Perspectives in Nutrigenomics for human health

Poonam C. Mittal, Biochemistry Department, University of Allahabad, Allahabad, India.

“Nutrition has often been the subject of conjectures and ingenious hypotheses—but our actual
knowledge is so insufficient that their only use is to try to satisfy our imagination. If we could
arrive at some more exact facts they could well have applications in medicine.”
Lavoisier (1743-1794).
Abstract:

The main concern of the science of human nutrition has been development of a
dietary regime that promotes optimum health for most of the population. However, as
data linking diet and disease accumulated, the response to dietary factors was found to be
individualistic. The same dietary factors were found to produce disease in a person who
had a genetic predisposition to that particular disease, but not in those, who seemingly
had a more efficient metabolic machinery to handle the nutrient in question. This led to
the discovery that nutrients can influence metabolic pathways through nutrient-gene
interactions, and thereby influence homeostasis. As information from the human genome
became available, rapid developments took place in this newly emerging science of
Nutrigenomics.

The study of nutrigenomics focuses on understanding the relationship between

nutrition, genetics and health. This requires application of genomics, transcriptomics and
metabolomics, which respectively help in understanding how dietary signals influence
gene expression, protein expression and metabolite production. The final outcome is a
pattern of these effects, which have been called the dietary signature of the metabolic
process.

This review will discuss the significance of studying these dietary signatures at the
level of the cell, the tissue and the organism as a whole. The use of genomic tools in
nutrition research, which can conduct millions of genetic screening tests, will be
explained and the modes by which nutrients affect the genome, proteome and
metabolome will be discussed.

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 Nutrigenomics has attracted media attention as the technology to prescribe tailored
dietary regimens specific to an individual’s genetic requirements. This is a distinct
possibility, as rapid strides have been made is the application of nutrigenomics for
disease management. A brief overview of these issues and what they mean for human
health will be provided in the present review.

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 INTRODUCTION:

The role of nutrition in human health: historical perspective

The earliest records linking the importance of specific substances in food to life can
be traced to scholars such as Hippocrates and Charaka, who lived more than 2400 yrs
back, even though, at that time, there was no knowledge of the chemical nature of foods.

The beginnings of modern concepts of food chemistry can be traced to the mid-1700s,
less than three centuries back, when Lavoisier discovered that oxidation of carbon is the
source of food energy. Chemical methods of analysis developed rapidly during the
‘chemical revolution’ in France at the end of the eighteenth century and became the
impetus for developments in food analysis and investigations linking consequences of
consuming various foods for human health and nutrition. Magendie and Liebig led
research through the 19th century to characterize macronutrients such as carbohydrates,
fat and protein. This was followed by characterization of more complex molecules such
as the vitamins, which are present in foods in smaller amounts, and required more
sophisticated techniques for determination.

Developments in nutritional sciences were also guided by observations linking poor

diets to diseases such as scurvy, beriberi, kwashiorkor, marasmus, anemia, night
blindness etc. Since war, famine and drought were common, the study of the science of
Human Nutrition concerned itself with the development of a dietary regime that
promoted optimum health for the entire population. The first recommended dietary
allowances (RDA) were developed during World War II by the United States National
Academy of Sciences (US-NAS) to provide populations with the knowhow to eat right.
Thereafter, many countries of the world developed their own RDA, based on data
obtained on local populations.

Throughout most of the twentieth century, the focus of research in nutritional science
was mainly on preventing undernutrition for the entire population. There was no
distinction between individual requirements: the approach was to treat everyone as
genetically identical.

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 The link between diet and disease

Towards the end of the twentieth century, data started accumulating to indicate the
involvement of diet in the etiology of several diseases, not hitherto recognized as directly
related to diet. Incidence of diseases such as cardiovascular disorders, diabetes mellitus
type 2, hypertension, cancer etc was linked to consumption of certain foods by
individuals or ethnic groups of distinct geographical regions. The issue received global
impetus when, in 1990, the World Health Organization (WHO) published the report of an
expert group appointed to look into the relationships between diet, nutrition and
prevention of chronic diseases [1].

This resulted in a paradigm shift in thinking: food is not only about preventing
undernutrition and deficiency diseases but also about optimum health and prevention of
common non-communicable diseases. Research in nutritional science started getting
focused on healthy diets for disease prevention [2], and in 2003, the WHO Expert
Committee published its report recognizing the link between several chronic diseases and
obesity, further emphasizing the link between diet, disease and optimum health [3]
Scientific evidence mounted that chronic disease could be modified, positively as well as
negatively, by dietary adjustments, and that these must begin early in the life of an
individual, because diet has a long-term bearing on later health.

Epidemiological as well as experimental approaches indicated that benefits of dietary

choices differ among individuals and dietary requirements may be individualistic. Diet
seemed to have a modifying effect on the genetics of a person which influenced the
phenotype. Thus, the same dietary factors were found to produce disease in a person who
had a genetic predisposition to that particular disease, but not in others with a different
genetics. The individual’s response to a food or nutrient could be traced to differences in
metabolic handling of a dietary component, involving complex interactions between
genotypes, metabolic phenotypes, other dietary factors, lifestyle and environmental
factors.

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 Thus arose the possibility of developing personal diets for disease prevention for an
individual. However, to unravel individual responses to food and diets, the requirement
was for satisfactory molecular approaches to study how metabolism of food differs with
regard to age, gender, lifestyle, phenotype such as body size, and genetics. This made the
issue very complex. It was further complicated by developments in epigenetics whereby
changes in phenotype or genetic expression were traced to mechanisms other than
changes in DNA sequence.

