A Synopsis of the "Influence of Epigenetics, Genetics, and Immunology" Session Part A at the 35th Annual Society of Toxicologic Pathology Symposium.

The overarching theme of the 2016 Society of Toxicology Pathology's Annual Symposium was "The Basis and Relevance of Variation in Toxicologic Responses." Session 4 focused on genetic variation as a potential source for variability in toxicologic responses within nonclinical toxicity studies and further explored how knowledge of genetic traits might enable targeted prospective and retrospective studies in drug development and human health risk assessment. In this session, the influence of both genetic sequence variation and epigenetic modifications on toxicologic responses and their implications for understanding risk were explored. In this overview, the presentations in this session will be summarized, with a goal of exploring the ramifications of genetic and epigenetic variability within and across species for toxicity studies and disseminating information regarding novel tools to harness this variability to advance understanding of toxicologic responses across populations.

Dynamic changes in 5-hydroxymethylation signatures underpin early and late events in drug exposed liver.

Aberrant DNA methylation is a common feature of neoplastic lesions, and early detection of such changes may provide powerful mechanistic insights and biomarkers for carcinogenesis. Here, we investigate dynamic changes in the mouse liver DNA methylome associated with short (1 day) and prolonged (7, 28 and 91 days) exposure to the rodent liver non-genotoxic carcinogen, phenobarbital (PB). We find that the distribution of 5mC/5hmC is highly consistent between untreated individuals of a similar age; yet, changes during liver maturation in a transcriptionally dependent manner. Following drug treatment, we identify and validate a series of differentially methylated or hydroxymethylated regions: exposure results in staged transcriptional responses with distinct kinetic profiles that strongly correlate with promoter proximal region 5hmC levels. Furthermore, reciprocal changes for both 5mC and 5hmC in response to PB suggest that active demethylation may be taking place at each set of these loci via a 5hmC intermediate. Finally, we identify potential early biomarkers for non-genotoxic carcinogenesis, including several genes aberrantly expressed in liver cancer. Our work suggests that 5hmC profiling can be used as an indicator of cell states during organ maturation and drug-induced responses and provides novel epigenetic signatures for non-genotoxic carcinogen exposure.

Comparative analysis of affinity-based 5-hydroxymethylation enrichment techniques.

The epigenetic modification of 5-hydroxymethylcytosine (5hmC) is receiving great attention due to its potential role in DNA methylation reprogramming and as a cell state identifier. Given this interest, it is important to identify reliable and cost-effective methods for the enrichment of 5hmC marked DNA for downstream analysis. We tested three commonly used affinity-based enrichment techniques; (i) antibody, (ii) chemical capture and (iii) protein affinity enrichment and assessed their ability to accurately and reproducibly report 5hmC profiles in mouse tissues containing high (brain) and lower (liver) levels of 5hmC. The protein-affinity technique is a poor reporter of 5hmC profiles, delivering 5hmC patterns that are incompatible with other methods. Both antibody and chemical capture-based techniques generate highly similar genome-wide patterns for 5hmC, which are independently validated by standard quantitative PCR (qPCR) and glucosyl-sensitive restriction enzyme digestion (gRES-qPCR). Both antibody and chemical capture generated profiles reproducibly link to unique chromatin modification profiles associated with 5hmC. However, there appears to be a slight bias of the antibody to bind to regions of DNA rich in simple repeats. Ultimately, the increased specificity observed with chemical capture-based approaches makes this an attractive method for the analysis of locus-specific or genome-wide patterns of 5hmC.

Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital.

