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Endocrinology, doi:10.1210/en.2008-0384
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Endocrinology Vol. 149, No. 9 4269-4275
Copyright © 2008 by The Endocrine Society

Sex Hormone-Binding Globulin in Fish Gills Is a Portal for Sex Steroids Breached by Xenobiotics

Solange Miguel-Queralt and Geoffrey L. Hammond

Department of Obstetrics and Gynaecology, University of British Columbia, and Child & Family Research Institute, Vancouver, British Columbia, Canada V5Z 4H4

Address all correspondence and requests for reprints to: Geoffrey L. Hammond, Ph.D., Child & Family Research Institute, 950 West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4. E-mail: ghammond{at}cw.bc.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
As in most vertebrates, plasma sex hormone-binding globulin (SHBG) is produced in fish liver and regulates sex steroid access to target tissues. Low levels of SHBG mRNA are present in zebra fish gills but are unlikely to account for the high amounts of immunoreactive SHBG in filaments and lamellae. Although the uptake of steroids by fish from water has been reported to correlate with their affinity for SHBG, it is not known how this occurs. Our studies of zebra fish SHBG have revealed its preference for biological active androgen (testosterone), as well as for androstenedione, a sex steroid precursor that also acts as a pheromone in some fish. In addition to natural steroids, zebra fish SHBG has a high affinity for synthetic steroids, such as ethinylestradiol and progestins (levonorgestrel and norethindrone), that are present in waste water systems. Because steroids can pass across fish gills, we examined whether SHBG serves as a portal for natural and synthetic steroids controlling their flux between the blood and aquatic environment. The results indicate that SHBG ligands are rapidly and specifically removed from water by the fish through their gills, whereas the accumulated steroids are released slowly. The capacity of fish to sequester SHBG ligands from water is similar between sexes, independent of size, and characterized by a wide dynamic range. We conclude that SHBG controls the flux of sex steroids across fish gills and that this highly specialized function can be hijacked by xenobiotic ligands of fish SHBGs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEX HORMONE-BINDING GLOBULIN (SHBG) is the major transport protein for sex steroids in the blood of most vertebrate species, including fish (1). Although SHBG is produced by hepatocytes in the liver and regulates the bioavailability and metabolic clearance of its steroid ligands in the blood (2), plasma SHBG can be sequestered by some tissue compartments where it likely exerts a more direct influence on the access of sex steroids to specific cell types (3). Expression of SHBG in tissues other than the liver also results in the presence of SHBG outside the blood circulation (4), and the best example of this is the production of an SHBG homolog in rodent Sertoli cells that is secreted into seminiferous tubules where it regulates androgen availability in the male reproductive tract (5).

The primary structures of SHBG are poorly conserved phylogenetically, but their tertiary and quaternary structures all comprise a homodimeric complex of tandem laminin G-like (LG) domains (6). Most importantly, the amino-terminal LG domains of all known SHBGs contain several invariably conserved residues that hydrogen bound with the functional groups of steroid ligands within a well-defined hydrophobic binding pocket (6, 7). As in mammals (1), the relative binding affinities of different fish SHBGs for androgens and estrogens vary, but the protein generally exhibits a preference for biologically active sex steroids in a given species (8). However, a characteristic feature of SHBGs in teleost species is that they bind the sex steroid precursor, androstenedione. This is remarkable because androstenedione in mammals does not bind SHBG (1) and functions only as an androgen precursor. By contrast, some male fish release androstenedione into the water during the spawning season where it acts as a pheromone (9). In addition to binding natural steroids, fish SHBGs can bind xenobiotic ligands (10, 11, 12, 13, 14, 15), including the synthetic estrogen, ethinylestradiol, a well-documented xenoestrogen in aquatic species (16, 17).

Small molecules pass across the gill into the aquatic environment, and this is considered a mechanism for the excretion of steroids (18, 19). However, sex steroids also appear to be preferentially taken up by fish from water in relation to their affinities for SHBG (20). In our ongoing studies of SHBG in several species of fish, an unexpected finding has been the remarkable accumulations of immunoreactive SHBG in their gills. Therefore, we performed a series of experiments to explore how SHBG in this location might act as a portal that controls the release and/or uptake of specific classes of sex steroids, and to address the obvious question of whether this portal could be breached by xenobiotic ligands of fish SHBG.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult zebra fish (Danio rerio) were obtained from commercial suppliers and held in aquarium water (distilled water supplemented with NutraFin Aquarium Salts, Rolf C. Hagen Inc., Montreal, Quebec, Canada) at room temperature. Experiments using zebra fish were approved by the animal care committee of the University of British Columbia under the guidelines of the Canadian Council of Animal Care.

