Toxicogenomics : Its scope and techniques

Dharmendra Kumar Meena^*  Kanti Meena¹, and Daryab Singh²

¹Central Inland Fisheries Research Institute, Barrackpore, Kolkata, 700120

*Central Research Institute on Jute & Allied Fibres, Barrackpore, 700121

²Project Directorate on Poultry Rajendranagar, Hyderabad, 500030

Corresponding Author: Dharmendra Kumar Meena

Email: Dkmeenafnb@gmail.com

Toxicogenomics: Toxicogenomics is a field of science that deals with the collection, interpretation, and storage of information about gene and protein activity within a particular cell or tissue of an organism in response to toxic substances. Toxicogenomics combines toxicology with genomics or other high throughput molecular profiling technologies such as transcriptomics, proteomics and metabolomics. Toxicogenomics endeavors to elucidate molecular mechanisms evolved in the expression of toxicity, and to derive molecular expression patterns i.e., molecular biomarkers that predict toxicity or the genetic susceptibility to it.

A growing concern over the presence of genotoxins in marine media, there is a rising need to elaborate sensitive methods for the assessment of genetic damage in indigenous organisms. There have been developed different methods for the detection of both double and single-strand breaks of DNA, DNA-adducts, micronuclei formation, and chromosome aberrations. Many environmental contaminants exert their effects via genotoxic and metabolically toxic mechanisms simultaneously causing carcinogenesis, embryotoxicity and implicit a long term alterations in organisms by being active through several generations (Jha et al., 2000). Toxicogenomics combines transcript, protein and metabolite profiling with conventional toxicology to investigate the interaction between genes and environmental stress in disease causation. The patterns of altered molecular expression that are caused by specific exposures or disease outcomes have revealed how several toxicants act and cause disease. Despite these success stories, the field faces noteworthy challenges in discriminating the molecular basis of toxicity. We argue that toxicology is gradually evolving into a systems toxicology that will eventually allow us to describe all the toxicological interactions that occur within a living system under stress and use our knowledge of toxicogenomic responses in one species to predict the modes of action of similar agents in other species.

Toxicogenomics: scope and methods

Toxicogenomics has three principal goals

1. To understand the relationship between environmental stress and human disease susceptibility

2. To identify useful BIOMARKERS of disease and exposure to toxic substances

3. To elucidate the molecular mechanisms of toxicity.

A typical toxicogenomics study might involve an animal experiment with three treatment groups: high dose and low dose treatment groups and a vehicle control group that has received only the solvent used with the test agent. These groups will be observed at two or three points in time, with three to five animal subjects per group. In this respect, a toxicogenomics investigation resembles a simple, acute-toxicity study. The two approaches differ in the scope of the response they aim to detect, and in the methods used. The highest-dose regimen is intended to produce an overtly toxic response that can be detected in a toxicogenomics study using the global measurement techniques that are described below.

1.Transcriptomics: cDNA microarray hybridization and analysis. Early gene- expression profiling experiments that were carried out for toxicogenomics studies used cDNA microarrays49. Although this cDNA technology is rapidly being supplanted by synthetic-oligonucleotide short and long microarrays the technological concepts underlying the two approaches are mostly analogous: cDNAs are derived from sequence verified clones representing the 3′ ends of the genes, which are either spotted onto glass slides using a robotic arrayer or synthesized in situ. Each RNA sample is labelled with dye conjugated dUTP (deoxyuridine triphosphate) by reverse transcription from an oligo-dT (deoxythymine) primer. The fluorescently labelled cDNAs are then hybridized to the microarray and the microarray is scanned using laser excitation of the fluorophores. Raw pixel intensity-images that are derived from the scanner are analysed to locate targets on the array, measure local background for each target and subtract it from the target intensity value.

2. Proteomics: An established proteomics strategy 90 uses global protein-stratification systems, such as PAGE, followed by protein identification through mass spectrometry. Two-dimensional PAGE separation, by charge and by mass, can resolve thousands of proteins to near homogeneity. This separation is a necessary prerequisite to enzymatic digestion and mass-spectrometry identification, which requires unique peptide fingerprint masses or amino acid sequence tags. Where proteins are separated by liquid chromatography instead of PAGE, a new and promising platform that involves multidimensional liquid chromatography can be used to fractionate and reduce the complexity of the protein mixture before peptide sequencing by mass spectrometry or TANDEM MASS.SPECTROMETRY. This approach is being augmented by SELDI (surface enhanced laser desorption/ionization) time-of-flight mass spectrometry; a method that results in the isolation of tens of thousands to hundreds of thousands of low molecular weight fragments that represent a proteome.

