ToxSci Advance Access originally published online on September 19, 2006
Toxicological Sciences 2006 94(2):428-438; doi:10.1093/toxsci/kfl111
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Hepatic Gene Downregulation following Acute and Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin
* Department of Pharmacology and Toxicology, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York 14214
School of Pharmacy and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705
1 To whom correspondence should be addressed at Department of Pharmacology and Toxicology, University at Buffalo, 102 Farber Hall, 3435 Main Street, Buffalo, NY 14214. E-mail: jolson{at}buffalo.edu.
Received September 8, 2006; accepted September 13, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to lead to the development of hepatotoxicity and carcinogenicity in the liver of female rats. In this study, we investigated hepatic gene downregulation in response to acute and subchronic TCDD exposure. We identified 61 probes which exhibited a downregulation of twofold or greater following subchronic (13 weeks) exposure to TCDD. Comparative analysis of the hepatic expression of these 61 probes was conducted with rats subchronically exposed to PeCDF, PCB126, PCB153, and a mixture of PCB126 and PCB153. PCB153 produced little or no alteration in these probes, while the binary mixture mimicked most closely the downregulation observed with TCDD. To discern if the repression of genes within this probe set occur as a primary response to TCDD exposure, we analyzed the early responsiveness of 11 genes at 6, 24, and 72 h following a single exposure to TCDD. We observed early repression of the 11 genes within this early time course, indicating that the repression of this subset of genes occurs as a primary response to TCDD exposure and not as a secondary response to 13 weeks of subchronic treatment. In addition, the gender, species, and AhR dependence of these responses were also investigated. Gender- and species-dependent repression was observed within this subset of genes. Furthermore, utilizing AhR knockout mice, we were able to determine the AhR-dependent downregulation of seven of 11 genes. Together these results assist efforts to understand the multitude of effects imposed by TCDD and AhR ligands on gene expression.
Key Words: TCDD; PCB126; PCB153; AhR; downregulation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a persistent organic pollutant (POP) which has been associated with the development of numerous adverse biological effects including dermatotoxicity, immunosuppresion, developmental toxicity, carcinogenesis, and hepatotoxicity (Birnbaum and Tuomisto, 2000
; NTPa, 2004; Sweeney and Mocarelli, 2000). In an effort to evaluate the toxic and carcinogenic effects of chronic exposure to TCDD and related dioxin-like compounds, the National Toxicology Program (NTP) conducted a 2-year cancer bioassay in which they observed a number of hepatotoxic effects, in addition to hepatocellular adenoma and cholangiocarcinoma, in female Harlan Sprague-Dawley rats following 2 years of chronic exposure to TCDD (100 ng/kg/day, po) (NTPa, 2004).
Most, if not all, of the toxic effects of TCDD are mediated through the binding and activation of the aryl hydrocarbon receptor (AhR). This cytosolic ligand-activated transcription factor belongs to the basic helix-loop-helix/Per, ARNT/AhR, SIM homology (bHLH/PAS) protein family (Denison et al., 2002). Following nuclear localization and heterodimerization with the AhR nuclear translocator (ARNT), the ligand-activated AhR/ARNT complex acts to regulate gene expression through the binding of the nucleotide recognition sequence, the dioxin response element (DRE) (Swanson, 2002). It is these changes in gene expression and function resulting from AhR activation which are believed to be a contributing factor to the development of hepatotoxicity, carcinogenicity, and other responses of TCDD exposure.
