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Scientists, regulators and the public all wish to have an efficient and precise approaches to measure the toxicological effects produced by various chemical, physical, and biological agents on living systems. But, till date there is no single approach exists to measures the adverse effects produced by various toxic substances. This may be due to complex nature of human and animal physiology and individual variations. Conventionally, testing of toxicity involves dosing of animals with toxic substance(s) or suspected toxicant(s) and subsequent observation of the treated group to explain the specific changes in physiology, cytology, metabolites and morphological endpoints such as death (Fokunang et al 2010). However, these approaches are time consuming, laborious, costly and impractical for tens of thousands of chemicals are used yearly in various industries (North and Vulpe 2010). Due to unprecedented advances in molecular biology and genetics; it would be possible to monitor potential hazardous effects of toxic substances on molecules, ultra structures and cell organelles before they manifest at phenotypic level.
Dr. William Greenlee defines toxicogenomics is the "application of global gene expression profiling, including DNA microarray technologies and proteomics, to study the relationship between exposure & disease and to understand gene-environment interactions & their impact on human health" (Fig1). National Center for Toxicological Research defined toxicogenomics as the "collection, interpretation and storage of information about gene and protein activity in order to identify toxic substances in the environment and to help treat people at the greatest risk of diseases caused by environmental pollutants or toxicants".
2. Technologies in Toxicogenomics
2.1 Gene Expression Profiling
Pharmacological or toxicological compounds alter gene expression patterns which in turn affect the signal pathways and these changes are compound specific (Hamadeh et al 2002). DNA microarray technologies have been used to assess the expression of virtually all genes in an organism. This opens a new path for global mapping gene expressions and modelling of genetic networks involved in various signal transduction pathways. Microarrays are used to predict gene functions and interactions, monitor therapeutic responses, classification of pharmaceuticals and toxicological compounds, discovery of new drug target and measure the toxicological properties of a compound (Neumann and Galvez 2002). There are two types of microarrays used in gene expression analyses: oligonucleotide-based arrays and cDNA arrays (Schena et al 1995) and both are yields comparable results.
Figure . Outline for systems toxicology. Indicates the paths from the first observation to an integrated toxicogenomics knowledgebase (blue drum), and so to systems toxicology (bottom right). The '-omics' data approach is shown by the clockwise from rat to knowledgebase; and the conventional toxicology approach shown in the anti-clockwise (Source: Waters and Fostel 2004).
Oligonucleotide arrays are prepared by using specific chemical synthesis steps to generate the specific sequence order in oligonucleotide synthesis and end result is the generation of high-density arrays of short or long oligonucleotide probes in predefined positions (Lipshutz et al 1998). In cDNA microarrays, large numbers of DNA sequences that correspond to the expressed gene sequences are usually printed on the surface of treated glass slides by using robotics. DNAs placed on the glass can stand for either sequenced genes of known or unknown function. Competitive, simultaneous hybridization with two colour fluorescence labelling approaches are used to measure the differential gene expression pattern for cDNA spotted on glass platforms (Yang and Speed 2002). Nowadays multi-color based labels are widely optimized for adequate utility. For toxicology studies, comparison of tissues from toxicant treated versus vehicle treated animals are necessary to decide the deferential genes expression (Hamadeh et al 2002).
DNA microarrays have certain drawbacks like these measurements are semi quantitative due to cross hybridization, anomalies in specific sequence binding and DNA sequence dependency for probe designing. In addition, optimization, incorporation of quality controls, collection, validation, processing, scanning of samples, skilled lab workers and data analysis are time consuming and expensive (Neumann and Galvez 2002, Miller and Tang 2009).
2.1.1 Quantitative Polymerase Chain Reaction (QPCR)
To overcome these limitations, combination of microarrays with quantitative polymerase chain reaction or Taqman and other technologies (Tokunaga et al 2000 read this paper) has been used to monitor the expression of hundreds of genes in a high throughput manner. This provides more quantitative output that may be vital for potential hazard screening and identification processes.
Toxicoproteomics deals with global protein profiling after exposure to toxins, chemicals, ecological stressors, therapeutics and any other materials to understand its involvement in the disease pathogenesis (Yu 2011). Currently, the most commonly used technologies for proteomics (Fig 2) research are gel-based proteomics and shotgun proteomics.
