and number of methyl groups. Histone methyltransferases (HMTs) transfer methyl groups from S-adenosylmethionine (SAM) to either lysine or arginine residues, whereas histone demethylases (HDMs) remove methyl groups. The HMTs and HDMs specifically catalyze particular lysine or arginine residues.
Methylation of lysine residues on histones was first identified in the 1960s. Histone lysines can have four states of methylation at different lysine sites. Histones H2B lysine 5 (H2BK5), H3K4, H3K9, H4K20, H3K27, H3K36, and H3K79 are subject to unmethylated, mono-methylation (me1), di-methylation (me2), or tri-methylation (me3) on the ε-amino groups of lysine residues. These lysine methylations change the chromatin structure and regulate gene transcription. Histone lysine methylation is a reversible modification and is maintained by the balance lysine methyltransferases (KMTs) and lysine demethylases (KDMs). The KMTs recruit SAM as a cofactor and catalyze the addition of methyl groups to lysine residues through the SET domain. The KMTs are grouped into the SET domain-containing enzyme families (KMT1–3 and KMT5–7), the KMT4/DOT1 family, and others. The KDMs include the flavin adenine dinucleotide- (FAD-) dependent monoamine oxidase family (KDM1/LSD), the Jumonji C domain-containing demethylase (JMJD) families (KDM2–6), and others. Methylation of H3K4, H3K36, and H3K79 usually correlate with gene activation, whereas methylation of H3K9, H3K20, H3K27, and H3K56 are associated with transcriptional silencing.
H3K4: H3K4 methylation (H3K4me) is present in euchromatic regions and is usually associated with transcriptional activation. H3K4me3 occurs principally at the 5′ end of actively transcribed genes, near the transcription start site (TSS). H3K4me2 is located throughout genes, but frequently found towards the middle of the coding region of transcribed genes, and H3K4me1 is more abundant at the 3′ ends [41]. H3K4me2 marks can be present at both active and inactive euchromatic genes, whereas H3K4me3 is present exclusively at active genes. H3K4me favors transcriptional activation by facilitating H3 acetylation and the recruitment of RNA polymerase II, but it also antagonizes gene repression by preventing the binding of nucleosome remodeling and deacetylase co-repressor complexes, such as NuRD, and interfering with substrate recognition by the variegation 3–9 (SUV39H) methyltransferases [42]. The balance between KMTs and KDMs is an important dynamics for H3K4me and the regulation of gene transcription. More than ten H3K4 KMTs have been identified, including the mixed-lineage leukemia (MLL)1–4 proteins, along with Set 1a and Set 1b, Ash1L, Set7/9, and also the SET and MYND domain-containing enzymes (SMYD) family members (SMYD1 and SMYD3). SMYD are involved in many cellular processes, including tumorigenesis and invasiveness. For example, SMYD3 is a novel histone H3K4-specific N-lysine di- and tri-methyltransferase, and highly characteristic active transcription. SMYD3 exerts its effects on initiation, invasion and metastasis of diverse tumors (e.g., esophageal squamous cell carcinoma (ESCC), gastric cancer, HCC, cholangiocarcinoma, breast cancer, prostate cancer, and leukemia). SMYD3 stimulates EZR and LOXL2 transcription to enhance proliferation, migration and invasion by directly binding to sequences of the promoter regions of these target genes [43]. SMYD3 is also capable of increasing cell migration through MMP-9 expression [44]. The MLL proteins (Trithorax homologs in Drosophila) are important for the regulation of developmental genes such as the Hox cluster, and deficiency of MLL1 or MLL2 causes embryonic lethality [45]. MLL1 coordinates with HIF1α and regulates hypoxia-induced HOTAIR expression to facilitate tumorigenesis [46]. In addition, MLL1 interacts with β-catenin to promote cervical carcinoma cell tumorigenesis and metastasis [47]. KMT2B/MLL2 is highly expressed in ESCC and promotes tumor progression by inducing EMT [48]. Another member of MLL family, MLL3 is reportedly mutated in multiple cancers. MLL3 regulates many migration-related genes and downregulation of MLL3 has a profound impact on the progression of ESCC [49]. Furthermore, Kim et al. [50] showed that KMT2D/MLL4 expression is associated with poor survival in breast cancer and regulates tumor proliferation and invasiveness.
Histone lysine methylation is a reversible process. H3K4 is demethylated by the KDM1 family (LSD1 and LSD2), the KDM2 family (FBXL10 and FBXL11), and the KDM5 family (JARID1A, JARID1B, JARID1C, and JARID1D) as well as JARID2 and NO66. The LSD subgroup of KDMs specifically targets the mono- and dimethylated lysines. This group demethylates substrates through a flavin adenine dinucleotide-dependent oxidative reaction, producing lysine and formaldehyde. KDM1A/LSD1 was the first H3K4 lysine-specific demethylase to be identified. We and others demonstrated that SNAIL recruits LSD1 to epithelial gene promoters with demethylation of H3K4me2 and subsequent silencing of target genes to enhance tumor metastasis [51]. SLUG also interacts with LSD1 to facilitate tumor metastasis [52]. In addition, both SNAIL and SLUG recruit LSD1 and bind to a series of E-boxes located within the BRCA1 promoter to repress BRCA1 expression. LSD1 overexpression promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of ESCC metastasis [53]. LSD1 and LSD2 act differently in the regulation of gene transcription and chromatin remodeling. However, both of KDM1A and KDM1B are overexpressed in invasive breast carcinoma, and depletion results in high levels of H3K4me1–2. The KDM5/JARID1 family is frequently found in the promoter region of transcriptionally active genes, and results in repressed expression of the target genes. KDM5A is highly expressed in ovarian cancer tissues and facilitates EMT and metastasis [54]. KDM5A promotes an increase in TNC expression, which augments breast cancer cell invasion and metastasis [55]. Reports indicate that, in gastric cancer cell, KDM5A is induced by TGF-β1 and recruited by p-SMAD3 to silence the E-cadherin promoter and promote tumor progression [56]. KDM5B plays a role in cell differentiation, stem cell self-renewal and other developmental progresses. Recent studies showed that KDM5B expression was increased in breast, bladder, lung, prostate and many other tumors and promote tumor initiation, invasion and metastasis. Mechanistically, KDM5B exerts its function through modulation of H3K4me3 at the PTEN gene promoter [57]. KDM5C/JARID1C is overexpressed in breast cancer, and its expression is significantly associated with metastasis. This demethylase modulates the status of H3K4 methylation in the breast cancer metastasis suppressor-1 (BRMS1) promoter, and thereby controls the expression of BRMS1 to inhibit tumor progression. Accordingly, the expression of KDM5C and BRMS1 are inversely correlated in human breast cancer [58].
H3K36: Because the level of H3K36me3 is high at the promoter site in active genes, H3K36me3 is involved in active transcription. In contrast, the H3K36me1 signal has a low association with active promoters. H3K36 is methylated by the KMT3 family (SETD2 and NSD1) as well as by NSD2, NSD3, SMYD1, SMYD2, SMYD3, SMYD4, and SMYD5. SETD2 plays a tumor suppressor role in tumor metastasis. Interestingly, SETD2 is frequently either deleted or mutated [59]. In contrast, H3K36 is demethylated by the KDM2 family (FBXL10 and FBXL11), the KDM4/JMJD family (JMJD2A, JMJD2B, and JMJD2C), and NO66. KDM2A expression is increased in breast cancer and associated with poor clinical outcomes [60]. KDM2A promotes lung tumorigenesis by epigenetically enhancing ERK1/2 signaling through demethylation of H3K36 [61]. In addition, KDM8/JMJD5 also demethylates H3K36me2, and overexpression of JMJD5 promotes cell invasion and is significantly correlated with clinical stage, histological grade and lymph node metastasis [62].
H3K79: H3K79me3 is associated with active transcription in yeast, whereas it is localized at both active and silent promoters in humans. H3K79me1 and H3K79me2 do not have any association with either active or silent promoters. H3K79 is methylated by the KMT4 family (DOT1L) and demethylated by PHF8 [63]. Methylation of H3K79 has been implicated in cell cycle regulation and the DNA damage response [63]. Disruption of this methylation can lead to cancers, making DOT1L a potential therapeutic target for cancers such as leukemia [64]. More recently, DOT1L has been implicated in the stimulation of proliferation, self-renewal, and metastatic potential of breast cancer cells [65]. DOTL1 cooperates with c-Myc-p300 complex to epigenetically activate EMT regulators in breast cancer progression. Clinically, DOTL1 expression is associated with poorer survival and aggressiveness of breast cancer [12]. PHF8 is highly expressed in metastatic prostate tissues and plays an important role in controlling invasion and metastasis [66]. PHF8 also interacts with β-catenin, and binds to the promoter region of vimentin, leading to the promotion of gastric cancer progression and metastasis [67].
H3K9: The methylation of H3K9 (H3K9me) was the first mechanism of gene repression to be linked to KMT. Studies in Drosophila showed that the gene Su(var)39, later shown to encode a H3K9 HMT, had an important role in the regulation of position-effect variegation [68] and similar enzymes were subsequently discovered in humans (SUV39H1/H2, G9a and Riz1 among others) [69]. H3K9 methylation is important for chromatin condensation and heterochromatin formation. H3K9me is recognized and bound by heterochromatin protein 1 (HP1), which recruits SUV39H, to reinforce the silencing process. H3K9 methylation plays a critical role in the formation of transcriptionally silent heterochromatin and the stable inheritance of the heterochromatin state. H3K9me1 and H3K9me2 are associated with euchromatic gene repression, whereas H3K9me3 is associated with stably silenced heterochromatin. H3K9me2 marks contribute to the maintenance of gene repression in differentiated tissues in large genomic regions known as ‘large organized chromatin K9–modifications (LOCKs)’, and require the activity of the methyltransferase G9a [70]. H3K9me is methylated by the KMT1 family (SUV39H1, SUV39H2, G9a, GLP, SETDB1, and SETDB2). H3K9 is demethylated by the KDM1 family (LSD1), the KDM3 family (JMJD1A, JMJD1B and JMJD1C), and the KDM4 family (JMJD2A, JMJD2B, JMJD2C, and JMJD2D) as well as PHF8 and KDM7A/JHDM1D. SUV39H1 generates H3K9me3, and is involved in breast carcinogenesis. In addition, we found that SUV39H1 cooperates with SNAIL to repress the expression of E-cadherin. Knockdown of SUV39H1 blocked the formation of H3K9me3 and DNA methylation and inhibited cell migration, invasion and metastasis of BLBC [71]. Furthermore, we demonstrated that knocking down G9a resulted in suppression of H3K9 methylation and inhibition of tumor cell migration or invasion [72]. Mechanically, we found that G9a interacted with SNAIL and is critical for SNAIL-mediated E-cadherin repression in human breast cancer. Consistent with our research, Huang et al. [73] demonstrated that knocking down G9a or pharmacological inhibition of its activity suppressed tumor cell growth, colony formation, invasion and migration in non-small-cell lung cancer cells (NSCLC). G9a is also associated with an increased expression in lung cancer [74]. SETDB1 is the most significantly up-regulated epigenetic regulator in human HCCs and prostate cancer [75, 76]. Knockdown of SETDB1 decreases cell migration and invasion and reduces EMT and CSC properties [77]. SETDB1 indirectly up-regulates STAT3 expression and induces TWIST. KDM3A catalyzes the demethylation of H3K9 associated with transcriptional repression, resulting in the derepression and activation of genes involved with invasion and metastasis [78]. Global gene expression profiling demonstrated KDM3A regulates genes and pathways that augment cell migration and metastasis. KDM3A promotes both migration in vitro and metastasis in vivo by targeting melanoma cell adhesion molecule (MCAM) [79]. Surprisingly, increased expression of KDM3B correlates with improved clinical outcomes [80]. Accordingly, JMJD1B and JMJD2B are associated with PRL-3, a gene crucial to metastasis in colorectal cancer (CRC). However, JMJD1B seems to be a candidate tumor suppressor while JMJD2B seems to be a potential oncoprotein for CRC metastasis and progression [81]. With respect to the breast cancer, KDM4A is a regulator of cancer cell growth and metastasis, which correlates with breast cancer progression, and is associated with the attenuation of the tumor suppressor ARHI [82]. KDM4B is physically associated with β-catenin and binds to the promoter of the β-catenin target gene vimentin to increase its transcription by inducing H3K9 demethylation [83]. Inhibition of JMJD2B attenuates migration and invasion of gastric cancer cells in vitro and metastasis in vivo. KDM4C expression correlates significantly with genes driving metabolic alterations in breast cancer; the mechanism involves an interaction between KDM4C and HIF1α, which is recruited to a subset of genes involved in metabolic remodeling and metastasis [84].
