ALPF Medical Research

Methods And Applications In Nutrigenomics

There is an increasing evidence indicating that antioxidants such as vitamin E, carotenoids, ascorbic acid, lipoic acids, bioflavonoids, and ginkgo biloba as well as trace elements (e.g., Zn, Fe, Se) affect differential gene expression in

Figure 1.4 Antioxidants as free-radical scavengers, metal chelators, and redox signaling molecules—both prevention of oxidative damage towards lipids, proteins, and DNA as well as redox signaling contributes to their potential beneficial effects.

Table 1.1 Studies on the Effect of Oxidants and Antioxidants on Differential Gene Expression in Cultured Cells, Laboratory Animals and in Humans

Cell/Tissue

Species

Number of genes monitored

Reference

Oxidants

Cigarette smoke

Hydrogen peroxide, menadione

/-butyl hydroperoxide Hydrogen peroxide 4-hydroxynonenal /-butyl hydroperoxide Oxidized LDL

Ozone

UVB radiation

Swiss 3T3 Breast cancer cells

Mouse Human

Retinal pigment epithelium cells Human

Aortic smooth muscle cells Human

Endothelial cells Human

Lung Mouse

Keratinocytes Human

513 17,000

1,176

35,932 588 4,000 6,000

Sukhanov et al. (13) Virgili et al. (14) Gohil et al. (15) Sesto et al. (16)

Antioxidants Ascorbic acid Coenzyme Q10 Copper

Epigallocatechin-3-gallate

Epigallocatechin-3-gallate, melatonin Fish oil

Keratinocytes Skeletal muscle Macrophages Cervical cancer cells Lung cancer cells Lung cancer cells Prostate carcinoma cells Neuroblastoma cells Brain Liver

Human 588 Catani et al. (17)

Human 12,000 Linnane et al. (18)

Human 6,800 Svensson et al. (19)

Human 588 Fujiki et al. (21)

Human 588 Okabe et al. (22)

Human 250 Wang and Mukhtar (23)

Human 25 Weinreb et al. (24)

Mouse 6,521 Takahashi et al. (26)

{continued)

	Cell/Tissue	Species	Number of genes monitored	Reference
Folic acid	Nasopharyngeal carcinoma cells	Human	2,200	Jhaveri et al. (27)
Ginkgo biloba	Brain	Rat	8,000	Li et al. (28)
	Brain	Mouse	7,000	Watanabe et al. (29)
Genistein	Prostate cancer cells	Human	557	Suzuki et al. (30)
Indole-3-carbinol	Prostate cancer cells	Human	22,215	Li et al. (31)
Lycopene, Vitamin E	Prostate	Rat	7,000	Siler et al. (32)
Melatonin	Retina	Rat	24,000	Wiechmann (33)
Methylseleninic acid	Premalignant breast cells	Human	316	Dong et al. (34)
Proanthocyanidin extract	Endothelial cells	Human	2,400	Bagchi et al. (35)
from grape seed
Procyanidins from pine bark	Keratinocytes	Human	588	Rihn et al. (36)
Resveratrol	Prostate cancer cells	Human	2,400	Narayanan et al. (37)
Selenium	Mammary epithelial organoids	Rat	588	Dong et al. (38)
	Intestine	Mouse	6,347	Rao et al. (39)
Sulphoraphane	Small intestine	Mouse	6,000	Thimmulappa et al. (40)
Vitamin A	Airway tissues	Human	30,000	Soref et al. (41)
Vitamin A and E, selenium	Skeletal muscle	Rat	800	Sreekumar et al. (42)
Vitamin D3	Osteosarcoma cells	Rat	5,000	Farach-Carson and Xu (43)
	Prostate cancer cells	Human	20,000	Krishnan et al. (44)
	Kidney	Mouse	12,422	Li et al. (45)
Vitamin E	Liver	Rat	7,000	Barella et al. (46)
	Aortic smooth muscle cells	Human	10,000	Villacorta et al. (47)
Vitamin E (Tocotrienol)	Fetal brains	Rat	8,000	Roy et al. (48)
Vitamin E and Selenium	Liver	Rat	465	Fischer et al. (49)
Zinc	Mucosa cells of small intestine	Rat	1,185	Blanchard et al. (50)
	Liver	Rat	2,500	torn Dieck et al. (51)

cultured cells, in laboratory animals, and in humans. In addition, oxidative stress, induced by oxidants such as ozone, cigarette smoke, UV radiation, and oxidized LDL, is associated with changes in differential gene expression. Recent studies on the effect of oxidants and antioxidants on differential gene expressions are summarized in Table 1.1.

The potential applications of transcriptomics in the field of antioxidant and free-radical research are manifold. Several methods have been developed for the quantitative and comprehensive analyses of changes in mRNA expression such as differential display, serial analysis of gene expression, DNA microarrays, and gene chips (Fig. 1.5). Gene-arrays have been used in order to analyze redox-sensitive signal transduction pathways, thereby getting more insights into the molecular functions of oxidants and antioxidants. A novel application of gene-array technology may be the development of new biomarkers of oxidative stress, which is still an open issue. Furthermore, differences in the bio-potency and bioavailability among different antioxidants may be detected by gene-array technology. Finally, gene-arrays can be applied in order to study the toxicity of oxidants and antioxidants as well as for screening new antioxidants.

Once candidate genes have been identified by array technology, the corresponding mRNA sequence has to be obtained from a data base (e.g., gene bank), and a blast of the sequence against the genome is performed in order to retrieve the DNA sequence, chromosomal loci, as well as the upstream sequence. This procedure is followed by a search for homology in regulatory elements and transcription factors (52) as summarized in Fig. 1.6.

Transcriptomics

Applications	Techniques
Screening ¡»id development of new antioxidants	Differential display
Assessing potential effects of antioxidants	cDNA arrays and Gene chips
Defining biomarkers of oxidative stress Evaluating the bioavailability of antioxidants	Serial Analysis of Gene Expression RT-PCR
Studying redox sensitive signal transduction pathways	Northern blotting

Figure 1.5 Analytical techniques and potential applications of transcriptomics in the field of free-radical research.

Figure 1.5 Analytical techniques and potential applications of transcriptomics in the field of free-radical research.

Search for Homology in Regulatory Elements and Transcription Factors

Search for Homology in Regulatory Elements and Transcription Factors

Describe potential signal transduction pathways

Figure 1.6 Major steps in the identification of redox-sensitive signal transduction pathways.

In order to obtain a comprehensive understanding of the molecular mechanisms of action of oxidants and antioxidants, rigorous study designs and statistical analysis are necessary. Time-point measurements need to be introduced in order to elucidate whether differences in the gene expression profile are manifested consistently over a prolonged period of time. In previous studies, differential changes in gene expression in response to dietary treatments

Figure 1.7 Schematic representation of the analytical steps involved in a gene chip experiment.

were often monitored only at one time-point and in pooled samples. Furthermore, differences in gene expression levels observed by gene chips technology should always be confirmed by independent methods such as real-time PCR and northern blotting, and then substantiated by functional parameters (Fig. 1.7). A set of guidelines (Minimum Information About a Micorarray Experiment, MIAME) have been established to outline the minimum information required for micro-array experiments.

Overall, gene expression profiling using array technology is rapidly becoming an important tool in nutrition and free-radical research. Array technology enables to study nutrient gene interactions on large scale that would be impossible using conventional analysis. Nutrigenomics has the potential to validate and to extend many of the strategies used in human nutrition on a molecular level, thereby improving human health.

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