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Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (Platichthys flesus L.)
Aquatic Toxicology 48 (2000) 549 – 559 www.elsevier.com/locate/aquatox
Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (Platichthys flesus L.) Pedro Ribeiro Rocha Monteiro *, Maria Armanda Reis-Henriques, Joa˜o Coimbra Laboratory of Applied Physiology, Institute of Biomedical Sciences Abel Salazar and Centre of Marine and En6ironmental Research, Rua do Campo Alegre, 823, 4150 -180 Porto, Portugal Received 19 January 1999; accepted 23 June 1999
Abstract The in vitro effect of polycyclic aromatic hydrocarbons (PAHs) on ovarian steroidogenesis of the flounder (Platichthys flesus L.) was determined. Fully vitellogenic ovary tissue was in vitro incubated in the presence of phenanthrene, benzo[a]pyrene or chrysene, using 17a-hydroxyprogesterone or androstenedione as precursors. Androstenedione (A), testosterone (T) and 17b-estradiol (E2) synthesised in the presence of PAHs were assayed by radioimmunoassay and results compared with control incubations. In order to establish the effect of PAHs on the steroidogenic enzyme systems cytochrome P450 17,20-lyase (P450-17,20l), 17b-hydroxysteroid dehydrogenase (17bHSD) and cytochrome P450 aromatase (P450-arom), results were also compared with the action of ketoconazole (KCZ) and aminoglutethimide (AMG), wich are, respectively, inhibitors of cytochrome P450 steroidogenic enzymes and of P450-arom. KCZ inhibited secretion of A and E2 in 65% and T in 40%, as a consequence of inhibited P450-17,20l and P450-arom. AMG inhibited P450-arom, which resulted in decreased E2 synthesis to approximately 50% of control incubations. All the three PAHs inhibited A secretion by approximately 50% and E2 from 10 to 40%. Because steroid conjugation was also inhibited by phenanthrene, it could be concluded that PAH action was mediated by an inhibitory effect over P450-17,20l, 17b-HSD and P450-arom. Except for 17b-HSD, PAHs resembled KCZ, and P450-17,20l was the most sensitive to their inhibitory effect. In conclusion, PAHs strongly blocked the activity of P450-17,20l, a rate-limiting enzyme for conversion of C21 to C19 steroids, and showed, therefore, the potential to disrupt the reproductive cycle of fish living in polluted environments, due to impairment of steroid biosynthesis. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Flounder; In vitro; PAHs; KCZ; AMG; Ovarian steroidogenesis; P450-17,20l; 17b-HSD; P450-arom
* Corresponding author. Tel.: +351-2-6060421; fax: + 3512-6060423. E-mail address: [email protected] (P.R. Rocha Monteiro)
1. Introduction In recent years, special attention has been given to the endocrine disruption of fish reproduction
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by chemical pollutants (Kime, 1995; Stahlschmidt-Allner et al., 1997; Arcand-Hoy and Benson, 1998) and the possible consequences on wildlife populations (Harries et al., 1997; Kovacs et al., 1997). Several studies have demonstrated the potential of a large number of man-made chemicals, like pesticides, polychlorinated biphenyls, alkylphenolic compounds and phthalates, to impair the reproductive function of fishes (e.g. Choudhury et al., 1993; Monosson et al., 1994; Singh et al., 1994; Jobling et al., 1995, 1996). Disruptions of the reproduction hormonal coordination can be induced by xenobiotics on various levels of the hierarchically-organised endocrine system of vertebrates and may encompass several different signalling pathways, including interactions with a variety of binding globulins, growth factors, different receptor systems, and/or steroidogenic enzymes (Schardein, 1993; Safe, 1995). Polycyclic aromatic hydrocarbons (PAHs) are major pollutants of the estuarine and coastal environments (Meador et al., 1995). Nevertheless, only a few studies have specifically focused on the disruptive potential of PAHs over the endocrine regulation of fish reproduction (Thomas, 1988; Singh, 1989; Thomas, 1990). Despite the paucity of knowledge about PAH effects on fish reproduction, these organic chemical pollutants are often referred to as estrogenic xenobiotics (Sumpter and Jobling, 1995; Arukwe et al., 1997). However, contradicting this alleged estrogenic capability, benzo[a]pyrene (BaP) and 3-methylcholanthrene (3-MC) decreased the circulating levels of 17bestradiol (E2), respectively in females Micropogonias undulatus (Thomas, 1988, 1990) and Monopterus albus (Singh, 1989). The same response was observed in the female flounder (Platichthys flesus) dietarily exposed to phenanthrene (PHN) or chrysene (CHR) (unpublished data). Plasma steroids may be reduced either by inhibition of hypothalamic gonadotropin-releasing hormone or pituitary gonadotropin, decreased ovarian cholesterol, stimulation of hepatic catabolism and excretion, or decreased activity of steroidogenic enzymes. In our previous study (unpublished data) some of the flounders dietarily
exposed to PHN showed decreased plasma E2 with concomitant increased plasma 17a-hydroxyprogesterone (17a-OHP) levels. One possible explanation for this observation is that PAH disruptive action could have been mediated by a selective inhibition of ovarian steroidogenic enzymes. Moreover, a recent work demonstrated that the alkyl and heteroatom-substituted PAHs, respectively 3-MC and 20b-naphthoflavone, inhibited the in vitro secretion of E2 by coho salmon ovarian follicles (Afonso et al., 1997). In this study, we evaluated the effects of PAHs on ovarian steroidogenesis of the flounder. In vitro ovarian steroid biosynthesis in the presence of PHN, BaP or CHR, using 17a-OHP or androstenedione (A) as precursors, was determined. The results were compared with the in vitro ovarian steroidogenesis in the presence of the antifungal ketoconazole (KCZ), an inhibitor of cytochrome P450 steroidogenic enzymes (Albertson et al., 1988; Gal et al., 1994), and aminoglutethimide, an aromatase inhibitor (Graves and Salhanick, 1979; Coster et al., 1990).
