Page 187 - The Toxicology of Fishes

Biotransformation in Fishes 167

expression of CYP2K1, using baculovirus Spodoptera frugiperda (Sf9)-infected insect cells, showed that
CYP2K1 catalyzed hydroxylation of lauric acid primarily at the (ω-1) position but also to a minor extent
at the (ω-2) position. In addition, CYP2K1 catalyzes the conversion of the procarcinogen aflatoxin B 1
to aflatoxin B -8,9-epoxide (Yang et al., 2000). Treatment of rainbow trout with 17β-estradiol decreased
1
CYP2K1 mRNA and protein levels as well as lauric acid hydroxylase activities and bioactivation of
aflatoxin B . Treatment with testosterone resulted in slightly decreased CYP2K1 mRNA levels, whereas
1
this treatment had no significant effect on CYP2K1 protein levels (Buhler et al., 2000). Thus, CYP2K1
is mainly expressed in the digestive tract and appears to be involved in metabolism of endobiotics such
as lauric acid as well as xenobiotics such as aflatoxin B . In addition, CYP2K1 also is expressed in, for
1
example, steroidogenic tissues; furthermore, hepatic CYP2K1 expression is affected by sex steroids,
particularly 17β-estradiol (Buhler et al., 2000; Cok et al., 1998). This may have toxicological implica-
tions, as many natural fish populations are exposed to endocrine-disrupting chemicals (EDCs), including
estrogenic compounds. In fact, treatment of rainbow trout with xenoestrogens (methoxychlor, diethyl-
stilbestrol, 4-tert-octylphenol, and biochanin A) decreased hepatic acid hydroxylase activity and reduced
CYP2K1 protein expression (Katchamart et al., 2002). CYP2K gene orthologs also have been cloned
from killifish, pufferfish, and zebrafish (see Table 4.2 and Table 4.3).

The CYP2M Subfamily—As mentioned earlier, rainbow trout LMC1, previously isolated from rainbow
trout liver (Miranda et al., 1989), was cloned and assigned as CYP2M1 (Yang et al., 1998). Highest
expression of CYP2M1 was observed in liver, but also trunk kidney expresses CYP2M1. Interestingly,
in trunk kidney a pronounced sexually dimorphic expression of CYP2M1 was seen, with juvenile females
expressing 20-fold higher levels than juvenile males. Expression decreased in trunk kidney in sexually
mature animals (Yang et al., 1998). In addition to liver and trunk kidney, CYP2M1 also is expressed,
though at lower levels, in head kidney, stomach, heart, gonads, and brain (Cok et al., 1998). Recombinant
CYP2M1, expressed in pSLV-transfected COS-7 and in baculovirus-infected Sf9 cells, catalyzed hydrox-
ylation of lauric acid primarily at the (ω-6) position (Yang et al., 1998). Although CYP2M1 appears to
be involved in the metabolism of fatty acids and fatty acid derivatives, the physiological function is
unknown. Treatment of rainbow trout with 17β-estradiol resulted in decreased CYP2M1 protein and
mRNA levels, whereas treatment with testosterone had no effect on CYP2M1 (Buhler et al., 2000). In
addition, treatment with xenoestrogens (methoxychlor, diethylstilbestrol, 4-tert-octylphenol, and biocha-
nin A) also reduced CYP2M1 protein levels as well as lauric acid hydroxylase activity (Katchamart et
al., 2002). It should be noted, however, that the lauric acid hydroxylase activity assay used could not
distinguish between CYP2K1, CYP2M1, and possible other CYP activities. So far, no CYP2M ortholog
genes have been reported in other species (Table 4.3); however, increased renal ω and ω-6 hydroxylation
of lauric acid was observed in male channel catfish treated with ciprofibrate, whereas in male bluegill
(Lepomis macrochirus) hepatic ω, ω-4, and ω-5 hydroxylation was induced by ciprofibrate (Haasch et
al., 1998). Although sequence data are needed to verify the identity of the CYP enzymes induced, these
data imply induction of CYP2M-like activities by peroxisome proliferators.

The CYP2N Subfamily—CYP2N1 and CYP2N2 were cloned from killifish liver and heart cDNA
libraries (Oleksiak et al., 2000). CYP2N1 mRNA expression was detected at high levels in liver and
intestine (mid gut) and at low levels in heart and brain. CYP2N2 mRNA levels were highest in heart
and brain and present at lower levels in liver and intestine. Heterologous expression of CYP2N1 and
CYP2N2 in Sf9 insect cells revealed transformation of arachidonic acid to epoxyeicosatrienoic acids.
CYP2N1 preferentially metabolized arachidonic acid at the 8,9- and 11,12-olefins and to a lesser degree
at the 14,15-olefin, and CYP2N2 preferentially metabolized arachidonic acid at the 8,9-olefin and to a
lesser degree at the 11,12- and 14,15-olefins. In addition to being arachidonic acid epoxygenases and
hydrolases, the CYP2Ns also metabolize xenobiotics. Thus, both CYP2N1 and CYP2N2 isoforms
metabolize benzphetamine and also exhibit minimal alkoxyresorufin-O-dealkylase activities (Oleksiak
et al., 2000). Intestinal CYP2N1 mRNA levels were decreased in starved animals and in animals treated
with 12-O-tetradecanoylphorbol-13-acetate (TPA) or starved and treated with TPA-treated killifish.
Intestinal CYP2N2 mRNA levels were diminished in animals treated with TPA, whereas starvation had
no significant effect on this response; however, increased CYP2N2 mRNA expression was observed in