ToxSci Advance Access originally published online on September 7, 2006
Toxicological Sciences 2006 94(2):398-416; doi:10.1093/toxsci/kfl100
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Comparative Toxicogenomic Analysis of the Hepatotoxic Effects of TCDD in Sprague Dawley Rats and C57BL/6 Mice
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* Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824
Center for Integrative Toxicology, National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
Wellington Laboratories Inc., Guelph, Ontario N1G 3M5, Canada
Department of Pathobiology and Diagnostic Investigation, National Food Safety and Toxicology Center, Michigan State University, East Lansing, Michigan 48824
¶ Gene Logic Inc., Gaithersburg, Maryland 20879
1 To whom correspondence should be addressed at: Department of Biochemistry and Molecular Biology, Michigan State University, 501 Biochemistry Building, Wilson Road, East Lansing, MI 48824-1319. Fax: (517) 353-9334. E-mail: tzachare{at}msu.edu.
Received July 12, 2006; accepted September 1, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAREFERENCES |
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In an effort to further characterize conserved and species-specific mechanisms of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)–mediated toxicity, comparative temporal and dose-response microarray analyses were performed on hepatic tissue from immature, ovariectomized Sprague Dawley rats and C57BL/6 mice. For temporal studies, rats and mice were gavaged with 10 or 30 µg/kg of TCDD, respectively, and sacrificed after 2, 4, 8, 12, 18, 24, 72, or 168 h while dose-response studies were performed at 24h. Hepatic gene expression profiles were monitored using custom cDNA microarrays containing 8567 (rat) or 13,361 (mouse) cDNA clones. Affymetrix data from male rats treated with 40 µg/kg TCDD were also included to expand the species comparison. In total, 3087 orthologous genes were represented in the cross-species comparison. Comparative analysis identified 33 orthologous genes that were commonly regulated by TCDD as well as 185 rat-specific and 225 mouse-specific responses. Functional annotation using Gene Ontology identified conserved gene responses associated with xenobiotic/chemical stress and amino acid and lipid metabolism. Rat-specific gene expression responses were associated with cellular growth and lipid metabolism while mouse-specific responses were associated with lipid uptake/metabolism and immune responses. The common and species-specific gene expression responses were also consistent with complementary histopathology, clinical chemistry, hepatic lipid analyses, and reports in the literature. These data expand our understanding of TCDD-mediated gene expression responses and indicate that species-specific toxicity may be mediated by differences in gene expression which may help explain the wide range of species sensitivities and will have important implications in risk assessment strategies.
Key Words: TCDD; toxicogenomics; liver; cross-species comparison.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAREFERENCES |
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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds are ubiquitous environmental contaminants that elicit a broad spectrum of toxic and biochemical responses in a tissue-, sex-, age-, and species-specific manner (Poland and Knutson, 1982
). These responses include a wasting syndrome, tumor promotion, teratogenesis, immunotoxicity, modulation of endocrine systems, and hepatotoxicity which are mediated by the aryl hydrocarbon receptor (AhR), a member of the basic-helix-loop-helix-PAS (bHLH-PAS) family (Denison and Heath-Pagliuso, 1998; Poland and Knutson, 1982). The proposed mechanism involves ligand binding to the cytoplasmic AhR and translocation to the nucleus where it forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT), another member of the bHLH-PAS family. This heterodimer binds specific DNA elements, termed dioxin response elements (DREs), in the regulatory regions of target genes leading to changes in gene expression (Hankinson, 1995). Evidence suggests that TCDD-mediated toxicity is due to the continuous and inappropriate AhR-mediated regulation of target genes (Denison et al., 2002).
The obligatory involvement of the AhR/ARNT signaling pathway in mediating the toxic and biochemical responses to TCDD has been well established by studies which reported decreased susceptibility to TCDD-mediated toxicity in mice with low-affinity AhR alleles (Okey et al., 1989) and resistance to toxicity in AhR-null mice (Gonzalez and Fernandez-Salguero, 1998; Peters et al., 1999; Vorderstrasse et al., 2001). Han/Wistar rats display a 1000-fold resistance to TCDD-mediated lethality when compared to the Long-Evans strain which is attributed to a genetic polymorphism in the AhR resulting in a 38 amino acid deletion from the transactivation domain (Pohjanvirta et al., 1999). More recent studies have shown that mice possessing mutations in the AhR nuclear localization/DRE-binding domain and mice harboring a hypomorphic ARNT allele fail to exhibit classical TCDD toxicities (Bunger et al., 2003; Walisser et al., 2004). Although the mechanism of AhR/ARNT-mediated changes in gene expression is well established, the gene expression responses involved in mediating the observed toxic and biochemical effects remain poorly understood.
Rodents exhibit a wide range of sensitivities to the toxic effects of TCDD with LD50 values ranging from 1 µg/kg in the guinea pig (Schwetz et al., 1973) to > 1000 µg/kg in the hamster (Olson et al., 1980). Sprague Dawley rats and C57BL/6 mice have been used extensively to study TCDD-mediated toxicity and exhibit oral LD50 values of 30 µg/kg and 120 µg/kg, respectively (Bickel 1982; Vos et al., 1974). Rats are also more sensitive to effects on body weight gain, liver weight, thymus weight, and vitamin A homeostasis, while effects on hepatic ethoxyresorufin O-deethylase activity are similar (Fletcher et al., 2001). AhR-binding affinity for TCDD is similar between these species and therefore does not explain the difference in sensitivity (Denison et al., 1986; Poland et al., 1976). Studies have indicated that the rat and mouse AhR are comparable but not identical molecular species and differ in their molecular weights (Denison et al., 1986). Comparison of amino acid sequences reveals high homology with the exception of a 42 amino acid truncation at the C-terminal end of the mouse AhR when compared to the rat. Differences in the AhR transactivation domain may be responsible for differential gene expression responses and altered sensitivity of these strains as proposed for Han/Wistar and Long-Evans rats (Okey et al., 2005). Alternatively, differences in genomic sequences at promoter and enhancer regions may result in species-specific gene expression responses which could also contribute to the differential sensitivity (Sun et al., 2004).
