Nutrigenomics and antioxidants - Future Medicine

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Nutrigenomics and antioxidants Nasséra Chalabi1,2, Dominique J BernardGallon1,2, Marie-Paule Vasson3,4 & Yves-Jean Bignon1,2,3† †Author

for correspondence Jean Perrin, Département d’Oncogénétique, 58 Rue Montalembert, BP 392, 63011 ClermontFerrand Cedex 01, France E-mail: [email protected] 2Centre de Recherche en Nutrition Humaine, 58 Rue Montalembert, BP 321, 63009 ClermontFerrand Cedex 01, France 3Université d’Auvergne, 28 Place Henri Dunant, BP 38, 63001 ClermontFerrand 1, France 4Centre Jean Perrin, Laboratoire de Biochimie, Biologie Moléculaire et Nutrition, EA2416, Faculté de Pharmacie, Unité de Nutrition, 63011, ClermontFerrand, cedex 01, France 1Centre

Keywords: antioxidant, gene expression, genomics, nutrition part of

Since the complete sequencing of the human genome, the era of the ‘omics’ has appeared. Among them, a new discipline called ‘nutrigenomics’ emerged from the interface of nutrition research and genetics. Its aim is to understand how nutrients modulate gene expression. This powerful tool allows determinination of new biomarkers and the molecular pathways by which our diet may have a potential protective effect against degenerative diseases such as cancer. On one hand, cellular metabolism produces continuous oxidative stress and reactive oxygen species with mutagenic and oncogenic effects. On the other hand, diet provides natural antioxidants present in various fruits and vegetables that may prevent diseases. This review first reports the main antioxidants provided by diet and the main results from epidemiological studies of their role in health. Second, we describe how nutrigenomics could provide new insights into nutrition research and innovative developments through neutraceutical products and a personalized medicine.

For the last few years, nutrition has been considered in a new light owing to newly developed technologies based on nutrient and gene interactions. This new discipline, named ‘nutrigenomics’, evaluates the effects of diet on gene expression, while ‘nutrigenetics’ evaluates how genotype can determine individual response to food. These approaches have emerged as a result of the deciphering of the complete human genome sequence. Indeed, genomic technologies have been developed that allow simultaneous determination of the expression of thousands of genes at transcriptomic and proteomic levels (Figure 1). DNA microarrays and proteomic analysis, such as 2D gel electrophoresis or tissue macroarrays, permit not only identification of new biomarkers but also understanding of how nutrition can influence human health. In this context, various micronutrients have been studied and, more precisely, their interaction at DNA, RNA and protein levels. The complexity of these studies stems from the fact that our diet consists of complex mixtures with various biological effects. Many disorders are related to reactive oxygen species (ROS). Indeed, free radicals such as ROS have been largely implicated in oxidative cell damage [1]. Three endogenous enzymatic systems protect our DNA from this oxidative stress: superoxide dismutase (SO0.D), glutathione peroxidase (GPx) and catalase. However, diet can also be a complementary source of cofactor minerals and exogenous antioxidants, provided in the main by fruits and vegetables. This review

10.2217/17410541.5.1.25 © 2008 Future Medicine Ltd ISSN 1741-0541

illustrates the relationship between nutrigenomics and antioxidants in order to suggest how genetics could contribute to a personalized medicine by understanding the biological effect of antioxidant micronutrients on gene expression. Antioxidants provided by diet (exogenous antioxidants) The best known antioxidants are β-carotene, ascorbic acid, tocopherol and polyphenols, including flavonoids, tannins (in coffee, tea or wine grapes) and phenolic acids (in cereals, fruits and vegetables). Fruits are rich in antioxidants, particularly red-blue fruits such as cherries and blueberries, which contain both vitamin C and polyphenols. The antioxidant power of the diet can be evaluated by oxygen radical absorbance capacity (Orac) unities, which represent the capacity of food to neutralize the peroxyl radical. Vegetables such as watercress, garlic, green cabbage, spinach, asparagus, Brussels sprouts, lucerne, broccoli, beets and red peppers are the richest in antioxidants. Indeed, they contain high amounts of vitamin C, carotenoids, flavonoids and phenolic compounds (Table 1). Carotenoids

Carotenoids are yellow or orange natural pigments present in fruits and vegetables and are synthesized by plants. Among the 600 known carotenoids, only a few are found in human plasma and tissues. These are essentially provided by diet. The best known is β-carotene, which can Personalized Medicine (2008) 5(1), 25–36

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REVIEW – Chalabi, Bernard-Gallon, Vasson & Bignon

Figure 1. ‘Omics’ tools used in nutrigenomic research.