To identify genes that may be contributing to the hepatotoxic effects of chronic dioxin exposure, we investigated global hepatic gene expression in female rats following subchronic (13 weeks) exposure to the AhR ligands TCDD (TEF = 1.0), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF, TEF = 0.5), and 3,3',4,4',5-pentachlorobiphenyl (PCB126, TEF = 0.1); and the non-AhR ligand 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153, TEF = 0) (Van den Berg et al., 1998; Vezina et al., 2004). Following 13 weeks of subchronic exposure to TCDD (100 ng/kg/day, po), rat livers were void of neoplastic lesions but exhibited a significant increase in liver hypertrophy (NTPa, 2004). In addition, there was a nonsignificant increase in the incidences of multinucleated hepatocytes and diffuse fatty change (NTPa, 2004). Gene array analysis of hepatic tissue of subchronically treated animals identified numerous genes, both classical- and novel-dioxin responsive genes, which exhibited altered expression (Vezina et al., 2004). A portion of these genes exhibited decreased expression following 13 weeks of subchronic exposure to TCDD. These genes include, but are not exclusive to, Serpina7 (serine peptidase inhibitor, clade A member 7), Cyp3a13 (cytochrome P450 3a13), and Ces3 (carboxylesterase 3) (Vezina et al., 2004). The downregulation of Ces3, Cyp3a13, and Serpina7 was confirmed utilizing real-time qPCR (Vezina et al., 2004). Ces3, a triglyceride hydrolyzing enzyme which is thought to play a role in basal lipid metabolism (Soni et al., 2004), exhibited a fivefold decrease in expression (Vezina et al., 2004). Cyp3a13, a female dominant steroid hormone–metabolizing enzyme (Wang et al., 2000) exhibited a 1000-fold decrease (Vezina et al., 2004). Serpina7 which encodes for the glycoprotein, thyroxine-binding globulin, the principle thyroid hormone carrier in human serum (Schussler, 2000) was decreased 27-fold (Vezina et al., 2004).
The absence of hepatic neoplasia in subchronically treated animals indicates that the downregulation in gene expression seen at this time point precedes the development of liver cancer, suggesting that altered regulation of these genes occurs as a response to dioxin exposure and not as a result of marked alterations to liver function resulting from carcinogenicity. These results, however, do not discern whether the downregulation of these genes occurs as a primary response to dioxin exposure or as a secondary response resulting from subsequent mild biological and toxicological responses occurring during the 13-week treatment period. The present study addresses this question by evaluating the early responsiveness of genes found to be down regulated following subchronic (13 weeks) exposure. To further elucidate the regulation of these responses, the AhR, species, and gender dependency of this dioxin-induced gene downregulation was also evaluated. Together, these studies will extend our understanding of the genes that are down regulated through the AhR signaling pathway and ultimately assist in efforts to better understand the mechanisms for the toxicological effects of dioxin and related compounds.
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MATERIALS AND METHODS |
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Animals and acute treatments.
Harlan Sprague-Dawley rats (200–249 g) were obtained from Harlan Inc (Indianapolis, IN). Female and male rats ranged from 65 to 75 days and 49 to 58 days of age, respectively. Sexually mature female C57BL/6J wild type (AhR+/+) and knockout (AhR–/–) mice were a generous gift from Richard Peterson (Univeristy of Wisconsin, Madison) and originally described by Schmidt et al. (1996)
. Female and male Sprague-Dawley rats were treated with a single dose of 5 µg/kg TCDD in corn oil, po. Control animals were administered an equivalent volume of corn oil. Mice were treated with a single dose of 25 µg/kg TCDD, po. Animals were maintained in normal dark and light cycles and received food and water ad libitum. Sprague-Dawley rats were sacrificed at 6, 24, and 72 h postexposure. Mice were sacrificed at 72 h postexposure. Hepatic tissue was harvested from animals and stored at – 80°C.
Subchronic NTP study.
Liver tissue obtained for gene array analysis was provided for by the NTP. The storage and treatment of these samples were as described in Vezina et al. (2004). Tissues were obtained from a 2-year cancer bioassay investigating the relative carcinogenic potencies of the AhR ligands TCDD, PeCDF, and 3,3',4,4',5-pentachlorobiphenyl (PCB126); the non-AhR ligand 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153); and the binary mixture of PCBs 126 and 153 (NTPa, 2004; NTPb, 2004; NTPc, 2004; NTPd, 2004; NTPe, 2004; Walker et al., 2005). Female Harlan Sprague-Dawley rats were exposed for 13 weeks, 5 days a week via oral gavage to toxic equivalent doses of TCDD (100 ng/kg/day), PeCDF (200 ng/kg/day), PCB126 (1000 ng/kg/day), PCB153 (1000 µg/kg/day), the binary mixture of PCBs 126 (1000 ng/kg/day) and 153 (1000 µg/kg/day), or a vehicle control of corn oil:acetone (99:1). The toxicologic dose equivalence was based on the World Health Organization toxic equivalence factor recommendations (Van den Berg et al., 1998). Harvested liver samples were removed, flash frozen in liquid nitrogen, and stored at – 80°C.
TCDD dosimetry analysis.
