Realisation of the promise of nutrient array systems is dependent on the following technological developments:
1. Physiological culture systems based on appropriate composition of the culture medium to reflect exactly the actual extracellular fluid composition in diverse tissue environments (e.g. plasma, cerebrospinal fluid) as well as the correct oxygen tension which can modify susceptibility and rate of nuclear and cell division.
2. The issue of nutrient requirements based on cell division kinetics could be significant but remains unexplored. Nutrient array systems will need to be developed for both dividing and non-dividing (confluent) cell cultures including 3 D cultures with mixed populations of dividing and non-dividing cells to test whether nutritional requirements for genome maintenance might vary depending on cell division status.
3. Because of the very large number of possible combinations of micronutrients (vitamins, minerals, phytonutrients, antioxidants and amino acids) a high throughput system is required that will be able to measure cell growth, cell death and DNA damage biomarkers in micro-cultures of cells using high content analyses with live cell-imaging systems that ideally will perform such measurements simultaneously in a manner that is not destructive of cells so that continuous measurements can be performed over several days with minimal cell number requirements (figure ). The ability to perform continuous measurements over several days or weeks is particularly relevant to nutrition because the effects of micronutrients are chronic rather than acute and their impact could drift as cells adapt to the different nutriome environments that they are exposed to in the nutrient array system.
Figure 6 Minimally invasive high-throughput nutrient array screening for genome-protective agents. A schematic diagram of the essential components of a high throughput high-content-analysis automated nutrient array system using human peripheral blood lymphocytes. (more ...)
4. Ideally such systems will be able to interrogate not only the optimal nutritional requirements for growth and genome maintenance of normal cells from an individual but also to verify that such a nutriome does not stimulate growth of cancer cells that the individual might have. Cancer cells are likely to have a markedly different genotype to that of the host's normal cells and could respond differently to the same nutriome environment. For example some cancer cells amplify the high affinity folic acid receptor [52
] giving them a distinct advantage over normal cells, when folate is limiting, in accessing folate from the surrounding fluid. The ideal nutriome for an ageing or cancer-prone individual would be the combination that not only sustains the replenishment of normal cells in a genetically integral manner but also inhibits the growth of cancer cells. It is conceivable that both normal cells and cancer cells from an individual could be simultaneously tested within a single nutrient array system.
5. Ultimately, although such systems can be readily implemented for optimising in vitro
culture conditions of cells, their practical use will be greatly enhanced once they have been validated for predicting the in vivo
nutritional requirements of an individual. The data from the optimal in vitro
nutriome, after comparison to plasma concentration, can then be used to estimate deficiencies or excesses of micronutrients in body fluids and appropriate dietary intervention can then be designed to make the necessary adjustments to optimise genome stability. This approach could be used in the emerging integrative and preventive medicine modality based on Genome Health Clinics in which developmental and degenerative diseases are prevented via diagnosis and nutritional prevention of DNA damage [11
]. Whether such attempts to optimise micronutrient status should be limited to those with above average DNA damage levels will remain an open question until we can find out what is the lowest DNA damage level achievable in vitro
or in vivo
6. Whether the nutrient array system can be adapted for use directly with an individual's sample of their own body fluids is an important question as this may be a better basis for in vitro testing of the efficacy of multiple nutritional adjustments under conditions that reflect exactly the individual's current physiological status. Although this approach seems attractive its feasibility has yet to be explored and could be limited by the difficulty of culturing cells in human serum.
7. There will always be a need to review and revise the DNA damage biomarkers that are most suitable for use in the nutrient array system based on their status of validation. At this point in time the cytokinesis-block micronucleus assay is the best validated with respect to its sensitivity to nutritional status and prospective association with cancer and cardiovascular disease mortality [1
]. Ultimately an automated high content analysis approach that integrates multiple complementary biomarkers of genome damage and instability (e.g. mitochondrial DNA mutations, telomere length, DNA methylation, micronuclei, nucleoplasmic bridges) would be required to achieve a deeper understanding of optimal nutritional requirements for genome maintenance on an individual basis.
8. Peripheral blood lymphocytes are ideal for use in the nutrient array system because they are easy to obtain and culture and have been used extensively to measure DNA damage in vitro
and in vivo
. Furthermore, because they travel throughout the body they experience fluctuations in micronutrient concentrations and nutriome profiles that may occur in different tissues and therefore can integrate the genomic impacts of sub-optimal nutrition throughout the body. Whether, the effects in lymphocytes might reflect what would happen in other tissues, such as epithelial tissues, is an important question because it is difficult to culture easily accessible epithelial tissues such as buccal mucosa. Two recent studies suggest that the level of micronuclei in lymphocytes correlates well with micronuclei in buccal cells and with DNA damage in sperm [58
]. However, despite these promising results more evidence is needed to justify the sole use of lymphocytes in the nutrient array system and ideally a practical epithelial cell culture alternative is also developed in the future.
In conclusion the use of nutrient array systems to interrogate genomic responses to multiple nutrient doses and combinations is in principle feasible and holds great promise to define the nutriome requirements of any cell type to either sustain its growth and reproduction in a genetically stable manner in the case of normal cells and stem cells or to suppress its growth and cause its death in the case of cancer cells.