* USEPA, RTD, NHEERL, Endocrinology Branch, Research Triangle Park, North Carolina;
University of Florida, Department of Zoology, Gainesville, Florida;
St. Mary's College, St. Mary's City, Maryland; and
U.S. Environmental Protection Agency, NHEERL, Mid-Continent Ecology Division, Duluth, Minnesota
Received February 1, 2001; accepted March 6, 2001
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ABSTRACT |
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Key Words: environmental androgens; Kraft pulp and paper mill effluent; masculinized female mosquitofish anal fin; gonopodium; androgen receptor; glucocorticoid receptor; AR binding; AR gene expression; in vitro; Fenholloway River, Florida.
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INTRODUCTION |
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In sexually dimorphic vertebrate species, males and females possess species-specific, androgen-dependent, and estrogen-dependent secondary sex characteristics. Although androgen-exposure induces the formation of a male-like anal fin in the female mosquitofish, the presence of masculinized female fish does not necessarily indicate that an endocrine-disrupting chemical (EDC) acts as an AR agonist, as other mechanisms of action could also produce these effects. For example, it has been hypothesized that inhibition of aromatase activity, the enzyme that converts testosterone to estradiol, could masculinize fish by causing an accumulation of testosterone (Orlando et al., 1999).
In the present study, male and female mosquitofish were collected simultaneously with some of the water samples from the Fenholloway and Econfina Rivers (Fig. 1). The Econfina River is an uncontaminated "reference" site that arises from the same headwaters as the Fenholloway River. We hypothesized that since mosquitofish are masculinized downstream from the discharge of the Buckeye-Kraft mill this water would display androgenic activity. In the current study we used in vitro mammalian cell-based assays containing the human androgen (hAR) or glucocorticoid (GR) receptors to determine if either androgenic- or glucocorticoid-like activity was present in the river water downstream from the plant. GR-induced gene expression was examined in PME samples, because it has been suggested that corticosteroids might be present and induce masculinization in the female fish, and secondly, because both GR and AR will induce luciferase gene expression via the MMTV promoter in vitro. Similar gene-expression assays with AR from fish have not yet been developed.
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MATERIALS AND METHODS |
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Collection and analysis of male and female mosquitofish.
Several hundred male and female mosquitofish were collected at 3 different times of the year from the Fenholloway (contaminated Site 3, near the Carlson Bridge on SR 361A) and Econfina (near the bridge on SR 98) Rivers. The data included herein are from female mosquitofish collected in May 1999 (n = 50 and n = 49 from the Econfina and Fenholloway Rivers, respectively) when water samples were taken for in vitro analysis of androgenic activity. Fish were transported in coolers and placed in aquaria with fresh water from their respective rivers. The following day, fish were anesthetized and the following morphometric data were collected: total body weight and length, anal fin length, number of segments in the longest ray of the anal fin, and liver, gonad, and brain weights. These data, the number of segments in the anal fin being the most robust, sexually dimorphic trait in this species, are presented here to confirm the abnormal, male-like sexual phenotype of the female fish in the Fenholloway River. Additional morphometric, endocrine (ovarian aromatase activity, body testosterone concentration, and ovarian histological data are presented in a companion paper, Orlando et al., in preparation).
Preparation of water samples by solid-phase extraction.
For some assays, site water was concentrated using solid-phase extraction (SPE) C-18 columns (J.T. Baker, #702006, 6-ml Bakerbond Octadecyl Extraction Columns). Columns were attached to a vacuum manifold and primed with 4 ml of methanol (Fisher Scientific), followed by 2 ml of distilled water. Columns remained wet during the entire priming process. After conditioning, 40 ml of site water was passed over a column. The column was then dried under vacuum for 1 h. The columns were eluted with 2 3-ml aliquots of methanol. In the CV-1 assay, the methanol was evaporated under vacuum and the sample was resuspended in 300 µl of phosphate-buffered saline (100 mM) plus 1% gelatin for the testosterone RIA; 500 microliters of serum-free DMEM (Gibco Brl.) for the COS assay; or 2 ml of DMEM plus 5% dextran charcoal-stripped serum (Hyclone).
Preparation of water samples: Dosing media made with site water.
For some assays, powdered DMEM (Gibco) with 3.7 g NaHCO3 (ICN Biochemicals) was reconstituted with 1 liter of site water from each site and adjusted to a pH of 7.4. Media were sterile filtered (0.2-micron, Nalgene bottle-top filters), supplemented with 5% dextran charcoal serum (HyClone) and antibiotics (Gibco), and stored at 4°C wrapped in aluminum foil in the dark until use in the CV-1 transcriptional-activation assay.
COS whole cell human androgen receptor (hAR) binding assay.
The COS whole cell-binding assay was used to evaluate the ability of the SPE concentrated water samples from 40 ml of each (dried down and resuspended in 500 µl of medium) from 5 sites to compete with [3H] R1881 (a synthetic androgen) for binding to the hAR. In 3 blocks, 2 replicates per block, COS cells (SV-40 transformed monkey kidney line ATCC # CRL-1650) were transiently transfected with the hAR expression vector pCMVhAR as described by Wong et al. (1995). COS cells were plated at 200,000 cells/well in 12 plates and transfected with 1 µg of pCMVhAR (from Dr. Elizabeth Wilson, UNC at Chapel Hill). After a 3-h transfection period, cells were washed with DPBS and incubated overnight in 2 ml 10% FBS DMEM. Twenty-four hours later, medium was aspirated and replaced with 200 µl of serum-free/phenol red-free DMEM with R1881 plus 200 microliters of medium made from SPE-extracted site water (400-µl incubation volume, with a concentration factor of 40x). Cells were incubated for 2 h with 5 nM [3H] R1881 at 37°C under an atmosphere of 5% CO2. Nonspecific binding (NSB) was determined by adding 100-fold molar excess of unlabeled R1881 to NSB tubes. Cells were washed in phosphate-buffered saline and lysed in 200 µl ZAP (0.13 M ethylhexadecyldimethylammonium bromide with 3% glacial acetic acid). The lysate was added to 5 ml OPTI-fluor scintillation cocktail (Packard Bioscience, The Netherlands) and radioactivity was counted using a Beckman LS 5000 TD counter (Beckman, Irvine, CA).
