ToxSci Advance Access originally published online on May 16, 2006
Toxicological Sciences 2006 92(2):537-544; doi:10.1093/toxsci/kfl015
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Effects of 4-n-Nonylphenol and Tamoxifen on Salmon Gonadotropin-Releasing Hormone, Estrogen Receptor, and Vitellogenin Gene Expression in Juvenile Rainbow Trout
* MRC Toxicology Unit, University of Leicester, Leicester LE1 9HN, United Kingdom; and Endocrinologie Moléculaire de la Reproduction, UMR-CNRS 6026, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, France
1 To whom correspondence should be addressed at MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester LE1 9HN, United Kingdom. Fax: +33 561 17 5871. E-mail: angelique.vetillard{at}ipbs.fr.
Received March 12, 2006; accepted May 8, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Alkylphenols such as nonylphenol (NP) are one of a wide variety of environmental chemicals reported to have estrogenic effects in both in vitro and in vivo studies. Induction of hepatic vitellogenin (Vg) gene expression is widely used as a biomarker for xenoestrogen exposure in fish. However, little work has been done to characterize the molecular effects of xenoestrogens on other potential target organs such as the brain. To evaluate brain and liver effects of 4-n-nonylphenol (4-NP), juvenile rainbow trout (Oncorhynchus mykiss) were exposed to waterborne 4-NP or 17ß-estradiol (E2). Changes in mRNA levels of salmon gonadotropin-releasing hormone (sGnRH) and estrogen receptor
(ER) isoforms in the brain and ER isoforms and Vg in the liver were measured after 3 and 6 days of exposure, with the help of a relative RT-PCR–based quantification method. Fish were treated with increasing doses of 4-NP ranging from 0.01 to 10µM (2.2 µg/l to 2.2 mg/l), and results were compared to the effect of E2 or tamoxifen, a specific ER modulator. In liver, E2 and the highest doses of 4-NP increased Vg and ER long-isoform mRNA levels within 3 or 6 days of exposure, but 4-NP did not have any effect on ER short-isoform transcription level. In the brain, 4-NP reduced sGnRH2 gene expression in a dose-dependent manner, but did not modify sGnRH1 or ER mRNA levels. Key Words: alkylphenol; endocrine disrupter; estrogen receptor; gonadotropin-releasing hormone; Oncorhynchus mykiss.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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During the last decade, abnormalities in the male reproductive systems of fish, reptiles, and mammals have been observed. It has been suggested that perturbations in our chemical and physiological environment may be responsible for the increasing incidence of reproductive abnormalities (Toppari et al., 1996
). Different groups of chemicals including the persistent degradation products of alkylphenol polyethoxylates (APEOs) have estrogenic activities (Nimrod and Benson, 1996).
APEOs are nonionic surfactants used primarily as industrial or agricultural cleaners or as detergents and emulsifiers for many products. The degradation of APEOs during anaerobic treatment of wastewater by activated sludge leads to the formation of alkylphenol mono- or diethoxylates which are further degraded to alkylphenols (Giger et al., 1984). After sewage treatment, alkylphenols end up in the aquatic environment. Nonylphenol (NP) polyethoxylates are the most common APEOs, constituting about 80% of the production. Thus NP and octylphenol (OP) are the major metabolites of APEOs, which have been identified as aquatic contaminants. In rainbow trout waterborne or dietary exposed to labeled NP, the radioactivity is rapidly taken up into most tissues and is widely distributed through the body including the edible tissues (Lewis and Lech, 1996; Thibaut et al., 1998; Uguz et al., 2003).
A number of compounds, such as OP, NP, or NP diethoxylates, appear to possess intrinsic estrogenic activity (White et al., 1994). In rainbow trout, NP produces estrogenic effects at concentrations significantly lower than the lethal concentration (Lech et al., 1996). The effective estrogenicity of NP reported in the literature is rather confusing because studies have been carried out with different isomers and different purities of the molecule. In a comparative study (Pedersen et al., 1999), a mixture of branched and linear NPs could stimulate vitellogenin (Vg) synthesis in rainbow trout (Oncorhynchus mykiss) while the linear form, 4-n-nonylphenol (4-NP), was inefficient alone. However, in male rainbow trout, both NP and 4-NP were found to be estrogenic in vivo on the basis of the induction of Vg synthesis (Andersen et al., 1999; Jobling et al., 1996; Xie et al., 2005). In vitro studies, using cultured hepatocytes of male rainbow trout, have shown that 4-NP is able to induce the expression of Vg and rainbow trout estrogen receptor (rtER) gene, both involved in reproductive functions (Flouriot et al., 1995; Jobling and Sumpter, 1993). Moreover, in recombinant yeast that constitutively express rtER, 4-NP has been shown to have an estrogenic activity on synthetic promoters carrying an estrogenic, responsive gene (Andersen et al., 1999; Petit et al., 1997).
