Gonzalo-Lumbreras and Izquierdo-Hornillos, 2003). The uptake and depletion of MT have been reported in several species of teleost fish, including cichlids (Fagerlund and Dye, 1979; Goudie et al., 1986; Curtis et al., 1991; Cravedi et al., 1993; Rinchard et al., 1999). As MT administrated orally is readily metabolized, research on the fate of MT and on its metabolites need to be addressed from human and environmental safety perspective. Chronic exposure of humans to MT can cause adverse health effects such as hepatotoxicity. Therefore, ingestion of MT residue in treated fish may be a potential hazard to human consumers. The quantity of MT residue in fish tissue will depend on its dosing history and its pharmacokinetics characteristics (Vick and Hayton, 2001).
Several methods have been reported to analyze MT and its many hydroxylated metabolites. Most researchers use the gradient elution with retention times for MT greater than 30 min followed by an additional, between-run, re-equilibration. Some of these methods require complex eluent gradient systems that often cause significant changes in the baseline that affect resolution and detection of later-eluted peaks (Testino Jr et al., 1999). Such problems have focused the analysis of steroids and metabolites by complex techniques based on combined detection methods such as HPLC-MS (high pressure liquid chromatography-mass spectrophotometry) (Stanley et al., 1997; Tsai and Wang, 1999; Clouet-Dumas et al., 2000; Williams et al., 2000; Lagana et al., 2001). We considered that with modifications of the existing detection techniques described in the literature it will be possible to detect 17 -methyltestosterone by using exclusively an HPLC technique.
A different approach to the use of methyltestosterone for sex reversal in fish may involve the use of isoflavonoids, flavonoids and saponins, which are natural estrogenic/androgenic compounds derived from soy, tea, fruits, and vegetables that present an anti-estrogenic activity. The known mechanisms include inhibition of several steroid metabolizing enzymes such as aromatase, a cytochrome P-450 hemoprotein that catalyzes the conversion of androgens, androstenedione, and testosterone via three hydroxylation steps to estrone and estradiol and other enzymes such as 5 -reductase and 17 -hydroxysteroid dehydrogenase (Brodie et al., 1999; Griffiths et al., 1999; Eng et al., 2001). Aromatase is an enzyme of particular interest in sexual differentiation in fish since inhibition of aromatase action mimics the sex-reversal effects of androgen treatments in some fish species (Kwon et al., 2001).
Flavonoids have been found to act as phytoestrogens since these compounds have structures that are recognized as estrogen mimics for the estrogen receptor. They can compete with endogenous estrogens for binding to the estrogen
receptor; therefore, they can act as antiestrogens or weak estrogens. Given that estrogen is the product of aromatase, it is not unexpected that some of these compounds can behave as inhibitors of aromatase, suppressing estrogen biosynthesis in cells (Geahlen et al., 1989; Pelissero et al., 1996; Eng et al., 2001).
Several experiments where phytoestrogens have been used either to inhibit aromatase or as steroid receptor antagonists are described in the literature. Isoflavonoids such as genistein act as estrogen agonists via estrogen receptors in cultured cells and also manifest estrogen-like effects in the female reproductive system (Miksicek, 1995; Santell et al., 1997). Some flavonoids, such as chrysin, are natural aromatase inhibitors (Chen et al., 1997) and may be used to boost low levels of testosterone in aging males. Aromatase affinity for flavonoids is generally lower than it is for steroidal derivatives (Seralini and Moslemi, 2001). 7-hydroxyflavone and apigenin used in microsomes of human placenta after normal full-term delivery were the most effective aromatase and 17 -hydroxysteroid dehydrogenase inhibitors. Le Bail et al. (1998) experiments showed that flavonoids with 7-methoxy or 8-hydroxyl groups in the ring A showed an important anti-aromatase activity, thus there is some of the structure-activity relations implied.
In contrast, results of the use of other non-steroidal aromatase inhibitors, such as tamoxifen, in tilapia are controversial. Hines and Watts (1995) are the only authors to report a masculinizing effect of this compound (100 mg kg-1 of food) in a hybrid tilapia. Guigue et al. (1999) has demonstrated that tamoxifen has no effect on the masculinization of all female tilapia or rainbow truot. New experimentation is required to validate the possibility of the use of phytosteroids as sex reversal agents in tilapia, in order to ensure that they are as effective as the common technique to produce monosex populations that involves the use of steroid hormones such as MT. We intend to develop a synthetic steroid-free method for producing all-male populations of tilapia using specific phytochemicals, including genistein, quercetin, and a cp,plex mixture of flavonoids provided by propolis and
