Indian Journal of Human Genetics Medknow Publications on behalf of Indian Society of Human Genetics ISSN: 0971-6866 EISSN: 1998-362x Vol. 11, Num. 2, 2005, pp. 61-75

Indian Journal of Human Genetics, Vol. 11, No. 2, May-August, 2005, pp. 61-75

Review Articles

Contribution of genomics, proteomics, and single-nucleotide polymorphism in toxicology research and Indian scenario

Patel S, Parmar D, Gupta YK, Singh MP

Industrial Toxicology Research Centre (ITRC), Lucknow, UP, India
Correspondence Address: Dr. MP Singh, Industrial Toxicology Research Centre (ITRC), Mahatma Gandhi Marg, Post Box 80, Lucknow – 226 001, UP, India, E-mail: singhmahendrapratap@rediffmail.com

Code Number: hg05016

Abstract

Keywords: Microarray; proteomics; single-nucleotide polymorphism (SNP); toxicogenomics.

Toxicogenomics is a combination of functional genomics and molecular toxicology to establish correlations between toxic responses elicited by the toxicants and changes in the genetic profiles of the exposed organism, organs, or tissues. It is rapidly developing and being committed to provide ventures for understanding the molecular responses of chemicals and global assessment of their effects in the biological system using molecular tools such as DNA microarray, high throughput nuclear magnetic resonance (NMR) and protein expression profiling. Toxicogenomics comprises of genomic-scale mRNA expression profiling (microarray), cell-wide or tissue-wide protein profiling (proteomics), genetic susceptibility of an individual or population (single-nucleotide polymorphism) and bioinformatics in order to understand the role of gene-environment interactions. Toxicogenomics predominantly has taken advantage over traditional toxicity testing and biochemical and molecular estimations. Gene expression profiles (GEP) potentially provide basic information about common regulatory mechanisms, biochemical pathways and broader cellular functions and hence a wider spectrum of the mechanism of toxicity.[1] The GEP approach facilitates the investigation of the molecular mode of action of toxins, prediction of the susceptibility of an individual for the toxic response and the role of modifying factors. Information obtained from GEP further improves the therapeutic strategies for the treatment of various diseases[2] such as cancer and neurological disorders caused by toxins. The major focus of toxicogenomics is the characterization of changes in gene expression in the cells or tissues following exposure to toxic substances.[3] Toxicogenomics has many advantages over the conventional techniques as it provides more detailed information regarding the molecular mechanisms of toxicity and faster screens for substance toxicity. Following toxicant exposure, gene expression is modulated in a specific and measurable way[4] and that is the main reason why toxicogenomics has advantage over the conventional toxicity testing procedures [Figure - 1].

Toxicoproteomics is positioned towards an expanded understanding of protein expression during toxicity and environmental disease for the advancement of public health and seeks to identify critical proteins and pathways affected by and respond to adverse chemical and environmental exposures using global protein expression technologies by integrating traditional toxicology, pathology, differential protein and gene expression analysis, and systems biology.[5] Genes or group of genes influence the outcome of any environmental exposure. An analysis of the toxicological properties of a chemical is estimated to cost huge amount of money, therefore, toxicological properties of approximately 70 000 known chemicals, require several years and thousands of dollars to complete. Development of cost effective methodologies has become an essential tool for screening the toxicity of drugs and chemicals[6] that has provided an information on the study of perturbation by chemicals and stressors, monitoring changes in molecular expression and iteratively integrating biological response data to describe the functioning of an organism.[7] Toxicogenomics has provided a tremendous amount of data to support the assessments of human health risk from environmental exposures and correlating them to health endpoints. The national center for toxicogenomics has started developing a public knowledge base called as chemical effects in biological systems (CEBS). The CEBS comprises of molecular expression data sets from transcriptomics, proteomics, metabonomics, and conventional toxicology with metabolic and toxicological pathway and gene regulatory network information relevant to environmental toxicology and human diseases (www.niehs.nih.gov). Council of Scientific and Industrial Research, Government of India has also launched a networked program on ′toxicogenomics of genetic polymorphism in Indian population to industrial chemicals for development of biomarkers′ (www.itrcindia.org) for the construction of databases of the gene expression profiles of exposed and unexposed animals, databases of protein profiles in Indian population, databases of single-nucleotide polymorphism (SNP) in the toxicant-responsive genes and protein fingerprints to identify the type of exposure to delineate the mode of action of the various test pollutants. In this review, the contribution of the tools and techniques used in molecular toxicology and its current status in India has been discussed.

