Per- and polyfluoroalkyl substances (PFAS) in mixtures show additive effects on transcriptomic points of departure in human liver spheroids.

Per- and polyfluoroalkyl substances (PFAS) are a wide range of chemicals that are used in a variety of consumer and industrial products leading to direct human exposure. Many PFAS are chemically nonreactive and persistent in the environment, resulting in additional exposure from water, soil, and dietary intake. While some PFAS have documented negative health effects, data on simultaneous exposures to multiple PFAS (PFAS mixtures) are inadequate for making informed decisions for risk assessment. The current study leverages data from previous work in our group using Templated Oligo-Sequencing (TempO-Seq) for high-throughput transcriptomic analysis of PFAS-exposed primary human liver cell spheroids; herein, we determine the transcriptomic potency of PFAS in mixtures. Gene expression data from single PFAS and mixture exposures of liver cell spheroids were subject to benchmark concentration (BMC) analysis. We used the 25th lowest gene BMC as the point of departure to compare the potencies of single PFAS to PFAS mixtures of varying complexity and composition. Specifically, the empirical potency of 8 PFAS mixtures were compared to predicted mixture potencies calculated using the principal of concentration addition (ie, dose addition) in which mixture component potencies are summed by proportion to predict mixture potency. In this study, for most mixtures, empirical mixture potencies were comparable to potencies calculated through concentration addition. This work supports that the effects of PFAS mixtures on gene expression largely follow the concentration addition predicted response and suggests that effects of these individual PFAS in mixtures are not strongly synergistic or antagonistic.

Potency Ranking of Per- and Polyfluoroalkyl Substances Using High-Throughput Transcriptomic Analysis of Human Liver Spheroids.

Per- and polyfluoroalkyl substances (PFAS) are some of the most prominent organic contaminants in human blood. Although the toxicological implications of human exposure to perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are well established, data on lesser-understood PFAS are limited. New approach methodologies (NAMs) that apply bioinformatic tools to high-throughput data are being increasingly considered to inform risk assessment for data-poor chemicals. The aim of this study was to compare the potencies (ie, benchmark concentrations: BMCs) of PFAS in primary human liver microtissues (3D spheroids) using high-throughput transcriptional profiling. Gene expression changes were measured using TempO-seq, a templated, multiplexed RNA-sequencing platform. Spheroids were exposed for 1 or 10 days to increasing concentrations of 23 PFAS in 3 subgroups: carboxylates (PFCAs), sulfonates (PFSAs), and fluorotelomers and sulfonamides. PFCAs and PFSAs exhibited trends toward increased transcriptional potency with carbon chain-length. Specifically, longer-chain compounds (7-10 carbons) were more likely to induce changes in gene expression and have lower transcriptional BMCs. The combined high-throughput transcriptomic and bioinformatic analyses support the capability of NAMs to efficiently assess the effects of PFAS in liver microtissues. The data enable potency ranking of PFAS for human liver cell spheroid cytotoxicity and transcriptional changes, and assessment of in vitro transcriptomic points of departure. These data improve our understanding of the possible health effects of PFAS and will be used to inform read-across for human health risk assessment.

High-Throughput Transcriptomic Analysis of Human Primary Hepatocyte Spheroids Exposed to Per- and Polyfluoroalkyl Substances as a Platform for Relative Potency Characterization.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focused on 4 model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at 4 time points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS, (2) identify similarities in biological responses, (3) compare PFAS potency through benchmark concentration analysis, and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (ie, was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Compendium of TCDD-mediated transcriptomic response datasets in mammalian model systems.