Cross-species comparisons of global gene expression responses represent a powerful approach to investigate the molecular mechanisms involved in TCDD-mediated toxicity. In order to further characterize the spectrum of AhR/ARNT-responsive transcripts and their relationship to hepatotoxicity, the present study has compared temporal and dose-dependent hepatic gene expression responses to TCDD in Sprague Dawley rats and C57BL/6 mice. Results indicate both conserved and species-specific gene expression responses which have extended our understanding of the AhR regulon and may help to explain the altered sensitivity in these species.
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MATERIALS AND METHODS |
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Animal handling.
Female Sprague Dawley rats and C57BL/6 mice, ovariectomized by the vendor on postnatal day (PND) 20 and all having body weights within 10% of the average body weight, were obtained from Charles River Laboratories (Raleigh, NC) on PND day 25. This animal model is utilized by our laboratory for a variety of studies and was employed in the present study for consistency and to facilitate future comparisons. Animals were housed in polycarbonate cages containing cellulose fiber chips (Aspen Chip Laboratory Bedding, Northeastern Products, Warrensberg, NY) in a 23°C high-efficiency particulate air-filtered environment with 30–40% humidity and a 12-h light/dark cycle (0700 h–1900 h). Animals were allowed free access to deionized water and Harlan Teklad 22/5 Rodent Diet 8640 (Madison, WI) and acclimatized for 4 days prior to dosing. On the fourth day, animals were weighed, and a stock solution of TCDD (provided by S. Safe, Texas A&M University, College Station, TX) was diluted in sesame oil (Sigma, St Louis, MO) to achieve the desired dose based on the average weight. All procedures were performed with the approval of the Michigan State University All-University Committee on Animal Use and Care.
Time course and dose-response studies.
For the time course studies, rats were treated by gavage with 0.1 ml of sesame oil for a nominal dose of 0 (vehicle control) or 10 µg/kg body weight of TCDD while mice received 30 µg/kg body weight of TCDD. A minimum of five animals were treated per dose group and time point, and all groups for each dose and time point were housed in separate cages. Both rats and mice were sacrificed 2, 4, 8, 12, 18, 24, 72, or 168 h after dosing. For the dose-response studies, rats were gavaged with 0.1 ml of vehicle or 0.001, 0.01, 0.1, 1, 10, 30, or 100 µg/kg TCDD while mice received 0.1 ml of vehicle or 0.001, 0.01, 0.1, 1, 10, 100, or 300 µg/kg TCDD, and both species were sacrificed 24 h after dosing. All treatments were staggered to ensure that exposure was within 5% of the desired duration. Doses were chosen to elicit moderate hepatotoxic effects while avoiding overt toxicity in longer term studies. Animals were sacrificed by cervical dislocation and tissue samples were removed, weighed, flash-frozen in liquid nitrogen and stored at – 80°C until further use. In each study, the right lobe of the liver was fixed in 10% neutral buffered formalin (NBF, Sigma), for histological analysis.
Clinical chemistry and histological analyses.
Blood samples were collected at sacrifice by cardiac puncture and placed in Vacutainer SST gel and clot activator tubes (Becton Dickinson, Franklin Lakes, NJ). Serum was separated by centrifugation at 1500 x g for 10 min and then stored at – 80°C until analysis. As sample was limiting, only select endpoints were monitored and included blood urea nitrogen (BUN), creatinine, free fatty acids (FFA), glucose (GLU), total bilirubin (TBIL), alanine aminotransferase (ALT), cholesterol (CHOL), and triglycerides (TRIG).
Formalin fixed hepatic tissues were sectioned and processed sequentially in ethanol, xylene, and paraffin using a Thermo Electron Excelsior (Waltham, MA). Tissues were then embedded in paraffin using a Miles Tissue Tek II embedding center after which paraffin blocks were sectioned at 5 microns with a rotary microtome. Sections were placed on glass microscope slides, dried, and stained with hematoxylin and eosin. All histological processing was performed at the Michigan State University Histology Laboratory (http://humanpathology.msu.edu/histology/index.html). For Oil Red O staining, liver cryosections were fixed in NBF, stained with Oil Red O solution, and washed and counterstained with hematoxylin.
Thin layer chromatography of liver lipid extracts.
To qualitatively characterize the lipid content of the liver, samples were homogenized in methanol, acidified with HCl, and lipids extracted with chloroform:methanol (2:1) containing 1mM butylated hydroxytoluene (BHT). The protein and aqueous phases were reextracted with chloroform, and the organic phases were pooled, dried under nitrogen, resuspended in chloroform and 1mM BHT, and stored at – 80°C. Lipid extracts were then fractionated by thin-layer chromatography (TLC; LK6D Silica G 60A; Whatman Inc., Florham Park, NJ) with hexane:diethyl ether:acetic acid (90:30:1) and developed with iodine (Sigma). The location of lipids was compared with authentic standards for triacylglycerol, diacylglycerol, and CHOL ester (Nu-Chek Prep, Elysian, MN).
Quantification of TCDD in liver samples.
Liver samples were processed in parallel with laboratory blanks and a reference or background sample at Wellington Laboratories Inc., (Guelph, ON, Canada). Samples were weighed, spiked with 13C12 TCDD surrogate, digested with sulfuric acid, and extracted. Extracts were cleaned, concentrated, and spiked with an injection standard. Analysis was performed on a high-resolution gas chromatograph/high-resolution mass spectrometer (HRMS) using a Hewlett Packard 5890 Series II GC interfaced to a VG 70SE HRMS. The HRMS was operated in the electron ionization/selective ion recording mode at 10,000 resolution. A 60-m DB5 column (J&W Scientific, Folsom, CA) with an internal diameter of 0.25 mm and film thickness of 0.25 µm was utilized. Injection volumes were 2 µl and used a splitless injection.
RNA isolation.
Frozen liver samples (approximately 70 mg) were transferred to 1.0 ml of Trizol (Invitrogen, Carlsbad, CA) and homogenized using a Mixer Mill 300 tissue homogenizer (Retsch, Germany). Total RNA was isolated according to the manufacturer's protocol with an additional phenol:chloroform extraction. Isolated RNA was resuspended in RNA storage solution (Ambion Inc., Austin, TX), quantified (A260) and assessed for purity by determining the A260/A280 ratio and by visual inspection of 1.0 µg on a denaturing gel.
cDNA microarray experimental designs.
