British Journal of Nutrition (2008), 99, Suppl.

3, S59S65 q The Author 2008

doi:10.1017/S0007114508006880

Transcriptomics and micronutrient research

British Journal of Nutrition

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 identied 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 prole 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. Nutritional genomics: Micronutrient: Transcriptomics: Microarray

Functional genomic approaches (e.g. transcriptomics, proteomics and metabolomics) have, for a number of years, been heralded as presenting enormous potential for advancement of nutritional research by providing new insights into dietgene interactions and for the development of new robust biomarkers of nutritional status(1 5). The technologies available for functional genomic research continue to expand and be rened. This progress constantly improves the scope for nutrigenomic research. But to what extent have the omics so far lived up to their hype in advancing our understanding of dietgene and nutrition-health interactions? This article will focus on technical progress with transcriptomic methods, their application to date for nutrition research, with particular emphasis upon micronutrients, and future needs and opportunities. Current status of transcriptomic technologies The rst microarrays were developed over 10 years ago(6) but the realisation of the full potential of transcriptomics has been held up to some extent by the inevitable need for adequate technology development. In the interim, there have been problems associated with data quality, data analysis methods, genome coverage and genome annotation. Now these issues appear to have been largely resolved. For example, concerns about the ability to obtain reproducible results across different array platform technologies no longer appear to be legitimate since the MicroArray Quality Control project demonstrated that is it possible to obtain high concordance in inter-site

and cross-platform comparisons using well established commercial array platforms(7). The range and scope of methods for analysing array data have also improved dramatically over the last few years so that it is now feasible to reliably identify even quite subtle alterations in the mRNA levels of discrete genes and to start to interpret alterations in gene expression proles in the context of pathways, biological processes and cell signalling mechanisms. Examples of such approaches are discussed below. Transcriptome coverage is no longer a genuine concern, since (i) the number of genes in mammalian genomes has proven to be far lower than initially expected and (ii) platform technologies have improved to a point at which the number of features that can be represented on a single array far exceeds the number of genes in a genome. Indeed, this has made it possible to extend the scope of microarrays to examine more than just mRNA levels for all genes. For example, array technologies exist that cover every individual gene exon, thus enabling expression analysis at the level of gene splice variants(8,9). In fact, DNA arrays are also being applied to perform a wider range of analyses including genome-wide DNA methylation proling, high-throughput genotyping and haplotying, comparative genomic hybridization for copy number determination and microRNA expression proling (Fig. 1). Another equally important issue has been the development and adoption of (i) standards for capturing microarray and associated study metadata (MIAME)(10), standards and systems for exchanging microarray data (MAGE-OM and

Abbreviations: GEO, gene expression omnibus; MIAME, minimum information about microarray experiments; MAGE-OM, microarray gene expression-object model; MAGE-ML, microarray gene expression-markup language. * Corresponding author: Dr Ruan Elliott, fax 01603 507723, email [email protected]

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Fig. 1. Schematic representation of the current range of applications for DNA microarray-based technologies in the broader context of structural and functional omic research areas.

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MAGE-ML)(11), (iii) open access resources, such as GEO and ArrayExpress(12,13), that make published microarray data freely available to all, and (iv) initiatives to provide research communities with common sets of descriptors (controlled vocabularies) arranged in ontologies (http://obi.sourceforge. net). Together, these efforts reect the viewpoint that, given the signicant level of resource that has to be put into microarray studies and the huge volume of data generated, this process should be considered a long-term investment for the benet of the whole research community. These standards, systems and structures provide key elements of a framework to make the most of the stored data: facilitating the development of new analytical methods, helping in the training of new researchers and providing the scope for detailed structured searches of stored data and mining of data combined from multiple studies(14). Thus, overall, it seems reasonable to assert that transcriptomic technology has nally achieved a level of technical maturity that means it is feasible for committed researchers to generate high quality experimental data and, with appropriate statistical and bioinformatics expertise, to extract robust biological information from that data. Current application of transcriptomics in nutrition research In the context of nutrition research, transcriptomic methods have already been applied fairly extensively, most commonly using the general study design format depicted in Fig. 2. Tables 1 and 2 illustrate the types and range of studies published in the context of micronutrient research, listing examples of microarray studies that have examined patterns of gene expression associated with varying iron and zinc exposure or status, respectively. Similar tables could equally be drawn up for many other nutrients, micronutrients and non-nutrient components of the diet.