2. Materials and methods
2.1. O6ary tissue Vitellogenic ovarian tissue was collected from female flounders (Platichthys flesus L.) captured in the Douro estuary, Porto, Portugal, during December–February. Only tissue of fully vitellogenic ovaries was used. After dissection of the ovary, small pieces were immediately frozen in liquid nitrogen and kept at − 70° C until used for incubations.
2.2. Chemicals Authentic steroids, PAHs, aminoglutethimide (AMG), b-glucuronidase from Helix pomatia, as well as N-2-hydroxyethylpiperazine-N%-2-ethanesulphonic acid (HEPES) sodium salt and the incubation medium, Minimum Essential Medium Eagle (MEM Eagle), were obtained from SigmaAldrich. KCZ was kindly offered by Janssen Pharmaceutica. Analytical grade diethyl ether,
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ethyl acetate, trifluoroacetic acid and propylene glycol were purchased from Merck. All other chemicals were of analytical grade and from either Merck or Riedel-de Hae¨n.
2.3. In 6itro incubations Frozen ovarian tissue thawed at room temperature (15 – 30 min), after which small pieces were minced together. In each incubation, approximately 1 g of minced ovarian tissue was incubated in vitro in 2 ml of MEM Eagle (HEPES 15 mM; pH 7.4) for 24 h, at 25°C in a shaking water bath. The in vitro effect on ovarian steroid biosynthesis of PHN, BaP, CHR, KCZ and aminoglutethimide (all at 15 mM) was evaluated using 17a-OHP (0.15 mM) as the precursor. In order to elucidate the sensitivity of each of the steroidogenic enzymes to PAHs, the in vitro effects of PHN, BaP and KCZ (15 mM for all) were also assessed, using androstenedione (0.15 mM) as the precursor. Incubations with or without 17a-OHP (0.15 mM) were carried out in order to compare flounder’s ovarian endogenous steroid biosynthesis with the steroidogenic capability in the presence of a steroid precursor. In vitro incubations were all carried out in triplicate, using tissue from three to seven different females. Steroid, PAH and KCZ solutions were made in propylene glycol and AMG in distilled water, using a Mettler H54 AR balance.
2.4. Steroid extraction Unconjugated steroids were extracted three times with 5 ml of diethyl ether. Tissue was vortexed for 60 s with the organic solvent and phases were separated by centrifugation at 2000× g (15 min). The upper diethyl ether layer (organic fraction) was recovered and evaporated under a stream of nitrogen, before dissolving it in a radioimmunoassay 0.1% gelatine buffer (sodium azide 0.1%, pH 7.4). In order to release steroid sulphates, the remaining aqueous fraction was lyophilised, 4 ml trifluoroacetic acid/ethyl acetate (1/100, v/v) were added and incubated overnight at 45°C (Scott and
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Canario, 1992). After that, the ethyl acetate was evaporated under a stream of nitrogen and the remaining fraction suspended in 2 ml of sodium acetate buffer (0.1 M, pH 5.0) containing 5000 U of b-glucuronidase. Incubation was carried out at 37°C, overnight, in a shaking water bath, in order to release steroid glucuronides. Finally, sulphate and glucuronide steroid fractions were extracted together as free steroids and suspended in the gelatine buffer.
2.5. Radioimmunoassay The steroids 17a-OHP, androstenedione, testosterone (T) and E2 were quantified by solid-phase 125 I radioimmunoassay, using kits from Diagnostic Products Corporation. The four antisera are highly specific and have an extremely low crossreactivity to other naturally occurring steroids. Gamma radiation was measured in an LKB-Wallac MiniGamma Counter 1275.
2.6. Statistics Values for the four different steroids are presented as mean9 standard error of the mean (SEM). Statistics were applied on log-transformed data, because some dependent variables did not satisfy parametric tests’ assumptions (Zar, 1974). Statistical differences between the level of steroids produced without precursor or in treated incubations, and respective controls, were analysed by a 2-way MANOVA. To determine which treatments significantly altered steroid biosynthesis, multiple comparisons were made using the Scheffe´ test, which provides a high protection against alpha inflation (Garcia-Marques and Azevedo, 1995). All statistics were analysed at the significance level of 5%, using the software Statistica 5.0 (StatSoft Inc., 1995).
