Natural progestogens play an important role in the stimulati

Natural progestogens play an important role in the stimulation of oocyte growth and maturation as well as in spermatogenesis and sperm maturation, and they act as sex pheromones in teleost fish (Kime, 1990, Kobayashi et al., 2002, Nagahama and Yamashita, 2008, Scott et al., 2010). A complex series of enzymes, located either in mitochondrial or endoplasmic reticulum membranes, are responsible for the biosynthesis of sex steroids in fish glutathione reductase (Fig. 1). The steroid precursor pregnenolone (P5) is converted to progesterone (P4), which is further metabolized to 17α-hydroxyprogestrone (17P4) by 17α-hydroxylase (CYP17). 17P4 is then converted to androstenedione (AD) by C17,20-lyase (CYP17) or to 17α,20β-dihydroxyprogesterone (17α,20β-DP) by 20β-hydroxysteroid dehydrogenases (20β-HSD); 17α,20β-DP has been identified as the maturation-inducing hormone (MIH) in several teleost species (Kime, 1990, Nagahama, 1997, Senthilkumaran et al., 2004, Scott et al., 2010). Androstenedione, on the other hand, is the precursor of testosterone (T) and, both T and AD can be transformed in testes to their respective 11β-hydroxylated metabolites (βT, βAD), a reaction catalyzed by 11β-hydroxylases (CYP11β) (Kime, 1993, Liu et al., 2000). βT and βAD can be further metabolized to 11-ketoandrogens (Kusakabe et al., 2003). 11-Ketotestrosterone (11-KT) is the most potent androgen in male teleost fish, being more effective than T in stimulating secondary sexual characters, influencing spermatogenesis and stimulating reproductive behaviour (Borg, 1994). Additionally, androgens (AD and T) are converted into estrogens (estrone and estradiol) through aromatization in female gonads; this reaction is catalyzed by cytochrome P450 aromatase (CYP19), a key enzyme regulating local and systemic levels of estrogens in the body (Cheshenko et al., 2008). Any interference with the normal biosynthesis of these steroids may alter the bioavailable amounts of active hormones within the organism and be a potential mechanism of endocrine disruption, affecting sexual differentiation, gamete growth and/or maturation of fish. Thus, given the important role of progestogens in regulating fish spawning processes, this study aimed at assessing the interference of four currently used synthetic progestogens – drospirenone (DRO), levonorgestrel (LNG), norethindrone (NOR) and cyproterone acetate (CPA) – with key enzymatic activities involved in the synthesis of steroids in fish. DRO is a third generation progestogen, an analogue of spironolactone with a biochemical and pharmacological profile similar to endogenous progesterone, especially regarding anti-mineralocorticoid and anti-androgenic activities. LNG and NOR are second- and first-generation progestins, respectively, derived from 19-nortestosterone and known to exhibit significant progestogenic and androgenic effects; NOR has also estrogenic action. CPA is a preganane derivative with a strong anti-androgenic effect (Besse and Garric, 2009). To address the hypothesis that synthetic progestogens directly interfere with the synthesis of androgens, estrogens and MIH in fish, a number of targeted bioassays were performed focusing mainly in the following enzymatic pathways: (a) CYP17 (C17,20-lyase) and CYP11β involved in the synthesis of AD and 11β-hydroxyandrostenedione (βAD), respectively, (b) CYP19 that plays a key role in the aromatization of androgens to estrogens, and (c) 20β-HSD that catalyzes the conversion of 17P4 to 17α,20β-DP. The assays were performed by incubating mitochondrial or microsomal fractions isolated from carp gonads at different stages of sexual maturation with 17P4 and AD as precursors.
Materials and methods
Results
Discussion Mitochondrial fractions isolated from gonads of male carp metabolized 17P4 to AD and βAD, by CYP17 (C17,20-lyase) and CYP11β enzymes, respectively (Fig. 1). As previously reported, the metabolism of androgens was elevated in testes classified as early-spermatogenic, which is consistent with the role that 11-oxygenated androgens have in testicular recrudescence in many teleost fish (Fernandes et al., 2007, Fernandes and Porte, 2013). All tested progestogens were capable of inhibiting both CYP17 and CYP11β activities, with the exception of LNG that had no effect on CYP17. Regarding CYP17 inhibition, DRO was a strong inhibitor, 10-fold more effective than the antifungal drug ketoconazole (IC50: 38μM) (Fernandes and Porte, 2013). Ketoconazole is a well-known inhibitor of several CYP enzymes that has been reported to inhibit C17,20-lyase activity in human adrenals, rat testis and flounder ovary, among others (Weber et al., 1991, Ahmed et al., 1995, Monteiro et al., 2000). In fact, DRO (IC50: 3.8μM for CYP17; 1.8μM for CYP11β), NOR (IC50: 0.4μM for CYP11β) and CPA (IC50: 183μM for CYP17; 87μM for CYP11β) showed higher ability to interfere with the synthesis of active androgens than other chemicals previously investigated. Thus, 9-hydroxyphenanthrene, a PAH metabolite, inhibited CYP17 and CYP11β enzymes in carp with IC50s of 11μM and 31μM, respectively (Fernandes and Porte, 2013). The anti-depressive drugs, fluoxetine and fluvoxamine inhibited CYP17 and CYP11β enzymes in carp with IC50s of 321–335μM and 244–550μM, respectively (Fernandes et al., 2011). Galaxolide and tonalide had IC50s in the range of 200μM for CYP17 (Schnell et al., 2009), while nonylphenol, a well-known endocrine disruptor, showed an IC50 of 100μM for CYP17, but had no detectable effect on CYP11β (Fernandes et al., 2007).