These effects of 4-NP are likely to be mediated by a direct interaction with estrogen receptor (ER) since it has been shown that alkylphenols such as 4-NP or OP can compete with 17ß-estradiol (E2) for the binding to rtER (Tollefsen et al., 2002). However, these xenobiotics have affinities for rtER that are about 1000- to 3000-fold lower than the natural ligand E2 (Flouriot et al., 1995; White et al., 1994).
The aim of this study was to investigate the acute effects of waterborne 4-NP on rtER and salmon gonadotropin-releasing hormone (sGnRH) mRNA levels in the brain of rainbow trout and to evaluate its impact on the expression of the short and long isoforms of rtER that have been recently characterized in rainbow trout liver (Pakdel et al., 2000). The effect of 4-NP has been compared with those of E2 and tamoxifen, a specific ER modulator. This should further improve our understanding on the wide range of effects of 4-NP on the reproductive function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fish
Rainbow trout (O. mykiss) were purchased from an experimental fish farm (SEMI, Le Drennec, France) and kept in 1000-l tanks in aerated recirculated freshwater at 12–13°C, under a natural photoperiod (SCRIBE INRA, Rennes, France). The study was conducted on 4-month postfertilization female fry (5–10 g) obtained from female monosex strains as a result of the fertilization of normal female (XX) oocytes with neomale (XX) semen.
Experimental Procedure
Experiments were carried out in accordance with the Guidelines of the European Union Council (86/609/EU) and with the French regulations (decret 87/848).
Fry were exposed to waterborne E2 (Steraloid, Wilton, NH) or 4-NP (PESTANAL; 99.9% linear form; LGC Promochem, Molsheim, France) in a 5-l aquarium. During exposure, fish were continuously maintained in their aquarium in aerated water cooled to 12°C. Substances were added at each water renewal (24 h).
NP treatments.
A time- and dose-effect study was performed. Increasing amounts of 4-NP were mixed to the water of the aquarium, in order to achieve a range of concentration from 0.01 to 10µM (2.2 µg/l to 2.2 mg/l). Fish were analyzed after 3 or 6 days of exposure. In parallel, a group of fish was exposed to E2 0.01µM (2.7 µg/l), a dose that was effective to induce Vg and rtER gene expression after 2–3 days in a previous experiment (Vetillard and Bailhache, 2005). Two different durations of exposure have been used in order to quantify the induction of rtER and Vg gene expression by E2, which is maximum after 3 and 6 days, respectively (Pakdel et al., 1989). Before the treatments, E2 and 4-NP were diluted in ethanol in order to distribute the same volume in each aquarium (1 ml for 5 l of freshwater). Control group aquariums received only 1 ml of ethanol.
Tamoxifen treatments.
Juvenile trout received an ip injection of tamoxifen (1 or 10 µg/g BW) or saline (0.9% NaCl, 10% ethanol) before they were exposed to E2 (0.01µM) via aquarium water during 3 days.
For both experiments, at the end of exposure, fish were sacrificed under deep anesthesia with 2-phenoxyethanol (0.4% in aquarium water; Merck, Darmstadt, Germany) and liver and brain dissected. Body and liver were weighed for hepatosomatic index (HSI) calculation. Dissected liver and brain were then stored at – 80°C for subsequent RNA extraction.
RNA Extraction
Total RNA was extracted from individual liver or brain with 1 ml TRIzol (GIBCO BRL-Invitrogen, Cergy Pontoise, France) as described by the manufacturer (n = 8 per treatment).
The quality of total RNA was checked by electrophoresis on agarose gel stained with ethidium bromide and quantification performed with a spectrophotometer at 260 nm. Samples that showed a partial degradation or a low extraction yield were discarded. Five samples per treatment were used for RT-PCR analysis.
RT-PCR Analysis
Changes in each mRNA species were quantified using a relative RT-PCR technique that allowed the semiquantitative determination of their abundance corrected by histone H3 (Connor et al., 1984) gene expression. The relative RT-PCR protocol and validation have been previously described for Vg, rtER (full-length rtER isoform [rtERL] and short rtER isoform [rtERS]), sGnRH1, and sGnRH2 mRNAs (Uzbekova et al., 2000; Vetillard and Bailhache, 2005).
Total RNA (1 µg) was reverse transcribed for 1 h at 42°C with random hexanucleotide primers and 50 units of EXPAND reverse transcriptase (GIBCO BRL-Invitrogen) in a total volume of 20 µl.
