Persistent, Low-Dose 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure: Effect on Aryl Hydrocarbon Receptor Expression in a Dioxin-Resistance Model

Toxicology and Applied Pharmacology 175, 43–53 (2001) doi:10.1006/taap.2001.9222, available online at http://www.idealibrary.com on

Persistent, Low-Dose 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure: Effect on Aryl Hydrocarbon Receptor Expression in a Dioxin-Resistance Model Monique-Andre´e Franc,* Raimo Pohjanvirta,† ,‡ ,§ Jouko Tuomisto,† and Allan B. Okey* *Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; †National Public Health Institute, Department of Environmental Medicine, Kuopio, Finland; ‡National Veterinary and Food Research Institute, Regional Laboratory of Kuopio, Kuopio, Finland; and §Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland Received March 5, 2001; accepted May 16, 2001

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) 1 is the most potent of the halogenated environmental organic pollutants collectively known as dioxins. Dioxin-like compounds include halogenated aromatic hydrocarbons, dibenzofurans, and certain polychlorinated biphenyls all of which exist as complex mixtures in the environment (Okey, 1990; Safe, 1995). At relatively low doses, TCDD produces a spectrum of adverse effects including reproductive, developmental, immunologic, and endocrine perturbations, carcinogenesis, and death (Pohjanvirta and Tuomisto, 1994; Birnbaum and Tuomisto, 2000). The subset of toxic outcomes in any one organism is dependent on the type of organism (species/strain/substrain, sex, developmental stage) and on the dose and duration of TCDD exposure (Hahn, 1998; Van den Berg et al., 1998; Hengstler et al., 1999). Human populations exposed to high levels of TCDD have been observed to manifest some acute toxic effects of TCDD, notably chloracne (Neuberger et al., 1991; Sweeney et al., 1997). Except for rare incidents of accidental high level exposure, human exposure to dioxins tends to be of chronic, low-dose nature, primarily through diet (Schecter et al., 1994; Wesp et al., 1996; Kiviranta et al., 2000). There remains great uncertainty regarding the risk of prolonged, low-level exposure to dioxins (Bertazzi et al., 1998; Neuberger et al., 1999; Sweeney and Mocarelli, 2000). Multiple lines of evidence indicate that virtually all toxic effects of TCDD are mediated by the aryl hydrocarbon receptor (AHR) (Poland and Glover, 1980; Okey et al., 1994; Fernandez-Salguero et al., 1996; Lahvis and Bradfield, 1998; Tuomisto et al., 1999). This cytosolic protein binds TCDD with high affinity and heterodimerizes with the AHR nuclear translocator (ARNT). In the nucleus, the ligand–AHR–ARNT

Persistent, Low-Dose 2,3,7,8-Tetrachlorodibenzo-p-dioxin Exposure: Effect on Aryl Hydrocarbon Receptor Expression in a Dioxin-Resistance Model. Franc, M.A., Pohjanvirta, R., Tuomisto, J., and Okey, A. B. (2001). Toxicol. Appl. Pharmacol. 175, 43–53. Most toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are mediated by the aryl hydrocarbon receptor (AHR). A single, acute dose of TCDD can alter its own receptor levels thus complicating evaluation of dose–response relationships for AHR-mediated events. Since environmental exposure to dioxins is typically of a repeated low-dose nature, we examined the effect of such exposure on AHR expression. Three rat strains differing greatly in their sensitivity to acute TCDD lethality, Long–Evans (Turku AB) (L-E) (LD50 ⬃10 ␮g/kg); Sprague Dawley (SD) (LD50 ⬃50 ␮g/kg); and Han/Wistar (Kuopio) (H/W) (LD50 > 9600 ␮g/kg), were administered TCDD intragastrically, biweekly for 22 weeks producing doses equivalent to 0, 10, 30, and 100 ng/kg/day. Changes in hepatic AHR levels were quantitated at the protein level by radioligand binding and immunoblotting and at the mRNA level by RT–PCR. Cytosolic AHR protein was elevated at 10 or 30 ng/kg/day TCDD in SD and L-E rats; AHR mRNA was also elevated at these doses, suggesting a pretranslational mechanism. There was no apparent relationship between TCDD-induced AHR regulation and strain sensitivity to TCDD. Overall, “subchronic” TCDD did not greatly perturb AHR expression. The maintenance of relatively constant receptor levels in the face of persistent agonist stimulation is in contrast to the sustained depletion of AHR by TCDD observed in cell culture and to the fluctuations in AHR observed hours to days following acute TCDD exposure in vivo. Changes in AHR levels may affect dose–response relationships; the effect of TCDD on its own receptor at environmentally relevant dosing schemes is therefore important to risk assessment. © 2001 Academic Press Key Words: aryl hydrocarbon receptor; subchronic; 2,3,7,8tetrachlorodibenzo-p-dioxin; rat; dioxin susceptibility; regulation of gene expression.

1 Abbreviations used: AHR, aryl hydrocarbon receptor; AP, antifluoresceinalkaline phosphatase; ARNT, aryl hydrocarbon receptor nuclear translocator protein; dNTP, 2⬘-deoxynucleoside 5⬘-triphosphate; DRE, dioxin response element; H/W, Han/Wistar (Kuopio) rat; L-E, Long–Evans (Turku AB) rat; PCB, polychlorinated biphenyl; SD, Sprague Dawley rat; TAE, Tris–acetate/ EDTA electrophoresis buffer; TBE, Tris/borate/EDTA electrophoresis buffer; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin.

