Kaput J, Ordovas JM, Ferguson L, van Ommen B, Rodriguez RL, Allen L, Ames BN, Dawson K, German B, Krauss R, Malyj W, Archer MC, Barnes S, Bartholomew A, Birk R, van Bladeren P, Bradford KJ, Brown KH, Caetano R, Castle D, Chadwick R, Clarke S, Clément K, Cooney CA, Corella D, Manica da Cruz IB, Daniel H, Duster T, Ebbesson SO, Elliott R, Fairweather-Tait S, Felton J, Fenech M, Finley JW, Fogg-Johnson N, Gill-Garrison R, Gibney MJ, Gillies PJ, Gustafsson JA, Hartman JL, He L, Hwang JK, Jais JP, Jang Y, Joost H, Junien C, Kanter M, Kibbe WA, Koletzko B, Korf BR, Kornman K, Krempin DW, Langin D, Lauren DR, Ho Lee J, Leveille GA, Lin SJ, Mathers J, Mayne M, McNabb W, Milner JA, Morgan P, Muller M, Nikolsky Y, van der Ouderaa F, Park T, Pensel N, Perez-Jimenez F, Poutanen K, Roberts M, Saris WH, Schuster G, Shelling AN, Simopoulos AP, Southon S, Tai ES, Towne B, Trayhurn P, Uauy R, Visek WJ, Warden C, Weiss R, Wiencke J, Winkler J, Wolff GL, Zhao-Wilson X, Zucker JD (2005) The case for strategic international alliances to harness nutritional genomics for public and personal health., Br J Nutr 94 (5) pp. 623-632
Nutrigenomics is the study of how constituents of the diet interact with genes, and their products, to alter phenotype and, conversely, how genes and their products metabolise these constituents into nutrients, antinutrients, and bioactive compounds. Results from molecular and genetic epidemiological studies indicate that dietary unbalance can alter gene-nutrient interactions in ways that increase the risk of developing chronic disease. The interplay of human genetic variation and environmental factors will make identifying causative genes and nutrients a formidable, but not intractable, challenge. We provide specific recommendations for how to best meet this challenge and discuss the need for new methodologies and the use of comprehensive analyses of nutrient-genotype interactions involving large and diverse populations. The objective of the present paper is to stimulate discourse and collaboration among nutrigenomic researchers and stakeholders, a process that will lead to an increase in global health and wellness by reducing health disparities in developed and developing countries.
de Roos B, Duthie SJ, Polley AC, Mulholland F, Bouwman FG, Heim C, Rucklidge GJ, Johnson IT, Mariman EC, Daniel H, Elliott RM (2008) Proteomic methodological recommendations for studies involving human plasma, platelets, and peripheral blood mononuclear cells., J Proteome Res 7 (6) pp. 2280-2290
This study was designed to develop, optimize and validate protocols for blood processing prior to proteomic analysis of plasma, platelets and peripheral blood mononuclear cells (PBMC) and to determine analytical variation of a single sample of depleted plasma, platelet and PBMC proteins within and between four laboratories each using their own standard operating protocols for 2D gel electrophoresis. Plasma depleted either using the Beckman Coulter IgY-12 proteome partitioning kit or the Amersham albumin and IgG depletion columns gave good quality gels, but reproducibility appeared better with the single-use immuno-affinity column. The use of the Millipore Filter Device for protein concentration gave a 16% ( p
Heaton SJ, Eady JJ, Parker ML, Gotts KL, Dainty JR, Fairweather-Tait SJ, McArdle HJ, Srai KS, Elliott RM (2008) The use of BeWo cells as an in vitro model for placental iron transport., Am J Physiol Cell Physiol 295 (5) pp. C1445-C1453
BeWo cells are a placental cell line that has been widely used as an in vitro model for the placenta. The b30 subclone of these cells can be grown on permeable membranes in bicameral chambers to form confluent cell layers, enabling rates of both nutrient uptake into the cells from the apical surface and efflux from the basolateral membrane to be determined. The aim of this study was to evaluate structural and functional properties of confluent b30 BeWo cell layers grown in bicameral chambers, focusing on the potential application for studying receptor-mediated uptake and transport of transferrin (Tf)-bound iron (Fe-Tf). While it proved extremely difficult to establish and maintain an intact BeWo cell monolayer, it was possible to grow the cells to a confluent multilayer. Iron, applied as Fe-Tf, was rapidly transported across this cell layer; 9.3 +/- 0.5% of the total dose was transported after 8 h, equivalent to 38.8 +/- 2.1 pmol.cm(-2).h(-1). Transfer of Tf across the cell layer was much more limited; 2.4 +/- 0.2% of the total dose was transported after 8 h, equivalent to 5.0 +/- 0.4 pmol.cm(-2).h(-1). Compartmental modeling of these data suggested that iron was transported across the cell layer predominantly, if not exclusively, via a transcellular route, whereas Tf taken up into the cells was predominantly recycled back to the apical compartment. The results suggest that these cells are very efficient at transporting iron and, under carefully controlled conditions, can be a valuable tool for the study of iron transport in the placenta.
