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Biotransformation in Fishes 175

multiple substrate molecules at any given time and results in unusual kinetic behaviors consistent with
allosteric interaction. The atypical kinetic behavior of CYP3A enzymes results in either sigmoidal or
convex rate–substrate concentration profiles indicative of positive or negative cooperativity (Houston and
Kenworthy, 2000). With human CYP3A4, homotropic cooperativity (i.e., one substrate) has been observed
with numerous chemicals and results in an initial lag in the rate–substrate concentration profile, thus
generating sigmoidal profiles (Harlow and Halpert, 1997). Addition of secondary substrates such as
α-naphthoflavone (ANF) can result in modification of testosterone hydroxylation activity, suggesting
heterotropic (two or more substrates) cooperative interaction (Harlow and Halpert, 1997, 1998). Activation
of BFC O-debenzyloxylase activity by ANF also was observed in rainbow trout liver microsomes
(Hegelund and Celander, unpublished data). In studies with CYP3A38 and CYP3A40, addition of higher
(>25 µM) BFC concentrations resulted in a decrease of catalytic activity and a convex rate–substrate plot.
This type of deviation from Michaelis–Menten kinetics is indicative of negative homotropic cooperativity
and is due to an inability to maintain V max at higher substrate concentrations. In separate experiments,
nonylphenol was used as a heterotropic effector. Results were biphasic, suggesting that nonylphenol acts
as a concentration-dependent cooperative activator and inhibitor of BFC catalysis. The rate substrate plot
demonstrates a sigmoidal curve at low concentrations (below 100 nM), indicative of heterotropic activa-
tion; however, at higher concentrations, activity levels are decreased, resulting in a convex rate–substrate
plot that is indicative of heterotropic inhibition (Kullman et al., 2004). Rainbow trout and killifish exposed
to ketoconazole, a potent antifungal agent, additionally exhibited significant decreases in CYP3A-medi-
ated BFC O-debenzyloxylase activity, suggesting that this pharmaceutical is a potent heterotropic inhibitor
of CYP3A activity (Hegelund et al., 2004). Alkylphenol additionally inhibited CYP3A activity in Atlantic
cod liver microsomes with an IC of 100 µM compared to ketoconazole, which had an IC in a
50
50
submicromolar range (Hasselberg et al., 2004). These studies demonstrate that teleost CYP3A enzymes
exhibit unusual kinetic behaviors consistent with allosteric interaction and cannot be described by hyper-
bolic kinetic models. Homotropic cooperative inhibition of BFC at high concentrations suggests that the
CYP3A protein is capable of binding multiple substrate molecules, which may result in autoactivation
or inhibition of catalysis. Interestingly, the addition of nonylphenol, alkylphenol, or ketokonazole results
in heterotropic cooperative inhibition at environmentally relevant concentrations (Hasselberg et al., 2004;
Hegelund et al., 2004). Given the putative role of CYP3A in maintaining the homeostatic balance for
numerous endobiotics, enzymatic activation and inhibition by xenobiotic compounds may represent a
(nongenomic) mechanism of altered metabolism and subsequent toxicity.

Flavin-Containing Monooxygenases
Overview
Flavin-containing monooxygenases (FMOs) are a multigene family of enzymes involved in the monooxy-
genation of primarily soft-nucleophilic-heteroatom-containing compounds (see Table 4.5) and some
inorganic compounds (Hines et al., 1994; Ziegler, 1988; Ziegler and Mitchell, 1972). Because FMOs
are located in the smooth endoplasmic reticulum (microsomes) and require NADPH and oxygen as
cofactors for catalysis, FMO-catalyzed reactions were thought at one time to merely be another mixed-
function oxidase reaction. FMOs were finally identified as unique enzymatic entities following purifi-
cation of the enzyme by Ziegler and Mitchell (1972). Through an elegant series of experiments, Poulsen
and Ziegler (1979) showed that FMOs have a distinct reaction mechanism that is not dependent on a
reductase coenzyme (such as CYP), but they are directly reduced by NADPH. The reduction of the
flavin by NADPH then allows binding of molecular oxygen, creating a hydroperoxyflavin that is very
susceptible to nucleophilic attack by soft-nucleophilic-heteroatom-containing compounds and various
inorganic species; thus, compounds with non-delocalized electronic features, such as tertiary amine- and
sulfur-containing compounds, are excellent substrates for FMOs (Ziegler, 1988; Ziegler and Mitchell,
1972). Numerous substrates have been identified in mammalian systems, but few have been examined
in fish (Schlenk, 1993, 1998).
One of the most surveyed, but more difficult to measure, FMO-catalyzed reactions in fish is that of
trimethylamine oxidase (or more accurately TMA oxygenase) (for reviews, see Baker et al., 1963;
Schlenk, 1998). Although FMO has yet to be purified to homogeneity in fish, numerous inhibition and