Page 196 - The Toxicology of Fishes

176 The Toxicology of Fishes

TABLE 4.5
Putative Substrates for Flavin-Containing
Monooxygenases in Fish
Substrate Product

Nitrogen-containing
Trimethylamine Trimethylamine N-oxide
N,N-Dimethylaniline N,N-Dimethylaniline N-oxide
2-Aminofluorene 2-Hydroxyaminofluorene a
Sulfur-containing
Thiobencarb Thiobencarb-S-oxide a
Thiourea Thiourea-sulfonic acid a
Methimazole Methimazole-sulfonic acid a
Aldicarb Aldicarb sulfoxide a
Selenium-containing
Dimethylselenide Dimethylselenoxide a
Selenomethionine Selenomethione-Se-oxide a
a More toxic metabolite than parent compound.

correlative studies have indicated that TMA oxidase is catalyzed by FMO (Agutsson and Strom, 1981;
Goldstein and Dewitt-Harley, 1973; Peters et al., 1995; Schlenk, 1994; Schlenk and Li-Schlenk, 1994;
Schlenk et al., 1995). A direct relationship also exists between TMA content in fish tissues and enzyme
expression (Larsen and Schlenk, 2001; Raymond, 1998; Raymond and DeVries, 1998; Schlenk, 1998).
Fish that possess high tissue (muscle, liver, blood) concentrations of TMA or TMA N-oxide have higher
levels of expression and enzyme activity than fish that do not have either of these biomolecules; for
example, TMA-lacking species, such as the channel catfish (Ictalurus punctatus), do not express FMO-
like protein or enzymatic activity, whereas TMA-containing rainbow trout or various elasmobranchs
possess relatively high levels of FMO activity and expression in various tissues (Schlenk and Buhler,
1991; Schlenk and Li-Schlenk, 1994; Schlenk et al., 1993). The relationship between TMA and FMO
has several implications regarding the evolution and possible physiological functions of these enzymes
which are discussed further below.
Xenobiotics that have been shown to be substrates of the enzyme in fish include tertiary amines such
as N,N-dimethylanaline (DMA) and TMA, thioether pesticides (e.g., aldicarb), thiocarbamates (e.g.,
eptam, thiobencarb), thiocarbamides (e.g., methimizole, thiourea), and thioamides (e.g., thiobenzamide)
(Table 4.5); for a review, see Schlenk (1998). Currently, the most characterized diagnostic substrates for
FMO activity in fish are thiourea and DMA (Schlenk, 1993, 1998). Enzymatic activities in fish appear
to be sensitive to temperature and have a relatively high pH optimum of 8.0 to 9.6 in various fishes
(Schlenk, 1993, 1998). Each of these assays is a simple spectrophotometric method; however, other
more elaborate high-performance liquid chromatography (HPLC) methods utilizing enantioselective
oxygenations have been identified in mammals to differentiate activities catalyzed by specific isoforms
(Rettie et al., 1995). Recent studies have indicated unique stereochemical sulfoxidation reactions in trout,
which have not been observed with any previous isoforms of mammalian FMOs (Schlenk et al., 2004).
Typically, to validate FMO activity for an uncharacterized xenobiotic, co-incubation of other FMO
substrates or CYP inhibitors is necessary, because FMO and CYP share many substrates. It is imperative
to note, however, that many putative inhibitors of mammalian enzymes are not as effective in fish and
few have been well-characterized, so care should be taken when interpreting data regarding the use of
enzyme inhibitors (especially CYP and FMO) in studies with fish.
Currently, 12 FMO genes have been identified in mammals (classified as FMO1 to FMO12), but none
has been fully characterized in any fish species (Hines et al., 1994). A close examination of accessible
genomic sequences of the pufferfish (Fugu sp.) has indicated a gene fragment that is 42% identical with
FMO4 and 56% identical to FMO5 (Dolphin, pers. commun.). Conservation of the secondary structure
of FMOs has been observed in fish using western blot analyses that employed antibodies raised against
mammalian forms (El-Alfy and Schlenk, 1998; Peters et al., 1995; Schlenk, 1998; Schlenk and Buhler,