Two key observations can be made from the information in these tables. The rst is that all the transcriptomic studies listed have been performed in animal models, mammalian cell lines or primary human cells ex vivo, but not in samples derived from human nutritional intervention studies. The second is that, whist these studies have served to identify a host of genes that are regulated at the level of mRNA by different nutritional components, this information has not yet been combined to provide a single coherent overview and has not yet led to the development of new biomarkers. These two observations hold true in general across the spectrum of nutrition research and are discussed in more detail in the following sections. The scope for transcriptomics-based nutritional studies in human subjects Proponents of transcriptomic approaches should particularly note the general absence of microarray data obtained from human nutritional intervention studies. Presumably this reects a general reticence to undertake such time-consuming and resource-intensive work without condence that it will yield useable and valuable data: limitations in access to target tissues, technical challenges of isolating sufcient high quality RNA and the analytical problems of dealing with inter-individual variation in human subjects are all valid concerns. Nevertheless, the limited evidence that is available suggests that transcriptomic analysis in nutritional intervention studies is feasible and can be highly informative. The most obvious source of cellular RNA for such studies is blood since this is readily obtained and, as such, ideally suited to gene-expression based biomarker development. However, just as with most solid tissues, blood contains a number of different cell types, each with their own unique gene transcript prole. This must be taken into consideration when performing gene transcript analyses with blood samples. Although red blood cells (erythrocytes) are by far the most abundant

Fig. 2. Schematic representation of the most common experimental format employed for nutritional studies employing transcriptomics analysis.

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Table 1. Examples of published transcriptomic studies examining the effects of iron on gene expression proles Reference Jones et al.
(33)

Experimental system Mouse hippcampus

Experimental conditions Variation in hippocampal Fe, Cu and Zn concentration Fe overload

Summary of study Conclusions Expression of 25 genes correlated with levels of at least 1 of the 3 metals

Transcriptomics and micronutrient research

Rodriguez et al. Coppin et al. (21)

Mouse skeletal muscle and heart

Mouse liver and duodenum

Hfe knockout

Abgueguen et al. (22) Collins et al. (35) Tamura et al. Bedrine-Ferran et al. (37) Martin et al. (38) Muckenthaler et al. (39) Muckenthaler et al. (40) Bilello et al. (41) Alcantara et al. (42)
(36)

Mouse duodenal epithelium Rat Duodenum Human hepatocytes Caco-2 cell line Mouse embryo Mouse duodenum/liver HeLa cells

Hfe knockout and iron overload Fe deprivation Lactoferrin transgene expression Differentiation Hecpidin transgene expression Hfe knockout Fe donors/chelators

Rat hepatocytes HL-60 promonocytes

Fe overload Fe deprivation during differentiation

Expression of 54 genes and 75 genes affected by iron overload in skeletal muscle and heart, respectively including genes included involved in glucose and lipid metabolism, gene transcription and cellular stress Expression of 1,343 and 370 genes in altered in liver and duodenum, respectively, of HfE2 /2 mice compared to wild-type. In liver HfE disruption up-regulated genes involved in antioxidant defence and down-regulated expression of genes involved in fatty acid b-oxidation, cholesterol catabolism and mitochondrial iron trafcking. In duodenum Hfe disruption associated with up-regulation of genes involved in iron transport Expression of 151 genes altered in Hfe(2 /2 ) mice compared with normal littermates, including genes involved in mucus production and cell maintenance 20 genes increased in all ages (e.g. DMT1, DcytB, TfR1, HO-1, ATP7a, MT); 4 genes decreased in all ages Expression of 9 genes increased and 19 genes decreased Expression of 80 genes down-regulated (including HEPH, DMT1, IREG/Fpn and TF) and 50 genes up-regulated (including ATP7B and ZIP1) Reduced TfR Altered expression of ZIP1, Cybrd1 and hepcidin Expression of 8 genes induced by loading (HO-1, Hsp70D, Hsp 105, mHsp70, c-myc, L-Fer, Gas-1 and Gas-3). Expression of 6 genes induced by iron deciency (TfR-1, DMT-1, c-jun, Mt-2, lysyl oxidase, Hif-1) Expression of 258 and 627 genes increased and decreased, respectively Expression of 11 genes suppressed (Rb, p21, bad, cdk2, cyclins A, D3 and E1, c-myc, egr-1, iNOS, FasL)