BACKGROUND: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of the dioxin class of environmental contaminants. Exposure to TCDD causes a wide range of toxic outcomes, ranging from chloracne to acute lethality. The severity of toxicity is highly dependent on the aryl hydrocarbon receptor (AHR). Binding of TCDD to the AHR leads to changes in transcription of numerous genes. Studies evaluating the transcriptional changes brought on by TCDD may provide valuable insight into the role of the AHR in human health and disease. We therefore compiled a collection of transcriptomic datasets that can be used to aid the scientific community in better understanding the transcriptional effects of ligand-activated AHR. RESULTS: Specifically, we have created a datasets package - TCDD.Transcriptomics - for the R statistical environment, consisting of 63 unique experiments comprising 377 samples, including various combinations of 3 species (human derived cell lines, mouse and rat), 4 tissue types (liver, kidney, white adipose tissue and hypothalamus) and a wide range of TCDD exposure times and doses. These datasets have been fully standardized using consistent preprocessing and annotation packages (available as of September 14, 2015). To demonstrate the utility of this R package, a subset of "AHR-core" genes were evaluated across the included datasets. Ahrr, Nqo1 and members of the Cyp family were significantly induced following exposure to TCDD across the studies as expected while Aldh3a1 was induced specifically in rat liver. Inmt was altered only in liver tissue and primarily by rat-AHR. CONCLUSIONS: Analysis of the "AHR-core" genes demonstrates a continued need for studies surrounding the impact of AHR-activity on the transcriptome; genes believed to be consistently regulated by ligand-activated AHR show surprisingly little overlap across species and tissues. Until now, a comprehensive assessment of the transcriptome across these studies was challenging due to differences in array platforms, processing methods and annotation versions. We believe that this package, which is freely available for download ( http://labs.oicr.on.ca/boutros-lab/tcdd-transcriptomics ) will prove to be a highly beneficial resource to the scientific community evaluating the effects of TCDD exposure as well as the variety of functions of the AHR.

A predictive toxicogenomics signature to classify genotoxic versus non-genotoxic chemicals in human TK6 cells.

Genotoxicity testing is a critical component of chemical assessment. The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the DNA damage response pathways involved in response, is becoming more common. In companion papers previously published in Environmental and Molecular Mutagenesis, Li et al. (2015) [6] developed a dose optimization protocol that was based on evaluating expression changes in several well-characterized stress-response genes using quantitative real-time PCR in human lymphoblastoid TK6 cells in culture. This optimization approach was applied to the analysis of TK6 cells exposed to one of 14 genotoxic or 14 non-genotoxic agents, with sampling 4 h post-exposure. Microarray-based transcriptomic analyses were then used to develop a classifier for genotoxicity using the nearest shrunken centroids method. A panel of 65 genes was identified that could accurately classify toxicants as genotoxic or non-genotoxic. In Buick et al. (2015) [1], the utility of the biomarker for chemicals that require metabolic activation was evaluated. In this study, TK6 cells were exposed to increasing doses of four chemicals (two genotoxic that require metabolic activation and two non-genotoxic chemicals) in the presence of rat liver S9 to demonstrate that S9 does not impair the ability to classify genotoxicity using this genomic biomarker in TK6cells.

Transcriptional profiling of rat white adipose tissue response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin.

Polychlorinated dibenzodioxins are environmental contaminants commonly produced as a by-product of industrial processes. The most potent of these, 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), is highly lipophilic, leading to bioaccumulation. White adipose tissue (WAT) is a major site for energy storage, and is one of the organs in which TCDD accumulates. In laboratory animals, exposure to TCDD causes numerous metabolic abnormalities, including a wasting syndrome. We therefore investigated the molecular effects of TCDD exposure on WAT by profiling the transcriptomic response of WAT to 100µg/kg of TCDD at 1 or 4days in TCDD-sensitive Long-Evans (Turku/AB; L-E) rats. A comparative analysis was conducted simultaneously in identically treated TCDD-resistant Han/Wistar (Kuopio; H/W) rats one day after exposure to the same dose. We sought to identify transcriptomic changes coinciding with the onset of toxicity, while gaining additional insight into later responses. More transcriptional responses to TCDD were observed at 4days than at 1day post-exposure, suggesting WAT shows mostly secondary responses. Two classic AHR-regulated genes, Cyp1a1 and Nqo1, were significantly induced by TCDD in both strains, while several genes involved in the immune response, including Ms4a7 and F13a1 were altered in L-E rats alone. We compared genes affected by TCDD in rat WAT and human adipose cells, and observed little overlap. Interestingly, very few genes involved in lipid metabolism exhibited altered expression levels despite the pronounced lipid mobilization from peripheral fat pads by TCDD in L-E rats. Of these genes, the lipolysis-associated Lpin1 was induced slightly over 2-fold in L-E rat WAT on day 4.