Historically, Jacob and Monad, in 1961, described how lactose acts as a nutrient
inducer and increases expression of three structural genes (lac operon) coding for lactose
metabolizing enzymes. Other classical experiments also indicated nutritional influences
on gene expression. For example, polyribosome formation was linked to the requirement
of essential amino acids; synthesis of ferritin was found to be iron-induced and a high
carbohydrate diet and fasting were found to regulate PEP carboxykinase. Indirect
influences, for example through mediators such as hormones or signaling systems, were
found to produce changes in transcription of specific genes to yield proteins that define
phenotypic expression [4].

Although it was clear that nutritional science could establish personal diet-health
relationships and predict disease in an individual, practical applications were limited due
to methodological constraints, and followed developments in genetics.

The evidence for nutrient-gene interactions: the birth of nutrigenomics

Genes are the segments of deoxyribonucleic acid (DNA) which code for a protein,
and direct the development of an organism. They are inheritable; and different forms of a
given gene, known as alleles, produce several characteristics: the phenotypes, of which
eye color and hair color are examples. Genetic diseases, such as phenylketonuria, sickle
cell anemia and scores of others, have traditionally been termed as those arising out of
effects in a single allele of a gene. 97 % of known genetic diseases are monogenic
diseases. More recently, it has been recognized that several genetic disorders are
multifactorial or polygenic in nature. Heart diseases and diabetes mellitus fall in this
category. The interaction between environmental factors such as nutrition and genes in

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the etiology of such diseases is also a subject of much recent study, involving scientists
from several disciplines.

The complete set of genes in an organism is known as its genome. The DNA in
the nucleus of the human cell has about 3 billion base pairs. Developments in sequencing
technologies and improved computing power led to the formal beginning, in October
1990, of the Human Genome Project (HGP) which was an international collaborative
effort at sequencing all these 3 billion bases to identify all human genes and make them
accessible for further biological study. It met its goal in April 2003 [5, 6].

Since the human body contains over a hundred thousand proteins, it was expected
that the genome would comprise of a commensurate number of genes. However,
according to estimates published in 2004 [7], the HGP has placed the number of genes in
the human genome at about 20 000 to 25 000, much less than expected at its outset. The
proteins coded by the genome determine the visible physical characteristics of the
organism and direct the metabolism of food and ensure that the body recognizes foreign
from self, thereby fighting the large variety of possible infecting agents. It also modulates
levels of various molecules which control behavior. The great complexity that is the
hallmark of the living system results to a large extent from thousands of chemical
modifications that these proteins undergo, and the regulatory processes that control them,
which ultimately manage to maintain homeostasis, despite the complexity of the
organism.

There has been a growing recognition that nutrients act as dietary signals in
controlling homeostasis by influencing the metabolic programming of cells [8]. The
establishment of this link between diet and gene interactions led to the shift in focus of
nutrition research to molecular biology and genetics; and nutrigenomics was born. The
aim of nutrigenomics is to regulate a person’s nutritional regime to suit his genotype, and
achieve optimal health.

For this, the need arose to obtain a holistic picture of the interaction between
genes and food, and consequent homeostatic state, which characterizes human health.

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Thus, nutrigenomics has emerged as a potential tool to develop tailored diets to recover
the homeostatic state and prevent or control diseases [9]. The premise underlying
nutrigenomics is that influence of diet on health depends on an individual’s genetic
make-up. Nutrigenomics tries to define the cause-effect relationship between specific
nutrients and diets on human health, leading to the idea of a personalized diet, based on
genotype. It seeks to provide a molecular understanding of how common chemicals in the
diet affect health by altering the expression of genes and the structure of an individual’s
genome.

However, the study of nutrient action at the molecular and subcellular levels is a
very complicated task. For the first time, due to emerging technologies associated with
the HGP, tools became available which could lead to a deeper understanding of
interactions among food, genes, protein structure, post-translational changes in protein
structure and consequent effects on metabolism. Thus, relationships among nutrients and
food components, genomic structure or function and molecular events have begun to be
established [9], [10].

To appreciate advances in nutrigenomics, it is essential to have a preliminary

understanding of the tools and techniques of molecular biology which are used to
understand gene expression, protein synthesis and finally metabolite production, through
the sciences of genomics, transcriptomics, proteomics and metabolomics.

Fundamental concepts in gene expression, protein synthesis and metabolite

production – Genomics; Transcriptomics; Proteomics and Metabolomics

Gene expression is the process that is described by the central dogma of

molecular biology. The information encoded in the strands of the DNA, in the sequence
of nucleotides, is used for its replication into two copies. It is then transcribed into a
complementary chain of messenger ribonucleic acid (mRNA), which is translated into
protein. This requires the mRNA to get anchored to another kind of RNA, the ribosomal
RNA (rRNA). A third kind of RNA, the transfer RNA (tRNA) specifically picks up

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amino acids based on the information in the nucleotide sequence of the mRNA, as
dictated by the genetic code. Amino acids are the building blocks of polypeptides which
organize to finally form the proteins. Proteins have a characteristic shape (conformation)
which determines their varied functions. For example, as biological catalysts, the
enzymes, they direct all metabolic activities. Thus, the final function of a protein depends
on the base sequence of the gene which directed its synthesis. Gene expression is an
extremely specific process, and maintains a very high level of fidelity with the
information coded by the nucleotide sequence in the parent DNA.

Genomics is the study of the genome of an organism, which is the sum total of all
genes of any individual. Genomics requires the study of all of the nucleotide sequences,
including structural genes, regulatory sequences, and non-coding DNA segments, in the
chromosomes of an organism. It requires determination of the entire DNA sequence of an
organism, which comprises of about 3 billion nucleotides. The foundation for sequencing
of nucleotides was laid by Fred Sanger [11], and by Allan Maxam and Walter Gilbert [12].
However, it became possible to consider large-scale sequencing only after the
development of high-throughput sequencing technologies that were capable of producing
millions of sequences at once, and formed the basis of the HGP.

