Estrogens counteract the masculinizing effect of tributyltin in zebrafish

Comparative Biochemistry and Physiology, Part C 142 (2006) 151 – 155 www.elsevier.com/locate/cbpc

Estrogens counteract the masculinizing effect of tributyltin in zebrafish M.M. Santos a,⁎, J. Micael a , A.P. Carvalho c , R. Morabito d , P. Booy e , P. Massanisso d , M. Lamoree e , M.A. Reis-Henriques a,b a

Centre of Marine and Environmental Research, Rua dos Bragas 289, 4050-123 Porto, Portugal Institute of Biomedical Sciences Abel Salazar, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal Department of Zoology and Anthropology, Faculty of Sciences, University of Porto, Pr. Gomes Teixeira, 4099-002 Porto, Portugal d ENEA C.R. Casaccia-Teinichim, Via Anguillarese, 301-00060 Rome, Italy e Institute for Environmental Studies, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands b

c

Received 17 July 2005; received in revised form 18 November 2005; accepted 20 November 2005 Available online 9 January 2006

Abstract Recently, it has been demonstrated that the biocide tributyltin (TBT) can interfere with fish sex differentiation, leading to a bias of sex toward males. On the contrary, it is well known that estrogenic compounds can induce fish feminization. Yet, the combined effects of mixtures of androgenic and estrogenic compounds on fish sex differentiation have never been investigated before, even though in the environment animals are frequently exposed to both groups of xenobiotics. Therefore, in order to investigate whether exposure to estrogenic compounds can block the masculinizing effect of TBT, 5 days post-fertilization zebrafish (Danio rerio) larvae were exposed for a four month period to TBT and to the synthetic estrogen–ethinylestradiol (EE2). The fish were fed a diet containing TBT at nominal concentrations of 25 and 100 ng TBT/g, and two groups of animals were also dosed with TBT plus EE2 at nominal water concentration of 3.5 ng/L, using a flow-through design. As expected, fish exposed to TBT showed a bias of sex toward males (62.5% males in control tanks and 86% and 82% in TBT 25 and TBT 100 ng TBT/g, respectively). Co-exposure to EE2 completely blocked the masculinizing effect of TBT, with 7% males in the TBT 25 ng/g + EE2 treatment and 0% in the EE2 alone and in the TBT 100 ng/g + EE2 exposed groups. These results clearly indicate that EE2, at environmentally relevant concentrations, can block the TBT masculinizing effects in zebrafish, which suggests that in the aquatic environment the presence of estrogens may neutralize the fish masculinizing effect of TBT. Our findings highlight the need of testing the combined effects of contaminants, as single exposure studies may not be sufficient to predict the effects of mixtures of xenobiotics with antagonistic properties. © 2005 Elsevier Inc. All rights reserved. Keywords: Tributyltin; Xenoandrogen; Ethinylestradiol; Xenoestrogen; Mixture; Masculinization; Sex ratio; Endocrine disruption; Fish

1. Introduction Recently, concerns have increased about chemicals in the environment which alter the normal endocrine function of animals and Humans, commonly termed endocrine disruptors (EDCs). The majority of the studies on EDCs in vertebrates have focused on the effects of estrogenic chemicals (EC) (natural steroid 17-β-estradiol (E2); alkylphenolic compounds; the synthetic estrogen ethinylestradiol (EE2), etc.) because many of the observed effects (reduce testicular development and

⁎ Corresponding author. Tel.: +351 22 3401824; fax: +351 22 340 18 38. E-mail address: [email protected] (M.M. Santos). 1532-0456/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.11.014

fertility, increase production of vitellogenin, the presence of male fish feminization) are believed to result from disruption of this axis (Jobling et al., 2003; Kinnberg et al., 2003; Andersen et al., 2003; Solé et al., 2003; Tollefsen et al., 2004; Rasmussen and Korsgaard, 2004; Angus et al., 2005). However, the best documented example of endocrine disruption in wildlife is the masculinization of female neogastropods (imposex) by the antifouling compound tributyltin (TBT) (Matthiessen and Gibbs, 1998; Santos et al., 2005). TBT is a ubiquitous persistent xenobiotic that can be found in freshwater, estuarine and costal ecosystems (Fent, 1996). Although its use has been recently restricted to all ship sizes (Anon., 2001), the levels of TBT in the aquatic environment are still a cause of great concern (Santos et al., 2002).