2.2.1 Gel-Based Proteomics
In the gel-based approach, proteins are resolved by electrophoresis and protein(s) of interest are selected for further analysis. In 2DE, first protein sample is separated by isoelectric focusing on immobilized pH gradient strip. After that, second dimension in SDS PAGE to separate proteins based their molecular mass, followed by visualization of proteins by various staining technique and scanning (Summeren et al 2012). Thereafter, selection of proteins of interest and its identification by mass spectrometry (MS) analysis, and database searching for further analysis to know the significance of the indentified proteins.
2.2.2 Shotgun Proteomics
In shotgun analyses direct digestion of protein samples to peptides and subsequent separation of peptide mixtures by reversed- phase high performance liquid-chromatography-coupled to mass spectrometry (LC-MS) (Summeren et al 2012). The resulting peptide tandem mass spectrometry (MS-MS) spectra are searched against databases to identify corresponding peptide sequences. Thereafter sequences are reassembled using computer software to know the proteins present in the original sample mixture
Figure: . Overview of the basic principles of DIGE and LC-MS/MS the two mainly used proteomics techniques (Summeren et al 2012)
Metabolomes includes different chemical compounds such are lipids, peptides, precursors of nucleic acid and degradation products to chemical intermediates of endogenously from biosynthesis and catabolism as well as metabolic product of exogenous compounds from the food, environmental exposures and drug treatments . NMR spectroscopy and gas chromatography MS (GC-MS) or LC-MS technology has been widely used for metabolomic studies of different bio fluids or in cell tissue extracts and both the techniques can detect metabolite profile to distinguish diverse toxicities (Beger et al 2010).
3. Environmental toxicology
The environment surrounds us and it provides everything to us such as air to inhale, water to drink and food to eat. At the same time it harbours vast amount of synthetic and natural chemicals, microbes, radiation and by products of industrial origins. Knowingly or unknowingly we are exposing ourselves to these agents in our daily life and at times these interactions are harmful leads to disease, disability and death. It is little known about the effects of atmospheric toxicants in disease production and it remains as a major challenge for the researchers to identify the toxicants, consequences of exposure, and disease pathogenesis from the toxicants of environmental origin. As a result, most of the environmental contaminants are identified after outbreaks of clinical poisoning. Usually low doses with long time exposure to large number of environmental toxicant are involved in disease initiation and progress and often difficult to determine the toxicity (Jayapal et al 2010). There are many types of interactions and connections in the environmental toxicology such are research programmes, scientific community, government and regulatory agencies, industry, ecological risk assessment and general public for the management of the ecological system (Landis and Yu 2003; Yu2004).
3.1 Frame work for environmental toxicology
Environmental toxicology can be simplified to understand three functions. First is the fate and distribution of the xenobiotic material after release in to the environment. Second the interaction of the material at the site of action. Third are impacts of these molecular interactions upon the function of an ecosystem. All these functions depends on the chemical and physical properties, bioaccumulation, biotransformation, biodegradation, population parameters and site of action of xenobiotic materials (Landis and Yu 2003; Yu2004).
3.2 Routes of Exposure and Modes of Action
A pollutant may get into an animal through a series of pathways. The routes may include exposure, entry into the host, transport, storage, metabolism and excretion. Exposure occurs through skin contact, eye contact, inhalation, ingestion leads to uptake of the pollutants. Once absorbed a rapid transportation of the substance throughout the body via circulation and distributed to various body tissues including those storage depots and metabolic sites. Then in the pollutants undergoes phase I and Phas II metabolism. It alters the solubility and detoxifies the xenobiotics and its subsequent excretion through kidneys, lungs, intestinal tracts, sweat, and saliva. Environmental pollutants cause an adverse effect on living organisms through disruption of cell structure (O3, SO2, NO2), direct chemical combination with a cell constituent (CO- binds with haemoglobin), influence on enzyme (Mercury, Cadmium, Lead- binds with SH group enzyme molecule; Fluoride- inactivates cofactor involved in enzyme active site; Beryllium- competes with cofactor for enzyme active site; Sodium fluoroacetate- inhibit enzyme activity), and initiation of a secondary action (cause release of certain substances which are injurious to cells) (Landis and Yu 2003; Yu2004).
3.3 Factors modifying the activity of toxins
Characteristics such as whether a pollutant is solid, liquid or gas; whether it soluble in water or lipid and whether it is organic or inorganic, ionized or nonionized etc can affects the toxicity of the pollutants. Duration of exposure and concentration are another determinant of toxic effects. Intermittent exposure to a toxicant is less injurious than continuous exposure. In addition, environmental factors such as temperature, humidity, light and PH also influences the pollutants toxicity. Besides that, various biological factors are also affects the toxicity like genetic factors, metabolic differences, age, sex, immune status and nutritional status (Jayapal et al 2010). Apart that interaction between different pollutions likes synergism, additive, potentiation and antagonism also plays a vital role in pollutants toxicity (Landis and Yu 2003).