3. Results
3.1. 17a-Hydroxyprogesterone incubations Ovarian steroid biosynthesis in the presence of an exogenous precursor (17a-OHP) is significantly
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increased when compared to the endogenous biosynthetic capability (P B0.001 for all steroids, Fig. 1). E2 (4.2 90.5 ng g − 1) is the main steroid produced by vitellogenic flounder’s ovarian tissue. However, in the presence of an exogenous steroid precursor, A (19.69 2.0 ng g − 1) becomes the principal steroid synthesised in vitro. In vitro A synthesis was significantly decreased by the three PAHs and KCZ, respectively to 50 and 35% of control (P B 0.001 for all, Fig. 2(A)). On the other hand, treatment with AMG resulted in a higher synthesis (140% of control) of A (PB0.001, Fig. 2(A)). It should be mentioned that the tendency observed in the five treatments occurred in all three replicates of each female. Results of T synthesis did not parallel those observed for A. In the incubations treated with KCZ, PHN and CHR there was a significant decrease in T production, respectively to 60, 65 and 85% of control (P B 0.001 for all, Fig. 2(B)), while treatment with BaP and AMG did not result in a significant alteration of its synthesis (P\0.05 for both, Fig. 2(B)). Caution should be taken when comparing the effect of PAHs on the biosynthesis of a specific
steroid using the parameter mean9 SEM as the reference. In some cases, the high variability in the steroidogenic capability of the different animals may mask the effect of a particular PAH. A good example can be observed in Fig. 2(B), when comparing the effect of BaP and CHR on T synthesis. The relation between mean values of control and treated incubations it is very similar for both PAHs. However, the mean value of the effect (dotted bar) it is significantly different between these two organic chemical pollutants, respectively 9796 and 859 6% of the control. Ovarian synthesis of E2 was significantly decreased by all five treatments (PB 0.001 for all, Fig. 2(C)), even though at different levels; respectively 35, 45, 60, 75 and 90% of control, for KCZ, AMG, PHN, BaP and CHR. The amount of unconverted precursor after the 24-hour incubation period in treatments with KCZ, AMG and PHN was higher than in the corresponding controls. However, differences were statistical significant only in treatments with KCZ and PHN (PB 0.01 for both, Fig. 2(D)). Ovarian biosynthesis of conjugated T and E2 was significantly decreased by KCZ, AMG and
Fig. 1. In vitro ovarian steroid synthesis with and without 17a-OHP as precursor. Incubations were carried out in triplicate using the tissue from four different females (n= 3×4). A: androstenodione; T: testosterone; E2: 17b-estradiol; c: conjugated. Significantly different: ***, PB 0.001.
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Fig. 2. In vitro ovarian steroid synthesis using 17a-OHP as precursor. Incubations were carried out in triplicate using the tissue from three (n= 3× 3) to seven (n =3× 7) different females. White bar: incubations only with precursor (control); black bar: incubations with precursor plus chemical (treated); dotted bar: percent of control (treated/control); KCZ: ketoconazole; AMG: aminoglutethimide; PHN: phenanthrene; BaP: benzo[a]pyrene; CHR: chrysene; c: conjugated. Significantly different: *, P B0.05; **, P B 0.01; ***, P B0.001.
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PHN, to approximately 60% of control (PB 0.001 for all, Fig. 2(E) and (F)).
3.2. Androstenedione incubations The amount of T synthesised in incubations with
KCZ was significantly higher than in respective controls (120% of control, PB 0.05, Fig. 3(A)), while the opposite occurred in PHN and BaP treated incubations (Fig. 3(A)), with percentages of respectively, 90 (PB 0.05) and 85% (P B 0.01) of the control.
Fig. 2. (Continued)
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Fig. 2. (Continued)
As previously observed when using 17a-OHP as precursor, the production of E2 in incubations treated with KCZ and both PAHs was significantly lower than in control incubations (65 and 80% of control, respectively, P B0.001 for all, Fig. 3(B)). Although highly significant, the inhibitory effect of PAHs on ovarian steroid biosynthesis in
androstenedione incubations it was not as prominent as the one detected in 17a-OHP incubations.
4. Discussion Endocrine disrupters, defined as exogenous substances that cause adverse health effects in an
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intact organism or in its progeny, consequently to changes in endocrine function (European Commission, 1996), can exert their effects at a variety of sites on the hypothalamus – pituitary–gonadal axis to alter reproductive endocrine function. Previously, female flounder dietarily exposed to PHN
or CHR showed a significant correlation between decreased plasma E2 levels and concomitant increased 17a-OHP (unpublished data), which could be a consequence of a direct action on the ovary. As a first approach to this hypothesis, in this study we compared the in vitro effects of
Fig. 3. In vitro ovarian steroid synthesis using androstenedione as precursor. Incubations were carried out in triplicate using tissue from three (n= 3× 3) to four (n=3× 4) different females. See Fig. 2 for legend.