Elliott RM (2005) Genes, genomes and molecular biology in nutrition research: A story of opportunities and challenges, Current Topics in Nutraceutical Research 3 (3) pp. 189-192
The wealth of freely available genetic information derived from genome mapping projects and the development of functional genomic and other powerful molecular biological tools have lead to profound changes in the scope of life science research and the way it is performed. As in many other disciplines, scientists in the fields of food science and nutrition are now beginning to fully realise the enormous potential these new resources and are seeking to integrate them into their research. However, there are still a number of issues specific to nutrition research that need to be addressed to ensure that this opportunity is exploited to the full. Copyright © 2005 by New Century Health Publishers, LLC.
van Ommen B, Bouwman J, Dragsted LO, Drevon CA, Elliott R, de Groot P, Kaput J, Mathers JC, Müller M, Pepping F, Saito J, Scalbert A, Radonjic M, Rocca-Serra P, Travis A, Wopereis S, Evelo CT (2010) Challenges of molecular nutrition research 6: the nutritional phenotype database to store, share and evaluate nutritional systems biology studies., Genes Nutr 5 (3) pp. 189-203
The challenge of modern nutrition and health research is to identify food-based strategies promoting life-long optimal health and well-being. This research is complex because it exploits a multitude of bioactive compounds acting on an extensive network of interacting processes. Whereas nutrition research can profit enormously from the revolution in 'omics' technologies, it has discipline-specific requirements for analytical and bioinformatic procedures. In addition to measurements of the parameters of interest (measures of health), extensive description of the subjects of study and foods or diets consumed is central for describing the nutritional phenotype. We propose and pursue an infrastructural activity of constructing the "Nutritional Phenotype database" (dbNP). When fully developed, dbNP will be a research and collaboration tool and a publicly available data and knowledge repository. Creation and implementation of the dbNP will maximize benefits to the research community by enabling integration and interrogation of data from multiple studies, from different research groups, different countries and different-omics levels. The dbNP is designed to facilitate storage of biologically relevant, pre-processed-omics data, as well as study descriptive and study participant phenotype data. It is also important to enable the combination of this information at different levels (e.g. to facilitate linkage of data describing participant phenotype, genotype and food intake with information on study design and-omics measurements, and to combine all of this with existing knowledge). The biological information stored in the database (i.e. genetics, transcriptomics, proteomics, biomarkers, metabolomics, functional assays, food intake and food composition) is tailored to nutrition research and embedded in an environment of standard procedures and protocols, annotations, modular data-basing, networking and integrated bioinformatics. The dbNP is an evolving enterprise, which is only sustainable if it is accepted and adopted by the wider nutrition and health research community as an open source, pre-competitive and publicly available resource where many partners both can contribute and profit from its developments. We introduce the Nutrigenomics Organisation (NuGO, http://www.nugo.org
) as a membership association responsible for establishing and curating the dbNP. Within NuGO, all efforts related to dbNP (i.e. usage, coordination, integration, facilitation and maintenance)
Elliott R, Ong TJ (2002) Science, medicine, and the future - Nutritional genomics, BRITISH MEDICAL JOURNAL 324 (7351) pp. 1438-1442 B M J PUBLISHING GROUP
Astley SB, Elliott RM (2005) How strong is the evidence that lycopene supplementation can modify biomarkers of oxidative damage and DNA repair in human lymphocytes?, J Nutr 135 (8) pp. 2071S-2073S
Astley SB, Elliott RM, Archer DB, Southon S (2004) Evidence that dietary supplementation with carotenoids and carotenoid-rich foods modulates the DNA damage: repair balance in human lymphocytes., Br J Nutr 91 (1) pp. 63-72
Epidemiological evidence has shown that the habitual consumption of diets high in fruits and vegetables is associated with reduced risk of cancers. The challenge is to identify causal mechanisms of effect. The aim of the current study was to determine whether an increase in rate of removal of DNA single-strand breaks (SSB) following oxidative challenge could be provoked ex vivo in peripheral blood lymphocytes (PBL). The PBL were isolated from apparently healthy volunteers following dietary intervention with: (1) a mixed carotene capsule; (2) a daily portion of cooked minced carrots; (3) a matched placebo; (4) a portion of mandarin oranges; (5) vitamin C tablets. Single-cell gel electrophoresis was employed to measure baseline levels of SSB and DNA susceptibility to oxidative damage, and to monitor the number of SSB over 4 h, in both unchallenged and H2O2-treated PBL. The enzymatic capacity for repair of different types of DNA oxidative lesions was also measured using two related cell-free assays. There was no evidence that any of the dietary supplementation regimens altered baseline levels of SSB, provided any direct antioxidant protection or altered DNA repair capacity, with two exceptions: the number of SSB following exposure to H2O2 decreased more rapidly in PBL from volunteers given the mixed carotene capsules and repair patch synthesis activity in PBL increased from volunteers given the cooked carrots. These results suggest that carotenoids and carotenoid-rich foods can influence DNA damage:repair by modulation of discrete stages in the DNA repair mechanisms.
Astley S, Elliott R, Archer D, Southon S (2000) DNA damage and repair: Relative responses to antioxidant nutrients in the diet, DIETARY ANTICARCINOGENS AND ANTIMUTAGENS (255) pp. 138-142 ROYAL SOC CHEMISTRY
Garosi P, De Filippo C, van Erk M, Rocca-Serra P, Sansone SA, Elliott R (2005) Defining best practice for microarray analyses in nutrigenomic studies., Br J Nutr 93 (4) pp. 425-432
Microarrays represent a powerful tool for studies of diet-gene interactions. Their use is, however, associated with a number of technical challenges and potential pitfalls. The cost of microarrays continues to drop but is still comparatively high. This, coupled with the complex logistical issues associated with performing nutritional microarray studies, often means that compromises have to be made in the number and type of samples analysed. Additionally, technical variations between array platforms and analytical procedures will almost inevitably lead to differences in the transcriptional responses observed. Consequently, conflicting data may be produced, important effects may be missed and/or false leads generated (e.g. apparent patterns of differential gene regulation that ultimately prove to be incorrect or not significant). This is likely to be particularly true in the field of nutrition, in which we expect that many dietary bioactive agents at nutritionally relevant concentrations will elicit subtle changes in gene transcription that may be critically important in biological terms but will be difficult to detect reliably. Thus, great care should always be taken in designing and executing microarray studies. This article seeks to provide an overview of both the main practical and theoretical considerations in microarray use that represent potential sources of technical variation and error. Wherever possible, recommendations are made on what we propose to be the best approach. The overall aims are to provide a basic framework of advice for researchers who are new to the use of microarrays and to promote a discussion of standardisation and best practice in the field.