Neurotoxicity may be an overlooked consequence of benzo[a]pyrene exposure that is relevant to human health risk assessment.

Benzo[a]pyrene (BaP) is a well-studied environmental compound that requires metabolic activation to have a carcinogenic effect. The neurotoxicity of BaP has received considerably less attention than its carcinogenicity. Environmental exposure to BaP correlates with impaired learning and memory in adults, and poor neurodevelopment in children. We carried out a comprehensive literature review to examine the neurotoxicity of BaP. The data were used to identify potential point of departure (POD) values for cancer and neurotoxicity endpoints using benchmark dose (BMD) modelling to compare the utility of both endpoints in the risk assessment of BaP. The POD for neurotoxicity in rodents, based on a standard behavioural test (Morris water maze), was 0.025 mg BaP/kg-bw-day compared to 0.54 mg BaP/kg-bw-day for rodent forestomach carcinogenicity, suggesting that neurotoxic endpoints are more sensitive than cancer endpoints for health risks associated with BaP exposure. Using the limited number of published studies on this topic, we propose a preliminary mode of action (MOA) to explain BaP-induced neurotoxicity in rodents. The MOA includes: (1) BaP binding to the aryl hydrocarbon receptor (AHR); (2) AHR-dependent modulation of the transcription of N-methyl-d-aspartate glutamate receptor (NMDAR) subunits; (3) NMDAR-mediated loss of neuronal activity and decreased long-term potentiation; and (4) compromised learning and memory. More data are needed to explore the proposed neurotoxic MOA. In addition, we consider alternative MOAs, including the hypothesis that BaP-mediated DNA damage may lead to either carcinogenicity or neurotoxicity, depending on the tissue. Our proposed MOA is intended to serve as a basis for hypothesis testing in future studies. We emphasise that further studies are needed to validate the proposed MOA, to evaluate its human relevance, and to explore other potential mechanisms of BaP neurotoxicity.

Technical guide for applications of gene expression profiling in human health risk assessment of environmental chemicals.

Toxicogenomics promises to be an important part of future human health risk assessment of environmental chemicals. The application of gene expression profiles (e.g., for hazard identification, chemical prioritization, chemical grouping, mode of action discovery, and quantitative analysis of response) is growing in the literature, but their use in formal risk assessment by regulatory agencies is relatively infrequent. Although additional validations for specific applications are required, gene expression data can be of immediate use for increasing confidence in chemical evaluations. We believe that a primary reason for the current lack of integration is the limited practical guidance available for risk assessment specialists with limited experience in genomics. The present manuscript provides basic information on gene expression profiling, along with guidance on evaluating the quality of genomic experiments and data, and interpretation of results presented in the form of heat maps, pathway analyses and other common approaches. Moreover, potential ways to integrate information from gene expression experiments into current risk assessment are presented using published studies as examples. The primary objective of this work is to facilitate integration of gene expression data into human health risk assessments of environmental chemicals.

Integration of metabolic activation with a predictive toxicogenomics signature to classify genotoxic versus nongenotoxic chemicals in human TK6 cells.