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PAHs and specific inhibitors on three different ovarian steroidogenic enzyme systems: cytochrome P450 17,20-lyase (P450-17,20l), 17b-hydroxysteroid dehydrogenase (17b-HSD) and cytochrome P450 aromatase (P450-arom). The addition of exogenous 17a-OHP to the medium significantly increased steroid secretion, when compared to endogenous steroidogenesis. This finding demonstrates that after being frozen and kept at − 70°C, flounder-minced ovarian tissue maintains a high catalytic capability of both P450-17,20l and P450-arom, necessary to synthesise the estrogens involved in vitellogenin production (Emmersen and Petersen, 1976; Janssen, 1996). Moreover, when 17a-OHP is used as the exogenous precursor, the main steroid produced in vitro becomes A instead of E2, which evidences the higher catalytic potential of P450-17,20l when compared to P450-arom. All chemicals used in this study inhibited in vitro ovarian steroidogenesis in the flounder. The P450arom inhibitor AMG reduced E2 secretion to approximately 50% of control incubations. Previous reports showed that AMG IC50 values towards rat granulosa cells P450-arom (Wouters et al., 1988) and human placental P450-arom (Ahmed et al., 1995), were, respectively, 6 and 20 mM. Therefore, flounder’s ovarian P450-arom shows a sensitivity to AMG similar to that of mammalian species. KCZ inhibited both A and E2 synthesis, demonstrating its potential to inhibit ovarian P450-17,20l and P450-arom. Androstenedione incubations showed that KCZ only inhibits cytochrome P450 steroidogenic enzymes and has no visible effect on ovarian 17b-HSD. In 17a-OHP incubations, both A and E2 synthesis were inhibited to approximately 35% of control incubations. However, when A was used as precursor, E2 synthesis inhibition exceeded 60% of control incubations, in this way demonstrating that P450-17,20l is more sensitive to KCZ than P450-arom. Also in human adrenals, testes and ovary, P450-17,20l was shown to be more sensitive to KCZ than P450-arom (Weber et al., 1991). Moreover, KCZ at 12 mM inhibited in 50% the activity of rat testicular P450-17,20l (Ahmed et al., 1995) and at the concentration of 10 mM inhibited in 68% the activity of human granulosa
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cells P450-arom (Gal et al., 1991), both studies using pregnenolone as precursor. Hence, flounder’s ovarian P450-17,20l and P450-arom showed also a sensitivity to KCZ similar to those from mammalian tissues. Concerning PAHs, at 15 mM, all three PAHs used in this study inhibited A secretion in 50% and E2 synthesis from 10 to 40%. Therefore, PAHs demonstrated the potential to inhibit ovarian steroidogenesis in the flounder. In a previous report, Afonso et al. (1997) have already shown that 3-MC and 20b-naphthoflavone inhibited in vitro secretion of E2 by ovarian follicles of coho salmon. However, these authors could not establish whether this was a result of inhibited steroidogenic enzymes activity or of increased catabolism of E2, as no conjugated metabolites were assayed in the incubation medium. On the contrary, in this study, both conjugated T and E2 were significantly decreased in PHN-treated incubations, showing that PAH action should have been mediated by an inhibition of steroidogenic enzymes. Combining the results obtained for 17a-OHP and androstenedione incubations, it was possible to conclude that PAHs inhibited all three steroidogenic enzyme systems evaluated in this work: P450-17,20l, 17b-HSD and P450-arom. From these, P450-17,20l was clearly the most sensitive to PAH action. Hence, except for 17b-HSD, PAHs effects resembled those from KCZ, although this last compound showed a more potent inhibitory capability. PAHs are lipophilic organic chemical pollutants that accumulate in lipid-rich tissues like the gonads (Varanasi et al., 1982; Meador et al., 1995) and because they are shown to strongly inhibit P45017,20l, the reproductive success of fish living in environments polluted by these chemical organic pollutants may be compromised. Previously, decreased plasma E2 and impaired ovarian growth has been observed in females of the Atlantic croaker (Thomas, 1988, 1990) and Monopterus albus (Singh, 1989) exposed, respectively, to BaP and 3-MC, as well as in English sole (Parophrys 6etulus) captured in areas contaminated with PAHs (Johnson et al., 1988). In addition, female flounder dietarily exposed to PHN and CHR displayed decreased plasma E2 with a concomitant increase in plasma 17a-OHP (unpublished data).
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Inducers of CYP1A1 gene expression, such as PAHs (Leighton et al., 1995; Campbell and Devlin, 1996), are thought to decrease plasma estrogens by enhancing its clearance, once cytochrome P450 1A1 (P450-1A1) plays an important role in E2 catabolism (Spink et al., 1992; Hammond et al., 1997). However, not only substrates of P4501A1, like PAHs, can bind to the active site of placental P450-arom and competitively inhibit aromatization of A (Toma et al., 1996), but also, in the present work, steroid conjugation was inhibited by PHN. Therefore, there is evidence for PAHs being able to disrupt teleost reproductive endocrine function by a direct inhibition of ovarian steroidogenic enzymes. Indeed, already two decades ago, Mattison et al. (1980) observed that BaP reduced the fertility and destroyed primordial oocytes of DBA/2N mice in a dose–dependent manner and suggested that the BaP mechanism of action resided within the ovary. In conclusion, ovarian steroidogenic enzyme systems of the flounder were shown to be as sensitive as those from mammalian tissues to aminoglutethimide and KCZ. PAH action resembled that of KCZ, because both inhibited androstenedione, testosterone and E2 synthesis. As steroid conjugation was also inhibited by PHN, it was concluded that PAH action is mediated by inhibition of steroidogenic enzymes, mainly of P450-17,20l. Acknowledgements This study was supported by the Portuguese Foundation for Science and Technology (Ph.D. grant BD/2651/93-IG). References Afonso, L.O.B., Campbell, P.M., Iwama, G.K., Devlin, R.H., Donaldson, E.M., 1997. The effect of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on sex steroid secretion by ovarian follicles of coho salmon. Gen. Comp. Endocrinol. 106, 169– 174. Ahmed, S., Smith, J.H., Nicholls, P.J., Whomsley, R., Cariuk, P., 1995. Synthesis and biological evaluation of imidazole based compounds as cytochrome P450 inhibitors. Drug Des. Discov. 13, 27–41.
