Page 852 - The Toxicology of Fishes

832 The Toxicology of Fishes

of several genes important for the regulation of follicular development (e.g., lhr, fshr, and all three
estrogen receptors) and estrogen biosynthesis (e.g., cyp19a1a) likely contribute to observed impacts of
TCDD on follicular development and reduced serum estradiol concentrations. Global changes in gene
expression demonstrate that TCDD impacts several integrated cellular pathways, resulting in alterations
in the expression of genes important for glucose and lipid metabolism, regulation of transcription, and
immune function. Their data also suggest that, although AhRE-mediated changes in gene expression
likely contribute to TCDD-induced ovarian toxicity, alterations in gene expression may be downstream
from AhR activation and AhRE-regulated transcription. Further, interference with estrogen-regulated
signal transduction by AhR signaling is likely a key component of the ovarian toxicity of TCDD.

ZfAHR2 Is Required for TCDD Developmental Toxicity in Zebrafish
It is not clear how activation of AhR signaling by TCDD produces such diverse biological responses in
zebrafish larvae; however, a variety of factors are probably involved, including AhR agonist potency,
duration of AhR activation, organism life stage, and differential responsiveness of cells and tissues to
AhR activation by TCDD (Walisser et al., 2004). In vitro assays suggest that zfAHR2 is the likely AhR
isoform that mediates responses to TCDD in zebrafish. Radioligand-binding assays demonstrate that
zfAHR2 specifically binds TCDD (Andreasen et al., 2002a), and exposure to the AhR ligand β-naph-
thoflavone causes zfAHR2 to translocate to the nucleus (Wentworth et al., 2004), where it can dimerize
with ARNT. ZfAHR2 and one form of zfARNT2 (zfARNT2b) have been shown to form a functional
heterodimer in vitro that specifically binds AhRE sequences in gel mobility shift assays and induces
AhRE-driven reporter gene activity in COS-7 cells exposed to TCDD (Tanguay et al., 2000). In contrast,
zfAHR1 is unable to bind TCDD in radioligand-binding studies and only weakly interacts with AhRE
sequences or promotes AhRE-driven transcription when expressed with ARNT2 proteins (Andreasen et
al., 2002a). To determine if zfAHR2 mediates TCDD developmental toxicity, antisense morpholino
oligonucleotides (MOs) were used to knock down levels of zfAHR2 protein expression in zebrafish
embryos, creating zfahr2 morphants, and the morphants were subsequently exposed to TCDD (Antkiewicz
et al., 2006; Bello et al., 2004; Carney et al., 2004; Dong et al., 2004; Hill et al., 2004a; Prasch et al.,
2003; Teraoka et al., 2003). The zfAHR2 morpholino results convincingly demonstrate that zfAHR2 is
required for mediating the hallmark endpoints of TCDD toxicity in developing zebrafish. Zfahr2 morphants
show decreased levels of zfCYP1A induction after TCDD exposure, demonstrating that induction of
zfCYP1A requires zfAHR2 (Carney et al., 2004; Dong et al., 2004; Prasch et al., 2003; Teraoka et al.,
2003). Zfahr2 morphants also show protection against certain cardiovascular endpoints of TCDD devel-
opmental toxicity, including reduced peripheral blood flow, reduced stroke volume, decreased cardiac
output, reduced number of cardiomyocytes in the heart, increased incidence of ventricular standstill,
impaired common cardinal vein migration, pericardial edema, and impaired erythrocyte maturation (Ant-
kiewicz et al., 2006; Bello et al., 2004; Carney et al., 2004; Prasch et al., 2003; Teraoka et al., 2003). The
zfahr2-MO also provides protection against the TCDD-induced increase in apoptosis in the dorsal midbrain
(Dong et al., 2004) and partial protection against reductions in lower jaw growth (Prasch et al., 2003). A
possible reason for why the TCDD-induced impairment in lower jaw growth is only partially protected
by treatment with zfahr2-MO is because impaired jaw growth can still be elicited by the TCDD that
remains in the fish after the effect of the zfahr2-MO has worn off and zfAHR2 levels return to normal
(Prasch et al., 2003). In contrast to the findings with zfAHR2, embryos injected with a morpholino targeted
against zfAHR1A do not show significant protection against any of the overt endpoints of TCDD
developmental toxicity (Carney, unpublished results); thus, zfAHR2 is the AhR isoform that mediates
TCDD developmental toxicity in zebrafish. Interestingly, a morpholino targeted against zfAHR1A does
provide partial protection against PAH toxicity in zebrafish embryos (Incardona et al., 2005, 2006).

ZfARNT1 but Not ZfARNT2 Is Required for
TCDD Developmental Toxicity in Zebrafish
To evaluate if the zfARNT2 protein is the functional dimerization partner for zfAHR2 in vivo, zfarnt2
morphants and a line of insertional mutant zebrafish lacking expression of all forms of zfARNT2 (Golling
et al., 2002; Prasch et al., 2004a) were assessed for responses to TCDD (Prasch et al., 2004b). Surprisingly,