Two microliters of single-strand cDNA were then amplified using 2.5 units of Taq DNA polymerase (PE Applied Biosystem, Courtaboeuf, France) in a total volume of 50 µl PCR buffer containing 1.5mM MgCl2, 200µM dNTPs, and 500nM specific primers. PCRs were performed in a Genamp PCR 9700 (PE Applied Biosystem), after a 5-min denaturing step at 94°C; 24–32 cycles were performed (Table 1). Each cycle consisted of 30 s of denaturation at 94°C, 30 s of annealing (see Table 1 for temperature), and 30 s of extension at 72°C. The last cycle was followed by a final 7-min extension at 72°C. Each PCR mixture was subjected to electrophoresis in 2% (wt/vol) agarose gel stained with ethidium bromide and photographed under UV illumination with an image analysis system (Gel-Doc1000, Bio-Rad, Marnes la coquette, France). The fluorescence intensity of the band, which is related to the concentration of DNA in the gel, was determined using the Molecular Analyst/PC software (Bio-Rad). For rtERS, both bands (164 and 204 bp) have been quantified together since they come from an alternative splicing at the acceptor splice site of exon 2 (Pakdel et al., 2000). The primers have been designed to span intron 1 so that the PCR generates two amplicons; one having an insertion of 40 bases compared with the other.
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To obtain meaningful results, the relative RT-PCR products have to be quantified when the reaction is in the exponential phase of amplification. The number of cycles that produces quantifiable signals was verified experimentally (Fig. 1), and Table 1 summarizes the PCR parameters used in our study.
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In all experiments, amounts of histone H3 mRNA were not modified by treatments (Figs. 2A, 3A, and 4A). Therefore, results are expressed as the ratio of the optical density of amplified mRNA (rtER
L, rtERS, Vg, sGnRH1, and sGnRH2) to that of histone H3, for each sample.
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Statistical Analyses
Data are expressed as means ± SEMs. Results were analyzed using a two-way ANOVA for 4-NP treatments and a one-way ANOVA for tamoxifen treatments followed by the Dunnett post hoc test when appropriate. In all the statistical tests, a difference was considered significant when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Effects of 4-NP on rtER
and Vg Genes Expression in LiverAfter 3 or 6 days of exposure to E2 or 4-NP, no change could be detected in liver weight or HSI (data not shown).
Exposure to 4-NP or E2 significantly enhanced rtERL mRNA contents in liver after 3 or 6 days (Fig. 2C, F = 4.68, p 
0.002). When fish were exposed during 3 days, the higher dose of 4-NP (10µM) was efficient, whereas a 6-day treatment further enhanced liver sensitivity since rtERL mRNA levels were significantly increased after a treatment with 1 and 10µM of 4-NP. The levels of rtERL mRNA in these 4-NP–treated groups reached a value similar to those of the E2-treated groups.
No change could be observed for rtERS mRNA levels after 3 or 6 days of exposure to 4-NP (Fig. 2D), whereas E2 treatments induced a significant increase in rtERS mRNA levels (F = 5.8, p 0.001).
Vg gene expression was enhanced by E2 and 4-NP treatments (Fig. 2B) for both exposure durations (F = 23.19, p 0.001). In a way similar to that for rtERL mRNA, only high doses of 4-NP were able to significantly enhance Vg mRNA contents. After 3 days of exposure, 10µM is efficient, whereas after 6 days, 1 and 10µM were equally efficient. However, the amounts of Vg mRNA in liver after the 4-NP treatments were 5- to 10-fold lower than those in the E2-treated groups.
Effects of Tamoxifen on rtER and Vg Genes Expression in Liver
When fish were injected with tamoxifen alone, the highest dose (10 µg/g) enhanced Vg mRNA levels (Fig. 3B). Treatments with E2 alone enhanced Vg and both isoforms of rtER mRNA levels in the liver. These mRNA levels were clearly reduced by injections of tamoxifen together with E2 exposure. The lower dose of tamoxifen (1 µg/g) was the most efficient since the E2-stimulated rtER gene expression is totally abolished by the injection (Figs. 3C and 3D), and Vg mRNA levels were significantly reduced (Fig. 3B). In each case, 10 µg/g tamoxifen failed to induce a significant inhibition compare to E2 exposure alone.
Effect of 4-NP and Tamoxifen on rtER and sGnRH Genes Expression in Brain
None of the treatments with E2 or 4-NP induced any change in rtERL and rtERS mRNA levels in the brain after 3 or 6 days of exposure (data not shown). In a similar way, tamoxifen alone or in combination with E2 did not induce any change in rtERL and rtERS mRNA levels in the brain (data not shown).
No change could be detected in sGnRH1 gene expression in the brain after 3 or 6 days of exposure to E2 or 4-NP (Fig. 4B) or after tamoxifen treatments (data not shown).
sGnRH2 gene expression was inhibited by 4-NP treatments (F = 3.72, p 0.006). After 3 days, only a high dose of 4-NP (10µM) was able to significantly reduce sGnRH2 mRNA levels (Fig. 4C), but after 6 days of exposure, a significant decrease was observed for treatments with 0.1µM of 4-NP or higher. Neither did the E2 treatments induce any change in sGnRH2 gene expression (Fig. 4C) nor did the tamoxifen treatments (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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APEOs and their degradation products such as alkylphenols are reported to have estrogenic effects (Nimrod and Benson, 1996
). The estrogenic effects of 4-NP and other xenoestrogens have been assessed using assays for plasma Vg and zona radiata proteins or their hepatic mRNA synthesis in juvenile and/or male fish (Arukwe et al., 1997; Jobling and Sumpter, 1993). More recently, it has been shown that 4-NP also mimics E2 in inducing the synthesis of hepatic ER in Atlantic salmon (Yadetie et al., 1999). Several in vitro studies have shown that 4-NP binds to ER and activates the transcription of E2-responsive genes (Celius et al., 1999; Flouriot et al., 1995; White et al., 1994). Thus, by mimicking E2 at the molecular level, xenoestrogens such as 4-NP could modulate gene expression in other potential target organs of the reproductive axis such as the brain.