The use of integrated approaches in genetic toxicology, including the incorporation of gene expression data to determine the molecular pathways involved in the response, is becoming more common. In a companion article, a genomic biomarker was developed in human TK6 cells to classify chemicals as genotoxic or nongenotoxic. Because TK6 cells are not metabolically competent, we set out to broaden the utility of the biomarker for use with chemicals requiring metabolic activation. Specifically, chemical exposures were conducted in the presence of rat liver S9. The ability of the biomarker to classify genotoxic (benzo[a]pyrene, BaP; aflatoxin B1, AFB1) and nongenotoxic (dexamethasone, DEX; phenobarbital, PB) agents correctly was evaluated. Cells were exposed to increasing chemical concentrations for 4 hr and collected 0 hr, 4 hr, and 20 hr postexposure. Relative survival, apoptosis, and micronucleus frequency were measured at 24 hr. Transcriptome profiles were measured with Agilent microarrays. Statistical modeling and bioinformatics tools were applied to classify each chemical using the genomic biomarker. BaP and AFB1 were correctly classified as genotoxic at the mid- and high concentrations at all three time points, whereas DEX was correctly classified as nongenotoxic at all concentrations and time points. The high concentration of PB was misclassified at 24 hr, suggesting that cytotoxicity at later time points may cause misclassification. The data suggest that the use of S9 does not impair the ability of the biomarker to classify genotoxicity in TK6 cells. Finally, we demonstrate that the biomarker is also able to accurately classify genotoxicity using a publicly available dataset derived from human HepaRG cells.

Integrating toxicogenomics into human health risk assessment: lessons learned from the benzo[a]pyrene case study.

The use of short-term toxicogenomic tests to predict cancer (or other health effects) offers considerable advantages relative to traditional toxicity testing methods. The advantages include increased throughput, increased mechanistic data, and significantly reduced costs. However, precisely how toxicogenomics data can be used to support human health risk assessment (RA) is unclear. In a companion paper ( Moffat et al. 2014 ), we present a case study evaluating the utility of toxicogenomics in the RA of benzo[a]pyrene (BaP), a known human carcinogen. The case study is meant as a proof-of-principle exercise using a well-established mode of action (MOA) that impacts multiple tissues, which should provide a best case example. We found that toxicogenomics provided rich mechanistic data applicable to hazard identification, dose-response analysis, and quantitative RA of BaP. Based on this work, here we share some useful lessons for both research and RA, and outline our perspective on how toxicogenomics can benefit RA in the short- and long-term. Specifically, we focus on (1) obtaining biologically relevant data that are readily suitable for establishing an MOA for toxicants, (2) examining the human relevance of an MOA from animal testing, and (3) proposing appropriate quantitative values for RA. We describe our envisioned strategy on how toxicogenomics can become a tool in RA, especially when anchored to other short-term toxicity tests (apical endpoints) to increase confidence in the proposed MOA, and emphasize the need for additional studies on other MOAs to define the best practices in the application of toxicogenomics in RA.

Comparison of toxicogenomics and traditional approaches to inform mode of action and points of departure in human health risk assessment of benzo[a]pyrene in drinking water.

Toxicogenomics is proposed to be a useful tool in human health risk assessment. However, a systematic comparison of traditional risk assessment approaches with those applying toxicogenomics has never been done. We conducted a case study to evaluate the utility of toxicogenomics in the risk assessment of benzo[a]pyrene (BaP), a well-studied carcinogen, for drinking water exposures. Our study was intended to compare methodologies, not to evaluate drinking water safety. We compared traditional (RA1), genomics-informed (RA2) and genomics-only (RA3) approaches. RA2 and RA3 applied toxicogenomics data from human cell cultures and mice exposed to BaP to determine if these data could provide insight into BaP's mode of action (MOA) and derive tissue-specific points of departure (POD). Our global gene expression analysis supported that BaP is genotoxic in mice and allowed the development of a detailed MOA. Toxicogenomics analysis in human lymphoblastoid TK6 cells demonstrated a high degree of consistency in perturbed pathways with animal tissues. Quantitatively, the PODs for traditional and transcriptional approaches were similar (liver 1.2 vs. 1.0 mg/kg-bw/day; lungs 0.8 vs. 3.7 mg/kg-bw/day; forestomach 0.5 vs. 7.4 mg/kg-bw/day). RA3, which applied toxicogenomics in the absence of apical toxicology data, demonstrates that this approach provides useful information in data-poor situations. Overall, our study supports the use of toxicogenomics as a relatively fast and cost-effective tool for hazard identification, preliminary evaluation of potential carcinogens, and carcinogenic potency, in addition to identifying current limitations and practical questions for future work.

