Page 267 - The Toxicology of Fishes

Receptor-Mediated Mechanisms of Toxicity 247

Ligand–Receptor Interactions and Receptor Function:
Approaches and Methods

The theoretical principles presented in the preceding section provide a foundation for considering
practical aspects of assessing receptor function in fishes. To understand the role of receptors in mecha-
nisms of toxicity, it is necessary to determine: (1) the biochemical characteristics of the receptors
themselves; (2) the levels, location, and timing of their expression; (3) their interaction with other proteins
and DNA; and (4) the genes or processes they regulate. A variety of experimental approaches and
techniques that were developed originally for use in mammalian systems also have utility in fish. Often,
however, modifications are required to account for physiological and other differences between fish and
mammals (e.g., poikilothermy vs. endothermy). In addition, some methods are especially suited for fish,
such as those that take advantage of the externally developing, transparent embryos. In this section, we
summarize a few of the key approaches and techniques that are used to investigate receptor-mediated
mechanisms of toxicity in fish, with examples from the well-studied AhR system.

Ligand-Binding Assays
The objective of investigating ligand–receptor interactions is to determine the physicochemical constants
that govern those interactions—principally, ligand-specific kinetic constants such as on and off rates
(association and dissociation rate constants), the equilibrium dissociation constant (K , a measure of
D
binding affinity), and binding capacity (R , also called B max ). The theoretical aspects of these
T
ligand–receptor interactions have been described earlier. To perform ligand-binding assays, one needs a
ligand labeled with a radioactive or fluorescent tag, a method to separate ligand free in solution from
that bound to proteins, and—because pure preparations of receptor are rarely available—a way to
distinguish binding of ligand to the receptor (specific binding) from its binding to other proteins
14
125
3
(nonspecific binding). Tritium ([ H])-labeled or [ I]-labeled ligands are commonly used, whereas [ C]-
labeled compounds usually do not have sufficiently high specific activity for binding assays.
Among the several ways to separate bound from free ligand is a classical method that uses dextran-
coated charcoal (Poland et al., 1976); the charcoal adsorbs free ligand while the dextran coating
minimizes the effect of charcoal on the proteins. The amount of charcoal must be determined empirically,
and for some receptors species-specific sensitivity to charcoal complicates the use of this technique
(Manchester et al., 1987; Nakai and Bunce, 1995). Rather than adsorb free ligand with charcoal, other
methods use hydroxylapatite (HAP) (Gasiewicz and Neal, 1982) or protamine sulfate (Denison et al.,
1984) to adsorb the proteins so free ligand can be washed away. Similarly, filter-binding assays collect
solvent-aggregated proteins on glass-fiber membranes and remove free ligand by several washes (Dold
and Greenlee, 1990). The latter can be performed either with cytosols prepared by differential centrif-
ugation or with whole cells that have been incubated with radioligand. Another classical method involves
velocity sedimentation on sucrose gradients (Okey et al., 1979; Tsui and Okey, 1981), which separates
ligand–receptor complexes based on their sedimentation coefficient; the latter is a function of the size
and shape of the protein complexes. Two types of centrifuge rotors can be used for this technique.
Swinging-bucket rotors allow separation along the length of the centrifuge tube but require long spins
(typically >18 hours). Vertical tube rotors permit much shorter spins (~2 hours) and thus facilitate analysis
of labile ligand–receptor interactions (Tsui and Okey, 1981). Finally, free and bound ligand can be
separated by denaturing gel electrophoresis, but only if the ligand has been covalently linked to the
receptor—for example, through use of a photoaffinity ligand (Poland et al., 1986).
All of the approaches described above have been used to investigate fish AhRs (Hahn, 1998), as well
as a variety of other receptors such as estrogen receptors (Hawkins and Thomas, 2004; Menuet et al.,
2002), androgen receptors (Wells and Van Der Kraak, 2000), and retinoid receptors (Alsop et al., 2001).
In some cases, multiple methods are used together to provide complementary information. Velocity
sedimentation using vertical tube rotors (Figure 5.8A–D) has been useful because it is the gentlest
procedure and provides ancillary information about receptor size; however, it is quite labor intensive
and thus not suitable for large numbers of samples, such as are required for generating competitive