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Polycyclic aromatic hydrocarbons inhibit in vitro ovarian steroidogenesis in the flounder (Platichthys flesus L.) Pedro Ribeiro Rocha Monteiro *, Maria Armanda Reis-Henriques, Joa˜o Coimbra Laboratory of Applied Physiology, Institute of Biomedical Sciences Abel Salazar and Centre of Marine and En6ironmental Research, Rua do Campo Alegre, 823, 4150 -180 Porto, Portugal Received 19 January 1999; accepted 23 June 1999
Abstract The in vitro effect of polycyclic aromatic hydrocarbons (PAHs) on ovarian steroidogenesis of the flounder (Platichthys flesus L.) was determined. Fully vitellogenic ovary tissue was in vitro incubated in the presence of phenanthrene, benzo[a]pyrene or chrysene, using 17a-hydroxyprogesterone or androstenedione as precursors. Androstenedione (A), testosterone (T) and 17b-estradiol (E2) synthesised in the presence of PAHs were assayed by radioimmunoassay and results compared with control incubations. In order to establish the effect of PAHs on the steroidogenic enzyme systems cytochrome P450 17,20-lyase (P450-17,20l), 17b-hydroxysteroid dehydrogenase (17bHSD) and cytochrome P450 aromatase (P450-arom), results were also compared with the action of ketoconazole (KCZ) and aminoglutethimide (AMG), wich are, respectively, inhibitors of cytochrome P450 steroidogenic enzymes and of P450-arom. KCZ inhibited secretion of A and E2 in 65% and T in 40%, as a consequence of inhibited P450-17,20l and P450-arom. AMG inhibited P450-arom, which resulted in decreased E2 synthesis to approximately 50% of control incubations. All the three PAHs inhibited A secretion by approximately 50% and E2 from 10 to 40%. Because steroid conjugation was also inhibited by phenanthrene, it could be concluded that PAH action was mediated by an inhibitory effect over P450-17,20l, 17b-HSD and P450-arom. Except for 17b-HSD, PAHs resembled KCZ, and P450-17,20l was the most sensitive to their inhibitory effect. In conclusion, PAHs strongly blocked the activity of P450-17,20l, a rate-limiting enzyme for conversion of C21 to C19 steroids, and showed, therefore, the potential to disrupt the reproductive cycle of fish living in polluted environments, due to impairment of steroid biosynthesis. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Flounder; In vitro; PAHs; KCZ; AMG; Ovarian steroidogenesis; P450-17,20l; 17b-HSD; P450-arom
* Corresponding author. Tel.: +351-2-6060421; fax: + 3512-6060423. E-mail address: [email protected] (P.R. Rocha Monteiro)
1. Introduction In recent years, special attention has been given to the endocrine disruption of fish reproduction
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by chemical pollutants (Kime, 1995; Stahlschmidt-Allner et al., 1997; Arcand-Hoy and Benson, 1998) and the possible consequences on wildlife populations (Harries et al., 1997; Kovacs et al., 1997). Several studies have demonstrated the potential of a large number of man-made chemicals, like pesticides, polychlorinated biphenyls, alkylphenolic compounds and phthalates, to impair the reproductive function of fishes (e.g. Choudhury et al., 1993; Monosson et al., 1994; Singh et al., 1994; Jobling et al., 1995, 1996). Disruptions of the reproduction hormonal coordination can be induced by xenobiotics on various levels of the hierarchically-organised endocrine system of vertebrates and may encompass several different signalling pathways, including interactions with a variety of binding globulins, growth factors, different receptor systems, and/or steroidogenic enzymes (Schardein, 1993; Safe, 1995). Polycyclic aromatic hydrocarbons (PAHs) are major pollutants of the estuarine and coastal environments (Meador et al., 1995). Nevertheless, only a few studies have specifically focused on the disruptive potential of PAHs over the endocrine regulation of fish reproduction (Thomas, 1988; Singh, 1989; Thomas, 1990). Despite the paucity of knowledge about PAH effects on fish reproduction, these organic chemical pollutants are often referred to as estrogenic xenobiotics (Sumpter and Jobling, 1995; Arukwe et al., 1997). However, contradicting this alleged estrogenic capability, benzo[a]pyrene (BaP) and 3-methylcholanthrene (3-MC) decreased the circulating levels of 17bestradiol (E2), respectively in females Micropogonias undulatus (Thomas, 1988, 1990) and Monopterus albus (Singh, 1989). The same response was observed in the female flounder (Platichthys flesus) dietarily exposed to phenanthrene (PHN) or chrysene (CHR) (unpublished data). Plasma steroids may be reduced either by inhibition of hypothalamic gonadotropin-releasing hormone or pituitary gonadotropin, decreased ovarian cholesterol, stimulation of hepatic catabolism and excretion, or decreased activity of steroidogenic enzymes. In our previous study (unpublished data) some of the flounders dietarily
exposed to PHN showed decreased plasma E2 with concomitant increased plasma 17a-hydroxyprogesterone (17a-OHP) levels. One possible explanation for this observation is that PAH disruptive action could have been mediated by a selective inhibition of ovarian steroidogenic enzymes. Moreover, a recent work demonstrated that the alkyl and heteroatom-substituted PAHs, respectively 3-MC and 20b-naphthoflavone, inhibited the in vitro secretion of E2 by coho salmon ovarian follicles (Afonso et al., 1997). In this study, we evaluated the effects of PAHs on ovarian steroidogenesis of the flounder. In vitro ovarian steroid biosynthesis in the presence of PHN, BaP or CHR, using 17a-OHP or androstenedione (A) as precursors, was determined. The results were compared with the in vitro ovarian steroidogenesis in the presence of the antifungal ketoconazole (KCZ), an inhibitor of cytochrome P450 steroidogenic enzymes (Albertson et al., 1988; Gal et al., 1994), and aminoglutethimide, an aromatase inhibitor (Graves and Salhanick, 1979; Coster et al., 1990).