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0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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complex acts as a transcription factor to alter expression of a battery of genes including some drug-metabolizing enzymes such as the cytochrome P450 enzymes CYP1A1, CYP1A2, and CYP1B1 (Whitlock, 1999; Nebert et al., 2000). These, and other genes, are under direct transcriptional control of the AHR by way of specific DNA consensus sequences, dioxin response elements (DREs) (Denison et al., 1998). The complex relationship between the level of AHR expression and biological responses remains unclear. Theoretical modeling of receptor–response relationships supports the belief that alterations in AHR levels may influence the ultimate response to AHR ligands by modifying the dose–response relationship (Ruffolo, 1982; Kenakin, 1997; Andersen and Barton, 1999). In cell culture, AHR levels are rapidly depleted following ligand activation due to proteolytic degradation (Pollenz, 1996; Giannone et al., 1998; Davarinos and Pollenz, 1999; Ma and Baldwin, 2000; Pollenz and Barbour, 2000); it has been suggested that proteolytic degradation may be a regulatory control point to limit the biological response to AHR agonists (Giannone et al., 1998; Davarinos and Pollenz, 1999; Ma and Baldwin, 2000). Preventing degradation of the AHR in cell culture results in superinduction of CYP1A1 mRNA (Ma et al., 2000), implying that AHR levels influence the degree of some biochemical responses. AHR expression levels have been shown to fluctuate with biological processes and with exposure to exogenous chemicals. Time of day (Richardson et al., 1998), stage of cellular differentiation (Hayashi et al., 1995; Wanner et al., 1995; Shimba et al., 1998), and hormonal status (Chaffin et al., 2000) have all been reported to modulate AHR expression. Exposure to exogenous chemicals, including AHR ligands (notably TCDD), has also been shown to modulate AHR expression (Sloop and Lucier, 1987; Bunce et al., 1990; Prokipcak and Okey, 1991; Giannone et al., 1995; Pollenz, 1996; Giannone et al., 1998; Pollenz et al., 1998; Roman et al., 1998; Abnet et al., 1999; Sommer et al., 1999; Tanguay et al., 1999; Franc et al., 2001). The effect of TCDD on AHR levels is dependent on dose, duration of exposure, and the tissue/organism studied. Sloop and Lucier (1987) provided experimental evidence that repeated, low-dose TCDD (10 –100 ng/kg/day; 22 weeks) might elevate AHR levels in vivo, introducing the possibility that, under typical dioxin exposure, animals may be gradually sensitized to dioxin toxicity. Dioxin toxicity is characterized by a wide range of sensitivity differences between species and among strains within the same species thus complicating risk assessment. In two rodent models, sensitivity of response to dioxins segregates with the AHR locus (Poland and Glover, 1980; Tuomisto et al., 1999). In mice, the greater resistance of the DBA/2 versus the C67BL/6 mouse has been attributed to a single nucleotide change in the ligand-binding domain of the AHR resulting in lowered ligand binding affinity (Poland et al., 1994). In rats, there exists a greater than 1000-fold difference in sensitivity to acute TCDD lethality between the resistant Han/Wistar (Kuo-

pio) (H/W) and sensitive Long–Evans (Turku AB) (L-E) rat strains. The exceptional resistance of H/W rats is associated with a deletion within the transactivation domain of the H/W AHR (Pohjanvirta et al., 1998; Tuomisto et al., 1999). The full consequences of this deletion are unknown but, in this model, the AHR (and another unidentified gene, “B”) determine the acute toxic response to TCDD (Tuomisto et al., 1999). The wide range of inherent susceptibility differences to TCDD among species and among strains likely is due to receptor structure rather than differences in AHR quantity (Birnbaum, 1994). Nevertheless, it remains to be determined whether factors that up- or down-regulate AHR expression can further influence susceptibility by sensitizing or desensitizing an organism to subsequent dioxin insult. Since AHR expression is known to be influenced by its own ligands and since typical exposure is of a persistent low-dose nature, the primary goal of this study was to comprehensively examine AHR regulation following “subchronic” agonist (TCDD) treatment in vivo. A supplementary goal was to compare receptor regulation in the highly resistant H/W and the sensitive L-E and Sprague–Dawley (SD) rat strains to determine if the AHR is differentially regulated by TCDD—the consequence of which could be a differential change in susceptibility to TCDD. Since the AHR mediates key biological and toxic effects of TCDD and since AHR expression levels may influence ultimate response, from the point of view of risk assessment it is important to consider the endogenous and exogenous factors that affect AHR expression in vivo. MATERIALS AND METHODS Reagents and Solutions The 2,3,7,8-TCDD used to treat animals was purchased from UFA-Oil Institute (Ufa, Russia) and was ⬎99% pure as determined by gas chromatography–mass spectrometry (Vartiainen et al., 1995). TCDD was dissolved in ether and added to corn oil; the ether was subsequently evaporated off. [ 3H]TCDD for binding assays (40 Ci/mmol, chemical purity ⬎97%) was purchased from Chemsyn Science Laboratories (Lenexa, KS). 2,3,7,8-Tetrachlorodibenzofuran was a kind gift from Dr. Stephen Safe (Texas A&M University, College Station, TX). HEGD buffer consisted of 25 mM Hepes, 1.5 mM EDTA, 10% v/v glycerol, and 1 mM dithiothreitol (pH 7.4); TNT consisted of 20 mM Tris base, 137 mM NaCl, and 0.5% Tween (pH 8.0); 10⫻ Taq DNA polymerase reaction buffer consisted of Tris–Cl, KCl, (NH 4)SO 4, and 15 mM MgCl 2 (pH 8.7). Antibodies Anti-AHR polyclonal antibody (from rabbit) was obtained from Biomol Research Laboratories Inc. (Plymouth Meeting, PA; catalog no. SA-210); this antibody was raised against amino acids 1– 402 of the mouse Ah b⫺1 allele (Pollenz et al., 1994). Animals Female Sprague Dawley rats, inbred Long–Evans (Turku AB) rats, and outbred Han/Wistar (Kuopio) rats (10 weeks old) were obtained from the breeding colony of the National Public Health Institute, Division of Environmental Health, Kuopio, Finland. Animals were kept in stainless-steel wire-