Genomics, proteomics, and SNP

A toxicological change at molecular level is accompanied by linking general toxicity with gene expression profiles to improve the sensitivity, accuracy and speed of toxicological investigations. Technologies of genomics and proteomics are being used at different stages of toxicity testing to assess the alteration in the expression of RNA and protein in mechanistic aspects, broader identification of target tissues, long-term exposure consequences to toxic chemicals and reducing ambiguities particularly when toxicities are seen in some but not in all test species. RNA and protein alternations in nonclinical toxicology provide a greater insight and better prediction of the performance of the products during the clinical phase of development. Toxicogenomics has also become critical for quality control of cell substrates for manufacturing biochemicals to improve the purity, safety and potency of vaccines, blood derivatives, and biological therapeutics. Toxicogenomic tools are used for the determination of potential toxicity of hundred of thousands of toxic chemicals, detection of the effect of such chemicals at lower doses, study of various interactions between the harmful chemicals, prediction of the toxic effects of drugs, relative contribution of environmental and natural courses of diseases, and development of biomarkers for a toxicant exposure. Toxicogenomics-based technologies identify critical regions of inherited somatic cell DNA sequence in patient-tailored therapy and patients at risk for developing life-threatening reactions. The functional toxicogenomics explains influences and effects measured at the level of the cellular genome and results obtained from histopathological and biochemical tests. Due to recent developments in molecular biology and bioinformatics, it has become possible to analyze protein transcripts (toxicogenomics) and profiles (toxicoproteomics).[8] Environmental and occupational toxic agents that influence cells at the level of transcription and translation have provided new opportunities to study gene sequencing and expression by DNA chip technique. DNA sequencing, transcription profiling and proteomics dealing with DNA, RNA, and proteins provided extensive mechanistic and predictive information about the toxicity of a particular compound. Although these techniques provide important information like identification of genes, the regulatory elements responsible for the precise environmental and developmental control of gene expression, snapshot of genes being expressed in a particular tissue at a given time, however, these do not always correlate directly with the level or activity of the corresponding protein at a single point of time.[9] Toxicoproteomics is the study of proteins to provide a comprehensive overview of the structure, function and post-translational modifications with special references to toxicology. Recent developments in the molecular tools have fueled an expansion of inventions from simple toxicological estimations of single proteins to measure set of proteins. Proteomics coupled with advances in bioinformatics has become an essential component of molecular biology. The SNPs-based studies are also essential component in understanding the actual expression or activity of a protein involved in the toxicity onset or progression.

Microarray

The advent of microarray has allowed toxicologists to analyze the differential expression of thousand and thousands of genes in a single experiment quickly and efficiently that were not possible with the conventional methods used in toxicology since conventional methods could assess the gene expression only in a relatively small number of toxicant-responsive genes at a given time. Microarrays are small, solid supports made up of glass microscope slides onto which the sequences from thousands of different genes are attached at fixed locations by spotting or synthesizing directly. The spots attached to the support in a fixed way in order to use the location of each spot in the array to identify a particular gene sequence. The kind of immobilized DNA used to generate the array and the kind of information that is derived from the chips, are the decisive factors. The target DNA may also determine the type of control and sample DNA to be used in the hybridization solution. A microarray works by exploiting the ability of a given mRNA molecule to bind specifically or hybridize to the DNA template from which it originates.

Researchers have identified a large number of novel toxicant-responsive genes within previously unknown sequences with the help of information available with the microarray. Microarray used for gene expression profiling represents a snapshot of the transcriptional responses at a given time in a particular tissue or organ. Microarray technology is used to understand fundamental aspects of toxicity as well as to explore the underlying genetic causes of toxicant-induced diseases and disorders.[10] Microarray is also used to characterize genetic diversity, drug responses, identify new drug targets, and assess the toxicological properties of chemicals and pharmaceuticals. The patterns and sequences of gene expressions constitute response ′signatures′ unique to specific toxicants or group of toxicants. Microarray has greatly facilitated the development of ′toxchip′ containing most of the human genes involved in toxicity responses such as DNA damage, replication and repair, apoptosis, and other cellular responses specific to polycyclic aromatic hydrocarbons (PAHs), dioxins, peroxisomes proliferators, estrogenic compounds, and oxidants.[11] Gene expression profile is used to monitor environmental exposure to chemicals in humans.[11] The mechanism of toxicants exposed to individuals can be clearly understood once the signatures are identified based on the patterns of altered gene expression.

Proteomics

SNP

More than 99% of human DNA sequences are the same across the population, however, variations in DNA sequence can have a major impact on how humans respond to environmental insults such as toxins. The SNPs or snips are DNA sequence variations that occur when a single nucleotide in the genome sequence is altered. A point mutation is considered as SNP if it occurs in at least in 1% of the population. The SNPs are responsible for about 90% of all human genetic variation and occur every 100-300 bases along the 3-billion-bases of human genome both in coding and noncoding regions. Two-third of known SNPs involve the replacement of cytosine with thymine. The SNPs that have no effect on cell function could predispose people to disease or influence their response to a toxicant.

