Page 853 - The Toxicology of Fishes

Reproductive Impairment of Great Lakes Lake Trout by Dioxin-Like Chemicals 833

it was discovered that zebrafish embryos with reduced or absent levels of zfARNT2 do not show
protection against endpoints of TCDD developmental toxicity. In TCDD dose–response experiments,
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wild-type and zfarnt2 knockout embryos still exhibited similar hallmark toxicity responses to TCDD,
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including pericardial edema, reduced blood flow, and reduced lower jaw growth. The zfarnt2 embryos
also exhibited zfCYP1A induction following TCDD treatment. This indicates that zfAHR2 signaling is
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functional in zebrafish embryos that lack zfARNT2. The sensitivity of zfarnt2 mutant embryos to
TCDD suggested that an alternative form of zfARNT must be expressed that can form a functional
heterodimer with zfAHR2. A search of the incomplete zebrafish genome revealed a likely zebrafish
ortholog of the mammalian arnt1, and multiple splice variants of zfARNT1 were subsequently amplified
and analyzed (Prasch et al., 2006). In vitro assays showed that zfARNT1 forms a functional heterodimer
with zfAHR2 that binds AhRE sequences and promotes AhRE-driven transcription. The in vivo role of
zfARNT1 in mediating TCDD developmental toxicity was then assessed using a morpholino designed
against the translation start site of zfARNT1. The zfarnt1-MO was observed to provide partial to complete
protection against endpoints of TCDD toxicity including pericardial edema, heart malformation, reduced
cardiac output, ventricular standstill, reduced peripheral blood flow, reduced lower jaw growth, and
zfCYP1A induction (Antkiewicz et al., 2006; Prasch et al., 2006). Thus, even though zfARNT2b can
function with zfAHR2 in vitro, all in vivo findings support a mechanism by which TCDD toxicity is
mediated by the zfAHR2 and zfARNT1 heterodimer.

Why Is Understanding AhR Signaling in Zebrafish Important?
Understanding AhR signaling in zebrafish is important because it provides a common mechanism for
extrapolating TCDD toxicity results from the zebrafish model to other fish species, including lake trout.
Recent findings on TCDD developmental toxicity in a marine fish species, red sea bream (Pagrus major),
revealed that many endpoints of TCDD developmental toxicity were similar to those in zebrafish (Yamau-
chi et al., 2006). Furthermore, it appeared in TCDD-exposed red sea bream larvae that rsAHR2, like
zfAHR2, mediated the induction of CYP1A and TCDD developmental toxicity (Yamauchi et al., 2006).
This suggests that functionally characterizing the AhR signaling pathway in zebrafish might ultimately
provide a foundation for both understanding and predicting differences between fish species in suscepti-
bility to TCDD toxicity. In support of this idea, the discovery in zebrafish that TCDD developmental
toxicity is mediated by zfAHR2 and zfARNT1 has caused scientists to focus on these transcription factors
as the genes most likely to be affected in populations of killifish (Fundulus heteroclitus) that have evolved
resistance to TCDD developmental toxicity. Similarly, functional understanding of the zebrafish AhR
signaling pathway also may shed light on why vertebrate embryos generally are more sensitive to TCDD
than adults. Finally, it is difficult to test for TCDD developmental and reproductive toxicity in a large
number of freshwater, estuarine and marine fish species. On the other hand, it is feasible to determine if
these species express AhR2 and ARNT1 and thereby have the capability of responding to TCDD; therefore,
knowledge of which ahr and arnt genes mediate TCDD toxicity in fish is important in determining any
effects TCDD and related AhR agonists may have on salmonine populations in the Great Lakes.

Induction of CYP1A Is Not Required for TCDD Developmental Toxicity in Zebrafish
A key question is whether or not induction of CYP1A is required for TCDD to cause early-life-stage
toxicity in fish. The transcription factors zfAHR2 and zfARNT1 are required for TCDD to cause
developmental toxicity in zebrafish; therefore, increased expression of one or more key genes mediated
by TCDD activation of the AhR2/ARNT1 signaling pathway may lead to a subsequent cascade of
molecular and cellular changes culminating in TCDD developmental toxicity in zebrafish. Cyp1a is the
most well-characterized TCDD-regulated gene in fish. It contains AhRE sequences in its promoter and
is directly regulated by the AhR/ARNT heterodimer (Abnet et al., 1999; Andreasen, 2002b; Powell et
al., 2004; Whitlock, 1999); however, there has been considerable speculation about whether or not
induction of cyp1a is required for TCDD developmental toxicity in fish.
Morpholino knock-down of zfcyp1a during embryonic development in zebrafish has provided an
exciting opportunity to gain new insight about the potential role of CYP1A in mediating TCDD toxicity.