Transcriptional profiling of rat hypothalamus response to 2,3,7,8-tetrachlorodibenzo-ρ-dioxin.

In some mammals, halogenated aromatic hydrocarbon (HAH) exposure causes wasting syndrome, defined as significant weight loss associated with lethal outcomes. The most potent HAH in causing wasting is 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD), which exerts its toxic effects through the aryl hydrocarbon receptor (AHR). Since TCDD toxicity is thought to predominantly arise from dysregulation of AHR-transcribed genes, it was hypothesized that wasting syndrome is a result of to TCDD-induced dysregulation of genes involved in regulation of food-intake. As the hypothalamus is the central nervous systems' regulatory center for food-intake and energy balance. Therefore, mRNA abundances in hypothalamic tissue from two rat strains with widely differing sensitivities to TCDD-induced wasting syndrome: TCDD-sensitive Long-Evans rats and TCDD-resistant Han/Wistar rats, 23h after exposure to TCDD (100µg/kg) or corn oil vehicle. TCDD exposure caused minimal transcriptional dysregulation in the hypothalamus, with only 6 genes significantly altered in Long-Evans rats and 15 genes in Han/Wistar rats. Two of the most dysregulated genes were Cyp1a1 and Nqo1, which are induced by TCDD across a wide range of tissues and are considered sensitive markers of TCDD exposure. The minimal response of the hypothalamic transcriptome to a lethal dose of TCDD at an early time-point suggests that the hypothalamus is not the predominant site of initial events leading to hypophagia and associated wasting. TCDD may affect feeding behaviour via events upstream or downstream of the hypothalamus, and further work is required to evaluate this at the level of individual hypothalamic nuclei and subregions.

Cross-species transcriptomic analysis elucidates constitutive aryl hydrocarbon receptor activity.

BACKGROUND: Research on the aryl hydrocarbon receptor (AHR) has largely focused on variations in toxic outcomes resulting from its activation by halogenated aromatic hydrocarbons. But the AHR also plays key roles in regulating pathways critical for development, and after decades of research the mechanisms underlying physiological regulation by the AHR remain poorly characterized. Previous studies identified several core genes that respond to xenobiotic AHR ligands across a broad range of species and tissues. However, only limited inferences have been made regarding its role in regulating constitutive gene activity, i.e. in the absence of exogenous ligands. To address this, we profiled transcriptomic variations between AHR-active and AHR-less-active animals in the absence of an exogenous agonist across five tissues, three of which came from rats (hypothalamus, white adipose and liver) and two of which came from mice (kidney and liver). Because AHR status alone has been shown sufficient to alter transcriptomic responses, we reason that by contrasting profiles amongst AHR-variant animals, we may elucidate effects of the AHR on constitutive mRNA abundances. RESULTS: We found significantly more overlap in constitutive mRNA abundances amongst tissues within the same species than from tissues between species and identified 13 genes (Agt, Car3, Creg1, Ctsc, E2f6, Enpp1, Gatm, Gstm4, Kcnj8, Me1, Pdk1, Slc35a3, and Sqrdl) that are affected by AHR-status in four of five tissues. One gene, Creg1, was significantly up-regulated in all AHR-less-active animals. We also find greater overlap between tissues at the pathway level than at the gene level, suggesting coherency to the AHR signalling response within these processes. Analysis of regulatory motifs suggests that the AHR mostly mediates transcriptional regulation via direct binding to response elements. CONCLUSIONS: These findings, though preliminary, present a platform for further evaluating the role of the AHR in regulation of constitutive mRNA levels and physiologic function.