152

M.M. Santos et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 151–155

In contrast to the extensive literature dealing with the negative impact of TBT in gastropods, only two articles have addressed the effects of TBT in fish sex differentiation (Shimasaki et al., 2003; McAllister and Kime, 2003). Both studies show that TBT can alter the sex ratio towards males at extremely low levels. This may indicate that many natural fish population may be negatively impacted by this organotin compound. Although estrogenic and androgenic compounds exist in combination in the aquatic environment, there are no available studies focusing on the combined effects of EDCs mixtures with antagonistic effects on fish sex differentiation. Clearly, studying the effects of single EDCs may not be sufficient to understand what is really occurring in the environment, which points to the need for a more robust approach which integrates the fact that animals are simultaneously exposed to both groups of xenobiotics. Therefore, the aim of this study was to investigate if an environmentally relevant concentration of a model xenoestrogen (EE2) could neutralise the masculinizing effect of TBT. To this end, we have exposed 5 days post-fertilization zebrafish (Danio rerio) larvae to environmentally low doses of TBT (25 and 100 ng TBT/g diet) for a period of four months (which includes the sex differentiation period), and concomitantly tested whether co-exposure to an environmentally relevant concentration (3.5 ng/L) of the synthetic estrogen—EE2— could neutralize the masculinising effect of the androgenic compound. The selection of zebrafish as the model species was based on the fact that it reaches sexual maturity in a short period of time (within 3–4 months) and also because TBT had previously been shown to induce masculinization of zebrafish at extremely low levels (nominal concentrations between 0.1–1 ng/L) in a life-cycle test (McAllister and Kime, 2003). 2. Material and methods 2.1. Chemicals Ethinylestradiol (EE2) (98% purity) was obtained from Sigma and tributyltin chloride (TBTCl) (96% purity) was obtained from Aldrich. All other chemicals were obtained from Sigma. 2.2. Experimental animals Adult zebrafish (D. rerio) kept in our laboratory for two generations were used as breeding stocks. Fish were initially kept at 27 °C with a 12 h light : 12 h dark photoperiod. Aeration and filtration were provided using sponge filters, and ammonia maintained below detection limit. Two days prior to breeding, fish were fed Artemia nauplii and a casein based diet 3 times a day (Carvalho et al., 2004). The day before breeding, four males and four females were housed in a 5 L breeding chambers. At 1 h past the start of the light phase the following day, viable eggs were collected and allowed to hatch in running water. At 5 days post-fertilization (pf), larvae started being

fed with a casein based diet (Carvalho et al., 2004). On day 16 pf, a 3 times a week Artemia nauplii supplement was introduced (prepared in artificial sea water); starting on day 60 pf, the Artemia nauplii supplement was given on a daily base, together with the casein diet. 2.3. Exposure studies Larvae (5 days pf) were assigned to 5 L aquaria provided with dechlorinated tap water (carbon activated filtration) at a flow rate of 50 L/ day, using a flow-through design. An initial density of 150 larvae per aquarium was used. At day 21, larvae were transferred to 30 L aquaria and at day 50 the density was reduced to 90 animals; the density was further reduced to 70 animals at day 65. Water was maintained at a temperature of 27 °C, and photoperiod at 14 h light : 10 h dark. The pH (7.9 ± 0.5), the dissolved oxygen (N6 mg O2/L) and ammonium (b 0.5 mg NH4/L) were weekly checked. Waste feed and faeces were daily siphoned from the aquaria. Larvae were exposed to TBT incorporated in the diet at nominal concentrations of 25 and 100 ng TBTCl/g wet mass. The selection of TBT doses was based on two previous studies which reported TBT masculinization of zebrafish and genetically female Japanese flounder at 1 ng TBT/L and 100 ng TBT/g wet mass in the diet, respectively (McAllister and Kime, 2003; Shimasaki et al., 2003). These levels of TBT incorporated in zebrafish diet are environmentally relevant, as they are found in animals from low to moderate TBTcontaminated areas (Albalat et al., 2002). EE2 was dissolved in DMSO and delivered to a mixing chamber by a peristaltic pump (ISMATEC IP-N 16) where it was further diluted in dechlorinated tap water to a final nominal aquarium concentration of 3.5 ng/L. Two treatments with the mixture of both compounds (TBT 25 ng/g + EE2 3.5 ng/L; TBT 100 ng/g + EE2 3.5 ng/L) were also performed, together with solvent control. The solvent (DMSO) concentration in the aquaria did not exceed 20 ng/L. Each treatment was run in duplicate for a four month period. At the end of the experiment, 30 animals per replicate (60 per treatment) were sacrificed, and the sex of animals annotated, after inspection of the gonads under a stereo microscope; animals were classified as males when a white gonad was present, as females when it was possible to observe the presence of oocytes and as undeveloped gonads when a hyaline tissue was present but no oocytes could be detected under the stereo microscope. Animals were then preserved for the study of other parameters. At the end of the experiment, one replicate only (n = 30) could be used to determine sex ratio in the TBT 100 treatment. As a routine analysis to confirm male sex identification, a simple methodological procedure was use to observe the presence of sperm in the gonads of those animals displaying white gonad (classified as males): after sacrificing the animal, one of the testis was collected and mixed in 0.5 mL of an extender solution (NaCl 5.8 g/L, KCl 0.2 g/L, CaCl2 0.22 g/L, MgCl2·6H2O 0.04 g/L, NaHCO3 2.10 g/L, NaH2PO4·2H2O 0.04 g/L, glycine 3.75 g/L in distilled water, pH 8.6) and 5 μL of 1% of Rose Bengal dye