Sequence of human genome has provided a great strength to toxicology by focusing on etiology and mechanism of toxicant-induced diseases. Scientists believe that SNP maps will help in identifying the multiple genes associated with complex etiology of toxicant borne diseases. These associations are difficult to establish with conventional methods because a single-altered gene make only a small contribution to the toxicant borne disease. Gene and environment play a critical role in many genetic disorders of multi-factorial origin. Epidemiological studies have shown statistically significant association of several factors among which genetic susceptibility is the common factor as indicated by familial aggregation and greater concordance in monozygotic twins with the onset of the diseases such as obesity, cancer, hypertension, and diabetes.[14] The PCR- and sequencing-based techniques have been used to assess the presence of SNPs in a particular gene. Genome-environment interaction among individuals for the susceptibility to a disease greatly varies and Fosmid library allele-specific haplotypes analysis (FLASH) and large insert genome analysis (LIGA) has improved the understanding of genetic variations in various populations.[15] FLASH allows to isolate and sequence fosmid clones that tile a given loci in a haplospecific manner over along genomic region of interest and LIGAN allows the use of end sequence and fingerprint data to identify the variant genomic regions from multiple individuals.[15] FLASH and LIGAN have overcome the limitations of PCR-based re-sequencing methods to screen thousands of individuals.[12] FLASH does not provide phase information for the underlying haplotypes structure of a diploid genome and LIGAN provides a limited resolution at the chromosome level.[15] Telomeric repeat amplification protocol (TRAP), a sensitive, high throughput PCR-based assay provides a reliable tool for the experiments that requires massive quantitation of telomerase activity, however, apyrase-mediated allele-specific extension reaction (AMASE) is a novel tool used for microarray-based mutation detection and allows the simultaneous, efficient, and accurate analysis of several samples from different stages of skin malignancies.[16]

′Human genome project SNP mapping goals′ established in 1998 by NIH, USA to develop technologies for rapid, large-scale identification, and scoring of SNPs and other DNA sequence variants, to identify common variants in the coding regions of most identified genes, create a SNP map of at least 100 000 markers, develop the intellectual foundations for studies of sequence variation and create public resources of DNA samples and cell lines (www.ornl.gov/sci/techresources/Human_Genome/faq/snps.shtml). In 1999, ten large pharmaceutical companies and the UK Wellcome Trust Philanthropy established a consortium to find 300 000 common SNPs with the aim to generate a publicly available SNPs map evenly distributed in the human genome (www.ornl.gov/sci/techresources/Human_Genome/faq/snps.shtml). In addition to toxicogenomics, CSIR, India has also launched a network program on SNPs with the aim to generate databases on the SNPs in many genes in Indian population.

International status

The National Institute of Environmental Health Sciences (NIEHS) in Research Triangle Park, NC has launched the Environmental Genome Project (EGP.http//www.niehs.nih.gov/envgenom/) with the aim to systematically identify and characterize common sequence polymorphisms in many genes and their contribution in determining chemical sensitivity. The knowledge gained is exploited to protect susceptible individuals from disease and subsequently to reduce adverse exposure and environmentally induced disease.[4],[5],[6],[7] The collection of genetic variations serves as a useful resource for analyzing potential association between genotypes and susceptibility to common diseases as well as efficacy and/or adverse reactions to drugs. The utilization of human cancer arrays to profile aberrant gene expression in arsenic-exposed populations in Guizhou, China have provided better understanding of the integral role of arsenic in hepatotoxicity and possibly carcinogenesis.[17] It is also reported that about 60 genes (10%) involved in cell-cycle regulation, apoptosis, DNA damage response and intermediate filaments are differentially expressed in arsenic-exposed human livers as compared with controls.[18] Over expression of endothelins and vascular endothelial growth factor B (VEGF-B) in arsenic-transformed rat liver epithelial cells has also suggested a correlation between arsenic-induced portal hypertension and cirrhosis.[18] The influence of gene-environment interaction in prostate cancer studied in Japanese (NJ) and Japanese-American (JA) men using tissue microarray have shown a historically similar, tissue biomarker expression, especially of lipoxygenase, and the caspase family (e.g., caspase-3) but different mechanism of carcinogenesis.[19] A number of clinically important substrate drugs involve CYP2C9, no 2C9 2(Arg144Cys), or 2C9 3(Ile359Leu) alleles have been reported in Chinese, however, or 2C9 3(Ile359Leu) alleles were found to occur in Canadian native Indian.[20] Expression profiling of such polymorphic enzyme could provide an explanation for variable susceptibility of individuals to different drugs or toxins and providing a better prospective for future drugs therapies.[20] The distribution of SNP in the retinoblastoma susceptibility locus (RBI) at 13q14 is being studied in normal Southeast Asian populations (Chinese, Malay, Javanese, Thai, Filipino), South Asian populations (Bangladeshi, Pakistani Pushtun, and Indian) in cases and control subjects and initial findings have suggested that RBI SNP appears to be an ethnic variant in South Asian populations and hence may be useful for studying RBI inheritance by pedigree analysis.[21]