Preparation of archival formalin-fixed paraffin-embedded mouse liver samples for use with the Agilent gene expression microarray platform.

INTRODUCTION: Tissue samples are routinely formalin-fixed and paraffin-embedded (FFPE) for long term preservation. Gene expression analysis of archival FFPE tissues may advance knowledge of the molecular perturbations contributing to disease. However, formalin causes extensive degradation of RNA. METHODS: We compared RNA quality/yield from FFPE samples using six commercial FFPE RNA extraction kits. In addition we compared four DNA microarray protocols for the Agilent 8×60K platform using 16year old FFPE mouse liver samples treated with phenobarbital or vehicle. RESULTS: Despite low quality RNA, archival phenobarbital samples exhibited strong induction of the positive control genes Cyp2b9 and Cyp2b10 by quantitative real-time PCR (qPCR). We tested one- and two-color microarray designs and evaluated the effects of increasing the amount of hybridized cDNA. Canonical gene responders to phenobarbital were measurably induced under each experimental condition. Increasing the amount of labeled cDNA did not improve the overall signal intensity. One-color experiments yielded larger fold changes than two-color and the number of differentially expressed genes varied between protocols. Gene expression changes were validated by qPCR and literature searches. Individual protocols exhibited high rates of false positives; however, pathway analysis revealed that nine of the top ten canonical pathways were consistent across experiments. Genes that were differentially expressed in more than one experiment were more likely to be validated. Thus, we recommend that experiments on FFPE samples be done in duplicate to reduce false positives. DISCUSSION: In this analysis of archival FFPE samples we were able to identify pathways that are consistent with phenobarbital's mechanism of action. Therefore, we conclude that FFPE samples can be used for meaningful microarray gene expression analyses.

Gene expression profiling to identify potentially relevant disease outcomes and support human health risk assessment for carbon black nanoparticle exposure.

New approaches are urgently needed to evaluate potential hazards posed by exposure to nanomaterials. Gene expression profiling provides information on potential modes of action and human relevance, and tools have recently become available for pathway-based quantitative risk assessment. The objective of this study was to use toxicogenomics in the context of human health risk assessment. We explore the utility of toxicogenomics in risk assessment, using published gene expression data from C57BL/6 mice exposed to 18, 54 and 162 µg Printex 90 carbon black nanoparticles (CBNP). Analysis of CBNP-perturbed pathways, networks and transcription factors revealed concomitant changes in predicted phenotypes (e.g., pulmonary inflammation and genotoxicity), that correlated with dose and time. Benchmark doses (BMDs) for apical endpoints were comparable to minimum BMDs for relevant pathway-specific expression changes. Comparison to inflammatory lung disease models (i.e., allergic airway inflammation, bacterial infection and tissue injury and fibrosis) and human disease profiles revealed that induced gene expression changes in Printex 90 exposed mice were similar to those typical for pulmonary injury and fibrosis. Very similar fibrotic pathways were perturbed in CBNP-exposed mice and human fibrosis disease models. Our synthesis demonstrates how toxicogenomic profiles may be used in human health risk assessment of nanoparticles and constitutes an important step forward in the ultimate recognition of toxicogenomic endpoints in human health risk. As our knowledge of molecular pathways, dose-response characteristics and relevance to human disease continues to grow, we anticipate that toxicogenomics will become increasingly useful in assessing chemical toxicities and in human health risk assessment.

mRNA levels in control rat liver display strain-specific, hereditary, and AHR-dependent components.

