Page 268 - The Toxicology of Fishes

248 The Toxicology of Fishes

2500 A [ 3 H]-TCDD 400 B [ 3 H]-TCDD
[ 3 H]-TCDD (dpm/fraction) 1500 300
2000
+ TCDF
+ TCDF

200
1000
500

0 100 0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
500 D FhAHR1
1200
C
[ 3 H]-TCDD (dpm/fraction) 800 300
[ 3 H]-TCDD
400
UPL
+ TCDF
1000
600
200
400
200
0 100
0
0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35
Fraction Number Fraction Number
– +
E
205 –

116 –
97 –
68 –

FIGURE 5.8 Data illustrating the specific binding of radioligands to AhR as measured using velocity sedimentation (A–D)
3
and photoaffinity labeling (E). Specific binding of [ H]-TCDD (~1 nM) to AhRs in cytosol from mouse Hepa-1 cells (A),
cytosol from fish PLHC-1 cells (B), cytosol from killifish liver (C), and killifish AhR1 expressed from an AhR1 expression
construct by in vitro transcription and translation using rabbit reticulocyte lysate (D). Samples were incubated with
3
[ H]-TCDD in the absence or presence of excess unlabeled TCDF (A–C) to determine total and nonspecific binding. In D,
lysate lacking the AhR1 cDNA (unprogrammed lysate [UPL]) was used to determining nonspecific binding. (E) Photoaffinity
125
labeling of AhRs in killifish hepatic cytosol using 2-azido-3-[ I]iodo-7,8-dibromodibenzo-p-dioxin in the absence (–) or
presence (+) of excess TCDF (for details, see Hahn et al., 1994). Numbers represent molecular weight standards. Data in
C were generously provided by Dr. Susan M. Bello (Bello, 1999).

3
binding curves. Despite the gentleness of this procedure, specific binding of [ H]-TCDD to fish AhRs
in cell lysates or tissue cytosol is difficult to measure as compared to mammalian AhRs (Figure 5.8A–C).
In contrast, expression of fish AhRs by in vitro transcription and translation from cloned cDNAs produces
robust peaks of specific binding in velocity sedimentation assays (Figure 5.8D); thus, this procedure has
been very useful for the initial characterization of cloned fish AhR cDNAs (Abnet et al., 1999a; Andreasen
et al., 2002; Karchner et al., 1999, 2005). Photoaffinity labeling (Figure 5.8E) is very sensitive and
provides an estimate of receptor molecular mass but cannot be used for determining binding constants.
The batch assays, such as filter-binding, hydroxylapatite, and dextran-coated charcoal, are best for