H4K20: H4K20 methylation is also associated with repressed chromatin. A recent genome-wide analysis demonstrated that H4K20me3 was associated with heterochromatin and played a pivotal role in chromatin integrity. In addition, loss of histone H4K20me3 predicts poor prognosis in breast cancer and is associated with invasive activity. On the other hand, H4K20me1 is located in the promoters or coding regions of active genes and co-localizes with H3K9me1, which suggest that H4K20me1 is associated with transcriptional activity. H4K20 is methylated by the KMT5 family (PR-Set7, SUV4-20H1, and SUV4-20H2) and the KMT7 family (SET7/9). KDMs that catalyze H4K20 demethylation have not been reported. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated invasion by normal mammary epithelial-cell in vitro [85]. Through its repressive H4K20me3 mark, SUV420H2 silences several key drivers of the epithelial state. Knockdown of SUV420H2 elicited MET on a molecular and functional level. An analysis of human pancreatic cancer biopsies suggests that high levels of SUV420H2 correlate with a loss of epithelial characteristics and progressively invasive cancer [86]. SET8 (also known as PR-Set7/9, SETD8, KMT5A), a member of the SET domain-containing methyltransferase family that specifically target H4K20 for monomethylation, physically interacts with TWIST to promote EMT and invasion by breast cancer cells [87]. Interestingly, SET8 acts as a dual epigenetic modifier on the promoters of E-cadherin and N-cadherin through its H4K20 monomethylation activity [88]. These bipolar roles of SET8 in EMT were also found in prostate cancer, which were mediated by ZEB1 [89]. A recent report indicates that the activation of the Shh pathway is required for EMT in NSCLCs [90]. SET7-mediated Gli3 methylations contribute to the tumor growth and metastasis in NSCLCs in vitro and in vivo [91].
H3K27: Another important repressive mark is H3K27 methylation which plays an essential role in embryogenesis, cell differentiation and organogenesis. H3K27me3 is associated with constitutive heterochromatin and maintenance of gene repression during early development. According to a genome-wide analysis, the levels of H3K27me2 and H3K27me3 are elevated in silent promoters and reduced in both active promoters and genic regions, whereas the level of H3K27me1 is high in promoters engaged in active transcription, especially downstream of the TSS [92, 93]. In embryonic stem cells (ESCs), H3K27 methylation usually overrides the effect of H3K4me3 in bivalent regions, maintaining them in a repressed state. Upon differentiation, these regions become exclusively marked by either of these modifications, leading to gene activation or repression [92]. H3K27 methylation is catalyzed by the polycomb repressive complex 2 (PRC2), which is composed mainly of suppressor of zeste 12 (SUZ12), embryonic ectoderm development (EED) and EZH2. H3K27 is demethylated by the KDM6 family (KDM6A/UTX and KDM6B/JMJD3), as well as UTY and JHDM1D [94, 95]. EZH2 is overexpressed in prostate and breast cancers and correlates with poor prognosis. Interestingly, EZH2 is essential for CSC self-renewal, and these CSCs provide the seeds for metastatic dispersal and differentiate into tumor-associated endothelial cells. Pre-clinical studies showed that EZH2 can silence several anti-metastatic genes (e.g., E-cadherin and tissue inhibitors of metalloproteinases), thereby favoring cell invasion and anchorage-independent growth. Accordingly, Tiwari and colleagues delineated an elegant pathway wherein TGF-β induces EZH2 expression to elicit EMT programs and metastasis of breast cancers by reprogramming the epigenome [96]. EZH2 represses TIMP2 transcription, which leads to increased activity of MMP-2 and MMP-9 and the invasive capacity of BLBC cells [97]. In pancreatic cancer cells, SNAIL recruits PRC to the E-cadherin promoter by binding to SUZ12 [98]. Increased KDM6A expression is associated with poor prognosis, along with derepression/activation of genetic programs that induce cell proliferation, luminal to basal-like transition, and metastasis. Furthermore, the function of KDM6A correlates with the activity of the MLL4, and increased expression of these epigenetic enzymes correlates with poor survival outcomes in breast cancer [50]. UTX interacts with the MLL4 complex to activate several pro-metastatic genes including MMP9 and SIX1, leading to increased EMT and metastasis of breast cancer [50]. In colon cancer, KDM6A not only demethylates H3K27me3 at the E-cadherin promoter but also recruits CBP to the E-cadherin promoter, resulting in increased H3K27ac [99]. However, it was also reported that KDM6A inhibited EMT by epigenetic repression of EMT genes in cooperation with LSD1 and HDAC1 [100]. Therefore, the role of KDM6A an EMT suppressor or enhancer requires further investigation. KDM6B expression is also increased in invasive breast carcinomas and enforcing KDM6B overexpression induces EMT, invasive migration, stem cell-like traits, and metastatic properties. The mechanism involves demethylation associated with increased SNAIL or SLUG expression mediating the EMT [101]. Interestingly, KDM6B also modulates the tumor microenvironment and promotes melanoma progression and metastasis through upregulation several targets of NF-κB and BMP signaling, including stanniocalcin 1 (STC1) and chemokine (C-C motif) ligand 2 (CCL2) [102].
In summary, histone lysine methylation modulates chromatin accessibility, transcriptional status, and control of tumor suppressor and oncogene expression in aberrant cell metastasis. Dynamic regulation of the either permissive or repressive histone methylation at different genomic loci and through different molecular mechanisms facilitates the dynamic EMT process.
Histone arginine methylation also occurs in many arginine sites, histone H3 arginine 2 (H3R2), H3R8, H3R17, H3R26, and H4R3 undergo monomethylation (me1), symmetrical dimethylation (me2s), or asymmetrical dimethylation (me2a) on the guanidinyl groups of arginine residues. The N-arginine methyltransferases (PRMTs) are a class of enzymes that transfer a methyl group from SAM to the guanidino nitrogen of arginine. PRMTs generate three arginine methylation forms: monomethylarginine (MMA), asymmetric dimethylarginine (aDMA), and symmetric dimethylarginine (sDMA). Human PRMTs are composed of nine members that are categorized into three groups based on the type of arginine methylation reaction each member catalyzes. Type I is comprised of PRMT1, PRMT2, PRMT3, CARM1/PRMT4, PRMT6, and PRMT8; these catalyze both mono-methyl and asymmetric dimethyl arginine reactions. The type II group is made up of two members, PRMT5 and PRMT9, which catalyze both mono-methyl arginine and symmetric dimethyl arginine. Finally, PRMT7 is, at this point, considered the only bona fide type III methyltransferase and can generate only mono-methyl arginines. Many studies demonstrated that PRMTs regulate a wide range of genetic programs and cellular processes including cell cycle, RNA splicing and differentiation. Although the consequence of lysine methylation is relatively well studied, the role of PRMT action in tumorgenesis is poorly understood. Here, we provide a description of these PRMTs regarding tumor metastasis.
PRMT1: PRMT1 has been extensively studied in many fields. Its activity is responsible for a substantial percentage of methylated arginine residues and modulates a wide range of cell types. Specifically, asymmetric dimethylation on H4R3 by PRMT1 is involved in transcriptional activation, thereby driving oncogenic pathways. PRMT1 is an important regulator of EMT, cancer cell migration, and invasion. PRMT1 can generate H4R3me2a on the promoter region of ZEB1 and TWIST, which play a critical role in EMT [103, 104]. Furthermore, PRMT1 is overexpressed in melanoma; silencing PRMT1 significantly suppresses tumor growth and metastatic ability by targeting activated leukocyte cell adhesion molecule (ALCAM) [105]. Similarly, downregulation of PRMT1 inhibits cell migration and invasion in HCC and oral squamous cell carcinoma (OSCC) [106, 107]. Because of complex alternative splicing in the 5′ region of its pre-mRNA, there are seven distinct PRMT1 isoforms [108]. Each of these isoforms, named PRMT1v1-v7, has distinct characteristics in terms of expression. PRMT1v1 is the most abundantly expressed isoform and likely represents the isoform that is described as PRMT1 in most reports. The expression of alternatively spliced PRMT1 (PRMT1v2) isoform, which is generated through inclusion of alternative exon 2, is significantly altered in breast cancer and promotes invasiveness. The RNA binding protein RALY regulates the PRMT1v2 isoform and promotes metastatic potential [109].
PRMT2: PRMT2 is also reported to be overexpressed in breast cancer [110]. PRMT2 interacts with many NRs, including ERα and ERβ in vitro [111]. Interestingly, the activation of these receptors within cells has both distinct and in some cases opposing effects, which suggests that the functional role(s) PRMT2 are quite diverse. Recently, four alternatively spliced PRMT2 isoforms (PRMT2L2, PRMT2α, β, and γ) in addition to the original PRMT2 isoform were identified [112]. Several splice variants (i.e., PRMT2-α, −β, −γ) were identified as induced in breast cancer, particularly in ER, PR-positive breast cancer [110]. PRMT2 directly binds and enhances estrogen-mediated transactivation of ERα, and enhances the promoter activity of the downstream target gene SNAIL. These findings suggest that the increased PRMT2 expression is associated with breast aggressiveness and metastasis [110].
PRMT4: PRMT4, more commonly known as coactivator-associated arginine methyltransferase 1 (CARM1), is involved in the regulation of a number of cellular processes including transcription, pre-mRNA splicing and cell cycle progression. The expression of CARM1 is dysregulated in colorectal, prostate and breast cancer. CARM1 methylates the chromatin-remodeling SWI/SNF core subunit, BAF155 in breast cancer [113]. The methylation of arginine 1064 residue of BAF155 is associated with breast cancer recurrence and metastasis, indicating that CARM1 plays an important role in tumorigenic activity through BAF155. Accordingly, CARM1-induced tumorigenic effects and its expression is increased in invasive breast cancer, and correlates with a high tumor grade [114]. Interestingly, the CARM1 gene also transcribes four isoforms: the primary isoform CARM1 (CARM1v1) and three alternative isoforms, v2, v3 and v4 [115]. Whether these isoform are responsible for the methylation of distinct substrates and their individual functions requires further study.