Transcriptomics Microarrays, RT-qPCR, RNAi

DNA

RNA

Protein

Genomics Sequencing, polymorphisms Epigenomics DNA methylation, histone modification

Metabolites

Metabolomics HPLC

Proteomics 2D elctrophoresis, mass spectrometry

HPLC: High pressure liquid chromatography; RT-qPCR: Real-time quantitative PCR.

be cleaved into two retinal molecules within intestinal mucous membrane. Nevertheless, the lycopene found in tomatoes is not provitamin A. Indeed, this carotenoid fulfils its biological activities in a vitamin A-independent pathway [2]. Carotenoids are divided into two major groups: xanthophylls and carotenes. Xanthophylls are a source of oxygenate compounds, such as lutein, zeaxanthin and cryptoxanthin. Conversely, carotenes, such as α-carotene, β-carotene and lycopene, do not contain oxygen. Fruits and vegetables contain specific proportions of carotenoids; apricots, melons, carrots, pumpkins and sweet potatoes are rich in α- and β-carotenes. Tomatoes, watermelons, mangos and papayas are a source of lycopene, whereas plums, oranges and green vegetables contain essentially lutein (kale, spinach and collard greens) and zeaxanthin (yellow peppers and corn). Carotenoids act as chimioprotector agents through diverse biological activities: retinoid metabolism, antioxidation, stimulation of the immune system and protection against cellular mutagenesis [3]. Lutein and zeaxanthin supplementation are particularly beneficial in the prevention of atrophic age-related macular degeneration [4]. B vitamins

The vitamin B complex consists of eight water-soluble vitamins: B1 or thiamine, B2 or riboflavin, B3 (PP) or nicotinamide, B5 or panthotenic acid, 26

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B6 or pyridoxine, B8 (H) or biotin, B9 or folic acid and B12 or cobalamin. However, only three of them are recognized for their antioxidant power (Figure 2). Vitamin B1, known as thiamine, serves as a catalyst in carbohydrate metabolism and helps to synthesize nerve-regulating substances. Deficiency can cause heart swelling, beriberi disease, leg cramps and muscular weakness. Rich food sources high in thiamine include liver, heart and kidney meats, eggs, leafy green vegetables, nuts, legumes, berries, wheat germs and enriched cereals. Vitamin B5, or pantothenic acid, has a role in the metabolism of fats, carbohydrates and proteins. It is the precursor of coenzyme A (CoA) and the acyl carrier protein [5]. It is most abundant in eggs, wholegrain cereals, legumes and meat, although it is found in some quantity in nearly every food. Deficiency can result in fatigue, allergies, nausea and abdominal pain. Indeed, altered homeostasis of CoA has been observed in diverse disease states, including starvation, diabetes, alcoholism, Reye syndrome, medium-chain acyl CoA dehydrogenase deficiency, vitamin B12 (cobalamine) deficiency and certain tumors [5,6]. Hormones such as glucocorticoids, insulin and glucagon, as well as drugs such as clofibrate, also affect tissue CoA levels. Vitamin B6, or pyridoxine, helps the body to absorb and metabolize amino acids, to use fats and to form red blood cells. Deficiency in the vitamin may result in smooth tongue, skin disorders, dizziness, nausea, anemia, convulsions, kidney stones, increased risk of cardiovascular disease, stroke and cancer [5,7–10]. Whole grains, bread, liver, green beans, spinach, avocadoes and bananas are food sources that are high in this vitamin. Amino acids: methionin & cysteine

Methionin and cysteine are two amino acids inducing synthesis of the amino acid homocysteine (Hcy). In normal circumstances, Hcy is converted to cysteine and partly remethylated to methionine with the help of vitamin B12 and folate. However, when normal metabolism is disturbed, due to deficiency of cystathionine-β-synthase, which requires vitamin B6 for activation, Hcy is accumulated in the blood with an increase in methionine, resulting in mental retardation. Decreased cysteine may cause eye diseases due to decrease in the synthesis of glutathione. Homocysteinemia is found in Marfan syndrome and some cases of Type I diabetes and is also linked to smoking and has a genetic basis as well. The harmful effects of homocysteinemia are due to production of oxidants future science group

