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,² , ‡ , § Jouko Tuomisto,² and Allan B. Okey* *Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada, M5S 1A8; ² 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 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 intra- gastrically, 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 appar- ent relationship between TCDD-induced AHR regulation and strain sensitivity to TCDD. Overall, “subchronic” TCDD did not greatly perturb AHR expression. The maintenance of rela- tively 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; 2,3,7,8- tetrachlorodibenzo-p-dioxin; rat; dioxin susceptibility; regula- tion of gene expression. 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 cer- tain 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; Fernan- dez-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 1 Abbreviations used: AHR, aryl hydrocarbon receptor; AP, antifluorescein- alkaline 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. Toxicology and Applied Pharmacology 175, 43–53 (2001) doi:10.1006/taap.2001.9222, available online at http://www.idealibrary.com on 43 0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.