All cells in the human body contain identical DNA, which can be sequenced by
these sequencing techniques. However, all genes are not expressed in every cell all the
time, which is the basis of differentiation of cells, leading to the varied functions of
different cells, which ultimately is responsible for the complexity of multicellular
organisms. Hence Genomics requires tools to understand which genes are being read and
to what extent, that is, the complete set of RNA transcripts produced by the genome at
any specific time in a cell type. This set of RNA is known as the transcriptome, and the
study of the transcriptome is known as transcriptomics. The genome is static but the
transcriptome is extremely dynamic and changing, due to varying patterns of gene
expression. In any organism, the transcriptome of various cells is never identical.

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 Hence there is a need to study which genes are active in a cell at any given time.
This has become possible due to the availability of DNA microarray technology which
has provided scientists with a powerful technique to obtain the transcriptome [13]. Unlike
sequencing techniques which describe the entire genome, this technique is capable of
determining which genes are turned on in a given cell by analyzing the mRNA in that cell
and obtaining the transcriptome. For this the mRNA in the cell under study is first
collected, then labeled by attaching a fluorescent dye and placed in a DNA array slide
containing a large number of DNA probes. The mRNA will attach to its complementary
DNA on the microarray and appear under a fluorescence scanner.

Microarrays obtained from a normal cell and a corresponding cell of a person

suffering from, say diabetes mellitus will appear different if there is a difference in the
activities of a particular gene in the two cells. This technique is used frequently to
examine the activities of various genes at different times. The technique is powerful
enough to examine how active thousands of genes are at any given time. Differences in
gene expression are studied by the process called expression analysis or expression
profiling. Thus, the science of transcriptomics is important for the identification of genes
that are differentially expressed in distinct cell populations or subtypes, to obtain data on
the likely proteins that will be found in a particular cell.

However, mRNA is not always translated into protein [14], so it does not always
correlate with the proteins produced in a cell [15, 16]. Translation, and the consequent set
of proteins in the cell, depends on the physiological state of the cell. So, the analysis of
relative mRNA expression levels can be complicated by the fact that relatively small
changes in mRNA expression can produce large changes in the total amount of the
corresponding protein present in the cell, and in any cell, the set of proteins actually
present at any given time depends on distinct requirements and stresses. It is also
important that the 25- 30 thousand or so genes code for at least 100,000 proteins, mainly
due to a variety of post translational modifications such as phosphorylation,
ubiquitination, methylation, acetylation, glycosylation, oxidation, nitrosylation etc. Thus,
the set of proteins present in any cell at any given time can vary to a large extent and the
utility of transcriptomics becomes limited. Genomics describes the blueprint, and

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transcriptomics tells us which part of the blue print is being transcribed. But both are
insufficient to obtain the entire set of proteins that are actually synthesized in a cell type
at any given time. Thus arose the need to obtain the entire complement of proteins
produced by a cell or an organism, leading to a new area of study called proteomics.

. The term proteomics was coined to rhyme with genomics, and denoted the study
of proteins within a cell or organism at a specific stage of the cell cycle and within a
given environment [17]. The entire complement of proteins in the cell at any given time is
known as the proteome [18]. Since the polypeptide is synthesized under instructions of the
genome, in conjunction with the transcriptome and is subjected to varying
posttranslational modifications, a large variety of particular sets of proteins are produced
by the cell. This set, the proteome, will depend on the distinct requirements of the cell
and the stresses that a cell or organism undergoes, because proteomics confirms the
presence of the protein and provides a direct measure of the quantity present. Since
proteins are involved in virtually every cellular function, control every regulatory
mechanism, and are modified in disease (as the cause or the effect), the proteome dictates
the phenotype of the cell and, collectively, of the tissue or organ that the cells comprise.
This phenotype varies with physiological state, such as cell cycle stage, differentiation,
function, and age. What is of relevance to nutrigenomics is that it can also be impacted by
the onset of or interventions in response to acute insults or chronic diseases [19].
Proteomic research aims to develop markers of disease expression and find therapeutic
solutions [17]. Proteomics also helps to obtain the changes in protein profile in response
to specific dietary interventions [20, 21].

The study of proteomics is much more complicated than genomics because the
proteome, but not the genome, varies with time and environment in the same cell line.
The tools of proteomics include obtaining the spectrum of different proteins in the cell
line, sequencing of amino acids in the polypeptide chains of the protein, their separation
using 2-D gel electrophoresis, detection by mass spectrometry, including matrix assisted
laser desorption/ionization (MALDI) mass spectrometry, ELISA based techniques for
antibody based detection and quantification etc. and informatics tools. Any proteomic
analysis is very costly, laborious, and time consuming. It yields a very large amount of

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data which is difficult to interpret. Hence the need is to design simple experiments with
clear-cut questions to make sense of data obtained from proteomic tools [19].

Proteins are not the only biomolecules in the cell. The cell also contains several
thousands of molecules of sugars, organic acids and amino acids. Most of these are the
result of metabolic processes which are the result of reactions catalyzed by some of the
cellular proteins, the enzymes [22], although some are externally acquired. The term
metabolome, to rhyme with genome, transcriptome and proteome, has been coined to
denote the complete set of metabolites in a cell or an organism. Metabolomics [23],
specifically nutritional metabolomics, is concerned with metabolic pathways and
networks and includes regulation of metabolic pathways and networks by nutrients and
other food components. It summates all the metabolites in body fluids, which are
impacted by endogenous factors such as age, sex, body composition, genetics, underlying
pathologies, circadian rhythms and resting metabolic rate and exogenous factors such as
diet, including all known and hitherto unknown nutrients as well as non-nutrients such as
dietary fiber, additives, pollutants, drugs etc., and the large number of signals from
hundreds of intestinal microflora. A very large number of compounds make the
metabolome, which can be likened to a metabolic fingerprint reflecting the balance of an
individual’s metabolism.

