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TOXICOLOGICAL SCIENCES 88(1), 1–3 (2005) doi:10.1093/toxsci/kfi321 TOXICOLOGICAL HIGHLIGHT The Implications of Low-Affinity AhR for TCDD Insensitivity in Frogs Adria A. Elskus United States Geological Survey, Department of Biological Sciences, University of Maine, Orono, Maine 04469 Received September 7, 2005; accepted September 8, 2005 The article by Lavine et al. (2005) in this issue provides a molecular explanation for the long-recognized but not understood dioxin-insensitivity of the amphibian-based toxicant screen FETAX (Frog Embryo Teratogenesis Assay—Xenopus). FETAX is the industry standard for identifying potential human developmental toxicants and teratogens (NIEHS, 2000), and it is widely used as a screening tool for evaluating amphibian sensitivity to environmental contaminants (Mann and Bidwell, 2000). By providing insights into the molecular mechanisms underlying frog response to environmental contaminants, the work of Lavine et al. (2005) will enable ecotoxicologists to refine, improve, and advance the use of frogs for toxicant screening methods for human developmental/teratogenic agents and the use of FETAX for ecotoxicological applications, an area of particular importance given the continuing rise in global occurrences of amphibian deformities. The crux of the findings reported by Lavine et al. (2005) is that TCDD, one of the most potent developmental toxicants known, binds with very low affinity to frog aryl hydrocarbon receptor (AhR). TCDD mediates most, if not all, of its toxic effects in vertebrates through the AhR. Upon ligand binding, cytosolic AhR releases hsp90 and other chaperone proteins, translocates into the nucleus, and heterodimerizes with ARNT (AhR nuclear translocator). The liganded AhR/ARNT complex transcriptionally activates a host of genes in the AhR gene battery via binding to DNA motifs (AhREs, also known as DREs and XREs) in the regulatory regions of phase I (CYP1A, CYP1B) and other xenobiotic metabolizing genes, as well as many genes involved in a variety of other cellular processes (Puga et al., 2002). Lavine et al. (2005) identify two distinct AhR1 genes in Xenopus laevis, AhR1a and AhR1b. Phylogenetic analysis using the amino acid sequences of the PAS domain revealed these AhRs to be orthologous to mammalian AhR and paralogous to fish AhR2 gene(s). Whereas mammals appear to have only one AhR form, fish have at least two, AhR1 and AhR2, with AhR2 believed to be responsible for the toxicologic functions of AhR in fish (Karchner et al., 2005; Prasch et al., 2003). While Lavine et al. (2005) found that both frog AhR forms bind TCDD, they bind with an affinity at least 20-fold lower than mouse AhRbÿ1 and are less responsive in TCDD-induced reporter gene assays using a mouse CYP1A1 promoter. Induction of CYP1A genes is a widely used, highly sensitive marker of AhR activation, with toxicant exposure inducing CYP1A expression levels over 400-fold in some species (Courtenay et al., 1999). In vivo studies with TCDDtreated X. laevis A6 cells confirmed endogenous AhR activation, although high concentrations of TCDD were required to achieve induction of CYP1A6 and CYP1A7 mRNAs relative to the concentrations needed to induce CYP1A in fish cell lines. The low affinity of TCDD for X. laevis AhRs in conjunction with the exceptionally low response to TCDD in the luciferase assay and the low response of endogenous CYP1As in the A6 cells, provide support for the contention (Lavine et al., 2005) that low affinity of the AhR for TCDD translates into significant reduction in AhR function with respect to CYP1A regulation. The low response of frogs to TCDD is similar to that found in TCDD-resistant fish populations, in certain strains of mice, and in some birds. As in X. laevis, the AhR machinery is present and functioning in TCDD-resistant fish, but it requires high levels of inducer (10–100-fold higher) to provoke CYP1A induction of a similar magnitude as that induced in responsive fish populations (Bello et al., 2001 and references therein). Whether the low sensitivity to TCDD found in resistant fish populations is the result of AhR(s) with reduced affinity for dioxin-like compounds is not yet clear (Hahn et al., 2004). However, numerous studies in birds and mammals report differences in AhR affinity for TCDD that reflect differences in the relative sensitivity to this compound (see citations in Lavine et al., 2005), suggesting that for compounds with similar efficacies, AhR affinity may determine the relative toxic potency of the AhR ligand. Differences in AhR affinity may not, however, completely explain differences in the biochemical response to toxicants. Recent studies with fish found that populations that have acquired