UNLABELLED: Acetaminophen overdose is a common reason for hospital admission and the most frequent cause of hepatotoxicity in the Western world. Early identification would facilitate patient-individualized treatment strategies. We investigated the potential of a panel of novel biomarkers (with enhanced liver expression or linked to the mechanisms of toxicity) to identify patients with acetaminophen-induced acute liver injury (ALI) at first presentation to the hospital when currently used markers are within the normal range. In the first hospital presentation plasma sample from patients (n = 129), we measured microRNA-122 (miR-122; high liver specificity), high mobility group box-1 (HMGB1; marker of necrosis), full-length and caspase-cleaved keratin-18 (K18; markers of necrosis and apoptosis), and glutamate dehydrogenase (GLDH; marker of mitochondrial dysfunction). Receiver operator characteristic curve analysis and positive/negative predictive values were used to compare sensitivity to report liver injury versus alanine transaminase (ALT) and International Normalized Ratio (INR). In all patients, biomarkers at first presentation significantly correlated with peak ALT or INR. In patients presenting with normal ALT or INR, miR-122, HMGB1, and necrosis K18 identified the development of liver injury (n = 15) or not (n = 84) with a high degree of accuracy and significantly outperformed ALT, INR, and plasma acetaminophen concentration for the prediction of subsequent ALI (n = 11) compared with no ALI (n = 52) in patients presenting within 8 hours of overdose. CONCLUSION: Elevations in plasma miR-122, HMGB1, and necrosis K18 identified subsequent ALI development in patients on admission to the hospital, soon after acetaminophen overdose, and in patients with ALTs in the normal range. The application of such a biomarker panel could improve the speed of clinical decision-making, both in the treatment of ALI and the design/execution of patient-individualized treatment strategies.

Mouse liver effects of cyproconazole, a triazole fungicide: role of the constitutive androstane receptor.

Cyproconazole, a triazole fungicide, causes hepatocellular adenomas and carcinomas in CD-1 mice at dose levels of 100 and 200 ppm. The constitutive androstane receptor (CAR) has been shown to play a significant role in the overall mode of action for several nongenotoxic rodent carcinogens such as phenobarbital. The liver effects of dietary cyproconazole or phenobarbital were investigated after 2, 7, or 14 days in male CD-1, C57BL/6J, and C3H/HeNClrBR mice. Cyproconazole produced similar, dose-responsive effects in all three strains of mice, and the response was similar to that of phenobarbital. Subsequently, Car-null and wild-type male mice on a C3H/HeNClrBR background were administered 200 or 450 ppm cyproconazole, or 850 ppm phenobarbital for up to 7 days. In wild-type mice, 200 ppm cyproconazole caused liver hypertrophy, increased liver weight and cell proliferation, single-cell necrosis and fat vacuolation, effects generally similar to those caused by 850 ppm phenobarbital. Plasma cholesterol was decreased by both compounds, but cyproconazole had a greater effect. The higher dose (450 ppm) of cyproconazole caused similar changes, but greater evidence of liver damage was observed, including a large increase in plasma transaminases. Induction of CAR target genes Cyp2b10 and Gadd45beta was observed with both compounds, whereas the cell cycle regulatory gene Mdm2 was unaffected. In Car-null mice, the effects noted with either cyproconazole or phenobarbital were absent or greatly diminished. These experiments demonstrate that short-term liver effects of cyproconazole in mice are CAR-dependent and similar to those of phenobarbital, a known nongenotoxic rodent liver carcinogen.

Defining baseline epigenetic landscapes in the rat liver.

AIM: Characterization of the hepatic epigenome following exposure to chemicals and therapeutic drugs provides novel insights into toxicological and pharmacological mechanisms, however appreciation of genome-wide inter- and intra-strain baseline epigenetic variation, particularly in under-characterized species such as the rat is limited. Material & methods: To enhance the utility of epigenomic endpoints safety assessment, we map both DNA modifications (5-methyl-cytosine and 5-hydroxymethyl-cytosine) and enhancer related chromatin marks (H3K4me1 and H3K27ac) across multiple male and female rat livers for two important outbred laboratory rat strains (Sprague-Dawley and Wistar). Results & conclusion: Integration of DNA modification, enhancer chromatin marks and gene expression profiles reveals clear gender-specific chromatin states at genes which exhibit gender-specific transcription. Taken together this work provides a valuable baseline liver epigenome resource for rat strains that are commonly used in chemical and pharmaceutical safety assessment.