Steroids
[3H]Testosterone (40 Ci/mmol), [3H]ethinylestradiol (60 Ci/mmol), [3H]cortisol (70 Ci/mmol), and [3H]5{alpha}-dihydrotestosterone (DHT) (44 Ci/mmol) were purchased from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ). Unlabeled steroids were purchased from Sigma-Aldrich Canada Ltd. (Oakville, Ontario, Canada) or Steraloids Inc. (Wilton, NH) and used as supplied.

Immunohistochemistry
Adult zebra fish tissues were fixed in PBS containing 4% paraformaldehyde at 4 C for 24 h, and subsequently dehydrated with a series of ethanol solutions before embedding in paraffin. Paraffin sections were dewaxed and treated with proteinase K (20 µg/ml) in PBS. The sections were then treated with 0.03% hydrogen peroxide solution for 7 min and blocked at room temperature for 1–2 h with BSA (5 mg/ml) in PBS. The primary antibody for zebra fish SHBG (zfSHBG) was first diluted (1:7500) in either culture medium harvested from wild-type Chinese hamster ovary (CHO) cells, or medium from CHO cells that express sufficient quantities of zfSHBG to block the anti-SHBG antibodies. This was done to confirm the specificity of the anti-zfSHBG antibodies. After an overnight incubation at 4 C with either the unblocked or blocked anti-zfSHBG antisera, immunoreactive zfSHBG was detected using the EnVision System, horseradish peroxidase (3'3-diaminobenzidine HCl) from Dako Corp. (Carpinteria, CA).

RT-PCR
To detect SHBG mRNA in adult zebra fish tissues, RT-PCR assays were performed using zfSHBG-specific forward (5'-TCTGTGCAGGAGAGCAGCAGGTG) and reverse (5'-CCAGGAACTGGAGTGGCTGTG) primers that amplify a 618-bp region within coding sequence for zfSHBG. Total RNA extracts were reverse transcribed into single-stranded DNA using oligo(dt) primer and SuperScript III, and the single-stranded cDNA product was used in a standard PCR using PCR SuperMix (Invitrogen Corp., Carlsbad, CA). After amplification, an aliquot of the PCR mixture was analyzed by agarose gel electrophoresis and staining with ethidium bromide to confirm the presence of a single amplicon of the expected size. Amplification of an 18S rRNA sequence was performed in parallel as an RT-PCR control.

Measurements of SHBG and its steroid-binding properties
Recombinant zfSHBG was produced in CHO cells (21), and its concentration and steroid-binding characteristics were determined using an established saturation ligand-binding assay (22). The relative binding affinities of zfSHBG for synthetic steroids were compared with known endogenous ligands using [3H]DHT as labeled ligand in the presence or absence of varying concentrations of unlabeled steroids as competitors (22).

Western blotting was used to assess SHBG concentrations in blood and tissue extracts, using a specific rabbit antiserum raised against pure recombinant zfSHBG (21), which was also used as a calibration standard based on its steroid-binding capacity measurement (22).

In vivo experiments
To measure the rate of steroid uptake by zebra fish, fish were placed into 20 ml freshly aerated aquarium water containing approximately 2 x 106 cpm [3H]steroids alone or in the presence of 10 µM unlabeled steroids. To monitor the nonspecific uptake of [3H]steroids, some fish were first placed in aquarium water at 4 C for 5 min. This results in almost instantaneous anesthesia and loss of gill function, from which the fish did not recover when they were placed in aquarium water containing radiolabeled steroids, as described previously. Aliquots (100–500 µl) of water were removed at timed intervals for radioactivity measurements.

