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BOR - Papers in Press, published online ahead of print August 18, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029520
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BIOLOGY OF REPRODUCTION 72, 119–126 (2005)
DOI: 10.1095/biolreprod.104.029520
© 2005 by the Society for the Study of Reproduction, Inc.

Cadmium: An Endocrine Disrupter That Affects Gene Expression in the Liver and Brain of Juvenile Rainbow Trout1

Angelique Vetillard2, and Thierry Bailhache3

Endocrinologie Moléculaire de la Reproduction, UMR-CNRS 6026, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes cedex, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An inhibition of vitellogenesis is observed in fish exposed to cadmium (Cd), either in natural or in experimental conditions. To investigate whether this correlates or not with modifications in the expression of several genes involved in reproduction, we have performed a study on juvenile rainbow trout (Oncorhynchus mykiss) exposed to waterborne Cd in combination with estradiol (E2). A relative reverse transcription-PCR protocol was used to evaluate the effect of Cd exposure on the expression of several genes. We quantified vitellogenin, rainbow trout estradiol receptor {alpha} (rtER{alpha}), short and long isoforms (rtER{alpha}S and rtER{alpha}L), mRNA levels in liver, and salmon GnRH1, salmon GnRH2, rtER{alpha}S, and rtER{alpha}L mRNA levels in the brain. In liver, Cd reduced the E2-stimulated mRNA levels of vitellogenin as well as these of both rtER{alpha} isoforms in a dose-dependent manner. In brain tissue, our results indicate that rtER{alpha} mRNA levels are not enhanced by E2. Cd treatments did not modify rtER{alpha}S isoform expression but reduced rtER{alpha}L expression in the brain. Focusing on the expression of salmon GnRH (sGnRH) genes, E2 did not affect mRNA levels, but experiments with Cd alone greatly enhanced sGnRH 1 as well as sGnRH 2 gene expression in a dose-dependant manner. This study supports the idea that Cd is an important endocrine disrupter that could act through an inhibition of E2-stimulated genes in the liver and also through a central effect on sGnRH gene expression. Cd may affect a number of E2 signaling pathways but could also affect the reproductive axis by nonestrogenic mechanisms.

estradiol receptor, gene regulation, gonadotropin-releasing hormone, neuroendocrinology, toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In many aquatic systems, metal concentrations are greater than natural background levels, due to a continuous release of metals from industrial, mineral mining, and agricultural sources. Numerous studies have examined the toxicity of heavy metals such as cadmium (Cd), zinc, mercury, copper, and others on freshwater fish under both field and controlled laboratory exposure (for review, see [1]). Most of these chemical contaminants target the central nervous system along with other organ systems, resulting in neuroendocrine and behavioral changes that may impair the subsequent survival or reproduction of exposed animals [2].

Cadmium is a persistent neurotoxic contaminant that was one of the most commonly used heavy metals in the 1970's [3] and is still present in North American rivers and lakes [4] as well as in European surface water [5]. This metal is not eliminated from water ecosystems; it accumulates in sediments and is released in water during heavy rainfall, snowmelt, and run-off episodes [6]. Over short periods, i.e., hours or days, metallic concentrations reach levels that may cause physiological stress and even kill organisms [7].

As with other heavy metals, Cd may enter the food chain and concentrate within organisms, but in fish, it may also penetrate through the gills as attested to by its rapid accumulation during waterborne exposure [8]. Cd also accumulates in other tissue, such as liver and kidney, as a result of dose and time exposure [9, 10].

Cadmium has been shown to induce heavy metal-binding proteins such as metallothionein in several tissues [6, 11]. These induced metallothioneins, together with other Cd-binding proteins [12], may participate in detoxification by sequestering the metal.

Toxicity of Cd to freshwater fish has been extensively studied [7] and salmonids seem to be among the most sensitive fish species to this metal [13–15]. The main physiological disruptions associated with Cd toxicity are related to modifications of enzymatic activities in several organs, i.e., liver, gills, kidney, and gut [16], to endocrine and metabolic changes [17, 18] or to a decrease in calcium uptake and blood levels [10, 19]. Other injuries of Cd have been reported, which include genotoxicity [15, 20] and reproductive disorders [21].