E2 is one of the essential sex steroids in fish and other vertebrates and is involved in many physiological functions. In this study, we aimed to examine if 4-NP which has been shown to have an estrogenic potency in several assays could also mimic E2 in fish brain and affect gene transcriptional activity. In addition, we have reevaluated its estrogenic effect on hepatic function not only to provide evidence for treatment efficiency but also to evaluate its impact on the expression of the two recently cloned rtER isoforms (Pakdel et al., 2000). Functional analysis of both rtER isoforms revealed that rtERS consistently exhibited a basal (E2 independent) transactivation activity that could be further increased in the presence of E2, when rtERL is characterized by a strict E2-dependent transcription activity.
In the liver, our results show a strong stimulatory effect of E2 on Vg, rtERL, and rtERS expression, confirming previous data (Menuet et al., 2001). Indeed, in the liver of rainbow trout, it is well established that rtER and Vg gene expression is upregulated by E2 as a result of an increase in transcriptional activity and mRNA stabilization (Flouriot et al., 1996). These two mechanisms were likely to be mediated by rtER itself, since several antagonists were able to reduce the effects of E2.
As far as 4-NP is concerned, we observed a significant stimulating effect on Vg and rtER long-isoform expression with 1 and 10µM after 6 days of exposure. When 4-NP was exposed for 3 days, only the latter was effective. Effects observed in our study agree with those in previous reports where 4-NP directly mimics E2 in inducing ER and Vg expression (Flouriot et al., 1995; Van den Belt et al., 2003; Yadetie et al., 1999; Yadetie and Male, 2002). The latter study (Yadetie and Male, 2002) showed that the ER mRNA synthesis is induced by 4-NP in a dose-dependent manner in juvenile Atlantic salmon liver and that the induction of ER mRNA synthesis is followed by induction of Vg mRNA synthesis. Moreover, the effect of 4-NP is likely to be mediated by a direct interaction with ER since it has been shown that alkylphenols such as NP compete with E2 for binding to ER (Danzo, 1997). However, since 4-NP has an affinity for the rtER that is about 1000-fold lower than that of the natural ligand E2 (Olsen et al., 2005), we could suggest that a longer exposure time could be necessary to observe a higher Vg gene expression stimulation. We observed a discrepancy in Vg gene induction when comparing 4-NP and E2, which has also been reported in similar studies (Arukwe et al., 1997; Van den Belt et al., 2003). In a comparative study (Andersen et al., 1999), 4-NP seems to be less efficient than E2 in inducing Vg, but seems as potent as E2 on synthetic promoter carrying an estrogen-responsive element. We also observed different efficiencies according to the gene, i.e., Vg versus rtER, or the promoter context, i.e., rtERS versus rtERL. This lesser efficiency of 4-NP to induce transcription from Vg genes compared to E2 is probably due to its weaker affinity for rtER and also due to a different conformation of the complex 4-NP–rtER which is not as efficient as the E2-rtER complex to induce gene transcription (Madigou et al., 2001).
In our experiments, 4-NP did not increase rtERS isoform expression, in contrast to E2. This could be the result of the conformation/transactivation properties of the 4-NP–rtER complex, associated to different promoter contexts for rtERS and rtERL. Such a difference in the transactivation capacity of 4-NP–rtER and E2-rtER complexes has been shown in vitro using recombinant yeast cells that contains various promoter/reporter plasmids, including the rtERS promoter (Madigou et al., 2001). Since 4-NP did not enhance rtERS mRNA levels, this must have produced a lower amount of rtER protein, which would result in the weaker effect of 4-NP on Vg gene expression compared to E2.
As previously shown in rainbow trout hepatocytes (Flouriot et al., 1996), tamoxifen has a partial E2-antagonistic activity on Vg and rtER genes expression, but it also has an agonistic effect on its own at a high dose (10 µg/g) on Vg gene expression. This agonistic activity could explain the nonsignificant inhibition of tamoxifen when a high dose is injected before E2 exposure.
Organ distribution studies in Atlantic salmon using radioactive 4-NP suggest that this compound can cross the blood-brain barrier (Arukwe et al., 2000). Thus, 4-NP is able to play a role at the brain level either by an estrogeno-mimetic activity or by another pathway.