Microarray technology is being applied in cancer research[22] due to its ability to assess the simultaneous expression of many genes. Gene expression profiles of 85 human cancer xenografts with their sensitivities to various drugs have been done and it is reported that 1578 genes were related to chemo-sensitivity; 333 of these genes were associated with two or more drugs and 32 genes were linked with sensitivity to six or seven drugs.[22] The potential involvement of certain signaling and biochemical pathways in drug resistance states of tumor cells could be obtained via ontology-based arrangement of differential gene expression.[22] The application of DNA microarray in the alteration of gene expression has been reported in rats administered with hepatotoxicants such as phenobarbital and carbon tetrachloride.[23] Microarray analysis of T-2 toxin-induced rat fetal brain lesions have also shown that the expression of oxidative stress-related genes, heat shock protein 70 (HSP70), and heme oxygenase (HO) is strongly induced at 12 h after the treatment.[24] In addition, the expression of mitogen-activated protein kinase (MAPK)-related genes (MEKK1 and c-jun) and apoptosis-related genes (caspase-2), and insulin-like growth factor binding protein-3 (IGF-BP3) were also induced by T-toxin treatment.[25] Calera′s amplified Fragment Length Polymorphism, Millennium Pharmaceutical′s Rapid analysis of Differential Expression, Gene Logic′s Restriction Expression Sequences are being used to identify unique transcripts of interest.[9] One method, the open method does not rely on previous knowledge of the sequence of the gene of interest, whereas the another method that is closed systems of transcription profiling rely on the defined elements whose function is well understood and for which only expressed sequence tag (EST) is known.[9]

cDNA microarray has revolutionized the toxicological studies to a great extent by providing correct assumptions of hazardous chemicals at molecular level.[26] Specific cDNA microarray chips designed for studying toxicant action has made possible to study its action in various organisms and allow simultaneous monitoring of gene expression changes for receptor-mediated responses, xenobiotic metabolizing enzymes,[27] cell cycle components, oncogenes, tumor suppressor genes, DNA repair genes, estrogen-responsive genes, oxidative stress genes,[27] and genes involved in apoptosis. The expression pattern of 12 000 genes in 3-month-old mice using microarray expression analysis has shown significant changes in the expression of 146 genes associated with UCH-1-dependent metabolic pathways in Parkinson′s disease.[27] Subtractive cDNA libraries and microarray analysis for the identification of gene expression profile has provided a way for better understanding of the molecular basis of hypoxia-tolerance by showing how engineered cells survive in the hypoxic environment of the brain parenchyma following transplantation and indicated that the hypoxia-tolerant phenotype is mediated by the active participation of more than hundred genes.[28] Application of DNA microarray to study alcohol-induced liver injury has given important information on the mechanism of action of alcohol in the liver and provided novel approaches to study liver disease.[29]

An accurate classification of compounds according to their toxicity mechanism is possible by the combination of well-designed database with appropriate bioinformatics tools.[30] Classification of toxic and non-toxic compounds on the basis of gene expression profiles in a study on male rats treated with various compounds has been done, wherein hepatotoxic and non-hepatotoxic compounds were discriminated on the basis of gene expression profiles.[30] Microarray-based studies have reported the hierarchies of signaling and gene regulatory networks and its disruption leading to birth defects in response to chemicals like antiepileptic drug valproic acid (VPA), a potent inducer of neural tube defects (NTD), and role of histone deacetylase (HDAC) in embryonic development.[31] Global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo [32] and in cultured P19 embryo carcinoma cells in vitro , have shown that among the VPA-responsive genes, vinculin, metallothioneins 1 and 2, keratin 1-18, etc., are related to NTD or VPA defects, whereas transgelin 2, galectin-1, fatty acid synthase etc., are found to be associated with processes relevant to neural tube formation and closure.[31] Microarray technology in developmental toxicity has been used to understand the mechanistic pathways associated with exposure to pharmaceuticals and environmental chemicals during pregnancy, a burning problem worldwide.[32] Gene expression patterns of chronic arsenic exposed skin, lung, urinary bladder, liver tumors in mice, and subchronic inorganic or organic arsenic exposed v-Ha- ras transgenic mice have shown alterations in toxic manifestations, hepatic arsenic accumulation, and global DNA hypomethylation.[33] The uses of microarray and gel-based expression technologies have shown the effect of phenobarbitol on more than 300 genes in rodent hepatocytes.[34]