Custom rat cDNA arrays, consisting of 8567 features representing 3022 unique genes (Unigene build no. 48), and mouse cDNA arrays, consisting of 13,361 features, representing 7885 unique genes (Unigene build no. 144), were used for gene expression analysis. Temporal changes in gene expression were assessed using an independent reference design in which samples from TCDD-treated animals were cohybridized with time-matched vehicle controls. Dose-response changes in gene expression were analyzed using a common reference design in which samples from TCDD-treated mice were cohybridized with a common vehicle control. All experiments were performed with a minimum of three biological replicates with two independent labelings of each sample (incorporating a dye swap) for each time point or dose group.
cDNA microarray analysis of differential gene expression.
Detailed protocols for microarray preparation, labeling of the cDNA probe, sample hybridization, and washing can be found at http://dbzach.fst.msu.edu/interfaces/microarray.html. Briefly, PCR-amplified DNA was robotically arrayed onto epoxy-coated glass slides (Schott-Nexterion, Duryea, PA) using an Omnigrid arrayer (GeneMachines, San Carlos, CA) equipped with Chipmaker 2 pins (Telechem) at the Research Technology Support Facility (http://www.genomics.msu.edu). Total RNA (30 µg) was reverse transcribed in the presence of Cy3- or Cy5-dUTP (Amersham, Piscataway, NJ) to create fluor-labeled cDNA which was purified using a Qiagen PCR purification kit (Qiagen, Valencia, CA). Cy3 and Cy5 samples were mixed, vacuum dried, and resuspended in 48 µl of hybridization buffer (40% formamide, 4x SSC, 1% SDS) and hybridized on the array under a 22 x 60 mm lifterslip (Erie Scientific Company, Portsmouth, NH) for 18–24 h in a 42°C water bath. Slides were then washed, dried by centrifugation, and scanned at 635 nm (Cy5) and 532 nm (Cy3) on an Affymetrix 428 Array Scanner (Santa Clara, CA). Images were analyzed for feature and background intensities using GenePix Pro 5.0 (Molecular Devices, Union City, CA).
Affymetrix analysis of TCDD-mediated gene expression responses in male Sprague Dawley rats.
Due to the limited coverage of the rat cDNA array and the immaturity rat genome annotation, Affymetrix GeneChip microarray data for TCDD-mediated hepatic gene expression responses in adult male Sprague Dawley rats were obtained to facilitate a more extensive cross-species comparison. Animal housing, treatments, and Affymetrix arrays were conducted according to good laboratory practice at Gene Logic Inc., laboratories as described previously (Fletcher et al., 2005). Briefly, rats were treated with 40 µg/kg TCDD and sacrificed 6, 24, or 168 h after treatment. Changes in gene expression were monitored using Affymetrix U34A arrays which consist of 8977 probe sets representing 4928 unique rat genes.
Microarray data normalization and analysis.
All data were normalized using a semiparametric approach (Eckel et al., 2005). Model-based t values were calculated from normalized data, comparing treated and vehicle responses per time point or dose group. Empirical Bayes analysis was used to calculate posterior probabilities (P1(t) value) of activity on a per-gene and time-point or dose-group basis using the model-based t value (Eckel et al., 2004). A P1(t) cutoff of 0.9999 combined with an absolute fold change greater than 1.5 was used to identify a subset of differentially regulated genes to initially focus analysis and data interpretation on the most reproducible differentially regulated genes. Normalization and empirical Bayes analysis were performed using SAS v9.1 (SAS Institute, Cary, NC) and R v2.0.1. Gene expression changes that passed the threshold were subsequently analyzed using agglomerative hierarchical and k-means clustering using a standard correlation distance metric (GeneSpring 6.0, Silicon Genetics, Redwood City, CA). Dose-response analysis was performed using Graph Pad Prism 4.0 (GraphPad Software, San Diego, CA). Functional categorization of differentially regulated genes was performed using an in-house developed Gene Ontology tool and GOMiner (Zeeberg et al., 2003).
Quantitative real-time PCR.
Quantitative real-time PCR (QRTPCR) verification of microarray responses was performed as described previously (Boverhof et al., 2005). Briefly, 1.0 µg of total RNA was reverse transcribed by SuperScript II using an anchored oligo-dT primer as described by the manufacturer (Invitrogen). The cDNA (1.0 µl) was used as a template in a 30 µl PCR reaction containing 0.1µM of forward and reverse gene-specific primers, 3mM MgCl2, 1.0mM dNTPs, 0.025 IU AmpliTaq Gold, and 1x SYBR Green PCR buffer (Applied Biosystems, Foster City, CA). Gene names, accession numbers, forward and reverse primer sequences, and amplicon sizes are provided in Supplementary Table 1. PCR amplification was conducted on an Applied Biosystems PRISM 7000 Sequence Detection System. cDNAs were quantified using a standard curve approach and the copy number of each sample was standardized to housekeeping genes to control for differences in RNA loading, quality, and cDNA synthesis. For graphing purposes, the relative expression levels were scaled such that the expression level of the time-matched control group was equal to one.
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Identification of DREs.
Gene regulatory regions (– 10,000 relative to the transcription start site [TSS] through the 5'-untranslated region [UTR]) were obtained from the University of California, Santa Cruz, Genome Browser for rat (assembly v3.4) and mouse (build 34) genes with a mature RefSeq accession. All sequences were deposited into dbZach, an in-house data management solution (Burgoon et al., 2006
). Core DRE sequences (5'-GCGTG-3') were identified using an in-house response element application developed in Java. For identification of conserved DREs, ClustalW was used to align the regulatory regions of rat and mouse genes, and the resultant consensus sequence was scanned to identify for DREs.
Statistical analysis.