In the brain, we did not demonstrate any effect of E2 or 4-NP on rtER mRNA levels. Our previous data (Vetillard et al., 2003; Vetillard and Bailhache, 2005) have shown that the rtER gene is not upregulated by E2 in the brain of juvenile or adult female rainbow trout. This is in agreement with a study performed in smolting sockeye salmon (Luo et al., 2005) in which E2 and NP did not modify the amount of ER in the brain. However, in Atlantic salmon, NP has been shown to stimulate ER in the brain (Meucci and Arukwe, 2005). The discrepancy observed in these studies could reflect a species-specific sensitivity to NP but could also result from the impurities of the NP mixture used by these authors compared to the 4-NP used in our study.
The present study also provides additional evidence that brain rtER gene expression is not regulated by E2 in the brain of rainbow trout. Tamoxifen injected alone or in combination with waterborne E2 did not further modify rtER gene expression in the brain, which tends to reinforce this idea. It is well documented in rat that E2 downregulates ER in the hypothalamus and midbrain, but tamoxifen fails to have any effect on ER mRNA levels (Zhou et al., 2002). Indeed, tamoxifen has been reported to have a tissue-specific agonistic or antagonistic activity in mammals (Diel, 2002), a phenomenon that is commonly observed for ER modulators (Miller, 2002). In our study, we analyzed rtER gene expression in the whole brain, and one could suggest that rtER gene could be differentially regulated in different brain structures as reported in the rat (Zhou et al., 2002). Experiments that differentiate brain structures (i.e., hypothalamus, preoptic area, and telencephalon) should answer this question.
The decapeptide sGnRH is issued from the expression of two different genes, sGnRH1 and sGnRH2 (Ferriere et al., 2001). The specific role of each of these genes is not yet known, but they show a differential regulation by sexual steroids (Vetillard et al., 2006). No effect of E2 and/or tamoxifen on sGnRH1 or sGnRH2 gene expression could be observed in our study. This confirms our previous finding in juvenile rainbow trout (Vetillard and Bailhache, 2005) where E2 treatments had no effects on sGnRH gene expression. The duration of the treatments seem to be important in trout since in 1-year-old triploid trout, long-term implants with E2 resulted in an increase in sGnRH peptide contents in the brain (Breton and Sambroni, 1996), whereas a 3-day treatment with E2 did not modify sGnRH gene expression (Vetillard et al., 2006).
Our study also reports a decrease in sGnRH2 gene expression under 4-NP treatment. Since waterborne E2 and/or tamoxifen treatments did not affect sGnRH gene expression in juvenile trout, we can suggest that the observed inhibition could be mediated by a nonestrogenic or by an indirect mechanism. Despite rtER being expressed in the brain of trout, it has never been found in sGnRH neurons (Navas et al., 1995). If the effect of 4-NP uses rtER as a pathway, this would be via an indirect mechanism that involves neuronal or glial neighbor cells to transduce the estrogenic signal as described in mammals (Buchanan et al., 2000). We demonstrated in a previous study (Vetillard et al., 2006) that sGnRH2 gene expression is controlled by an androgenic inhibitory feedback. Indeed, testosterone as well as 5-dihydrotestosterone strongly decreased sGnRH2 gene expression in immature and adult rainbow trout brain. An androgenic effect of 4-NP is unlikely to be mediated by a direct binding on androgen receptors since NP does not compete with DHT in binding studies (Danzo, 1997), but this androgeno-mimetic activity could be mediated by indirect mechanisms.
NP has been shown to modify the expression of steroidogenic enzymes in the brain of Atlantic salmon (Arukwe, 2005). This could be an indirect way to regulate the expression of steroid-sensitive genes such as sGnRH2. In female Daphnia magna, it has been shown that 4-NP leads to an inhibition of the excretion of glucose-conjugated testosterone and consequently elevated testosterone levels (Baldwin et al., 1997). 4-NP treatments in rats also modify hepatic testosterone metabolism by a reduction of 16alpha-OH-, 2alpha-OH-testosterone metabolites (Laurenzana et al., 2002). A similar mechanism could occur in trout since NP has been shown to inhibit hepatic biotransformation enzymes, which resulted in a significant inhibition of the 6beta- and 16beta-hydroxylation of testosterone (Sturm et al., 2001). However, waterborne 4-NP has been shown to inhibit steroidogenesis in female rainbow trout (Harris et al., 2001), which could lead to a decrease in testosterone plasma levels. In the absence of precise data on androgen plasma levels after 4-NP exposure, the mechanism by which 4-NP is able to reduce sGnRH2 gene expression still remains unknown. A study using various steroid antagonists together with 4-NP would answer the question whether this effect uses an estrogenic, an androgenic, or another pathway.