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Table 2. Examples of published transcriptomic studies examining the effects of zinc on gene expression proles Reference Jones et al.
(33)

Experimental system Mouse hippcampus Human peripheral blood mononuclear cells

Experimental conditions Variation in hippocampal Fe, Cu and Zn concentrations Ex vivo Zn exposure

Summary of study Conclusions Expression of 25 genes correlate with levels of at least 1 of the metals Expression of 68 and 61 genes affected in cells from young and old donors, respectively. Pathways affected included tryptophan metabolism, eicosanoid signalling, p38 MAPK signalling, integrin signalling, purine metabolism, G-protein coupled receptor signalling and PPAR signalling Expression of 62 and genes affected by Zn deciency and excess, respectively Expression of 7 genes increased and 4 genes decreased Expression of several hundred genes affected by Zn including genes related to pro-inammatory cytokines and cellular survival. Seven genes (all involved in Zn homoeostasis) regulated by zinc in 3 cell lines Expression of 323 genes affected by over-expression of ZIP1 including cell matrix adhesion proteins, metalloproteinases, membrane transporters, signalling molecules, regulators of transcription/translation, proliferation/differentiation, nucleic and amino acid metabolism, metal ion homeostasis, apoptosis and motility Expression of 268 gene transcripts altered including 43 involved in lipid metabolism Expression of 30 genes affected (21 up-regulated and 9 down-regulated) Expression of 309 genes altered, including genes involved in intermediary metabolism, signalling; cell cycle control and growth, vesicular trafcking, cell-cell interaction, cytoskeleton and transcription control 1,045 genes signicantly altered Expression of 12 genes altered, immediate-early genes Ranked lists of up-and down-regulated genes including genes involved in signalling, growth, transcription, redox and energy utilization Expression of genes affected

Mazzatti et al. (23)

Sun et al. (43) Yamada et al. (44) Haase et al. (45) Tang et al. (46)

Rat liver HeLa cells Human monocyte, T cell and B cell lines

Zn deciency and excess Zn supplementation or deciency Zn supplementation or deciency

R. M. Elliott

Human mesenchymal stem cells directed into the osteogenic lineage

Over-expression of ZIP1 Zn transporter

Tom Dieck et al. (47) Andree et al. (48) Kindermann et al. (49) Cousins et al. (50) Inoue et al. (51) Blanchard et al. (52) Moore et al. (53)

Rat liver Human lymphoblastoid cell line HT-29 cells

Zn deciency Zn supplementation Zn depletion

THP-1 cells Mouse heart Rat small intestine Mouse thymus

Zn depletion/supplementation ZNT5 null mice Zn deciency Zn deciency

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cell type in blood, they pose a number of technical problems for gene expression analysis. Firstly, mature erythrocytes are anucleate and, therefore, are unable to modify gene transcription in response to environmental changes. Instead, gene transcript proles of erythroytes are effectively set in the immature reticulocyte. Secondly, the quantity and quality of RNA in an erythrocyte will depend on its age since no new mRNA can be synthesized after the cell loses its nucleus and RNA present at this stage will inevitably undergo some degradation over time. Finally, haemoglobin mRNA predominates in reticulocytes, reecting the highly specialised function of red cells. This predominance of a single message can hamper microarray analysis. For these reasons, white blood cells (leukocytes) are commonly used for gene transcript analysis. Leukocytes have been described as scouts, maintaining continuous surveillance for signs of infection or other threats(15). Equally, they are exposed to acute and chronic uctuations in plasma composition arising from the sporadic and varied nature of the diet and the metabolic consequences of exposure to different levels and types of dietary components. Thus they represent a potentially valuable and widely used tool for nutritional intervention studies with human subjects both as direct targets for studying effects on immune and inammatory processes and as surrogates for direct sampling of less accessible tissues. There are a number of different types of leukocytes (generally categorised into neutrophils, eosinophils, basophils, lymphocytes and monocytes although some of these categories can be further sub-divided). The relative proportions of these different cell types can vary somewhat from sample to sample, with knock-on effects on the average gene expression proles(16). This noise can be reduced by isolation and analysis of specic leukocyte sub-sets although care should be taken to avoid, or at least minimise, the risk of introducing artefacts in gene expression that could arise from environmental stresses applied to the cells during the additional isolation steps(17). Analyses of normal variation in leukocyte gene expression suggest that it is best to design studies in which subjects act as their own controls, since the extent of intra-individual variation, in the absence of any intervention, is markedly lower than that of inter-individual variation(15 17). Using such an approach, van Erk and co-workers were able to characterise the specic acute effects of a high-protein and a high-carbohydrate breakfast on the expression of a range of genes in blood leukocytes obtained from human volunteers(18). The response to the high carbohydrate meal was notably associated with changes in expression of genes involved in glycogen metabolism, whereas the response to the high protein breakfast was noted to include altered expression of genes involved in protein biosynthesis. By employing microarray analysis of RNA obtained from blood lymphocytes of post-menopausal women before and after dietary supplementation with high-dose puried soy isoavones, Niculescu and co-workers were able to detect altered expression of a large number of genes, including induction of genes associated with cyclic cAMP signalling and cell differentiation and decreased expression of genes associated with cyclin-dependent kinase activity and cell division. They also found that the response to isoavone supplementation was dependent on the volunteers capacity to produce equol. This observation is interesting since equol is a bacterial

