Download TOXICOLOGICAL HIGHLIGHT The Implications of Low

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
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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. A re-examination of
previous toxicant results with X. laevis in light of the current
findings could prove insightful.
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