An analysis of the metabolome can be expected to lead to an understanding of the

dynamic behavior of metabolism and consequent cellular function. For this, the
compounds comprising the metabolome need to be identified, quantified and their
relative proportions analyzed and interpreted. This has led to the development of
metabonomics, which is concerned with the quantitative measurement of the
metabolome. [23].

However, the measurement of such a large number of metabolites requires

advanced methodology such as nuclear magnetic resonance, functional magnetic
resonance imaging and high performance liquid chromatography, and handling of a large
amount of mathematical data. Since many metabolites are small, mass spectroscopy (MS)

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is found to be suitable to measure compounds with mol wt. 70-500. But this technique
cannot distinguish compounds with similar molecular weights. Hence it needs to be
combined with other techniques such as liquid chromatography (LC) and gas
chromatography (GC), denoted as LC/MS and GC/MS. Analysis of data thus obtained
requires sophisticated tools of information technology (IT) [24]. More recently, a new
technique which combines capillary electrophoresis (CE) with MS, known as CE/MS has
been found suitable for obtaining the metabolome. Metabolomes can be used to compare
profiles of cancer cells or other diseased cells with a normal cell and study modifications
by drugs and similar interventions. The major goal of metabolomics is to model
intracellular metabolism in its entirety [24].

An important difference between data obtained from metabolomics and other

molecular biology techniques is that the latter employs reductionist approaches, while the
former focuses on understanding complex biological systems from a holistic systems
point of view [24]. Metabolomics amplifies changes in the proteome, and represents the
phenotype of an organism more closely [25]. It includes both signaling and structural
molecules [26]. However, metabolomics as a science is still in its infancy, while the fields
of genomics and proteomics are relatively more advanced. The need is to integrate these
areas of research and follow a systems approach to the understanding of health and
disease.

Application of metabolomics in the fields of pharmacology and toxicology has led

to some success. However, most research in these fields have been conducted on
laboratory animals which are genetically and nutritionally more homogenous than
humans. More recently, attempts are being made to study the impact of nutrients on the
metabolome [27]. But such studies have to be performed on humans, which makes it more
difficult to formulate an experimental design that yields meaningful data. Thus
application of metabolomics to nutrition research is more complicated. The human
nutritional metabolome is a sum of all endogenous and exogenous metabolites [23],
which depends on extrinsic factors such as all nutrient and non-nutrient constituents of
diet, drugs, physical activity, colonic flora, stress and on intrinsic factors such as body

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composition, tissue turnover, resting metabolic rate, age, genotype, health status,
reproductive status and diurnal cycles.

Various biofluids used to obtain the human metabolome are blood, urine and saliva,
of which urine appears to be a suitable because it is easily available [23]. Some
compounds identified in spectra obtained by nuclear magnetic resonance spectrum
of human urine are: lactate, alanine, citrate, dimethylamine, creatinine,
trimethylamine-N-oxide, glycine, hippurate, urea, etc. There are several other
compounds that may be identifiable. So, the first priority for research in this area is
consensus on the definition of the human metabolome. The metabolome will be a
complex set of a large number of compounds, found in varying concentrations at
different points of time due to variations in metabolic rates. Analysis of such a
dynamic and large body of data is another priority for research in this area.
Techniques involving mathematical modeling and pattern recognition emphasize
the multidisciplinary nature of this field.

The foregoing provides preliminary insights into the utility of genomics,

transcriptomics, proteomics and metabolomics for understanding basic genetic and
cellular processes. They are the tools of nutrigenomics that allow study of the
effects of nutrients as dietary signals on gene expression, which includes genomic
structure, function and molecular events.

Interplay between diet and gene expression: Some examples

High-throughput genomic tools and the HGP provided the theoretical framework
required to compare the nucleotide sequences of the entire genome of individuals. But
this is no mean task. The human DNA, which forms the basis of all gene expression,
comprises of 3 billion base pairs. However, genes comprise only about 2 per cent of these
nucleotides, called the coding part or the exon. The remaining 98 per cent is the non-
coding portion called the introns. Tools of molecular biology and genomics have
identified genes responsible for production of nutritionally important proteins such as
digestive enzymes, transport molecules responsible for ferrying nutrients and cofactors to

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their site of use, and numerous other molecules responsible for metabolism and
utilization of our dietary components, including macronutrients, vitamins, minerals, and
phytochemicals [28]. These tools have also enabled study of the ‘non-coding DNA’, the
introns, also erroneously called ‘junk DNA’; and have assigned to them important uses
such as regulation of gene expression, evolutionary importance [29] and manual dexterity
[30].

99.9 per cent of the genes of any two individuals are the same. The tiny 0.1
percent difference, roughly one nucleotide in 1000, is what makes up all the diversity
seen in the approximately 10 billion humans on earth. The most common individual
genomic variations (alleles) in humans include single nucleotide polymorphisms (SNPs,
pronounced ‘snip’), which is a single base substitution of one nucleotide with another.
SNPs make up about 90% of all human genetic variation, since SNPs are estimated to
occur in about 1 of every 1000 nucleotides [10]. According to one estimate, a total of at
least 1.42 million SNPs are found at a density of one SNP per 1.91 kilobases [31]. An SNP
may be of two types: the Adenine (A) may be replaced by Guanine (G), or cytosine (C)
may be replaced by thymine (T), so that the polymorphism is known as A/G or C/T
respectively. C/T polymorphism is more common than the A/G polymorphism [32].