Female Sprague-Dawley rats (n = 3) were administered 2 µCi of 3H-TCDD at a dose of 5 µg/kg TCDD via oral gavage. At 6 h postexposure, animals were sacrificed and tissues were harvested. Tissues (50–100 mg) were digested in 1.0 ml of 0.5 N NaOH at 50°C, mixed with 100 µl of 30% H2O2, and neutralized with 5 N HCl. TCDD-derived 3H was measured in tissue digests using a Wallac 1409 liquid scintillation counter and expressed as the percentage of the administered dose per gram of tissue.
Gene microarray data analysis.
RNA was isolated and hybridized as described previously (Vezina et al., 2004). Cell intensity files (.CEL) for treated (n = 3) and control (n = 6) groups were generated with Affymetrix Microarray Suite (MAS) 5.0 software (Affymetrix Inc., Santa Clara, CA). Probe-level data were background subtracted and normalized by the GC-Robust Multiarray Analysis (gcRMA) method using GeneTraffic software (Iobion Informatics LLC). Probe-level intensity values were used to calculate changes in gene expression between treated and control groups. Hierarchical clustering and Principle Component Analysis (PCA) were conducted using TIGR Microarray Experiment Viewer (Saeed et al., 2003). Gene annotation and gene symbols were obtained through the Affymetrix NetAffx Analysis Center. A complete summary of gene microarray data is available through the Gene Expression Omnibus at the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/geo/index.cgi, as accession number GSE5789.
RNA isolation and cDNA synthesis for real-time qPCR.
Hepatic tissue (30–50 mg) was transferred from – 80°C storage to 1 ml of RNAlater Ice (Ambion Inc, Austin, TX) and stored at – 20°C for 24 h. RNA was isolated from tissue samples following the instructions provided for the Qiagen RNeasy Mini kit (Qiagen Inc, Valencia, CA). RNA was subjected to RT-PCR. A total of 20 µg RNA was mixed with 4 µl of 10mM dNTPs, 12 µl of random primers 100 ng/µl (Invitrogen Inc, Carlsbad, CA), and incubated at 60°C for 5 min followed by immediate incubation on ice. To this nucleotide mixture, 16 µl of 5x first strand buffer, 8 µl of 0.1M DTT, 0.66 µl of RNAout (40 U/µl), 4 µl of superscript II reverse transcriptase (Invitrogen Inc) and 3.34 µl H2O were added and incubated at 42°C for 60 min followed by 72°C for 15 min. Hepatic cDNA was then stored at – 80°C.
Quantitative real-time qPCR analysis.
Real-time qPCR primers were selected from GenBank database sequences with Primer3 software (Rozen and Skaletsky, 2000). The parameters for primer selection as well as the rat primer sequences used for Ces3, Cyp1b1, Cyp3a13, Serpina7, and 18s were described previously (Vezina et al., 2004). Mouse orthologs of rat genes were identified using NCBI HomoloGene (http://www.ncbi.nlm.nih.gov). Primer sequences used for the analysis of other rat genes as well as their mouse orthologs are listed in Table 1. Real-time qPCR was conducted on hepatic cDNA using the IQ SYBR green supermix kit (Bio-Rad Laboratories, Hercules, CA). The PCR mixture was as follows, 2 µl of diluted cDNA, 5.5 µl H2O, 5.0 µl 500nM primers, and 12.5 µl IQ SYBR green supermix. The amplification reactions were initiated with a 95°C hold for 3 min followed by 50 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. Fluorescence measurements were made during the 60°C annealing extension step. Following the amplification, protocol melt-curve analysis was performed to confirm the formation of a single amplicon. The melt-curve protocol was as follows; 2 min of denaturation at 95°C followed by a ramp down to 55°C and then a incremental increase of 0.5°C every 15 s for a total of 80 cycles. Changes in relative gene expression were calculated using the comparative threshold cycle (Ct) method as described in User Bulletin #2 for the ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA). The difference between Ct for the target gene and the 18s ribosomal RNA housekeeping gene (
Ct = Ct Target – Ct 18s) was determined for control and treated animals. The difference in the normalized data between control and treated animals (Ct = Ct TCDD – Ct Corn Oil) was determined and used to calculate the magnitude of gene downregulation (–2(Ct)) and gene upregulation (2(–Ct)). 18s ribosomal RNA did not exhibit a difference in average Ct between control and treated animals.
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Analysis of transcription factor–binding sites.