Juvenile salmonid fish are commonly used as model organisms to study in vivo effects of xenoestrogens. In the present study, we have demonstrated that 4-NP stimulates rtERL and Vg gene expression but does not modify rtERS mRNA levels in the liver. To understand the molecular mechanisms underlying this effect, it would be interesting to perform a promoter study. This would indicate if the lack of stimulation of rtERS expression is the result of a lower sensitivity of this promoter or if it involves another level of regulation linked to the differential splicing of exon 2. By showing that 4-NP clearly decreases sGnRH2 mRNA levels in rainbow trout, we have pointed out another level of potential endocrine disruption. Since GnRH is a key factor for reproductive function in all vertebrates including mammals, a decrease in GnRH neuron activity could lead to a major disturbance of reproductive activity. There is a need to include the central nervous system in the potential targets for endocrine-disruptive compounds because studies focusing exclusively on the peripheral effects might indeed underestimate the potential risk of these compounds on the populations.
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ACKNOWLEDGMENTS |
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This work was supported by Centre National de la Recherche Scientifique and Institut National de la Recherche Agronomique. A.V. was supported by a grant from Conseil Régional de Bretagne. We are grateful to D. Read for proofreading the manuscript.
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REFERENCES |
|---|
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Andersen, H. R., Andersson, A. M., Arnold, S. F., Autrup, H., Barfoed, M., Beresford, N. A., Bjerregaard, P., Christiansen, L. B., Gissel, B., Hummel, R., et al. (1999). Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals. Environ. Health Perspect. 107(Suppl. 1), 89–108.[CrossRef]
Arukwe, A. (2005). Modulation of brain steroidogenesis by affecting transcriptional changes of steroidogenic acute regulatory (StAR) protein and cholesterol side chain cleavage (P450scc) in juvenile Atlantic salmon (Salmo salar) is a novel aspect of nonylphenol toxicity. Environ. Sci. Technol. 39, 9791–9798.[Medline]
Arukwe, A., Knudsen, F. R., and Goksoyr, A. (1997). Fish zona radiata (eggshell) protein: A sensitive biomarker for environmental estrogens. Environ. Health Perspect. 105, 418–422.[Web of Science][Medline]
Arukwe, A., Thibaut, R., Ingebrigtsen, K., Celius, T., Goksoyr, A., and Cravedi, J. P. (2000). In vivo and in vitro metabolism and organ distribution of nonylphenol in Atlantic salmon (Salmo salar). Aquat. Toxicol. 49, 289–304.[CrossRef][Web of Science][Medline]
Baldwin, W. S., Graham, S. E., Shea, D., and LeBlanc, G. A. (1997). Metabolic androgenization of female Daphnia magna by the xenoestrogen 4-nonylphenol. Environ. Toxicol. Chem. 16, 1905–1911.[CrossRef]
Breton, B., and Sambroni, E. (1996). Steroid activation of the brain-pituitary complex gonadotropic function in the triploid rainbow trout Oncorhynchus mykiss. Gen. Comp. Endocrinol. 101, 155–164.[CrossRef][Web of Science][Medline]
Buchanan, C. D., Mahesh, V. B., and Brann, D. W. (2000). Estrogen-astrocyte-luteinizing hormone-releasing hormone signaling: A role for transforming growth factor-beta(1). Biol. Reprod. 62, 1710–1721.
Celius, T., Haugen, T. B., Grotmol, T., and Walther, B. T. (1999). A sensitive zonagenetic assay for rapid in vitro assessment of estrogenic potency of xenobiotics and mycotoxins. Environ. Health Perspect. 107, 63–68.[CrossRef][Web of Science][Medline]
Connor, W., Mezquita, J., Winkfein, R. J., States, J. C., and Dixon, G. H. (1984). Organization of the histone genes in the rainbow trout (Salmo gairdnerii). J. Mol. Evol. 20, 227–235.[CrossRef][Medline]
Danzo, B. J. (1997). Environmental xenobiotics may disrupt normal endocrine function by interfering with the binding of physiological ligands to steroid receptors and binding proteins. Environ. Health Perspect. 105, 294–301.[Web of Science][Medline]
Diel, P. (2002). Tissue-specific estrogenic response and molecular mechanisms. Toxicol. Lett. 127, 217–224.[CrossRef][Web of Science][Medline]
Ferriere, F., Uzbekova, S., Breton, B., Jego, P., and Bailhache, T. (2001). Two different messenger RNAs for salmon gonadotropin-releasing hormone are expressed in rainbow trout (Oncorhynchus mykiss) brain. Gen. Comp. Endocrinol. 124, 321–332.[CrossRef][Web of Science][Medline]
Flouriot, G., Pakdel, F., Ducouret, B., and Valotaire, Y. (1995). Influence of xenobiotics on rainbow trout liver estrogen receptor and vitellogenin gene expression. J. Mol. Endocrinol. 15, 143–151.