4. Use of transcriptomics in toxicogenomics and environmental toxicology
Analysis of the gene expression pattern by high density micro arrays technology is useful to characterize alerted functional changes in tissue response to therapeutic interventions and chemicals. In addition, transcriptomics are useful to identify mode of action of unknown compounds toxicity, classification of toxic substances and to identify pathways involved in toxicity (Bulera et al 2001, Cui and Paules 2010). It was reported that one third of drug failures due to ADRs during therapeutic development and have been implicated for major stumbling blog in drug discovery and clinical use (Ge and He 2009). A large number of gene expression studies were carried out in order to create transcription `fingerprints' to classify or predict chemical agents with different toxic mechanisms (Cui and Paules 2010). Gene expression studies can be used in predictive toxicology to identify a compound as being potentially toxic or responsible for ADRs and mechanistic toxicology to understand biochemical and biological reposes to a particular category of toxicity (Ge and He 2009).
4.1 Transcription profile of Saccharomyces cerevisiae for methyl methanesulfonate toxicity
Exposure of Saccharomyces cerevisiae whole genome to the alkylating agent methyl methanesulfonate revealed that the transcription of more than 300 genes were induced and the expression of 76 genes were decreased. This effort established that a global gene-expression profile is important in the discovery of new genes and pathways responsible for toxicity (Cui and Paules 2010).
4.2 Arsenic toxicity
Gene expression arrays of liver biopsies obtained from chronically exposed humans displaying typical signs of arsenic toxicity compared with healthy individuals. People chronically exposed to arsenic revealed altered gene expression for those genes involved in numerous cell-cycle regulation (E2F3, E2F5, and E2F dimerization-partner 2), apoptosis (caspase 3, 4, 6, 8, 9 and 10 and tumour necrosis factor), DNA damage response (Cu,Zn-SOD) and repair ( ERCC2, ERCC5, topoisomerase II, replication factor C) and cytokeratins. In addition, altered gene expression of cellular regulators and growth factors were also observed. This study revealed the molecular mechanism (s) behind arsenic induced hepatotoxicity and carcinogenesis (Lu et al 2001).
Gene expression pattern in rat models treated with six well-known hepatotoxic compounds like carbon tetrachloride (CCL4), chloroform, 1-naphthylisothiocyanate (ANIT), tetracycline (TE), erythromycin (EE) and acetaminophen(AAP); and nonhepatotoxicÂ compounds like cloï¬brate, theophylline, naloxone, estradiol, quinidine, or dexamethasone to classify compounds with respect to toxicity prediction was conducted by Zidek et al (2007). They found all the hepatotoxic compounds showed compound-speciï¬c differences in their gene expression. Five genes were found to be deregulated by all six liver toxicants namely ATP-binding cassette subfamily B (MDR/TAP) member 11, flavin containing monooxygenase (Fmo1), monoamine oxidase (Maob), liver glycogen phosphorylase (Pygl), and thioredoxin reductase (Txnrd1). The oxidative stress enzyme, Txnrd1, was significantly up regulated by hepatotoxic compounds leads to DNA and protein damage. Down regulation Fmo1 enzyme leads to hepatotoxicity due to defect in xenobiotic metabolism and its elimination. In addition, down regulation of Maob, affects the catalyzation of oxidative deamination of biogenic and xenobiotic amines leads to hepatotoxicity. The carbohydrate metabolism enzyme Pygl, was down regulated by all liver toxicants indicating a reduction in glycogenolysis and this affects energy metabolism in liver (Zidek et al 2007).They concluded, that it is possible to differentiate hepatotoxic compounds and non hepatotoxicants based on their gene expression proï¬les.