Rat is a major model organism in toxicogenomics and pharmacogenomics. Hepatic mRNA profiles after treatment with xenobiotic chemicals are used to predict and understand drug toxicity and mechanisms. Surprisingly, neither inter- and intra-strain variability of mRNA abundances in control rats nor the heritability of rat mRNA abundances yet been established. We address these issues by studying five populations: the popular Sprague-Dawley strain, sub-strains of Long-Evans and Wistar rats, and two lines derived from crosses between the Long-Evans and Wistar sub-strains. Using three independent techniques--variance analysis, linear modelling, and unsupervised pattern recognition--we characterize extensive intra- and inter-strain variability in mRNA levels. We find that both sources of variability are non-random and are enriched for specific functional groups. Specific transcription-factor binding-sites are enriched in their promoter regions and these genes occur in "islands" scattered throughout the rat genome. Using the two lines generated by crossbreeding we tested heritability of hepatic mRNA levels: the majority of rat genes appear to exhibit directional genetics, with only a few interacting loci. Finally, a comparison of inter-strain heterogeneity between mouse and rat orthologs shows more heterogeneity in rats than mice; thus rat and mouse heterogeneity are uncorrelated. Our results establish that control hepatic mRNA levels are relatively homogeneous within rat strains but highly variable between strains. This variability may be related to increased activity of specific transcription-factors and has clear functional consequences. Future studies may take advantage of this phenomenon by surveying panels of rat strains.

Hepatic transcriptomic responses to TCDD in dioxin-sensitive and dioxin-resistant rats during the onset of toxicity.

The dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes a wide range of toxic effects in rodent species, all of which are mediated by a ligand-dependent transcription-factor, the aryl hydrocarbon receptor (AHR). The Han/Wistar (Kuopio) (H/W) strain shows exceptional resistance to many TCDD-induced toxicities; the LD50 of > 9600 µg/kg for H/W rats is higher than for any other wild-type mammal known. We previously showed that this resistance primarily results from H/W rats expressing a variant AHR isoform that has a substantial portion of the AHR transactivation domain deleted. Despite this large deletion, H/W rats are not entirely refractory to the effects of TCDD; the variant AHR in these animals remains fully competent to up-regulate well-known dioxin-inducible genes. TCDD-sensitive (Long-Evans, L-E) and resistant (H/W) rats were treated with either corn-oil (with or without feed-restriction) or 100 µg/kg TCDD for either four or ten days. Hepatic transcriptional profiling was done using microarrays, and was validated by RT-PCR analysis of 41 genes. A core set of genes was altered in both strains at all time points tested, including CYP1A1, CYP1A2, CYP1B1, Nqo1, Aldh3a1, Tiparp, Exoc3, and Inmt. Outside this core, the strains differed significantly in the breadth of response: three-fold more genes were altered in L-E than H/W rats. At ten days almost all expressed genes were dysregulated in L-E rats, likely reflecting emerging toxic responses. Far fewer genes were affected by feed-restriction, suggesting that only a minority of the TCDD-induced changes are secondary to the wasting syndrome.

Aryl hydrocarbon receptor (AHR)-regulated transcriptomic changes in rats sensitive or resistant to major dioxin toxicities.

BACKGROUND: The major toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to result from dysregulation of mRNA levels mediated by the aryl hydrocarbon receptor (AHR). Dioxin-like chemicals alter expression of numerous genes in liver, but it remains unknown which lie in pathways leading to major toxicities such as hepatotoxicity, wasting and lethality. To identify genes involved in these responses we exploited a rat genetic model. Rats expressing an AHR splice-variant lacking a portion of the transactivation domain are highly resistant to dioxin-induced toxicities. We examined changes in hepatic mRNA abundances 19 hours after TCDD treatment in two dioxin-resistant rat strains/lines and two dioxin-sensitive rat strains/lines. RESULTS: Resistant rat strains/lines exhibited fewer transcriptional changes in response to TCDD than did rats with wildtype AHR. However, well-known AHR-regulated and dioxin-inducible genes such as CYP1A1, CYP1A2, and CYP1B1 remained fully responsive to TCDD in all strains/lines. Pathway analysis indicated that the genes which respond differently to TCDD between sensitive and resistant rats are mainly involved in lipid metabolism, cellular membrane function and energy metabolism. These pathways previously have been shown to respond differently to dioxin treatment in dioxin-sensitive versus dioxin-resistant rats at a biochemical level and in the differential phenotype of toxicologic responses. CONCLUSION: The transactivation-domain deletion in dioxin-resistant rats does not abolish global AHR transactivational activity but selectively interferes with expression of subsets of genes that are candidates to mediate or protect from major dioxin toxicities such as hepatotoxicity, wasting and death.