Flouriot, G., Pakdel, F., and Valotaire, Y. (1996). Transcriptional and post-transcriptional regulation of rainbow trout estrogen receptor and vitellogenin gene expression. Mol. Cell. Endocrinol. 124, 173–183.[CrossRef][Web of Science][Medline]
Giger, W., Brunner, P. H., and Schaffner, C. (1984). 4-Nonylphenol in sewage sludge: Accumulation of toxic metabolites from nonionic surfactants. Science 225, 623–625.
Harris, C. A., Santos, E. M., Janbakhsh, A., Pottinger, T. G., Tyler, C. R., and Sumpter, J. P. (2001). Nonylphenol affects gonadotropin levels in the pituitary gland and plasma of female rainbow trout. Environ. Sci. Technol. 35, 2909–2916.[Medline]
Jobling, S., Sheahan, D., Osborne, J. A., Matthiessen, P., and Sumpter, J. P. (1996). Inhibition of testicular growth in rainbow trout (Oncorhynchus mykiss) exposed to estrogenic alkylphenolic chemicals. Environ. Toxicol. Chem. 15, 194–202.[CrossRef]
Jobling, S., and Sumpter, J. P. (1993). Detergent components in sewage effluent are weakly estrogenic to fish—An in-vitro study using rainbow-trout (Oncorhynchus mykiss) hepatocytes. Aquat. Toxicol. 27, 361–372.[CrossRef]
Laurenzana, E. M., Weis, C. C., Bryant, C. W., Newbold, R., and Delclos, K. B. (2002). Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression. Food Chem. Toxicol. 40, 53–63.[CrossRef][Medline]
Lech, J. J., Lewis, S. K., and Ren, L. F. (1996). In vivo estrogenic activity of nonylphenol in rainbow trout. Fundam. Appl. Toxicol. 30, 229–232.[CrossRef][Web of Science][Medline]
Leguellec, K., Lawless, K., Valotaire, Y., Kress, M., and Tenniswood, M. (1988). Vitellogenin gene-expression in male rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 71, 359–371.[CrossRef][Medline]
Lewis, S. K., and Lech, J. J. (1996). Uptake, disposition, and persistence of nonylphenol from water in rainbow trout (Oncorhynchus mykiss). Xenobiotica 26, 813–819.[Medline]
Luo, Q., Ban, M., Ando, H., Kitahashi, T., Kumar Bhandari, R., McCormick, S. D., and Urano, A. (2005). Distinct effects of 4-nonylphenol and estrogen-17 beta on expression of estrogen receptor alpha gene in smolting sockeye salmon. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 140, 123–130.[Medline]
Madigou, T., Le Goff, P., Salbert, G., Cravedi, J. P., Segner, H., Pakdel, F., and Valotaire, Y. (2001). Effects of nonylphenol on estrogen receptor conformation, transcriptional activity and sexual reversion in rainbow trout (Oncorhynchus mykiss). Aquat. Toxicol. 53, 173–186.[Medline]
Menuet, A., Anglade, I., Flouriot, G., Pakdel, F., and Kah, O. (2001). Tissue-specific expression of two structurally different estrogen receptor alpha isoforms along the female reproductive axis of an oviparous species, the rainbow trout. Biol. Reprod. 65, 1548–1557.
Meucci, V., and Arukwe, A. (2005). Transcriptional modulation of brain and hepatic estrogen receptor and P450arom isotypes in juvenile Atlantic salmon (Salmo salar) after waterborne exposure to the xenoestrogen, 4-nonylphenol. Aquat. Toxicol. 77, 167–177.[CrossRef]
Miller, C. P. (2002). SERMs: Evolutionary chemistry, revolutionary biology. Curr. Pharm. Des. 8, 2089–2111.[CrossRef][Medline]
Navas, J. M., Anglade, I., Bailhache, T., Pakdel, F., Breton, B., Jego, P., and Kah, O. (1995). Do gonadotrophin-releasing hormone neurons express estrogen receptors in the rainbow trout? A double immunohistochemical study. J. Comp. Neurol. 363, 461–474.[CrossRef][Medline]
Nimrod, A. C., and Benson, W. H. (1996). Environmental estrogenic effects of alkylphenol ethoxylates. Crit. Rev. Toxicol. 26, 335–364.[Web of Science][Medline]
Olsen, C. M., Meussen-Elholm, E. T., Hongslo, J. K., Stenersen, J., and Tollefsen, K. E. (2005). Estrogenic effects of environmental chemicals: An interspecies comparison. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 141, 267–274.[Medline]
Pakdel, F., Le Guellec, C., Vaillant, C., Le Roux, M. G., and Valotaire, Y. (1989). Identification and estrogen induction of two estrogen receptors (ER) messenger ribonucleic acids in the rainbow trout liver: Sequence homology with other ERs. Mol. Endocrinol. 3, 44–51.
Pakdel, F., Metivier, R., Flouriot, G., and Valotaire, Y. (2000). Two estrogen receptor (ER) isoforms with different estrogen dependencies are generated from the trout ER gene. Endocrinology 141, 571–580.