Rocca-Serra P, Elliott RM (2006) Data storage: bringing us a step closer to data sharing?, Br J Nutr 95 (6) pp. 1237-1239
Wortley GM, Elliott RM, Harvey LJ, Fairweath-Tait SJ (2006) Copper homeostasis: use of in vitro cell systems and transcriptional analysis to detect markers of copper regulation in human subjects., PROCEEDINGS OF THE NUTRITION SOCIETY 65 pp. 103A-103A CAMBRIDGE UNIV PRESS
Astley SB, Elliott RM, Archer DB, Southon S (2002) Increased cellular carotenoid levels reduce the persistence of DNA single-strand breaks after oxidative challenge., Nutr Cancer 43 (2) pp. 202-213
Dietary antioxidants, such as the carotenoids, may protect DNA from oxidative damage. This has been proposed to explain the epidemiological association between higher consumption of fruits and vegetables, which are rich in antioxidants, and lower incidence of cancer. However, this remains to be demonstrated conclusively. The effects of carotenoid supplementation on 1) baseline DNA damage, 2) susceptibility of cellular DNA to oxidative attack, and 3) DNA repair were measured in the human lymphocyte cell line Molt-17. Baseline DNA damage, susceptibility to oxidant attack (100 mumol/l H2O2 for 5 min at 4 degrees C), and disappearance of DNA single-strand breaks (SSB) after oxidative challenge were monitored by single-cell gel electrophoresis. DNA repair patch synthesis activity in cell extracts was determined using assays that measure nucleotide incorporation during repair of oxidative lesions in template DNA. Unlike single-cell gel electrophoresis, the parameters measured with these assays are not dependent on strand break religation. There was no evidence that beta-carotene, lutein, or beta-cryptoxanthin supplementation protected cellular DNA from oxidation under basal conditions or after oxidative challenge. However, only carotenoid-supplemented cells exhibited a significant decrease in numbers of SSB over a 2-h period after treatment with H2O2. Carotenoid supplementation did not provoke any detectable change in repair patch synthesis activity. We conclude that supplementation with carotenoids at 8 mumol/l does not provide significant antioxidant protection for DNA in Molt-17 lymphocytes but may enhance recovery of cells from oxidative challenge, as measured by loss of SSB. We argue that these data are most consistent with carotenoids acting to enhance DNA strand break repair.
Elliott RM (2008) Transcriptomics and micronutrient research., Br J Nutr 99 Suppl 3 pp. S59-S65
This review examines the extent to which transcriptomic methods have lived up to their promise in the context of nutrition research, placing particular emphasis on examples from micronutrient research. A case is made that the high quality platform technologies now available, together with established standards and systems for data storage and exchange and powerful new methods of data analysis, mean that microarrays have reached a level of technical maturity at which they can be exploited to their full potential. In the context of nutrition and micronutrient research, transcriptomic methods have already been widely applied, albeit primarily in studies using cell lines and animal models. Using this type of approach, a multitude of genes regulated at the mRNA level by dietary components has been identified and this, in turn, has provided new insights into the biological processes affected by nutritional parameters. Evidence from the very limited number of published transcriptomics-based nutritional studies performed in human volunteers suggests that, with appropriate study design, it is feasible to apply transcriptomic methods successfully in dietary intervention trials. On the other hand, gene expression-based biomarker development still poses a major challenge. Here the use of expression profile 'signatures', rather than single genes, may provide a solution. Approaches designed to identify such 'signatures' are being developed and tested widely, primarily in the context of medical research. The applicability and power of such approaches should also be evaluated in the context of nutrition.
Hesketh J, Wybranska I, Dommels Y, King M, Elliott R, Pico C, Keijer J (2006) Nutrient-gene interactions in benefit-risk analysis., Br J Nutr 95 (6) pp. 1232-1236
Individuals respond differently to nutrients and foods. This is reflected in different levels of benefits and risks at the same intake of a nutrient and, consequently, different 'windows of benefit' in terms of nutrient intake. This has led recently to the concept of 'personalised nutrition'. Genetic factors such as single nucleotide polymorphisms may be one source of this inter-individual variation in benefit-risk response to nutrients. In 2004 a European Union-funded network of excellence in the area of nutrigenomics (European Nutrigenomics Organisation; NuGO) organised a workshop on the role of nutrient-gene interactions in determining benefit-risk of nutrients and diet. The major issues discussed at the workshop are presented in the present paper and highlighted with examples from the presentations. The overall consensus was that although genetics provides a new vision where genetic information could in the future be used to provide knowledge on disease predisposition and nutritional requirements, such a goal is still far off and much more research is required before we can reliably include genetic factors in the risk-benefit assessment of nutrients and diets.