In mammals, Cd may interfere with steroidogenesis [22] or with gametogenesis [23]. In fish, Cd alters hormone synthesis in rainbow trout testes [24], and it inhibits gonadotropin-stimulated steroidogenesis and ovarian maturation in female common carp (Cyprinus carpio) [25]. Trout oogenesis appeared to be delayed by extensive exposition to Cd [26], while eggs obtained from females exposed to Cd did not develop to the fry stage. A direct exposition of rainbow trout fertilized eggs to Cd induced premature hatching, mortality, and developmental abnormalities [27]. Vitellogenesis has been shown to be altered in winter flounder (Pleuronectes americanus) populations collected from Cd-contaminated areas [28] or after experimental Cd exposure [29]. In rainbow trout, Cd treatments inhibited estradiol-stimulated transcription and translation of vitellogenin (Vg) [30, 31].

In oviparous species, during vitellogenesis, Vg is produced by the liver, released, and transported by blood to the ovaries, then it is incorporated and processed into oocytes to form the major yolk protein [32]. The first step of this process is controlled by circulating estradiol (E2) [33], the action of which is mainly mediated by the estrogen receptor (ER), a nuclear receptor that functions as a ligand-dependant transcription factor. Cd has been shown to inhibit rainbow trout ER (rtER) biological activity by diminishing its interaction with DNA [34]. These data suggest that Cd could have a wide range of effects and could interfere with other E2-controlled gene expression.

In rainbow trout liver, rtER{alpha} gene expression is upregulated by E2 [35, 36], which is likely due to the presence of an estrogen-responsive element in the promoter region [37]. Thus, rtER{alpha} gene could be a major target of Cd inhibitory action as well as the Vg gene.

Recently, the existence of two rtER{alpha} isoforms having different estrogen dependencies and issuing from the same gene was reported in rainbow trout [36]. Functional analysis of both rtER{alpha} isoforms revealed that the short rtER{alpha} isoform (rtER{alpha}S) consistently exhibited a basal (E2-independent) transactivation activity that could be further increased in the presence of E2, when the full-length rtER{alpha} isoform (rtER{alpha}L) is characterized by a strict E2-dependent transcription activity.

The present study was performed to investigate effects of Cd on E2-stimulated Vg and rtER{alpha} gene expression in the liver, as well as on sGnRH and rtER{alpha} gene expression in the brain, genes that are strongly involved in reproduction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fish

Rainbow trout (Oncorhynchus mykiss) were purchased from an experimental fish farm (SEMI, Le Drennec, France), and kept in 1000-L tanks in aerated recirculating freshwater at 12–13°C, under a natural photoperiod (SCRIBE INRA, Rennes, France). The study was conducted on 4-mo 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 on animal care during scientific experiments (décret 87/848).

Fry were exposed to waterborne E2 (17ß-estradiol; steraloid, Wilton, NH) and/or cadmium chloride (CdCl2) via aquarium water. During exposition, fish were maintained in a 5-L aquarium in aerated water cooled to 12°C. Substances were added at each water renewal (24 h). At the end of exposition, fish were killed under deep anesthesia and liver was dissected. Body and liver weight were measured and hepatosomatic index (HSI) was calculated with the formula: HSI (%) = liver weight (g) x 100/all body weight (g). According to experiments, liver and brain were dissected out and stored at –80°C until RNA extraction.

Experiment 1 In experiment 1, a time- and dose-effect study was performed. Increasing concentrations of E2, ranging from 1 to 1000 nM, were applied to fry, which were analyzed after 24, 48, or 72 h of exposure. Prior to treatments, E2 was diluted in ethanol to distribute the same volume in each aquarium (1 ml per 5 L freshwater). Control groups received only 1 ml ethanol.

This preliminary experiment aimed to determine the best concentration and exposure time to be used in experiment 2. Thus, liver alone was dissected out and processed for RNA extraction.