SUBCHRONIC TCDD EXPOSURE IN RATS: EFFECT ON AHR EXPRESSION mesh cages, five rats per cage, and allowed unlimited access to standard pelleted animal feed (R36, Ewos, So¨derta¨lje, Sweden) and tap water. Lights were on between 7 a.m. and 7 p.m. Ambient room temperature and humidity were maintained at 21.5 ⫾ 1°C and 55 ⫾ 10%, respectively. Animal Treatment and Tissue Harvest TCDD (or vehicle alone) was administered in corn oil every 2 weeks by oral gavage at doses equivalent to 0, 10, 30, or 100 ng/kg/day. Actual doses were 140, 420, or 1400 ng/kg once every 2 weeks for 22 weeks. The 22-week time point was selected as it falls between times reported for steady-state TCDD kinetics (Rose et al., 1976) and times for development of hepatic preneoplastic and neoplastic lesions (in tumor promotion models) (Pitot et al., 1980). The final dose was administered 10 days before euthanasia. Animals were weighed and euthanized by decapitation (n ⫽ 8/treatment group). Approximately 1 g of tissue from two lobes of liver was removed, weighed, and immediately submerged in liquid nitrogen for RNA isolation. Remaining liver was weighed and homogenized with a Potter–Elvehjem glass-Teflon homogenizer in 4 volumes of ice-cold HEGD buffer (20% homogenate) for preparation of subcellular fractions. Subcellular Fractions Cytosol and nuclear extracts were prepared as described by Mason and Okey (1982) with the following modifications: nuclear proteins were extracted with 0.5 M NaCl–HEGD (pH 8.5), subcellular fractions were stored in liquid nitrogen, and total protein was measured in triplicate by the Bradford protein assay (Bradford, 1976) using bovine serum albumin as the protein standard. Radioligand Binding Radioligand binding and protein separation on sucrose density gradients were performed as previously described (Pohjanvirta et al., 1999). Briefly, cytosols (5 mg/ml protein in HEGD buffer) were incubated on ice for 1 h with 10 nM [ 3H]TCDD in the presence or absence of a 100-fold molar excess of 2,3,7,8-tetrachlorodibenzofuran as the competitor. Radioligand concentration was selected based on our previous studies in this animal model (Franc et al., 2001). Excess ligand was removed with dextran-coated charcoal (1 mg/mg cytosolic protein) and proteins were separated by velocity sedimentation on sucrose density gradients. Specific ligand binding was calculated per milligram of total protein as measured in cytosol postcharcoal treatment. Immunoblotting Proteins from cytosol and nuclear extract were separated by Tris– glycine sodium dodecyl sulfate polyacrylamide (6%) gel electrophoresis (SDS– PAGE). Protein loading per lane was 75 ␮g for SD and L-E cytosol, 150 ␮g for H/W cytosol, and 100 ␮g for all nuclear extracts. Gels were run at constant voltage and then electroblotted onto a polyvinylidine difluoride membrane (Immobilon-P, Millipore Corp; Bedford, MA) (100 V; 1 h). Protein transfer was assessed by Ponceau S staining and membranes were blocked in 5% (w/v) blocking reagent in TNT (Vistra ECF Western Blotting Kit, Amersham). Membranes were sequentially incubated with each antibody and with the alkaline phosphatase conjugate with gentle agitation at ambient temperature. Primary and secondary antibodies were diluted in blocking reagent as follows: cytosols: AHR antibody ⫽ 0.0050 ␮g/ml; fluorescein-linked anti-rabbit antibody ⫽ 1:10,000; nuclear extracts: AHR antibody ⫽ 0.02 ␮g/ml; fluoresceinlinked anti-rabbit antibody ⫽ 1:2500. The antifluorescein-alkaline phosphatase (AP) conjugate was diluted in TNT at 1:2500. Three changes of TNT buffer for a total of 30 min were used to wash membranes between incubations. The AP-catalyzed fluorescence was initiated by addition of approximately 50 ␮l/cm 2 of AttoPhos fluorescent substrate (Amersham) (10 min). Fluorescent emissions were captured using a Storm Phosphorimager (Molecular Dynamics, Sunnyvale, CA). Signal strength (with subtraction of background at band perimeter) was calculated with the IPLab Gel H band quantitation software. To ensure the quantitative nature of the immunoblotting assay, experimental

