Page 371 - The Toxicology of Fishes

Liver Toxicity 351

activation of the AhR pathway and AhR-dependent CYP1A induction, this biochemical response has been
used extensively as a biomarker of exposure of fish to dioxin-like contaminants (Hahn, 2002; van der Oost
et al., 2003; Whyte et al., 2000) and has been used in a range of field biomonitoring studies (Behrens and
Segner, 2005; Collier et al., 1995; Goksøyr and Förlin, 1992; Payne and Penrose, 1975). Typically, CYP1A
induction, measured as catalytic 7-ethoxyresorufin-O-deethylase (EROD) activity, as CYP1A protein, or
as CYP1A mRNA, is assessed in the liver because specific CYP1A expression is highest in this organ.
Although CYP1A is induced in various resident liver cell types, responding cells appear to differ in
their induction response to xenobiotics. Environmental exposure led to a particularly strong increase of
cytochrome P4501A immunoreactivity in the biliary and endothelial cells of cod and flounder, while
hepatocyte staining remained weak (Husoy et al., 1996). In contrast, in lemon sole (Microstomus kitt),
the cytochrome P450 induction response was stronger in hepatocytes than in vascular cells (Husoy et
al., 1996). In scup, 3,3′,4,4′-tetrachlorobiphenyl treatment strongly increased CYP1A immunoreactivity
in both hepatocytes and nonhepatocytes (Singh et al., 1996). It is possible that the cell-specific induction
response varies with the type of inducer (readily vs. slowly metabolized compounds), dose of inducer,
or fish species (Anulacion et al., 1998; Goksøyr and Husoy 1998; Ortiz-Delgado et al., 2005). Another
relevant factor influencing hepatic CYP1A response could be the exposure route. A stronger increase of
CYP1A immunostaining occurred in hepatocytes of mummichog (Fundulus heteroclitus) when exposure
took place via the water than by the dietary route (Van Veld et al., 1997).
Hepatic CYP1A induction is a measure of the exposure of fish to dioxin-like chemicals; it is not a
direct measure of the ability of a species to metabolize these compounds. A direct correlation was
demonstrated in mammals and birds between ability to metabolize 3,3′,4,4′-tetrachlorobiphenyl and their
hepatic EROD activity; however, no such relation was seen in rainbow trout and flounder (Murk et al.,
1994). This finding possibly reflects species differences in the participation of different CYP isoenzymes
in the metabolism of the toxicant. In line with this speculation is the observation that the liver of turbot
exposed to benzo(a)pyrene produced different metabolites before and after induction of hepatic EROD
activity (Telli-Karakoc et al., 2002).

Phase I Enzymes Other than CYP1A
Phase I metabolic enzymes have not been as intensively studied in fish liver, but recent advances are
noteworthy. With the completion of genome sequences for several teleosts, including medaka, zebrafish,
pufferfish, and stickleback, full complements of CYP gene families are being discovered (Nelson, 2003;
http://drnelson.utmem.edu/CytochromeP450.html). Although the functionality for many of these
enzymes remains to be determined, several have been investigated using recombinant systems such as
CYP3A38/40 (Kashiwada et al., 2005) , CYP2N (Oleksiak et al., 2000), CYP2K1 (Yang et al., 2000),
CYP2M1 (Yang et al., 1998), and CYP3A27 and CYP3A45 (Lee and Buhler, 2002, 2003). In general,
the catalytic activities and substrate specificity of teleost CYPs are highly similar to their mammalian
homologs. Examples include the preference of CYP1A for aromatic hydrocarbons, CYP3A for steroidal
compounds, and CYP2 for arachidonic acid, lauric acid, and sex steroid hormones. In some instances,
however, activities may be specific to teleosts. Mammalian forms of CYP3A preferentially catalyze
6β-hydroxylation of testosterone with lower levels of the 2β-, 15β-, and 1β- hydroxides (Krauser et al.,
2004). In comparison, teleost CYP3A forms catalyze testosterone hydroxylation at the 6β-, 2β-, and
16β- positions (Kullman and Hinton, 2001; Lee and Buhler, 2002, 2003), illustrating an inherent
difference in catalytic profiles that exists between teleost and mammalian species. Any physiological
significance of this difference has yet to be determined. Often, the optimal requirements for cofactors,
including CYPOR, cytochrome b , lipids, and temperature, differ among teleosts. This is not surprising
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given the fact that these organisms inhabit a range of environmental conditions from extreme cold to
tropical temperatures and fresh to seawater (Kashiwada et al., 2005). The availability of modern molecular
techniques has made it much easier to identify specific isoforms and has thus stimulated research on the
CYP isoenzymes in fish. With these new technical developments, a more in-depth characterization of
the phase I metabolism of xenobiotics in fish liver will become possible. Of great interest is a further
understanding of CYP temporal and spatial expression, the mechanisms of gene regulation, and catalytic
function, much of which remains unknown for many of these enzymes.