Elliott RM, Morgan LM, Tredger J, Wright J (1994) The effects of cereal source and processing on the metabolic responses to commercially available breakfast cereals and breads, International Journal of Food Sciences and Nutrition 45 (3) pp. 217-222
Baccini M, Bachmaier EM, Biggeri A, Boekschoten MV, Bouwman FG, Brennan L, Caesar R, Cinti S, Coort SL, Crosley K, Daniel H, Drevon CA, Duthie S, Eijssen L, Elliott RM, van Erk M, Evelo C, Gibney M, Heim C, Horgan GW, Johnson IT, Kelder T, Kleemann R, Kooistra T, van Iersel MP, Mariman EC, Mayer C, McLoughlin G, Müller M, Mulholland F, van Ommen B, Polley AC, Pujos-Guillot E, Rubio-Aliaga I, Roche HM, de Roos B, Sailer M, Tonini G, Williams LM, de Wit N, For the NuGO PPS Team (2008) The NuGO proof of principle study package: a collaborative research effort of the European Nutrigenomics Organisation.,Genes Nutr 3 (3-4) pp. 147-151
We are exposed to a complex mixture of food compounds in the womb and eat a composite mixture of foods throughout life. Our taste changes as we grow and mature, and we become influenced by external factors such as holidays and advertising by the food industry. Intricate biochemical processes extract energy and other useful components, enabling us to grow and live and keep our bodies and minds functioning effectively. There are, however, many food compounds that have biological effects within our bodies, and diet and disease are intimately associated. To address the fractured nature of nutrigenomic research, leading centres in nutritional research from across Europe have put together a Network of Excellence, the European Nutrigenomics Organisation (NuGO): linking genomics, nutrition and health research. Led by Dr Ben van Ommen of the Dutch Centre for Human Nutrigenomics, NuGO has been awarded ¬17.3 million over a period of 6 years. The project has been over a year, in preparation and currently has 22 partners from 10 EU member states. © 2004 British Nutrition Foundation.
Heaton SJ, Eady JJ, Fairweather-Tait SJ, MCardle HJ, Srai KS, Elliott RM (2007) BeWo cells as an in vitro model for iron transport across the placenta, PROCEEDINGS OF THE NUTRITION SOCIETY 66 pp. 17A-17A CAMBRIDGE UNIV PRESS
Thompson B, Sharp P, Elliott RM, Al-Mutairi S, Fairweather-Tait SJ (2010) Development of a modified Caco-2 cell model system for studying iron availability in eggs, J Agric Food Chem 58 (6) pp. 3833-3839
Rubio-Aliaga I, de Roos B, Duthie SJ, Crosley LK, Mayer C, Horgan G, Colquhoun IJ, Le Gall G, Huber F, Kremer W, Rychlik M, Wopereis S, van Ommen B, Schmidt G, Heim C, Bouwman FG, Mariman EC, Mulholland F, Johnson IT, Polley AC, Elliott RM, Daniel H (2011) Metabolomics of prolonged fasting in humans reveals new catabolic markers, METABOLOMICS 7 (3) pp. 375-387 SPRINGER
Watson AJ, Worley J, Elliott RM, Jeenes DJ, Archer DB (2000) Cloning stress-induced genes from Aspergillus niger using polymerase chain reaction-augmented subtractive hybridization, Analytical Biochemistry 277 (1) pp. 162-165
Elliott R (2005) Mechanisms of genomic and non-genomic actions of carotenoids., Biochim Biophys Acta 1740 (2) pp. 147-154
Carotenoids are highly bioactive dietary compounds that have the potential to have significant effects on human health. It is becoming increasingly clear that the various biological effects that carotenoids exert could be driven via a number of different mechanisms. These include direct pro- and antioxidant effects, redox sensitive cell signalling, vitamin A signalling pathways and other as yet unidentified mechanisms. This article provides an overview of the known effects of carotenoids and discusses the use of model systems and functional genomic approaches further to elucidate their modes of action.
Hurst R, Elliott RM, Goldson AJ, Fairweather-Tait SJ (2008) Se-methylselenocysteine alters collagen gene and protein expression in human prostate cells., Cancer Lett 269 (1) pp. 117-126
The anti-cancer activity of selenium is dose-dependent and species-specific but the mechanism is unclear. Se-methylselenocysteine (MSC), found in selenium-enriched alliums, is one of the most potent forms. We exposed two human prostate cell lines (LNCaP clone FGC and PNT1A) to nutritionally relevant doses of MSC and selenite, ranging from deficient to the equivalent of selenium supplementation in humans. The cells were adapted for one month to attain steady-state selenium status. Two microarray platforms, an in-house printed microarray (14,000 genes) and the Affymetrix U133A array (22,000 genes) were used to probe the molecular effects of selenium dose and form and several selenium-responsive genes were identified, many of which have been ascribed to cancer cell growth and progression. In response to MSC supplementation, the expression of 23 genes changed significantly, including several collagen genes. Quantitative RT-PCR assays were designed and optimized for four of the collagen genes to validate array data. Significant decreases in expression of collagen type I alpha 1 (COL1A1), COL1A2 and COL7A1 genes were observed in cells adapted to MSC supplementation compared to the control and selenite exposed cells. There were significant increases in genes encoding other types of collagen, including significant increases in COL6A1 and COL4A5 in response to MSC dose. Functional changes in collagen type I protein expression in response to MSC were confirmed by ELISA. This study reveals for the first time that MSC can alter the expression of several types of collagen and thus potentially modulate the extracellular matrix and stroma, which may at least partially explain the anti-cancer activity of MSC.
Elliott R, Pico C, Dommels Y, Wybranska I, Hesketh J, Keijer J (2007) Nutrigenomic approaches for benefit-risk analysis of foods and food components: defining markers of health., Br J Nutr 98 (6) pp. 1095-1100
To be able to perform a comprehensive and rigorous benefit-risk analysis of individual food components, and of foods, a number of fundamental questions need to be addressed first. These include whether it is feasible to detect all relevant biological effects of foods and individual food components, how such effects can confidently be categorised into benefits and risks in relation to health and, for that matter, how health can be quantified. This article examines the last of these issues, focusing upon concepts for the development of new biomarkers of health. Clearly, there is scope for refinement of classical biomarkers so that they may be used to detect even earlier signs of disease, but this approach defines health solely as the absence of detectable disease or disease risk. We suggest that the health of a biological system may better be reflected by its ability to withstand and manage relevant physiological challenges so that homeostasis is maintained. We discuss the potential for expanding the range of current challenge tests for use in conjunction with functional genomic technologies to develop new types of biomarkers of health.