metabolite of the isoavone daidzein to which some of the benecial effects of isoavones have been tentatively attributed. The capacity to metabolise daidzein to equol is dependent the microora of the gut, such that approximately 3040 % of humans produce it. Thus the data from this study could provide new insights into functional effects of equol in relation to the apparent benecial effects of isoavones(19). The recent study of Pagmantidis and co-workers, demonstrated that a 6 week dietary supplementation of human volunteers with moderate levels of selenium elicited small but detectable changes in the expression of blood lymphocyte genes, in particular of genes encoding proteins that function in protein biosynthesis(20). Overall, these data clearly support the potential value of transcriptomic analysis of human tissue samples in human nutritional intervention studies. Analytical methods for transcriptomic studies and the development of new nutritional biomarkers As noted above, microarray analysis of samples from nutritionally relevant studies in animal and cell models have led to the identication of many genes that are regulated at the mRNA level by exposure to different dietary components. This information has helped to provide new insights into the range of biological processes that can be affected by different nutritional parameters but a great deal more can and should be done in this regard. Closer inspection of the literature suggests that, in the past, there has been a widespread tendency to allocate insufcient time and resources for the data analysis phase of transcriptomic studies (phase 2 in Fig. 1), with many studies achieving little more than generating lists of up and downregulated genes. This is entirely understandable, since the complexity of the analytical process is often not fully appreciated until it is attempted rsthand. Such vast datasets require time, unique expertise and advanced computing tools to interpret properly. However, the number of people with relevant expertise and the range of analytical tools have increased and become more widely accessible and this, in turn, is reected in the more detailed analyses performed in the more recent studies. Functional and pathway analysis, literature mining tools, are now employed routinely, providing a clearer biological context for the results obtained and, just as importantly, facilitating cross-study comparisons(21 26). Improvements in data quality and data normalisation strategies together with the development of powerful statistical approaches, such as gene set enrichment analysis, have combined to enhance the sensitivity of transcriptomic methods to such an extent that they genuinely compete with real-time RT-PCR, widely considered as the gold standard, in detecting subtle changes in gene expression(26,27). This is particularly important for nutrition research where only small changes in gene expression are generally expected to be brought about by dietary modication but, nevertheless, such changes may have profound effects in the long term on health. In terms of gene expression-based biomarker development, progress to date has been more disappointing. In a few cases, results from nutritionally-relevant transcriptomic studies performed in vitro or in animal models have been taken forward into human studies to the test the effects of nutritional interventions on the mRNA levels of individual genes in circulating blood leukocytes using techniques such as real time