To assess the rate of SHBG-ligand release from zebra fish, fish were first placed for 1 h in aquarium water containing [3H]ethinylestradiol, as described previously. They were then briefly washed in a net before being placed in 20 ml freshly aerated aquarium water to monitor the release of [3H]ethinylestradiol by the fish over time (1–2 h). In this experiment, some fish were transferred after 1 h in the fresh water to water containing 10 µM androstenedione for an additional 1 h to assess how this influences the rate of [3H]ethinylestradiol release from the fish.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization of SHBG in adult zebra fish gills
Comparisons of the immunohistochemical content and localization of SHBG in adult zebra fish liver (Fig. 1AGo), gills (Fig. 1CGo), and muscle (Fig. 1EGo) revealed that the gills and muscle contain significant amounts of immunoreactive SHBG. The specificity of the immunochemical staining in these tissues was demonstrated using recombinant SHBG to block the rabbit anti-zfSHBG antibodies (Fig. 1Go, B, D, and F). Given that the liver is likely the major site of plasma SHBG synthesis in fish, and has a rich blood supply, it is remarkable that SHBG immunoreactivity in the gills and muscle appears to exceed that of the liver when examined under the same immunohistochemical conditions. In the gills, intense immunostaining of SHBG was observed in the filament arteries as well as in the lamellae (Fig. 1CGo), thus providing a huge surface area rich in SHBG that is in intimate contact with the water. We also observed lower levels of immunoreactive SHBG in the stroma of the filament, suggesting that SHBG may either originate from blood or is produced locally in this tissue compartment. Intense staining was also observed under similar conditions in muscles and particularly in the fibers of fin muscles (Fig. 1EGo).


Figure 1
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  		FIG. 1. Comparison of SHBG immunoreactivity (brown staining) in adult zebra fish liver (A and B), gills (C and D), and muscle (E and F). Serial sections of an entire zebra fish were probed with rabbit anti-zfSHBG antisera (A, C, and E) or the same dilution of the antisera blocked with recombinant zfSHBG (B, D, and F). Controls using preimmune rabbit serum were also performed, and these were uniformly negative. All images were acquired at x40 magnification. Bars, 25 µm.

 
Relative abundance of SHBG mRNA and SHBG in zebra fish gills and muscle
To determine whether the gills or muscle is capable of producing SHBG locally, we compared the relative abundance of SHBG mRNA in total RNA extracts of these zebra fish tissues with those in the liver. The results of an RT-PCR analysis demonstrate that zebra fish gills contain relative low levels of SHBG mRNA when compared with liver, whereas SHBG mRNA is undetectable in muscle (Fig. 2AGo). To assess the amount of SHBG in the gills and muscle, we compared the relative amounts of SHBG in whole blood with those in soluble extracts of these tissues from the same fish using a Western blot calibrated against a standard amount (5 nM) of pure zfSHBG (Fig. 2BGo). These data indicate that there is no difference in the molecular sizes of zfSHBG in the gill and muscle extracts when compared with the plasma protein. Although the gills contain much more immunoreactive SHBG than the muscle in relation to the protein content of the tissue extracts, it should be appreciated that the gill extracts also contain substantial levels of blood contamination, when compared with muscle.


Figure 2
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  		FIG. 2. Relative abundance of SHBG mRNA and SHBG in zebra fish tissues. A, RT-PCR analysis of zfSHBG mRNA in total RNA extracts of liver, gills, and muscle. Aliquot reverse-transcriptase reactions were used for PCR analysis using oligonucleotide primers for zfSHBG and 18S RNA. The negative control represents an RT-PCR performed in the absence of RNA template. B, Western blot of SHBG in homogenates of gill and muscle (adjusted to a protein concentration of 5 mg/ml) diluted (1:100) plasma, and a standard concentration (5 nM) of pure zfSHBG.

 
Affinity of zfSHBG for synthetic steroids and other xenobiotics
In the present study, we used [3H]DHT as the labeled ligand to assess the affinities of zfSHBG for potential xenobiotics, in relation to its preferred endogenous steroid ligands (Table 1Go). As noted previously (21), zfSHBG displays a preference for testosterone over estradiol and binds androstenedione almost as well as testosterone. However, what is most remarkable is that the affinity of zfSHBG for ethinylestradiol far exceeds that of any natural steroid, and that two of the most commonly used synthetic progestins (levonorgestrel and norethindrone) also bind zfSHBG with affinities that exceed those of the fish androgen, 11 keto-testosterone (Table 1Go).