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parameters were optimized such that extremes in AHR levels were within the linear range of the assay. Antibody concentrations and incubation times, total amount of sample protein loaded, and fluorescence detection parameters were optimized accordingly. All samples of each strain (n ⫽ 32/strain) were immunoblotted simultaneously. To allow comparison between gels, signal strength was normalized to the sum of signal intensities of duplicate normalization standards [prepared by pooling control cytosols (or nuclear extracts) of the corresponding rat strain] loaded onto each gel. AHR immunoreactive protein was not normalized to an internal standard (such as ␤-actin) since total protein has previously been shown to be an adequate denominator for similar AHR measurements (Pollenz, 1996; Holmes and Pollenz, 1997; Pollenz et al., 1998; Sommer et al., 1999). Assays were performed in duplicate with satisfactory agreement between duplicate normalized data and results are expressed as mean of the duplicates. The AH receptor band was positively identified as previously described (Franc et al., 2001). RNA Isolation Total RNA was isolated from frozen tissue with TRIzol Reagent per the manufacturer’s instructions. Briefly, frozen tissue was homogenized with a Polytron in TRIzol Reagent (7% w/v) (on ice; 60 s) (Gibco BRL/Life Technologies; Burlington, Ontario, Canada) and allowed to dissociate (room temp; 3 min). Addition of chloroform (20% v/v) combined with vigorous manual agitation (15 s) and centrifugation (12,000g; 15 min; 4°C) forced the RNA into the aqueous phase. RNA was recovered by isopropyl alcohol precipitation (1:1 v/v) and centrifugation (12,000g; 10 min; 4°C). The pellet was washed with 75% ethanol, collected by centrifugation (7,500g; 5 min; 4°C) and redissolved in diethylpyrocarbonate-treated water (55°C; 15 min). Contaminating DNA was enzymatically degraded with 15 U DNaseI (37°C, 20 min ⫹ 55°C, 10 min) (FPLCpure, Pharmacia Biotech). Total RNA yield was calculated from the spectrophotometric absorbance at 260 nm (A 260). An A 260/280 ⬎1.7 was deemed an acceptable measure of RNA purity from tissue. RNA integrity was estimated by visual examination of two distinct ribosomal RNA bands (28S and 18S) on an ethidium bromide–1% agarose gel. Total RNA was reisolated from samples not meeting quality criteria. RNA was stored at ⫺80°C and integrity reconfirmed immediately prior to RT–PCR. Semiquantitative RT–PCR mRNA was reverse transcribed to cDNA in a two-step approach using Maloney Murine Leukemia Virus Reverse Transcriptase primed by an oligo(dT) primer as previously described (Li et al., 1998). Sufficient cDNA was synthesized to PCR amplify all genes (targets and internal standard) from the same source of cDNA. AHR, CYP1A1, and ARNT cDNAs were PCR amplified and signals were normalized to the internal reference standard, ␤-actin. Each cDNA was amplified in a separate tube using Qiagen Taq DNA polymerase (Mississauga, Ontario, Canada). The product of amplification was labeled for detection by incorporation of [␣- 32P]dCTP. The 50-␮l reactions contained cDNA derived from 125 ng RNA, 2.5 U Taq polymerase; PCR reaction buffer (containing 1.5 mM MgCl 2); 0.2 ␮M of each primer; 0.2 mM of each dNTP, and 1 ␮Ci [␣- 32P]dCTP (Amersham Pharmacia Biotech Inc., Montreal, Quebec, Canada). Reaction tubes were introduced into a 90°C GeneAmp PCR System 9600 (Perkin Elmer). Temperature cycling (Table 1) was flanked by an initial denaturation step (4 min; 94°C) and a final extension step (7 min; 72°C). PCR product was separated on 10% nondenaturing polyacrylamide gels. Radioactive emissions were captured on a Phosphor screen and digitized by the Storm Phosphorimager SF system. Signal intensity was quantitated using IPLab Gel H software. RT–PCR was performed in triplicate (from the same total RNA isolate). Relative rather than absolute levels of gene expression were sought since (i) the input RNA concentration was not accurately known, (ii) variation in efficiency of RT and PCR reactions for each sample was undefined, and (iii) decay of radioisotope was variable between assays. ␤-Actin was used as an internal reference standard to control for variability in all steps leading up to PCR amplification. The quantitative nature of the polymerase chain reaction to

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TABLE 1 Primer Sequences and Thermal Cycling Conditions for the Four Genes Amplified by PCR

Target AHR 1A1 ARNT

␤-Actin

Thermal cycling parameters (s/°C) (denature) (anneal) (extend) ⫻ cycles

Amplimer sequences 5⬘-714-AGGGAGGTTAAAGTATCTTCATGGAC-739-3⬘ 5⬘-1630-TCCCTAGGTTTCTCATGATGCTATAC-1605-3⬘ 5⬘-627-ACGTTATGACCACGATGACC-646-3⬘ 5⬘-1299-AGGCCGGAACTCGTTTG-1283-3⬘ 5⬘-877-CTTGGCTCTGTGAAGGAAGG-896-3⬘ 5⬘-1289-CGGAATCGGAACATGACAG-1271-3⬘ 5⬘-344-ACCGTGAAAAGATGACCCAG-363-3⬘ 5⬘-1031-GAGCCACCAATCCACACAG-1011-3⬘

(20⬙; 94°C)(20⬙; 54°C)(40⬙; 72°C) ⫻ 24 (20⬙; 94°C)(20⬙; 52°C)(40⬙; 72°C) ⫻ 17 (20⬙; 94°C)(19⬙; 53°C)(40⬙; 72°C) ⫻ 25 (20⬙; 94°C)(20⬙; 51°C)(40⬙; 72°C) ⫻ 16

Note. Accession numbers are as follows: AHR, U09000; CYP1A1, X00469; ARNT, U61184; and ␤-actin, J00691. Sites of primer design are designated according to 1 ⫽ A in the first codon (ATG).

measure relative gene expression was ensured as validated by Murphy et al. (1990). Cycle number and template concentration were selected such that minimum and maximum mRNA levels for each gene were within the exponential range of amplification (data not shown). For CYP1A1 mRNA in untreated animals, levels were virtually undetectable by RT–PCR within the exponential range of any of the TCDD treatment groups. The ratio of radiolabeled amplicon of the target to that of the reference standard (from the same cDNA source) was used as the measure of steady-state levels of target mRNA. From RNA isolation to signal detection, all individuals within a rat strain were processed simultaneously. Comparisons of AHR levels were made between treatment groups within each strain. Primer design. Amplimers were designed with the Primer software program (Whitehead Institute of Biomedical Research, Cambridge, MA; version 0.5) and, when possible, pairs were designed to contain intron– exon boundaries to distinguish potential genomic DNA contamination (Table 1). AHR primers did not span the exon 10/intron 10 mutation in H/W AHR, which results in three splice variants of the receptor in this strain. (Pohjanvirta et al., 1998). The Blast program (National Center for Biotechnology Information) was used to ensure specificity of the primers. Primers were commercially synthesized by ACGT Corp. (Toronto, Ontario, Canada) and concentrations were confirmed spectrophotometrically. For each reverse transcription, diethylpyrocarbonate-treated water was used as a negative control for contamination. The expected product sizes were AHR, 917 bp; CYP1A1, 672 bp; ARNT, 413 bp; and ␤-actin, 688 bp. Statistical Analyses The results are given as group means ⫾ SD. A strain-wise comparison of treated values with control values was performed for the means of treated groups across all variables analyzed at doses of 10, 30, and 100 ng/kg/day. Variables with homogeneous variances (directly or after logarithmic transformation; assessed by the Levene test) were analyzed by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range post-hoc test. In the case of nonhomogeneous variances, the Kruskal–Wallis nonparametric oneway ANOVA was used followed by the Mann–Whitney U test. The limit for statistical significance was set at 0.05. Body weights were analyzed over the entire TCDD exposure period by repeated measures ANOVA.