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R. M. Elliott impact of biomics technology on nutrition research. Ann Nutr Metab 49, 355 365. Kaput J (2007) Developing the promise of nutrigenomics through complete science and international collaborations. Forum Nutr 60, 209223. van Ommen B & Stierum R (2002) Nutrigenomics: exploiting systems biology in the nutrition and health arena. Curr Opin Biotechnol 13, 517521. Kussmann M, Raymond F & Affolter M (2006) Omics-driven biomarker discovery in nutrition and health. J Biotechnol 124, 758 787. Schena M, Shalon D, Davis RW & Brown PO (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270, 467 470. Guo L, Lobenhofer EK, Wang C, et al. (2006) Rat toxicogenomic study reveals analytical consistency across microarray platforms. Nat Biotechnol 24, 1162 1169. Xing Y, Kapur K & Wong WH (2006) Probe selection and expression index computation of affymetrix exon arrays. PLoS ONE 1, e88. Gardina PJ, Clark TA, Shimada B, et al. (2006) Alternative splicing and differential gene expression in colon cancer detected by a whole genome exon array. BMC Genomics 7, 325. Brazma A, Hingamp P, Quackenbush J, et al. (2001) Minimum information about a microarray experiment (miame)-toward standards for microarray data. Nat Genet 29, 365 371. Spellman PT, Miller M, Stewart J, et al. (2002) Design and implementation of microarray gene expression markup language (mage-ml). Genome Biol 3, RESEARCH0046. Parkinson H, Sarkans U, Shojatalab M, et al. (2005) Arrayexpress a public repository for microarray gene expression data at the ebi. Nucleic Acids Res 33, D553 D555. Barrett T, Suzek TO, Troup DB, et al. (2005) Ncbi geo: mining millions of expression proles database and tools. Nucleic Acids Res 33, D562 D566. Rocca-Serra P & Elliott RM (2006) Data storage: bringing us a step closer to data sharing? Br J Nutr 95, 1237 1239. Whitney AR, Diehn M, Popper SJ, Alizadeh AA, Boldrick JC, Relman DA & Brown PO (2003) Individuality and variation in gene expression patterns in human blood. Proc Natl Acad Sci U S A 100, 1896 1901. Radich JP, Mao M, Stepaniants S, et al. (2004) Individualspecic variation of gene expression in peripheral blood leukocytes. Genomics 83, 980988. Eady JJ, Wortley GM, Wormstone YM, Hughes JC, Astley SB, Foxall RJ, Doleman JF & Elliott RM (2005) Variation in gene expression proles of peripheral blood mononuclear cells from healthy volunteers. Physiol Genomics 22, 402411. van Erk MJ, Blom WA, van Ommen B & Hendriks HF (2006) High-protein and high-carbohydrate breakfasts differentially change the transcriptome of human blood cells. Am J Clin Nutr 84, 1233 1241. Niculescu MD, Pop EA, Fischer LM & Zeisel SH (2007) Dietary isoavones differentially induce gene expression changes in lymphocytes from postmenopausal women who form equol as compared with those who do not. J Nutr Biochem 18, 380 390. Pagmantidis V, Meplan C, van Schothorst EM, Keijer J & Hesketh JE (2008) Supplementation of healthy volunteers with nutritionally relevant amounts of selenium increases the expression of lymphocyte protein biosynthesis genes. Am J Clin Nutr 87, 181 189. Coppin H, Darnaud V, Kautz L, Meynard D, Aubry M, Mosser J, Martinez M & Roth MP (2007) Gene expression proling of hfe2/2 liver and duodenum in mouse strains with differing susceptibilities to iron loading: identication of transcriptional regulatory targets of hfe and potential hemochromatosis modiers. Genome Biol 8, R221.

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RT-PCR(28). Such studies provide further evidence that nutrient regulated changes in the expression of specic genes can be detected in vivo in samples from human subjects. But, although a number of potential gene expression-based nutrient sensitive biomarkers have been identied, these suffer from potential confounding effects that undermine their value. This reects a fundamental problem with the specicity of single genes as biomarkers since expression of most (perhaps all) individual genes can be regulated by more than one environmental factor. An interesting alternative that may help to overcome this problem is the development of markers based on expression prole signatures rather than single genes. Such signatures (characteristic patterns of differential gene expression) are effectively measures of cell phenotype, and can be used to look for novel biomarkers in cells that have been exposed to different levels of micronutrients. Approaches designed to identify characteristics gene expression signatures are being developed and tested widely in the context of medical research, in particular cancer research(29 32). Although still in a comparatively early developmental stage, the applicability and power of such approaches should also be evaluated in the context of nutrition. Conclusions Even during their initial developmental phase, transcriptomic approaches have already provided many valuable new insights into diet-gene interactions. Now the technology has reached a sufcient level of maturity that it can be used as a routine tool by all researchers who wish to exploit it to produce high quality data. The emphasis needs to shift towards more extensive, disciplined and detailed examination of the data generated by using advanced statistical, data- and literature-mining tools. The development of universal standards and infrastructures for microarray data capture, storage and sharing will be vital in ensuring that data produced can used to it full potential. Although not yet comprehensively tested, the scope for human nutritional intervention studies, which employ transcriptomic analysis of accessible tissues, appears very promising. This kind of study represents an exciting avenue of investigation for the development of new nutritional biomarkers.