2. Materials and methods
2.1. O6ary tissue Vitellogenic ovarian tissue was collected from female flounders (Platichthys flesus L.) captured in the Douro estuary, Porto, Portugal, during December–February. Only tissue of fully vitellogenic ovaries was used. After dissection of the ovary, small pieces were immediately frozen in liquid nitrogen and kept at − 70° C until used for incubations.
2.2. Chemicals Authentic steroids, PAHs, aminoglutethimide (AMG), b-glucuronidase from Helix pomatia, as well as N-2-hydroxyethylpiperazine-N%-2-ethanesulphonic acid (HEPES) sodium salt and the incubation medium, Minimum Essential Medium Eagle (MEM Eagle), were obtained from SigmaAldrich. KCZ was kindly offered by Janssen Pharmaceutica. Analytical grade diethyl ether,
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ethyl acetate, trifluoroacetic acid and propylene glycol were purchased from Merck. All other chemicals were of analytical grade and from either Merck or Riedel-de Hae¨n.
2.3. In 6itro incubations Frozen ovarian tissue thawed at room temperature (15 – 30 min), after which small pieces were minced together. In each incubation, approximately 1 g of minced ovarian tissue was incubated in vitro in 2 ml of MEM Eagle (HEPES 15 mM; pH 7.4) for 24 h, at 25°C in a shaking water bath. The in vitro effect on ovarian steroid biosynthesis of PHN, BaP, CHR, KCZ and aminoglutethimide (all at 15 mM) was evaluated using 17a-OHP (0.15 mM) as the precursor. In order to elucidate the sensitivity of each of the steroidogenic enzymes to PAHs, the in vitro effects of PHN, BaP and KCZ (15 mM for all) were also assessed, using androstenedione (0.15 mM) as the precursor. Incubations with or without 17a-OHP (0.15 mM) were carried out in order to compare flounder’s ovarian endogenous steroid biosynthesis with the steroidogenic capability in the presence of a steroid precursor. In vitro incubations were all carried out in triplicate, using tissue from three to seven different females. Steroid, PAH and KCZ solutions were made in propylene glycol and AMG in distilled water, using a Mettler H54 AR balance.
2.4. Steroid extraction Unconjugated steroids were extracted three times with 5 ml of diethyl ether. Tissue was vortexed for 60 s with the organic solvent and phases were separated by centrifugation at 2000× g (15 min). The upper diethyl ether layer (organic fraction) was recovered and evaporated under a stream of nitrogen, before dissolving it in a radioimmunoassay 0.1% gelatine buffer (sodium azide 0.1%, pH 7.4). In order to release steroid sulphates, the remaining aqueous fraction was lyophilised, 4 ml trifluoroacetic acid/ethyl acetate (1/100, v/v) were added and incubated overnight at 45°C (Scott and
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Canario, 1992). After that, the ethyl acetate was evaporated under a stream of nitrogen and the remaining fraction suspended in 2 ml of sodium acetate buffer (0.1 M, pH 5.0) containing 5000 U of b-glucuronidase. Incubation was carried out at 37°C, overnight, in a shaking water bath, in order to release steroid glucuronides. Finally, sulphate and glucuronide steroid fractions were extracted together as free steroids and suspended in the gelatine buffer.
2.5. Radioimmunoassay The steroids 17a-OHP, androstenedione, testosterone (T) and E2 were quantified by solid-phase 125 I radioimmunoassay, using kits from Diagnostic Products Corporation. The four antisera are highly specific and have an extremely low crossreactivity to other naturally occurring steroids. Gamma radiation was measured in an LKB-Wallac MiniGamma Counter 1275.
2.6. Statistics Values for the four different steroids are presented as mean9 standard error of the mean (SEM). Statistics were applied on log-transformed data, because some dependent variables did not satisfy parametric tests’ assumptions (Zar, 1974). Statistical differences between the level of steroids produced without precursor or in treated incubations, and respective controls, were analysed by a 2-way MANOVA. To determine which treatments significantly altered steroid biosynthesis, multiple comparisons were made using the Scheffe´ test, which provides a high protection against alpha inflation (Garcia-Marques and Azevedo, 1995). All statistics were analysed at the significance level of 5%, using the software Statistica 5.0 (StatSoft Inc., 1995).