3. Metabolomics and metabonomics: Quantitative analytical methods have been developed to identify metabolites in pathways or classes of compounds. This collective directed approach has been called metabolite profiling or metabolomics. Semiquantitative, nuclear agneticresonance (NMR) based metabolic fingerprinting has also been applied to high-abundance metabolites and has been termed 'metabonomics'. Peaks detected in NMR spectra carry information regarding the structure of the metabolites, whereas peaks detected by mass spectrometry have associated molecular weights. In addition, specific massspectrometry methods can be established to fragment the parent molecule, allowing metabolites to be identified through investigation of fragmentation patterns.

Phenotypic anchoring: Conventional toxicology has used surrogate markers that are correlated with toxic responses to monitor adverse outcomes in inaccessible tissues (Loeb et al.,1999 ). For example, the liver enzymes alkaline phosphatase (ALT) and aspartate aminotransferase (AST) are released after hepatic damage has occurred, and concentrations of these enzymes that are found in serum correlate with histopathological changes in the liver (Loeb et al., 1999;Travalos et al., 1996). These serum enzyme markers, in conjunction with histopathology, facilitate the 'phenotypic anchoring' of molecularexpression data (Tennant, 2002; Waters et al ., 2003; Paules et al., 2003). Phenotypic anchoring is the process of determining the relationship between a particular expression profile and the pharmacological or toxicological phenotype of the organism for a particular exposure or dose and at a particular time ( Tennant, 2002). The dose and time alone are often insufficient to define the toxicity experienced by an individual animal, so another measure of toxicity is needed for the full interpretation of the data obtained during a toxicogenomics study. Conversely, the phenotype alone might be insufficient to anchor the molecular profile, because an elevated value for ALT in serum can be observed both before peak toxicity (as it rises) and after peak toxicity (as it returns to baseline). Therefore, anchoring the molecularexpression profile in phenotype, dose and time helps to define the sequence of key molecular events in the modeofaction of a toxicant. Phenotypic anchoring can also be used in conjunction with lower doses of the toxicant to classify agents and to explore the mechanisms of toxicity that occur before histopathological changes are seen.

Biomarkers: Some toxicities lack conventional biomarkers, which leads to increased risk in clinical trials and motivates the search for new pre-clinical biomarkers to support drug development. A class of Lead Compounds identified in a discovery programme based on γ-secretase inhibition as therapy for Alzheimer disease also have been found to have an undesirable effect of inhibiting cleavage of the Hes1 gene-product by Notch; a process that is important for the differentiation of intestinal epithelial cells. Through the use of geneexpression profiling and subsequent protein analysis. Searfoss et al. (2003) identified adipsin as a biomarker for this toxicity.

References:

The National Academies Press: Communicating Toxicogenomics Information to Nonexperts: A Workshop Summary (2005).

Hisham K. Hamadeh; Cynthia A. Afshari. (2004). In Hamadeh HK, Afshari CA. ed. Toxicogenomics: Principles and Applications. Hoboken, NJ: Wiley-Liss. ISBN 0-471-43417-5.

Jha A.N., Cheung V.V., Foulkes M.E., Hill S.J. and Depledge M.H. (2000). Detection of genotoxins in the marine environment: adoption and evaluation of an integrated approach using the embryolarval stages of the marine mussel, Mytilus edulis. Mutation Research, 464: 213-228.

Loeb, W. F. & Quimby, F. W. (eds) The Clinical Chemistry of Laboratory Animals (Taylor and Francis,1999).

Tennant, R. W. (2002). The National Center for Toxicogenomics:using new technologies to inform mechanistic toxicology. Environ. Health Perspect. 110, A8 — A10.

Waters, M. D. et al (2003). Systems toxicology and the chemical effects in biological systems knowledge base. Environ. Health Perspect. 111, 811 — 824

Paules, R. (2003). Phenotypic anchoring: linking cause and effect. Environ. Health Perspect. 111, A338 — A339