Nutrigenomics and antioxidants – REVIEW

Table 1. Antioxidant nutrients, dietary sources and health benefits. Antioxidant nutrients

Dietary sources

Health benefits

Carotenoids

Carrots, tomato products, spinach, leafy vegetables, green cabbages and French beans

Prostate cancer, breast cancer and age-related macular degeneration

Vitamins

Liver, heart, eggs, cereals and citrus fruits

Immunomodulation, atherosclerosis, hypertension, eye degenerative disorders, cancer and memory functions

Polyphenols

Soybeans (isoflavones), flaxseed and wholegrain products (lignins), green tea, coffee, wine and grapefruits

Prostate cancer, breast cancer and cardiovascular diseases

Trace elements 2n 5è

Seafood, calf liver and cereals

Immune system and DNA repair

Thiol aminoacids (methionin, cystein)

Meat, fish (cod, sardines), eggs, milk, cheese and shellfish

Epilepsy and heart diseases

(ROS) generated during oxidation of Hcy to homocystine and disulphides in the blood in cardiovascular diseases and stroke [11]. Indeed, there are significant associations between moderate increases in serum homocysteine and three cardiovascular diseases: ischemic heart disease, deep vein thrombosis and pulmonary embolism and stroke [12]. Vitamins C & E

Vitamins are essential to maintain normal metabolic processes in human health. Most vitamins are synthesized by plants and have to be obtained by diet. Vitamin C, also known as ascorbic acid, works by helping to form and maintain collagen. It has many benefits, supports a variety of the body’s structures and is essential to the formation of bones and teeth. Humans, unlike most animals, do not create their own vitamin C and they need to consume it through foods rich in the vitamin. A deficiency in vitamin C can cause scurvy, which manifests as loss of teeth, hemorrhages, bruising, inability to fight off infection, mild anemia and bleeding [13]. The best sources of vitamin C are fruits and vegetables. Citrus fruits are most well known for their vitamin C content but vitamin C can also be found in vegetables such as red peppers, cauliflower, mushrooms, broccoli and cereals. Vitamin C may play an anticarcinogenic role through its action as a redox agent (Figure 3) [14,15], as well as have an effect on atherosclerosis, hypertension and degenerative eye disorders [13,16–18]. Hickey et al. explain that the half-life of vitamin C is rapid and, as a water-soluble nutrient, effective supplementation requires repeated dosing – 500 mg five-times daily for optimal blood levels [19]. We know that there are groups that are deficient, such as smokers who destroy 25 mg of the body’s pool of vitamin C for every cigarette smoked. future science group

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Moreover, an estimated 16–23% of Americans are deficient in vitamin C and virtually all Americans are deficient in vitamin C if compared with animals that synthesize their own vitamin C in their liver or kidneys. Vitamin E represents a group of potent, lipidsoluble, chain-breaking antioxidants. There are four tocopherols (α-, β-, γ- and δ-) and four tocotrienols (α-, β-, γ- and δ-). α-tocopherol is the most abundant in nature and it prevents the propagation of free radical reactions, suggesting a role in prevention of chronic diseases, such as cardiovascular disorders, atherosclerosis and cancer [20–22]. Moreover, intake of dietary supplements containing vitamins and trace elements was associated with lower C-reactive protein (CRP) levels in women [23]. In particular, vitamin E in combination with other vitamins, such as vitamins C, B1, B2, B6, B12, niacin, folic acid, pantothenic acid and selenium, was significantly associated with lower CRP levels. Intake of these micronutrients may influence the inflammatory process underlying the pathogenesis of atherosclerosis [23]. The exact role of selenium in cataract development is still unclear. Both protective and toxic effects and mechanisms have been postulated in the past. Indeed, selenium in excess of the tiny amounts required for selenoprotein synthesis is toxic in general and causes cataracts in experimental animals [24]. Deficiency in vitamin E can lead to mild hemolytic anemia or spinocerebellar disease. Absolute vitamin E deficiency, however, is very rare in humans, except for supplement users. The US population does not consume the Recommended Daily Allowance for vitamin E. People who cannot absorb dietary fat, premature infants and individuals with genetic abnormalities are susceptible. Too much vitamin E (more than 1g per day), however, can increase the risk 27


