Page 182 - The Toxicology of Fishes

162 The Toxicology of Fishes

regions are identical to all mammalian CYP1A2 but not CYP1A1 (Stegeman and Hahn, 1994). This
homology pattern can be explained by a single CYP1A gene in fish that is ancestral to both mammalian
CYP1A1 and CYP1A2.
More recently, CYP1A has been cloned from killifish (Morrison et al., 1998) and medaka (Oryzias
latipes) (Kim et al., 2004; Ryu et al., 2004) and assembled from the pufferfish (Fugu rubripes) genome,
although the pufferfish sequence is missing much of its N-terminal half (Nelson, 1999). Morrison and
coworkers (1998) conducted a phylogenetic analysis of CYP1 genes (mammalian, avian, and fish) that
highlighted the problem with CYP1 nomenclature, particularly with trout CYP1A. Following the isolation
by Heilmann et al. (1988) of the trout CYP1A1 cDNA, Berndtson and Chen (1993) reported a CYP1A2
gene cloned in rainbow trout; however, the sequence of this clone was only 4% different from the
CYP1A1 clone isolated from the same species. Furthermore, the fish CYP1A2 was not orthologous to
the mammalian 1A2 and was coordinately induced with CYP1A1 in trout treated with 3-MC (Berndtson
and Chen, 1993). Later, the “CYP1A1” was renamed “CYP1A3,” and the original “CYP1A2” was
renamed “CYP1A1.” Until functional data are provided for these various forms (Cao et al., 2000; Carvan
et al., 1999), the nomenclature remains confusing and somewhat arbitrary, and, accordingly, CYP1As
are not being provided a number following the subfamily until this issue is resolved (see cytochrome
P450 homepage).
Multiple CYP proteins have been purified. Those corresponding to CYP1A include P450LM4b in
rainbow trout (Oncorhynchus mykiss) (Williams and Buhler, 1984); P450E in scup (Stenotomus chrysops)
(Park et al., 1986), perch (Perca fluviatilis) (Forlin and Celander, 1993), and rainbow trout (Celander
and Forlin, 1991); and P450c in the Atlantic cod (Gadus morhua) (Goksøyr, 1985). The activity of the
monoclonal antibody (mAb) developed against scup CYP1A (mAb 1-12-3) supports the conservation
of CYP1A through evolution because this antibody recognizes presumptive CYP1A proteins in mammals,
birds, amphibians, reptiles, and nearly 100 different fish species (Stegeman and Hahn, 1994). A study
by Goksøyr and coworkers (1991) investigated the immunochemical cross-reactivity of the three anti-
bodies prepared against the β-naphthoflavone (BNF)-inducible CYP1A proteins from rainbow trout, cod,
and scup. Microsomes from induced hagfish (Myxine sp.), herring (Clupea harengus), rainbow trout,
perch, scup, plaice (Pleuronectes platessa), and rat were tested. Western blot results indicate that all
three antibodies recognize the same antigens in the microsomes, and, as expected, the antibodies react
most strongly with their conspecific microsomes. Of the microsomes tested, perch and hagfish were the
only microsomes not recognized by all three antibodies. The molecular mass of the immunoreactive
proteins ranged from 52 kDa for hagfish to 59 kDa in rainbow trout (Goksøyr et al., 1991).
Since the early reports on the effects of crude oil on brown trout (Salmo trutta) (Payne and Penrose,
1975), research correlating environmental pollution with induction of CYP1A-mediated activities in fish
has been used as a biomarker. The induction of hepatic CYP1A mRNA, immunoreactive protein, and
EROD or AHH enzyme activities in fish have been extensively studied in both controlled laboratory and
field experiments. Some of these studies are summarized in a review by Bucheli and Fent (1995), which
found that 93% of the field studies (68/76) showed that CYP1A induction in fish was related to
contaminant levels in the environment. It should also be noted that fish adapted to living in highly
contaminated habitats (e.g., Superfund sites with PAH or HAH contamination) are also refractory to
CYP1A induction (Bello et al., 2001; Brammell et al., 2004; Meyer et al., 2002; Prince and Cooper,
1995). Prolonged exposure of rainbow trout to a PCB mixture resulted in unresponsiveness to 3-MC
treatment and decreased CYP1A expression upon additional dosage of PCB (Celander and Forlin, 1995).
The mechanisms of this inhibition are not entirely clear (Meyer et al., 2003; Powell et al., 2000). For
more on the use of CYP1A as a biomarker, refer to Chapter 16.

Inhibitors—The type, time dependence, and degree of inhibition caused by various CYP inhibitors are
also species dependent. Selective inhibitors of CYP isozymes have been used to characterize substrate
specificity and modulation of toxicity and carcinogenicity of xenobiotics and in biochemical mechanistic
studies of CYP enzymes. Both AHH activities and the mutagenicity of 3-MC are inhibited by ellipticine
and its derivatives. Structural analysis of ellipticine derivatives reveled that methyl substitution in the 5
and 11 positions is essential for inhibitory responses (Lesca et al., 1980). Some environmental contam-
inants are also CYP1A inhibitors in fish; for example, 2-aminoanthracene (2-AA) caused a 67% inhibition