Serum proteomic pattern diagnostics is a new type of proteomic platform in which patterns of proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier and has shown tremendous promise in early detection of cancers, detection of drug-induced toxicity.[2] Analysis of serum from rat models of anthracycline and anthracenedione induced cardiotoxicity has indicated the potential clinical utility of diagnostic proteomic patterns where low molecular weight peptides and protein fragments have higher accuracy than traditional biomarkers of cardiotoxicity.[35] A novel and relatively simple analytical method for the separation, characterization, and semi-quantitation of phospholipids (PLs) from extracts of complex biological samples has been developed that has allowed PL extracts from cells and tissues to be analyzed by liquid chromatography (LC) coupled to electrospray ionization mass spectrometry (ESI-MS).[36] Selection of a potential metabolic marker of phospholipidosis (PLD) has been identified as a lyso-bis-phosphatidic acid (LBPA) derivative, also known as bis(monoglycero) phosphate (BMP), in quantifiable amounts in the samples from amiodarone-treated rats.[36]

Interaction between genetic and environmental factors play a role in many cancers and the genetic make up of an individual affects the vulnerability of an individual to cancer, whereas SNPs contribute to functional variations. The SNPs occur at a frequency of approximately 1 SNP/kb throughout the genome. The frequency range varies from one in 1000 bases to 1 in 100 bases.[37] The SNP typing is a powerful tool for genetic analysis enabling to uncover the association of loci at specific sites in the genome with many disease traits. The establishment of a SNP map of the genome has been possible to detect and map SNPs in the human genome.[38] A SNP, C419A in glyoxalase 1 as autism susceptibility factor has been identified.[39] Acetaminophen (APAP), is hepatotoxic in high doses and a reactive metabolite of APAP reacts with cellular macromolecules causing mitochondrial dysfunction and centrilobular necrosis in the liver that leads up-regulation of stress-related genes such as heme-oxygenase and metallothionein indicating a disruption of energy metabolism and normal metabolic processes.[40] Liver insufficiency is a major cause of death that results from exposures to environmental toxins, specific combinations or doses of pharmaceuticals, and microbial metabolites.[41] It is reported that low nanomolar levels of dexamethasone and diluted concentrations of extra cellular matrix overlay are critical for maintaining hepatocyte differentiation, normal hepatocyte physiology, promoting expression of hepatic nuclear factors such as c/EBPa, c/EBPb, c/EBPg, HNF 1-a, HNF b, etc., expression of liver prototypical markers such as albumin and transferrin.[41] Proteomic and genomic profiling in the acute and chronic brain injuries due to methylazoxymethanol (MAM), a neurotoxicant implicated in Western Pacific ALS, Parkinsonism, Alzheimer-like dementia (ALS/PDC) has been studied.[42] Generation of basic data on sequence polymorphism and its putative influence on gene expression are being extensively used to detect the frequency of (TG/CA)n repeats in human genome. The frequency of (TG/CA)n repeats is known to decrease with the length and longer (TG/CA) is found to be associated with lower expression in a fewer individual genes (IFN-g, EFGR, HSD11B2).[43],[44] The enormous variability in the sequence of cytochrome P450 gene (CYP) in human liver is the major cause for unpredictable drug response and expression of hepatic gene CYP3A4 in women is reported twice than in men.[45] Pharmacokinetic findings of faster metabolism of CYP3A4 drug substrates in women, further illustrated the contribution of various factors in the expression of CYP in human liver.[45] The SNP-based studies have revealed the correlation of ER-alpha polymorphisms with various aspects and emphasized that ER-alpha genotype represent a surrogate marker for predicting breast cancer lymph node studies.[39] Age-incidence patterns of suspected toxicant-induced breast cancer in Asian differ from those in Caucasians.[39] The SNPs found in Spanish and Chueta familial Mediterranean patients with fever and case controls have shown a common feature in all Mediterranean polpulations.[46] Six novel UDP-glucuronosyl transferase (UGT1A3) polymorphisms are capable of affecting the steady state levels of estrogens, and may increase sensitivity to adverse drug affects, as studied in Japanese population.[47] The genetic variation in TNF-SF5 was not found to affect tuberculosis susceptibility in Western Africa[48] and a few cases of identical short tandem repeat (STR) haplotypes and SNPs have been detected in Germans, Chinese, and Thais.[49] The SNPs of the NOD2/CARD15 have also shown to be associated with the development of Crohn′s disease in Caucasians but not in Chinese patients.[50] Polymorphism in secreted protein acidic and rich in cysteine (SPARC) in systemic sclerosis (SSc) has been investigated in skin fibroblast.[51] About 0.05% of the Western population is affected with systemic lupus erythematosus and a number of susceptibility loci for SLE have been investigated in an intronic enhancer region.[52] The population differences and marker location within the gene is an important factor in selection of SNPs for use in the study of toxicant-induced complex diseases with linkage or association mapping methods.[53] The microarray-based genotyping has become an alternative for parallel and simultaneous analysis of many SNPs in Finno-Ugric populations.[54]