Figure 2. Vitamins B1, B5, B6, C and E. HO N+ HO

S

H2N

N

N

O

HO OH

O N H

H

Vitamin B6

CH3 HO

CH3

H3C

CH3

Vitamin C

CH3

CH3 CH3

O CH3

Vitamin E

of bleeding problems. Vitamin E is found in almonds, hazelnuts, leafy green vegetables, whole grains, cereal and bread, wheat germ, liver, vegetable oils and margarine. Polyphenols: flavonoids, anthocyanes & phenolic acids

Polyphenols are bioactive compounds found in fruits, vegetables, grains, nuts and oils. These molecules, synthesized by plants as secondary metabolites, represent a large family of organic molecules, ranging from simple phenolic acid to highly polymerized compounds. Polyphenols are divided into different groups according to the chemical structure: phenolic acids, flavonoids including flavonols, flavones, isoflavones, flavanones, anthocyanidins, flavanols, proanthocyanidins, lignans and stilbenes (Table 2). These groups present various biological activities linked to different molecular pathways. The epigallocatechin-3-gallate, known as catechin, derived from green tea has been one of the most studied polyphenol compounds. Indeed, green tea is one of the most consumed beverages, especially in Asian countries, whereas black tea is usually consumed in Western countries. Flavonols, including quercetin, kaempferol, myricitin and their glycosides, are also present in tea. There is evidence of the anticarcinogenic properties of tea through various molecular mechanisms, such as inhibition of nuclear factor-κB, induction of apoptosis and cell cycle [25]. Last year, the Journal of the American Medical 28

O

Vitamin B5

O

HO

OH

OH

S

HN

Vitamin B1

OH

H

O

N


Association reported that green tea consumption lowers all-cause mortality rates, a health benefit that increases with increasing intake of green tea (from one to five cups a day) [26]. Green tea also binds iron. Our laboratory studied the effects of various micronutrients on breast cancer, including resveratrol, a polyphenolic compound found in plants. This molecule was involved at the three major stages of breast carcinogenesis: initiation, promotion and progression. It has antioxidant and anti-inflammatory properties, induces apoptosis and modulates cell cycle and estrogen receptor function in breast cancer cell lines [27]. Moreover, it has been found that resveratrol is also a copper chelator and reduces mortality by 31% and for women by 18%. Wine drinkers have a 20–30% reduced mortality rate compared with alcohol abstainers [28]. Zinc: superoxide dismutase constituent

Zinc contributes to an effective immune system. It plays an important role in cell-mediated immune functions and also functions as an anti-inflammatory and antioxidant agent [29]. Zinc is also known to be an essential component of DNA-binding proteins with zinc fingers, as well as copper/zinc SOD and several proteins involved in DNA repair. Thus, zinc plays an important role in transcription factor function, antioxidant defense and DNA repair. Dietary deficiencies in zinc can contribute to singleand double-strand DNA breaks and oxidative future science group


Figure 3. Possible chemopreventive mechanisms of vitamin C as an antioxidant in carcinogenesis. Carcinogenic stress

Antioxidant enzyme systems

ROIs RNIs

Inactive products

Direct scavenger

Normal cell

Initiated cell

Preneoplastic cell

Modification of epigenetic events: e.g., anti-inflammation, restoration of cell-tocell communication

Neoplastic cell

ROI: Reactive oxygen intermediate.

modifications to DNA that increase the risk of cancer development [30]. Moreover, dysregulation of copper and zinc homeostasis in the brain plays a critical role in Alzheimer’s disease. It has been found that zinc-thiolate clusters in Zn7MT-3 cells can efficiently silence the redox-active free Cu2+ ions [31]. Those that are at increased risk of zinc deficiency include people who do not consume an adequate intake of calories, alcoholics and those who suffer from digestive disorders. Vegetarians can require 50% more zinc than those who are not vegetarians. Indeed, with elimination of meat and increased intake of phytate-containing legumes and whole grains, the absorption of both iron and zinc is lower with vegetarian than with nonvegetarian diets [32]. Pregnant women, mothers who are breastfeeding and young children should ensure that sufficient zinc is consumed. Zinc is present in high amounts in oysters, calf and pig liver and also in cereals such as wheat. Selenium: cytosolic glutathion peroxydase constituent