Nephrotoxicity is one of the common adverse effects produced by many therapeutic drugs. cDNA microarrays to measure altered gene expression patterns in the rats kidney after dosing with three nephrotoxicants namely cisplatin, gentamicin, and puromycin were examined in a by Amin et al 2004. They noticed deregulated gene expression pattern for genes involved in "creatinine biosynthesis (L-arginine-glycine amidinotransferase and guanidinoacetate methyltransferase), renal transporters, osmoregulation, kinase signalling, cell cycle-related genes, renal damage, and regeneration after cisplatin treatment. Gentamicin down regulates the kallikerin gene and it leads to phospholipidosis in the kidney due to inactivation of phospholipase A pathway. Puromycin treatment up regulated transporter genes such are serum amyloid p-component and dentin sialophosphoprotein, dihydropyrimidinase, cathepsin B and H, alcohol dehydrogenase, solute carrier family 4, (SLC4A4), macrophage inflammatory protein 1-alpha (Mip-1-alpha), interferon, retinol binding protein and glucose-6-phosphatase. These up regulated genes aid in reuptake of solutes and small molecules
by injured glomerulus and are responsible for solute homeostasis during the renal injury. Besides, there were number of genes down regulated namely acidic nuclear phosphoprotein 32, L-glycine arginine amidinotransferase and cytosolic epoxide hydrolase have been suspected for glomerulonephritis. Dose and time dependent up regulation of KIM-1, osteopontin, clusterin, several ESTs, LCN2 and TNF receptor superfamily, member 12a genes may serve as potential biomarkers for renal injury by drug induced hepatotoxicity (Amin et al 2004).
5. Toxicoproteomics in toxicology and Environmental pollutions
A number of toxicoproteomic analyses have been performed for environmental toxicants including air pollutants, pesticides, chemicals in water systems, and so on. The main objectives of toxicoproteomics are the detection of potential biomarkers or toxicity signatures during pre-clinical safety consideration or risk assessment, early diagnosis and therapeutic intervention to cure the diseases. In addition, it is useful to improve our understanding on molecular mechanisms of toxicity and combing of toxicogenomics databases using bioinformatics, imaging techniques and computation tools for a systems biology approach (George et al 2010).
The liver samples from acetaminophen (APAP) treated mice contained 35 modified proteins. Number of these proteins (N-10 formyl tetrahydrofolate dehydrogenase, proteasome proteins, GST, glutathione peroxidase, sorbitol dehydrogenase andcalreticulin) was well-known target for covalent modification such as arylation and oxidation by fÂ N-acetyl-p-benzoquinoneimine, which is the most toxic metabolite of APAP. Other down regulated proteins (Catalse, thioredoxin peroxidase 1 and 2, superoxide dismutase, and S adenosylmethionine) was involved in oxidative stress responses. These observations are whispered to be APAP induced hepatotoxicity (Fountoulakis et al 2000).
Proteomic analysis of the microsomal fractions isolated from carbon tetrachloride (CCl4)-treated male rat liver showed altered protein expression for 17 CYP450 proteins. Among these, down regulation of CYP2C11, CYP 3A2, and CYP2E1 proteins was observed. Other hand up regulation of CYP2C6, CYP 2B2, and CYP2B1 proteins was noticed. These altered proteins were involved in xenobiotic metabolism, liver toxicity and carcinogenesis (Jia et al 2007). They concluded that proteomic approaches can be useful in toxicological assessment of chemicals. Â
The nephrotoxic effects of gentamicin on protein expression in rat kidneys revealed that more than 20 proteins were significantly altered based on the dose dependency. These proteins are involved in gluconeogenesis and glycolysis (Fructose 1,6-bisphosphatase and Î±-enolase), fatty acid metabolism (Serum albumin, fatty acid transport protein, acetyl-CoA carboxylase and methylacyl-CoA racemase), the citric acid cycle (succinyl CoA synthetases ), and stress responses(Î±-Tubulin, actin, DNA polymerases, serine protease inhibitor 1) (Charlwood et al 2002). They concluded that gentamicin induces nephrotoxicity through impairment of energy metabolism and mitochondrial dysfunction.
Exposure of mouse skin to cypermethrin a synthetic pyrethroid, revealed altered expression for 27 proteins. Up regulation of six proteins such as Hsp-27, S100A6, S100A9, S100A11, carbonic anhydrase 3 and galectin-7 were noticed. In addition down-regulation of one protein namely superoxide dismutase (SOD 1) also noticed. These altered proteins plays a vital role in tumorigenesis through the disturbances of the various biological pathways which are involved in oxidative stress response, cell cycle, apoptosis and binding of calcium ions (George et al 2011).
Proteome profiling pentachlorophenol (organochloride) exposed liver from male and female rare minnow (Gobiocypris rarus) revealed altered proteins and these proteins are involved in transport, metabolism, response to oxidative stress and other cellular pathways. These changes may be responsible for pentachlorophenol induced hepatoxicity and hepatocellular carcinoma in humans (George and Shukla 2011).