Statistical analysis, unless otherwise defined, was performed using SAS v9.1. Data were analyzed using analysis of variance followed by Dunnett or Tukey post hoc tests. Differences between treatment groups were considered significant when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAREFERENCES |
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Organ and Body Weights
Effects on liver, thymus, spleen, and body weight gain are classic rodent responses to TCDD exposure (Poland and Knutson, 1982
). Treatment of immature female Sprague Dawley rats with TCDD resulted in a significant (p < 0.05) increase in liver weight relative to time-matched vehicle controls after 72 and 168 h (Table 1). Thymus weights were significantly decreased at 72 and 168 h while spleen weights were significantly reduced at 72 h only (data not shown). Although there was no statistically significant effect on body weight, there was a significant decrease in absolute and relative body weight gain compared to vehicle controls at 72 and 168 h (Table 1). Effects on body weight are consistent with that observed in mature male Sprague Dawley rats exposed to TCDD for 168 h (Fletcher et al., 2005). In the 24-h dose-response study, TCDD induced significant increases in liver weight at 30 and 100 µg/kg and significant decreases in body weight gain at 1.0, 10, 30, and 100 µg/kg (Table 2).
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Immature female C57BL/6 mice treated with TCDD also exhibited a significant (p < 0.05) temporal increase in relative liver weight at 24, 72, and 168 h (Table 1). In the dose-response study, relative liver weights were significantly (p < 0.05) increased at 100 and 300 µg/kg (Table 2). No significant effects were noted on spleen weights, while effects on the thymus were not monitored. Unlike rats, mice did not exhibit any significant alterations in body weight or body weight gain in either the time course or dose-response studies.
Histopathology
Rats exposed to TCDD exhibited minimal to moderate hepatocellular hypertrophy in centriacinar regions at 24, 72, and 168 h. The cytoplasm of these enlarged hepatocytes was more granular and eosinophilic and less vacuolated compared to centriacinar hepatocytes of control rats (Fig. 1A and B). The severity of these lesions increased with time after exposure and are consistent with reported effects in male Sprague Dawley rats treated with TCDD (Fletcher et al., 2005). In the dose-response study, minimal to mild hepatocellular hypertrophy was observed at 30 and 100 µg/kg. No inflammatory, degenerative, or other hepatocellular lesions were microscopically evident in either study.
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In the mouse time course study, cytoplasmic vacuolization was observed in the periportal and midzonal regions with extension into the centriacinar regions at later time points. Minimal vacuolization was observed at 18 h with severity progressing from mild to moderate at 24 and 72 h, respectively. Marked cytoplasmic vacuolization was noted at 168 h and was accompanied by individual cell apoptosis and foci of mixed populations of inflammatory cells consisting mainly of mononuclear cells and a smaller number of neutrophils (Fig. 1D and E). In the dose-response study, minimal cytoplasmic vacuolization was noted in two of five mice at 0.1 µg/kg with mild to moderate vacuolization observed in mice at higher doses. Oil Red O staining indicated that the vacuolization was due to lipid accumulation (Fig. 1C and F), and TLC analysis of liver lipid extracts revealed a 2.5-fold increase in liver TRIG at 168 h in mice while no change was observed in rats (data not shown).
Clinical chemistry
TCDD treatment resulted in a significant (p < 0.05) increase in rat serum CHOL (30%), FFA (73%), and TRIG (200%) at 24 h only (Fig. 2). Serum GLU levels were decreased at 72 and 168 h, although this did not achieve statistical significance. Effects on these endpoints are consistent with effects reported in male Sprague Dawley rats treated with TCDD (Fletcher et al., 2005). There were no treatment-related alterations in serum ALT, BUN, or TBIL.
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In the mouse, significant treatment related alterations were noted for serum ALT, CHOL, FFA, and TRIG (Fig. 2). ALT levels increased steadily after 24 h to a maximum of 260% relative to time-matched vehicle controls at 168 h, indicative of mild liver injury. Serum CHOL was significantly (p < 0.05) decreased by 33 and 28% at 72 and 168 h, respectively, while serum FFA were increased 33, 16, and 28% at 24, 72, and 168 h, respectively. TRIG levels were elevated by 24, 15, and 40% in TCDD-treated mice at 24, 72, and 168 hrs, respectively. No significant treatment related effects were noted on serum BUN, GLU, or TBIL.
Hepatic Concentrations of TCDD
Hepatic levels of TCDD were determined in hepatic samples from the time course study, in which rats and mice were dosed with 10 and 30 µg/kg TCDD, respectively, in order to relate tissue concentrations to molecular responses. At the 4- and 12-h time points, hepatic concentrations were similar in rats and mice (Table 3). Hepatic levels in rats plateaued within 12 h while levels in mice continued to increase and were maximal at 72 h. Both species exhibited 50% decreases in tissue levels between 72 and 168 h. Differences between the rat and the mouse in this study are likely due to differences in the dose administered as well as differences in absorption and hepatic elimination or sequestering; however, the overall tissue levels of TCDD were comparable. Hepatic levels in these studies are comparable to other reports using similar exposure regimens. For example, 102 ppb TCDD was reported in rat hepatic tissue 24 h after an oral dose of 10 µg/kg while we observed levels of 131 ppb in rats (Wang et al., 1997). In mice, 54 ppb TCDD was detected in the liver 168 h following acute administration of 10 µg/kg (Diliberto et al., 1995), while we report 60 and 103 ppb TCDD in rats and mice, respectively.
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Identification of Differentially Expressed Genes
Examination of temporal hepatic gene expression responses to TCDD in the rat was performed using a custom rat cDNA microarray containing 8567 features representing 3022 unique genes. Empirical Bayes analysis identified 467 features, representing 221 unique genes, which were differentially expressed (P1(t) > 0.9999 and |fold change| > 1.5) relative to vehicle controls, at one or more time points (Fig. 3). TCDD-mediated hepatic gene expression responses in the mouse were monitored using a cDNA microarray containing 13,361 features representing 7885 unique mouse genes. Analysis of these data identified 669 microarray features, representing 542 unique differentially expressed genes (P1(t) > 0.9999 and |fold change| > 1.5) (Fig. 3). Comparison of temporal expression patterns for differentially regulated genes in each species revealed similar categories which included upregulated early, upregulated sustained, downregulated early, and downregulated late responses (data not shown). The exception was an upregulated late category which was primarily observed in the mouse consisting of numerous genes involved in lipid accumulation and inflammatory responses.