Selenium plays an important role in human health owing to its presence within at least 25 proteins, named selenoproteins, including future science group


GPx involved in redox homeostasis [33]. Moderate selenium deficiency has been linked to many conditions, such as increased cancer and infection risk, male infertility, decrease in immune and thyroid functions, and several neurologic conditions, including Alzheimer’s and Parkinson’s diseases [34]. By contrast, a supplementation in selenium seems to have a preventional effect on the incidence of prostate cancer [35]. Selenium can be found in seafood such as salmon or mussels, but also in calf liver, asparagus, carrots, broccoli and cereals, whose contents depend on the soil in which they grow. Antioxidant & health: epidemiological studies Numerous epidemiological studies considered the impact of intakes of antioxidant supplements on the prevention of various diseases. In April 1994, a randomized, double-blind, placebo-controlled primary-prevention trial was performed to determine whether daily supplementation with α-tocopherol, β-carotene or both would reduce the incidence of lung cancer and other cancers. A total of 29,133 male smokers 50–69 years of age from South Western Finland were randomly assigned to one of four regimens: α-tocopherol (50 mg/day) alone, β-carotene (20 mg/day) alone, both αtocopherol and β-carotene, or placebo. Follow-up continued for 5–8 years. Results showed no reduction in the incidence of lung cancer among male smokers after 5–8 years of dietary supplementation with α-tocopherol or β-carotene. This study rather showed that these supplements may have harmful as well as beneficial effects [36]. In China, a study conducted by both the Cancer Institute of the Chinese Academy of Medical Sciences and the American National Cancer Institute investigated the effect of daily multiple micronutrients (retinol-zinc, riboflavin-niacin, vitamin C-molybdenum or β-carotene-vitamin E-selenium) on the incidence and mortality rates for esophagus or stomach cancers among the high-risk population of Chinese Linxian communes. The trial included more than 30,000 people who received one of the four combinations of vitamins/minerals daily for 5 years. Results indicated a 9% reduction in deaths from all causes and a 13% reduction in cancer mortality for people who received a β-carotene-vitamin E-selenium combination, mainly due to a 21% decrease in stomach cancer mortality [37]. In the 1980s, the relation between fruit and vegetable intakes and prevention of cancer was comprehensively studied. Over 7.5 years, a 29

REVIEW – Chalabi, Bernard-Gallon, Vasson & Bignon Table 2. Polyphenol groups and dietary sources. Phytochemical classes

Bioactive compounds

Dietary sources

Phenolic acids

Benzoic acid

Cranberries, apples, black radishes, onions, tea

Cinnamic acid

Coffee, blueberries, kiwis, plums

Caffeic acid

Pears, basil, thyme, oregano, coffee

Ferulic acid

Rice, wheat, oats, coffee, apples, artichokes, peanuts, oranges, pineapples

Flavonols

Quercetin

Onions

Flavones

Kaempferol

Tea, broccoli

Tangeretin

Citrus fruits

Naringenin

Grapefruits

Hesperetin

Oranges

Eriodictyol

Lemons

Genistein, daidzein

Soya

Catechins

Gallocatechin, epigallocatechin

Apricots, red wine, green tea, chocolate

Proanthocyanidins

Tannins

Peaches, raspberries, apples, pears, wine, cider, tea, beer

Lignans

Secoisolariciresinol

Flaxseed

Pinoresinol, lariciresinol

Sesame seeds, brassica vegetables

Resveratrol

Red wine

Flavonoids

Flavanones

Isoflavones: phytoestrogens Flavanols

Stilbenes

French study was conducted on more than 13,017 adults aged from 35 to 60 years [38]. This randomized, double-bind study was called Supplementation en Vitamins et Minéraux Antioxidants (SU.VI.MAX). All participants took a single daily capsule of a combination of 120 mg of ascorbic acid, 30 mg of vitamin E, 6 mg of βcarotene, 100 µg of selenium and 20 mg of zinc, or a placebo. Results showed a 30% decrease in cancer risk and mortality in men but no significant effect on women. These results were explained by a lower intake of fruits and vegetables in men and a lower consumption of tobacco in women. Moreover, it has been shown that high doses of β-carotene supplementation may be deleterious in individuals in whom the initial phase of cancer development has already started and could be ineffective in well-nourished individuals with adequate antioxidant status [39]. The relationship between intake of antioxidant and cancer risk incidence has also been explored in a prospective cohort study: the European Prospective Investigation into Cancer and Nutrition (EPIC). This study investigated the relationship between food, nutritional status and various lifestyle and environmental factors, and incidence of

