Page 192 - The Toxicology of Fishes

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testosterone at 1.48, 0.043, and 0.034 nmol/min/nmol, respectively, as well as dehydrogenation of
nifedipine at 50 pmol/min/nmol (Lee and Buhler, 2002). Although turnover rates were significantly
higher with the recombinant enzyme, hydroxylase profiles were similar to that observed with purified
CYP3A27 enzyme. Heterologous expression of the rainbow trout paralog CYP3A45 additionally exhibits
testosterone hydroxylase activity with 6β-hydroxytestosterone as the major metabolite. Activity was
higher than that observed with CYP3A27 and was significantly enhanced by the addition of cytochrome
b to the reconstitution assays (Lee and Buhler, 2003).
5
Recombinant medaka CYP3A38 and CYP3A40 catalyzed hydroxylation of testosterone, as well as
the O-debenzylation of benzyloxyresorufin (BR) and 7-benzyloxy-4-(trifluoromethyl)-coumarin (BFC);
however, efficiencies and specificities were significantly different between the two isoforms. Thus, K m
and V max activities based on BFC O-debenzyloxylase were estimated to be 0.116 and 0.363 µM and 7.95
and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. Medaka CYP3A38 preferen-
tially catalyzed testosterone hydroxylation at the 6β- and 16β-positions, with minor hydroxylation at
other positions within the steroid nucleus, whereas CYP3A40 catalysis was predominantly limited to
the 6β- position (Kashiwada, unpublished data). Putative identification of CYP3A SRS1 to SRS6
indicated that 12 of the 49 amino acid differences between CYP3A38 and CYP3A40 occur in SRS1,
SRS3, and SRS5, previously known to be associated with steroid hydroxylation (Kashiwada, unpublished
data; Kullman and Hinton, 2001). Functional analysis of teleost and mammalian CYP3A paralogs has
demonstrated that gene-duplication events are tied to acquisition of new function and that convergent
evolution of CYP3A function may be frequent among independent gene copies (McArthur et al., 2003).
The physiological role of CYP3A is yet to be determined. Given the robust steroid hydroxylase activity,
there is speculation that CYP3A forms play an important part in steroid (hormones as well as bile acids)
homeostasis. Although the physiological function of CYP3A is still unknown, the fact that these genes
are expressed in tissues that act as barriers to the environment (i.e., digestive and respiratory tracts)
together with the broad substrate specificities of CYP3A enzymes suggest that they evolved as biochem-
ical defense to prevent bioaccumulation of xenobiotics. In addition, the presence of CYP3A enzymes
in steroidogenic tissues implies a role in steroid biotransformation.

Regulation—CYP3A are the major constitutive CYP forms expressed in the liver and intestine of most
mammals and other species, including fish (Celander et al., 1996, 1989; Hegelund and Celander, 2003;
Hegelund et al., 2004; Husoy et al., 1994). Numerous studies have demonstrated, however, that consti-
tutive expression of CYP3A genes between species, and paralogous CYP3A forms within species, are
highly variable with age, gender, development, tissue localization, and between individuals. Variations
in gene expression may be due to external environmental factors, including temperature, salinity, diet,
or other environmental stressors; biological factors, such as circulating hormone levels; or tissue-specific
factors associated with development or reproductive cycle. Depending on species, sexual dimorphic
expression of CYP3A has been observed. Lee et al. (1998) reported higher levels of CYP3A expression
in juvenile female rainbow trout intestine when compared to males. Gender differences were additionally
observed in kidney and liver but to a lesser degree than in intestine. A similar trend was demonstrated
in winter flounder (Pleuronectes americanus) hepatic microsomes and in Atlantic cod, where CYP3A
expression was sevenfold higher in females than males (Gray et al., 1991; Hasselberg et al., 2004). In
contrast, adult killifish males displayed up to 2.5-fold higher levels of hepatic and extrahepatic CYP3A30
and CYP3A56 mRNA and protein levels compared to females (Hegelund and Celander, 2003). This
finding was in agreement with higher hepatic testosterone 6β-hydroxylase activities in male killifish
(Gray et al., 1991; Stegeman and Woodin, 1984). Other species, including medaka and sexually mature
rainbow trout, also exhibit a higher level of CYP3A expression in male liver microsomes compared to
females (Aoyama et al., 1990; Celander et al., 1989, 1996; Gillam et al., 1993; Gotoh, 1992; Harlow
and Halpert, 1998; Kullman and Hinton, 2001; Lee and Buhler, 2003; Li et al., 1995; Smith et al., 1996;
Szklarz and Halpert, 1997; Waxman et al., 1998). Variations in CYP3A expression in Atlantic salmon
and turbot (Scophthalmus maximus) during reproductive cycles have been reported, implying a regulatory
role of sex steroids on CYP3A expression (Arukwe and Goksøyr, 1997; Larsen et al., 1992). Steroid
reproductive hormones and plasma growth hormones have been shown to influence the sexual dimorphic
expression of CYP3A in rodents (Park et al., 1999; Sakuma et al., 2002; Wang and Strobel, 1997).