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  		TABLE 1. Relative binding affinities of natural steroids and xenobiotics for zfSHBG

 
Zebra fish rapidly and specifically sequester SHBG ligands from water
When adult fish were placed in water containing radioactive testosterone or ethinylestradiol, as examples of both natural and synthetic high-affinity zfSHBG ligands, or radioactive cortisol, as an example of a steroid that does not bind this protein (21), we were astonished by the speed at which testosterone and ethinylestradiol disappeared from the water, whereas no cortisol was removed from the water by live fish over the same time frame (Fig. 3Go). These data indicate that the fish were capable of removing about 70% of the radiolabeled testosterone or ethinylestradiol from the water within 90 min. This rate of steroid removal from the water suggests that gills must play a key role in this process, and this is supported by the fact that fish exposed to hypothermic anesthesia were unable to sequester radiolabeled SHBG ligands from the water (Fig. 3Go).


Figure 3
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  		FIG. 3. Zebra fish specifically sequester SHBG ligands from water. [3H]Testosterone (bullet) or [3H]ethinylestradiol ({circ}) is rapidly taken up from water by live zebra fish, whereas [3H]cortisol ({blacksquare}) is not. Fish exposed to hypothermic anesthesia were unable to take up [3H]testosterone ({triangledown}) or [3H]ethinylestradiol ({blacktriangledown}). Data points are the means of three to five animals, with bars indicating SD values.

 
The relatively low concentrations of radiolabeled steroids used in these studies mimic those reported to be present in water systems (23, 24). To explore further the specificity and capacity of zebra fish to remove steroids from water, we conducted an experiment in which an excess concentration (10 µM) of different steroids was added to water containing the same trace amount of [3H]testosterone, as in the first experiment. When the rate of removal of [3H]testosterone was monitored over the same 90-min time frame, we observed that the ability of C19 steroids to retard the uptake of [3H]testosterone (Fig. 4Go) generally mirrored their relative binding affinities for zfSHBG (Table 1Go). However, it should also be noted that estradiol and ethinylestradiol were less effective than the C19 steroids in their ability to limit the uptake of [3H]testosterone in relation to their relative binding affinities. Nevertheless, this experiment further demonstrated the specificity of this uptake system, and revealed that fish SHBG has an enormous capacity to remove steroids from the aqueous environment. This can be appreciated by the fact that 10 µM testosterone is only capable of reducing the uptake of 4.5 nM [3H]testosterone by about 30% over a 90-min period. In other words, assuming that rates of uptake of radiolabeled testosterone and cold testosterone are equivalent, the zebra fish are capable of taking up more than 7 µM testosterone within this time frame. This is astonishing given that the blood concentrations of SHBG in zebra fish are about 200 nM (21), and it suggests that steroids sequestered from the water are rapidly dispersed from the gills and taken up by peripheral tissues. This was further explored by performing an experiment in which the rate at which the [3H]ethinylestradiol sequestered by zebra fish is released back into the water over a short 1-h time interval. The results indicate that only about 3–4% of the [3H]ethinylestradiol sequestered by fish returns to water within 1 h (Fig. 5Go). The small size of the fish and the difficulty in collecting uncontaminated blood samples preclude measurements of the plasma concentrations and distribution of [3H]ethinylestradiol. However, we assume that the [3H]ethinylestradiol released from the animals over this time interval is lost from the SHBG, as it returns to the gills, because the presence of 10 µM androstenedione in water doubles the amount of [3H]ethinylestradiol released from the animals during 1 h (Fig. 5Go).


Figure 4
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  		FIG. 4. Uptake of 4.5 nM [3H]testosterone (T) from water is rapidly blocked by an excess (10 µM) of unlabeled zfSHBG ligands, when compared with the uptake of [3H]testosterone in the absence of unlabeled steroid (no competitor) or 10 µM cortisol as an example of a steroid that does not bind zfSHBG. Data points are the means of two animals.