Genomic DNA sequences spanning 3000 bp above and 100 bp below the transcriptional start site of mouse and rat target genes were surveyed using MatInspector (Genomatix Software GmbH, Munchen, Germany). DNA sequences were scanned for AhR/ARNT heterodimer–binding sites with a core similarity of 1.0 and matrix similarity equal to or greater than the optimized matrix threshold (Cartharius et al., 2005
).
Statistical analysis.
Statistical analysis of real-time qPCR data for treatment, gender, and genotype comparisons was performed with a two-sample t-test using Minitab 14 statistical software (Minitab Inc, State College, PA). Statistical comparisons of microarray gene array data were performed using a t-test with an adjusted Bonferroni correction by TIGR Microarray Experiment Viewer (Saeed et al., 2003).
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RESULTS |
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Gene Downregulation in Hepatic Tissue following Subchronic (13 Weeks) Exposure to TCDD and Related Compounds
Global hepatic gene expression was evaluated in liver tissue from female Sprague-Dawley rats that were exposed subchronically for 13 weeks as part of an NTP-dioxin equivalents study. Utilizing gene array analysis, we identified 61 probes which exhibited a twofold or greater decrease in hepatic expression following 13 weeks of subchronic exposure to TCDD (100 ng/kg/day, po) (Table 2). Comparative analysis of the hepatic expression of these 61 probes were conducted for rats subchronically exposed to toxic equivalent doses (Van den Berg et al., 1998
) of PeCDF (200 ng/kg/day, po) and PCB126 (1000 ng/kg/day, po), PCB153 (1000 µg/kg/day, po), and a binary mixture of PCB126 (1000 ng/kg/day, po) and PCB153 (1000 µg/kg/day, po) (NTPa, 2004; NTPb, 2004; NTPc, 2004; NTPd, 2004; NTPe, 2004). The AhR ligand PeCDF caused a twofold or greater decrease in expression of 23 probes whereas the other AhR ligand PCB126 caused at least a twofold decrease in the expression almost half of the probes repressed by TCDD (Table 2). The non-AhR ligand PCB153 caused a twofold or greater downregulation in the expression of the genes Alcam, Adrp3, Fdx1, Fmo1, and Ywhae, suggesting that these genes may be down-regulated through an AhR independent mechanism (Table 2). Interestingly, subchronic exposure to the binary mixture of PCB126 and PCB153 caused a twofold or greater repression in 49 of the 61 probes (Table 2), whereas individually, exposure to PCB126 and PCB153 caused decreased expression of 27 and five probes, respectively. Hierarchical clustering and principle component analysis of the 61 probes show that compared to PeCDF, PCB126, and PCB153, the binary mixture of PCB126 and PCB153 altered the expression of the 61 probes in a manner most similar to that of TCDD (Fig. 1).
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Time Course of TCDD-Mediated Gene Downregulation in Hepatic Tissue from Female Sprague-Dawley Rats
In attempting to understand the manner by which dioxin causes a downregulation in the expression of these genes, it is important to determine whether they are down regulated as a primary response to dioxin exposure or rather as a secondary response due to dioxin-induced changes in liver morphology. To better understand the nature of this dioxin-induced gene downregulation, we evaluated the early responsiveness of a subset of genes following acute exposure to TCDD. This group of genes was limited to 11 genes which exhibited repressed expression of threefold or greater in response to subchronic TCDD exposure and included the metabolizing enzymes Ces3, Cyp3a13, Srd5a1, Cyp2j9, Dao1, and Ca2 (Hamase et al., 2005
; Li et al., 2006; Mahendroo et al., 2001; Moran et al., 2000; Soni et al., 2004; Wang et al., 2000); the solute transporters Slc6a6, Slco1a4, and Slc13a3 (Markovich and Murer, 2004; van Montfoort et al., 2002; Warskulat et al., 2006); the thyroxine carrier Serpina7 (Schussler, 2000); and the neurotrophin Ntf3 (Cassiman et al., 2001). To determine if the downregulation of these subchronically repressed genes occurs as an early response to dioxin exposure, hepatic expression of these genes was assessed in female Sprague-Dawley rats at 6, 24, and 72 h following a single acute exposure to TCDD (5 µg/kg, po).