Individuals with polymorphisms in xenobiotic metabolizing enzymes like cytochrome P450s (CYPs), glutathione S-transferase (GSTs), or N-acetyl transferases (NATs) and DNA repair greatly altered susceptibility to various environmentally induced diseases such as cancer, asthma, and central nervous system diseases.[55] It is also reported that DNase1 prevents systemic lupus erythematosus (SLE) by removing DNA from nuclear antigens at sites of high cell turnover.[26] Reports have suggested that the mutation associated with DNase1 is not present in Caucasian, African and Asian on the basis of linkage disequilibria among SNPs, their frequency, location, and haplotype tagging status.[26] Tumor necrosis factor (TNF) and its receptor (TNF-TNFR) super family are known to be involved in the onset of various toxicant-induced diseases and SNPs in these genes have shown different patterns of allele frequency among various populations and thereby indicating varying susceptibility and progression.[56] Schizophrenia, a neurological disease is linked with activation of the immune system and an association between ethnic heterogeneity in 308G/A polymorphisms in TNF gene has been reported in Caucasian, however, no significant association was found in the cases and controls in Asian population.[57] About 2% of the population is affected with psoriasis (OMIM 177900), a chronic inflammatory skin disorder of unknown pathogenesis and SNP lying between SLC9A3R1 and NAT9 leads to its defective regulation by RUNX1, a susceptibility factor for psoriasis.[58] About 20 novel SNPs in the guanine nucleotide binding protein alpha 12-gene locus have been identified in Japanese population.[59] The SNP of growth hormone receptor (GHR) are unevenly distributed in various ethnic groups as evidenced by a study conducted in Chinese Hans ethnic population.[60] Signals of differential demographic history in African-American, East-Asian and European-American populations have been reported by the mathematical formulation for the SNP allele frequency spectrum.[61] The impact of SNP density on fine-scale patterns of linkage disequilibrium (LD) has reflected in a high-resolution study in African-American, Asian, and Caucasian populations.[62] The SNPs are not absolute indicators of disease development as anyone who has inherited two E4 alleles never develops Alzheimer′s disease, while another who has inherited two E2 alleles develops Alzheimer′s disease. Alzheimer′s disease caused by variations in several genes and the polygenic nature of the disorders make genetic testing so complicated. The genomics, proteomics, and SNPs have now become essential components of toxicology research even for the identification of newer and less toxic drugs/biomolecules worldwide.

Indian senario

Multidisciplinary approaches of microarray, SNP, and proteomics to understand gene-environment interaction in response to toxins at the global level have become routine practice in toxicology, even in India. After a grand beginning of genomics, proteomics, and SNPs researches worldwide, these areas are expected to become most popular areas of research in toxicology and pharmacology in India in near future. Many studies based on these tools have also provided evidence that how early changes as a result of toxic outcomes serve early and sensitive indicators of potential toxicity. Keeping in view, the importance of toxicogenomics in molecular toxicity, differential genome and proteome-wide analysis and genetic susceptibility assessment in Indian populations, Council of Scientific and Industrial Research (CSIR), Government of India, has launched a networked program on ′toxicogenomics of genetic polymorphism in Indian population to industrial chemicals for development of biomarkers.′ Industrial Toxicology Research Center (ITRC), Lucknow is the coordinating laboratory and Center for Cellular and Molecular Biology (CCMB), Hyderabad, Institute of Genomics and Integrated Biology (IGIB), Delhi, National Environmental Engineering Research Institute (NEERI), Nagpur, and Indian Institute of Chemical Biology (IICB), Kolkata are the participating laboratories in this program. It has two major objectives, one, how the chemical or mixture of chemicals acts for toxic response and most particularly identification of individuals genetically predisposed towards increased toxic stress and second, how genome interacts with toxicants. Differential display of proteins in human blood exposed to lead, mercury, environmental estrogens and cytotroxic agents, and biomass fuels are being initiated at ITRC. Studies related to manganese- and PAH-exposed individuals are being initiated at NEERI, however, arsenic-exposed samples are being analyzed at ITRC and scientists at IICB, Kolkata are involved in the collection of blood samples for this study. Transcription profiling in experimental animals exposed to lead, mercury, environmental estrogens, and carcinogens are being initiated at ITRC and studies involving pesticides are at the final stage. NEERI is working on manganese and PAH exposed animal experimentation and IICB has initiated studies related to arsenic-exposed animals and human skin biopsies. The SNP in toxicant-responsive genes such as cytochrome P-450 1A1 (CYP1A1), CYP 2E1, glutathione - s-tranferases (GSTs), epoxide hydrolase, p53 and cyclins are extensively being done at ITRC, IICB, and IGIB. Scientists working in this program are trying to develop a database on the gene and protein expression profiles of exposed and unexposed animals and human beings respectively for different classes of chemicals that include petroleum products, chlorinated pesticides, biomass fuels, lead, mercury, arsenic, and several other heavy metals. The program is being used to identify genetically predisposed human population in India who are at the higher risk for toxicant-induced diseases. It has been observed that certain individuals show differential response to a given toxicant and studies related to such observations are being extensively carried out in India. The goal of SNP part of this program is to identify the sequence polymorphism that could make some human populations either more or less sensitive to toxicant exposure and once the adequate database of polymorphism for various toxicant-responsive genes is developed, screening of individuals in Indian population can be initiated for evaluation of their susceptibility to different chemicals.