Elliott RM, Astley SB, Southon S, Archer DB (2000) Measurement of cellular repair activities for oxidative DNA damage., Free Radic Biol Med 28 (9) pp. 1438-1446
Two related assays capable of determining cell extract repair activities for different oxidative lesions in DNA are described. Both assays measure the incorporation of radiolabeled nucleotides during repair of an oxidatively damaged template in a cell-free system. The assays differ in the type of oxidative damage present in the DNA. In one, singlet oxygen is used to generate predominantly 8-oxo-2'-deoxyguanosine lesions. In the other, hydroxyl radicals are used to generate a broad spectrum of damage including oxidized bases and strand breaks. Assay conditions were adjusted to ensure that radiolabel incorporation was directly proportional to cell extract repair activity. These assays represent sensitive tools for investigating the regulation of repair systems for oxidative DNA damage.
Elliott R, Astley S, Southon S, Archer D (2000) The development of DNA repair assays which show that dietary carrots stimulate DNA repair activity, DIETARY ANTICARCINOGENS AND ANTIMUTAGENS (255) pp. 125-128 ROYAL SOC CHEMISTRY
Astley SB, Elliott RM (2005) The European Nutrigenomics Organisation: linking genomics, nutrition and health research (NuGO), TRENDS IN FOOD SCIENCE & TECHNOLOGY 16 (4) pp. 155-161 ELSEVIER SCIENCE LONDON
Dawes LJ, Elliott RM, Reddan JR, Wormstone YM, Wormstone IM (2007) Oligonucleotide microarray analysis of human lens epithelial cells: TGFbeta regulated gene expression., Mol Vis 13 pp. 1181-1197
PURPOSE: Transforming growth factor beta (TGFbeta), a pro-fibrotic cytokine has been proposed a causative factor in the progression of lens pathologies including posterior capsule opacification (PCO), a condition that occurs after cataract surgery. This study employs oligonucleotide microarrays to provide a global profile of gene expression in FHL 124 cells, to identify changes in gene expression following treatment with TGFbeta1 and TGFbeta2, and to enable putative genes relating to TGFbeta regulation and PCO to be identified. METHODS: Routinely cultured FHL 124 cells maintained in serum free Eagle's Minimum Essential Medium (EMEM) were treated with either TGFbeta1 or TGFbeta2 at 10 ng/ml for 24 h then total RNA extraction was carried out. Total RNA (16 microg) was used to analyze gene expression by spotted oligonucleotide microarray hybridization. The spotted oligonucleotide microarrays employed contained 13,971 oligonucleotide probes, each designed to be specific for an individual gene. Array images were analyzed using GenePix Pro 3.0, followed by raw data import into GeneSpring 7.0 where a cross gene error model (CGEM) filter was applied. Data was subjected to LoWess normalization prior to comparison of the different treatment groups. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to validate the oligonucleotide microarray data, using a select number of genes exhibiting differential expression. RESULTS: A total of 301 genes were up-regulated by more than 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these up-regulated genes had biological functions relevant to lens epithelial cells including roles in contraction, transdifferentiation and as extracellular matrix (ECM) components. A total of 164 genes were down-regulated by more that 1.5 fold in FHL 124 cells by both TGFbeta1 and TGFbeta2. Many of these down-regulated genes have biological functions including roles in apoptosis, signaling, and as anti-oxidants. Following treatment with TGFbeta1 and TGFbeta2, QRT-PCR successfully validated the differential changes in gene expression detected by oligonucleotide microarrays. CONCLUSIONS: TGFbeta1 and TGFbeta2 regulate the gene expression of genes that have important roles in human lens epithelial cell biology. Most importantly, TGFbeta induces the gene expression of a number of fibrotic markers which may have a role in promoting the development of PCO such as transdifferentiation markers, contractile factors, and ECM
Bouwman FG, de Roos B, Rubio-Aliaga I, Crosley LK, Duthie SJ, Mayer C, Horgan G, Polley AC, Heim C, Coort SL (2011) 2D-electrophoresis and multiplex immunoassay proteomic analysis of different body fluids and cellular components reveal known and novel markers for extended fasting.,BMC Med Genomics 4
Proteomic technologies applied for profiling human biofluids and blood cells are considered to reveal new biomarkers of exposure or provide insights into novel mechanisms of adaptation.
van Ommen B, El-Sohemy A, Hesketh J, Kaput J, Fenech M, Evelo C, McArdle H, Bouwman J, Lietz G, Mathers J, Fairweather-Tait S, van Kranen H, Elliott R, Wopereis S, Ferguson LR, Meplan C, Perozzi G, Allen L, Revero D, The Micronutrient Genomics Project Working Group (2010) The micronutrient genomics project: creating a community driven knowledge base for micronutrient research,Genes Nutr 5 (4) pp. 285-296
Crosley LK, Duthie SJ, Polley AC, Bouwman FG, Heim C, Mulholland F, Horgan G, Johnson IT, Mariman EC, Elliott RM, Daniel H, de Roos B (2009) Variation in protein levels obtained from human blood cells and biofluids for platelet, peripheral blood mononuclear cell, plasma, urine and saliva proteomics., Genes Nutr 4 (2) pp. 95-102
Blood cells and biofluid proteomics are emerging as a valuable tool to assess effects of interventions on health and disease. This study is aimed to assess the amount and variability of proteins from platelets, peripheral blood mononuclear cells (PBMC), plasma, urine and saliva from ten healthy volunteers for proteomics analysis, and whether protein yield is affected by prolonged fasting. Volunteers provided blood, saliva and morning urine samples once a week for 4 weeks after an overnight fast. Volunteers were fasted for a further 24 h after the fourth sampling before providing their final samples. Each 10 mL whole blood provided 400-1,500 mug protein from platelets, and 100-600 mug from PBMC. 30 muL plasma depleted of albumin and IgG provided 350-650 mug protein. A sample of morning urine provided 0.9-8.6 mg protein/dL, and a sample of saliva provided 70-950 mug protein/mL. None of these yields were influenced by the degree of fasting (overnight or 36 h). In conclusion, in contrast to the yields from plasma, platelets and PBMC, the protein yields of urine and saliva samples were highly variable within and between subjects. Certain disease conditions may cause higher or lower PBMC counts and thus protein yields, or increased urinary protein levels.
