Page 188 - The Toxicology of Fishes

168 The Toxicology of Fishes

heart (but not in brain) in killifish treated with TPA (Oleksiak et al., 2000). Hepatic CYP2N2 mRNA
levels were increased in killifish exposed to anthracene in the lab and in killifish collected from a PAH-
contaminated site (Peterson and Bain, 2004); thus, CYP2N genes are expressed in hepatic and extrahe-
patic organs and appear to be regulated by environmental agents. CYP2N gene orthologs also have been
cloned from scup, butterfly fish (Chaetodon xanthurus), pufferfish, and zebrafish (Table 4.3).

The CYP2P Subfamily—CYP2P1, CYP2P2, and CYP2P3 were cloned from a killifish genomic library
(Oleksiak et al., 2003). CYP2P genes are predominantly expressed in liver and intestine. Recombinant
CYP2P3 expressed in baculovirus-infected Sf9 insect cells catalyzed benzphetamine N-dealkylation and
arachidonic acid oxidation. Arachidonic acid was oxidized to 14,15-, 11,12-, and 8,9-epoxyeicosatrienoic
acids and 19-hydroxyeicosatetraenoic acid. The regiospecificity was similar to human CYP2J2 and rat
CYP2J3 isozymes. Similar to that observed for CYP2N1 and CYP2N2, decreased levels of intestinal
CYP2P2 and CYP2P3 mRNA were observed in killifish treated with TPA. Prolonged starvation or fasting
(20 days) resulted in decreased levels of CYP2P2 and CYP2P3. Neither TPA nor fasting had any
significant impact on CYP2P1 mRNA levels; however, fasting followed by refeeding resulted in increased
levels of CYP2P1 mRNA but not CYP2P2 and CYP2P3 transcription levels. In rat, fasting resulted in
reduced intestinal CYP2J3 and CYP2J4 protein levels; thus, intestinal expression of killifish CYP2P2
and CYP2P3 genes and arachidonic acid regiospecificity of the recombinant CYP2P3 isozyme show
great similarities to mammalian CYP2J forms. Relatedness to mammalian CYP2J genes was further
confirmed by phylogenetic analysis, using the minimum evolution criterion. Killifish CYP2P genes
clustered with mammalian CYP2J genes, separate from the piscine CYP2N and CYP2K subfamilies
(Oleksiak et al., 2003). CYP2P gene orthologs also have been cloned from scup, butterfly fish, pufferfish,
zebrafish, and largemouth bass (Micropterus salmoides) and as a partial sequence from Atlantic salmon
(Table 4.3).

The CYP2X Subfamily—The presence of two CYP2-like isozymes in channel catfish was indicated
based on immunoreactivity with antibodies against both rainbow trout CYP2K1 and CYP2M1. Further-
more, treatment with ethanol (Perkins and Schlenk, 1998) or the insecticide fenitrothion (Perkins, 1999)
specifically decreased expression of the lower (47-kDa) protein. Clofibrate treatment specifically
increased the upper (51-kDa) protein. Furthermore, female catfish displayed higher levels of the 47-kDa
protein compared to males (Perkins, 1998). A CYP2-immunoreactive protein, denoted CM-HA3, was
next isolated from channel catfish liver. N-Terminal amino acid analyses followed by a BLAST search
revealed sequence identity to both CYP2K1 and CYP2M1 (Perkins et al., 2000). By using degenerate
PCR primers designed against this N-terminal followed by RACE, a CYP clone was isolated from catfish
liver and designated as CYP2X1 (Schlenk et al., 2002); however, the derived amino acid sequence was
different from the N-terminus of CM-HA3. CYP2X1 expressed in Sf9 cells demonstrated benzphetamine
demethylase activity, but testosterone, fenthion, and p-nitrophenol metabolism was not observed. CYP2X
gene orthologs also have been cloned from pufferfish and zebrafish (Table 4.3).

CYP3
Phylogeny — Analyses of the complete sequences of several teleost genomes indicate that fish species
contain a complement of CYP gene families similar to those found in mammals. To date, however, tissue
distribution, mechanisms of gene regulation, and the catalytic function of many of these enzymes remain
unknown. The CYP3 gene family is believed to have diverged between 800 and 110 MYA (Maurel,
1996), and four subfamilies have been identified, including CYP3A to CYP3D (see CYP home page).
To date, 13 teleost CYP3A genes have been identified by sequence homologies. Additional subfamilies
(CYP3B to CYP3D) have been discovered by data mining of both the pufferfish and zebrafish genome
databases (see Table 4.4). The CYP3 identity of these genes was based on gene sequence homologies
of less than 55% when compared to members of the CYP3A family. Current nomenclature for the CYP3
gene family, however, does not reflect orthologous relationships between organisms due to the presence
of multiple CYP3A-like sequences in individual species; thus, CYP3A diversity is thought to include
both orthology (diversification due to speciation) and paralogy (diversification due to gene duplication)
(McArthur et al., 2003; Nelson et al., 1996). In a previous study, orthologous relationships between