Xenobiotic CAR Activators Induce Dlk1-Dio3 Locus Noncoding RNA Expression in Mouse Liver.

Derisking xenobiotic-induced nongenotoxic carcinogenesis (NGC) represents a significant challenge during the safety assessment of chemicals and therapeutic drugs. The identification of robust mechanism-based NGC biomarkers has the potential to enhance cancer hazard identification. We previously demonstrated Constitutive Androstane Receptor (CAR) and WNT signaling-dependent up-regulation of the pluripotency associated Dlk1-Dio3 imprinted gene cluster noncoding RNAs (ncRNAs) in the liver of mice treated with tumor-promoting doses of phenobarbital (PB). Here, we have compared phenotypic, transcriptional ,and proteomic data from wild-type, CAR/PXR double knock-out and CAR/PXR double humanized mice treated with either PB or chlordane, and show that hepatic Dlk1-Dio3 locus long ncRNAs are upregulated in a CAR/PXR-dependent manner by two structurally distinct CAR activators. We further explored the specificity of Dlk1-Dio3 locus ncRNAs as hepatic NGC biomarkers in mice treated with additional compounds working through distinct NGC modes of action. We propose that up-regulation of Dlk1-Dio3 cluster ncRNAs can serve as an early biomarker for CAR activator-induced nongenotoxic hepatocarcinogenesis and thus may contribute to mechanism-based assessments of carcinogenicity risk for chemicals and novel therapeutics.

Induction of iron homeostasis genes during estrogen-induced uterine growth and differentiation.

We have previously used genome-wide transcript profiling to investigate the relationships between changes in gene expression and physiological alterations during the response of the immature mouse uterus to estrogens. Here we describe the identification of a functionally inter-related group of estrogen-responsive genes associated with iron homeostasis, including the iron-binding protein lactotransferrin, the ferroxidase ceruloplasmin, the iron delivery protein lipocalin 2 and the iron-exporter ferroportin. Quantitative real-time PCR revealed that the expression of these genes increases with time during the uterotrophic response, reaching maximal levels in the post-proliferative phase (between 48 and 72 h). In contrast, the heme biosynthesis genes aminolevulinic acid synthase 1 and 2 were maximally induced by estrogen at 2 and 4 h, respectively, prior to increased cell proliferation. Together, these data reveal that estrogen induces the temporally coordinated expression of iron homeostasis genes in the mouse uterus, and suggest an important role for iron metabolism during sex steroid hormone-induced uterine cell growth and differentiation.

Identification of early molecular pathways affected by paraquat in rat lung.

We have used global gene expression profiling, combined with pathway analysis tools, to identify in rats the molecular events associated with paraquat toxicity in the lung. Early (2, 8 and 18h) gene expression changes induced following intraperitoneal (i.p.) exposure to paraquat were measured in the caudal lobe of lungs using Affymetrix rat genome GeneChips (31,042 probe sets). A single high dose of paraquat dichloride (20mg/kg) was used that has been shown previously to cause in rats extensive lung fibrosis after 10 days. Hierarchical clustering of 543 paraquat-responsive genes (false discovery rate<0.05) revealed that under these conditions of exposure paraquat induces a staged transcriptional response in the rat lung that precedes the appearance of lung damage. We report here that many of the transcriptional responses to paraquat were rapid (being maximal at 2h post-dose), and that the predominant molecular functions and biological processes associated with these genes include membrane transport, oxidative stress, lung development, epithelial cell differentiation and transforming growth factor beta (TGF-beta) signalling. These data provide novel insights into the molecular pathways that lead to toxicity after exposure of the rat lung to paraquat.

Analysis of DNA repair and chromatin assembly in vitro using immobilized damaged DNA substrates.