Elliott RM, Southon S, Archer DB (1999) Oxidative insult specifically decreases levels of a mitochondrial transcript, Free Radical Biology and Medicine 26 (5-6) pp. 646-655
DNA repair capacity varies greatly between individuals, and evidence has begun to link this variation to cancer risk, obesity and related chronic diseases. There is also emerging evidence that dietary components can affect DNA repair, but research to date has been restricted by methods for measuring DNA repair. This study made use of newly developed microplate-based assays for the direct determination of DNA repair enzyme activities. Lipid loading of the HepG2 human hepatocellular carcinoma cell line was employed as a model to test the hypothesis that hepatic steatosis affects DNA repair activity via induction of oxidative stress.
Background: Allergic rhinitis is one of the most prevalent manifestation
of allergy, affecting over 15% of the population worldwide. Recent published
clinical studies have shown that specific probiotics can improveallergic rhinitis
Findings: In this study, thirty one adult volunteers suffering from allergic
rhinitis were enrolled in a crossover study evaluating the efficacy of the
consumption of a product containing either L. paracasei-fermented milk or
the placebo. Transcriptomic analysis was performed on unstimulated PBMC
after each treatment period and analysis was adjusted for the crossover
design. No differences were observed between PBMCs from probiotic treated
allergen challenged allergic patients and PBMCs from placebo treated allergen
challenged allergic patients.
Conclusion: This study shows that, in the blood compartment, PBMCs
mRNA levels are too stable to mirror the changes of symptoms and alteration of
cytokine expressions observed after a treatment with L. paracasei.
It is estimated that 10,000 lesions arise in the genome of a cell every day. Cells have therefore also evolved ways to protect the integrity of their genomes using direct DNA repair enzymes and multi-step pathways including base excision repair and nucleotide excision repair. Alkylating agents are reactive chemicals that transfer alkyl groups to biological molecules, including DNA. The base excision repair pathway mainly repairs non-bulky lesions produced by alkylation, oxidation or deamination of bases. This pathway is initiated by alkyladenine DNA glycosylase (Aag).
Antioxidants neutralise free radicals including reactive oxygen specied (ROS), and have been widely reported to protect against disease. However, some studies have also reported that anti-oxidants may instead make disease progression worse. This thesis aims at evaluating the role of antioxidants in the cellular response to the alkylating agent, methylmethane sulfonate.
WT and Aag-deficient mouse embryonic fibroblasts (MEFs) were pre-treated with the antioxidant N-acetylcysteine (NAC) and exposed to MMS. NAC increased MMS-induced cell death in both Aag-deficient and wild-type (WT) MEFs. These were further confirmed with embryonic stem cells (ESc) being also sensitized to MMS-induced cell death by the anti-oxidant 2-mercaptoethanol (2-ME); and with 661W photoreceptor cells being sensitised to MMS-induced cell death by a commercial antioxidant mixture and NAC.
MEFs exhibited ROS generation when exposed to MMS, which was abrogated with NAC. The mitochondrial superoxide probe MitoSox proves that the MMS-induced ROS generation did not originate from the mitochondria. The NADPH oxidase inhibitor Diphenyleneiodonium (DPI) abrogated MMS-induced ROS generation and also sensitised cells to MMS in a similar fashion to NAC. Collectively, we conclude that cells generate ROS as a response to MMS treatment, and that this ROS generation is essential for cell survival.
We also show by using different glucose concentrations, ATP levels appear to be irrelevant to MMS-induced cell death, and that higher basal NAD levels correlates with higher amount of MMS-induced cell death.
Tripkovic Laura, Wilson LR, Hart Kathryn, Johnsen Sigurd, de Lusignan Simon, Smith CP, Bucca G, Penson S, Chope G, Elliott Ruan, Hypponen E, Berry J L, Lanham-New Susan (2017) Daily supplementation with 15 mg vitamin D2 compared with vitamin D3 to increase wintertime 25-hydroxyvitamin D status in healthy South Asian and white European women: a 12-wk randomized, placebo-controlled food-fortification trial,American Journal of Clinical Nutrition 106 (2) pp. 481-490
American Society for Nutrition
Background: There are conflicting views in the literature as to whether vitamin D2 and vitamin D3 are equally effective in increasing and maintaining serum concentrations of 25-hydroxyvitamin D [25(OH)D], particularly at lower doses of vitamin D.
Objective: We aimed to investigate whether vitamin D2 or vitamin D3 fortified in juice or food, at a relatively low dose of 15 ¼g/d, was effective in increasing serum total 25(OH)D and to compare their respective efficacy in South Asian and white European women over the winter months within the setting of a large randomized controlled trial.
Design: A randomized, double-blind, placebo-controlled food-fortification trial was conducted in healthy South Asian and white European women aged 20?64 y (n = 335; Surrey, United Kingdom) who consumed placebo, juice supplemented with 15 ¼g vitamin D2, biscuit supplemented with 15 ¼g vitamin D2, juice supplemented with 15 ¼g vitamin D3, or biscuit supplemented with 15 ¼g vitamin D3 daily for 12 wk. Serum 25(OH)D was measured by liquid chromatography?tandem mass spectrometry at baseline and at weeks 6 and 12 of the study.