Application of DNA microarray to study pesticide-induced Parkinson′s disease has given important information on the mechanism of the onset of PD and provided novel approaches to study chemically induced neurological disorders by our group at ITRC (unpublished data). Microarray-based research in toxicology is only recently been realized in India and now it has been widely accepted by the toxicologists. Such studies will be helpful in developing the databases that may be used for delineation of molecular mechanism and extrapolation of data for predicting the toxicity of newer chemicals. Differential gene expression profiling and proteome analysis of many types of cells and tissues has already been extensively done in various parts of India. Serum proteomic has become a diagnostic tool where proteomic signatures from high dimensional mass spectrometry data are used as a diagnostic classifier and this approach has shown tremendous promise in early detection of environmental exposure and chemical-induced toxicity. Two dimensional polyacrylamide gel electrophoresis and mass spectrometry facilities have been extensively used in various laboratories in many parts of the country sponsored by CSIR and other national and international government and private funding agencies. Serum proteome profile in toxicant exposed humans and animals are being conducted in various CSIR laboratories. We are doing serum proteome profiling at ITRC in respective case controls and arsenic, pesticides and PAHs-exposed animals or humans for the identification of biomarkers of toxicant exposure (unpublished data). Similarly, tissue proteome profiling is also being initiated at ITRC to understand the mechanism of the onset of chemically induced Parkinson′s disease in animals that may be extrapolated for humans to understand the mechanistic aspect of the disease (unpublished data). Many other investigators in India are also involved in conducting proteomics-based researches in toxicology [Figure - 3]. The relationship between the functional proteomics of receptor-Ck and developmental stages of human atherosclerotic aortic wall to study gene expression profiles of 25 aortas have revealed that cholesterol-specific receptor-Ck-dependent gene regulation may be of crucial importance in atherogenesis.[63] The proteome analysis have indicated that GnRH in the olfactory system of Cirrhina mrigala contribute a major role in translation of the environmental clues and influence the downstream signals leading to the stimulation of the brain-pituitary-ovary axis.[64]

Toxicogenomics has become an unprecedented power of technology and is greatly facilitating the underlying genetic factors of relevance that are encoded in the spectrum of genetic variations. Indian population is rich in diverse genetic pool and provides a good model for SNP-based studies. Our group at ITRC is investigating sNPs in various toxicant-responsive genes and their association with various neurological diseases. Combination of genome sequence analysis and population polymorphism scanning in Indian population for cerebella ataxia and Schizophrenia has given a clue for origin and mechanism of tri-nucleotide repeat instability, identification of predisposed alleles, possible involvement of Alu elements in diseases and the utility of defining gene structure and LD blocks for identifying susceptible individuals.[65] Genetic profile based on polymorphisms is of great significance as it varies greatly amongst different populations of the same ethnic group. The oxidative stress has been shown to play an important role in various neurological disorders and pulmonary disorders and the involvement of eNOS gene 786 T/C and 4B/4A polymorphisms have been shown by a study in chronic obstructive pulmonary disease (COPD). A significant over-representation of mutant alleles 786C and 4A was observed in the patients as compared to control presumed that 786T/C and 4B/4A conversions in the eNOS gene predispose COPD subjects to the overload of oxidative stress, at least to the reactive nitrogen species.[66] Angiotensin converting enzyme (ACE) and endothelial nitric oxide synthase (eNOS) genes are of particular importance in COPD and the genetic predisposition to lower ACE activity and higher NO level by virtue of the inter-chromosomal cross talk between ACE I and eNOS G alleles involves a mechanism that may avert the pathophysiological severity of COPD.[67] Genetic polymorphisms of GST M1 and GST T1 on susceptibility to cervical cancer in Indian population have revealed a significant difference between the cases and controls in the distribution of the null genotype of GST M1 in individuals of above 45 years; however, no such difference exists for GST T1 null genotype.[44] Imbalance between pro- and anti-inflammatory cytokines during inflammation plays a role in pathogenesis of inflammatory bowel disease (IBD) and polymorphism in intron 2 of IL-1RA gene is involved in pathogenesis of IBD in North Indian population has been reported.[68] Alternation in polymorphic frequency of detoxifying genes GSTT1, GSTM1, and GSTP1 have suggested polymorphism in these genes as a potential risk factor for development of bladder cancer. In patients with bladder cancer of North India, frequencies of null genotypes in GSTT1, GSTM1, and GSTP1 were found to be higher in patients as compared with controls.[68],[69] A detailed RFLP study on 86 patients with cervical cancer and 76 healthy females revealed that CYP2D6 polymorphic genotype is significantly higher in smokers as compared to nonsmokers in cervical cancer.[70] The brain-specific cytochrome P450 2D8 in 50% of human autopsy brains explained the variable analgesic responses to codeine due to genetic polymorphism.[71]