 

Figure 5
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  		FIG. 5. Rates of [3H]ethinylestradiol (EE) released from zebra fish. A, Zebra fish were exposed to 4 nM [3H]ethinylestradiol in water for 60 min, and then transferred to fresh water in the absence ({square}) or presence of excess cortisol ({blacktriangleup}) or androstenedione ({blacksquare}). B, Zebra fish were exposed to 4 nM [3H]ethinylestradiol in water for 60 min, and then transferred to fresh water in the absence of competitor steroids for 60 min, and the same fish were then transferred again into fresh water in the absence ({square}) or presence of androstenedione ({blacksquare}) for 60 min. Data points are the means of four animals, with bars indicating the SD values.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our studies of zebra fish (21) and European sea bass (Dicentrarchus labrax L) SHBGs (25) have indicated that the liver is the major site of SHBG gene expression and the main source of plasma SHBG in these teleosts. Despite this, the amounts of immunoreactive SHBG in the hepatocytes of these fish are modest (21, 25), and are consistent with the rapid secretion and release of SHBG into the blood. By contrast, SHBG immunoreactivity in zebra fish gills is greater than in the liver, and we have also observed this in European sea bass (unpublished data). Although our RT-PCR analyses of SHBG mRNA in fish gills indicate that they may have some capacity to produce SHBG locally, it is unclear where this occurs because our attempts to locate SHBG mRNA within gill structures by in situ hybridization were unsuccessful, most likely because of its relatively low abundance in this tissue. Nevertheless, we also observed low-level SHBG immunoreactivity in the filament stroma, and this may be where SHBG mRNA is located. However, most of the immunoreactive SHBG in gills is concentrated in the filament arteries and lamella blood, and does not accumulate outside the blood as it does in muscle fibers.

The accumulations of immunoreactive SHBG in muscle fibers are substantial, and this was verified by Western blot analysis of muscle extracts that contain little blood contamination. Because muscle lacks SHBG mRNA, we conclude that the SHBG in this tissue must be sequestered from the blood. This could occur through interactions between SHBG and tissue-specific extracellular proteins, as in the mammalian endometrial stroma, where the sequestration of plasma SHBG is mediated by ligand-dependent interactions between SHBG and the fibulin family of extracellular matrix-associated proteins (3).

The preference of SHBGs in different fish for androgens vs. estrogens varies, but all fish SHBGs studied so far bind the sex steroid precursor, androstenedione, which distinguishes them from mammalian SHBGs. The obvious question is whether this might be related to a functional property of fish SHBGs that is not shared with mammals, and/or the role SHBG appears to play in controlling the brachial flux of sex steroids between the blood circulation and aqueous environment. Although our in vivo studies all point to a mechanism that serves to limit the brachial release of sex steroids rather than the sequestration of relatively low concentrations of steroids from water, this may occur under some circumstances, e.g. in relation to reproductive behaviors during the spawning season (19).

The brachial uptake and release of steroids by fish are influenced by a variety of factors (opercular beat rate, stress, etc.) that our experimental model does not accommodate, but it does provide a very simple system to monitor SHBG ligand uptake that is remarkably consistent between animals regardless of size or sex. Others have reported that the blood levels of endogenous steroids in fish can be monitored by their concentrations in water (19). Therefore, in pilot experiments we tried to detect SHBG ligands in the water in which zebra fish had been kept under the same experimental conditions used to monitor radiolabeled steroid uptake, and this was done by subjecting water samples directly to an SHBG ligand-binding assay. However, despite the pM sensitivity of this type of assay, we found no evidence of SHBG ligands in the water.

It has recently been reported that the stickleback (Gasterosteus aculeatus) has an enormous capacity to rapidly uptake testosterone and estradiol from water (26). Our results confirm this in another teleost and demonstrate that the capacity of zebra fish to specifically uptake SHBG ligands far exceeds the release of these steroids back into water. Therefore, we conclude that the high SHBG concentration in filament blood normally functions to retrieve any free ligands that happen to diffuse passively across the gills. This might also explain why fish SHBG binds androstenedione with such high affinity because it would provide a mechanism to limit its inappropriate release as a pheromone. However, what intrigued us most was the possibility that this extraordinarily efficient mechanism, which appears to have evolved to control the loss of endogenous SHBG ligands, might serve as a portal for trace amounts of anthropogenic compounds that happen to bind fish SHBGs with high affinity. The fact that trace amounts of radiolabeled SHBG ligands can be sequestered from water by live fish within minutes suggests this might be true.