PRMT5: PRMT5 is a type II enzyme that generates symmetric dimethylarginine (sDMA). The PRMT5 symmetrically methylates H3R8 site and functions in gene silencing. H3R8me2s strongly associates with H4R3me2s, because both modifications are catalyzed by PRMT5. However, acetylation of H3K9 and H3K14 prevents H3R8 methylation. PRMT5 also acts as a novel cofactor of SHARPIN (Shank-associated RH domain interacting protein), which plays a central role in controlling lung cancer cell metastasis. SHARPIN-PRMT5 is essential for the monomethylation of histones at key metastasis-related genes [116]. PRMT5 has another distinct function; PRMT5 coordinates with multiple Mediator complex subunits to dimethylate H4R3 at the promoter regions of immune response genes and C/EBPβ target genes [117]. Conversely, PRMT5 methylation of histone H3R2 recruits WDR5 and the MLL complex, stimulating H3K4 methylation and euchromatin maintenance [118]. In the context of cancer metastasis, PRMT5 is involved in TGF-β-WDR77 signaling, which induces cancer cell invasion [119]; this report indicates PRMT5 interacts with the WDR77 complex to catalyze arginine methylation. With respect to the acquisition of EMT via TGF-β signaling, epigenetic PRMT5-WDR77 activity is necessary for tumor invasion and metastasis. Furthermore, PRMT5 appears to be recruited by AJUBA to SNAIL and functions as a co-repressor. PRMT5, AJUBA and SNAIL form ternary complex to repress E-cadherin, concomitant with increase arginine methylation at the locus [120]. PRMT5 also modulates metastasis by methylating KLF4. Methylation blocks ubiquitylation of KLF4 by the von Hippel–Lindau tumor suppressor, and as a result, arginine methylation of KLF4 via PRMT5 increases the level of KLF4 protein and increases the probability of breast carcinogenesis [121].
PRMT6: PRMT6 primarily catalyzes asymmetric dimethylation of H3R2. H3R2me2a counter-correlates with the methylation of H3K4, which suggests that H3R2me2a is a repressive marker. However, PRMT6 also methylates H3K4 since both H3R2me2a and H3K4me3 markers are likely to coexist. Furthermore, genome-wide analyses indicate that both H3R2me1 and H3R2me2a are associated with active genes [93]. Thus, the data on the H3R2me2a marker are contradictory, and further studies are required to resolve this issue. There is also emerging evidence of an oncogenic role of PRMT6 in cancer. Overexpression of PRMT6 is associated with several cancer types, including breast, cervix, prostate, and lung cancers, indicating that PRMT6 might play an important role for the onset, incidence, and metastasis of cancer [122]. Furthermore, Dowhan et al. [123] demonstrated a PRMT6-dependent signature that influences long-term survival in patients with breast cancer.
PRMT7: The oncogenic role of PRMT7 has been emerging over the past few years. There are two isoforms, PRMT7α and β, which are active and have slightly different methylation profiles and locations. PRMT7α localizes to the cytoplasm and nucleus, whereas PRMT7β is exclusively cytoplasmic. R531 of PRMT7 is self-methylated and loss of PRMT7 automethylation leads to a reduced recruitment to the E-cadherin promoter by YY1, which consequently derepresses E-cadherin expression by decreasing the H4R3me2’s level [124]. In terms of the functional role of PRMT7, this methytransferase is highly expressed in breast cancer and induces EMT by inhibiting E-cadherin. Baldwin et al. [125] also showed that PRMT7 promotes a well-known metastasis mediator, MMP9 and induces breast cancer cell invasion. Importantly, a gene expression analysis of independent data sets of more than 1200 breast tumors identified PRMT7 expression as significantly increased. In addition, this gene is located 16q22, where the chromosomal region was correlated with an increased metastatic potential of breast cancer [126].
PRMT9: PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming sDMA residues as type II PRMTs. However, the specificity of these enzymes for their substrates is distinct and not redundant. Interestingly Yang et al. [127] showed that PRMT9 is also nonhistone methyltransferase. For example, it methylates the arginine 508 site of the alternative splicing factor SAP145. Given that alternative splicing is of paramount importance in RNA processing, PRMT9 might play a key role in many cellular programs including cancer biology. Recent reports demonstrate that overexpression of PRMT9 strongly promotes HCC invasion and metastasis through EMT by regulating SNAIL expression via activation of the PI3K/Akt/GSK-3β/SNAIL signaling pathway [128].
Many HMTs and HDMs inhibitors have been developed and evaluated in clinical trials, such as chaetocin, BIX-01294, BIX-01338, UNC0638 and DZNep. Chaetocin, a natural fungal substance, is the first inhibitor of an HMT, which targets SUV39H1 without high selectively [129]. Treatment with Chaetocin induces expression of E-cadherin while reducing H3K9me3 but does not produce a global H3K9 methylation on its promoter in multiple tumor cells [130]. By the contrast, BIX-01294 specifically reduces the dimethylation of H3K9me2 through an inhibition of the enzymatic activities of G9a and GLP [131]. Treatment of BIX-01294 activates E-cadherin expression and reverse EMT phenotypes in a variety of cancer cells, and is accompanied by reduced H3K9me2 and increased H3K9 acetylation on the E-cadherin promoter [132]. Another G9a/GLP inhibitor, UNC0638, was developed with higher potency and selectively [133]. UNC0638 treatment not only resulted in lower global H3K9me2 levels but also markedly reduced the abundance of H3K9me2 marks at promoters of known G9a-regulated endogenous genes. UNC0638 treatment activates E-cadherin expression and reverses EMT in PANC-1 pancreatic cancer cells and triple negative breast cancer (TNBC) and suppresses migration and invasion [134]. Because of the importance of H3K27 methylation in cancer, several highly specific EZH2 inhibitors have been developed, such as GSK2816126 and EPZ-6438, which are currently being evaluated in clinical trials for lymphoma and solid tumor/lymphoma respectively [135]. Another EZH2 inhibitor, 3-deazaneplanocin A (DZNep), selectively inhibits H3K27me3 and H4K20me3 [136]. DZNep dampens TGF-β-induced EMT signals and reduces tumor metastasis in pancreatic cancer and colon cancer [136, 137]. We found that Parnate, an LSD1 inhibitor, activates E-cadherin expression and suppresses motility and invasiveness in breast cancer cells [51]. Two highly specific LSD1 inhibitors, GSK2879552 and ORY-1001 are employed to clinical trials for the treatment of small cell lung cancer and acute leukemia [135]. Several inhibitors targeting HDMs also have been developed as well. For example, JIB-04, a specific inhibitor targeting the JMJC-domain, inhibits the activity of H3K4 and H4K9 and attenuates lung cancer cell proliferation [138]. The first reported small molecule PRMT inhibitors, including AMI-1 and AMI-5 were identified through virtual screening and high throughput screening [139]. AMI-1 was reported as type I PRMT and PRMT5 inhibitor [140]. AMI-1 inhibits proliferation and decreases cell migratory activity of CRC cells in vitro and in xenograft mouse models [141].
Sometimes, histone modifications can directly regulate the chromatin dynamic. However, in most cases, the modifications are recognized by proteins containing distinct recognition domains, which act as “readers” and bind to different histone modifications. For example, bromodomain acts as lysine acetylation “readers” of modified histones that mediate signaling transduction changes in gene regulatory networks. In the human genome, there are 61 bromodomains found within 46 proteins that can be divided into eight families based on structure/sequence similarity. Among them, bromodomain and the extra-terminal domain (BET) family recognize acetylated lysine residues in histones H3 and H4. BRD4 is a member of the BET family that carries two bromodomains. Recently, our studies revealed that the di-acetylated TWIST, mediated by Tip60, recruits BRD4 and related transcriptional components to the super-enhancer of its targeted genes during tumor progression in BLBC [20]. In addition, pharmacologic inhibition of BRD4 with the BET-specific bromodomain inhibitors, JQ1 and MS417, effectively reduces WNT5A expression and suppresses invasion, CSC-like properties and tumorigenicity of breast cancer cells in vitro and in vivo [20]. Given the extensive cancer-related functions of BRD4 and the proof-of-concept demonstrated by disruption of the BRD4–acetyl-lysine interactions as a therapeutic target, significant efforts have thus been made to develop BRD4 inhibitors from both pharmaceutical and academic settings. BRD4 inhibitors have several chemical classifications including azepines, 3,5-dimethylisoxazoles, pyridones, triazolopyrazines, tetrahydroquinolines (THQs), 4-acyl pyrroles and 2-thiazolidinones [142]. BET inhibitor treatment results in AMIGO2 silencing and changes in PTK7 proteolytic processing, and thus inhibit melanoma metastasis [143].
Histone methylation provides docking sites and is recognized by specific reader proteins that contain a methyllysine binding protein, which has emerged as a focus of epigenetic research due to its critical role in gene regulation and oncogenesis. This reader harbors specific motifs, including Chromodomain (CD), MBT, WD40 repeat, PHD finger, PWWP, Tudor and Ankyrin repeat. Methyllysine binding proteins distinguish methylation marks on different residues as well as different methylation states on the same residue and in turn mediate distinct downstream functions [144]. CD-containing HP1 proteins were the first identified methyl-lysine binding proteins and recognize methylated-H3K9 (methyl-H3K9) [145]. HP1α was down-regulated in metastatic cells of colon cancer and thyroid carcinomas relative to non-metastatic cells, indicating HP1α may be directly involved in the silencing of genes that potentiate cancer cell invasive potential and metastasis. Recent evidence implicate HP1α in EMT. The association of HP1α to major satellite repeat sequences located in pericentric heterochromatin decreased during the initial steps of TGF-β-induced EMT in a SNAIL/LOXL2-dependent manner [146]. In addition, HP1α post-translational modifications could participate in the heterochromatin dynamics associated with EMT. In a different set of modifications, four MBT-repeats domain of SFMBT1 recognize H3K4me2/3 and form a stable complex with LSD1. SFMBT1 is essential for SNAIL-dependent recruitment of LSD1 to chromatin, demethylation of H3K4me2, transcriptional repression of epithelial markers, and induction of EMT by TGF-β [147]. H3K4me2/3 is also recognized by the WD40 repeat domain of WDR5, which is also important for the assembly and activity of the SET1 protein complex catalyzing H3K4me3 [148]. Under hypoxic conditions, WDR5 is induced, interacts with HDAC3 and further recruits SET1 complex to activate mesenchymal gene expression to promote EMT [149]. Furthermore, the PRC2 component, EED, also contains a WD40 repeat that recognizes H3K27me3. EED recruits PRC2 to chromatin with pre-existing H3K27me3 to spread the same methylation into adjacent regions [150]. Intriguingly, G9a and GLP itself contain a methyl-lysine binding module (the ankyrin repeat domains), which generates and reads the same epigenetic mark [151]. Several small molecule compounds targeting the lysine methylation reader domain have been developed, including UNC1215 and UNC3866 that block the methyl-lysine binding mediated by the MBT domain-containing protein L3MBTL3, and the CD-containing protein CBX4/7 respectively [152, 153]. However, whether these inhibitors reverse EMT and tumor progression remains unknown.