resistance to CYP1A induction by dioxin-like polychlorinated biphenyls (PCBs) remain responsive to polynuclear aromatic hydrocarbons (Courtenay et al., 1999), even though both compound classes exert their effects through the AhR (Fernandez-Salguero et al., 1996; Shimizu et al., 2000). Whether this reflects differences in the balance of AhR Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please email: [email protected] 2 ELSKUS variants with differing ligand affinities (Hahn et al., 2004; Karchner et al., 2005), or altered regulation of AhR(s), remains unknown. The findings put forth by Lavine et al. (2005) prompt several questions, including whether or not FETAX is an appropriate screen for chemicals that exert their effects through the AhR. For humans, known to have a low affinity AhR (Silkworth et al., 2005), FETAX may remain an appropriate screen. However for amphibians, whose AhR affinities for dioxin-like chemicals are not known, the relevance of FETAX for screening AhR ligands is uncertain. FETAX relies on the response of only one organism, the African clawed frog, Xenopus laevis. The relative insensitivity of ranid frogs to the model developmental toxicant, TCDD, has been known for nearly 30 years (Beatty et al., 1976), yet there have been only a handful of studies comparing the relative sensitivity of X. laevis to that of other frog species (see Mann and Bidwell, 2000). Such studies reveal that X. laevis is not always the most sensitive species. Do AhRs with low affinity for dioxin-like compounds benefit X. laevis? Ahr-null mice and fish with low inducibility of CYP1A show resilience to the toxic effects of TCDD and the procarcinogen, benzo[a]pyrene (Bello et al., 2001; FernandezSalguero et al., 1996; Shimizu et al., 2000), suggesting that reduced AhR function may protect against the deleterious effects of these chemicals. Indeed, if all amphibians are shown to have AhRs with low affinity for dioxin, it would suggest that lowaffinity AhR may confer a selective advantage for amphibians. Alterations in AhR may affect a multitude of AhR-regulated processes, making it difficult to predict whether low affinity for TCDD translates into low affinity for endogenous ligands and whether low affinity for TCDD influences other AhR-mediated processes. Mediation of processes such as development, signal transduction, endocrine homoestasis, or cell cycle control, among others, involves AhR ‘‘cross-talk’’ with other receptors and proteins via signal-transduction cascades (Oesch-Bartlomowicz et al., 2005; Pocar et al., 2005; Puga et al., 2002). For example, TCDD can have antagonist or agonist effects on reproductive processes due to indirect cross-talk between the estrogen receptor (ER) and the AhR (reviewed in Pocar et al., 2005). Because hormones are also involved in developmental processes, hormone mimics and/or antagonists (endocrine disruptors) whose actions require a fully functional AhR may not be properly ‘‘detected’’ by FETAX. Similar complexity is seen in the apparent contribution of AhR to processes leading, paradoxically, to both cell cycle arrest and cell proliferation (Puga et al., 2002). Altered affinity of AhR for TCDD could affect its utility as an ‘‘environmental sensor,’’ signaling cell cycle arrest in response to contaminant-induced DNA damage (Puga et al., 2002). A recent report implicating cyclic AMP as an endogenous activator of AhR contends that cAMP activation of AhR provides AhR with a role in development and differentiation that may serve to ‘‘balance’’ AhR activation by toxicants (Oesch-Bartlomowicz et al., 2005). These numerous roles for AhR reveal both its importance in homeostasis and the challenge of trying to predict how altered AhR(s) may affect these many processes. The aryl hydrocarbon receptor mediates not only the toxicity of several classes of compounds, including the model developmental toxin TCDD, it is also involved in endocrine homeostasis, developmental processes, cell cycle regulation, and signal transduction (Pocar et al., 2005; Puga et al., 2002). As evidenced from the above discussion, the variety and numbers of pathways in which AhR is directly or indirectly involved make it difficult, if not impossible, to predict what effects the low TCDD affinity of X. laevis AhR might have on various AhR-mediated processes in frogs. The findings of Lavine et al. (2005) prompt several questions, including whether or not FETAX is an appropriate screen for chemicals that exert their effects through the AhR. Further work characterizing the functionality of X. laevis AhR in these various realms is needed to fully evaluate the extent to which FETAX may be compromised as a tool for evaluating amphibian response to certain toxicants. 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