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Due to the limited coverage of our rat cDNA microarray and the immaturity of the annotation for the rat genome, TCDD-mediated hepatic gene expression responses from male Sprague Dawley rats were incorporated to facilitate a more comprehensive cross-species comparison (Fletcher et al., 2005
). TCDD-mediated gene expression responses for this study were performed using the Affymetrix U34A GeneChip microarray consisting of 8977 probe sets representing 4928 unique genes. Empirical Bayes analysis identified 169 probe sets, representing 130 unique genes, which were differentially expressed (P1(t) > 0.9999 and |fold change| > 1.5) (Fig. 3). Complete data sets for each study can be found in Supplementary Tables 2–4
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Cross-Species Comparison of Gene Expression Responses
In order to effectively compare TCDD-mediated gene expression responses in the rat and mouse, orthologous genes were first identified using HomoloGene (http://www.ncbi.nlm.nih.gov/HomoloGene/). Collectively, the cDNA and Affymetrix platforms represented 6423 unique rat genes which were compared to the 7885 unique genes represented on the mouse cDNA array to identify 3087 unique orthologous genes. Examination of this list of orthologous genes for TCDD-mediated responses (P1(t) > 0.9999 and |fold change| > 1.5) identified 201 and 238 unique rat and mouse genes, respectively. Comparison of these responses identified 33 differentially expressed genes which were common between the two species while 185 and 225 were specific to the rat and mouse, respectively (Fig. 4). A discrepancy exists in the number of genes reported in the Venn diagram (i.e., the sum of the genes in the Venn diagram is greater than the input) due to the fact that unique genes were represented by multiple cDNAs or probe sets on a given array, some of which passed the filtering criteria while others did not. For example, histidine ammonia lyase (Hal), which was downregulated on the mouse cDNA array, was represented on the rat Affymetrix GeneChip by three different probe sets, all of which showed a downregulated pattern of expression; however, only one passed the filtering criteria (P1(t) > 0.9999 and |fold change| > 1.5). Therefore, Hal was included in the list of common responses as well the list of mouse-specific genes as the mouse response is compared to each rat probe set. This example also indicates that although a gene may not pass the filtering criteria it may exhibit a similar response to its active ortholog. Therefore, to be more inclusive, genes classified as species specific were investigated for cross-species similarity, and if the response in the alternate species approached our filtering criteria (P1(t) > 0.99 and |fold change| > 1.25), the genes were reclassified as common. These efforts resulted in the identification of 111 unique genes which were classified as common responses to TCDD in hepatic tissue from rats and mice (Supplementary Table 5). Although these 111 genes were differentially expressed in both species, they did not necessarily display a similar directional or temporal pattern of regulation. In total, 79 genes displayed similar directional responses, whereas 32 displayed divergent responses.
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Functional Categorization of Microarray Data
Functional annotation of the TCDD-mediated changes in gene expression revealed that many of the commonly regulated genes were associated with responses to chemical/xenobiotic stimuli, nitrogen/amino acid metabolism, and lipid metabolism (Table 4). Genes involved in a chemical/xenobiotic stimulus response included a number of phase I and II metabolizing enzymes such as the well-characterized TCDD-inducible genes cytochrome P450 1a1 (Cyp1a1) and NAD(P)H dehydrogenase-quinone 1 (Nqo1) as well as more novel genes including abhydrolase domain-containing 5 (Abhd5), carbonic anhydrase 3 (Car3), epoxide hydrolase 1 (Ephx1), P450 cytochrome oxidoreductase (Por), thioredoxin reductase 1 (Txnrd1), and UDP-glucose dehydrogenase (Ugdh). Genes involved in nitrogen/amino acid metabolism included asparagine synthetase (Asns), glutamate-cysteine ligase (Gclc), glutamate dehydrogenase (Glud1), glutamate ammonia ligase (Glul), glutamic pyruvic transaminase 1 (Gpt1), and Hal. The regulation of these genes is consistent with previous reports of TCDD-mediated alterations in circulating amino acids (Viluksela et al., 1999
). Conserved lipid metabolism responses included fatty acid–binding proteins 4 and 5 (Fabp4 and 5), fatty acid synthase (Fasn), fatty acid desaturase 1 (Fads1), elongation of long-chain fatty acids 5 (Elovl5), and hepatic lipase (Lipc). Collectively, alterations on amino acid and lipid metabolism may be involved in the effects of TCDD on intermediary metabolism and inhibition of gluconeogenesis (Christian et al., 1986; Viluksela et al., 1999). Functional categorization of rat-specific responses revealed a number of genes involved in cellular growth and lipid metabolism (Table 5). Cellular growth genes included cyclin-dependent kinase 4 (Cdk4), fibroblast growth factor receptor 3 (Fgfr3), p21-activated kinase 1 (Pak1), protein phosphatase 2a (Ppp2ca), and sphingosine kinase 1 (Sphk1). Deregulated expression of these genes may be involved in the observed hepatocyte hypertrophy specific to the rat. Despite the lack of lipid accumulation, a number of lipid metabolism genes were specific to the rat including branched chain ketoacid dehydrogenase (Bckdha), carnitine palmitoyltransferase 1a (Cpt1a), guanidinoacetate methyltransferase (Gamt), diacylglycerol kinase (Dgka), forkhead box A3 (Foxa3), and paraoxonase 1 (Pon1).
Mouse-specific gene expression responses were involved in lipid metabolism/binding and immune responses (Table 6). Genes involved in lipid metabolism/binding included the upregulation of acyl-CoA thioesterase 7 (Acot7), Cd36 antigen (Cd36), lipoprotein lipase (Lpl), sterol-C4-methyl oxidase-like (Sc4mol), and very low-density lipoprotein receptor (Vldlr). Regulation of these genes may be involved in mediating the observed liver TRIG/fatty acid (FA) accumulation. Inflammatory response genes included CD53 antigen (Cd53), CD3 antigen (Cd3d), complement component 1 polypeptides (C1qa and C1qb), granzyme A (Gzma), integrin beta 1 (Itgb1), and histocompatibility 2 antigens A and E (H2-Aa, H2-Ab1, and H2-Eb1). Induction of these genes is coincident with the hepatic inflammatory response which was only observed in the mouse.