Because different chromatin modifying enzymes coexist in the same protein complex, and because diverse catalyzed modifications have been implicated in regulating the same set of genes, it is likely that these processes act in concert to orchestrate transcriptional regulation during EMT. For example, HDAC1/2, G9a/GLP, LSD1, HP1 and ZEB1/2 were co-purified in the CtBP1 co-repressor complex [154, 155]. ZEB1/2 could first target the complex to E-cadherin promoter to initiate repression. Next, HADC1/2 would deacetylate histones while the primed H3K9 was methylated by G9a/GLP. Meanwhile, LSD1, which removes H3K4me1/2, whereby the un-methylated H3K4 could also prevent H3K9 from re-acetylation [156, 157]. An affinity purification of Flag-TWIST identified several components of the NuRD chromatin remodeling complex. Among them, TWIST directly interacts with Mi2β, MTA2 and RbAp46 and likely targets the NuRD complex for histone deacetylation and chromatin remodeling on E-cadherin promoter. Together, these epigenetic events lead to gene silencing and promote EMT and breast cancer metastasis [158]. In addition, TWIST was also co-purified with SET8, BRCA1-associated protein (BRAP), NF-kB subunit RelA, PPP2CA and HES6 in MCF7 breast cancer cells [88]. SET8 interacts with TWIST. However, SET8 and TWIST are functionally interdependent in promoting EMT. SET8 mediates E-cadherin repression and N-cadherin activation simultaneously via its H4K20 monomethylation to promote cell invasion and EMT. However, the molecular mechanism that underlies the same repressive protein complex that contributes to opposite functions on different genomic loci remains an open question. Our recent study found that TWIST is diacetylated by Tip60, which was further recognized by BRD4, thereby constructing an activated TWIST/BRD4/P-TEFβ/RNA-Pol II complex at the WNT5A promoter and enhancer to promote EMT and breast cancer cell metastasis [20]. In breast cancer cells, the UTX-MLL4 forms a complex with LSD1/HDAC1/DNMT1 on the promoter of several EMT-TFs and decreases H3K4mes and H3 acetylation. UTX facilitates epigenetic silencing of EMT-TFs by inducing competition between MLL4 and the H3K4 demethylase LSD1, which results in inhibition of EMT and CSC-like properties [100].
MPP8, another methy-H3K9 binding protein, bridges DNMT3A and G9a/GLP to assemble a repressive trimeric protein complex on chromatin by binding to different methyl-lysines. MPP8 also couples H3K9 methylation and DNA methylation to silence epithelial genes and EMT [159, 160]. Interestingly, MPP8 also cooperates with the SIRT1 in this process through a physical interaction [161]. SIRT1 and MPP8 reciprocally promote each other’s function and coordinate epithelial gene silencing and EMT. SIRT1 antagonizes PCAF-catalyzed MPP8-K439 acetylation to protect MPP8 from ubiquitin-proteasome-mediated proteolysis. Conversely, MPP8 recruits SIRT1 for H4K16 deacetylation after binding to methyl-H3K9 on target promoters. Therefore, MPP8 not only promote DNA-methylation but also H4K16 deacetylation to fine-tune the transcriptional regulation of EMT.
Increasing evidences show that aberrant profiles of histone modifications contribute to a dysregulation those results in the metastatic cascade. The biochemically reversible nature of histone modifications provides a platform for rapid changes in a variety of epithelia and mesenchymal genes during EMT and MET. In concert with different ETM-TFs and oncogenic signaling, pleiotropic histone modifications form a sophisticated and regulated network to coordinate the plasticity and dynamic change required for EMT.
Recent research identifies the critical role of histone modifications in metastasis, but leaves many important, open questions. First, do tumor microenvironmental signals trigger the formation of histone modification enzyme complexes present on different EMT-TFs? Whether these extrinsic signals affect enzyme activity indirectly through intracellular signaling pathways or directly through the EMT-TFs remains to be determined. Second, how do these EMT-TFs form distinct complexes that coordinate the epigenetic regulation of gene expression programs during EMT? Third, EMT is usually activated only transiently and partially. Therefore, which and how do different histone modifying enzymes and the catalyzed modifications contribute to these dynamic changes? Finally, what consequences do epigenetic instabilities have on cancer cell fitness? Do these activities increase plasticity and/or lead to vulnerabilities that it could influence the metastasis?
We know that histone modification enzymes are highly correlated with tumor progression and a poor clinical outcome. Therefore, these enzymes can serve not only as effective biomarkers for earlier diagnosis, but also present multiple therapeutic opportunities. Over the last decade, considerable progress has been made in the discovery and development of potent and selective small molecule inhibitors targeting specific histone modifiers. Many of these molecules are currently under extensive preclinical testing or being evaluated in clinical trials. These inhibitors show great potential as clinically useful drugs. Additionally, inhibitors to specific histone modifying enzymes could serve as useful chemical probes to characterize the function of different epigenetic pathways in EMT in vivo as well as many other important pathological diseases.
In all, advances in our understanding of the landscape of histone modifications in metastasis will provide a better sense of the molecular mechanisms associated with metastasis and thus help speed the development of new therapeutic strategies and biomarkers for metastasis.
We thank Dr. Cathy Anthony for the critical editing of this manuscript. Our research was supported by the Shared Resources of the University of Kentucky Markey Cancer Center (P30CA177558). Our research was also supported by grants from American Cancer Society Research Scholar Award (RSG13187) and NIH (P20GM121327 and CA230758) (to Y Wu).
The authors have declared that no conflict of interests exists.
The history of food additives goes back to ancient times. As great civilisations developed, populations grew and so did the demand for food. In ancient Egypt, where the climate was not conducive to food storage, especially due to the heat, people started looking for ways to extend the usability life of products. Common practices included the addition of salt, drying in the sun, curing/corning, meat and fish smoking, pickling, and burning sulphur during vegetable preservation. The earliest preservatives included sulphur dioxide (E220), acetic acid (E260), and sodium nitrite (E250), while turmeric (E100) and carmine (E120) were among the first colours. Food preservation was also of immense importance during numerous armed conflicts. Both during the Napoleonic wars in Europe and during the American Civil War, seafarers and soldiers needed food. Limited access to fresh food at the front motivated the armed forces to transport their food with them. This is when cans were introduced for food preservation purposes. In the subsequent centuries, ammonium bicarbonate (E503ii), also known as salt of hartshorn, used as a rising agent for baked goods, and sodium hydroxide solution (E524), used in the production of salty sticks, rose to prominence [1, 2].
\nThe nineteenth century saw considerable advancements in the fields of chemistry, biology, and medicine. A name that needs to be mentioned here is Louis Pasteur, a French scientist, who studied microbiology, among other things. He was the first to prove that microorganisms were responsible for food spoilage. At the same time, new chemical compounds were discovered that were able to inhibit the growth of microbes. Some substances, such as picric acid, hydrofluoric acid, and their salts, often had disastrous consequences when added to food. Insufficient knowledge of toxicology resulted in consumer poisonings and even deaths [1, 3]. At that time, food preservation was the number one priority, which was achieved, for instance, by using salicylic acid, formic acid (E236), benzoic acid (E210), boric acid (E284), propionic acid (E280), sorbic acid (E200) and its potassium salt (E202), and esters of p-hydroxybenzoic acid. Later, food concerns also focused on improving the organoleptic properties of their products and started to enhance food with colours, flavours, and sweeteners, without first researching their effects on human health. For example, such practices involved the use of synthetic colours used in fabric dyeing. This desire to make money on beautiful-looking products led to adulterating food with copper and iron salts, which have a negative impact on the human body. It was as late as in 1907 that the United States studied 90 of the synthetic colours used at that time for food dyeing and found only 7 to be acceptable for further use. Detailed studies and strict regulations on the use of food additives were created almost a century later [1, 4].
\nGlobally, food safety is ensured by the World Health Organization (WHO) and the Food and Agriculture Organization (FAO). In 1962, these organisations established a special agenda—the Codex Alimentarius Commission. The Commission has prepared and updated the Codex Alimentarius, which is not a legal Act per se, but provides a reference for standards on raw materials and food products, acceptable contamination levels, hygienic processing, research methods, and food additives for almost all countries worldwide [5]. In the European Union, the body responsible for improving human health protection and food safety risk mitigation, as well as for taking care of purchaser interests, is the European Food Safety Authority (EFSA). It is a scientific agency established in 2002 pursuant to the Regulation of the European Parliament and of the Council of 28 January 2002. European legislation is based on the Codex Alimentarius but conducts its own complementary research. Therefore, the list of food additives permitted by the European Union is different from the American one [5].
\nThe primary legal Act governing food in Poland is the Food and Nutrition Safety Act of 25 August 2006 (as amended). It specifies the requirements applicable to food and nutrition, concerning product labelling, hygienic conditions throughout the production process, and product replacement rules, as well as requirements concerning the use of food additives. The key document that pertains specifically to food additives is the Regulation of the European Parliament and of the Council of 16 December 2008 on food additives. The EU-approved list of food additives is presented in the Commission Regulation (EU) of 11 November 2011 [4, 5].
\nA food additive (additional substance) is any substance that is not a food in itself or an ingredient in food, but when added to a product for processing purposes, it becomes part of the food [5]. The following are not considered to be food additives: ingredients in food or chemicals to be used in other products, i.e. in particular sweeteners, such as monosaccharides, disaccharides, and oligosaccharides; substances with flavouring, dyeing, and sapid properties (such as dried fruit); glazing and coating substances, which are not intended to be consumed; and chewing gum bases, dextrin, modified starch, ammonium chloride, edible gelatine, milk protein and gluten, blood plasma, casein, and inulin. The law forbids the use of food additives in unprocessed food, honey, non-emulsified oils and fats of an animal or vegetable origin, butter, milk, fermented milk products (unflavoured, with living bacteria cultures), natural mineral and spring water, unflavoured leaf tea, coffee, sugar, dry pasta, and unflavoured buttermilk [5]. Any marketed additive must comply with the requirements of the European Food Safety Authority, i.e. it has to be technologically justified. It must not put consumers’ life or health at risk; its use should not mislead the purchaser; its acceptable daily intake (ADI), or quantum satis, the smallest amount which is needed to achieve a specific processing objective for the substance, must be calculable; and, last but not least, such an additive must not adulterate the product it is to be added to. Producers are also required to include information on any food additives on product labelling [6, 7].
\nEU legislation has approved approximately 330 food additives for use. The primary objectives behind the use of additives are to extend the shelf life and freshness of products, prevent product quality impairment, make the product more attractive to customers, achieve the desired texture, ensure specific product functionality, facilitate production processes, reduce production costs, and enrich the nutritional value of products. In order to harmonise, effectively identify any additives, and ensure smooth exchange of goods, each food additive has its own, standardised, code. This code is consistent with the International Numbering System (INS) and comprises the letter “E” and three or four digits. There are several food additive classifications. One is based on the regulation and differentiates between colours (approx. 40), sweeteners (approx. 16), and other additives (approx. 277) [8, 9].
\nAdditional substances can also be categorised on the basis of code numbers:
Colours—E100–E199
Preservatives and acidity regulators—E200–E299
Antioxidants and synergists—E300–E399
Stabilising, thickening, emulsifying, coating, and bulking substances—E400–E499
Other substances—E500 and above
Food additives can also be divided into four major groups, based on their processing purpose. These are substances that prevent food spoilage, those which improve sensory features, firming additives and excipients. The most numerous group among additives that slow down food spoilage are preservatives. These are either natural or synthetic chemical compounds added to food to restrict as much as possible the biological processes that take place in the product, e.g. the development of microflora and pathogenic microbes, and the effects of enzymes that affect food freshness and quality. In food products, preservatives change the permeability of cytoplasmic membranes or cell walls, damage the genetic system, and deactivate some enzymes. Food is preserved using antiseptics or antibiotics. The former are synthetically produced simple compounds that often have natural correlates, and they make up no more than 0.2% of the product. Antibiotics, or substances produced by microorganisms, are used in very small, yet effective, doses. The effectiveness of preservatives depends primarily on their effect on a specific type of microorganism, which is why it is vital to select the appropriate preservative based on the microbes found in the product (bacteria, mould, or yeast). Other factors that determine the effectiveness of preservatives include the pH value (a low pH is desirable), temperature, the addition of other substances, and the chemical composition of the product. Preservatives constitute an alternative to physical and biological product freshness stabilisation methods, such as drying, pickling, sterilising, freezing, cooling, and thickening. Consumer objections concerning the widespread use of chemical preservatives and their effects on human health have motivated producers to develop new food preservation procedures. These include radiation, packaging, and storing products in a modified atmosphere, using aseptic technology. Products that are most commonly preserved include ready-made dishes and sauces, meat and fish products, fizzy drinks, and ready-made deserts [9, 10].
