Page 854 - The Toxicology of Fishes

834 The Toxicology of Fishes

Although an initial study of zfcyp1a morphants indicated possible protection against TCDD-induced
pericardial edema and reduced intersegmental blood flow at 72 hpf in zebrafish larvae (Teraoka et al.,
2003), a subsequent study that examined, far more comprehensively, the developmental responses to
TCDD at both 72 and 96 hpf in zfcyp1a morphants showed no protection whatsoever against several
endpoints of TCDD developmental toxicity (Carney et al., 2004). More specifically, induction of
zfCYP1A by TCDD was blocked to the same extent in zebrafish zfahr2 morphants and zebrafish zfcyp1a
morphants; however, in sharp contrast to the zfahr2 morphants that were rescued from TCDD develop-
mental toxicity, zfcyp1a morphants exposed to the same concentration of waterborne TCDD were not
protected against any endpoint of TCDD developmental toxicity including the increase in pericardial
edema, reduction in peripheral blood flow, inhibition of jaw cartilage growth, and impaired erythropoiesis.
These latter findings are highly significant and mean that the key molecular events that lead to endpoints
of TCDD developmental toxicity in zebrafish are not downstream of CYP1A induction; thus, the
induction of zfCYP1A is merely a biomarker of TCDD exposure in zebrafish, and zfCYP1A is not
involved in TCDD developmental toxicity (Figure 21.4). This realization has led aquatic toxicologists
to search for other genes in zebrafish larvae whose expression is directly regulated by AhR2/ARNT1
heterodimeric binding to AhRE. Such genes would be required for certain endpoints of TCDD devel-
opmental toxicity. Three general approaches have been used: (1) the candidate gene approach, (2)
microarray technology, and (3) computational approaches based on predicting functional AhRE
sequences in promoter regions. These three approaches have revealed the complexity of the transcriptional
response to TCDD, but no single, strong hypothesis has emerged about the identity of genes, other than
zfahr2 and zfarnt1, that lead to toxicity.
Recently research in zebrafish larvae that is focused on identifying genes that mediate specific
endpoints of TCDD cardiac toxicity is beginning to change this picture (Carney et al., 2006b; Handley-
Goldstone et al., 2005). AhR hyperactivation by TCDD during zebrafish larval development impairs
heart growth, morphology, and function, culminating in mortality. Carney and coworkers (2006b) exam-
ined the transcriptional response to TCDD in the same adversely affected zebrafish larval hearts and
compared it to the transcriptional response in the rest of the larval body. Zebrafish larvae were exposed
to TCDD for 1 hour at 72 hpf. Hearts were extracted for microarray analysis at 1, 2, 4, and 12 hours
after exposure (73, 74, 76, and 84 hpf). The remaining body tissue was also collected at each time for
comparison. TCDD rapidly induced the expression of 42 genes within 1 to 2 hours of exposure. These
genes function in xenobiotic metabolism, cell proliferation, heart muscle contractility, and pathways that
regulate heart development. Importantly, these TCDD-induced changes in cardiac gene expression
preceded the endpoints of cardiac toxicity, characterized by decreased stroke volume, reduced peripheral
blood flow, and a halt in heart growth. The significance of this study is that it has identified candidates
for AhR target genes in the fish heart of which we were previously unaware (Carney et al., 2006b).
Other exciting results are also now being obtained with molecular approaches in adult zebrafish
exposed to TCDD; for example, TCDD inhibits caudal fin regeneration in the adult zebrafish, and
microarray analysis of the regenerated tissue has revealed a profile of misexpressed genes suggesting
an impairment of cellular differentiation and extracellular matrix composition that is potentially regulated
by SOX9b (Andreasen et al., 2006). Taken together, the importance of this study and the previous studies
on developmental cardiac toxicity (Carney et al., 2006b; Handley-Goldstone et al., 2005) are significant
because they provide the first real glimpse into downstream cellular and molecular effects of zfAHR2
and zfARNT1 signaling that may ultimately lead to specific endpoints of TCDD toxicity (Figure 21.4).

Is TCDD Developmental Toxicity Dependent on AhRE Binding?
An alternative hypothesis for the mechanism of TCDD toxicity is that it requires nuclear localization of
AhR but not AhRE binding followed by transcriptional activation of target genes. TCDD toxicity,
according to this hypothesis, might be due to cross-talk between AhR signaling and the signaling of
other nuclear proteins. This could occur either through direct interaction of the AhR with components
of other pathways or through competition for shared transcription factors or cofactors. In either case,
AhR activation may cause altered regulation of other signaling pathways resulting in toxicity; however,
research findings obtained so far in zebrafish do not support a cross-talk mechanism of TCDD toxicity.