Following 13 weeks of subchronic exposure to TCDD (100 ng/kg/day), the mean hepatic tissue concentration of dioxin was 18.3 ± 0.8 ng/g (NTPa, 2004). To ascertain if the liver tissue concentration of TCDD following an acute exposure is similar to that observed with subchronic exposure, the hepatic tissue concentration of dioxin was evaluated at 6 h postexposure to TCDD. Rats were treated with 2 µCi of 3H-TCDD (5 µg/kg, po) and the tissue distribution of dioxin-derived 3H was analyzed. At 6 h following a single exposure to 3H-TCDD, the liver contained 2.7 ± 0.7% of the dose per gram tissue. This represents a liver concentration of dioxin of 29.7 ± 0.8 ng/g tissue, which is similar to that observed following subchronic exposure.
Utilizing real-time qPCR, the hepatic downregulation of the 11 target genes was evaluated in female rats at 6, 24, and 72 h postexposure to TCDD (5 µg/kg, po). As a positive control, the expression of Cyp1b1 was assessed at 6, 24, and 72 h postexposure. The hepatic expression of Cyp1b1 was increased 21-, 121-, and 411-fold at 6, 24, and 72 h, respectively (data not shown). Analysis of the down-regulated genes show that the metabolizing enzymes Ces3, Srd5a1, and Cyp2j9; the solute transporters Slc6a6 and Slco1a4; the carrier protein Serpina7 and the neutrophic factor Ntf3 all exhibited a twofold or greater decrease in expression as early as 6 h postexposure to TCDD (Table 3). Although the genes Cyp3a13 and Slc13a3 did not exhibit a decrease of twofold at 6 h, they exhibited a trend toward downregulation at 6 h and decreases of threefold or greater at 24 and 72 h following acute TCDD exposure (Tables 3 and 4). The genes Ca2 and Dao1 did not exhibit a twofold decrease in expression at either 6 or 24 h postexposure (Table 3), but did show a twofold or greater decrease in expression at 72 h postexposure (Table 4). Analysis for the presence of putative AhR/ARNT heterodimer–binding sites within these early responsive genes show that eight of 11 genes contain putative AhR/ARNT-binding sites (Fig. 2). These data indicate that all 11 genes are repressed as a primary response to TCDD exposure and not as a secondary response to the subchronic (13-week) treatment regimen, however, the delayed downregulation of Ca2, Cyp3a13, Dao1, and Slc13a3 suggests that the repression of these genes may occur subsequent to a principle TCDD-mediated event.
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Gender Dependence of TCDD-Mediated Changes in Hepatic Gene Expression in Female and Male Sprague-Dawley Rats
TCDD toxicity has been reported to cause a spectrum of biological effects that exhibit age, species, and gender dependence. To determine if gender plays a role in the downregulation of the 11 target genes, we evaluated their expression in female and male Sprague-Dawley rats at 72 h following a single exposure to TCDD (5 µg/kg, po). As a positive control, Cyp1b1 expression was monitored in female and male rats at this time point. Cyp1b1 exhibited a 411- and 236-fold increase in expression in female and male rats, respectively (data not shown). With the exception of the gene Slc13a3, the magnitude of gene downregulation was greater in female rats than in male rats, however, the disparity in gene downregulation between male and female rats was not statistically significant for all evaluated genes (Table 4). At 72 h postexposure to TCDD all 11 genes exhibited a twofold or greater decrease in expression in female rats, whereas in male rats only Serpina7, Srd5a1, and Slc13a3 exhibited decreased expression of twofold or greater (Table 4). Together these data suggest that there is gender dependence exhibited in the downregulation of these genes, however, the gender dependence is not uniform across all down-regulated genes.