RESULTS

AHR Levels Measured by Radioligand Binding The AHR population was quantitated by measuring the apparent number of specific [ 3H]TCDD binding sites in liver

cytosol from TCDD-treated and untreated rats under standard assay conditions; this technique detects unoccupied ligand binding sites only. Representative sucrose density gradient profiles for control and TCDD-treated rats of all three strains are displayed in Fig. 1A. The shaded area highlights the total specific binding and reflects the AHR density. The visually discernible differences in peak areas illustrate the effect of TCDD treatment on AHR levels in each strain. Mean values for the area of the specific binding peak are graphically displayed in Fig. 1B. Control levels of AHR, as measured by radioligand binding, were similar in SD and H/W rats, which had approximately 90 fmol/mg protein of AHR; control AHR levels in L-E rats were almost twofold greater (all 32 weeks old). Since control levels were different among strains, comparison of treatment effects of TCDD among strains is facilitated by the Fig. 1B inset which displays results as a percentage of control. Radioligand binding was dose-dependently elevated at all doses up to twofold that of controls in SD rats. The effect of TCDD on AHR in SD rats observed by this technique is in accord with that previously observed by Sloop and Lucier (1987) under similar conditions. The only other statistically significant increase in specific radioligand binding was in L-E rats receiving 30 ng/kg/day. AHR Levels Measured by Immunoblotting Immunoblotting was used as an independent approach to measure changes in AHR protein in cytosol and nuclear extract. This technique detects immunoreactive protein regardless of functional integrity of the receptor. The specific AHR bands (⬃106 kDa for SD and L-E and ⬃98 kDa for H/W) are indicated by arrows on the representative immunoblot in Fig. 2A. The band identity was confirmed as described under Materials and Methods. The variation in band intensity illustrates the effect of TCDD on cytosolic AHR protein levels. No AHR was detectable in nuclear extracts in any treatment group (data not shown). Mean values for each treatment group are plotted in Fig. 2B. The 10 and 30 ng/kg/day doses produced the

SUBCHRONIC TCDD EXPOSURE IN RATS: EFFECT ON AHR EXPRESSION

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FIG. 1. Radioligand binding in hepatic cytosol of TCDD-treated rats. Cytosols (5 mg/ml protein) were incubated with 10 nM [ 3H]TCDD as the radioligand in the absence (■) or presence (䊐) of a 100-fold molar excess of 2,3,7,8-tetrachlorodibenzofuran as the competitor. Unbound ligand was removed with dextran-coated charcoal and proteins were separated by velocity sedimentation on sucrose density gradients. Gradients were fractionated and radioactivity was measured in each fraction. (A) Representative radioligand binding profiles from untreated and TCDD-treated (30 and 100 ng/kg/day ⫻ 22 weeks) rats. The shaded area highlights the specific binding peak. Specific binding was calculated by subtracting binding in the presence of competitor from binding in the absence of competitor. (B) Dose effect of 22-week TCDD treatment on hepatic AHR measured by radioligand binding. Œ, SD; ƒ, L-E; 䡬, H/W. Specific binding is expressed per mg total protein (measured in duplicate) in samples analyzed on sucrose gradients. Plotted values are means of eight animals per treatment group ⫾ Standard Deviation with unidirectional error bars for clarity. *Statistically significant difference from control ( p ⬍ 0.05). (Inset) Results are expressed as percentages of control for each strain (n ⫽ 8/strain). Individual values were divided by the mean of control (shown to be normally distributed) for each strain. Error bars have been omitted for clarity.

greatest increases in cytosolic receptor protein: statistically significant elevations in AHR immunoreactive protein were produced by 30 ng/kg/day TCDD in SD rats and by 10 and 30 ng/kg/day TCDD in L-E rats. At the 100 ng/kg/day dose, a marginal, though statistically significant depletion in AHR protein was observed in H/W rats but not in the other strains. Cross-strain comparisons are facilitated by the Fig. 2B inset, which displays results as a percentage of control. The pronounced increase in SD AHR with 30 and 100 ng/kg/day observed by radioligand binding was not observed by immunoblotting. Other laboratories have measured AHR levels in whole tissue lysates (Pollenz et al., 1998; Roman et al., 1998; Sommer et al., 1999) rather than in cytosol and nuclear extract as in our study. We have previously shown that receptor levels in cytosols from TCDD-treated animals were highly correlated with those in whole tissue lysates from the same animal (r 2 ⫽ 0.945, n ⫽ 8), indicating that quantitation of AHR in either preparation is reliable (Franc et al., 2001).

Effect of TCDD on mRNA Levels The effect of TCDD on AHR expression was assessed at the mRNA level using RT–PCR to measure relative levels of steady-state mRNA. AHR, ARNT, and CYP1A1 target mRNAs were normalized to the internal control, ␤-actin. Since all measurements were derived within the exponential range of amplification, normalized band intensity is a measure of steady-state mRNA levels of each target gene. Representative PCR amplicons for the target and control mRNAs are displayed in Fig. 3A. Mean normalized values are plotted in Fig. 3B. The three rat strains responded similarly to TCDD treatment at the mRNA level. Ten or 30 ng/kg/day resulted in significant up-regulation of AHR mRNA in all rat strains. The 100 ng/kg/day dose also elevated AHR mRNA in L-E rats. Increases in AHR mRNA did not always translate into statistically significant elevations in AHR protein; however, all groups exhibiting elevated protein levels had a corresponding increase in mRNA. Changes in ARNT mRNA were observed

