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The implications of low-affinity AhR for TCDD insensitivity in frogs.

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 AhR 1 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