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different types of cancer. The cohort included 23 centers in ten European countries: Denmark, France, Germany, Greece, Italy, The Netherlands, Norway, Spain, Sweden and the UK [40]. This cohort included 519,978 participants, which enabled comparisons between countries with very different rates of cancer occurrence and distribution of lifestyle and food habits. Indeed, there were considerable differences in food group consumption and dietary patterns among the EPIC study populations. This large heterogeneity was an advantage when investigating the relationship between diet and cancer and formulating new etiological hypotheses related to dietary patterns and disease [41,42]. Results were exploited by cancer type and different conclusions were obtained: for breast cancer there was no association with total or specific vegetable and fruit intakes [43], whereas a significant inverse association between fruit consumption and lung cancer was observed [44,45]. Opportunities provided by genomics in nutrition research Classical methods used in nutrition research were based on analytical chemistry and biochemistry for isolation and structural characterization of


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bioactive food components. Epidemiological studies allow nutritionists to better understand the relationship between intake of micronutrients and diseases. Nevertheless, most degenerative diseases, such as cancer, cardiovascular disease and diabetes, implicate complex interactions between diet and several genes. Therefore, these classical dietary methods are insufficient and molecular genomics could contribute to elucidation of how common dietary components could affect the balance between health and disease. Nutrigenomics emerged from the interface between different disciplines, including nutrition, molecular biology, medicine, genomics and bioinformatics. Indeed, this is the association between nutrition tools and the new ‘omics’ disciplines, which allow identification of molecular targets for nutritional pre-emption [46]. Nutrients modulate molecular processes, such as DNA structure, gene expression and metabolism, resulting in usefulness of ‘omics’ tools, such as genomics (gene analysis), transcriptomics (gene expression analysis), proteomics (global protein analysis) and metabolomics (metabolite profiling) [47]. Transcriptomics

Transcriptomics using oligonucleotide microarray technology is a powerful tool for gene expression study. In nutrition research, it has been used for identifying diet-regulated genes that cause or contribute to disease processes by measurement of gene-relative expression between healthy cells and diseased cells before and after exposure to different antioxidant dietary components. Our team investigated lycopene, the major tomato carotenoid, and its effects on breast cancer. These studies were designed to characterize the differences between lycopene-treated and -untreated cells on 202 genes interacting with the two major breast cancer genes, BRCA1 and BRCA2 [48], and on the whole genome [49]. Results showed an effect of lycopene on cell cycle, apoptosis and DNA repair mechanisms. Another study conducted by Siler et al. was conducted using microarray technology on prostate cancer to gain insight into the in vivo action of lycopene and vitamin E. Results showed that lycopene and vitamin E contribute to the reduction of prostate cancer by interfering with internal autocrine or paracrine loops of sex steroid hormone and growth factor activation/synthesis and signaling in the prostate [50]. future science group