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indicating that the orally administered TCDD was effectively absorbed and received by the liver. Body Weight Changes Temporal body weight changes over the 22-week TCDD exposure period are displayed in Fig. 4. L-E rats were most sensitive to suppression of normal body weight gain as reflected by a highly significant ( p ⬍ 0.001) strain effect as well as a highly significant ( p ⬍ 0.001) time ⫻ strain ⫻ TCDD interaction term in the repeated measures ANOVA. Tissue Weight Changes

FIG. 2. Immunoblotting for AHR protein in hepatic cytosols of TCDDtreated rats. Cytosolic proteins were separated by SDS–PAGE (L-E and SD ⫽ 75 ␮g total protein; H/W ⫽ 150 ␮g total protein per lane), electrotransferred onto polyvinylidine difluoride membranes, and immunoblotted with an antiAHR antibody as described under Materials and Methods. (A) Representative immunoblots for AHR: The illustrated blots were digitally reconstructed from the original blots; signal and background intensities remained intact in the process. Arrows indicate the specific AHR band; the AHR migrated to ⬃106 kDa for L-E and SD and ⬃98 kDa for H/W. (B) Dose effect of 22-week TCDD treatment on AHR protein levels measured by immunoblotting. Œ, SD; ƒ, L-E; E, H/W. Band intensities were normalized to the mean of two normalization standards loaded on the same gel for comparison between gels. Plotted values are means of normalized band intensities ⫾ Standard Deviation (n ⫽ 8/treatment group) with unidirectional error bars for clarity. Assays were performed in duplicate. *Statistically significant difference from control ( p ⬍ 0.05). (Inset) Results are expressed as percentages of control for each strain (n ⫽ 8/strain). Individual values were divided by the mean of controls (shown to be normally distributed) for each strain. Error bars have been omitted for clarity.

in some treatment groups, notably, an increase with 30 ng/kg/ day in L-E and H/W and a decrease below control levels with 100 ng/kg/day in SD and H/W. The effect of TCDD on ARNT protein levels was not investigated. For each strain, the greatest degree of up-regulation for both AHR and ARNT mRNA resulted from the 30 ng/kg/day dose. The well-characterized induction of CYP1A1 served as a biomarker for the presence of TCDD in the liver. No CYP1A1 mRNA was detectable in control animals within the exponential range of PCR amplification for TCDD-treated animals; consequently, a fold induction could not be calculated for CYP1A1. However, TCDD produced a dose-dependent induction of CYP1A1 in all strains,

Since TCDD influences body weight gain, changes in tissue weights with treatment are expressed relative to body weight (Fig. 5). Liver. TCDD produced liver hypertrophy in all animals. SD rats receiving the highest dose (100 ng/kg/day) exhibited a notable increase in liver/body weight ratio up to 160% of controls. Liver/body weight ratio was comparable in L-E and H/W rats at all doses despite the greater dose-dependent suppression of body weight gain in L-E rats. Thymus. The 10 ng/kg/day dose of TCDD had no significant impact on thymus in any strain. At the higher doses, a similar extent of thymic weight loss (relative to controls) was observed in all strains, though absolute thymus weights were greater at all doses in H/W rats, which had a larger control thymus weight. DISCUSSION

The ultimate mechanisms that produce TCDD toxicity and lethality remain unknown but are well recognized as being initiated by the AHR. Classical receptor theory predicts that changes in receptor density (B max) will influence the subsequent response to receptor agonists. The extrapolation of receptor density to tissue response is often difficult. However, changes in receptor density can influence both the potency and maximal response of an agonist depending on the nature of the agonist–receptor– effector system (Ruffolo, 1982; Kenakin, 1997). In intact organisms, regulation of receptor density by its own agonists has been observed in many receptor systems (Lefkowitz, 1981). For some of these, up-regulation has been linked to the enhanced potency (sensitization) and down-regulation has been linked to tolerance (desensitization) of an organism to subsequent agonist insult. Our comprehensive examination of AHR expression in vivo indicates that AHR levels are not greatly perturbed by persistent, low-dose exposure to the most potent AHR agonist, TCDD. The complex relationships between AHR concentration and various biological responses to TCDD remain unknown in vivo and may depend on the specific endpoint. The maintenance of a relatively steady receptor density in the presence of persistent agonist activation demonstrated in our

SUBCHRONIC TCDD EXPOSURE IN RATS: EFFECT ON AHR EXPRESSION

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FIG. 3. RT–PCR amplification of AHR, ARNT, and CYP1A1 mRNA from liver of TCDD-treated rats. One microgram of total RNA per sample was reverse transcribed and PCR amplified to incorporate radiolabeled ␣-[ 32P]dCTP. PCR product was separated by polyacrylamide gel electrophoresis and radioactive emissions were captured on a Phosphor screen for detection by a phosphorimager (see Materials and Methods). (A) Representative digitized images of radiolabeled PCR product for AHR, ARNT, CYP1A1, and ␤-actin mRNA. PCR product for ␤-actin was used as an internal normalization standard for the target genes. Images shown were digitally reconstructed from original images; band and background intensities were not altered in the process. (B) Dose effect of 22-week TCDD treatment on AHR, ARNT, and CYP1A1 mRNA levels. Œ, SD; ƒ, L-E; E, H/W. Values are group means ⫾ Standard Deviation (n ⫽ 8/treatment group) of normalized band intensities with unidirectional error bars for clarity. Assays were performed in triplicate from the same RNA isolate. *Statistically significant difference from control ( p ⬍ 0.05).