Experiment 2 In experiment 2, fry were exposed to E2 and CdCl2 in a combined dose regime. E2 concentrations ranged from 1 to 100 nM and two doses of CdCl2 were used to achieve final [Cd++] concentrations of 1 and 5 µg/L (ppb). The latter correspond to the maximal concentration of Cd allowed in European public rivers and lakes.

The control groups for each substance received only the corresponding vehicle.

According to results of experiment 1, fish were exposed to E2 during 48 h. To enhance Cd inhibition on E2-induced gene expression in the liver, a 24-h pretreatment with Cd was necessary, as assessed in a preliminary experiment (data not shown). Fish were exposed to CdCl2 for 24 h followed by a mix of E2 and CdCl2 for 48 h.

At the end of exposition, liver and brain were dissected out and processed for RNA extraction or Cd content determination.

RNA Extraction

Total RNA was extracted from individual liver or brain by 1 ml Trizol reagent (Gibco BRL) 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 was performed by spectrophotometry at 260 nm. Samples that show a partial degradation or a low extraction yield were discarded; finally, five samples per treatment were used for reverse transcription-PCR (RT-PCR) analysis.

Dot Blot Analysis

RNA samples (5 µg) were spotted onto nylon Biodyne A (Pall) membrane, using a Biorad dot blot apparatus. Duplicate membranes were made with the same samples to allow the quantification of Vg and ß-actin mRNAs. The membranes were prehybridized at 42°C for 6 h, hybridized at 42°C for 16 h with Vg cDNA [38] or ß-actin cDNA [35] probes labeled with 32P dCTP (~3000 Ci/mmol) and washed under stringent conditions, as previously described [39]. Vg and ß-actin mRNA levels were quantified by counting the remaining radioactivity on dot blots using Instantimager (PE Applied Biosystem, Courtaboeuf, France). Membranes were then exposed at –70°C with a Biomax film (Kodak) for autoradiography. Amounts of Vg mRNA were corrected with amounts of ß-actin mRNA.

Development of a Semiquantitative RT-PCR Analysis

The low amount of rtER or sGnRH mRNAs in the brain of fry did not allow their quantification by dot blot. So changes in each mRNA species were quantified using a relative RT-PCR technique that allows a semiquantitative determination of abundance. The relative RT-PCR protocol has been previously described for sGnRH 1 and sGnRH 2 [40, 41] and adapted to our experimental conditions in fry and to rtER mRNAs (rtER{alpha}L and rtER{alpha}S).

Total RNA (1 µg) was reverse transcribed for 1 h at 42°C with random hexanucleotide primers and 50 U of EXPAND reverse transcriptase (Gibco BRL) in a total volume of 20 µl.

Two microliters of single-strand cDNA were then amplified using 2.5 U Taq DNA polymerase (PE Applied Biosystem) in a total volume of 50 µl PCR buffer that contained 1.5 mM MgCl2, 200 µM dNTPs, and 500 nM specific primers (Table 1). PCRs were performed in a Genamp PCR 9700 (PE Applied Biosystem), after a 5-min denaturing step at 94°C, 24– 32 cycles was performed according to the cDNA amplified (see Table 1 for details). Each cycle consisted of 30 sec denaturing at 94°C, 30 sec annealing (see Table 1 for temperature) and 30 sec extension at 72°C. The last cycle was followed by a final 7-min extension at 72°C. Each PCR reaction mixture was subjected to electrophoresis in 2% (w/v) agarose gel stained with ethidium bromide and photographed under ultraviolet illumination with an image-analysis system (Gel-Doc1000; Bio-Rad). Intensity of the fluorescence, which is related to the concentration of DNA in the gel, was determined using Molecular Analyst/PC software (Bio-Rad).


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  		TABLE 1. Oligonucleotide primers and settings for relative RT-PCR amplification

To obtain meaningful results, the relative RT-PCR reaction products have to be quantified when the reaction reaches the linear phase of amplification. Experiments were performed to determine the number of cycles that produced quantifiable signals with an amplification factor, which remains constant over a number of cycles. The number of cycles was determined experimentally for each pair of primers (Fig. 1).