M.M. Santos et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 151–155

2.4. Statistical analysis The Chi-square test was used to identify significant differences in the percentage of males between control and each of the other treatments, using the software Statistica 5.0. 3. Results Fig. 1 shows the proportion of each sex in the different treatments. The percentage of males was significantly altered (p b 0.01, Chi-squared test) in all treatments in comparison with control. The TBT treatments displayed a higher proportion of males when compared with control; in contrast, all EE2 and TBT + EE2 exposed groups displayed a lower percentage of males than control tanks; TBT 25 + EE2 was the only treatment among the EE2 exposed groups that showed the presence of males (7%). The TBT dosed groups showed a lower percentage

100

M F UD

* *

80

Sex ratio (%)

(CI45440) was added to make sperm more visible under the microscope (400×) (McAllister and Kime, 2003). This allowed us to confirm our identification, as all animals with white gonads displayed a large number of sperm cells. In order to assure that actual concentrations of EE2 were similar to nominal concentrations, the aquaria were allowed to equilibrate for two weeks before the beginning of the experiment, and the actual EE2 concentrations in the aquaria were checked twice during the experiment. Briefly, water samples were collected in solvent rinsed glass bottles that were silylated prior to use. Samples underwent solid phase extraction (SPE) on a DVB Speedisk (Baker, Deventer, The Netherlands), and after clean up of the extracts by solid phase C18 cartridges, EE2 was derivatized using a silyl reagents before analysis using gas chromatography combined with ion trap detection (GCITD–MS) (adapted from Belfroid et al., 1999; Houtman et al., 2004). The detection limit of the technique was 1 ng EE2/L. Likewise, at the end of the experiment, the concentrations of TBT in the diet and in fish body were analysed by GC–MS. The full description of the method can be found in Morabito et al. (1995). Briefly, freeze-dried tissue (0.5 g) was taken as sample and extracted with 15 mL of 0.03% (w/v) tropolone in methanolic solution and 1 mL of concentrated hydrochloric acid. The supernatant was transferred to a separation funnel and the extraction procedure was repeated. After solvent exchange, extracts were pentylated by Grignard reagent and purified on a silica gel column. After solvent concentration down to 0.5 mL, 1 μL of the final solution was injected for GC–MS analysis using TPrT as internal standard. A certified reference material, a mussel sample (CRM 477), obtained from BCR was used for validation of the procedures. The analysis of the reference material showed a good performance as TBT results overlapped the certified values ± their uncertainty (the concentration founds for TBT in the two replicates was 2.18 and 2.02 as cations mg kg− 1 respect to a certified value of 2.20 ± 0.19). Moreover, the laboratory (ENEA) has participated in one intercomparison exercises on organotin compounds in mussel tissues (QUASIMEME, rounds 16), which did not reveal any significant deviation with the accepted values.