SNPs are more commonly found within the coding region of genes, but also may
be found within the introns. They are evolutionarily stable, they do not change much
from generation to generation; hence they can be followed in population studies. SNPs
have been recognized as precise gene markers. Since they can be affected by specific
nutritional factors, proper levels of nutrients can be worked out and supplied exogenously
or withheld, to ensure adequate expression of the gene to prevent disease.

Scientists believe SNP maps will help them identify the multiple genes associated
with complex ailments such as cancer, diabetes, vascular disease, and some forms of
mental illness. These associations are difficult to establish with conventional gene-
hunting methods because a single altered gene may make only a small contribution to the
disease. This has made SNPs an intensive area of study, and led to the establishment of

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The SNP Consortium (TSC) in 1999 as a collaboration of several companies and
institutions to produce a public resource of single nucleotide polymorphisms (SNPs) in
the human genome [33 , 34].

However, mapping such a large number of SNPs is a difficult task. This became
easier with the discovery that SNPs are not inherited independently but sets of adjacent
SNPs are present on alleles in a block pattern. They are called haplotype. Entire blocks of
SNPs are transmitted through generations. This makes it easier to identify them. So if
about 10 million SNPs have been estimated in human populations, all of them need not
be identified. Just a few from a block are sufficient to characterize the presence of the
entire block. This discovery led to the formation of The International HapMap
Consortium in Oct 2002 to create a haplotype map of the human genome. An important
objective of this endeavor was to guide the design and analysis of medical genetic studies
and create a resource that would accelerate the identification of genetic factors that
influence medical traits and open a new area in population genetics [35].

The importance of SNPs for nutrigenomics is emerging. Individual response to

dietary factors consequent to differences in metabolic imbalances have been traced to
SNPs. An SNP in the gene coding for the enzyme, methylenetetrahydrofolate reductase
(MTHFR) is an example of its application in nutrigenomics. MTHFR is a cytoplasmic
enzyme involved in processing of amino acids. It is required for proper utilization of
dietary folic acid, because it catalyzes the reduction of 5, 10-methylenetetrahydrofolate to
5-methyl tetrahydrofolate. This reaction is required for the multistep process that converts
the amino acid homocysteine to another amino acid, methionine, which is used by the
body to make proteins and other important compounds, including neurotransmitters.

An SNP occurs at base pair 677of the gene for MTHFR. It is a C/T
polymorphism, designated as MTHFR677T. The versions with C (CC and CT)
function normally while the TT version is thermolabile and its activity is reduced. People
with this variant accumulate homocysteine so that their methionine levels are reduced,
which leads to increased risk for vascular disease, coronary heart disease, stroke,
preeclampsia, certain kinds of birth defects and cognitive decline. This can be reversed

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by supplementing their diet with folate. The TT variant is relatively common in many
populations worldwide [36]. Thus the MTHFR SNP is an example to show how
requirement of a dietary constituent, in this case folate, depends on the polymorphism.
There are about 20 genes that have polymorphisms that appear to confer a significant
disadvantage, which may be overcome with a specific dietary modification which may
require modulation of just one single compound, such as folate in the case of the MTFHR
SNP. [37].

Similar mechanisms involving other SNPs form an intensive area of research. For
example, an SNP in the gene for the polypeptide angiotensinogen has been found to be
associated with essential hypertension and response to dietary modulations such as the
Dietary Approaches to Stop Hypertension (DASH) trial [38].

Gene expression can also be affected by another type of nutrient-gene interaction

that involves transcription factors. Genes are flanked by untranscribed regions called
Promoters. These are DNA sequences near the beginning of genes that signal RNA
polymerase where to begin transcription. Transcription is mediated through binding of
transcription factors to response element sequences which can modulate gene expression.
Transcription factors are one of the groups of proteins that read and interpret the genetic
"blueprint" in the DNA. Nutrients can bind to the transcription factors to further modulate
this gene expression. Several macro- and micro-nutrients have been found to affect
various transcription factors, thus mediating nutrient-gene interactions [39]. This is the
main mode of nutrient influence on gene expression. There are approximately 2600
proteins in the human genome that contain DNA-binding domains and most of these are
presumed to function as transcription factors [40].

The nuclear hormone receptor superfamily of transcription factors, with 48

members in the human genome, is the most important group of nutrient sensors [8, 39, 41,
42
]. Numerous receptors in this superfamily bind nutrients and their metabolites. These
include retinoic acid (retinoic acid receptor (RAR) and retinoid X receptor (RXR)), fatty
acids (peroxisome proliferator activated receptors (PPARs) and liver X receptor (LXR)),

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vitamin D (vitamin D receptor (VDR)), oxysterols (LXR), bile salts (farnesoid X receptor
(FXR), also known as bile salt receptor) or other hydrophobic food ingredients
(constitutively active receptor (CAR) and pregnane X receptor (PXR)) [8, 41, 42, 43].

To illustrate, the mode of action of one such nutrient-gene interaction is detailed

here. The PPARs are lipid-activated transcription factors, associated with expression of
genes involved in fatty acid metabolism [44]. The gene transcription first requires the
binding of the PPAR to ω-3 and ω-6 fatty acids, after which it must bind to another
ligand activated transcription factor, the retinoid X-receptor (RXR). This complex of
PPAR-fatty acid + RXR-retinoid binds to the receptor response element and alters gene
expression such that fatty acid synthesis is reduced and fatty acid oxidation is increased,
leading to a lowering of fat storage. The effect of ω-3 fatty acids on increasing fatty acid
oxidation has been found to be more than that of ω-6 fatty acids. Hence nutritionists now
advocate a high ω-3 fatty acid intake to improve lipid profile.