Significant advances have been made in identifying a complex network of proteins that could play a role in the repair of DNA damage in the context of chromatin. Insights into this process have been obtained by combining damaged DNA substrates with mammalian cell-free systems that contain both DNA repair and chromatin assembly activities. The methods described in this chapter provide a powerful approach for the detection of proteins recruited during the recognition and repair of DNA lesions, including repair proteins and chromatin associated factors. Substrates for the recruitment assay consist of DNA containing damage that is immobilized on magnetic beads. A human cell-free system that supports both DNA repair and chromatin assembly is incubated with the immobilized DNA-damaged substrates, and proteins associated with the DNA are then isolated and subjected to analysis. We present here protocols for preparing bead-linked DNA substrates containing different types of lesions, for the reaction of the damaged DNA with cell-free systems, and for the subsequent analysis of proteins that are recruited to the immobilized damaged DNA substrates.

Assaying for the dual incisions of nucleotide excision repair using DNA with a lesion at a specific site.

Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily specifically measured by detecting the 24-32 nucleotide products of the dual-incision reaction. This chapter describes several methods for detection of repair of a specific lesion in closed-circular DNA. As a model lesion, we use the well-repaired 1,3-intrastrand d(GpTpG)-cisplatin crosslink. Three methods are given for analysis of repair. One is to incorporate a radioactive label internally near the lesion and measure excision by detecting radioactive excised oligomers. Two other methods use DNA that is not internally labeled so that it can be stored and used when convenient. The first method for detection of repair of such unlabeled DNA is to detect excision products with a labeled complementary oligonucleotide by Southern blot hybridization. The second method is to 3'- end-label the excised oligonucleotide directly with radiolabeled dNTP and a DNA polymerase, using a complementary oligonucleotide with a 5'-overhang that serves as a template. This protocol is fast and sensitive, but relies on accurate foreknowledge of the site of 3'-incision for the particular lesion being used.

Derisking Drug-Induced Carcinogenicity for Novel Therapeutics.

Assessing the carcinogenic potential of innovative drugs spanning diverse therapeutic modalities and target biology represents a major challenge during drug development. Novel modalities, such as cell and gene therapies that involve intrinsic genetic modification of the host genome, require distinct approaches for identification of cancer hazard. We emphasize the need for customized weight-of-evidence cancer risk assessments based on mode of action that balance multiple options for preclinical identification of cancer hazard with appropriate labeling of clinical products and risk management plans. We review how advances in molecular carcinogenesis can enhance mechanistic interpretation and preclinical indicators of neoplasia, and recommend that drug targets be systematically assessed for potential association with tumorigenic phenotypes via genetic models and cancer genome resources.

Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens.

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.

Loss of Tet1-Associated 5-Hydroxymethylcytosine Is Concomitant with Aberrant Promoter Hypermethylation in Liver Cancer.

Aberrant hypermethylation of CpG islands (CGI) in human tumors occurs predominantly at repressed genes in the host tissue, but the preceding events driving this phenomenon are poorly understood. In this study, we temporally tracked epigenetic and transcriptomic perturbations that occur in a mouse model of liver carcinogenesis. Hypermethylated CGI events in the model were predicted by enrichment of the DNA modification 5-hydroxymethylcytosine (5hmC) and the histone H3 modification H3K27me3 at silenced promoters in the host tissue. During cancer progression, selected CGIs underwent hypo-hydroxymethylation prior to hypermethylation, while retaining H3K27me3. In livers from mice deficient in Tet1, a tumor suppressor involved in cytosine demethylation, we observed a similar loss of promoter core 5hmC, suggesting that reduced Tet1 activity at CGI may contribute to epigenetic dysregulation during hepatocarcinogenesis. Consistent with this possibility, mouse liver tumors exhibited reduced Tet1 protein levels. Similar to humans, DNA methylation changes at CGI in mice did not appear to be direct drivers of hepatocellular carcinoma progression, rather, dynamic changes in H3K27me3 promoter deposition correlated strongly with tumor-specific activation and repression of transcription. Overall, our results suggest that loss of promoter-associated 5hmC in liver tumors licenses reprograming of DNA methylation at silent CGI during progression. Cancer Res; 76(10); 3097-108. ©2016 AACR.