AhR Dependence of TCDD-Mediated Downregulation in Hepatic Tissue of Female AhR+/+ and AhR–/– Mice
Many of the effects of TCDD are known to be dependent on the binding and activation of the AhR. TCDD induced changes in gene expression, however, have been reported to occur independent of the presence of a functional AhR (Tijet et al., 2006). AhR-dependent changes in gene expression involve the binding of AhR/ARNT heterodimers to nucleotide sequences known as DREs. DREs consist of a minimal core pentanucleotide "GCGTG," and binding to this site by the heterodimeric transcription factor is influenced by flanking nucleotides (Swanson, 2002). Sequences spanning 3000 bp above and 100 bp below the mRNA start sites of the mouse orthologs of the target genes were surveyed for putative AhR/ARNT heterodimer–binding sites using MatInspector (Genomatix Software GmbH) (Cartharius et al., 2005). Mouse Cyp3a13, Ces3, Serpina7, and Slco1a4 did not contain a putative AhR/ARNT heterodimer–binding site in the surveyed region. The other examined target genes, however, did contain one or more putative AhR/ARNT heterodimer–binding sites within the surveyed region (Fig. 2). To determine if the downregulation of the 11 target genes is dependent on the presence of a functional AhR, we examined hepatic gene expression in female wild type (AhR+/+) and knockout (AhR–/–) mice at 72 h postexposure to TCDD (25 µg/kg, po). As a positive control, Cyp1b1 expression was monitored in AhR+/+ and AhR–/– mice. A 350- and twofold increase in Cyp1b1 expression was observed in AhR+/+ and AhR–/– mice, respectively (data not shown). A dioxin-induced downregulation of at least twofold was observed for Cyp3a13, Serpina7, Dao1, Ces3, and Srd5a1 in AhR+/+ mice, but was absent in AhR–/– mice (Table 5). Ntf3 and Slc6a6 exhibited a repression of –1.8 and –1.7, respectively, in AhR+/+ mice, which was significantly different from their expression in AhR–/– mice (Table 5). The genes Cyp2j9, Slco1a4, and Slc13a3 exhibited no change in AhR+/+ mice but did exhibit increased expression of twofold or greater in AhR–/– mice (Table 5). Car2, the mouse ortholog of rat Ca2 did not exhibit a significant change in either AhR+/+ or AhR–/– mice (Table 5). The lack of repression of Car2, Cyp2j9, Slco1a4, and Slc13a3 in AhR+/+ mice suggests that there is species dependence in hepatic gene downregulation by TCDD, however, there is a need for a functional AhR in the dioxin-induced downregulation of hepatic Cyp3a13, Serpina7, Dao1, Ces3, Slc6a6, Srd5a1, and Ntf3 in mice (Table 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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POPs such as TCDD represent a potential health risk because of their potent toxicological activities, long biological half-life and long environmental half-life. The exceptional potency and wide range of adverse biological effects associated with dioxin exposure has led many to investigate the mechanisms of action of this prototypical POP. The downregulation of gene expression in response to TCDD exposure has not been as extensively characterized as the association between TCDD exposure and gene upregulation. There are, however, studies which have identified both transcriptional and epigenetic mechanisms of dioxin-induced gene repression in extrahepatic systems. The downregulation of the estrogen responsive genes pS2, c-fos, cathepsin D, and HSP27 in MCF-7 human breast adenocarcinoma cells was observed to be dependent on the presence of a proposed inhibitory dioxin response element (iDRE), a DRE involved in the downregulation of genes (Duan et al., 1999
; Gillesby et al., 1997; Safe et al., 1998). The iDRE is also thought to play a role in the repression of the glycosylphophatidylinositol anchored T-cadherin in rat aortic vascular smooth muscle cells (Niermann et al., 2003). Epigenetic mechanisms such as hypermethylation of gene promoters have been reported to be associated with dioxin-induced gene downregulation. In human keratinocytes, dioxin-induced downregulation of p16, the stabilizing protein of the tumor suppressor p53, was shown to be associated with an increase in methylation of the p16 promoter (Ray and Swanson, 2004). Similarly, a decrease in the expression of H19 and Igf2 in mouse embryos was associated with increased methylation and an overall increase in embryonic methyltransferase activity following exposure to TCDD (Wu et al., 2004).
In this study, we investigated the repression of hepatic genes following acute and subchronic exposure to TCDD. Following subchronic exposure to TCDD, the livers of female rats are void of the neoplastic lesions which manifest themselves with chronic exposure to dioxin. Mild hypertrophy was observed following 13 weeks of subchronic exposure to TCDD, however, liver hypertrophy was also observed with female rats exposed to the non-AhR ligand PCB153 (NTPa, 2004; NTPd, 2004). Following 13 weeks of subchronic exposure to TCDD, a small but nonsignificant increase in the incidence of multinucleated hepatocytes and fatty diffuse change were also observed (NTPa, 2004). Changes in gene expression occurring with acute and subchronic exposure to dioxin could be contributing to the ultimate manifestation of hepatotoxicity and/or carcinogenicity.