However, findings with regard to effects of transcription factors are based on

laboratory studies. Mutant mice, transgenic mice, knockout mice and cell cultures are
generally used to study how a particular transcription factor mediates the effect of a
particular nutrient. However, sometimes cell lines display large differences in the
expression of important transcription factors compared with primary cells or in vivo [39].

Nutrient-gene interactions also lead to varying metabolomic patterns. Since

metabolomics is the science that analyzes metabolites which are the end products that
depend on the genomics, transcriptomics and proteomics of an individual, the
metabolome represents the outcome of the nutrient-gene-environment interaction.
However, the analysis of the small molecules that comprise a metabolome is no easy task.
To make metabolomics work for nutrigenomics, there is a need to have a library of small
molecules to enable their identification. For example, in a detailed study of deproteinized
plasma, 38 compounds were identified with the use of 1H NMR but 14 (25%) could not
be identified [45]. Newer methods are more sensitive, yielding a larger number of
metabolites but their identification is even more difficult.

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 To assist scientists in the analysis of the metabolome, the National Institutes of
Health (NIH) has established a consortium of small molecule screening centers called the
Molecular Libraries Probe Production Centers Network (MLPCN) which performs high
throughput screening (HTS) to identify small compounds. The MLPCN has established a
collection of 300,000 chemically diverse small molecules, generally with molecular
weight of 500 or less [46]. The challenges for the nutritional sciences will be to create a
consensus of small molecules that are important for the study of metabolomics and then
to create the standards needed for their identification with MS, NMR, and other emerging
technologies [23].

Epigenetics is another area of research which has recently found bearing on

nutrigenomics [10]. Epigenetic modifications influence gene expression so that only
genes useful to a given kind of cell are activated, and this information can be transmitted
to daughter cells. Variable environmental conditions can influence epigenetics, through
the selective use and silencing of genes as cells develop.

As is evident from the foregoing, most methodologies used in biological sciences,

especially in the area of nutrigenomics, have adopted the reductionist approach to
knowledge. This has catalyzed large strides in our understanding of biology. However,
when studying complex life forms, reductionist approaches have their limitations [47, 48]
and the need is to apply methodologies which allow a holistic view of the biological
system being studied.

Methodological challenges: the growth of Systems Biology and the dietary

signature

Any system is likely to behave differently when impacted by diverse stimuli, than
when it is in a controlled environment. One of the great current debates in biology
concerns whether the observed behavior of a system can be accounted for in terms of the

18
behaviors of its subcomponents, and it has been suggested that holistic approaches may
be more predictive and make for better understanding of the functioning of the body.

Systems biology is the term that envelops the various ‘-omics’ technologies. It
develops the concept of complexity and attempts to understand the integrated function of
complex, multicomponent biological systems, ranging from interacting proteins that carry
out specific tasks to whole organisms [48]. This requires the study of the entire system
under defined conditions by defining all the constituents in the system and determining
the interactions among them [49, 50].

The systems biology approach has reported a fair amount of success in the area of
pharmacogenomics, which is the study of genetic variability with regard to response to a
drug [10]. But the methodological challenges of nutrigenomics are much more
demanding because food comprises several thousands of varying nutrient and non-
nutrient substances that, individually and collectively, impact the final outcome of
metabolic processes and consequent health. Food is hundreds of folds more complex than
any drug, and is constantly varying. It is the only input into the body, apart from the
respiratory gases, which is responsible for the growth of a 3 kg newborn to a 70 kg adult,
yet foods with widely varying nutrient compositions produce comparable body
composition and functioning. Moreover, the system being affected, that is the human
body, is the sum total of about a hundred trillion cells working in unison to maintain a
homeostatic (homeodynamic?) entity.

Food components interact with our body at system, organ, cellular, and molecular
levels, depending on their absorption, bioavailability, metabolism and bioefficacy.
Modern nutritional and health research focuses on promoting health, preventing or
delaying the onset of disease, and optimizing performance. It is important that the
beneficial action of a particular food component at the molecular level does not cause a
deleterious effect at some other level. Deciphering the molecular interplay between food
and health requires therefore holistic approaches because nutritional improvement of
certain health aspects must not be compromised by deterioration of others. In other

19
words, in nutrition, we have to get everything right. [21]. Innumerable studies are
available which report the effect of various food components on health, yet consensus
with regard to the beneficial or detrimental effects of even a single component is elusive,
for various reasons [47], mainly because they lack the systems biology approach.

Applying the systems biology approach, nutrigenomics seeks to establish dietary

signatures which are characteristic outcome of a person’s nutrient-environment-gene
interaction. Thus, diseases with a genetic predisposition can lead to varied types of
dietary signatures, which can be examined at various levels, such as cell culture, tissue
culture and whole organisms.

Diet, environment and habits influence the development of common chronic

diseases such as coronary heart disease (CHD), diabetes, cancer, hypertension and
obesity. As described earlier, SNPs have been reported to trigger or provide protection
against these diseases. Thus SNPs can be used as biomarker for early disease diagnosis as
well as for preventive medicine. One such application, through comparison of two
microarrays signifying ‘healthy’ versus ‘stress’ signatures, describes how nutrigenomic
experiments can be used to identify individuals with sensitive genotypes. Individuals
showing a ‘stress signature’ in the microarray would be at higher risk for developing
serious conditions such as cirrhosis or insulin resistance under sustained metabolic and
pro-inflammatory stress. With enough early warning, dietary intervention might reverse
this process, regain homeostatic control and prevent these conditions in at-risk groups
[39, 51]. However, such studies must be conducted on non-human models, because it is
not possible to obtain human tissue for such work.

