Page 184 - The Toxicology of Fishes

164 The Toxicology of Fishes

2-hydroxylation. Similarly, an elevated ratio of 4-/2-hydroxyestradiol formation in human mammary
microsomes has been used as a risk factor for malignant breast cancer (Liehr and Ricci, 1996). Catechol
estrogens, such as 4-hydroxyestradiol, are capable of undergoing metabolic redox cycling between
hydroquinone and quinone forms, generating potentially mutagenic free radicals and oxidative stress.
In channel catfish the 4-/2-hydroxyestradiol ratio was statistically higher in microsomes from BaP-treated
fish compared to controls (0.2 and 0.04, respectively) (Butala et al., 2004). The shift toward more
4-hydroxyestradiol production in exposed fish suggests induced production of the redox active estrogen
metabolite. As with PAH metabolism, lower levels of CYP1B1 and less associated formation of 4-hydroxy-
estradiol may be indicative of lower genotoxicity. More studies are necessary to determine the physio-
logical significance of CYP1B in fish.

CYP2
Phylogeny — CYP2 and CYP1 genes belong to the CYP2 clan (Figure 4.4). The CYP1 gene family
is believed to have diverged from the CYP2 gene family more than 420 MYA (Nelson, 2003). CYP2 is
the most diverse CYP gene family, with 13 known CYP2 subfamilies in fish: 2K, 2M, 2N, 2P, 2R, 2U,
2V, 2X, 2Y, 2Z, 2AA, 2AD, and 2AE (see cytochrome P450 homepage). Two of these, CYP2R and
CYP2U, also have mammalian representatives; thus, these subfamilies probably represent more con-
served CYP2 genes that emerged over 420 million years of evolution. No functional data are available
on CYP2R and CYP2U isozymes, although it is proposed that these earlier emerging CYP2 forms are
more likely involved in the metabolism of endobiotics than xenobiotics (Nelson, 2003).
Although mammalian and piscine CYP2 gene families have structurally diverged during vertebrate
evolution, there are still some conserved structures as well as catalytic functions among certain CYP2
subfamilies; for example, fish CYP2N, CYP2P, CYP2V, and CYP2Z genes are related to mammalian
CYP2D and CYP2J genes (Nelson, 2003). Furthermore, phylogenetic analyses suggest that the killifish
CYP2P subfamily is more closely related to the mammalian CYP2J subfamily, compared to the CYP2K
and CYP2N subfamilies in fish; thus, the piscine CYP2P and the mammalian CYP2J subfamilies may
have arisen from a common ancestral gene (Oleksiak et al., 2003). The fish CYP2 genes and suggested
pseudogenes reported to the P450 nomenclature committee are listed in Table 4.3.
Members of the CYP2 gene family in fish are involved in the metabolism of endobiotics, such as
arachidonic acid, lauric acid, and sex steroid hormones, as well as xenobiotics such as aflatoxin,
alkoxyresorufins, and benzphetamine (Buhler and Wang-Buhler, 1998; Oleksiak et al., 2003; Yang et al.,
1998, 2000). The following paragraphs summarize highlights from functional studies of some of these
CYP2 subfamilies.
CYP2B-Like Forms—Over the last decades, fish liver microsomes have been shown to metabolize
prototypical mammalian CYP2B substrates, including aldrin, benzphetamine, ethylmorphine, aminopy-
rine, and alkoxyresorufins (Buhler and Williams, 1989; Eisele et al., 1984; Elskus and Stegeman, 1989;
Goksøyr et al., 1987; Haasch et al., 1994; Kleinow et al., 1990; Stegeman, 1981). The existence of
piscine CYP2B-like enzymes further was supported by protein purification and immuno-cross-reactivity
studies; however, a piscine CYP2B gene ortholog has so far not been reported. In the scup, five different
CYP isozymes (P450A to P450E) were isolated from liver microsomes. Reconstitution of scup P450B
demonstrated oxidation of testosterone at the 15α-position (Klotz et al., 1986). N-terminal analysis of
scup P450B showed 50% sequence identity with rat CYP2B1 and CYP2B2; furthermore, proteins from
different taxa, including several fish species, show cross-reactivity with antibodies against both scup
P450B and rat CYP2B1 (Stegeman and Hahn, 1994).
In rainbow trout, five different CYP isozymes (LMC1 to LMC5) were isolated from liver microsomes
(Miranda et al., 1989). Reconstituted LMC1 was shown to catalyze lauric acid hydroxylase activity and
to cross-react with rat CYP2B1 antibodies (Miranda et al., 1989, 1990). Rainbow trout LMC1 was later
assigned as CYP2M1 (Yang et al., 1998). Further studies are needed to elucidate whether scup P450B
and CYP2M1 are homolog genes and whether the mammalian CYP2B and the piscine CYP2M sub-
families have arisen from a common ancestral gene.
The presence of hepatic CYP2B immunoreactive proteins and comparatively high activities toward
mammalian prototypical CYP2B substrates (i.e., aminopyrene and pentoxyresorufin) were observed in