A total of 61 probes exhibited a twofold or greater downregulation in female rat liver following 13 weeks of subchronic exposure to TCDD (Table 2). Comparative analysis of the expression of this probe set in rats subchronically exposed to the AhR ligands PeCDF and PCB126, the non-AhR ligand PCB153 and a binary mixture of PCB126 and PCB153 led us to determine that simultaneous exposure to PCB126 and PCB153 caused a downregulation in the probe set that most mimicked that of TCDD (Fig. 1). A combined total of 30 probes were down regulated by subchronic exposures to PCB126 alone and PCB152 alone. Concurrent exposure to PCB126 and PCB153 causes a repression in 49 probes, suggesting a synergistic effect by these two compounds (Table 2). Future studies examining the relationship between gene expression profiles and hepatotoxic effects of PCB126 and PCB153 individually and in combination are being conducted to further elaborate on the effects of this binary mixture.
In order to better understand the temporal dependence of this downregulation, we examined the early responsiveness of the 11 subchronically repressed genes in the livers of female rats following acute exposure to TCDD (5 µg/kg, po) (Tables 3 and 4). All 11 genes exhibited early responsiveness to TCDD indicating that their downregulation occurs as a response to dioxin exposure and not as a result of the subchronic (13-week) treatment regimen. A single acute TCDD exposure (5 µg/kg, po) resulted in a hepatic tissue level (29.7 ng/g liver) at 6 h postexposure similar to that achieved following 13 weeks of subchronic exposure in the NTP study (18.3 ng/g liver). Thus, gene expression studies in rat liver following acute and subchronic TCDD exposures were conducted at comparable tissue levels of TCDD. Of the 11 genes examined, seven were decreased twofold or greater at 6 h postexposure to TCDD (5 µg/kg, po) (Table 3). Cyp1b1, a known transcriptionally up-regulated gene also exhibited an effect at 6 h, suggesting that the seven repressed genes are likely to be primary genomic targets of dioxin-mediated downregulation. Interestingly, the gene Serpina7, which exhibited early downregulation at 6 h postexposure, did not contain a putative AhR/ARNT-binding site (Fig. 2). It should also be noted that these seven genes also exhibited downregulation at 24 and 72 h following acute exposure to TCDD. While the metabolizing enzyme Cyp3a13 only exhibited a decrease of 1.8-fold at 6 h, a decrease in expression of 6- and 10-fold was observed at 24 and 72 h, respectively. Following 13 weeks of subchronic exposure to TCDD, an 85-fold decline in Cyp3a13 hepatic expression was observed through gene array analysis (Table 2). In addition, we observed an 86-fold decrease in hepatic Cyp3a13 expression through real-time qPCR at 14 days postexposure to a single acute dose of TCDD (5 µg/kg, po) (data not shown). These observations indicate that there is a marked temporal-dependent effect of dioxin on hepatic Cyp3a13 expression, with prolonged exposure resulting in greater Cyp3a13 repression.
The toxic effects induced by TCDD exposure have been shown to be dependent on multiple factors including age, gender, tissue type, and species. Kociba et al. (1978) reported that following chronic exposure to TCDD (100 ng/kg/day), female rats exhibited a higher incidence of hepatocellular inflammatory, necrotic, and degenerative changes when compared to male rats. In addition, neoplastic lesions were observed in female but not male rats following chronic exposure to TCDD. At 13 weeks into the study, it was shown that female and male rats contained comparable hepatic TCDD tissue concentrations, suggesting that the more pronounced hepatotoxic and carcinogenic effects observed in females were not due to increased hepatic retention of dioxin by female rats (Kociba et al., 1976). We observed that for the majority of the 11 target genes, a greater repression was observed in TCDD-exposed female rats when compared to their male counterparts (Table 4). In male rats, only three of the 11 target genes exhibited a twofold or greater decrease in expression following TCDD exposure, whereas in female rats all 11 genes exhibited this repression at 72 h postexposure (Table 4). The gene Serpina7, which was decreased threefold in male rats exposed to TCDD, has been shown to be repressed by dioxin in male C57BL/6J mice (Tijet et al., 2006). Following a single large dose of 1000 µg/kg TCDD, male mice exhibited a 20-fold decrease in hepatic Serpina7 expression at 19 h postexposure (Tijet et al., 2006). In female C57BL/6J mice, we observed a downregulation in Serpina7 of 20-fold following a single dose of 25 µg/kg TCDD. Together the data from these two reports corroborate the observation that Serpina7 is down regulated in both genders. However, given the disparity in dosing regimens between the genders, it seems that