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Receptor-Mediated Mechanisms of Toxicity 249

saturation binding analysis (see Figure 5.6); of these, the filter-binding assay has been most useful for
studies of fish AhRs (Hestermann et al., 2000).
The assays described above are typically done using cell lysates or the soluble fraction of tissues or
cells (cytosols); however, as noted above, they can also be performed on receptor proteins expressed
from cloned cDNAs in bacteria, in mammalian cells, or by in vitro transcription and translation. An
advantage of expression from cloned cDNAs is the ability to perform parallel assays in the same system
but in the absence of receptor (Figure 5.8D); this provides a much more realistic assessment of nonspecific
binding than that obtained by using an excess of unlabeled ligand.

Cell Culture
Cultured cells have shown great utility in characterizing receptors and receptor-dependent processes in
fish, as they have in mammals. Fish cells or cell lines have been used for the direct assessment of ligand–
receptor binding interactions (Hestermann et al., 2000; Lorenzen and Okey, 1990; Pollenz and Necela,
1998; Swanson and Perdew, 1991), for assessing reporter gene activation by ligand-activated transcription
factors (Carvan et al., 2000), and for determining relative potencies of chemicals acting through receptor-
dependent mechanisms by measuring the expression of endogenous target genes such as CYP1A (AhRs)
(Clemons et al., 1996; Henry et al., 2001) or vitellogenin (ERs) (Petit et al., 1997; Smeets et al., 1999).
An alternative way to characterize fish receptors is to express them in heterologous systems, such as
bacteria, yeast, or mammalian cells. Cloned receptor cDNAs are inserted into an appropriate expression
vector (sometimes as a fusion protein) and then are used to transform bacteria or transfect mammalian
cells. Receptors expressed in this way can be characterized by the ligand-binding methods described
above, as well as by reporter gene assays in which the ability of the receptor to activate transcription is
assessed in the presence of different ligands. One advantage of expressing receptors in heterologous
cells is that it facilitates comparative studies by providing a common cellular background on which
receptors from different species can be examined (see, for example, Abnet et al., 1999b; Matthews et
al., 2000).

In Vivo Assays

One of the great advantages of using fish to study mechanisms of toxicity is the ability to carry out in
vivo experiments to assess receptor functions, especially those involved in developmental toxicity. Two
of the most powerful approaches involve: (1) targeted gene knock-down to reduce or eliminate specific
gene products, and (2) gene expression or overexpression by transgenesis. Currently, these are used
primarily in zebrafish and other small fish models, although their use is expanding to other species as well.

Gene Knock-Down
The ability to perform targeted inactivation of mouse genes by homologous recombination (gene knock-
out) has been a valuable tool in toxicological research, permitting an assessment of gene function through
observation of the phenotype of animals lacking specific gene products. Gene knock-outs are not yet
possible in fish. Analogous to gene knock-out, but with key differences, an anti-sense approach using
morpholino-modified oligonucleotides (MOs) has been developed for producing targeted gene knock-
downs in developing zebrafish (Anon., 2000; Ekker, 2004; Nasevicius and Ekker, 2000; Sumanas and
Larson, 2002). Morpholino-modified oligonucleotides inhibit protein synthesis either by blocking the
translational start sites of mature mRNAs (Figure 5.9A) or by altering pre-mRNA splicing (Figure 5.9B).
MOs targeted to the 5′-UTR inhibit translation of maternal and zygotic transcripts by binding to mRNA
between the 5′ cap and the start codon (Ekker and Larson, 2001; Summerton, 1999; Summerton and
Weller, 1997); MOs targeted to exon–intron splice sites block the processing of zygotic (but not maternal)
RNA (Draper et al., 2001; Ekker and Larson, 2001). By targeting the splice donor or splice acceptor
site (or both), one can obtain mis-spliced mRNAs that have skipped exons or retained introns or reveal
cryptic splice sites. If these modified splice products cause a frameshift or deletion of a critical part of
the encoded protein, the product is inactive and the embryos are deficient for this protein. Incorrectly