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Verification of Microarray Responses
QRTPCR was used to verify changes in transcript levels for a selected subset of differentially expressed genes (Fig. 5). In total, 16 rat and 27 mouse genes were verified by QRTPCR, all of which displayed temporal expression patterns consistent with the microarray data (See Supplementary Table 1 for complete list of genes). Conserved rat-mouse responses for Cyp1a1, Nqo1, Fabp5, Por, solute carrier family 20, member 1 (Slc20a1), and Ugdh were verified by QRTPCR. In addition, divergent or oppositely regulated gene expression responses were verified including cathepsin L (Ctsl) and glutamate oxaloacetate transaminase 1 (Got1) both of which were downregulated in the mouse and upregulated in the rat. Species-specific responses were also verified by QRTPCR including Cpt1a in the rat and Cd36 and Lpl in the mouse. In general, there was a good agreement between the temporal gene expression patterns of the microarray and QRTPCR data. The microarray induction profile of Por was confirmed by QRTPCR and approached, but did not reach, statistical significance due to temporal variability in the vehicle group. Microarray data compression was evident for genes such as Cyp1a1 due to the smaller dynamic fluorescence intensity range (0–65,535) of the microarrays which resulted in signal saturation and compression of the true induction. Cross-hybridization of homologous probes to a given target sequence on the microarray may also be a contributing factor especially in comparison to other, more gene-specific, measurement techniques such as QRTPCR (Yuen et al., 2002
).
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Dose-Response Analysis
Comparison of the dose-response and temporal data within each species revealed a high correlation (r > 0.95) between gene expression responses at their respective doses and time points. These data indicate the reproducibility of these responses across independent experiments for each species. Comparison of dose-response data across species for commonly regulated genes did not reveal any overall differences in the sensitivity to gene expression regulation across species which was verified by QRTPCR for Cyp1a1 and Nqo1. Both genes were similarly induced in each species with Cyp1a1 displaying ED50 values of 0.49 and 0.38 µg/kg in rats and mice, respectively, while Nqo1 exhibited ED50 values of 4.85 and 8.81 µg/kg, respectively (Fig. 6). These results suggest similar sensitivity for common TCDD-mediated gene expression responses across these species.
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Identification of Conserved Putative DREs
The 111 genes classified as commonly regulated between the rat and mouse were scanned for the DRE core sequence (5'-GCGTG-3') in the range of – 10,000 relative to the TSS through the 5'UTR. The gene regulatory sequences were obtained for all 111 mouse genes but only 95 rat genes due to the incomplete annotation for this genome. The analyses revealed that 94 rat and 110 mouse genes possessed one or more DRE core elements with 94 possessing a DRE in both the rat and mouse. Cross-species alignments revealed that 53 of the 94 genes contained one or more positionally conserved DREs (Table 7 and Fig. 7) which have a higher likelihood of being functional due to their evolutionary conservation (Frazer et al., 2003
; McGuire et al., 2000). This included a number of genes previously shown to be regulated in response to AhR ligands including Car3 (Ikeda et al., 2000), Igf1 (Croutch et al., 2005), insulin-like growth factor–binding protein 1 (Igfbp1) (Marchand et al., 2005), Pck1 (Stahl et al., 1992), and Ugdh (Sun et al., 2004). The conserved putative DREs in these genes represent important starting points for investigation into their TCDD- and AhR-mediated regulation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAREFERENCES |
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The present study used a comparative toxicogenomic approach to assess the physiological and hepatic gene expression responses to TCDD in Sprague Dawley rats and C57BL/6 mice. The results indicate a number of conserved and species-specific gene expression responses which were consistent with the observed physiological responses and published data. As previous studies have reported the TCDD-mediated hepatic gene expression responses of rats and mice individually (Boverhof et al., 2005
; Fletcher et al., 2005), the focus of this report is on the similarities and differences between these species.
Conserved Gene Expression Responses between Rats and Mice
Several conserved changes in gene expression were associated with responses to chemical or xenobiotic exposure and included known members of the AhR gene battery such as Aldh3a1, Cyp1a1, Nqo1, and Gsta2 (Nebert et al., 2000) as well as novel TCDD-mediated gene expression responses. For example, Por, which transfers electrons from NADPH to P450 enzymes (Wang et al., 2005), was induced in both rats and mice consistent with the AhR-mediated induction of a wide range of cytochrome P450 enzymes. Ephx1 and Ugdh were commonly upregulated and encode enzymes involved in phase I and II detoxification reactions (Miyata et al., 1999; Vatsyayan et al., 2005). Hmox1 and Txnrd1 were also commonly induced, consistent with their roles in protecting cells from oxidative damage and their regulation by oxidative stress (Malaguarnera et al., 2005; Xia et al., 2003). TCDD suppressed Car3 in both rats and mice, in agreement with its regulation by PCB126 and 3-MC in the rat hepatic tissue and primary hepatocytes, respectively (Ikeda et al., 2000; Ishii et al., 2005). Car3 overexpression has been shown to reduce hydrogen peroxide–induced ROS formation and apoptosis (Raisanen et al., 1999), and downregulation by AhR ligands may create an environment more susceptible to oxidative stress. Furthermore, recent comparative studies in rats indicate that Car3 is downregulated in the TCDD-sensitive Long-Evans strain but not in the resistant Han/Wistar strain, suggesting a role in susceptibility to toxicity (Pastorelli et al., 2006).
The regulation of each of these genes occurred within 8 h of exposure suggesting primary AhR-mediated responses. Many also possess one or more conserved DREs which may be targets for AhR binding. The exceptions were Ephx1 and Hmox1 which may be regulated through an alternate mechanism such as the antioxidant response element via Nrf2 (Lee et al., 2003). Alternatively, their genomic regulatory regions may currently be improperly annotated which would preclude proper genomic alignments and conserved DRE identification, as is the case for Cyp1a1 (Burgoon and Zacharewski, 2006). Regardless, their conserved regulation suggests that they are not likely to be involved in the species-specific responses to TCDD. This is supported by the dose-response behavior for Cyp1a1 and Nqo1 which displayed no difference in sensitivity to TCDD across these species. Collectively, the regulation of this class of genes is consistent with the biological role of the AhR in mediating an adaptive metabolic response (Bunger et al., 2003) and has expanded our understanding of the AhR gene battery and its conservation across species.