Gene ontology mapping as an unbiased method for identifying molecular pathways and processes affected by toxicant exposure: application to acute effects caused by the rodent non-genotoxic carcinogen diethylhexylphthalate.

Toxicogenomics has the potential to reveal the molecular pathways and cellular processes that mediate the adverse responses to a toxicant. However, the initial output of a toxicogenomic experiment often consists of large lists of genes whose expression is altered after toxicant exposure. To interpret gene expression changes in the context of underlying biological pathways and processes, new bioinformatics methods must be developed. We have used global gene expression profiling combined with an evaluation of Gene Ontology (GO) and pathway mapping tools as unbiased methods for identifying the molecular pathways and processes affected upon toxicant exposure. We chose to use the acute effects caused by the non-genotoxic carcinogen and peroxisome proliferator (PP) diethylhexylphthalate (DEHP) in the mouse liver as a model system. Consistent with what is known about the mode of action of DEHP, our GO analysis of transcript profiling data revealed a striking overrepresentation of genes associated with the peroxisomal cellular component, together with genes involved in carboxylic acid and lipid metabolism. Furthermore we reveal gene expression changes associated with additional biological functions, including complement activation, hemostasis, the endoplasmic reticulum overload response, and circadian rhythm. Together, these data reveal potential new pathways of PP action and shed new light on the mechanisms by which non-genotoxic carcinogens control hepatocyte hypertrophy and proliferation. We demonstrate that GO mapping can identify, in an unbiased manner, both known and novel DEHP-induced molecular changes in the mouse liver and is therefore a powerful approach for elucidating modes of toxicity based on toxicogenomic data.

Molecular responses to xenoestrogens: mechanistic insights from toxicogenomics.

The xenoestrogen group of endocrine disruptors has the potential to cause reproductive and developmental effects through stimulation or disruption of sex steroid nuclear receptor signalling pathways. A more detailed understanding of the ways in which xenoestrogens interact with biological systems at the molecular level will provide a mechanistic basis for improved safety assessment. The recent sequencing of mammalian genomes has driven the development of toxicogenomic technologies, including microarray based gene expression profiling, which allow the expression levels of thousands of genes to be measured simultaneously. Since the cellular responses to xenoestrogens are predominantly mediated by estrogen receptors, which function as ligand-activated transcription factors to regulate gene expression, the application of toxicogenomics has great potential for providing insights into the molecular mechanisms of xenoestrogen action. A major challenge in applying toxicogenomics to the field of endocrine disruption is the need to define how xenoestrogen-induced changes in gene expression relate to conventional physiological and toxicological endpoints. Gene Ontology Mapping, Pathway Mapping and Phenotypic Anchoring of xenoestrogen-induced gene expression changes to cellular pathways and processes represent key steps in defining these relationships. Mechanistic insights into how xenoestrogens target specific genes and into the functional significance of xenoestrogen-induced alterations in gene expression can be further enhanced by combining transcript profiling with transgenic animal models or cell-based systems in which the estrogen receptor signalling pathways have been modified experimentally. This review illustrates how these toxicogenomic approaches are providing an unprecedented amount of mechanistic information on the molecular responses to xenoestrogens and how they are likely to impact on hazard and risk assessment.

Mapping molecular responses to xenoestrogens through Gene Ontology and pathway analysis of toxicogenomic data.

The recent sequencing of mammalian genomes has driven the development of genomic technologies, including microarray-based gene expression profiling, that allow simultaneous measurement of the expression levels of thousands of genes. Gene expression profiling applied to toxicology (toxicogenomics) has the potential to reveal, holistically, the molecular pathways and cellular processes that mediate the adverse responses to a toxicant. However, the initial output of a toxicogenomics experiment consists of a list of genes whose expression is altered upon toxicant exposure. In order to interpret these data in a biological context, new bioinformatic methods must be developed to place gene expression changes in the context of the underlying pathways and processes affected. One emerging approach is the application of Gene Ontology (GO) mapping and pathway analysis to gene expression profiling data. The utility of this in mechanistic toxicology will be illustrated using examples in which GO mapping of toxicogenomic data has provided novel insights into the molecular mechanisms induced by exposure to xenoestrogens.