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  		FIG. 1. Establishment of relative RT-PCR analysis in juvenile trout tissues. A) Determination of the linear range of amplification of histone H3, vitellogenin (Vg), rtER{alpha}S, and rtER{alpha}L mRNA from liver. B) Determination of the linear range of amplification of histone H3, sGnRH 1, sGnRH 2, rtER{alpha}S, and rtER{alpha}L mRNA from brain. The mean optical densities of PCR products were plotted against the number of PCR cycles

In all experiments, the expression of histone H3 [42] was used to normalize the results. The amount of histone H3 mRNA was not modified by any treatments (data not shown), and results are expressed as the ratio of optical density of amplified mRNA (rtER{alpha}L, rtER{alpha}S, Vg, sGnRH 1, or sGnRH 2) on that of histone H3, for each sample.

Cd Assays in Tissues and Water

For Cd determination, tissue samples were digested in 65% HNO3 for 3 h at 80°C. HNO3 was evaporated and the mineral residue was solubilized in acidified water (2% HNO3). Accumulated Cd was quantified by graphite furnace atomic absorption spectrometry (Perkin Elmer 4110ZL, PE Applied Biosystem). Aquarium water was sampled at the end of a 24-h exposure before renewal of the tank water. The water replacement was combined with the renewal of the metal contamination. Water samples were stabilized by addition of 2% of HNO3 and analyzed by the same atomic absorption spectroscopic method described for the tissue samples. The detection limit in the samples was 1.2 ng/g (ppb) in tissues and 0.15 µg/ L (ppb) in water. Tissue Cd concentrations were expressed as ng/g wet tissue (ppb), metal concentrations in the water as µg/L freshwater (ppb).

Statistical Analyses

Data are expressed as means ± SEM. Results were analyzed using ANOVA followed by a Fisher projected least significant difference post hoc test when appropriate. In all the statistical tests, difference was considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of E2 Exposure on HSI

HSI was not significantly different between treatment groups, whatever the dose and duration of exposure to E2 were. In our experimental conditions, in vivo E2 treatments did not induce hepatic growth (Fig. 2A).



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  		FIG. 2. Time and dose effect of waterborne E2 treatments on (A) hepatosomatic index (HSI), (B) ß–actin, and (C) vitellogenin (Vg) mRNA levels in the liver of juvenile rainbow trout. Values are expressed as mean ± SEM (n = 5 fish per treatment). Different letters above columns indicate a significant difference (P < 0.05). ns, nonsignificant difference

Time and Dose Effect of E2 Exposure on Liver Vg Gene Expression

This preliminary experiment aimed to determine the best exposure time to be chosen in experiment 2. Thus, only liver Vg mRNAs were analyzed by a dot blot hybridization protocol.

We first verified that E2 treatments did not affect the housekeeping gene ß-actin expression in the liver (Fig. 2B), and this experiment showed that RNA extraction and dot blotting were homogeneous among samples.

Expression of the Vg gene was upregulated within as early as 24 h of exposure to E2 and for all the doses (Fig. 2C). After a 48-h exposure, Vg mRNA levels were significantly increased in a dose-dependent manner for doses from 1 to 100 nM. A higher dose did not significantly enhance Vg gene expression. Another 24-h exposure did not significantly change Vg expression pattern.

These experiments led us to the conclusion that experiments with Cd have to be carried out with several concentrations of E2 (1–100 nM) and requiring an exposure time of 48 h.

Dose-Related Accumulation of Cd in Brain and Liver

Cd concentrations in the water after 24 h were close to what was expected (Fig. 3A). In the water of control aquaria, Cd concentrations were low (<1 ppb), they raised to values over 1 ppb in the lower dose treatment, but the difference was not significant. In the higher dose treatment, values were significantly increased but did not reach the expected 5 ppb, which could be the result of adsorption on organic matter and on aquaria walls or assimilation by fish.