\nOther substances used as preservatives are acids and acidity regulators. These substances lower the pH level and slow down the growth of enzymes, which hampers the development of microbes. They are used mainly in the production of marinades. For a specific acid or acidity regulator to fulfil its role as a preservative, it needs to be added in highly concentrated form, but acetic acid, for instance, can irritate mucous membranes when its concentration exceeds 3%. Acids and acidity regulators are also used to enhance flavour (usually in fruit or vegetable products, or beverages, to bring out their sour taste) or to facilitate gelatinisation and frothing during food processing [11, 12].
\nNot only microorganisms but also oxygen is responsible for food spoilage. Products such as oils, fats, and dry goods (flour, semolina) oxidise when they come into contact with atmospheric oxygen. Fat oxidisation (rancidification) occurs in oils, lard, flour, and milk powder. The browning of fruit, vegetables, and meat, on the other hand, is the result of non-fat substance oxidisation. These oxidisation processes can be slowed down or eliminated completely using antioxidants. There are natural and synthetic antioxidants and synergists. Synthetic antioxidants are primarily esters (BHA, BHT, propyl gallate). These are used to stabilise fats used to fry, e.g. crisps and chips. The most common natural antioxidants are tocopherols, i.e. vitamin E. Other antioxidants include phenolic compounds, such as flavonoids and phenolic acids. Synthetic antioxidants are more potent and resistant to processing. Synergists are substances that support and extend the functioning of antioxidants. They can form complexes with heavy metal ions, which retard the oxidisation process. The most frequently used synergists are EDTA, citric acid, and ascorbic acid. Antioxidants do not pose a risk to human health. In fact, they can be beneficial. Antioxidants prevent unfavourable interactions between free radicals and tissue and slow down ageing processes and the development of some diseases [12, 13].
\nIn order to extend the freshness of consumer goods, products are also packaged in a modified atmosphere. As part of this process, the oxygen content inside the packaging is reduced and replaced with other gases, such as nitrogen, argon, helium, and hydrogen. Furthermore, products in the form of aerosol sprays, such as whipped cream, have nitrous oxide, butane, or propane added to them. All these gases are also food additives with their own E codes [5, 11].
\nThe organoleptic properties of consumer goods are very important to consumers. Visual appeal is considered to be as important as taste or smell. This is where food colours come into play. These are used to add colour to transparent products (e.g. some beverages), intensify or bring out product colour (beverages, sweets), preserve or reproduce colours that have faded as a result of processing, ensure that all product batches have a specific colour, and provide the products that are diluted after purchase with strong colour. In order to add colour to a product, manufacturers use natural, nature-identical, synthetic, and inorganic colours. Natural colours are produced from edible plant parts (fruits, flowers, roots, leaves) and from animal raw materials, such as blood, chitinous exoskeletons of insects, and muscle tissue. New technologies have also made it possible to obtain colours from algae, fungi, and mould. Natural colouring substances include carotenoids that provide a spectrum of yellow and orange colours (carrot, citrus fruit skin), flavonoids that give products blue and navy-blue colours (grapes, currants, chokeberry, elder), betalains that give products a red colour (beetroot, capsicum), and chlorophyll that lends green colours (salad, parsley), as well as riboflavin (vitamin B2), curcumin, and caramel. Natural colours are desirable for consumers, as they do not show any negative effects on health. However, a significant drawback to using natural colours is that they are very sensitive to environmental factors, such as pH, ambient temperature, oxygen content, or sun exposure, which is why they are not durable when it comes to processing and storage. Moreover, the cost of obtaining such colouring substances is rather high. The list of additives contains 17 natural colours, and their market share in 2012 was approx. 31% and was subject to an upward trend [6, 8].
\nSynthetic food colours are very competitive compared to natural ones. They offer a wide spectrum of colours, including those that are not available in nature, provide strong colouring, and are resistant to environmental factors, so they do not fade during processing. Furthermore, they are not expensive to produce, which contributes to low end-product prices. Synthetic colours can be divided into organic and inorganic, with organic constituting the considerable majority in terms of food colouring. In the past, chemical colours were made of coal, while now crude oil is used for this purpose. EU law approves 15 synthetic colours, including the so-called Southampton colours. A study conducted in 2007 in the United Kingdom (in Southampton, hence the name) showed the particularly negative effects of six colours on children’s health [10]. Specifically, tartrazine (E102), quinoline yellow (E104), sunset yellow (E110), azorubine (E122), cochineal red (E124), and Allura red AC (E129) were found to cause hyperactivity. As a result, since 2010, manufacturers which add at least one of their products have been required to provide label information about their negative effects on concentration and brain functioning in children. Acceptable daily doses of these colours have also been reassessed and updated. Moreover, research conducted on lab animals has shown that the long-term use of synthetic colours, and especially the three that account for 90% of the use of all synthetic colours (Allura red, tartrazine, and sunset yellow), can cause cancer, allergies, and chromosome mutations. Products that are most often synthetically coloured include candy, wine gums, ready-made desserts, and refreshing beverages [8, 10].
\nDuring consumption, one can experience product taste, smell, and consistency. These three sensations are referred to as palatability and are caused by flavours. Taste is experienced by taste buds located in the tongue. Adult individuals have approximately 10,000 such receptors. There are four primary tastes, namely, salty, sweet, bitter, and sour. There is also an additional type, referred to as umami, which is Japanese for “savoury, meaty”. This taste experience is provided by monosodium glutamate. Smell is experienced through volatile compounds that go directly through the nasal or oral cavity and throat to smell receptors. Taste and smell provide a ready source of information on whether the product is fresh, whether it has specific characteristics, and whether it has been adulterated. Flavours are mixtures of many compounds, in which the specific characteristic smell is produced by a single compound or several indispensable compounds. These are added to enhance the taste or smell of the product or to give something the flavour or aroma that has been lost during product processing [6, 7, 11]. There are natural, nature-identical, and synthetic flavours. Natural flavours are obtained from parts of fruits and vegetables, spices, and their flavouring compounds, such as lactones (found in fruits and nuts), terpenes (in essential oils, found in almost every plant), and carbonyl compounds (fermented dairy products). Nature-identical flavours are compounds originally found in a given raw material that can be recreated in the lab. Synthetic flavours are compounds that have been chemically created and produced and do not have their equivalent in nature. Similarly to natural colours, natural flavours are easily degraded during processing, and their extraction is costly, which is why the food industry generally uses synthetic substances to provide products with specific taste and odour. Moreover, synthetic compounds are capable of giving products much stronger flavours than natural ones [6, 7, 13].
\nA separate group that enhances the sensory properties of food are sweeteners. Formerly, in order to make products sweet, manufacturers used sucrose, commonly known just as sugar, obtained from sugar beet or sugarcane. Now large-scale methods are commonly used, such as chemical production and the extraction of intensively sweetening substances, known as sweeteners, from specific plants. What is characteristic about such substances is that they are much more potent as sweeteners compared to sucrose, and, at the same time, their calorific value is close to zero. Natural sweeteners include glucose-fructose syrup (or syrup based on one of those sugars), thaumatin, neohesperidin DC, stevia, and xylitol. Synthetic sweeteners include acesulfame K, aspartame (and the salts of these two compounds), sucralose, cyclamates, saccharin, and neotame. Sweeteners are used in the production of beverages, juices, dairy products, spirits, sweets, marmalade, and chewing gum [14, 15]. In contrast to sucrose, the majority of synthetic sweeteners do not increase blood sugar level and do not cause tooth decay. These substances are attractive for producers because the cost of their production is low, and even small amounts of such compounds are able to ensure the desired sweetness of the product, so these are economical to use. In addition, most sweetener additives remain functional during processing, although some compounds are not resistant to high temperatures. A study conducted in 2010 on lab animals raises some concerns when it comes to sweetener safety in relation to human health [20]. Its findings showed that regular consumption of sweeteners in large quantities caused obesity and neoplasms in animals. Sweetener additives in consumer goods have been considered safe for humans [10]. Each such additive has a specific ADI value and amount (in milligrammes) that can be added to 1 kg (or 1 dm3) of product [13, 14, 15].
\nThe additives that are vital in terms of processing are firming additives. They create or stabilise the desirable product structure and consistency. Firming agents include gelling, thickening, emulsifying, bulking, binding, and rising agents, humectants, and modified starches. The highest status among these substances is enjoyed by hydrocolloids. Hydrocolloids, known as gums, are polysaccharides of plant, animal, or microbiological origin. There are natural (guar gum, agar, curdlan), chemically and physically modified (modified starches), and synthetic gums. With their macromolecular structure, they are able to bind water, improve solution viscosity, and create gels and spongiform masses. Hydrocolloids are used as gelling (e.g. in the production of jelly, desserts, pudding, and fruit-flavoured starch jelly), thickening (ready-made sauces, vegetable products), water-binding (powdered products to be consumed with water, frozen food), and emulsifying agents (to create oil-in-water-type emulsions). They also act as emulsion stabilisers. Hydrocolloids are considered safe for human health, although some of them can cause allergies. Consumed in large quantities, they can have laxative effects [12].
\nWhat is also important in creating product structure are emulsifiers and the emulsification method. Emulsifiers are compounds which facilitate emulsification. There are water-in-oil (margarine) and oil-in-water (mayonnaise) type of emulsions. Emulsifiers position themselves at the interface between two different phases to stabilise the emulsion. There are natural emulgents, with lecithin as the most common, and synthetic emulgents (glycerol and its esters) [1]. Product consistency and texture are also adjusted using modified starches. Such starches are usually obtained from potatoes or corn (also genetically modified one) with chemically altered composition. Similarly to hydrocolloids, such substances can bind water and produce gels and are also resistant to high temperatures [11, 12]. Modified starches are added to ready-made sauces and dishes (such as frozen pizza), frozen goods, bread, and desserts (also powdered) to thicken and maintain product consistency after thermal processing. In order to enhance starch properties, phosphates are often added during starch modification. The human body needs phosphorus, but its excess can negatively affect the bones, kidneys, and the circulatory system [7, 11, 12].
\nNowadays, consumer goods are widely available, and consumers are provided with a broad range of products to choose from. The continuously growing number of world population (approximately 7 billion in 2011) has made supply on the food market exceed demand. This situation is characteristic of countries with a high GDP. Food producers examine consumer behaviour patterns to see what encourages them to make a purchase, and also the purchase itself and its consequences, and then analyse these processes to launch a new product or a substitute for an already existing one. To sum up, the market has provided more food products than consumers are able to purchase, which results in unimaginable food wastage. Each year, approximately 100 million tonnes of food goes to waste in Europe. This quantity does not include agricultural and food waste or fish discards [13].
\nThe methodology of this study was based on the information contained on the labels. The chemical composition of the investigated food products was presented. Interview with the store’s seller concerned the popularity and frequency of sales listed in the product tables. It should be noted that the examined store is representative when it comes to this type of stores in the majority of small towns in south-eastern Poland.