Genomics can be used to identify the populations of various ethnic groups that may have different response for toxicity for the same chemical entity. The SNP-based Y-haplotypes are able to distinguish South Indian populations based on Y-specific short repeat tandem markers[72] and such study may be used for the identification of various ethnic groups in Indian population. The SNPs are also important in understanding the mechanism of the pathogenesis of various cardiac diseases and its population-specific prevalence. A possible role of MTHFR A1298C SNP in the pathogenesis of heart diseases has also been found in Tamilians.[73] The SNP in combination with STRs, haplotype based on Y-chromosome-specific short tandem (STR) loci across caste-tribe boundaries in South India has been done and 27 SNPs in the non-recombining region of the Y chromosome has been investigated in 204 individuals belonging to three caste groups (Vizag Brahmins, Peruru Brahmins, Kammas), three tribes (Bagata, Poroja, Valmiki), and an additional group (the Siddis) of African ancestry.[72] The mutation frequency of the MYOC gene is 2% in the Indian population affected with primary open angle glaucoma (POAG), a less studied ethnic group of the Asian continent and a nonsense mutation Gln 368 stop was not found in Indian population as in Japanese population, however, reports exist in Western population.[74] Phase II detoxification genes play a crucial role in the identification of individualistic response for the detoxification of chemicals responsible for the onset or progression of various disorders. Oral cancer is one of the most common cancers in India and phase II enzymes such as glutathione S-transferase M1 and T1 null genotypes (GSTM1 and GSTT1) are considered as the major risk factors. The metabolically generated epoxide intermediates of PAHs are known substrates for GSTM1 and alkyl halides found in cigarette smoke and lipid peroxides.[75] Transcription of the insulin gene involved in putative type 2 diabetes mellitus (T2DM), is regulated by LIM-homeodomain containing protein LMXIA present on 1q21-q23 in Pima Indians and variant screening in Pima Indians has identified seven SNPs throughout the LMXIA locus, however, no evidence for association of any LMXIA SNPs with T2DM was found giving the idea that LMXIA does not contribute significantly to T2DM etiology in Pima Indians.[76]

In India, use of genomics and proteomics are only just being realized and there is a long and arduous way to achieve success in this direction. Transcription profiling and proteome analysis in India are at the beginning stage, however SNPs-based studies have become indispensable part of research in each and every Indian laboratories involved in biological sciences. Toxicogenomics permits global gene expression analysis at a time and provides an opportunity to identify early and more sensitive biomarkers of a toxicant exposure and has become one of the most important areas of research for a country like India. The possibility of making decisions on the basis of detailed toxicity, mechanistic and exposure data in which many of the uncertainties can be eliminated to improve the quality of human health in the developing countries. Microarray, proteomics, SNPs analysis in combination will definitely provide better opportunities to make appropriate decisions for the improvement of human health in India.

Challenges in toxicogenomics

Toxicogenomics, a tool of unprecedented power in toxicology for understanding and predicting the body′s response to toxic exposures has sorted out most of the disputes and problems in toxicology, however, there are still many obstacles and uncertainties remain to be resolved before the toxicogenomic data can get widespread practical and legal applications beyond the research laboratories. Validation of toxicological significance of gene expression changes is required that includes the robustness and reproducibility of toxicogenomic assays between or across different laboratories, species, individuals, tissues, development stages, exposure levels and exposure durations, all of which in one way or another affect gene expression patterns.[77] This technology gave an opportunity to define at unprecedented levels of detail, the molecular events that precede and accompany toxicity, opening the path for better understanding of toxic mechanisms.[27] The major challenge for toxicogenomics in predictive toxicology is the discrimination of changes due to interindividual variation and experimental background noise in transcription profiling, however identification of a toxic fingerprint is possible for various compounds if compounds result in same toxicological end point. Validation of microarray data is a very complicated process and available technologies are not up to the mark. The data can be used for the development of new products to assess the safety and efficacy only if microarray technology moves faster than the validation technology. Many 2D tools and databases are available but still it is not capable of detecting the hydrophobic proteins and proteins present in very high or very minute quantities. The probability of detecting low-copy-number proteins are being increased by fractionating proteins into organelle components, each containing less number of components as comparison with the starting material prior to 2D analysis.

Nevertheless, application of diagnostic genes and proteins expression profiling and SNPs identification in a predictive context is still a challenge and needs immediate attention by the toxicologists.

References

Copyright 2005 - Indian Journal of Human Genetics

The following images related to this document are available:

Photo images