Perturbation of Critical Metabolic and Control Pathways: Genomic, Proteomic, and "Metabonomic" Technologies Can Provide a Global View
The availability of the new "global" technologies of genomics (Aardema and MacGregor, 2002), expression profiling (Hamadeh et al., 2002), proteomics (Anderson et al., 2000; Bichsel et al., 2001; Hermann et al., 2001; Huang et al., 2001; Steiner and Anderson, 2000; Wolters et al., 2001; Yates, 1998, 2001), and metabonomics (Nicholson et al., 1999, 2002) promises to make routine the monitoring of many, or in some cases all, of the components of key control and metabolic pathways. These powerful technologies, termed "-omic" technologies because of their potential to monitor complete classes of structural or functional molecules within tissues or organisms (Lederberg and McCray, 2001), provide the potential to assess the functional activity of biochemical pathways through a single simultaneous analysis of the many cellular components controlled by a particular pathway. The potential of these "-omic" technologies to revolutionize the current approach to toxicological assessment has recently been addressed (Aardema and MacGregor, 2002). Among the classes of molecules that are currently thought to be addressable through "-omics" technologies are mRNAs, proteins and peptides, and small molecular weight intermediary metabolites.
Each of the technologies available currently has particular advantages and disadvantages for specific applications. DNA arrays are powerful tools for direct monitoring of increases or decreases in gene transcripts from large numbers of genes in comparative samples. Thus, this technology will play an important role in identifying those genes induced in response to specific types of damage or to identify global shifts in gene expression that result from pathological alterations within cells and tissues. However, it is likely that this technology will play mainly a "discovery" role with respect to biomarkers for in vivo monitoring, because invasive procedures are required to obtain sufficient nucleic acid samples from internal tissues and organs. Thus, it is likely that proteins or peptides, or small molecules controlled by gene expression, will emerge as those biomarkers of functional status or damage response used in routine toxicological practice. When key gene products are identified using nucleic acid array technologies, methods are now available to construct protein-based assays for monitoring those products. These methods include phage-antibody libraries coupled with high-throughput selection of antigen-antibody interactions that can identify high-affinity binding molecules to almost any protein (Holt et al., 2000). Once suitable antibodies or other binding substrates are identified, protein-binding arrays can be constructed for efficient analysis of the protein products (Huang, 2001).
Proteomic and metabonomic approaches are suitable for identification of gene products and cellular constituents in accessible body fluids and tissue compartments, and will likely lead to new biomarkers for in vivo monitoring. Among the advances in the technologies of identifying proteins and peptides are improvements in classical 2-dimensional (2D) gel electrophoresis coupled with sophisticated mass spectroscopic identification of protein sequences (Anderson et al., 2000; Steiner and Anderson, 2000), matrix-assisted (MALDI) or surface-enhanced laser desorption ionization (SELDI) techniques that allow rapid characterization of proteins, protein fragments, or polypeptides (e.g., Bichsel et al., 2001; Hermann et al., 2001), and multidimensional chromatographic/mass spectroscopic methodologies (Wolters et al., 2001; Yates, 1998, 2001). These technologies have the potential to identify accessible markers in body fluids as well as measures of functional and structural proteins and peptides within tissues and cells. Protein and antibody arrays (Cahill, 2001; Huang et al., 2001) and bead-capture methodologies (Nolan and Mandy, 2001) also offer advantages for certain applications.
Metabonomics employs NMR technology to identify intermediary metabolites that provide an index of metabolic state (Nicholson et al., 1999). This technology has proven effective at characterizing metabolic shifts associated with a variety of pathologies and functional alterations (Nicholson et al., 2002), including renal and hepatic toxicity (Robertson et al., 2000). This technology has the major advantage that it is based on non- or minimally-invasive measurements in urine or plasma, making it directly applicable to studies in humans or animals in vivo.
Various strategies to identify inducible (and suppressible) biomarkers of pathology are possible. Whatever the strategy employed, effects of specific well-characterized pathologies on genes, proteins, and small molecules within the cell will need to be characterized to determine the relationship between these potential markers and specific types of damage. Once key elements of damage response and/or pathological perturbation are characterized, appropriate low-cost technologies that allow these identified changes to be monitored inexpensively on a routine basis will then need to be validated for regulatory purposes.