3. Results
3.1. 17a-Hydroxyprogesterone incubations Ovarian steroid biosynthesis in the presence of an exogenous precursor (17a-OHP) is significantly
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increased when compared to the endogenous biosynthetic capability (P B0.001 for all steroids, Fig. 1). E2 (4.2 90.5 ng g − 1) is the main steroid produced by vitellogenic flounder’s ovarian tissue. However, in the presence of an exogenous steroid precursor, A (19.69 2.0 ng g − 1) becomes the principal steroid synthesised in vitro. In vitro A synthesis was significantly decreased by the three PAHs and KCZ, respectively to 50 and 35% of control (P B 0.001 for all, Fig. 2(A)). On the other hand, treatment with AMG resulted in a higher synthesis (140% of control) of A (PB0.001, Fig. 2(A)). It should be mentioned that the tendency observed in the five treatments occurred in all three replicates of each female. Results of T synthesis did not parallel those observed for A. In the incubations treated with KCZ, PHN and CHR there was a significant decrease in T production, respectively to 60, 65 and 85% of control (P B 0.001 for all, Fig. 2(B)), while treatment with BaP and AMG did not result in a significant alteration of its synthesis (P\0.05 for both, Fig. 2(B)). Caution should be taken when comparing the effect of PAHs on the biosynthesis of a specific
steroid using the parameter mean9 SEM as the reference. In some cases, the high variability in the steroidogenic capability of the different animals may mask the effect of a particular PAH. A good example can be observed in Fig. 2(B), when comparing the effect of BaP and CHR on T synthesis. The relation between mean values of control and treated incubations it is very similar for both PAHs. However, the mean value of the effect (dotted bar) it is significantly different between these two organic chemical pollutants, respectively 9796 and 859 6% of the control. Ovarian synthesis of E2 was significantly decreased by all five treatments (PB 0.001 for all, Fig. 2(C)), even though at different levels; respectively 35, 45, 60, 75 and 90% of control, for KCZ, AMG, PHN, BaP and CHR. The amount of unconverted precursor after the 24-hour incubation period in treatments with KCZ, AMG and PHN was higher than in the corresponding controls. However, differences were statistical significant only in treatments with KCZ and PHN (PB 0.01 for both, Fig. 2(D)). Ovarian biosynthesis of conjugated T and E2 was significantly decreased by KCZ, AMG and
Fig. 1. In vitro ovarian steroid synthesis with and without 17a-OHP as precursor. Incubations were carried out in triplicate using the tissue from four different females (n= 3×4). A: androstenodione; T: testosterone; E2: 17b-estradiol; c: conjugated. Significantly different: ***, PB 0.001.
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Fig. 2. In vitro ovarian steroid synthesis using 17a-OHP as precursor. Incubations were carried out in triplicate using the tissue from three (n= 3× 3) to seven (n =3× 7) different females. White bar: incubations only with precursor (control); black bar: incubations with precursor plus chemical (treated); dotted bar: percent of control (treated/control); KCZ: ketoconazole; AMG: aminoglutethimide; PHN: phenanthrene; BaP: benzo[a]pyrene; CHR: chrysene; c: conjugated. Significantly different: *, P B0.05; **, P B 0.01; ***, P B0.001.
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PHN, to approximately 60% of control (PB 0.001 for all, Fig. 2(E) and (F)).
3.2. Androstenedione incubations The amount of T synthesised in incubations with
KCZ was significantly higher than in respective controls (120% of control, PB 0.05, Fig. 3(A)), while the opposite occurred in PHN and BaP treated incubations (Fig. 3(A)), with percentages of respectively, 90 (PB 0.05) and 85% (P B 0.01) of the control.
Fig. 2. (Continued)
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Fig. 2. (Continued)
As previously observed when using 17a-OHP as precursor, the production of E2 in incubations treated with KCZ and both PAHs was significantly lower than in control incubations (65 and 80% of control, respectively, P B0.001 for all, Fig. 3(B)). Although highly significant, the inhibitory effect of PAHs on ovarian steroid biosynthesis in
androstenedione incubations it was not as prominent as the one detected in 17a-OHP incubations.
4. Discussion Endocrine disrupters, defined as exogenous substances that cause adverse health effects in an
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intact organism or in its progeny, consequently to changes in endocrine function (European Commission, 1996), can exert their effects at a variety of sites on the hypothalamus – pituitary–gonadal axis to alter reproductive endocrine function. Previously, female flounder dietarily exposed to PHN
or CHR showed a significant correlation between decreased plasma E2 levels and concomitant increased 17a-OHP (unpublished data), which could be a consequence of a direct action on the ovary. As a first approach to this hypothesis, in this study we compared the in vitro effects of
Fig. 3. In vitro ovarian steroid synthesis using androstenedione as precursor. Incubations were carried out in triplicate using tissue from three (n= 3× 3) to four (n=3× 4) different females. See Fig. 2 for legend.