Another ‘signature’, which is more holistic and can be obtained from humans, is
the metabolome, which is characteristic of a person, and is the outcome of his genomics,
transcriptomics and proteomics. The impact of a nutrient or any molecule on one’s
genome and transcriptome can lead to a modified proteome which will impact utilization
of nutrients due to alterations in metabolism and lead to characteristic metabolomes. The
metabolome, thus is the dietary signature of the dietary signal.

20
 However, although metabolomics has been used successfully in pharmacology
and toxicology, where the number of exogenous compounds are few and well
characterized, the challenges of the metabolomic approach for nutrigenomics is much
more complex and requires handling of very large datasets, pattern analysis techniques,
mathematical modeling and extensive interdisciplinary research [23].

There is a concerted effort to identify small molecules likely to be found in the

metabolome and make the data available in the public domain. The Human Metabolome
Database (HMDB) is a freely available electronic database containing detailed
information about small molecule metabolites found in the human body. It is intended to
be used for applications in metabolomics, clinical chemistry, biomarker discovery and
general education. The database (version 2.0) contains over 6500 metabolite entries
including both water-soluble and lipid soluble metabolites as well as metabolites that
would be regarded as either abundant (> 1 uM) or relatively rare (< 1 nM). Additionally,
approximately 1500 protein (and DNA) sequences are linked to these metabolite entries.
Each MetaboCard entry contains more than 100 data fields with 2/3 of the information
being devoted to chemical/clinical data and the other 1/3 devoted to enzymatic or
biochemical data. Many data fields are hyperlinked to other databases (KEGG, PubChem,
MetaCyc, ChEBI, PDB, Swiss-Prot, and GenBank) and a variety of structure and
pathway viewing applets. The HMDB database supports extensive text, sequence,
chemical structure and relational query searches. Two additional databases, DrugBank
and FooDB are also part of the HMDB suite of databases. DrugBank contains equivalent
information on ~1500 drugs while FooDB contains equivalent information on ~2000 food
components and food additives [52, 53].

Another methodological issue is that most nutrients may be weak but

chronic dietary signals, acting on polygenic diet related diseases. Their
detection would poses problems which are distinct from those faced in
pharmacogenomics.

21
 Thus, there are two main strategies of nutrigenomics. The first,
molecular nutrition and genomics, allows study of how food can impact
target genes, mechanisms and pathways, using a reductionist
approach, requiring relatively affordable methodologies. The other,
which entails development and study of dietary signatures, profiles
and early biomarkers requires the more desirable holistic nutritional
systems biology approach, but which is much more complex and
requires much larger funding.

Tailor-made diets - Panacea or distant dream

From the foregoing, it can be said that nutrigenomic tools have been instrumental
in establishing that gene-nutrient-environmental interactions exist and produce specific
stress signals. The question is whether specific medical foods and supplements can be
tailored for alleviating the stress, and for early detection and management of various
diseases. Some success has been achieved with regard to understanding the genetic basis
of many common polygenic diseases and the impact of isolated dietary components on
their etiology at the molecular level, which is required to obtain an assessment of the
feasibility of tailor-made diets.

Obesity is a major health problem which predisposes the body to several diseases.
Living beings have evolved through food shortages, so genetically, we are predisposed to
assimilate and store as much energy as possible in times of food surplus, but we are not
genetically tuned to losing it, which complicates body weight management. Overweight,
obesity and related medical complications can occur as a result of genetic or acquired
changes in a large number of processes, including, cardiac diseases, Diabetes mellitus,
cancer [3, 54] etc. According to one estimate, food components may be acting on genetic
variants in more than 400 genes, thereby the predisposition to develop several obesity-
associated medical problems becomes a very complex issue [55]. Hence, though limited
success has been achieved in understanding obesity at the molecular level, more in depth

22
studies are required to understand the nutrient-gene interactions for various disorders
before nutrigenomics provides a panacea for this problem.

There are many challenging issues concerning diet and genetic polymorphism in
relation to cardiac diseases that need to be discussed. Single-nucleotide polymorphisms in
several genes have been linked to differential effects in terms of lipid metabolism;
however, even a simple model of benefit and risk is difficult to interpret in terms of
dietary advice to carriers of the various alleles because of conflicting interactions
between different genes. For example, the reported benefit of polyunsaturated fatty acids
in studies of various polymorphisms may be due to the n-3 family of polyunsaturated
fatty acids which is high in fish and is likely to have been high in primitive diets but is
deficient in our modern diet [56]. Predisposition to cardiac diseases has been discussed
earlier in relation to another SNP, the MTHFR677T SNP.

Osteoporosis is another widespread disease that has been found to have a genetic
basis. Numerous candidate genes for osteoporosis susceptibility have been identified over
the last two decades, and the effect of certain nutrients and dietary components on bone
health-related parameters. The issue is very complex, and has not yet reached a stage
where it is scientifically possible to advise people to alter their diet on the basis of their
genotype (i.e. personalised nutrition for osteoporosis prevention) [57]. Another disease
which seems to have a genetic basis and whose incidence and severity can be modulated
by diet is Crohn’s disease, which is one of the causes of Inflammatory Bowel Disease
(IBD). The Nutrigenomics NZ is attempting to identify SNPs involved in the
development of IBD as a first step on determining how food components can affect the
disease at the molecular genetic level. [58].

These are just a few of the large quantum of research which have found links
between various dietary components and most common health conditions such as obesity,
cardiac diseases, hypertension, cancer, osteoporosis, IBD etc. Thus, the prospects appear
to be good for nutrigenomics as the technology to prescribe tailored dietary regimens
specific to an individual’s genetic requirements.

