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Biotransformation in Fishes 177

1993; Schlenk and Li-Schlenk; Schlenk et al., 1995). At least two microsomal proteins in rainbow trout
liver were recognized by antibodies raised against porcine FMO1 (Schlenk and Buhler, 1993). Subsequent
studies with antibodies raised against human FMO1 only recognized one of the two bands (Schlenk et
al., 2004). Although a faint band was noted in Japanese medaka liver microsomes with anti-FMO3, a
stronger signal from anti-FMO1 was observed (El-Alfy and Schlenk, 2002); however, catalytic activities
(stereochemical analyses) in trout and shark do not correspond to FMO1 or FMO3 but seem to resemble
FMO4-like activities, which tends to support genetic observations in Fugu (Schlenk et al., 2004).

Regulation, Function, and Toxicological Relevance
Regulation of FMO expression appears to be extremely complex, with the enzyme often being expressed
in a random manner (Baker et al., 1963). As mentioned earlier, FMO has been observed in all marine
fish species, some euryhaline, and virtually no freshwater species. Strong correlations have been observed
between FMO1-like proteins, enzymatic activity, and mRNA recognized by FMO1 cDNA in juvenile
Atlantic flounder (Platichthys flesus) and turbot (Scophthalmus maximus) (Peters et al., 1995; Schlenk
et al., 1996a,b), but catalytic activity did not correspond with mRNA expression in sexually mature adult
flounder (Schlenk, unpublished data). In fact, although FMO1-like mRNA was observed in all sexually
mature animals, hepatic FMO activity was lacking in more than 40% of the animals, with males having
more frequent expression. Although evidence suggests that various hormones may modulate the expres-
sion of FMO (El-Alfy and Schlenk, 2002; El-Alfy et al., 2002; Schlenk et al., 1997), no consistent
induction of enzyme expression has been observed following xenobiotic treatment. In medaka, estradiol
downregulated the hepatic FMO3-like protein but induced the gill FMO1-like form (El-Alfy and Schlenk,
2002). Testosterone downregulated both forms of FMO and activity in medaka. Arterial infusion of
cortisol induced expression of FMO1 in rainbow trout gill and liver (El-Alfy et al., 2002). However,
infusion of growth hormone failed to alter FMO activity or expression (Schlenk, unpublished data).
As mentioned above, FMO activity and protein in several tissues, particularly the gill, appear to be
directly correlated with serum osmolality or the salinity regime in which the fish resides (Daikoku and
Sakaguchi, 1990; El-Alfy and Schlenk, 2002; Schlenk, 1998; Schlenk and El-Alfy, 1998; Schlenk et al.,
1996a,b). Several hypotheses have been put forth to explain this relationship. Four possibilities are: (1)
TMA N-oxide is produced as a cellular defense to prevent the enzyme inactivation by high cellular ion
(Na, K) or urea that occurs in fish residing in subartic or subantarctic environments (Raymond, 1998;
Raymond and DeVries, 1998); (2) TMA N-oxide is produced to counterbalance high tissue and serum
levels of urea that are present regardless of temperature (e.g., in sharks) (Van Waarde, 1988; Yancey et
al., 1982); (3) TMA N-oxide may be produced in muscle of deep-sea gadiform teleosts (which also
produce urea) as an adaptive defense against high pressure (Gillett et al., 1997); and (4) TMA N-oxide
may be formed by FMOs as a secondary organic osmolyte in response to shifts in salinity regimes
(Schlenk, 1993). Each scenario is interrelated, with the common similarity being hyperosmolality. In
some euryhaline fish, FMO activity and expression are directly related to the salinity regime in which
the animal resides (Charest et al., 1988; Daikoku and Sakaguchi, 1990; Daikoku et al., 1988; El-Alfy
and Schlenk, 2002; Lange and Fugelli, 1965; Schlenk and El-Alfy, 1998; Schlenk et al., 1996a,b). FMO
activity and protein expression is higher in gills and kidneys than liver in several species of euryhaline
fish such as the Atlantic flounder (Platichthys flesus), Japanese medaka (Oryzias latipes), and rainbow
trout (Larsen and Schlenk, 2001; Schlenk, 1998; Schlenk and El-Alfy, 1998; Schlenk et al., 1995). In
addition, FMO activity in trout and medaka is downregulated following steroid treatment, which also
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downregulates osmoregulatory function (i.e., Na /K -ATPase) (McCormick, 1995; Schlenk et al., 1997).
FMO activity and expression in rapid osmoconformers such as striped bass and tilapia do not respond
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to changes in salinity (Wang et al., 2001), which is also consistent with mechanisms of Na /K -ATPase
regulation in these species. Testing scenarios 1 and 3, recent studies in rainbow trout have indicated that
urea infusion or reductions in temperature induce FMO activity and increase muscular TMA N-oxide
concentrations (Larsen and Schlenk, 2001). Given induction following cortisol treatment, several factors
may be involved in FMO regulation in rainbow trout, including stress resulting from alterations in cellular
redox potential due to urea or hyperosmotic conditions (El-Alfy et al., 2002; Larsen and Schlenk, 2001).
Clearly, more studies are necessary to better understand the regulation of this enzyme system given its
role in xenobiotic biotransformation and toxicology.