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PAHs and specific inhibitors on three different ovarian steroidogenic enzyme systems: cytochrome P450 17,20-lyase (P450-17,20l), 17b-hydroxysteroid dehydrogenase (17b-HSD) and cytochrome P450 aromatase (P450-arom). The addition of exogenous 17a-OHP to the medium significantly increased steroid secretion, when compared to endogenous steroidogenesis. This finding demonstrates that after being frozen and kept at − 70°C, flounder-minced ovarian tissue maintains a high catalytic capability of both P450-17,20l and P450-arom, necessary to synthesise the estrogens involved in vitellogenin production (Emmersen and Petersen, 1976; Janssen, 1996). Moreover, when 17a-OHP is used as the exogenous precursor, the main steroid produced in vitro becomes A instead of E2, which evidences the higher catalytic potential of P450-17,20l when compared to P450-arom. All chemicals used in this study inhibited in vitro ovarian steroidogenesis in the flounder. The P450arom inhibitor AMG reduced E2 secretion to approximately 50% of control incubations. Previous reports showed that AMG IC50 values towards rat granulosa cells P450-arom (Wouters et al., 1988) and human placental P450-arom (Ahmed et al., 1995), were, respectively, 6 and 20 mM. Therefore, flounder’s ovarian P450-arom shows a sensitivity to AMG similar to that of mammalian species. KCZ inhibited both A and E2 synthesis, demonstrating its potential to inhibit ovarian P450-17,20l and P450-arom. Androstenedione incubations showed that KCZ only inhibits cytochrome P450 steroidogenic enzymes and has no visible effect on ovarian 17b-HSD. In 17a-OHP incubations, both A and E2 synthesis were inhibited to approximately 35% of control incubations. However, when A was used as precursor, E2 synthesis inhibition exceeded 60% of control incubations, in this way demonstrating that P450-17,20l is more sensitive to KCZ than P450-arom. Also in human adrenals, testes and ovary, P450-17,20l was shown to be more sensitive to KCZ than P450-arom (Weber et al., 1991). Moreover, KCZ at 12 mM inhibited in 50% the activity of rat testicular P450-17,20l (Ahmed et al., 1995) and at the concentration of 10 mM inhibited in 68% the activity of human granulosa
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cells P450-arom (Gal et al., 1991), both studies using pregnenolone as precursor. Hence, flounder’s ovarian P450-17,20l and P450-arom showed also a sensitivity to KCZ similar to those from mammalian tissues. Concerning PAHs, at 15 mM, all three PAHs used in this study inhibited A secretion in 50% and E2 synthesis from 10 to 40%. Therefore, PAHs demonstrated the potential to inhibit ovarian steroidogenesis in the flounder. In a previous report, Afonso et al. (1997) have already shown that 3-MC and 20b-naphthoflavone inhibited in vitro secretion of E2 by ovarian follicles of coho salmon. However, these authors could not establish whether this was a result of inhibited steroidogenic enzymes activity or of increased catabolism of E2, as no conjugated metabolites were assayed in the incubation medium. On the contrary, in this study, both conjugated T and E2 were significantly decreased in PHN-treated incubations, showing that PAH action should have been mediated by an inhibition of steroidogenic enzymes. Combining the results obtained for 17a-OHP and androstenedione incubations, it was possible to conclude that PAHs inhibited all three steroidogenic enzyme systems evaluated in this work: P450-17,20l, 17b-HSD and P450-arom. From these, P450-17,20l was clearly the most sensitive to PAH action. Hence, except for 17b-HSD, PAHs effects resembled those from KCZ, although this last compound showed a more potent inhibitory capability. PAHs are lipophilic organic chemical pollutants that accumulate in lipid-rich tissues like the gonads (Varanasi et al., 1982; Meador et al., 1995) and because they are shown to strongly inhibit P45017,20l, the reproductive success of fish living in environments polluted by these chemical organic pollutants may be compromised. Previously, decreased plasma E2 and impaired ovarian growth has been observed in females of the Atlantic croaker (Thomas, 1988, 1990) and Monopterus albus (Singh, 1989) exposed, respectively, to BaP and 3-MC, as well as in English sole (Parophrys 6etulus) captured in areas contaminated with PAHs (Johnson et al., 1988). In addition, female flounder dietarily exposed to PHN and CHR displayed decreased plasma E2 with a concomitant increase in plasma 17a-OHP (unpublished data).
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Inducers of CYP1A1 gene expression, such as PAHs (Leighton et al., 1995; Campbell and Devlin, 1996), are thought to decrease plasma estrogens by enhancing its clearance, once cytochrome P450 1A1 (P450-1A1) plays an important role in E2 catabolism (Spink et al., 1992; Hammond et al., 1997). However, not only substrates of P4501A1, like PAHs, can bind to the active site of placental P450-arom and competitively inhibit aromatization of A (Toma et al., 1996), but also, in the present work, steroid conjugation was inhibited by PHN. Therefore, there is evidence for PAHs being able to disrupt teleost reproductive endocrine function by a direct inhibition of ovarian steroidogenic enzymes. Indeed, already two decades ago, Mattison et al. (1980) observed that BaP reduced the fertility and destroyed primordial oocytes of DBA/2N mice in a dose–dependent manner and suggested that the BaP mechanism of action resided within the ovary. In conclusion, ovarian steroidogenic enzyme systems of the flounder were shown to be as sensitive as those from mammalian tissues to aminoglutethimide and KCZ. PAH action resembled that of KCZ, because both inhibited androstenedione, testosterone and E2 synthesis. As steroid conjugation was also inhibited by PHN, it was concluded that PAH action is mediated by inhibition of steroidogenic enzymes, mainly of P450-17,20l. Acknowledgements This study was supported by the Portuguese Foundation for Science and Technology (Ph.D. grant BD/2651/93-IG). References Afonso, L.O.B., Campbell, P.M., Iwama, G.K., Devlin, R.H., Donaldson, E.M., 1997. The effect of the aromatase inhibitor fadrozole and two polynuclear aromatic hydrocarbons on sex steroid secretion by ovarian follicles of coho salmon. Gen. Comp. Endocrinol. 106, 169– 174. Ahmed, S., Smith, J.H., Nicholls, P.J., Whomsley, R., Cariuk, P., 1995. Synthesis and biological evaluation of imidazole based compounds as cytochrome P450 inhibitors. Drug Des. Discov. 13, 27–41.
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