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.

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.

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.

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.

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.

Genome-wide effects of acute progressive feed restriction in liver and white adipose tissue.

Acute progressive feed restriction (APFR) represents a specific form of caloric restriction in which feed availability is increasingly curtailed over a period of a few days to a few weeks. It is often used for control animals in toxicological and pharmacological studies on compounds causing body weight loss to equalize weight changes between experimental and control groups and thereby, intuitively, to also set their metabolic states to the same phase. However, scientific justification for this procedure is lacking. In the present study, we analyzed by microarrays the impact on hepatic gene expression in rats of two APFR regimens that caused identical diminution of body weight (19%) but differed slightly in duration (4 vs. 10 days). In addition, white adipose tissue (WAT) was also subjected to the transcriptomic analysis on day-4. The data revealed that the two regimens led to distinct patterns of differentially expressed genes in liver, albeit some major pathways of energy metabolism were similarly affected (particularly fatty acid and amino acid catabolism). The reason for the divergence appeared to be entrainment by the longer APFR protocol of peripheral oscillator genes, which resulted in derailment of circadian rhythms and consequent interaction of altered diurnal fluctuations with metabolic adjustments in gene expression activities. WAT proved to be highly unresponsive to the 4-day APFR as only 17 mRNA levels were influenced by the treatment. This study demonstrates that body weight is a poor proxy of metabolic state and that the customary protocols of feed restriction can lead to rhythm entrainment.

microRNAs in adult rodent liver are refractory to dioxin treatment.

Dioxin-like chemicals are well known for their ability to upregulate expression of numerous genes via the AH receptor (AHR). However, recent transcriptomic analyses in several laboratories indicate that dioxin-like chemicals or AHR genotype itself also can downregulate levels of mRNAs encoded by numerous genes. The mechanism responsible for such downregulation is unknown. We hypothesized that microRNAs (miRNAs), which have emerged as powerful negative regulators of mRNA levels in several systems, might be responsible for mRNA downregulation in dioxin/AHR pathways. We used two miRNA array platforms as well as quantitative reverse transcriptase-polymerase chain reaction to measure miRNA levels in wild-type (WT) versus Ahr-null mice, in dioxin-sensitive Long-Evans (L-E; Turku/AB) rats versus dioxin-resistant Han/Wistar (H/W; Kuopio) rats and in rat 5L and mouse Hepa-1 hepatoma cells in culture. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo caused few changes in miRNA levels in mouse or rat livers, and those changes that were statistically significant were of modest magnitude. Hepatoma cells in culture also exhibited few changes in miRNA levels in response to TCDD. AHR genotype had little effect on hepatic miRNA levels, either in constitutive expression or in response to TCDD-only a few miRNAs differed in expression between Ahr-null mice compared to mice with WT AHR or between L-E rats (that have WT AHR) compared to H/W rats (whose AHR has a large deletion in the transactivation domain). It is unlikely that mRNA downregulation by dioxins is mediated by miRNAs, nor are miRNAs likely to play a significant role in dioxin toxicity in adult rodent liver.

Aryl hydrocarbon receptor splice variants in the dioxin-resistant rat: tissue expression and transactivational activity.

The AHR locus encodes the aryl hydrocarbon receptor (AHR), a transcriptional regulator of multiple drug-metabolizing enzymes and mediator of toxicity of dioxin-like chemicals. The Han/Wistar (Kuopio) rat strain (H/W) is remarkably resistant to lethal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) because of a point mutation in the exon/intron 10 boundary in AHR genomic structure that leads to use of 3 alternative cryptic splice sites, potentially creating 3 alternative transcripts and 2 protein products. The deletion variant (DV), which lacks 43 amino acids in the transactivation domain, has the highest intrinsic transactivation activity in vitro; amino acids 766 to 783 suppress transactivation function. However, DV expression levels in H/W rats in vivo are low in liver, lung, thymus, kidney, and testis; insertion variant mRNAs (IVs) are the dominant mRNA forms in H/W rats in which wild-type AHR mRNA is undetectable. In dioxin-sensitive rat strains and lines that are homozygous for wild-type AHR alleles, wild-type AHR mRNA is the most abundant transcript but some IV transcripts are detectable. TCDD treatment in vivo increases transcript levels for both the DV and IVs in H/W rats and increases wild-type transcript levels in dioxin-sensitive rats but does not alter which transcript forms are expressed. In silico modeling indicates that the DV mRNA has lost considerable secondary structure, whereas at the protein level, the transactivation domain of the IV in the dioxin-resistant H/W rat has greater alpha-helical content and a more hydrophobic terminus than wild-type AHR, which may produce a protein conformation that is less amenable to interaction with other regulatory proteins.

Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.

One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance.

Evaluation of various housekeeping genes for their applicability for normalization of mRNA expression in dioxin-treated rats.

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is an extremely sensitive, convenient and rapid method to measure mRNA levels in cells and tissues, and is gaining popularity in toxicology. To correct for sample-to-sample variation, normalization of the expression data is required. The conventional way to perform normalization is to select a reference gene whose expression is believed to remain stable across all experimental conditions, then relate the concentrations of gene(s) of interest to those of this housekeeping gene. Since recent evidence shows that some housekeeping genes are actually not as refractory to experimental manipulations as previously thought, we validated a large number (18) of commonly used housekeeping genes for acute toxicity studies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an extremely potent environmental toxin known to regulate a wide variety of genes. Microarray and qRT-PCR analyses coherently demonstrated that about 50% of the housekeeping genes examined were responsive to TCDD in rat liver with the magnitudes of change up to nearly 10-fold. Extension of the study to spleen and hypothalamus verified that phosphoglycerate kinase 1 (Pgk1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) retained their basal expression levels in all experimental settings, although body weight loss-generated repression may mask a slight induction of GAPDH by TCDD in liver. These findings show that normalization genes for qRT-PCR must be carefully validated in advance, especially if the study involves a potent modifier of gene expression.

Aryl hydrocarbon receptor regulates distinct dioxin-dependent and dioxin-independent gene batteries.

Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of AHR-dependent genes in male adult liver using expression arrays to contrast mRNA profiles in Ahr-null mice (Ahr(-/-)) with those in mice with wild-type AHR (Ahr(+)(/)(+)). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 microg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members of the classic AHRE-I gene battery, such as Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the AHR has multiple functions in normal physiology. In Ahr(-/-) mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible, were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor alpha as well as two estrogen-receptor-related genes (alpha and gamma) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either for constitutive expression or for response to TCDD.

Toxicological implications of polymorphisms in receptors for xenobiotic chemicals: the case of the aryl hydrocarbon receptor.

Mechanistic toxicology has predominantly been focused on adverse effects that are caused by reactive metabolites or by reactive oxygen species. However, many important xenobiotics exert their toxicity, not by generating reactive products, but rather by altering expression of specific genes. In particular, some environmental contaminants target nuclear receptors that function as regulators of transcription. For example, binding of xenobiotic chemicals to steroid receptors is a principle mechanism of endocrine disruption. The aryl hydrocarbon receptor (AHR) mediates toxicity of dioxin-like compounds. In mice, a polymorphism in the AHR ligand-binding domain reduces binding affinity by about 10-fold in the DBA/2 strain compared with the C57BL/6 strain; consequently, dose-response curves for numerous biochemical and toxic effects are shifted about one log to the right in DBA/2 mice. In the Han/Wistar (Kuopio) (H/W) rat strain, a polymorphism causes a deletion of 38 or 43 amino acids from the AHR transactivation domain. This deletion is associated with a greater than 1000-fold resistance to lethality from 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Genes in the conventional AH gene battery (e.g. CYP1A1, CYP1A2, CYP1B1, ALDH3A1, NQO1 and UGT1A1) remain responsive to TCDD in H/W rats despite the large deletion. However, the deletion may selectively alter the receptor's ability to dysregulate specific genes that are key to dioxin toxicity. We are identifying these genes using an expression array approach in dioxin-sensitive vs. dioxin-resistant rat strains and lines. Polymorphisms exist in the human AH receptor, but thus far they have not been shown to have any substantial effect on human responses to AHR-ligands.

Dioxin-responsive AHRE-II gene battery: identification by phylogenetic footprinting.

We identified a set of genes that respond to dioxins through the recently discovered AHRE-II ("XRE-II") enhancer element. A total of 36 genes containing AHRE-II motifs conserved across human, mouse, and rat gene orthologs were identified by genome-wide transcription-factor binding-site searches and phylogenetic footprinting. Microarray experiments on liver from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin revealed statistically significant changes in mRNA levels for 13 of these 36 genes after three hours and 15 genes after 19h. The set of responsive genes was functionally characterized by ontological analysis and found to be enriched in ion-channels and transporters. Our identification of 36 putatively AHRE-II-regulated genes highlights the regulatory versatility of the aryl hydrocarbon receptor (AHR) and the ability of the AHR and its dimerization partner, ARNT, to act both as a ligand-activated transcription-factor (on AHRE-I) and as a ligand-activated coactivator (on AHRE-II). Collectively, these results demonstrate that the AHRE-II induction mechanism is employed by multiple genes and provide the first categorization of the gene battery of a ligand-activated coactivator.