experiments suggests that sensitivity is not likely to be appreciably altered by a dioxin regimen mimicking typical environmental exposure. Body burden rather than daily intake is currently considered the best dose metric for interspecies comparisons and extrapolations (DeVito et al., 1995). The lowest dose in our experiments, 10 ng/kg/day, is well above (10,000fold) the intake level experienced from general “background” environmental exposure (Grassman et al., 1998). However, this corresponds to a body burden of approximately 200 ng/kg. Although this is about 10 times greater than the mean value in the general human population, only marginal effects on AHR expression were observed in three strains of rat. Dose and duration of exposure appear to be the most im-

portant factors determining the magnitude and the direction of change in tissue AHR concentrations by TCDD; these factors appear to be valid for both acute and prolonged exposures to TCDD. However, the pattern of agonist-induced regulation following persistent long-term exposure (months) is clearly different from that previously observed in acute short-term exposures (hours to days) both in vivo and in cell culture. It has been previously shown that, following short-term treatment of cultured cells with TCDD, AHR levels are depleted immediately following treatment (Pollenz, 1996; Giannone et al., 1998) and that this depletion persists up to 72 h—the maximum time-frame tested. In vivo, an acute 10 or 50 ␮g/kg dose of TCDD produces a similar pronounced depletion in AHR levels

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short-term TCDD exposure, there was a notable lack of depletion of AHR levels in vivo; a single treatment group (H/W; 100 ng/kg/day) exhibited statistically significant depletion by immunoblotting. By multiple technical approaches and in multiple rat strains, we found that persistent, low-dose TCDD did not have a substantial impact on AHR levels in vivo. This implies that little, if any, change in the dose–response behavior should be expected following “subchronic” TCDD exposure. For two of the three strains, the measured changes in AHR levels by radioligand binding were paralleled by those obtained by immunoblotting. The two techniques provide a measurement of AHR protein by different endpoints: radioligand binding detects unoccupied, functional ligand binding sites only whereas immunoblotting detects immunoreactive protein regardless of functional integrity of the receptor. The power of receptor quantitation is greatly improved by combination of the two techniques. In our previous short-term experiments, results from radioligand binding and immunoblotting were well correlated (Franc et al., 2001). In the current study, the TCDD-

FIG. 4. Temporal body weight change of female rats treated with TCDD. Body weight was measured every 2 weeks for the 22-week duration of TCDD treatment. Plotted values are means of eight animals per treatment group; error bars have been omitted for clarity of presentation, but Standard Deviations were less than 12% of the mean for all groups. ■, control; ƒ, 10 ng/kg/day; E, 30 ng/kg/day; ‚, 100 ng/kg/day.

at early time points (hours) (Pollenz et al., 1998; Franc et al., 2001). However, within days (10 –14 days), hepatic tissue recovers from this initial depletion and AHR levels are restored (Franc et al., 2001). A similar transient depletion followed by recovery is also evident in other tissues (Pollenz et al., 1998). Recovery of cytosolic AHR protein is accompanied by an increase in steady-state AHR mRNA, suggesting that regulation occurs at the pretranslational level (Franc et al., 2001). It remains unclear whether the failure of recovery in cell culture is due to a true inability of cultured cells to restore AHR protein levels or whether 72 h simply is insufficient time for recovery. Lower doses (5 ␮g/kg) in vivo produce yet a different pattern of regulation: AHR mRNA and protein levels increase without an initial transient depletion phase; this results in a net overall increase in cytosolic AHR levels (Franc et al., 2001). In our current long-term investigation, hepatic AHR levels remained either unchanged or were modestly increased, depending on dose, after 22 weeks of TCDD exposure. In contrast to

FIG. 5. Dose-related impact of 22-week TCDD treatment on liver and thymus weights. Tissue weights, as ratios of body weight at the time of euthanasia, are expressed as percentages of control. Individual values were divided by the mean of the respective control (shown to be normally distributed) for each strain. Œ, SD; ƒ, L-E; E, H/W. Plotted values are means (n ⫽ 8/treatment group) ⫾ Standard Deviation with unidirectional error bars for clarity. *Statistically significant difference from control ( p ⬍ 0.05). Control values for tissue/body weight ratios were SD, 0.02820 ⫾ 0.0015; L-E, 0.03010 ⫾ 0.0012; H/W, 0.0290 ⫾ 0.0018 for liver and SD, 0.00053 ⫾ 0.00016; L-E, 0.00020 ⫾ 0.00004; H/W, 0.00069 ⫾ 0.00009 for thymus.

SUBCHRONIC TCDD EXPOSURE IN RATS: EFFECT ON AHR EXPRESSION

induced increase in radioligand binding in one strain (SD) was proportionately greater than the corresponding immunoblot would indicate; the reason is unclear. This apparent up-regulation in SD when assessed by radioligand binding is, nevertheless, in accord with that of Sloop and Lucier (1987) who measured AHR by radioligand binding in SD rats in a similar treatment regimen. Altered AHR protein levels are effectively the result of the dynamics of change of protein synthesis versus degradation. In the current study, all increases in protein were accompanied by a corresponding increase in mRNA, though increases in mRNA were not always accompanied by an increase in AHR protein. A change in steady-state mRNA could be due to effects on transcription and/or mRNA turnover with or without any change in receptor synthesis. A lack of change in receptor protein (in the presence of elevated mRNA levels) might reflect either lack of translation into new protein or replenishment of previously depleted receptor protein. In cell culture it has been shown that, following receptor activation by ligand, the AHR is rapidly degraded via the ubiquitin–proteasome pathway (Ma and Baldwin, 2000). Although it has not been investigated, it is probable that this proteolytic pathway also functions in vivo. In the current study we observed that cytosolic AHR levels either remain unchanged or are marginally elevated by TCDD. Overall it appears that, with persistent TCDD exposure in vivo, a balance between degradation and synthesis is established so that AHR levels remain relatively constant in the long term. It has been proposed that the mechanisms of toxicity after short- and long-term exposure to TCDD are the same, although the manifestation of toxic response is different depending on the toxicokinetic/toxicodynamic time scales (Li and Rozman, 1995; Viluksela et al., 1998a,b). Dose–response relationships for diverse endpoints after acute, subchronic, and chronic administration of TCDD are reported to be very similar if not identical and a possibly generalizable rule holds: “tissue concentration ⫻ time ⫽ toxicity” (Rozman et al., 1993). Our previous examination of the effect of acute TCDD exposure on AHR regulation was also conducted in dioxin-sensitive L-E and SD rats versus dioxin-resistant H/W rats. Acute lethality has been extensively examined in the L-E versus H/W model, but less is known about the effects of prolonged, low-dose TCDD exposure in this model. There is evidence that L-E and H/W rats differ in their susceptibility to some of the toxic effects from chronic TCDD exposure as they do from acute exposure (Viluksela et al., 2000). Here, we compared the TCDD-induced regulation of AHR expression in resistant versus sensitive rat strains to determine if there was a differential pattern of chronic TCDD-induced AHR regulation between these. Such a difference could preferentially influence the dose–response relationship resulting in a shift in susceptibility among strains. All three rat strains showed some response of AHR levels to persistent, low-dose TCDD. Although there were some differences in AHR regulation among the strains tested, strain sensitivity to TCDD toxicity/lethality proved to