Transcriptomic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver: comparison of rat and mouse.

BACKGROUND: Mouse and rat models are mainstays in pharmacology, toxicology and drug development -- but differences between strains and between species complicate data interpretation and application to human health. Dioxin-like polyhalogenated aromatic hydrocarbons represent a major class of environmentally and economically relevant toxicants. In mammals dioxin exposure leads to a broad spectrum of adverse affects, including hepatotoxicity of varying severity. Several studies have shown that dioxins extensively alter hepatic mRNA levels. Surprisingly, though, analysis of a limited portion of the transcriptome revealed that rat and mouse responses diverge greatly (Boverhof et al. Toxicol Sci 94:398-416, 2006). RESULTS: We employed oligonucleotide arrays to compare the response of 8,125 rat and mouse orthologs. We confirmed that there is limited inter-species overlap in dioxin-responsive genes. Rat-specific and mouse-specific genes are enriched for specific functional groups which differ between species, conceivably accounting for species-specificities in liver histopathology. While no evidence for the involvement of copy-number variation was found, extensive inter-species variation in the transcriptional-regulatory network was identified; Nr2f1 and Fos emerged as candidates to explain species-specific and species-independent responses, respectively. CONCLUSION: Our results suggest that a small core of genes is responsible for mediating the similar features of dioxin hepatotoxicity in rats and mice but non-overlapping pathways are simultaneously at play to result in distinctive histopathological outcomes. The extreme divergence between mouse and rat transcriptomic responses appears to reflect divergent transcriptional-regulatory networks. Taken together, these data suggest that both rat and mouse models should be used to screen the acute hepatotoxic effects of drugs and toxic compounds.

Patterns of dioxin-altered mRNA expression in livers of dioxin-sensitive versus dioxin-resistant rats.

Dioxins exert their major toxicologic effects by binding to the aryl hydrocarbon receptor (AHR) and altering gene transcription. Numerous dioxin-responsive genes previously were identified both by conventional biochemical and molecular techniques and by recent mRNA expression microarray studies. However, of the large set of dioxin-responsive genes the specific genes whose dysregulation leads to death remain unknown. To identify specific genes that may be involved in dioxin lethality we compared changes in liver mRNA levels following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in three strains/lines of dioxin-sensitive rats with changes in three dioxin-resistant rat strains/lines. The three dioxin-resistant strains/lines all harbor a large deletion in the transactivation domain of the aryl hydrocarbon receptor (AHR). Despite this deletion, many genes exhibited a "Type-I" response-that is, their responses were similar in dioxin-sensitive and dioxin-resistant rats. Several genes that previously were well established as being dioxin-responsive or under AHR regulation emerged as Type-I responses (e.g. CYP1A1, CYP1A2, CYP1B1 and Gsta3). In contrast, a relatively small number of genes exhibited a Type-II response-defined as a difference in responsiveness between dioxin-sensitive and dioxin-resistant rat strains. Type-II genes include: malic enzyme 1, ubiquitin C, cathepsin L, S-adenosylhomocysteine hydrolase and ferritin light chain 1. In silico searches revealed that AH response elements are conserved in the 5'-flanking regions of several genes that respond to TCDD in both the Type-I and Type-II categories. The vast majority of changes in mRNA levels in response to 100 microg/kg TCDD were strain-specific; over 75% of the dioxin-responsive clones were affected in only one of the six strains/lines. Selected genes were assessed by quantitative RT-PCR in dose-response and time-course experiments and responses of some genes were assessed in Ahr-null mice to determine if their response was AHR-dependent. Type-II genes may lie in pathways that are central to the difference in susceptibility to TCDD lethality in this animal model.