Page 848 - The Toxicology of Fishes

828 The Toxicology of Fishes

The zebrafish is ideally positioned phylogenetically to make it a useful model for investigating TCDD
developmental toxicity. Zebrafish, as vertebrates, are closely related to humans, so results on TCDD
toxicity in zebrafish embryos and larvae are of human health relevance. Zebrafish, as fish, are useful for
identifying adverse effects of TCDD exposure in fish and for functionally characterizing the AhR
signaling pathway in fish that mediates TCDD toxicity. Like trout, the zebrafish is an oviparous species
that has external fertilization and development; however, the time required for zebrafish to undergo early
development is much shorter than lake trout and rainbow trout, making the zebrafish ideal for hypothesis
testing in research on developmental toxicity. Also, unlike trout, the zebrafish embryo is transparent,
which permits easy detection of morphological alterations caused by TCDD. In addition to their short
generation time, zebrafish exhibit rapid growth and high egg yield and their genetics and developmental
biology are well known. The sequenced zebrafish genome is now being assembled and annotated, and
many molecular approaches exist to study gene function in zebrafish, including the production of
transgenic and mutant fish and the ability to selectively block the expression of certain genes in zebrafish
embryos using morpholino oligonucleotide (MO) knock-down technology (Nasevicius and Ekker, 2000;
Udvadia and Linney, 2003). Thus, the zebrafish model has great appeal for developmental toxicologists.
Among all environmental contaminants evaluated for developmental toxicity in zebrafish (Carney et al.,
2006a,b; Hill et al., 2005; Spitsbergen and Kent, 2003; Tanguay et al., 2003), TCDD is the most
extensively studied. Target organs of TCDD developmental toxicity have been identified and components
of the AhR signaling pathway functionally characterized in zebrafish. Progress has been made in
understanding how hyperactivation of the AhR pathway by TCDD leads to developmental toxicity in
zebrafish by a CYP1A-independent mechanism.

AhR Signaling in Fish
The AhR is a member of the basic helix–loop–helix PAS family of transcription factors (Huang et al.,
1993). Proteins in the α-class, such as AhR and hypoxia-inducible factor-1α (HIF-1α), function as sensor
proteins. Once activated, α-class proteins dimerize with β-class proteins, such as ARNT1 and ARNT2,
and the heterodimer (i.e., AhR/ARNT) binds specific DNA sequences termed AhR response elements
(AhREs), leading to altered gene expression. The AhR has three functions: (1) adaptation signaling,
leading to upregulation of xenobiotic metabolizing enzymes; (2) toxic signaling, causing adverse effects
from persistent, high-affinity ligands such as TCDD; and (3) developmental signaling, resulting in normal
development of certain organs and tissues (Denison and Nagy, 2003; Fernandez-Salguero et al., 1996;
Gu et al., 2000; Mimura et al., 1997; Peters et al., 1999; Walisser et al., 2004). All three types of signaling
require sequential ligand activation of the AhR, translocation of the liganded receptor to the nucleus and
heterodimerization of AhR with ARNT.
To establish zebrafish as a model for investigating TCDD developmental toxicity in fish, it was essential
that components of the AhR signaling pathway in zebrafish (Figure 21.4) be identified and functionally
characterized (Andreasen et al., 2002a; Carney et al., 2004, 2006a; Evans et al., 2005; Prasch et al.,
2006; Tanguay et al., 1999, 2000; Zodrow et al., 2001). Zebrafish express three forms of AhR: zfAHR1A,
zfAHR1B, and zfAHR2 (Andreasen et al., 2002a; Karchner et al., 2005; Tanguay et al., 1999). The three
zfAHRs exist as a result of gene duplications that occurred during vertebrate evolution as well as a
genome duplication specific to the fish lineage. Multiple ahr genes are also found in other fish species
(Hahn et al., 1997, 2006). Likewise, there are two arnt genes in zebrafish. ZfARNT2, which exists as
multiple splice variants, was the first zebrafish ARNT identified as a possible dimerization partner for
zfAHRs (Hsu et al., 2001; Tanguay et al., 2000; Wang et al., 2000). Later, a second arnt gene, zfarnt1,
was identified in zebrafish and the zfARNT1 protein was determined to be the dimerization partner for
zfAHR2 involved in mediating TCDD developmental toxicity (Antkiewicz et al., 2006; Prasch et al.,
2006). Additional components of the zebrafish AhR signaling pathway include two AhR repressor genes,
zfahrr1 and zfahrr2 (Evans et al., 2005). Zebrafish embryo exposure to TCDD upregulated in vivo
expression of zfAHRs and repressed in vitro transactivation of the AhR/ARNT heterodimer (Evans et
al., 2005). The in vivo role of the zfAHRs in modulating TCDD developmental toxicity in zebrafish,
however, has yet to be established. An AhR interacting protein (AIP) has been identified in zebrafish
(Zodrow et al., 2001), and its role in zfAHR signaling is being investigated.