Results: Postintervention in the 2 ethnic groups combined, both the vitamin D3 biscuit and the vitamin D3 juice groups showed a significantly greater absolute incremental change () in total 25(OH)D when compared with the vitamin D2 biscuit group [ (95% CI): 15.3 nmol/L (7.4, 23.3 nmol/L) (P
Conclusions: With the use of a daily dose of vitamin D relevant to public health recommendations (15 ¼g) and in vehicles relevant to food-fortification strategies, vitamin D3 was more effective than vitamin D2 in increasing serum 25(OH)D in the wintertime. Vitamin D3 may therefore be a preferential form to optimize vitamin D status within the general population. This trial was registered at www.controlled-trials.com as ISRCTN23421591.
Base excision repair (BER) is a pathway that repairs DNA damage inflicted by oxidative stress, certain alkylating agents, and spontaneous hydrolysis. Assays to measure the BER pathway are usually limited by being low-throughput, time consuming, and unable to measure each step of the pathway in addition to complete repair. The aim of the present work was to develop an assay format to overcome these limitations, and use this to develop a mathematical
model to examine differences between cell types on flux through the pathway. A novel assay format was developed and optimised to measure uracil DNA glycosylase, AP endonuclease, DNA polymerase ², DNA ligase, AP site repair and complete base repair. Data was generated for each enzyme activity in HepG2, Caco-2, and peripheral blood mononuclear cells (PBMCs), and the effects on the pathway were predicted using a mathematical model. In addition, the assay was used to examine variability in enzyme activity between the two cell lines, correlations between enzyme activities in primary human cells, and the effects of caloric restriction on BER in a human weight loss intervention trial. The model revealed marked differences in the response to aberrant uracil between the two immortalised cell lines and the PBMCs, with PBMC nuclear extract in general excising the base more slowly and causing a smaller and slower accumulation of harmful intermediates, such as abasic sites and single strand breaks, compared to the immortalised cell lines. The model underestimated complete repair when compared to the biological data for all cell types, potentially due to cooperativity between BER enzymes not captured when repair steps were measured individually under experimental conditions. There were also significant correlations found between enzyme activities in PBMC extracts from different individuals, and a significant predictive effect of weight loss method on polymerase ² activity in the caloric restriction trial. In conclusion, the research described here uncovered novel information regarding the effects of weight loss on DNA repair, and correlations between BER enzyme activities in healthy volunteers. This is also the first work to compare BER profiles of different cell types using biological data and mathematical modelling.
DNA repair is essential for the maintenance of genomic integrity, and evidence suggest that interindividual variation in DNA repair efficiency maycontribute to disease risk. However, robust assays suitable for quantitative determination of DNA repair capacity in large cohort and clinical trials are needed to evaluate these apparent associations fully. We describe here a set of microplate-based oligonucleotide assays for high-throughput, non-radioactiveand quantitative determination of repair enzyme activity at individual steps and over multiple steps of
the DNA base excision repair pathway. The assays are highly sensitive: using HepG2 nuclear extract, enzyme activities were quantifiable at concentrationsof 0.0002 to 0.181 g per reaction, depending on the enzyme being measured. Assay coefficients of variation are comparable with other microplate-based assays. The assay format requires no specialist equipment and has the potential to be extended for analysis of a wide range of DNA repair enzyme activities. As such, these assays hold considerable promise for gaining new mechanistic insights into how DNA repair is related to individual genetics, disease status or progression and other environmental factors and investigating whether DNA repair activities can be used a biomarker of disease risk.
Shah Nikunj, Meira Lisiane B., Elliott Ruan M., Hoole Stephen P., West Nick E., Brown Adam J., Bennett Martin R., Garcia-Garcia Hector M., Kuku Kayode O., Dan Kazuhiro, Kolm Paul, Mariathas Mark, Curzen Nick, Mahmoudi Michael (2019) DNA damage and repair in patients with coronary artery disease: Correlation with plaque morphology using optical coherence tomography (DECODE study),Cardiovascular Revascularization Medicine 20 (9) pp. 812-818
The aim of this study was to examine DNA ligase activity and expression of DNA damage response pathway (DDR) genes in patients with stable angina (SA) and non-ST elevation myocardial infarction (NSTEMI) and determine whether they correlate with plaque morphology.
Patients with coronary artery disease (CAD) have evidence of deoxyribonucleic acid (DNA) damage in peripheral blood mononuclear cells (PBMCs). It is unclear whether this represents excess damage or defective DNA repair activity.
DNA ligase activity and the expression of 22 DDR genes were measured in PBMCs of patients (both SA (n/=/47) and NSTEMI (n/=/42)) and in age and gender-matched controls (n/=/35). Target lesion anatomical assessment was undertaken with frequency domain optical coherent tomography.
DNA ligase activity was different across the three groups of patients (control/=/119/±/53, NSTEMI/=/115.6/±/85.1, SA/=/81/±/55.7/units/g of nuclear protein; ANOVA p/=/0.023). Pair wise comparison demonstrated that this significance is due to differences between the control and SA patients (p/=/0.046). Genes involved in double strand break repair and nucleotide excision repair pathways were differentially expressed in patients with SA and NSTEMI. In SA patients, fibrocalcific plaques were strongly associated with GTSE1, DDB1, MLH3 and ERCC1 expression. By contrast, in NSTEMI patients the strongest association was observed between fibrous plaques and ATM and XPA expression.
PBMCs from patients with CAD exhibit differences in DNA ligase activity and expression of DDR genes. Expression levels of certain DDR genes are strongly associated with plaque morphology and may play a role in plaque development and progression.