Energy metabolism in the liver involves the interconversion of lipids, carbohydrates, and amino acids which is regulated by multiple factors including allosteric effectors, substrate availability, and hormones (She et al., 2000). TCDD disrupts intermediary metabolism, and comparative studies indicate a common alteration of genes associated with amino acid metabolism. This is consistent with previous reports of TCDD-mediated alterations in circulating amino acids (Viluksela et al., 1999) and included the downregulation of a number of genes involved in glutamate metabolism. Glutamate plays a central role in intermediary metabolism (Yang and Brunengraber, 2000), and the downregulation of these genes may be a contributing factor to TCDD's inhibitory effects on gluconeogenesis. Additional genes associated with amino acid metabolism were also downregulated in rats and/or mice, and many are involved in glutamate, cysteine, and glycine metabolism including Glud1, Glul, Gpt1, cytolosic cysteine dioxygenase (Cdo1), D-amino acid oxidase (Dao1), glycine N-methyltransferase (Gnmt), and glycine decarboxylase (Gldc). In addition to mediating effects on gluconeogenesis, these genes may be downregulated to conserve the amino acid building blocks of glutathione (GSH). Consistent with this, the enzymes required for GSH synthesis, Gclc and glutathione synthetase (Gss), were both upregulated in rats and mice, as were a number of glutathione S-transferase conjugating enzymes. The importance of amino acids and GSH in cellular redox status has been well characterized (Mates et al., 2002), and the downregulation of these amino acid metabolizing genes combined with the upregulation of enzymes involved in GSH synthesis and conjugation would create an adaptive environment to TCDD-mediated oxidative stress. This is consistent with AhR-dependent increases in GSH and decreases in the GSH/glutathione disulfide ratio after TCDD exposure (Shen et al., 2005). However, few of these genes possess conserved DREs suggesting that they are secondary to TCDD-mediated oxidative stress which is supported by toxicogenomic studies that indicate similar gene regulation by diverse chemical inducers of hepatic oxidative stress (Heijne et al., 2004, 2005; Huang et al., 2004; McMillian et al., 2004). These results strongly suggest that the alteration of amino acid metabolizing genes may be related to TCDD-mediated oxidative stress.
In addition to the effects on amino acid metabolism, a number of genes involved in lipid metabolism were commonly regulated including the induction of Fabp4 and 5 and Elovl5. Fabp4 and 5 are lipid-binding proteins which play key roles in promoting FA uptake and metabolism (Simpson et al., 1999). Recent reports also suggest that Fabp5 may function as a protective antioxidant protein by scavenging reactive lipids, consistent with its early induction by TCDD (Bennaars-Eiden et al., 2002). Elovl5 encodes FA elongase, and additional research has indicated that the activity of this enzyme is also induced by TCDD (data not shown).
A number of genes in this category were also commonly downregulated including Fasn, Fads1, Lipc, and phosphoenolpyruvate carboxykinase 1 (Pck1). Fasn is involved in the de novo synthesis of FA, thereby playing an important role in energy homeostasis and its activity has previously been reported to be repressed by TCDD (Lakshman et al., 1989). Lipc is a lypolytic enzyme found at hepatic sinusoidal surfaces which influences lipid metabolism and uptake by affecting the phospholipid, TRIG, and CHOL content of lipoproteins (Perret et al., 2002). Pck1 is a key gluconeogenic enzyme; however, recent studies indicate that mice with diminished Pck1 activity display profound abnormalities in lipid metabolism characterized by increases in circulating FFA and TRIG and hepatic TRIG accumulation (She et al., 2000). In combination, the dysregulation of these genes may play a role in the observed alterations in serum TRIG, FFA, and CHOL and, ultimately, in the toxic manifestations of TCDD such as the wasting syndrome. The common regulation of these genes also supports a biological role for the AhR in FA and lipid homeostasis consistent with the microvesicular fatty metamorphosis phenotype observed in AhR-null mice (Schmidt et al., 1996).
Several responses which did not fit into an overrepresented functional category were also commonly regulated including the downregulation of Igf-1 and upregulation of Igfbp1. The regulation of these genes is consistent with previous studies in human hepatoma cells and rats in vivo which suggested that these responses may contribute to alterations in growth, reproduction, and GLU homeostasis (Croutch et al., 2005; Marchand et al., 2005). Comparative genomics revealed that each gene possesses a single conserved DRE which may be involved in mediating the response to TCDD.
Although not orthologous genes, rat Notch2 and mouse Notch1 exhibited comparable temporal expression patterns in response to TCDD which were verified by QRTPCR. Notch genes encode transmembrane proteins involved in controlling cell fate decisions during embryonic development (Lai, 2004), and their deregulated expression may contribute to the teratogenic effects of TCDD. Examination of rat Notch1 by QRTPCR revealed a similar but nonsignificant temporal induction when compared to mouse Notch1, while mouse Notch2 was not induced by TCDD. Therefore, genes within this family are commonly regulated by TCDD although gene orthologs are not similarly responsive. Dissimilar expression patterns between orthologous genes have previously been reported and may indicate that these genes are not true functional orthologs (Zhou and Gibson, 2004).
Rat-Specific Responses
Functional annotation of rat-specific responses identified genes involved in cellular growth responses including Cdk4, Fgfr3, Pak1, Ppp2ca, and Sphk1. Cdk4 and Ppp2ca were upregulated and play important roles in the regulation of cell cycle and growth. Fgfr3 was repressed and is involved in the negative regulation of bone growth (Nakajima et al., 2003) and has been implicated in the etiology of hepatocellular carcinoma (Shao et al., 2005). Pak1, a regulatory enzyme involved in cell growth and morphogenesis as well as stress responses, was induced early (Sells and Chernoff, 1997). Sphk1, which catalyzes the synthesis of sphingosine-1-phosphate, a signaling molecule involved in cell growth, proliferation, survival, and morphogenesis, was also upregulated (Allende et al., 2004). Although evidence of hyperplasia was not observed in the rat liver, the regulation of these genes, combined with hepatic stress, may contribute to the observed hypertrophic response as well as the carcinogenic potential of TCDD.