\nThis study was based on data on the most frequently chosen consumer goods in a store in a small town in Poland. The town is located in a commune that has 5300 residents. Data were obtained by monitoring the sales over the course of 12 months. These products are presented in Tables 2, 3, 4, 5, 6 and classified into the following categories: (i) meat and fish; (ii) beverages; (iii) condiments; (iv) ready-made sauces, soups, and dishes; and (v) sweets and desserts. The main classification criterion was segregation into primary food groups. The chemical composition of each product, as listed on the packaging, was included in a table and then assessed against the presence of any food additives. Sixteen most common additives were selected in all the investigated products; only chemical compounds that were found in at least four food products were taken into consideration. The most common food additives were highlighted in Holt in the “product composition” column and presented in Table 1, together with their E codes. Then, based on the literature, the study described the most common additional substances.
\nName | \nSymbol | \nNumber of products | \n
---|---|---|
Citric acid | \nE330 | \n15 | \n
Monosodium glutamate | \nE621 | \n10 | \n
Guar gum | \nE412 | \n8 | \n
Sodium nitrite | \nE250 | \n7 | \n
Disodium 5′-ribonucleotides | \nE635 | \n6 | \n
Sodium erythorbate | \nE316 | \n5 | \n
Glucose-fructose syrup | \nNot considered an additive | \n5 | \n
Soy lecithin | \nNot considered an additive | \n5 | \n
Maltodextrin | \nNot considered an additive | \n5 | \n
Triphosphates | \nE451 | \n4 | \n
Xanthan gum | \nE415 | \n4 | \n
Carrageenan | \nE407 | \n4 | \n
Tocopherols | \nE306 | \n4 | \n
Glucose syrup | \nNot considered an additive | \n4 | \n
Sodium benzoate | \nE211 | \n4 | \n
Ammonia caramel | \nE150c | \n4 | \n
The most common food additives and ingredients.
Table 1 shows 16 of the most popular substances found in food. The majority of these substances are food additives; four other substances are not considered in the European Union as food additives. The additives that are the most frequently found in the food products examined in this study are citric acid (E330), monosodium glutamate (E621), and guar gum (E412). In Ref. [16] it is reported that the most popular preservatives found in food are the mixture of sodium benzoate and potassium sorbate, or potassium sorbate (E202) and sodium benzoate (E211) used separately, and also ulphur dioxide (E220). Data presented in Table 1 shows that, compared to citric acid, another preservative, sodium benzoate, is used rarer. No potassium sorbate was found in any of the products examined in this study. In Ref. [13] it can be concluded that the most commonly used preservatives and antioxidants are sorbic acid and its salts (E200-203), benzoic acid and its salts (E210-213), sulfur dioxide (E220), sodium nitrite (E250), lactic acid (E270), citric acid (E330) and tocopherols (E306). The majority of the additives listed in Ref. [13] can be found in Table 1.
\nTable 2 shows 10 meat and fish products and their composition, as specified on the label. Each of the investigated items contained at least 1 of the 16 most common food additives (Table 1). As much as 50% of meat and fish products contained four or more of such additives. The highest number of additives (seven) was found in “Z doliny Karol” mortadella. “Masarnia u Józefa” crispy ham and “Lipsko” Śląska sausage contained six different food additives. Seventy percent of the examined products had had sodium nitrite (E250) added. This means that this preservative is frequently added to meat products, as confirmed in Ref. [9]. Other widespread preservatives mentioned in Ref. [9] include lactic acid (E270), sodium benzoate (E211), sorbic acid (E200), and sulphur dioxide (E220). In Ref. [9] it also mentions other additives frequently added to meat and fish products; these include carrageenan, gum arabic, and xanthan gum. In this study, 50% of the examined items contain one or two gums, and carrageenan is present in only three in ten products. A study in Ref. [17] demonstrates that fish products are the second leading food (after edible fats) in terms of preservative content.
\nProduct | \nIngredients | \nProduct | \nIngredients | \n
---|---|---|---|
Szynka krucha (ham) Masarnia u Józefa | \nPork ham, salt, pork protein, carrageenan, potassium acetate, potassium lactate, smoke flavouring, monosodium glutamate, diphosphates, triphosphates, flavourings, sodium erythorbate, tocopherols, sodium nitrite | \nPasztet podlaski (pâté) 155 g Drosed | \nWater, mechanically separated chicken meat, rapeseed oil, chicken liver and skin, cream of wheat, salt, soy protein, potato starch, dried vegetables, spices, powdered milk, (milk) whey, sugar, maltodextrin, plant protein hydrolysate, yeast extract | \n
Kiełbasa śląska (sausage) Lipsko | \nPork 60%, pig fat 17%, water, mechanically deboned chicken meat, fibre, pork skin emulsion, potato starch, milk proteins, triphosphates, tara gum, xanthan gum, sodium erythorbate, aluminium ammonium sulphate, salt, glucose, flavourings, carmine, spice extracts, maltodextrin, monosodium glutamate, soy protein, sodium nitrite | \nŁuków przysmak kanapkowy (tinned meat) 300 g | \nPork meat 30%, water, beef meat 18%, pig fat, soy protein, salt, beef fat, triphosphates, spices, pork gelatine, flavouring, sodium nitrite, tinned high-yield luncheon meat | \n
Mortadela doliny (mortadella) Karol | \nWater, pork 20%, mechanically separated chicken meat 15%, pig fat, pork connective tissue, cream of wheat, acetylated starch, polyphosphates, triphosphates, diphosphates, sodium citrate, calcium lactate, sodium lactate, salt, soy protein concentrate, pork protein, wheat fibre, spices (including mustard seeds, corn, and legumes), spice extracts, yeast extract, flavourings, glucose syrup, glucose, vinegar, sodium erythorbate, ascorbic acid, guar gum, disodium 5′-ribonucleotides, monosodium glutamate, sodium nitrite | \nAgrovit duże porcje konserwa tyrolska (tinned meat) 400 g | \nWater, mechanically separated chicken meat 23%, pork raw materials 23%, modified (corn) starch, wheat fibre, pea fibre, salt, carrageenan, processed Eucheuma seaweed, spices, spice extracts, monosodium glutamate, sodium erythorbate, sodium nitrite | \n
Mięso mielone wieprzowe (ground pork) Adrian | \nPork meat 65%, pig fat 34%, salt, xanthan gum, carrageenan, konjac, starch, sodium nitrite | \nEuro Fish szprot w sosie pomidorowym (sprat in tomato sauce) 170 g | \nFish—sprat without heads—tomato sauce, water, tomato concentrate, sugar, rapeseed oil, salt, modified starch, dried onion, guar gum, xanthan gum, spice extracts, acetic acid | \n
Parówki (frankfurters) Indykpol | \nChicken meat 25.9%, mechanically separated turkey meat 17%, mechanically separated chicken meat 17.3%, water, poultry fat, pork, corn flour, chicken skins, pig fat, pork skins, potato starch, soy protein, salt, spices, spice extracts, flavourings, monosodium glutamate, acetylated distarch adipate, guar gum, potassium acetate, potassium lactate, diphosphates, ascorbic acid, sodium erythorbate, sodium nitrite | \nGraal Flet z makreli w sosie pomidorowym (mackerel fillet in tomato sauce) 170 g | \nMackerel fillets 60%, tomato sauce, water, tomato concentrate, sugar, rapeseed oil, modified starch, spirit vinegar, salt, powdered tomatoes, dried onion, spice extract, spices, guar gum, xanthan gum, pepper extract, maltodextrin | \n
Food additives and ingredients in the studied meat and fish products.
Table 3 shows ten non-alcoholic beverages, six of which contain at least one common food additive (Table 1). Foreign substances that are most frequently found in this food group are citric acid (E330), sodium benzoate (E211), and glucose-fructose syrup. A study in Refs. [18, 19] shows that the most popular sweeteners in non-alcoholic beverages are glucose, fructose, and glucose-fructose syrups. As shown on product label, 100% juice by brands such as “Hortex” and “Tymbark”, as well as “Cisowianka” and “Kubuś” mineral waters, is additive free. Pursuant to the Regulation of the European Parliament and of the Council (EC) of 16 December 2008, no food additives may be used in mineral and spring bottled water. The beverage to contain the largest number of additive substances was white orangeade by “Hellena”.
\nProduct | \nIngredients | \nProduct | \nIngredients | \n
---|---|---|---|
Woda mineralna gazowana (carbonated mineral water) Cisownianka 1.5 L | \nNatural mineral water, unsaturated with carbon dioxide, moderately mineralised | \nWoda mineralna niegazowana (non-carbonated mineral water) Kubuś water 0.5 L | \nWater, cane sugar, apple juice from concentrated apple juice, lemon juice from concentrated lemon juice, flavouring | \n
Sok jabłko (apple juice) 100% 1 L Hortex | \n100% apple juice from concentrated apple juice | \nCoca cola 1.5 L | \nWater, sugar, carbon dioxide, sulphite ammonia caramel, phosphoric acid, natural flavourings, including caffeine | \n
Sok multiwitamina (multivitamin juice) 100% 1 L Tymbark | \nJuices from concentrated apple juice 60% and orange juice 22%, carrot juice from concentrated juice 12%, purées from banana 3%, peach, guava, papaya, juices from concentrated pineapple juice 2%, mango juice 0.5%, passion fruit juice 0.1%, lychee juice 0.05%, cactus fig juice, kiwi fruit juice and lime juice, vitamins A, C, E, B6, and B12, thiamine, riboflavin, niacin, biotin, folic acid, pantothenic acid | \nTymbark 2 L jabłko-pomarańcza (apple-orange) | \nWater, orange juice from concentrated juice 19%, glucose-fructose syrup, sugar, peach juice from concentrated juice 1%, lemon concentrate, flavourings, ascorbic acid, carotenes | \n
Volcano 2 L cola | \nSpring water, carbon dioxide, sulphite ammonia caramel, phosphoric acid, citric acid, sodium citrates, flavourings (including caffeine), gum arabic, aspartame, saccharin, sodium benzoate, potassium sorbate | \nVolcano 2 L pomarańcza (orange) | \nSpring water, carbon dioxide, orange juice 0.3% from concentrated orange juice, citric acid, gum arabic, glycerol and plant resin esters, flavouring, cyclamates, saccharin, aspartame, acesulfame K, sodium benzoate, potassium sorbate, ascorbic acid, carotenes, beta-apo-8′-carotenal | \n
Hellena 1.25 L oranżada biała (white orangeade) | \nSugar, water, glucose-fructose syrup, carbon dioxide, citric acid, flavouring, sodium benzoate | \nKubuś marchew, jabłko, pomarańcza, sok (carrot, apple, and orange juice) 330 mL | \nPurées and juices (59%), water, glucose-fructose syrup, citric acid, vitamin C, flavouring | \n
Food ingredients in the studied non-alcoholic beverages.
Table 4 shows 12 food items, such as ketchup, mustard, herbs and spices, and tomato concentrates, together with their composition. Only four products in this group contain a food additive, of which three are preserved using citric acid (E330). In this group of products, the products to contain the most common additive substances were the ketchup and the Kucharek seasoning by “Prymat”. Pursuant to the Regulation of the European Parliament and of the Council (EC) of 16 December 2008, tomato products (such as concentrates) must not contain food colours. They may, however, contain other additives. The ketchup has no colours, but contains other food additives. Studies in Ref. [17] demonstrate that mayonnaises and mustards are the fourth most often preserved product group, with ready-made concentrates ranking seventh. One of the two mustards examined in this paper contained a preservative, and two of the presented tomato concentrates had not had any food additives added to them.