153

60

40

20 * *

*

0 cont

TBT25

TBT100

EE2

EE2+TBT25 EE2+TBT100

Treatments Fig. 1. Sex ratios of adult zebrafish (120 days pf, n = 60 fish per treatment; TBT 100 n = 30) exposed to 25 and 100 ng TBT/g (TBT 25, TBT 100, respectively), 3.5 ng EE2/L (EE2), the mixture of TBT 25 or TBT 100 with 3.5 ng EE2/L (EE2 + TBT 25, EE2 + TBT 100, respectively), and solvent control (Cont). M—male, F—female, UD—undeveloped gonad. Data are mean ± S.E. (n = 2). *Significantly different from control males (P b 0.01).

of females in comparison with control tanks, while all EE2 exposed groups displayed a higher percentage of females. Animals with undeveloped gonads were observed in all aquaria, with exception of those groups exposed to TBT 25 and TBT 100 only. The EE2 alone exposed group displayed the highest percentage of animals with undeveloped gonads (33%). Concurrent exposure to TBT and EE2 led to a dose-dependent decrease in the percentage of animals with undeveloped gonad in comparison with EE2 only exposed groups. Our control groups also showed a low proportion of individuals (8 animals out of 60) that had undeveloped gonads, although the external features of these animals clearly resembled the adult female phenotype. Mortality was annotated during the exposure period, and it was below 30% in all treatments; no differences were observed in the mortality rate between the control tanks and the other groups (data not shown). This mortality rate is within the normal expected values for a life-cycle test with zebrafish (Hill and David, 2003). The TBT concentration in all body zebrafish tissues ranged from 15 ng TBT/g wet mass for control and 47 ng TBT/g wet mass for the TBT 100 group. TBT was also detected in the diets at 45 ng TBT/g in the control, 90 ng TBT/g in the TBT 25 and 150 ng TBT/g in TBT 100 diet. Levels of EE2 in water were monitored twice during the exposure study; the EE2 dosed aquaria displayed a mean concentration of 3.85 ± 0.95 ng EE2/L, whereas in those aquaria that were not dosed with EE2, the compound was below the analytical detection limit of the technique (b 1 ng EE2/L). 4. Discussion The results of our study demonstrate that the masculinization of zebrafish due to exposure to low doses of TBT can be blocked if the larvae are co-exposed, from 5 days pf to four months of age (which includes the sex differentiation period), to an environmentally relevant concentration of EE2 ( 3.5 ng/L). Based on the integration of our results with those of previous

154

M.M. Santos et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 151–155

studies, it is possible to put forward a hypothetical explanation for the present observations. It is well known that the steroid hormones, testosterone and estradiol, play an important role in fish sex differentiation (Baroiller and D'Cotta, 2001; Strussmann and Nakamura, 2002; Maack and Segner, 2004). In genetically female Chinook salmon (Onchorhynchus tshawytscha) exposure for 2 h to the aromatase inhibitor— fadrozole—during sex differentiation, induces all males phenotype (Piferrer et al., 1994), and similar results were observed when genetic female zebrafish larvae were dietexposed to fadrozole during the sex differentiation period (Uchida et al., 2004); all males phenotype were also observed in zebrafish when exposed to fadrozole between days 35 and 71 pf (Fenske and Segner, 2004). Likewise, fadrozole or 17-α methyltestosterone caused suppression of the P450 aromatase gene expression in the gonads of female Japanese flounder (Paralichthys olivaceus) and induced sex reversal of genetic females into males (Kitano et al., 2000). In a study where TBT was administered through the diet (similar to the present study), Shimasaki et al. (2003) have demonstrated that TBT induces masculinization in genetically female Japanese flounder, and concomitantly leads to the suppression of the expression of the P450 aromatase gene. On the contrary, exposure of several fish species to estrogens, during the sex differentiation period, induces an increase in the proportion of females (Iguchi et al., 2001; Hill and David, 2003); in zebrafish, exposure to 2 ng/L of EE2 during the first 2 months of age induces approximately 85% of females (Örn et al., 2003), and exposure to 3 ng/L from 43 to 71 days pf induces 100% females (Maack and Segner, 2004). Taken together, these studies support a role of testosterone and/or estradiol in fish sex differentiation. Although the mechanism of TBT-induce masculinization in some fish species is still not fully understood, the evidences suggest the involvement of P450 aromatase or the suppression of its gene expression (Shimasaki et al., 2003). In fact, several studies have shown that TBT acts as an aromatase inhibitor (or down-regulates the enzyme synthesis) in human tissues (Saitoh et al., 2001) and molluscs (Curieux-Belfond et al., 2001). As previously suggested, if the balance of testosterone/estradiol or the levels of estrogens itself are essential for the definition of the fish sex, inhibiting the production of estrogens, during sex differentiation, could balance the sex toward males (as seems to be the case in the TBT-exposed fish). However, if those animals are externally administered with estrogens, the decrease in the rate of conversion of androgens to estrogens, due to a decrease in aromatase activity, should not limit the amount of available estrogens. Therefore, fish may be able to show the normal pattern of sex differentiation, or, if the estrogen levels are high enough to induce feminisation, they may develop predominantly as females (as seems to be the case in our study in the TBT plus EE2 exposed groups). This hypothesis clearly needs to be addressed in the near future. Our results on the effects of TBT on sexual development are similar to those reported by McAllister and Kime (2003), when zebrafish larvae were water exposed, from hatch to 70 days, to 1 and 10 ng TBT/L (80% to 90% of the animals developed as males). However, in contrast to the McAllister and Kime (2003)