23
 This has attracted extensive media attention, and has triggered the introduction of
large numbers of functional foods, which are claimed to have health-promoting / disease-
preventing properties. ‘Nutrigenetic services’ are also available over the internet without
the involvement of a health care professional. Among the genetic variants most
commonly assessed by these companies are those found in genes that influence
cardiovascular disease risk [59]. Several commercial companies are already offering
personalized nutrition services based on testing. Personalized foods are predicted to be
launched on the market in 3 to 10 years. It is predicted that everyone would have the
opportunity to choose, among a large set of recommended products, the foods that are
best adapted to his/her personal metabolic profile, as described by the metabolome.

However, caution is warranted in the interpretation of DNA-based data, which is

very complex. There is need to carefully examine nutritional genomics as a potential tool
for targeted medical nutrition therapy [59]. Clear-cut yes or no answers to whether
personalized diets are ‘doable’ needs to be assessed at the level of the experiment, the
laboratory and the relevant social worlds [60]. Attempts are being made to review
nutritional genomics research and policy for nutrition practice and policy and for
maximizing benefit and minimizing adverse outcomes within genetically diverse human
populations [61]. However, until the scientific evidence concerning diet-gene interactions
is much more robust, the provision of personalized dietary advice on the basis of specific
genotype remains questionable [62].

Even if the applications of currently available information are useful for

individualized dietary advice, there is a dearth of personnel who can educate the end-user
of nutrigenomic technologies; about its benefits, utilizing social research and addressing
consumers’ hopes and concerns [63], others who can provide genetic counseling [64]), yet
others who can develop products, handle patenting issues, create regulations and conduct
clinical trials, in a wide variety of industries such as the pharmaceutical industry, food
industries and those involved in diagnostics [65].

24
 It is important that applications of findings in nutrigenomics are accompanied by
appropriate information for the consumer, because scientific claims about the benefits of
a food ingredient may be based on reductionist approaches where confounding variables
are controlled [47]. Nutritional needs are complex, and apart from genetic make up, are
also dependent on age, gender, lifestyle, exercise, phenotype, and epigenomic imprinting.
What may work for one person may not work for another. The benefits are limited by
laws of probability, and there are no universal magic bullets [66].

Another area that is very complex but equally important must deal with the
ethical, legal and social implications (ELSI) of genomic research [10]. The bioethical
issues concerning human nutrigenomics include issues of privacy, social stigmatization,
despair in affected individuals and their family and friends, discriminatory practices by
employers and insurance agencies, and several related problems [10, 62].

The question has been raised whether nutrigenomics can provide the panacea that
it propounds to do [55], because living cells are complex, dynamic chemical plants with
redundant pathways and feedback systems which respond to every change, and
translating laboratory findings to practical diet plans is not a simple task. The diversity of
dietary compounds is enormous; there are an estimated 5000 types of flavanoids alone 67,
in addition to scores of other compounds. Moreover, there are difficulties associated with
finding biomarkers that quantify health status. Health is more difficult to define than
disease, so most biomarkers define disease rather than health. Nutrients have relatively
minor effects on health endpoints, as compared to those of drugs in pharmacodynamic
studies, and there are large intra and inter individual variations not related to diet.
Biological systems can be treated in isolated pieces only for purposes of study. However,
they form a complex and delicate network of feedback loops and interplays that defies
simplification [55], and ‘…one thing is clear: to understand the whole one must study the
whole.’ [68].

CONCLUSION

The present and future of nutrigenomics:

25
 Less than two centuries after Magendie discovered the essentiality of dietary
protein and his student, Claude Bernard gave the concept of homeostasis, and about half a
century since Watson and Crick discovered the structure of DNA, incredibly rapid
technological advances have led to in depth knowledge about the complexity that
underlines the interactions among diet, genes, environment and the tools to study them.

Scientists have unraveled the entire human genome of 3 billion base pairs,
identified the 25 thousand genes among these base pairs, assessed the presence of about
100 thousand proteins in the human proteome, studied thousands of the approximately 10
million SNPs, and some HapMaps, that exist in human populations, identified about 2600
proteins which function as transcription factors and created a molecular library of about
300 thousand small molecules, most of which are metabolites. They have also identified
about 5000 naturally occurring flavonoids in various plants used as food [67] and a large
number of other nutrient and non nutrient compounds in foods.

Thus, an enormous amount of data has been generated and efforts continue to
obtain meaningful information from it. However, given the complexity of both the
systems involved, that is the human body and the dietary compounds, compounded by
other variables, it is an enormous task to make sense of the data that will emerge from
current technologies. Hence, it will be necessary to develop nutrigenomics on the lines of
nutritional systems biology to address current challenges [69]. Data analysis by super
computers, capable of complex mathematical modeling will be required, especially if the
more favored holistic perspective is to be adopted.

Despite these complexities, nutrigenomics holds promise for improving the life of
future generations; and personalized assessments based on metabolomics will, some day
in the not too distant future, replace the traditional tools used to evaluate nutritional
status. They will lead to the ability to make nutrition recommendations uniquely suited to
the individual according to their genetic makeup and metabolic profile [70], thus making
personalized, customized nutrition counseling a reality.

26
 However, new approaches to handle the complexities and social consequences of
the emerging technologies will be required, and the major challenges for nutrigenomic
research in the next decade will continue to be identification of cause/effect relationships
among multiple genome variants, diet and other environmental factors, and the main
chronic diseases. The technology is directionally correct but we still have a long way to
go.

The future must also address the need to provide both specialized genomics-
related education and training and general public information to enhance awareness, build
competencies, make informed decisions, and ensure continuity of access to health
services. The controversies related to personalized diets and benefits of functional foods
must be presented to the public in their correct perspective. Scientists and the media must
synergize to ensure that the issue is publicized in its correct perspective, without undue
euphoria, nor unwarranted caution, because as a science, nutrigenomics is still in its
infancy, but holds the promise to offer the panacea in times to come.

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