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  		FIG. 3. Concentration of cadmium (Cd) found (A) in aquarium water, (B) in the brain, and (C) in the liver of juvenile rainbow trout exposed during 72 h to waterborne CdCl2. Values are expressed as mean ± SEM (n = 5 samples per treatment). Different letters above columns indicate a significant difference (P < 0.05)

A high level of Cd accumulation was observed in the liver after a 72-h exposure to the higher dose (Fig. 3C). Increase in Cd levels in the brain is less (Fig. 3B) but is also highly significant (P < 0.001), and accumulation ratios are similar among tissues: 4.9 and 5.26 in brain and liver, respectively.

The lower dose of treatment did not produce any significant accumulation of Cd in tissues.

Effects of Cd on E2-induced rtER{alpha} and Vg gene expression in liver

After exposure to Cd and/or E2, no change could be detected in liver weight or HSI (Fig. 4A).



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  		FIG. 4. Combined dose effect of waterborne estradiol and CdCl2 treatments on (A) hepatosomatic index, (B) rtER{alpha}S, (C) rtER{alpha}L, and (D) vitellogenin mRNA levels in the liver of juvenile rainbow trout. Values are expressed as mean ± SEM (n = 5 fish per treatment). Different letters above columns indicate a significant difference (P < 0.05). ns, nonsignificant difference

Results of relative RT-PCR quantification of rtER{alpha}L, rtER{alpha}S and Vg mRNAs in the liver are plotted in Figure 4.

For each gene studied and whatever the Cd concentration, results provide evidence that Cd did not have any effect on basal gene expression. In control fish, rtER{alpha} showed a basal gene expression for both isoforms (rtER{alpha}L and rtER{alpha}S), but Vg mRNA was nearly undetectable even after RT-PCR amplification.

Both rtER isoforms and Vg gene expression were upregulated by E2 treatments with doses of 10 and 100 nM. After treatments with 1 nM E2, levels of rtER{alpha}S and Vg mRNAs were weakly increased, but this raise was not significant.

Cd treatments reduced E2-stimulated rtER and Vg mRNA levels in a dose-dependent way. This inhibition is highly effective because Cd (5 µg/L) totally abolished the effects of a medium dose (10 nM) of E2. However, when fry were exposed to a higher dose of E2, Cd only partly inhibits rtER isoforms expression and, consequently, was not able to prevent Vg mRNA increase.

From these results in the liver, we can conclude on the efficiency of treatments and on the reliability of the semiquantitative RT-PCR to quantify gene expression.

Effects of E2 and Cd on rtER{alpha} gene expression in brain tissue

First, we noticed that rtER{alpha}S mRNA was detectable in the brain of fry but levels were very low (Fig. 5A). As a consequence, 32 cycles of PCR have been performed to allow a reliable quantification (Fig. 1).



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  		FIG. 5. Effects of cadmium alone or in combination with estradiol on (A) rtER{alpha}S and (B) rtER{alpha}L mRNA levels in the brain of juvenile rainbow trout. Values are expressed as mean ± SEM (n = 5 fish per treatment). Different letters above columns indicate a significant difference (P < 0.05). ns, nonsignificant difference

Prior to analyzing the effect of Cd in combination with E2, we have investigated the action of increasing concentrations of E2, from 1 nM to 1 µM, on rtER{alpha}L and rtER{alpha}S mRNAs levels. Whatever the concentration, E2 did not have any effect on rtER{alpha}L and rtER{alpha}S expression when compared with the control group (data not shown). E2 does not seem to be involved in the regulation of rtER{alpha} gene expression in the brain.

However, we analyzed the effect of increasing doses of Cd in combination or not with a high dose of E2 (100 nM). We did not observe any changes in rtER{alpha}S mRNA levels in the brain of Cd-treated fish, in combination or not with E2 (Fig. 5A), but we found a clear inhibition of rtER{alpha}L expression by Cd, which is independent from E2 treatments (Fig. 5B).

Effects of E2 and Cd on sGnRH 1 and sGnRH 2 Gene Expression in Brain Tissue

Increasing concentrations of E2, ranging from 1 nM to 1 µM, did not modify sGnRH mRNA levels (data not shown). So we could not expect for an antagonistic effect of Cd, and we focused on analyzing the effect of increasing doses of Cd in combination or not with a high dose of E2 (100 nM).