study, we did not find a dose-dependent effect of TBT; this may be due to the fact that our actual highest TBT dose (150 ng TBT/ g diet) was only twice the lowest dose (90 ng TBT/g diet). Perhaps a 10-fold difference between doses, as in the MacAllister and Kime study, would have produced an effect. Similar to our study, Shimasaki et al. (2003) have exposed Japanese flounder to TBT through diet (100 and 1000 ng TBT/g wet weight), during sex differentiation, but they could not find a dose-dependent effect of TBT, although both doses induced masculinization of genetically females. The percentage of males in our controls is slightly higher than the expected in zebrafish reared under normal laboratory conditions (typically a 1 : 1 ratio) (McAllister and Kime, 2003; Hill and David, 2003). This was probably the reflection of low, but detectable levels of TBT in the control diet that led to some accumulation of TBT in the animal’s tissues. Similar to our study, Shimasaki et al. (2003) also reported some masculinization of control animals of genetically female Japanese flounder that were attributed to low, but detectable levels of TBT in their control diets. Although our results should be interpreted with caution, as it will be important to demonstrate that our observations also apply to other fish species, they may indicate that in the environment fish masculinization due to low levels of TBT can be neutralized if the animals are co-exposed to an environmentally relevant concentration of EE2 (and perhaps other estrogenic compounds). Fenske et al. (2005) have recently investigated the effects of zebrafish exposure to EE2 (3 ng/L) from fertilisation until 118 days pf. After 118 days exposure to EE2 all individuals possessed ovaries, although 52% had immature ovaries. Likewise, in our study, 33% of zebrafish exposed to EE2 showed undeveloped gonads, although our experimental approach does not allow us to confirm if these were immature ovaries. Similarly, it would have been interesting to investigate if the low proportion of animals (6–15%) with undeveloped gonads in the TBT + EE2 exposed tanks had immature ovaries or testis, particularly because in the TBT 25 + EE2 tanks, 7% animals were still able to develop as males. Because of the particular nature of zebrafish gonad development (undifferentiated gonochorists), Fenske et al. (2005) postulated that those animals with immature ovaries after 118 days EE2 exposure were in fact genetic males at the protogynic stage. One of the arguments to support this hypothesis was the fact that 56 days depuration at EE2 free medium, led to 26% of animals with fully differentiated testis in the EE2 exposed group. Based on these results, future more in-depth work should evaluate if the capability of EE2 to rescue TBT masculinization still persists after animals are allowed to depurate in clean water. As our study was planned to investigate if EE2 exposure could counteract the fish masculinizing effects of TBT, a different experimental design had to be performed to investigate if xenoandrogens can neutralise the feminization effects of estrogenic compounds. This is particularly important, because most of the studies dealing with endocrine disruption in aquatic ecosystems have pointed out that fish feminization seems to be the main ecological problem involving endocrine disruptors (Jobling et al., 2003). Therefore, future studies should evaluate the effects of

