Page 271 - The Toxicology of Fishes

Receptor-Mediated Mechanisms of Toxicity 251

Transgenics
Transgenic technologies are well developed in zebrafish (Udvadia and Linney, 2003) and well suited for
studying receptor-mediated mechanisms of toxicity. Transient and stable (germline) expression of trans-
genes can be used to screen for chemical effects on gene expression (Blechinger et al., 2002; Perz-
Edwards et al., 2001), test promoter function (Jessen et al., 1998; Long et al., 1997), and map regulatory
elements (Barton et al., 2001; Meng et al., 1997, 1999), all in vivo. Heterologous promoters and proteins
have been shown to function faithfully in zebrafish, recapitulating native expression patterns (Barton et
al., 2001) or rescuing mutant phenotypes (Porcher et al., 1999). The coupling of green fluorescent protein
(GFP)-based reporters (Amsterdam et al., 1995; Finley et al., 2001; Gibbs and Schmale, 2000) and
transparent zebrafish embryos provides a powerful system for visualizing in vivo gene expression.
Transgenic fish also can be used to investigate gene function by assessing the phenotype of fish in which
specific proteins have been overexpressed through injection of mRNA or DNA. Such gain-of-function
experiments provide a valuable complement to loss-of-function approaches such as MO-based gene
knock-downs (Malicki et al., 2002). One advantage of overexpression is its flexibility to test the function
of heterologous proteins (i.e., from other species) and the ability to test the effect of specific mutations
on protein function. Gain-of-function experiments have been used to examine the effects of overexpress-
ing hypoxia-inducible factor-1α (HIF-1α) (Kajimura et al., 2006), estrogen receptor-related receptor α
(ERRα) (Bardet et al., 2005), CYP26D1 (Gu et al., 2006), and ARNT2X (Hsu et al., 2001).

Chromatin Immunoprecipitation
Chromatin immunoprecipitation (ChIP) has found widespread utility as a method for measuring the
ability of receptors, other transcription factors, and associated protein complexes (including coactivators
and chromatin-modifying enzymes) to occupy gene promoter and enhancer sequences in vivo. ChIP is
replacing gel mobility shift assays as the preferred method for measuring protein–DNA interactions.
ChIP has been applied most widely in mammalian cell culture, but it also has been used with fish cells
(Dann et al., 2004; Hirayama et al., 2005) and embryos (Havis et al., 2006).

Genomics and Gene Expression Profiling
The emergence of genome-scale approaches and techniques has provided new opportunities for progress
in a variety of fields, including comparative toxicology. Genomic approaches can be classified in a variety
of ways; one useful distinction is between structural genomics and functional genomics, which provide
distinct yet complementary information of relevance to mechanistic toxicology.

Structural Genomics
Structural genomics concerns gene sequences (coding and noncoding), gene structure, and gene organi-
zation—information that is usually obtained through whole-genome sequencing efforts. Genome sequences
permit the description of the complete set of genes in a particular gene family, illuminating phylogenetic
aspects of gene family diversity and providing information to help distinguish orthologous and paralogous
genes among species. As an example, the genome sequences of the pufferfish Takifugu rubripes (Aparicio
et al., 2002) and Tetraodon nigroviridis (Jaillon et al., 2004) have been useful in defining the complete
sets of cytochrome P450s (Nelson, 2003) and nuclear receptors (Maglich et al., 2003) in fishes. The
identification of several AhR genes in the Fugu genome helped resolve the orthologous and paralogous
relationships between fish AhR1 and AhR2 forms (Karchner and Hahn, 2004; Karchner et al., 2005). In
addition, sequenced genomes allow the identification of conserved noncoding sequences involved in
regulating gene expression (Dickmeis et al., 2004). This latter approach has not yet been widely applied
in fish toxicology but has great potential for understanding the regulation of toxicologically important genes.

Functional Genomics and Proteomics
The term functional genomics refers primarily to genome-scale assessment of gene and protein expression
and interactions. There are several approaches for this; each has its own advantages and disadvantages.