Glyphosate, a genotoxic/carcinogenic organophosphate and widely used to control weed in agriculture field. 2DE and MS analysis of proteins from mouse skin cells exposed to glyphosate revealed altered protein profile for SOD 1, calcyclin (S100A6) and calgranulin-B proteins (S100A9). These proteins are involved in tumor promotion and it can be used as biomarkers (George and Shukla 2011).
6. Metabolomics in toxicology
Metabolomic analysis of rat urine sample after exposure to acetaminophen (APAP) showed significant reduction of S- adenosylmethionine (SAMe). SAMe is a precursor for sulfur containing antioxidants glutathione and taurine. These antioxidants are responsible for detoxification of N-acetyl-p-benzoquinone imine (NAPQI), the toxic metabolite of APAP. APAP affects the transulfuration pathway from SAMe to glutathione and taurine. Analysis of reactive metabolites and metabolites of transulfuration pathways are useful in evaluation of drug induced hepatoxicity and serves as a potential biomarkers (Berger et al 2010).
Metabolomics of bio fluids and tissues from rats exposed to an anti-hepatitis B virus compound (Bay41-4109) by using NMR- for based metabolomics revealed that metabolites of fatty acid metabolism and mitochondrial function were altered which might contribute to drug induced hepatotoxicity (Berger et al 2010).
The metabolic profiling of urine from the rodent model after administration Cyclosporin A revealed elevated levels glucose, acetate, trimethylamine, and succinate along with decreased levels of urinary trimethylamine-N-oxide by NMR. Urinary metabolomic analysis by HPLCTOF/ MS showed reduced levels of kynurenic acid, xanthurenic acid, citric acid and riboflavin. These finding are linked with initiation of nephrotoxicity. Metabolomics has the potentiality in the biomarker discovery for drug efficacy and toxicity. In addition, it is useful in identification drug metabolites. But this technology is very young when compared to other omic technologies. It needs lot of quality control measures and protocols to be accepted by scientific community and regulatory agencies for routine metabolomic analysis (Berger et al 2010).
7. Limitations and challenges
In spite of all of these significant developments, this technique is not ideal to address all the issues related to toxicities of various origins. It requires lot of efforts to eliminate invalid figures, unnoticed gene expressions, complexity in data analysis and interpretation, biological variations, and experimental errors of human origin (Hummelen and Sasaki 2010, Jayapal et al 2010). It is imperative to correlate the data originates from transcriptomic, proteomic and metabolomics studies and this provide comparative information to assess the genetic and molecular basis of gene-environment interactions. This type of toxicogenomics analysis should be performed regularly.
Presence of a bio marker may indicate exposure to a certain environmental chemical, but how that marker relates other events in the exposure and disease process is poorly understood. It is necessary to validate the biomarkers for consistency, sensitivity and specificity before accepted by regulatory bodies as a substitute endpoint. In addition there are other issues including such as cost, requirement of skilled personnel, legal and ethical issues while handling human samples need to be addressed (Jayapal et al 2010).
8. Future prospects and Conclusions
Toxicogenomic analysis provides a new path to improve and transform the way by which environmental pollutants and drugs are presently examined. Data obtained from properly designed toxicogenomics studies of exposed human population promises in risk assessment, discovery of novel biomarkers of exposure or early effect for disease diagnosis, off target effects, biological pathways, gene networks, prognosis, disease pathogenesis underlying exposure, gene networks, pathways, drugs screening, classification of toxicant and hazard identification (Jayapal et al 2010, McHale et al 2010). It in turns leads to the development of safer therapeutics to prevent, and treat the disabilities caused by toxicants (Jayapal et al 2010). It is necessary to incorporate precise individual exposure measurements, conventional toxicological markers, different doses and time of exposure in toxicogenomics studies. In addition, the issues like such as large sample size, exposure circumstances, social and legal problems, experimental costs are needs to be tackled based on available funds and prioritization of experiments (McHale et al 2010).
Though the toxicogenomics is rationally challenging field but it is currently developing. Integrated strategic approaches like cell based assays to identify biomarkers at in vitro and subsequent validation and quantification of these biomarkers in suitable in vivo models. Thereafter, proteomics and metabolomics analysis of bio fluids for additional biomarkers discovery for follow-up for human and animal studies for the risk assessment can be performed. Combing of in vitro and in vitro measurement for biomarkers evaluation should complement each other and human studies. The unprecedented growth in bioinformatics and biostatics are helping in data analysis, identification, evaluation and application of biomarkers (Jayapal et al 2010).