In the absence of E2 treatment, we observed a concentration-dependent stimulatory effect of Cd on sGnRH 1 and sGnRH 2 mRNA levels (Fig. 6). E2 treatments did not significantly modify the effect of Cd, but the increase was less important and was not significantly different from the controls.



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  		FIG. 6. Effects of cadmium alone or in combination with estradiol on (A) sGnRH 1 and (B) sGnRH 2 mRNA levels in the brain of juvenile rainbow trout. Values are expressed as mean ± SEM (n = 5 samples per treatment). Different letters above columns indicate a significant difference (P < 0.05)


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Fish exposed to the highest dose of Cd experienced a significant accumulation of the metal in liver and brain tissue. It is well documented that Cd and other heavy metals accumulate in liver, gills, and kidney [10, 43]. In our study, Cd levels found in liver follow the predictive linear pattern of metal accumulation in this tissue [9, 44] and agree with observations on trout coming from contaminated rivers [6].

Cd not only accumulated in the liver but was also increased in the brain, even if levels were far lower than those found in the liver. Accumulation in the brain seems to be dependent on the administration route. Oral treatments failed to induce any significant increase in trout [45], but Cd has been shown to be taken up by olfactory epithelium and transported to the brain in pikes [46], a route that could fit with the waterborne exposition used in our study. In rainbow trout, after waterborne exposure, Cd accumulated in the olfactory rosettes, nerves, and bulbs but not in the brain [47]. In our study, the brain samples include the olfactory bulbs among other brain structures. Therefore, it was not possible for us to know if Cd accumulation was restricted to the olfactory system or if it was also present in other brain structures.

In rainbow trout liver, it is well established that rtER and Vg gene expression are upregulated by E2 as a result of both an increase in transcription and mRNA stabilization [48], two mechanisms that involve the activation of rtER by E2.

Our results demonstrated that, in liver, E2 stimulates in vivo the expression of both rtER{alpha} isoforms with similar amplitudes and that a Cd pretreatment reduces this stimulation in a dose-dependent manner. Although Cd inhibition of E2-stimulated Vg expression is only significant with a medium dose of E2, our results confirm previous in vivo [31] and in vitro [30] studies on Cd-inhibitory activity. Cd can affect Vg gene expression by a direct binding to rtER followed by the inhibition of its transactivation function [34]. In addition to this direct inhibition of rtER transactivation function, we show that Cd also reduces rtER mRNA levels in the liver, which is likely to lower the amount of rtER, the main regulatory factor for Vg gene expression [49]. Both mechanisms could be involved in the strong inhibition of Vg gene expression observed in our experiments.

However, when fish were exposed to a high dose of E2, Cd was not able to totally inhibit rtER upregulation and did not modify Vg gene expression. This could explain why Cd is able to delay ovulation or to disrupt vitellogenesis in trout [26] but does not totally abolish vitellogenin production.

Several studies have reported that Cd can interact with transcription factors [50, 51] or with nuclear receptors [34, 52] and therefore can affect the biological properties of these proteins. Cd has been shown to inhibit the transcription activity of E2-activated rtER in recombinant yeast. This inhibitory action of Cd is not restricted to rtER because human ER was also inhibited to a lesser extent [34]. However, the latter effect seems to be dependant on the cellular context because Cd stimulates human ER activity in MCF7 cells [53]

The putative site of action on rtER is located in the DNA-binding domain [34], where Cd could reduce the interaction between activated rtER and DNA, thus reducing rtER transcription activity. This characteristic could explain why Cd could display inhibitory properties only when rtER is activated by E2. Our results in the liver reinforce this hypothesis because Cd inhibits E2-stimulated gene expression but does not affect the basal amounts of rtER{alpha} mRNA.

In the present work, we report that brain rtER{alpha} gene expression is not affected by E2 treatments. This agrees with our recent study in sexually mature female trout, where rtER gene expression in the preoptic area was not regulated by E2 [54]. But in triploid female rainbow trout, brain rtER{alpha} mRNA levels are increased by a single injection of E2 [39, 55]. Indeed, this rise in brain rtER{alpha} mRNA levels is likely to correspond to rtER{alpha}S mRNA, of which brain levels are increased by E2 treatments in triploid trout (unpublished results).