Importance of investigating epigenetic alterations for industry and regulators: An appraisal of current efforts by the Health and Environmental Sciences Institute.

Recent technological advances have led to rapid progress in the characterization of epigenetic modifications that control gene expression in a generally heritable way, and are likely involved in defining cellular phenotypes, developmental stages and disease status from one generation to the next. On November 18, 2013, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) held a symposium entitled "Advances in Assessing Adverse Epigenetic Effects of Drugs and Chemicals" in Washington, D.C. The goal of the symposium was to identify gaps in knowledge and highlight promising areas of progress that represent opportunities to utilize epigenomic profiling for risk assessment of drugs and chemicals. Epigenomic profiling has the potential to provide mechanistic information in toxicological safety assessments; this is especially relevant for the evaluation of carcinogenic or teratogenic potential and also for drugs that directly target epigenetic modifiers, like DNA methyltransferases or histone modifying enzymes. Furthermore, it can serve as an endpoint or marker for hazard characterization in chemical safety assessment. The assessment of epigenetic effects may also be approached with new model systems that could directly assess transgenerational effects or potentially sensitive stem cell populations. These would enhance the range of safety assessment tools for evaluating xenobiotics that perturb the epigenome. Here we provide a brief synopsis of the symposium, update findings since that time and then highlight potential directions for future collaborative efforts to incorporate epigenetic profiling into risk assessment.

The need to decide if all estrogens are intrinsically similar.

We used gene expression profiling to investigate whether the molecular effects induced by estrogens of different provenance are intrinsically similar. In this article we show that the physiologic estrogen 17-beta-estradiol, the phytoestrogen genistein, and the synthetic estrogen diethylstilbestrol alter the expression of the same 179 genes in the intact immature mouse uterus under conditions where each chemical has produced an equivalent gravimetric and histologic uterotrophic effect, using the standard 3-day assay protocol. Data are also presented indicating the limitations associated with comparison of gene expression profiles for different chemicals at times before the uterotrophic effects are fully realized. We conclude that the case has yet to be made for regarding synthetic estrogens as presenting a unique human hazard compared with phytoestrogens and physiologic estrogens. Key words: diethylstilbestrol, estrogen, gene expression, genistein, microarray, phytoestrogen, toxicogenomics, uterus.

Phenotypic anchoring of gene expression changes during estrogen-induced uterine growth.

A major challenge in the emerging field of toxicogenomics is to define the relationships between chemically induced changes in gene expression and alterations in conventional toxicologic parameters such as clinical chemistry and histopathology. We have explored these relationships in detail using the rodent uterotrophic assay as a model system. Gene expression levels, uterine weights, and histologic parameters were analyzed 1, 2, 4, 8, 24, 48, and 72 hr after exposure to the reference physiologic estrogen 17 beta-estradiol (E2). A multistep analysis method, involving unsupervised hierarchical clustering followed by supervised gene ontology-driven clustering, was used to define the transcriptional program associated with E2-induced uterine growth and to identify groups of genes that may drive specific histologic changes in the uterus. This revealed that uterine growth and maturation are preceded and accompanied by a complex, multistage molecular program. The program begins with the induction of genes involved in transcriptional regulation and signal transduction and is followed, sequentially, by the regulation of genes involved in protein biosynthesis, cell proliferation, and epithelial cell differentiation. Furthermore, we have identified genes with common molecular functions that may drive fluid uptake, coordinated cell division, and remodeling of luminal epithelial cells. These data define the mechanism by which an estrogen induces organ growth and tissue maturation, and demonstrate that comparison of temporal changes in gene expression and conventional toxicology end points can facilitate the phenotypic anchoring of toxicogenomic data.