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be unrelated to susceptibility to agonist-induced AHR regulation. The characteristic wide range of inherent organism susceptibility to TCDD is a major complication in the assessment of human risk of dioxins. External factors that alter susceptibility either by sensitizing or desensitizing organisms to TCDD further complicate risk assessment. Currently, much dioxin risk assessment is based on induction of liver tumors in rats. Tumor promotion is a known AHR-mediated event. The finding that AHR levels are not greatly perturbed by environmentally relevant TCDD exposure and that this can be generalized to several strains of rat is important to risk assessment. From the perspective of risk, it may be reassuring that persistent exposure to TCDD does not cause a substantial alteration of AHR levels in liver. The balance between synthesis and degradation of AHR seems to be maintained at an equilibrium in the face of TCDD exposure such that the animal is not likely to be sensitized nor desensitized to this important environmental toxicant. ACKNOWLEDGMENTS This research was supported by the Medical Research Council of Canada/ Canadian Institutes of Health Research (A.B.O. and M-A.F.); the Academy of Finland, Research Council for Health (Grant 43984); the Finnish Research Program for Environmental Health (SYTTY) (Grant 42386); and the European Commission (Contract ENV4-CT96-0336) (J.T. and R.P.). We thank Ms. Arja Tamminen and Ms. Minna Voutilainen for excellent technical assistance.

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SUBCHRONIC TCDD EXPOSURE IN RATS: EFFECT ON AHR EXPRESSION and Birnbaum, L. S. (1998). Female Sprague–Dawley rats exposed to a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin exhibit sustained depletion of aryl hydrocarbon receptor protein in liver, spleen, thymus, and lung. Toxicol. Sci. 42, 117–128. Pollenz, R. S., Sattler, C. A., and Poland, A. (1994). The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy. Mol. Pharmacol. 45, 428 – 438. Prokipcak, R. D., and Okey, A. B. (1991). Downregulation of the Ah receptor in mouse hepatoma cells treated in culture with 2,3,7,8-tetrachlorodibenzop-dioxin. Can. J. Physiol. Pharmacol. 69, 1204 –1210. Richardson, V. M., Santostefano, M. J., and Birnbaum, L. S. (1998). Daily cycle of bHLH–PAS proteins, Ah receptor and Arnt, in multiple tissues of female Sprague–Dawley rats. Biochem. Biophys. Res. Commun. 252, 225– 231. Roman, B. L., Pollenz, R. S., and Peterson, R. E. (1998). Responsiveness of the adult male rat reproductive tract to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure: Ah receptor and ARNT expression, CYP1A1 induction, and Ah receptor down-regulation. Toxicol. Appl. Pharmacol. 150, 228 –239. Rose, J. Q., Ramsey, J. C., Wentzler, T. H., Hummel, R. A., and Gehring, P. J. (1976). The fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36, 209 –226. Rozman, K., Roth, W. L., Greim, H., Stahl, B. U., and Doull, J. (1993). Relative potency of chlorinated dibenzo-p-dioxins (CDDs) in acute, subchronic and chronic (carcinogenicity) toxicity studies: Implications for risk assessment of chemical mixtures. Toxicology 77, 39 –50. Ruffolo, R. R., Jr. (1982). Review: important concepts of receptor theory. J. Auton. Pharmacol. 2, 277–295. Safe, S. H. (1995). Modulation of gene expression and endocrine response pathways by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol. Ther. 67, 247–281. Schecter, A., Furst, P., Furst, C., Papke, O., Ball, M., Ryan, J. J., Hoang, D. C., Le, C. D., Hoang, T. Q., Cuong, H. Q., et al. (1994). Chlorinated dioxins and dibenzofurans in human tissue from general populations: A selective review. Environ. Health Perspect. 102(Suppl. 1), 159 –171. Shimba, S., Todoroki, K., Aoyagi, T., and Tezuka, M. (1998). Depletion of arylhydrocarbon receptor during adipose differentiation in 3T3–L1 cells. Biochem. Biophys. Res. Commun. 249, 131–137. Sloop, T. C., and Lucier, G. W. (1987). Dose-dependent elevation of Ah receptor binding by TCDD in rat liver. Toxicol. Appl. Pharmacol. 88, 329 –337. Sommer, R. J., Sojka, K. M., Pollenz, R. S., Cooke, P. S., and Peterson, R. E. (1999). Ah receptor and ARNT protein and mRNA concentrations in rat prostate: Effects of stage of development and 2,3,7,8-tetrachlorodibenzo-pdioxin treatment. Toxicol. Appl. Pharmacol. 155, 177–189. Sweeney, M. H., Calvert, G. M., Egeland, G. A., Fingerhut, M. A., Halperin, W. E., and Piacitelli, L. A. (1997). Review and update of the results of the

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