Page 194 - The Toxicology of Fishes

174 The Toxicology of Fishes

of juvenile Atlantic cod to ketoconazole resulted in slight increases in CYP3A expression but marked
decreases in CYP3A-specific catalytic activity. Similar discrepancies have been noted in medaka, for
which significant decreases in catalytic activity cannot be accounted for by corresponding reductions in
CYP3A expression (Kashiwada, unpublished data). As noted in the section on CYP1A, these discrep-
ancies between message level and catalytic activity necessitate determination of expression at several
levels prior to making conclusions about regulatory mechanisms.

Localization—Localization of CYP3A expression is similar to that observed in mammalian species, with
high levels of expression found in the liver and intestinal mucosa. Some differences are observed between
specific CYP3A paralogs and may represent putative regulatory and functional differences that occurred
during gene duplication events. Immunohistochemical and mRNA analysis of CYP3A27 has demon-
strated strong responses in intestinal ceca, proximal descending intestine, and liver, with minor expression
occurring in brain (Cok et al., 1998; Lee et al., 2001). The rainbow trout paralog CYP3A45 was
predominantly expressed in the gastrointestinal tract, with weak expression occurring in the liver (Lee
and Buhler, 2003). In killifish, CYP3A30 and CYP3A56 were coexpressed in intestine and liver, consistent
with that observed for rainbow trout CYP3A27. In both species, CYP3A expression is prominent in the
digestive tract (intestinal mucosa and liver), suggesting a role for these enzymes in first-pass metabolism
of xenobiotics. Extrahepatic CYP3A expression is observed in gill, kidney, brain, spleen, and ovary,
suggesting a possible role for CYP3A enzymes in the fine-tuning of endogenous substrates at the site of
synthesis or action as well as metabolic defense against xenobiotics (Hegelund and Celander, 2004).

Development—Biotransformation systems for embryonic and fetal tissues of vertebrates are not as well
characterized as those for adults. Embryogenesis is a dynamic process that presents a continuously
changing metabolic profile as enzymes are induced and repressed (Miller et al., 1996). In general,
developing organisms lack many CYP forms present in adults; however, in some instances, they contain
certain forms that are only expressed during development. These differences in total CYP content may
explain developmental processes and altered sensitivities to toxic exposures during development. Teleost
embryos are often sensitive to xenobiotics, and prolonged exposures result in numerous developmental
malformations, early mortality, and delayed hatch (Cooke and Hinton, 1999; Villalobos et al., 1996). The
nonspecific onset of many of these aberrations may reflect an inability of embryos to sufficiently
metabolize and detoxify many of these xenobiotics. CYP3A27 cDNA was cloned from both embryonic
and adult hepatic mRNA, suggesting this gene is expressed early during rainbow trout development (Lee
et al., 1998). Kullman and Hinton (2001) demonstrated that CYP3A38 and CYP3A40 genes are differ-
entially regulated during embryonic development. Analysis of CYP3A38 and CYP3A40 mRNA and
protein demonstrated the presence of a single CYP3A transcript for early and late embryonic stages and
two CYP3A transcripts in larvae and adult liver in medaka. Using gene-specific probes, results demon-
strated that CYP3A40 is expressed early in embryonic development and continues throughout adult
stages. CYP3A38, however, is tightly suppressed during embryonic development and is only expressed
post-hatch. Given the role of CYP enzymes in maintaining steady-state levels of morphogenic ligands,
it is not surprising to find these enzymes in the earliest stages of life. Previous studies have provided
strong evidence for the presence of multiple forms of CYP in the developing fish embryo (Buhler et al.,
1997; Chen and Cooper, 1999). In general, fetal CYP forms are present in low levels and exhibit stringent
temporal expression patterns that diminish following birth or hatching (Juchau et al., 1998). CYP3A40
mRNA expression was detected as early as stage 11, representing an early multicellular stage of devel-
opment. Liver formation does not occur until stage 26, demonstrating that initial CYP3A40 expression
occurs prior to organogenesis. Functionally, expression of CYP3A40 during early embryonic development
may serve multiple purposes including xenobiotic metabolism, hydroxylation of steroid or other mor-
phogenic ligands, and metabolism of yolk. As with human CYP3A7, CYP3A may be the only constitu-
tively expressed cytochrome P450 during embryogenesis, although some adults do express CYP3A7.

Mechanisms of Stimulation and Inhibition—Mutation and docking studies have demonstrated that
CYP3A proteins have a large substrate-binding pocket in comparison to other members of the CYP
superfamily (Khan and Halpert, 2000). This large pocket is thought to enable CYP3A enzymes to bind