Genes uniquely regulated in the rat were also involved in lipid metabolism responses including Cpt1a, Foxa3, and Gamt. Liver Cpt1a catalyzes the rate-controlling transfer of long-chain FA into mitochondria for beta-oxidation (Louet et al., 2002), and its deregulated expression is associated with altered food intake and body weight (Bonnefont et al., 2004; Pocai et al., 2006). QRTPCR verified the early induction of Cpt1a in the rat while it was not induced in the mouse. Gamt is involved in creatine biosynthesis and was downregulated by TCDD. Deficiency of this gene is associated with metabolic disorders including decreased body weight due to reduced body fat mass (Schmidt et al., 2004). Foxa3, which was also downregulated, plays a key role in GLU homeostasis during fasting through the regulation of Glut2 expression with null mutations resulting in decreased blood GLU concentrations (Shen et al., 2001). Collectively, the deregulated expression of these genes may play a contributing role in the alterations in serum GLU and decreases in body weight gain which were confined to the rat.
Mouse-Specific Responses
Histological examination of mouse hepatic tissue revealed vacuolization due to TRIG/FA accumulation and inflammatory cell accumulation with apoptosis. Consistent with this, a number of mouse-specific gene expression responses were associated with lipid binding and metabolism including the upregulation of Acot7, Cd36, Lpl, Sc4mol, and Vldlr. Acot7 is a member of a group of enzymes that catalyze the hydrolysis of acyl-CoAs to the FFA and coenzyme A to regulate their intracellular levels (Hunt et al., 2002). Cd36, also known as FA transporter, is a receptor for high-affinity uptake of long-chain FA. Null mutations of this gene result in reduced FA uptake, while overexpression increases FA uptake and metabolism (Bonen et al., 2004; Febbraio et al., 1999). Lpl functions in TRIG and chylomicron metabolism and as a bridging factor for lipoprotein uptake (Weinstock et al., 1995). Previous reports have shown that TCDD reduces Lpl activity in guinea pig adipose tissue (Brewster and Matsumura, 1984), suggesting a potential tissue- or species-specific effect. Mutations in Sc4mol result in altered lipid metabolism and the accumulation of FA and TRIG (Li and Kaplan, 1996) while Vldlr mediates the internalization and degradation of TRIG-rich lipoproteins and is required for optimal Lpl activity (Yagyu et al., 2002). Collectively, the regulation of these genes may play an important role in mediating the increased uptake and accumulation of hepatic TRIG and FA.
A second functional category confined to the mouse involved genes associated with immune responses. The regulation of this category was primarily observed at the late time points consistent with histological observations of immune cell accumulation and apoptosis at 168 h. These genes included several cluster of differentiation and lymphocyte antigens (Cd and Ly antigens), complement components (C1qa and C1qb), and major histocompatabilty complex (MHC) molecules. Cd and Ly antigens are surface molecules on hematopoietic cells important for immune signaling functions (Lai et al., 1998; Sumoza-Toledo and Santos-Argumedo, 2004). C1q components are members of the classical pathway of the immune complement response involved in apoptotic cell clearance. H2-Ab1 and H2-Eb1 belong to the MHC class II and are involved in antigen presentation and processing (Alfonso et al., 2001). These changes in gene expression are likely a secondary response to hepatic damage mediated by ROS or fatty accumulation as induction was concurrent with histological detection of immune cell infiltration and apoptosis. This is consistent with the increases in serum ALT which were observed in the mouse and were unaltered in the rat.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAREFERENCES |
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The present study identified several conserved and species-specific hepatic gene expression responses which were phenotypically anchored to the physiological, histopathological, and clinical chemistry effects elicited by TCDD. Conserved gene expression responses were associated with xenobiotic and chemical stress consistent with the role of the AhR in mediating adaptive metabolic responses. Common responses were also associated with alterations in intermediary metabolism including amino acid and lipid metabolism. Rat-specific responses were related to cellular growth and lipid metabolism which may be involved in the observed physiological alterations on hepatocyte hypertrophy and body weight. Meanwhile, mouse-specific responses were phenotypically anchored to TRIG and FA accumulation and immune responses. Overall, despite the conservation in AhR biology across the mouse and rat, these data indicate that differences in TCDD-mediated hepatotoxicity may be mediated by different gene expression profiles, potentially through species-specific AhR regulons.
Many factors complicate comparative toxicology studies including differences in absorption, distribution, metabolism, and elimination as well as age and gender. Comparative toxicogenomic studies face additional difficulties which can limit a complete and comprehensive assessment of the data. One obvious difficulty stems from differences in genes represented on array platforms for each species which limits the number of comparisons that can be made. This is further complicated by the incomplete and unstable nature of annotation for the rat genome (Boverhof and Zacharewski, 2006). In addition, array probes for orthologous genes may represent different transcript regions which can limit the ability to detect and compare expression responses. Different responses may also be due to different levels of basal gene expression between these species which could dictate the overall magnitude and direction (induced or repressed) of TCDD's modulating effect. Furthermore, genes currently annotated as orthologs may not be functional orthologs and, as such, may not exhibit similar expression responses. All these factors are confounding variables in the definitive assignment of these genes as species-specific responses to TCDD. Examination of additional target tissues, more extensive cross-species comparisons, and meta-analysis of existing data will further highlight and substantiate TCDD's species-specific gene expression responses which should be characterized in light of the physiological, molecular, and genomic variations between species when deciphering their roles in toxicity. As the technology advances, toxicogenomic comparisons between rodent and human models of toxicity will help explain species-specific toxicity and susceptibility, thereby decreasing the uncertainties in current risk assessment extrapolation practices.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSSUPPLEMENTARY DATAREFERENCES |
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Supplementary Tables 1–5 are available online at http://toxsci.oxfordjournals.org./
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ACKNOWLEDGMENTS |
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We thank Jeremy Burt, Ed Dere, and Josh Kwekel for critical reading of this article. This work was supported by funds from National Institute of Health grant R21-GM75838.
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