\nProduct | \nIngredients | \nProduct | \nIngredients | \n
---|---|---|---|
Koncentrat pomidorowy (tomato concentrate) Aro 190 g | \n30% tomato concentrate | \nKoncentrat pomidorowy (tomato concentrate) Pudliszki | \n30% tomato concentrate | \n
Ketchup łagodny (mild ketchup) 470 g | \n37% tomato concentrate, water, sugar, vinegar, modified starch, salt, citric acid, sodium benzoate, thyme, oregano, savoury, sage, coriander, flavouring | \nKetchup Pudliszki łgodny (mild ketchup) 480 g | \nTomatoes, sugar, vinegar, salt, modified starch, natural flavouring | \n
Musztarda Parczew kremska (Krems mustard) 180 g | \nWater, mustard seeds, vinegar, sugar, salt, spices | \nMusztarda Roleski stołowa (table mustard) | \nWater, mustard seeds, sugar, spirit vinegar, salt, spices, turmeric extract, citric acid, natural flavouring | \n
Zioła prowansalskie (Herbes de Provence) Prymat | \nBasil, marjoram, rosemary, savoury, sage, thyme, oregano, mint | \nPrzyprawa do kurczaka (chicken seasoning) Goleo | \nSalt, garlic, white mustard seeds, sweet pepper, carrot, coriander, fenugreek, caraway, chilli, turmeric, cinnamon | \n
Przyprawa Tzatziki (tzatziki seasoning) Prymat | \nGarlic, salt, sugar, onion, citric acid, onion extract, dill extract, dill leaves, pepper extract, black pepper | \nKucharek Prymat 250 g | \nSalt, died vegetables, monosodium glutamate, disodium 5′-ribonucleotides, sugar, starch, black pepper, riboflavin | \n
Food ingredients in the studied condiments.
Table 5 shows 12 products categorised into ready-made dishes, soups and sauces, and their chemical composition. Each of these products contains at least one common additive. Citric acid (E330) was added to nearly 67% of the products in this category. Only five in twelve items (including four instant soups and stock cubes) contain the three most popular food additive substances (Table 1). A study in Ref. [13] shows that the most common additives in ready-made dishes are citric acid (E330), sunset yellow (E110), guar gum (E412), disodium guanylate (E627), disodium inosinate (E631), and monosodium glutamate (E621).
\nProduct | \nIngredients | \nProduct | \nIngredients | \n
---|---|---|---|
Rosół drobiowy kucharek (chicken soup) 60 g | \nSalt, palm fat, partially hydrogenated, starch, monosodium glutamate, disodium 5′-ribonucleotides, rapeseed oil, dried vegetables, sugar, flavourings, chicken fat, turmeric, citric acid, dried chicken meat | \nRosół drobiowy Winiary (chicken soup) 60 g | \nSalt, monosodium glutamate, disodium 5′-ribonucleotides, starch, fully hydrogenated palm fat, flavourings, sugar, chicken fat, spices, dried vegetables, citric acid, dried chicken meat | \n
Vifon kurczak Carry (curry chicken) | \nNoodles (92.1%), wheat flour, plant fat, tapioca, modified starch, acetylated starch, sugar, stabilisers (pentasodium triphosphate, guar gum, rising substances: sodium carbonate, potassium carbonate, turmeric), flavouring additives (7.9%) (refined palm oil, salt, sugar), flavour enhancers (monosodium glutamate, disodium guanylate, disodium inosinate, dried vegetables (carrot, green onion, coriander), powdered curry (flavour additive content 6%), turmeric, aniseed, clove, coriander seed, cinnamon, pepper, garlic, chilli, lemongrass, flavouring), colour (beta-carotene, antioxidant tocopherols) | \nAmino zupa błyskawiczna gulaszowa (instant goulash soup) | \nNoodles (85%), wheat flour, palm fat, modified starch, salt, rapeseed oil, tocopherols, fatty acid and ascorbic acid esters; flavouring mix: salt, starch, paprika, monosodium glutamate, disodium guanylate and disodium inosinate, tomato concentrate, onion, flavourings, palm fat, Cayenne pepper, garlic, caraway, hydrolysed plant protein, dried pork, parsley, ammonia caramel | \n
Sos Winiary Italia boloński (Bolognese sauce) | \nDried vegetables, modified starch, sugar, salt, spices, flavourings, sunflower oil, citric acid, spices, beetroot juice concentrate, olive oil | \nSos Winiary pieczeniowy ciemny (dark roasting sauce) | \nPotato starch, modified starch, salt, dried vegetables, flavourings, sugar, yeast extracts, fully hydrogenated palm fat, palm oil, rice flour, ammonia caramel, wheat protein hydrolysate, spices, citric acid | \n
Sos Winiary borowikowy (bolete sauce) | \nCorn starch, wheat flour, powdered cream, palm oil, sunflower oil, maltodextrin, dried mushroom, salt, flavourings, lactose, yeast extract, sugar, dried fried onion, dried onion, milk proteins, spices, wheat protein hydrolysate, ammonia caramel, bolete extract | \nZupa Winiary barszcz biały (white borscht) | \nWheat flour, skimmed powdered milk, salt, potato starch, sugar, smoked pig fat, citric acid, dried vegetables, yeast extract, herbs, spices, smoke flavour | \n
Zupa Winiary jak u mamy pieczarkowa (champignon soup) | \nCorn starch, skimmed powdered milk, wheat flour, powdered cream, dried champignons, yeast extracts, salt, potato starch, dried vegetables, flavourings, sunflower oil, wheat protein hydrolysate, parsley, black pepper, citric acid | \nŁowicz sos boloński (Bolognese sauce) 350 g | \nTomatoes, water, vegetables, glucose-fructose syrup, apple purée, modified corn starch, salt, sugar, guar gum, citric acid, rapeseed oil, spices, herbs, flavourings, ground dried parsley, garlic and paprika, leek and carrot extracts | \n
Danie gotowe Flaczki (ready-made tripe) Pamapol | \nWater, beef rumen 305, wheat flour, carrot, parsley, celeriac, tomato concentrate, onion, salt, pork gelatine, sugar, soy protein hydrolysate, dried vegetables, yeast extract, spices, disodium 5′-ribonucleotides, ammonia caramel, flavourings, partially hydrogenated palm and rapeseed fats | \nPomysł na soczystą karkówkę z ziemniakami (pork shoulder with potatoes seasoning) Winiary | \nWheat flour, vegetables, salt, modified starch, yeast extract, herbs, maltodextrin, plant oil, spices, flavourings, wheat protein hydrolysate, citric acid | \n
Food ingredients and additives in the studied ready-made dishes, soups, and sauces.
Table 6 shows 10 food items classified as sweets and desserts. As many as nine products in this group contained at least one of the most common food additives (Table 1). Glucose-fructose or glucose syrups were found in six of the examined items. A study in Ref. [19] shows that sweets often include the so-called Southampton colours, such as quinoline yellow and tartrazine. However, the study reports that the amounts of these substances added to sweets are much lower than the maximum values allowed by the applicable law.
\nProduct | \nIngredients | \nProduct | \nIngredients | \n
---|---|---|---|
Lód Top milker (ice cream) Koral | \nSkimmed reconstituted milk, sugar, cocoa oil, glucose syrup, skimmed powdered milk, mono- and diglycerides of fatty acids, locust bean flour, guar gum, powdered cream, natural vanilla, flavourings | \nBaton 3bit (candy bar) | \nSugar, biscuit 14% [wheat flour, sugar, plant fat, powdered whey, glucose-fructose syrup, whole powdered milk, salt, rising agents (sodium bicarbonate, ammonium bicarbonate), acidity regulator (citric acid), skimmed powdered milk (13. 5% in filling), plant fat, cocoa fat, cocoa paste, powdered whey, plant oil, milk fat, emulsifiers (soy lecithin, polyglycerol polyricinoleate), flavourings, salt. Cocoa mass in chocolate—minimum 30% | \n
7 days | \nWheat flour, cocoa filling 25% [(sugar, partially hydrogenated plant fats, water, low-fat powdered cocoa 7%, skimmed powdered milk, ethyl alcohol, emulsifier (lactic acid esters of mono- and diglycerides of fatty acids), vanilla flavouring, gelling agent (sodium alginate), preservative (potassium sorbate 0.1%)], margarine [partially hydrogenated plant fats, water, salt, emulsifier (mono- and diglycerides of fatty acids), acidity regulator, flavouring, preservative (potassium sorbate 0.1%)], sugar, stabiliser (mono- and diglycerides of fatty acids), glucose-fructose syrup, yeast, skimmed powdered milk, salt, vanilla flavouring, preservative (calcium propionate 0.1%), soy flour, emulsifier (soy lecithin) | \nLód rożek truskawkowy (ice cream cone) Koral | \nSkimmed reconstituted milk, cornet 14% [wheat flour, sugar, palm fat, potato starch, emulsifier (soy lecithin, wheat fibre, salt), colour (sulphite ammonia caramel], sugar, coconut oil, strawberry sauce 7% [strawberries 42%, sugar, glucose syrup, water, thickening agent (hydroxypropyl distarch glycerol), acidity regulator (citric acid, flavouring], coating for cornet waterproofing [sugar, coconut and palm fats, reduced-fat powdered cocoa (10–12%), emulsifier (soy lecithin)], water, glucose syrup, strawberry purée 1%, emulsifier (mono- and diglycerides of fatty acids), stabilisers (Guar gum, cellulose gum, carrageenan, locust bean flour), acidity regulator (citric acid), colours (betanin, annatto, flavourings) | \n
Baton Milky way (candy bar) | \nSugar, glucose syrup, skimmed powdered milk, cocoa fat, palm fat, cocoa mass, milk fat, lactose, powdered (milk) whey, barley malt extract, salt, emulsifier (soy lecithin), powdered egg white, hydrolysed milk protein, natural vanilla extract | \nMlekołaki Lubella muszelki (cereal) 250 g | \nWholemeal wheat, wheat, and corn flours, sugar, glucose, reduced-fat cocoa, cocoa, barley malt extract, milk chocolate, palm fat, salt, soy lecithin, flavourings, vitamin C, niacin, pantothenic acid, vitamin B, riboflavin, thiamine, folic acid, vitamin B12, calcium, iron | \n
Nestlé Corn Flakes 600 g | \nCorn grits, sugar, salt, glucose, brown sugar, invert sugar syrup, cane sugar molasses, sodium phosphates, niacin, pantothenic acid, riboflavin, vitamin B6, folic acid | \nNestlé Frutina 250 g | \nWheat flakes (wholemeal wheat, sugar, wheat bran, barley malt extract, invert sugar syrup, salt, cane sugar molasses, glucose syrup, sodium phosphates, tocopherols), raisins, cut dried apples, sodium metabisulphite, niacin, pantothenic acid, vitamin B6, riboflavin, folic acid, calcium, iron | \n
Lays zielona cebulka (crisps) 150 g | \nPotatoes, palm oil, sunflower oil, flavouring, powdered onion, powdered milk whey, powdered milk lactose, sugar, powdered milk, monosodium glutamate, disodium 5′-ribonucleotides, flavourings, powdered milk cheese, citric acid, malic acid, annatto, pepper extract, powdered garlic, maltodextrin, salt | \nStar chips paprika (crisps) 170 g | \nPotatoes, palm fat, flavourings, wheat breadcrumbs, glucose, sugar, monosodium glutamate, pepper extract, citric acid, salt | \n
Food additives and ingredients in the studied sweets.