One of the most well-studied xenobiotics in waste water systems is ethinylestradiol, and this potent synthetic estrogen binds SHBGs from several fish species (15, 25). Our data now demonstrate that fish very rapidly sequester trace amounts of ethinylestradiol from water most likely through binding to SHBG in the brachial filaments. Moreover, very little of the ethinylestradiol taken up by the fish in this way is released back into the water. Although additional studies are required to determine exactly where the ethinylestradiol accumulates within the animals, preliminary studies of the tissues of animals exposed to radiolabeled ethinylestradiol have shown that it accumulates in the brain, ovaries/eggs, and muscle (data not shown). The latter is of interest, given the high amounts of immunoreactive SHBG we observed in muscle, and this suggests that other anthropogenic SHBG ligands could accumulate in the muscle of fish.

Pharmaceutical use of ethinylestradiol accounts for its presence in waste water systems. What is less appreciated is that synthetic progestins are almost always used in combination with ethinylestradiol, and in significantly greater concentrations. These progestins are usually extensively metabolized, sometimes to more biologically active progestogenic compounds (27), before being excreted in urine. Although knowledge of the concentrations of synthetic progestins in waste water systems is limited, norethindrone levels have exceeded those of ethinylestradiol (23). Therefore, it is of particular interest that zfSHBG has a relatively high affinity for at least two of the most common used progestins in pharmaceutical preparations, i.e. levonorgestrel and norethindrone. Although we have not studied the uptake of radiolabeled progestins by zebra fish directly, norethindrone limits the uptake of radiolabeled testosterone in the same way as natural steroid ligands with similar affinity for SHBG and is, therefore, likely to be sequestered effectively from water through binding SHBG in the brachial filaments.

Like human SHBG (28, 29), fish SHBGs are capable of binding nonsteroidal ligands, and we examined whether some of the more widely recognized xenoestrogens bind to zfSHBG. Among these, benzanthracene and dichlorodiphenyl dichloroethylene (DDE) bind zfSHBG poorly, whereas its affinity for bisphenol A is negligible. It remains to be determined if low-affinity ligands such as benzanthracene and DDE could be as effectively sequestered as ethinylestradiol via SHBG in gills, but these experiments will be difficult to perform in the absence of radiolabeled compounds or sensitive methods for measuring them. However, it is possible that other anthropogenic compounds in aquatic environments may be even better ligands for fish SHBGs, and innovative methods for screening for such compounds are warranted. For instance, we have recently explored through the use of in silico modeling methodologies that have identified novel nonsteroidal ligands that bind zfSHBG with nanomolar affinities (unpublished data). Although the latter studies demonstrate the feasibility of this approach, the ligand-binding properties of fish SHBG vary considerably between species, especially in relation to their binding of synthetic compounds. Moreover, it has also recently been reported that a second SHBG gene is expressed in a very distinct tissue-specific manner in salmonids, and this SHBG paralogue has a very different steroid-binding specificity when compared with the SHBG produced by salmonid liver (30). Therefore, this diversity in fish SHBG needs to be examined in greater detail because it represents a potential route for bioaccumulation of anthropogenic compounds in food chains that could adversely impact ecosystems and human health.

In conclusion, we have demonstrated that SHBG in fish gills controls the flux of natural sex steroids between fish and their environment, and is a portal that can be breached by synthetic compounds with potential xenobiotic activities. Therefore, it will be important to extend these studies to determine how changes in plasma SHBG (31) influence the gill SHBG content and ability to control the uptake and release of both natural and synthetic ligands of SHBG in specific fish species.


   Acknowledgments
 
We thank Caroline Underhill for technical assistance.


   Footnotes
 
This work was supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada. G.L.H is a Tier I Canada Research Chair in Reproductive Health.

Disclosure Statement: The authors have nothing to declare.

First Published Online May 15, 2008

Abbreviations: CHO, Chinese hamster ovary; DDE, dichlorodiphenyl dichloroethylene; DHT, 5{alpha}-dihydrotestosterone; SHBG, sex hormone-binding globulin; zfSHBG, zebra fish SHBG.

Received March 20, 2008.

Accepted for publication May 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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