In the absence of E2 regulation of rtER gene expression in the brain, we did not expect any Cd inhibitory effect on mRNA levels. Moreover, Cd has been shown to accumulate in the olfactory bulbs but not in other regions of the brain [47], where rtER{alpha} is expressed (i.e., preoptic area and ventrolateral hypothalamus) [56]. However, a Cd treatment alone or in combination with E2 was able to decrease rtER{alpha}L mRNA levels in the brain without affecting rtER{alpha}S expression. The mechanism involved in that effect of Cd is still unknown; it seems to be E2 independent and is likely to be indirect.

Indeed, part of Cd toxicity is due to the alteration of calcium homeostasis [57]. In rainbow trout, Cd inhibits unidirectional calcium uptake by the gills from water and induces hypocalcemia in the plasma [10], and Cd is used as a blocker for voltage-dependent calcium channels. Because calcium ions are essential for neuronal activity, a reduced plasma calcium concentration could induce a drop in brain calcium levels that could modify neuronal biosynthesis activity. Different promoter contexts and/or different cofactors could explain that it may have different effects on each rtER{alpha} isoform expression.

We also demonstrated that E2 treatments did not modify sGnRH 1 or sGnRH 2 gene expression in the brain. However, Cd treatment alone or in combination with E2 stimulated sGnRH 1 and sGnRH 2 gene expression in a dose-dependent manner. To analyze such an effect, we have to take into account that part of the sGnRH neurons are located in the olfactory bulbs and have processes running along the olfactory nerves [58] and also that sGnRH neurons all originate from the olfactory placode. In pike, the uptake of Cd by primary olfactory neurons is followed by an axonal transport up to the olfactory bulbs [59]. An uptake of Cd by sGnRH neuron endings in the olfactory rosette or in the olfactory bulbs could be responsible for the changes in sGnRH gene expression.

Cd has been shown to inhibit gamma aminobutyric acid-A (GABA-A) receptor activity in snail neurons by increasing intracellular calcium [60], and GABA-A activation can negatively regulate GnRH gene expression in rats [61]. A similar mechanism could also be present in trout brain, where Cd could reverse such an inhibition and enhance sGnRH genes expression.

Cd has also been reported to induce accumulation of gonadotropin in the pituitary of catfish, Clarias batrachus [62], which could be the consequence of GnRH gene expression activation.

In conclusion, our study showed that both rtER{alpha} short and long isoforms are equally upregulated by E2 in the liver of juvenile rainbow trout and that Cd is able to antagonize E2 stimulatory effects on Vg as well as rtER{alpha} gene expression. In addition, rtER{alpha} gene expression does not seem to be regulated by E2 in the brain, but rtER{alpha}L mRNA levels are greatly reduced by Cd. As it accumulates in the brain, Cd enhances sGnRH 1 and sGnRH 2 gene expression, but the interpretation of such an effect remains open. Taken together, our data provide evidence that Cd is an important endocrine disrupter that may act on different tissues and alter genetic programs that may or may not be controlled by E2.


   ACKNOWLEDGMENTS
 
We acknowledge Dr. G. Gruau, laboratoire de Géosciences, UMR 6118 Université de Rennes, France, for his help in Cadmium assays. We thank Mrs. Joan Riley for her help in proofreading the manuscript.


   FOOTNOTES
 
1 This work was supported by CNRS, INRA, and EU (BIO4 CT 97-0554). A.V. was supported by a grant from Conseil Regional de Bretagne (France). Preliminary results were presented at the 20th Conference of European Comparative Endocrinologists (Faro, Portugal). Back

2 Correspondence: Angelique Vetillard, MRC Toxicology Unit, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK. FAX: +44 116 252 5616; av33{at}le.ac.uk Back

3 Current address: Department of Biology, University of Leicester, University road, Leicester LE1 7RH, UK Back

Received: 12 March 2004.

First decision: 4 April 2004.

Accepted: 17 August 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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