This high-throughput technique allows determination of a ‘gene signature’ under specific conditions of thousands of genes at the same time in a single-step study. Nutrigenomicsbased signature analysis is a promising strategy for learning more about phenotypic responses to nutrition prevention [51]. Indeed, effects on the phenotype from a genetic variation due to dietary components have been described and referred to as gene-nutrient effects [52]. Among genetic variations, the single nucleotide polymorphisms (SNPs) in genes are involved in the response to antioxidant nutrients. Available SNP microarrays have been used in associated studies to identify new candidate loci for a disease or diet individual responses [53]. Usually, validation of results from trancriptomic microarrays is necessary through real-time quantitative PCR. Indeed, this technique enables measurement of PCR product accumulation through a dual-labeled fluorogenic probe [54]. Like microarrays, it could be possible to quantify a single RNA expression or multiple before and after nutrient exposure [55]. Using the quantitative real-time PCR technology, we investigated the effects of resveratrol on breast cancer gene expression. Three human breast tumor cell lines (HBL-100, MCF-7 and MBA-MB-231) and one breast cell line (MCF-10a) derived from a fibrocystic disease were used to study in vitro the effect of resveratrol on the transcription of a group of genes whose proteins interact in different pathways with BRCA1, the major gene involved in breast cancer. Thus, the expression of BRCA1, BRCA2, ERa, ERβ, p53, p21waf1/cip1, CBP/P300, RAD51, pS2 and Ki67 mRNA were quantified with an ABI 7700 (Applied Biosystems®). Results indicated that resveratrol at 30 mM increased expression of genes involved in the aggressiveness of human breast tumor cell lines through an estrogen receptor-dependent pathway [56,57]. Moreover, we used this same technology to evaluate the effect of two soya phytoestrogens (genistein and daidzein) on breast cancer gene expression [58,59]. We also carried out an in vivo study in ovariectomized Wistar rats to estimate Brca1 and Brca2 expressions after different phytoestrogen-rich diets. A total of 200 mammary glands were harvested in three independent experiments. We showed that isoflavones, given in the diet at different doses, increased Brca1 and Brca2 mRNA in ovariectomized female Wistar rat [60]. All these results provided important conclusions for an antioxidant effect on breast cancer gene expression. 31


Thus, oligoarrays and quantitative real-time PCR could be used in nutritional trials in a high-risk breast cancer population in order to define new biomarkers and potential neutraceuticals. This is our aim with a European program called ‘Prediction to Prevention’ (P2P) performed in order to understand whether a specific dietary lifestyle could reduce or influence the incidence of breast cancer in patients with a BRCA1 or BRCA2 germline mutation. Among transcriptomic technologies, an siRNA method can also be used to induce a specific gene inhibition. This technology has been used to investigate which genes are involved in explaining the actions of bioactive food components and characteristics of diseases and conditions [46,61]. This technique enabled identification of molecular targets for different diseases, such as cholesterolemy or cancer [62,63]. Genomics

Epigenetic events are also modified by antioxidant food components. These events are modulated by an epigenetic code through various enzymes (methylation, acetylation and phosphorylation) allowing chromatin remodeling. Several dietary factors may modify DNA methylation patterns. A study conducted in the agouti mouse was designed to determine whether maternal dietary methyl supplements increase DNA methylation and methylation-dependent epigenetic phenotypes in mammalian offspring [64]. The results showed that in utero exposure to dietary factors influences embryonic development with lifelong consequences. Moreover, DNA methylation is dependent on B vitamins as enzymatic cofactors, including folate, B6 and B12 vitamins, whereas cytosine methylation is highly sensitive to cellular folate status [8,65]. Proteomics

Dietary food components can also alter protein expression. Proteomic tools such as bi-dimensional electrophoresis or mass spectrometry are used to identify abnormal proteins in response to food [66,67]. In one study, extracts of six popularly consumed berries (blackberry, black raspberry, blueberry, cranberry, red raspberry and strawberry) were evaluated for their phenolic constituents using high-performance liquid chromatography with ultraviolet and electrospray ionization mass spectrometry detection. The major classes of berry phenolics were anthocyanins, flavonols, flavanols, ellagitannins, gallotannins, proanthocyanidins and phenolic 32


acids. The berry extracts were evaluated for their ability to inhibit the growth of human oral (KB, CAL-27), breast (MCF-7), colon (HT-29, HCT116) and prostate (LNCaP) tumor cell lines, and increasing inhibition of cell proliferation in all of the cell lines was observed, with different degrees of potency between cell lines [68]. Proteomic analysis provides important insights into the possible mechanisms by which antioxidant components could regulate our health. For example, in our team, we used the affinity chromatography to evaluate the effect of micronutrients on breast cancer cells. With these two technologies we were able to demonstrate that lycopene regulated protein expression of several genes involved in breast carcinoma [69]. Metabolomics