M.M. Santos et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 151–155

a wide range of concentrations of both compounds to find out if the capacity of EE2 to rescue TBT induced fish masculinization is concentration dependent, and whether xenoandrogens can neutralise the feminization effects of estrogenic compounds. Previous studies with zebrafish (McAllister and Kime, 2003) have shown that TBT not only increased the proportion of males, but also led to sperm abnormalities. Although this was not investigated in the present work, future studies involving concurrent exposure of fish to TBT and xenoestrogens should address this endpoint as it is of great ecological relevance. Additionally, other aspects such as reproduction success should be investigated, as they are essential for the understanding of the population-level impact of mixtures containing androgenic and estrogenic compounds. Acknowledgements Hugo Santos is acknowledged for helping in the maintenance of the aquaria. The Portuguese Foundation for Science and Technology is acknowledged for financial support under the project POCI/MAR/60895/2004. We also acknowledge the comments of an anonymous referee which helped us improve the manuscript. References Albalat, A., Potrykus, J., Pempkowiak, J., Porte, C., 2002. Assessment of organotin pollution along the Polish coast (Baltic Sea) by using mussels and fish as sentinel organisms. Chemosphere 47, 165–171. Andersen, L., Holbech, H., Gessbo, A., Norrgren, L., Petersen, G.I., 2003. Effects of exposure to 17α-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio). Comp. Biochem. Physiol. C 134, 365–374. Angus, R.A., Stanko, J., Jenkins, R.L., Watson, R.D., 2005. Effects of 17αethinylestradiol on sexual development of male western mosquito fish (Gambusia affinis). Comp. Biochem. Physiol. C 140, 330–339. Anon, 2001. International Convention on the Control of Harmful Anti-fouling Systems on Ships. IMO, London. 5 October 2001. Baroiller, J.F., D'Cotta, H., 2001. Environment and sex determination in farmed fish. Comp. Biochem. Physiol. C 130, 399–409. Belfroid, A.C., Van der Horst, A., Vethaak, A.D., Schafer, A.J., Rijs, G.B.J., Wegener, J., Cofino, W.P., 1999. Analysis and occurrence of estrogenic hormones and their glucuronies in surface water and waste water in the Netherlands. Sci. Total Environ. 225, 101–108. Carvalho, A.P., Sá, R., Oliva-Teles, A., Bergot, P., 2004. Solubility and peptide profile affect the utilization of dietary protein by common carp (Cyprinus carpio) during early larval stages. Aquaculture 234, 319–333. Curieux-Belfond, O., Moslemi, S., Mathieu, M., Séralini, G.E., 2001. Androgen metabolism in oyster Crassostrea gigas: evidence for 17β-HSD activities and characterization of an aromatase-like activity by pharmacological compounds and a marine pollutant. J. Steroid Biochem. 78, 359–366. Fenske, M., Segner, H., 2004. Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquat. Toxicol. 67, 105–126. Fenske, M., Maack, G., Schafers, C., Segner, H., 2005. An environmentally relevant concentration of estrogen induces arrest male gonad development in zebrafish, Danio rerio. Environ. Toxicol. Chem. 24, 1088–1098. Fent, K., 1996. Ecotoxicology of organotin compounds. Crit. Rev. Toxicol. 26, 1–117. Hill, R.L., David, J.D.M., 2003. Developmental estrogenic exposure in zebrafish (Danio rerio). I. Effects on sex ratio and breeding success. Aquat. Toxicol. 63, 417–429. Houtman, C.J., Van Oostveen, A.M., Brouwer, A., Lamoree, M.H., Legler, J., 2004. Identification of estrogenic compounds in fish bile using bio-assay directed fractionation. Environ. Sci. Technol. 38, 6415–6423.