Pedersen, S. N., Christiansen, L. B., Pedersen, K. L., Korsgaard, B., and Bjerregaard, P. (1999). In vivo estrogenic activity of branched and linear alkylphenols in rainbow trout (Oncorhynchus mykiss). Sci. Total Environ. 233, 89–96.[CrossRef][Medline]
Petit, F., Le Goff, P., Cravedi, J. P., Valotaire, Y., and Pakdel, F. (1997). Two complementary bioassays for screening the estrogenic potency of xenobiotics: Recombinant yeast for trout estrogen receptor and trout hepatocyte cultures. J. Mol. Endocrinol. 19, 321–335.
Sturm, A., Cravedi, J. P., Perdu, E., Baradat, M., and Segner, H. (2001). Effects of prochloraz and nonylphenol diethoxylate on hepatic biotransformation enzymes in trout: A comparative in vitro/in vivo-assessment using cultured hepatocytes. Aquat. Toxicol. 53, 229–245.[CrossRef][Web of Science][Medline]
Thibaut, R., Debrauwer, L., Rao, D., and Cravedi, J. P. (1998). Characterization of biliary metabolites of 4-n-nonylphenol in rainbow trout (Oncorhynchus mykiss). Xenobiotica 28, 745–757.[CrossRef][Medline]
Tollefsen, K. E., Mathisen, R., and Stenersen, J. (2002). Estrogen mimics bind with similar affinity and specificity to the hepatic estrogen receptor in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 126, 14–22.[CrossRef][Medline]
Toppari, J., Larsen, J. C., Christiansen, P., Giwercman, A., Grandjean, P., Guillette, L. J., Jegou, B., Jensen, T. K., Jouannet, P., Keiding, N., et al. (1996). Male reproductive health and environmental xenoestrogens. Environ. Health Perspect. 104, 741–803.
Uguz, C., Iscan, M., Erguven, A., Isgor, B., and Togan, I. (2003). The bioaccumulation of nonyphenol and its adverse effect on the liver of rainbow trout (Onchorynchus mykiss). Environ. Res. 92, 262–270.[Medline]
Uzbekova, S., Chyb, J., Ferriere, F., Bailhache, T., Prunet, P., Alestrom, P., and Breton, B. (2000). Transgenic rainbow trout expressed sGnRH-antisense RNA under the control of sGnRH promoter of Atlantic salmon. J. Mol. Endocrinol. 25, 337–350.[Abstract]
Van den Belt, K., Verheyen, R., and Witters, H. (2003). Comparison of vitellogenin responses in zebrafish and rainbow trout following exposure to environmental estrogens. Ecotoxicol. Environ. Saf. 56, 271–281.[CrossRef][Web of Science][Medline]
Vetillard, A., Atteke, C., Saligaut, C., Jego, P., and Bailhache, T. (2003). Differential regulation of tyrosine hydroxylase and estradiol receptor expression in the rainbow trout brain. Mol. Cell. Endocrinol. 199, 37–47.[CrossRef][Medline]
Vetillard, A., and Bailhache, T. (2005). Cadmium: An endocrine disrupter that affects gene expression in the liver and brain of juvenile rainbow trout. Biol. Reprod. 72, 119–126.
Vetillard, A., Ferriere, F., Jego, P., and Bailhache, T. (2006). Regulation of salmon gonadotrophin-releasing hormone gene expression by sex steroids in rainbow trout brain. J. Neuroendocrinol. 18, 445–458.[Medline]
White, R., Jobling, S., Hoare, S. A., Sumpter, J. P., and Parker, M. G. (1994). Environmentally persistent alkylphenolic compounds are estrogenic. Endocrinology 135, 175–182.[Abstract]
Xie, L., Thrippleton, K., Irwin, M. A., Siemering, G. S., Mekebri, A., Crane, D., Berry, K., and Schlenk, D. (2005). Evaluation of estrogenic activities of aquatic herbicides and surfactants using an rainbow trout vitellogenin assay. Toxicol. Sci. 87, 391–398.
Yadetie, F., Arukwe, A., Goksoyr, A., and Male, R. (1999). Induction of hepatic estrogen receptor in juvenile Atlantic salmon in vivo by the environmental estrogen, 4-nonylphenol. Sci. Total Environ. 233, 201–210.[CrossRef][Medline]
Yadetie, F., and Male, R. (2002). Effects of 4-nonylphenol on gene expression of pituitary hormones in juvenile Atlantic salmon (Salmo salar). Aquat. Toxicol. 58, 113–129.[CrossRef][Web of Science][Medline]
Zhou, W., Koldzic-Zivanovic, N., Clarke, C. H., de Beun, R., Wassermann, K., Bury, P. S., Cunningham, K. A., and Thomas, M. L. (2002). Selective estrogen receptor modulator effects in the rat brain. Neuroendocrinology 75, 24–33.[CrossRef][Web of Science][Medline]
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