Finally, diet is also associated with changes in metabolic pathways. Metabolomics is the quantitative analysis of metabolites in an isolated cell system, tissue or biological fluid [70]. For example, a study conducted in 2007 showed that green tea consumption resulted in enhanced enzyme activities of carbohydrate metabolism and antioxidant defenses [71]. In the field of nutrition, ‘omics’ tools provide important opportunities for better understanding environmental and behavioral factors such as antioxidants. In this way, nutrigenomics helps to define new prevention strategy and their follow-up by the identification of new biomarkers. Toward a personalized medicine The main aim of nutrigenomics is to understand how nutrients act at the molecular level regarding individual response to diet and, more precisely, “the scientific study of the way specific genes and bioactive food components interact” [46]. Indeed, individual genetic variation can influence how nutrients are assimilated, metabolized, stored and excreted by the body [72]. This is the concept of personalized medicine, which means the prescription of specific nutrients best suited to an individual based on nutrigenetic and nutrigenomic information [73]. This new concept emerged from the Human Genome Project, which enabled development of high-throughput technology analyses, such as mutation determination, SNP genotyping, haplotype mapping, microarrays, proteomics and metabolomics. Molecular biology is a powerful tool for developing a personalized nutrition plan and a personalized preventive and therapeutic medicine. Nutrition research combined to genetics could future science group


optimize the efficacy of pharmaceutical agents to genetic background [74]. Indeed, nutrigenomics and pharmacogenomics defined as “an approach to pharmacology that takes into account the genotype of the patient” [75] both used the ‘omics’ high-throughput technologies to discover new biomarkers. The aim of these two disciplines is to introduce individualized medicine where there is an inter-individual variation in drug or food response among patients and no biomarkers can predict which group of patients will or will not respond positively to a treatment [75]. Indeed, drug administration can interfere with nutrient absorption, digestion, metabolism and secretion, and diet can also affect pharmacological effects of drugs [75]. For example, in cancer treatment the use of methotrexane could induce a folate deficiency [76]. Another study conducted on premenopausal breast cancer patients showed that lignan-rich flaxseed interacted adversely with tamoxifen but induced beneficial effects on bone, both alone and when combined with tamoxifen [77]. With regard to antioxidants, oxidative stress is a significant challenge in nutrition research in view of its important role in many chronic human diseases. With this new technology it would be possible to develop specific neutraceuticals, for example, in cancer therapy, without undesirable side effects. The cost of personalized therapy is a potential issue: will it reduce the costs of drug development by shortening the drug development cycle? The introduction of individualized medicine into clinical trials will reduce the likelihood of failed clinical trials and increase the prospects of safer and more effective therapies for specific groups of patients [73]. The food industries recognize the need for nutrigenomic research as a basis for developing the concept of

‘personalized diets’ for identifying molecular biomarkers or new bioactive food ingredients, and for validating the effectiveness of these bioactive ingredients as functional food components or nutraceuticals [51]. Conclusion Nutrigenomics provides new important answers and insights in antioxidant research. Combined with nutrigenetics it could contribute to the expected new era of a personalized medicine. Indeed, through individually adapted diet and treatments, it could enhance new therapies in order to reduce the resistance or sensitivity of patients or healthy people. Recent work in cancer research has shown death-from-cancer signature, predicting therapy failure in patients with multiple types of cancer [78]. Nutrigenomics could also help in determining, for each individual, the negative or positive development of a therapy on the anti-free radical action [79]. Despite this exciting potential, the ethical, legal and social implications of such research should be borne in mind. Future perspective From classical epidemiological intervention studies, nutrition research has mainly evolved by including high-throughput genetic technologies in order to discover new bioactive food components. ‘Nutrition’ and ‘genomics’ became ‘nutrigenomics’. In the area of oxidative stress, antioxidants represent a significant challenge to the understanding of how treatment will be efficient in chronic diseases, such as cancer, cardiovascular disease, diabetes or cholesterolemy. In this way, diet is becoming a potential ‘natural’ protective therapy through high consumption of fruits and vegetables, the richest sources of antioxidant micronutrients.

Executive summary • Nutrigenomics studies the effects of nutrients on gene expression. • Nutrigenomics has emerged from genetic high throughput technologies (genomics, epigenetics, transcriptomics, proteomics and metabolomics) and classic nutrition research. • Understanding antioxidant effects on human health is a significant challenge in finding new biomarkers for the treatment of degenerative diseases, such as cancer, obesity or cardiovascular diseases. • Fruits and vegetables are the best source of antioxidants in the human diet. • Nutrigenomics and pharmacogenomics are two complementary disciplines that will confer development of neutraceuticals. • Diet interferes in our health through individual polymorphisms. • Nutrigenomics will enhance the new era of personalized medicine.



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