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5.

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7.

8.

9.

10.

11.

12.

13.

14. 15.

16.

17.

Acknowledgements The Institute of Food Research is funded by the Biotechnology & Biological Sciences Research Council (BBSRC), UK. Dr Elliott is a member of NuGO The European Nutrigenomics Organisation: linking genomics, nutrition and health research (NuGO, CT-2004-505944) a Network of Excellence funded by the European Commissions Research Directorate General under Priority Thematic Area 5 Food Quality and Safety Priority of the Sixth Framework Programme for Research and Technological Development. The content of this article presents no conicts of interests. References
1. 2. Elliott R & Ong TJ (2002) Nutritional genomics. BMJ 324, 1438 1442. Corthesy-Theulaz I, den Dunnen JT, Ferre P, Geurts JM, Muller M, van Belzen N & van Ommen B (2005) Nutrigenomics: the 18.

19.

20.

21.

Transcriptomics and micronutrient research 22. Abgueguen E, Toutain B, Bedrine H, et al. (2006) Differential expression of genes related to hfe and iron status in mouse duodenal epithelium. Mamm Genome 17, 430 450. 23. Mazzatti DJ, Malavolta M, White AJ, Costarelli L, Giacconi R, Muti E, Cipriano C, Powell JR & Mocchegiani E (2007) Differential effects of in vitro zinc treatment on gene expression in peripheral blood mononuclear cells derived from young and elderly individuals. Rejuvenation Res 10, 603 620. 24. Scherf M, Epple A & Werner T (2005) The next generation of literature analysis: integration of genomic analysis into text mining. Brief Bioinform 6, 287 297. 25. Gentleman RC, Carey VJ, Bates DM, et al. (2004) Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5, R80. 26. Mootha VK, Lindgren CM, Eriksson KF, et al. (2003) Pgc-1alpharesponsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet 34, 267 273. 27. Subramanian A, Tamayo P, Mootha VK, et al. (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression proles. Proc Natl Acad Sci U S A 102, 15545 15550. 28. Aydemir TB, Blanchard RK & Cousins RJ (2006) Zinc supplementation of young men alters metallothionein, zinc transporter, and cytokine gene expression in leukocyte populations. Proc Natl Acad Sci U S A 103, 1699 1704. 29. Naderi A, Teschendorff AE, Barbosa-Morais NL, et al. (2007) A gene-expression signature to predict survival in breast cancer across independent data sets. Oncogene 26, 1507 1516. 30. Lyman GH & Kuderer NM (2006) Gene expression prole assays as predictors of recurrence-free survival in early-stage breast cancer: a metaanalysis. Clin Breast Cancer 7, 372 379. 31. Marchionni L, Wilson RF, Wolff AC, Marinopoulos S, Parmigiani G, Bass EB & Goodman SN (2008) Systematic review: gene expression proling assays in early-stage breast cancer. Ann Intern Med 148, 358 369. 32. Xu L, Tan AC, Winslow RL & Geman D (2008) Merging microarray data from separate breast cancer studies provides a robust prognostic test. BMC Bioinformatics 9, 125. 33. Jones LC, Beard JL & Jones BC (2008) Genetic analysis reveals polygenic inuences on iron, copper, and zinc in mouse hippocampus with neurobiological implications. Hippocampus 18, 398 410. 34. Rodriguez A, Hilvo M, Kytomaki L, Fleming RE, Britton RS, Bacon BR & Parkkila S (2007) Effects of iron loading on muscle: genome-wide mrna expression proling in the mouse. BMC Genomics 8, 379. 35. Collins JF, Franck CA, Kowdley KV & Ghishan FK (2005) Identication of differentially expressed genes in response to dietary iron deprivation in rat duodenum. Am J Physiol Gastrointest Liver Physiol 288, G964 G971. 36. Tamura T, Nozaki A, Abe K, Dansako H, Naka K, Ikeda M, Tanaka K & Kato N (2005) Cdna microarray analysis of lactoferrin expression in non-neoplastic human hepatocyte ph5ch8 cells. Biochim Biophys Acta 1721, 73 80. 37. Bedrine-Ferran H, Le Meur N, Gicquel I, et al. (2004) Transcriptome variations in human caco-2 cells: a model for enterocyte differentiation and its link to iron absorption. Genomics 83, 772 789. 38. Martin ME, Nicolas G, Hetet G, Vaulont S, Grandchamp B & Beaumont C (2004) Transferrin receptor 1 mRNA is downregulated