female mice are more sensitive to Serpina7 downregulation than male mice, a trend that was seen in the treated rats. Cyp3a13, an estrogen responsive gene which exhibits female dominance in rats but not mice (Anakk et al., 2003; Wang and Strobel, 1997), exhibited a 10-fold decrease in female rats but no change in male rats (Table 4). Exposure to TCDD has been shown to modulate estrogen response pathways (Safe et al., 1998), which may in part explain the mode of Cyp3a13 downregulation by TCDD in hepatic tissue. The importance of ovarian hormones such as estrogen on TCDD-induced hepatocarcinogenesis was investigated by Clark et al. (1991) in a two-stage tumor-promotion model which showed that ovariectomized female rats exhibited a decreased incidence of preneoplastic foci when compared to intact sham-operated female rats. Promoter analysis for the presence of palindromic estrogen response elements (ERE) indicated that rat Dao1 and Slc6a6 and mouse Cyp3a13 and Ces3 contained EREs (data not shown). Of these four genes, only mouse Ces3 contained a putative AhR/ARNT-binding site within close proximity to the ERE. This does raise the possibility that the DRE in mouse Ces3 could be acting as an iDRE and invoking an antiestrogenic effect that is resulting in mouse Ces3 repression. The more pronounced effect of TCDD on hepatic gene repression in female rats may begin to explain the increased incidence of hepatotoxicity and carcinogenicity observed in female rats when compared to male rats. While the downregulation of Ces3 was observed in both rats and mice, downregulation of Car2, Cyp2j9, Slco1a4, and Slc13a3 was not observed in mice, indicating that the dioxin-induced downregulation of these genes exhibits species specificity. Together these data indicate that early gene downregulation following acute dioxin exposure is influenced by factors such as species and gender.
Changes to gene expression incurred by exposure to dioxin are commonly mediated through the AhR, however, AhR-independent changes in gene regulation have recently been reported in male AhR knockout mice (Tijet et al., 2006). To determine if the downregulation of the 11 target genes is dependent on the presence of the AhR, hepatic gene expression was investigated in AhR wild type and knockout mice following acute exposure to TCDD (Table 5). We observed that the downregulation of Cyp3a13, Serpina7, Dao1, Ces3, Slca6a6, Srd5a1, and Ntf3 in wild type mice was abolished in mice which lacked an AhR. Interestingly, Cyp3a13, Ces3, and Serpina7 did not contain putative AhR/ARNT heterodimer–binding site within 3000 bp upstream and 100 bp downstream from the transcriptional start site (Fig. 2), suggesting that the heterodimer may not be directly binding to the promoter region of these genes. Contrary to these observations was the lack of responsiveness of the genes Cyp2j9 and Slc13a3 in wild type mice, both of which contain putative AhR/ARNT heterodimer–binding sites. This indicates that the presence or lack of a putative AhR/ARNT-binding site does not solely determine the response to acute dioxin exposure.
A toxicogenomic approach was used in the present study to investigate the effects of acute and subchronic exposure to dioxin on hepatic gene downregulation. While most previous studies focus on dioxin-mediated gene upregulation, very few studies have investigated hepatic gene repression following dioxin exposure. Identifying acute responses in gene expression can assist efforts to discern primary temporal events following dioxin exposure from responses that may be secondary to other dioxin-mediated responses. The temporal, gender, species, and AhR dependence of these responses, along with further investigation of the function of primary responsive genes will assist efforts to relate those changes to the pathological end points observed with chronic dioxin exposure. In addition, these data will assist efforts to elucidate the network of events which occur following exposure to this prototypical POP. Further investigations into the mechanisms of action of dioxin that allow for the downregulation of these genes and the their role in the development of hepatotoxicity will lead to a better understanding of the multitude of effects imposed by TCDD and other AhR ligands on liver function.
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SUPPLEMENTARY DATA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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These studies were supported in part by National Institute of Environmental Health Sciences ES09440, the University at Buffalo, and the Environment and Society Institute, University at Buffalo. We would like to acknowledge Leighton Stein and Gene Expression Facility at Roswell Park Cancer Institute.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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