Citric acid (E330) is a natural compound found in citrus fruits. It is also the by-product of digestive processes in the human body. However, on the industrial scale, the substance is produced using the Aspergillus niger mould. Citric acid is used in food as an acidity regulator, preservative, and flavour enhancer. Outside the food industry, the acid is added to cleaning agents and acts as a decalcifying agent. Citric acid in food is a safe additive and is added to food on the quantum satis basis; nevertheless its widespread use constitutes a risk. This substance is found in many food products, such as beverages, juices, lemonades, sweets, ice creams, canned goods, and even bread, so customers consume it in large quantities everyday [20]. When consumed frequently in excess, citric acid can lead to enamel degradation and teeth deterioration. This additive also supports the absorption of heavy metals, which, in turn, might lead to brain impairment. It can also affect the kidneys and liver [13, 15].
\nMonosodium glutamate (E621) is the most widespread flavour enhancer. It is even considered to be one of the five basic tastes (umami). Glutamic acid and its (magnesium, potassium, and calcium) salts lend a meaty flavour to products. The substance was first extracted from algae by a Japanese scientist, but now it is generally produced by biotechnological means using microorganisms that can be genetically modified [6]. Another commonly used flavour enhancer is chemically produced disodium 5′-ribonucleotides (E635). These additives can be found in ready-made dishes, sauces, meat and fish products, instant soups, crisps, and cakes. These flavour enhancers are the not inert in relation to the neurological system [16]. This can affect brain cells and lead to headaches, heart palpitations, excessive sweating, listlessness, nausea, and skin lesions. Such anomalies, which could have been caused by the excessive consumption of products rich in glutamates, are referred to as the Chinese restaurant syndrome [20]. Flavour enhancers can also serve a positive function by increasing appetite in the sick or the elderly [20]. Other additional substances commonly found in foodstuffs are polysaccharides:
\nGuar gum (E412) and xanthan gum (E415). These are referred to as hydrocolloids, i.e. substances that bind water, are easily soluble in both cold and warm water, and improve mixture viscosity. Guar gum is a polysaccharide obtained from guar, a leguminous plant grown in India and Pakistan [14]. Xanthan gum is a polysaccharide of microbiological origin. On the industrial scale, it is obtained as a result of Xanthomonas campestris bacteria fermenting the sugar contained in corn (often genetically modified). Both these additives are approved for use in all food products as thickening, firming, and stabilising agents, on the quantum satis basis. Guar gum and xanthan gum can be found mainly in bread, cakes, ready-made sauces and dishes, and powdered food, where they ensure the appropriate consistency. Moreover, they prevent the crystallisation of water in ice cream and frozen food and the separation of fluids in dairy products and juices. The human body is not capable of digesting, breaking down, or absorbing these gums. These substances swell in the intestines, which can cause flatulence and stomach ache. In addition, guar gum can cause allergies [13, 14, 15].
\nA commonly found preservative is sodium nitrite (E250). It is a salty and white or yellowish crystalline powder, obtained by the chemical processing of nitric acid or some lyes and gases [9]. This additive is generally used in the meat industry to inhibit botulinum toxin and Staphylococcus aureus bacteria, slow down fat rancidification, maintain the pink red colour of meat, and provide meat with a specific flavour. It does not, however, prevent the growth of yeast or mould. Sodium nitrite is toxic, oxidising, and dangerous to the environment, so it must not be added to food in its pure form. This additive is used in very small doses (0.5–0.6%) in the form of a mixture with domestic salt [9] in amounts up to 150 mL per L or mg kg−1. When consumed in large quantities, nitrites can cause cyanosis, whose symptoms include blue coloration of the skin, lips, and mucous membranes. During digestion, nitrites are transformed into carcinogenic nitrosamines. Moreover, they are particularly dangerous for children, since they stop erythrocytes from binding oxygen, which can lead to death by suffocation [11].
\nA common ingredient in food is maltodextrin, which in the European Union is not considered as a food additive, but as an ingredient. Therefore, within the community, maltodextrin has no E code, while in Sweden it is considered an additive and identified as E1400 [18]. Maltodextrin is a disaccharide obtained from corn starch, but it is not sweet in taste. Nevertheless, it provides greater sweetness than normal sugar or grape sugar (the glycaemic index of maltodextrin is 120, that of normal sugar is 70, and that of grape sugar is 100). It is used as a thickening agent, stabiliser, bulking agent, and even as a fat substitute in low-calorie products. It is added to products for athletes and children, to instant soups, sweets, and meat products [10]. Maltodextrin does not affect the natural product taste or flavour, while it provides human body with carbohydrates and energy. Due to the fact that glucose particles in maltodextrin are broken down only in the intestines, it can also support metabolism. A negative aspect of its use is tooth decay [10, 18].
\nWhat frequently occurs in consumer goods is glucose-fructose syrup. Similarly to maltodextrin, it is not considered to be a food additive, but, due to its widespread application, it is important to mention it here. Glucose-fructose syrup, also known as high-fructose corn syrup (HFCS), replaces traditional sugar in many products, such as beverages, sweets, jams, fruit products, and liqueurs, and in the United States and Canada is the dominant sweetener [19]. Sucrose is a disaccharide composed of glucose and fructose, which are joined with alpha-1,4-glycosidic bond, and HFCS contains free fructose and free glucose in specific proportions. The name of this substance depends on the proportion of its ingredients. When the syrup contains more fructose, it is referred to as fructose-glucose syrup [12]. It is obtained mainly from corn starch as a result of acid or enzymatic hydrolysis. Glucose-fructose syrup is much sweeter and cheaper than traditional sugar, it does not crystallise, and it has a liquid form, which makes it functional during processing. Nevertheless, there are some disturbing aspects of using this substance. During the consumption of products with glucose-fructose syrup, the body receives unnatural amounts of fructose, which is broken down in the liver in a manner similar to alcohol. Therefore, its excessive amounts can cause fatty liver and overburden this organ. This has even been named “non-alcoholic fatty liver disease”. In addition, heavy consumption of monosaccharides has been found to contribute to obesity, which, in turn, can cause high blood pressure and diabetes. Fructose affects the lipid metabolism and disrupts the perception of hunger and satiety. Labels do not provide the exact HFCS content, but it is estimated that the consumption of a single product with this substance satisfies the acceptable daily monosaccharide intake [5, 6, 11, 13].
\nAnother frequently added substance is sodium erythorbate (E316). This synthetic compound is used as an antioxidant and stabiliser in meat and fish products and is useful for ham and sausage pickling [13]. It has similar properties to ascorbic acid, but it is not effective as vitamin C. Sodium erythorbate is considered to be noninvasive in the human body [12, 13].
\nThe most widespread natural emulsifier is soy lecithin. Etymologically, the word “lecithin” can be traced back to lekythos, Greek for egg yolk, but this compound is actually found in any plant or animal cell. Lecithin is produced from eggs, sunflower and rapeseed oils, and soybeans [11, 12, 13]. This additive is identified as E322 and is used for the production of mayonnaise, ice creams, margarine, ready-made desserts, sauces, and instant soups. Products with added lecithin dissolve in water more easily. EU law does not impose any limits on the use of E322. Only in products for children, lecithin content must not exceed 1 g per L.
\nTriphosphates (E451), as well as diphosphates and polyphosphates, are used as preservatives, flavour enhancers, stabilisers, and rising and water-binding agents. Triphosphates are produced chemically and have a broad application. They are added to sauces, meats and meat products, desserts, bread, pâtés, fish products, ice creams, and non-alcoholic beverages [21]. The human body needs phosphorus in specific amounts, but the widespread use of phosphoric acids and phosphates in food makes people likely to consume this element in excess. When consumed regularly, increased doses of phosphates can lead to osteoporosis or contribute to kidney dysfunction and affect the circulatory system [13, 21]. A popular hydrocolloid found in food is carrageenan (E407). This substance is extracted from Eucheuma, a tribe of red algae. Carrageenan is highly soluble in water and is used as a bulking agent in dietary products, and it is also added to beverages, ice creams, sauces, marmalades, and powdered milk [6, 7]. Carrageenan can be used on the quantum satis basis. Usually, it is combined with other hydrocolloids. This additive is not digestible by the human body. There are certain objections concerning the consumption of carrageenan, e.g. it can cause intestinal cancer and stomach ulcers [11, 12, 13].
\nTocopherols (E306) are commonly known as vitamin E, insoluble in water and soluble in fats. It is used as a preservative, stabiliser, and potent antioxidant in such products as oils, margarines, desserts, meat products, and alcoholic beverages. Tocopherols are produced synthetically or obtained from plant oils, but natural vitamin E is twice as easily absorbed by the human body [21].
\nCommon preservatives include benzoic acid and its salts, of which the most frequently used is sodium benzoate (E211). Negligible amounts of these substances are naturally found in berries, mushrooms, and fermented milk-based drinks. On an industrial scale, it is produced synthetically from toluene obtained from crude oil [3, 12]. What is characteristic of sodium benzoate is that it slows down the growth of mould and yeast, but does not prevent the growth of bacteria, which is why it is often used with other preservatives, such as sulphur dioxide (E220). It is commonly used in products with acidic pH, such as marinades, fruit juices, and products with mayonnaise, such as vegetable salads. Sodium benzoate can cause allergies [6, 13]. Our own study (see “Results and discussion”) showed that ammonia caramel (E150c) and sulphite ammonia caramel (E150d) are fairly common colours. It adds brown to black colours to products. Under natural conditions, this substance is created when sugar is heated. As a food additive, it is produced chemically using ammonia, as well as phosphates, sulphates, and sulphites (sulphite ammonia caramel is produced) [19]. This substance is approved for use under EU law [5]; however, there are studies that have confirmed that it negatively affects human health. It has been proven that this colour can cause hyperactivity and liver, thyroid, and lung neoplasms and also impair immunity. Ammonia caramel is used to dye non-alcoholic beverages, such as cola and marmalades [10, 11].
\nThe external aspect that is most crucial for buyers when it comes to food selection is its freshness. Buyers assess the best before date against the possibility of consuming the food quickly or storing it for future use. Another determinant is the value of the item. Any consumer will pay attention to the price of the product they buy. Another factor is the product ingredients specified on the packaging. Buyers have been observed to have developed a habit of reading labels before buying anything. Some customers also pay attention to the country of origin or brand [22]. Men and women who are determined to stay fit will also consider nutritional value. The factors that are not considered that are relevant include net product weight, information about any genetically modified raw material content, and notices about any implemented quality management systems. Moreover, consumers are likely to be affected by marketing devices, such as advertisements or special offers, used by producers. A temporary reduction in price, or the opportunity to buy two items for the price of one, encourages customers to make a purchase [3, 4]. What is also vital is whether the food is functional. Many people live at a fast pace, work a lot, or get stuck in traffic jams, and the lack of free time pushes them to buy ready-made dishes to be heated up at home or food that can be prepared in an instant [4, 13, 22].
\nNowadays, food additives are very widespread in the everyday human diet, but not all of them are synthetic and invasive to human health. Products which must not contain foreign substances do not contain food additives. The explorations undertaken by this and other studies confirm the widespread use of the investigated additives, except for citric acid, which is less popular an additive than sodium benzoate and potassium sorbate. This study shows that when adopting a healthy lifestyle, consumers can choose from a range of food and pharmaceutical products that either contain a limited amount of unconventional substances or do not contain such substances at all.
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