Colorectal cancer (CRC) is the third most common type of cancer and the fourth most common cause of cancer-related death worldwide. The incidence and mortality of CRC are higher in more developed regions than in less developed regions and they are also higher in males than in females from 45.7% to 7% and from 16.1% to 5.5%, respectively. These and other data suggest CRC may be amenable to improve prevention by suitable lifestyle interventions, including dietary modification. Quercetin (QC) is a flavonoid obtained from plants that can reach concentrations in the gastrointestinal tract in the range of 0.16?1.30 µM as determined by LC-MS analysis of faecal water. However, many other compounds are also present in faecal water, including those from other plants (e.g. SFN released from brassicas) and DHCA (3,4-dihydroxyphenylpropionic acid), which is a colonic microflora catabolite of the major dietary phenolic acids, derived from the consumption of fruits, vegetables, coffee, and tea.
The investigation was done to assess the cytotoxic effect of individual components in human colon adenocarcinoma (Caco-2) cells and primary human colonic epithelial cells (HCoEpiC). It also investigates whether synergistic interactions or additive interactions occur between mixtures of QC, DHCA, and SFN in terms of potential cytotoxic activity in Caco-2 and HCoEpiC and it compares the effects observed in the cancer cell line with those in HCoEpiC.
The study demonstrated that Caco-2 cells or HCoEpiC were treated with various concentrations of QC (0-150 µM), DHCA (0-500 µM) or SFN (0-200 µM) individually and in combination, to determine the half maximal inhibitory concentration (IC50) value using the methylthiazol tetrazolium (MTT) assay.
This has resulted in that QC and SFN had both concentration and time-dependent cytotoxic effects on Caco-2 cells (IC50 50 µM, p > 0.001 and 32 µM, p > 0.0001 for QC and 45 µM, p > 0.05 and 20 µM, p > 0.0001 for SFN after 24 and 48 h, respectively). DHCA only showed detectable cytotoxic effect in Caco-2 cells at the highest concentration tested. QC had no detectable cytotoxic effect on HCoEpiC, while SFN showed a very similar cytotoxic effect in HCoEpiC (IC50 19.21 µM, p > 0.0001), DHCA had no cytotoxic effect. However, SFN supplementation increased QC cytotoxicity in Caco-2 and HCoEpiC at low concentrations. Moreover, DHCA appeared to cause an increase in viability or cell number at all SFN concentrations tested in HCoEpiC but not in HCoEpiC. DHCA supplementation had a clear influence on QC-induced cytotoxicity in Caco-2 and but not in HCoEpiC.
In conclusion, combinations of the phytochemicals at low concentrations exhibited even greater cytotoxic effects than phytochemicals individually in CRC cells and they do not have an effect on primary. These data suggest the three phytochemicals each exhibit unique and distinct effects in CRC and primary colon cells. It is clear from the present studies that some combinations of phytochemicals can have an additive interaction effect rather than a synergistic interaction in CRC growth cells. In summary, evidence suggests that a combination of phytochemicals is a good candidate for further anticancer studies.
Mas Claret Eduard, Al Yahyaei Balqees, Chu Shuyu, Elliott Ruan M., Imperato Manuel, Lopez Arnaud, Meira Lisiane B., Howlin Brendan J., Whelligan Daniel K. (2020) An aza-nucleoside, fragment-like inhibitor of the DNA repair enzyme alkyladenine glycosylase (AAG),Bioorganic & Medicinal Chemistry 28 (11) 115507
The DNA repair enzyme AAG has been shown in mice to promote tissue necrosis in response to ischaemic reperfusion or treatment with alkylating agents. A chemical probe inhibitor is required for investigations of the biological mechanism causing this phenomenon and as a lead for drugs that are potentially protective against tissue damage from organ failure and transplantation, and alkylative chemotherapy. Herein, we describe the rationale behind the choice of arylmethylpyrrolidines as appropriate aza-nucleoside mimics for an inhibitor followed by their synthesis and the first use of a microplate-based assay for quantification of their inhibition of AAG. We finally report the discovery of an imidazol-4-ylmethylpyrrolidine as a fragment-sized, weak inhibitor of AAG.
Alhumaydhi Fahad A., de O. Lopes Debora, Bordin Diana L., Aljohani Abdullah S. M., Lloyd Cameron B., McNicholas Michael D., Milano Larissa, Charlier Clara F., Villela Izabel, Henriques João Antonio P., Plant Kathryn E., Elliott Ruan M., Meira Lisiane B. (2020) Alkyladenine DNA glycosylase deficiency uncouples alkylation-induced strand break generation from PARP-1 activation and glycolysis inhibition,Scientific Reports 10 (1)
Nature Publishing Group
DNA alkylation damage is repaired by base excision repair (BER) initiated by alkyladenine DNA
glycosylase (AAG). Despite its role in DNA repair, AAG-initiated BER promotes cytotoxicity in a process
dependent on poly (ADP-ribose) polymerase-1 (PARP-1); a NAD+-consuming enzyme activated by
strand break intermediates of the AAG-initiated repair process. Importantly, PARP-1 activation has
been previously linked to impaired glycolysis and mitochondrial dysfunction. However, whether
alkylation affects cellular metabolism in the absence of AAG-mediated BER initiation is unclear.
To address this question, we temporally profiled repair and metabolism in wild-type and Aag?I?
cells treated with the alkylating agent methyl methanesulfonate (MMS). We show that, although
Aag?I? cells display similar levels of alkylation-induced DNA breaks as wild type, PARP-1 activation
is undetectable in AAG-deficient cells. Accordingly, Aag?I? cells are protected from MMS-induced
NAD+ depletion and glycolysis inhibition. MMS-induced mitochondrial dysfunction, however, is AAG-independent. Furthermore, treatment with FK866, a selective inhibitor of the NAD+ salvage pathway
enzyme nicotinamide phosphoribosyltransferase (NAMPT), synergizes with MMS to induce cytotoxicity
and Aag?I? cells are resistant to this combination FK866 and MMS treatment. Thus, AAG plays an
important role in the metabolic response to alkylation that could be exploited in the treatment of
conditions associated with NAD+ dysregulation.