S65

39.

40.

41.

42.

43.

British Journal of Nutrition

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

in placenta of hepcidin transgenic embryos. FEBS Lett 574, 187191. Muckenthaler M, Roy CN, Custodio AO, Minana B, deGraaf J, Montross LK, Andrews NC & Hentze MW (2003) Regulatory defects in liver and intestine implicate abnormal hepcidin and cybrd1 expression in mouse hemochromatosis. Nat Genet 34, 102107. Muckenthaler M, Richter A, Gunkel N, et al. (2003) Relationships and distinctions in iron-regulatory networks responding to interrelated signals. Blood 101, 3690 3698. Bilello JP, Cable EE & Isom HC (2003) Expression of e-cadherin and other paracellular junction genes is decreased in iron-loaded hepatocytes. Am J Pathol 162, 1323 1338. Alcantara O, Kalidas M, Baltathakis I & Boldt DH (2001) Expression of multiple genes regulating cell cycle and apoptosis in differentiating hematopoietic cells is dependent on iron. Exp Hematol 29, 1060 1069. Sun JY, Wang JF, Zi NT, Jing MY & Weng XY (2007) Gene expression proles analysis of the growing rat liver in response to different zinc status by cdna microarray analysis. Biol Trace Elem Res 115, 169 185. Yamada H, Suzuki K & Koizumi S (2007) Gene expression prole in human cells exposed to zinc. J Toxicol Sci 32, 193196. Haase H, Mazzatti DJ, White A, Ibs KH, Engelhardt G, Hebel S, Powell JR & Rink L (2007) Differential gene expression after zinc supplementation and deprivation in human leukocyte subsets. Mol Med 13, 362 370. Tang Z, Sahu SN, Khadeer MA, Bai G, Franklin RB & Gupta A (2006) Overexpression of the zip1 zinc transporter induces an osteogenic phenotype in mesenchymal stem cells. Bone 38, 181198. Tom Dieck H, Doring F, Fuchs D, Roth HP & Daniel H (2005) Transcriptome and proteome analysis identies the pathways that increase hepatic lipid accumulation in zinc-decient rats. J Nutr 135, 199205. Andree KB, Kim J, Kirschke CP, Gregg JP, Paik H, Joung H, Woodhouse L, King JC & Huang L (2004) Investigation of lymphocyte gene expression for use as biomarkers for zinc status in humans. J Nutr 134, 1716 1723. Kindermann B, Doring F, Pfaf M & Daniel H (2004) Identication of genes responsive to intracellular zinc depletion in the human colon adenocarcinoma cell line ht-29. J Nutr 134, 57 62. Cousins RJ, Blanchard RK, Popp MP, Liu L, Cao J, Moore JB & Green CL (2003) A global view of the selectivity of zinc deprivation and excess on genes expressed in human thp-1 mononuclear cells. Proc Natl Acad Sci U S A 100, 6952 6957. Inoue K, Matsuda K, Itoh M, et al. (2002) Osteopenia and malespecic sudden cardiac death in mice lacking a zinc transporter gene, znt5. Hum Mol Genet 11, 1775 1784. Blanchard RK, Moore JB, Green CL & Cousins RJ (2001) Modulation of intestinal gene expression by dietary zinc status: effectiveness of cdna arrays for expression proling of a single nutrient deciency. Proc Natl Acad Sci U S A 98, 13507 13513. Moore JB, Blanchard RK, McCormack WT & Cousins RJ (2001) Cdna array analysis identies thymic lck as upregulated in moderate murine zinc deciency before t-lymphocyte population changes. J Nutr 131, 3189 3196.