155

Iguchi, T., Watanabe, H., Katsu, Y., 2001. Developmental effects of estrogenic agents on mice, fish, and frogs: a mini-review. Horm. Behav. 40, 248–251. Jobling, S., Casey, D., Rodgers-Gray, J., Oehlmann, J., Schulte-Oehlmann, U., Pawlowski, S., Baunbeck, T., Turner, A.P., Tyler, C.R., 2003. Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent. Aquat. Toxicol. 65, 205–220. Kinnberg, K., Korsgaard, B., Bjerregaard, P., 2003. Effects of octylphenol and 17β-estradiol on the gonads of guppies (Poecilia reticulata) exposed as adults via the water or as embryos via the mother. Comp. Biochem. Physiol. C 134, 45–55. Kitano, T., Takamune, K., Nagahama, Y., Abe, S.I., 2000. Aromatase inhbitor and 17α-methyltestosterone cause sex-reversal from genetical females to phenotypic males and suppression of P450 aromatase gene expression in Japanese flounder (Paralichthys olivaceus). Mol. Reprod. Dev. 56, 1–5. Maack, G., Segner, H., 2004. Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure. Comp. Biochem. Physiol. C 139, 47–55. Matthiessen, P., Gibbs, P., 1998. Critical appraisal of evidence for tributyltinmediated endocrine disruption in molluscs. Environ. Toxicol. Chem. 17, 37–43. McAllister, B.G., Kime, D.E., 2003. Early life exposure to environmental levels of the aromatase inhibitor tributyltin causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio). Aquat. Toxicol. 65, 309–316. Morabito, R., Chiavarini, S., Cremisini, C., 1995. GC–MS for the speciation of organotin compounds in environmental samples. In: Quevauviller, Ph., Maier, E.A., Griepink, B. (Eds.), Quality Assurance for Environmental Analysis. Elsevier Science, Amsterdam, pp. 435–464. Örn, S., Holbech, H., Madsen, T.H., Norrgren, L., Petersen, G.I., 2003. Gonad development and vitellogenin production in zebrafish (Danio rerio) exposed to ethinylestradiol and methyltestosterone. Aquat. Toxicol. 65, 397–411. Piferrer, F., Zanuy, S., Carillo, M., Solar, I., Devlin, R.H., Donaldson, E.M., 1994. Brief treatment with aromatase inhibitor during sex differentiation causes chromosomally female salmon to develop as normal functioning males. J. Exp. Zool. 270, 255–262. Rasmussen, T.H., Korsgaard, B., 2004. Estrogenic octylphenol affects seminal fluid production and its biochemical composition of eelpout (Zoarces viviparus). Comp. Biochem. Physiol. C 139, 1–10. Saitoh, M., Yanase, T., Morinaga, H., Tanabe, M., Um, Y.M., Nishi, Y., Nomura, M., Okabe, T., Goto, K., Takayanagi, R., Nawata, H., 2001. Tributyltin or triphenyltin inhibits aromatase activity in the human granulosa-like tumor cell line KGN. Biochem. Biophys. Res. Commun. 289, 198–204. Santos, M.M., Ten Hallers-Tjabbes, C.C., Santos, A.M., Vieira, N., 2002. Imposex in Nucella lapillus, a bioindicator for TBT contamination: resurvey along the Portuguese coast to monitor the effectiveness of EU regulation. J. Sea Res. 48, 217–223. Santos, M.M., Castro, L.F.C., Vieira, M.N., Micael, J., Morabito, R., Massanisso, P., Reis-Henriques, M.A., 2005. New insights into the mechanism of imposex induction in the dogwhelk Nucella lapillus. Comp. Biochem. Physiol. C 141, 101–109. Shimasaki, Y., Kitano, T., Oshima, Y., Inoue, S., Imada, N., Honjo, T., 2003. Tributyltin causes masculinization in fish. Environ. Toxicol. Chem. 22, 141–144. Solé, M., Raldua, D., Piferrer, F., Barceló, D., Porte, C., 2003. Feminization of wild carp, Cyprinus carpio, in a polluted environment: plasma steroid hormones, gonadal morphology and xenobiotic metabolizing system. Comp. Biochem. Physiol. C 136, 145–156. Strussmann, C.A., Nakamura, M., 2002. Morphology, endocrinology, and environmental modulation of gonadal sex differentiation in teleost fishes. Fish Physiol. Biochem. 26, 13–29. Tollefsen, K.-E., Øvrevik, J., Stenersen, J., 2004. Binding of xenoestrogens to the sex steroid-binding protein in plasma from Arctic charr (Salvelinus alpinus L.). Comp. Biochem. Physiol. C 139, 127–133. Uchida, D., Yamashita, M., Kitano, T., Iguchi, T., 2004. An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sexreversal. Comp. Biochem. Physiol. A 137, 11–20.