CV-1 AR and GR 40-dependent transcriptional activation assays.
Three experiments, each with several replicates, were conducted to determine if PME induced AR- or GR-dependent gene expression in CV-1 cells (monkey kidney line, ATCC # CCL-70). In these experiments, 200,000 CV-1 cells were plated in a 60-mm dish and then transiently cotransfected with 1 µg pCMVhAR and 5 µg MMTV-luciferase reporter using 5 µl Fugene reagent in 95 µl serum-free medium as per the manufacturer's protocol (Boehringer Mannheim, Germany). Twenty-four h after transfection, medium was aspirated and replaced with 2 ml of DMEM plus 5% DCC (1) reconstituted extracted sample (20-fold concentration factor) or (2) with 2 ml medium prepared with water from each of the 5 sites (without any concentration). Cells were then incubated at 37°C under 5% carbon dioxide. After 5 h of exposure, medium was removed, and cells were washed once with phosphate-buffered saline and harvested with 500 µl lysis buffer (Promega). Relative light units of 0.05-ml aliquots of lysate were determined using a Monolight 2010 luminometer (Analytical Luminesce).
Experiments 1 and 2 examined AR agonist activity using extracted and unextracted site water. In the first experiment, cell media were made with site water (4 replicates), while in a second experiment, 20 ml SPE extracted site water (prepared as described above from 40 ml divided into 2 duplicates, 3 replicates) was used to determine if the androgenic activity eluted with the more lipophilic fraction. In the first experiment, 1 µM hydroxyflutamide was added to half of the samples with PME to see if this potent antiandrogen would block the PME-induced luciferase activity. Cells also were exposed to 1 nM DHT as a positive control. A third experiment was conducted to determine if PME would induce glucocorticoid-dependent transcriptional expression. CV-1 cells were transfected as above with 1 µg of pCMVhGR instead of pCMVhAR. CV-1 cells containing the glucocorticoid receptor (GR) were then dosed with media made with site water (2 ml, concentration factor 0.5 x) from all 5 sites, incubated, and luciferase activity measured as above for CV-1 cells transfected with the hAR. In addition, CV-1 cells were incubated with 1 nM dexamethasone, a potent GR agonist, which induces MMTV-luc gene expression as a positive control (3 replicates).
COS cell immunocytochemistry.
The following experiment was conducted to visualize, by immunofluorescence, the ligand-induced nuclear translocation of hAR in COS cells. Two chamber slides (Nunc) were seeded with 100,000 cells/chamber in 2 ml DMEM (Gibco) supplemented with 10% FBS (HyClone) after which cells were transfected with 0.5 µg pCMVhAR (as per Wong et al., 1995). Following transfection, 2 ml DMEM plus 5% DCC medium was prepared from site water (without concentration), added to slides, and incubated for 24 h at 37°C under 5% CO2. The next day, medium was removed, cells washed once with DPBS (Dulbecco's phosphate buffered saline), allowed to dry for 45 min at room temperature, fixed for 10 min with 95% ethanol (-20°C), blocked with 5% BSA (Sigma, in DPBS), and incubated overnight with primary AR antibody (Affinity Bioreagents, 1:1000) at 4°C. The following day, cells were washed once with DPBS and incubated with fluorescently labeled secondary antibody (Molecular Probes) for 30 min at room temperature. To visualize the nuclei, cells were counter-stained with DAPI (Sigma), a DNA stain, mounted with fluoromount (Electron Microscopy Sciences), and the slides examined using a Nikon Optiphot-2 Microscope at 200 x magnification. The localization of the AR was classified as either perinuclear or nuclear in a blinded fashion from 10 randomly selected fields from a slide for each site. In another experiment, cells were exposed to 1 nM DHT, as a positive control.
Testosterone (T) radioimmunoassay (RIA).
A testosterone RIA was conducted as previously described (Kelce et al., 1997 modifed from Cochran et al., 1981; Ewing et al., 1984; and Schanbacher and Ewing, 1975) with the water samples from the 5 sites (3 replicates per sample). Forty ml of water was extracted using an SPE protocol described above (100% methanol elution). After evaporation of the methanol, samples were resuspended in 300 µl PBS-G (100mM), vortexed 30 s and placed in a 45°C water bath. These samples have been concentrated 133-fold (from 40 ml to 0.3 ml). After 10 min, samples were removed from the water bath, at which time [3H] testosterone (10,000 DPM in 100 µl, 1 mCi/ml, DuPontNEN), T-antibody ((1:10,000) ICN in 100 µl) and 400 µl PBS-G were added to each sample. Samples were then vortexed and incubated overnight at 4°C. On the following day, a charcoal mixture (1 ml) was added to each sample for 20 min, and then each sample was vortexed (30 s) and centrifuged (10 min at 2000 x g). After centifugation, the supernatant was combined with 15 ml of OPTI-fluor scintillation cocktail (Packard Bioscience, The Netherlands) in a 20-ml plastic scintillation vial and the level of radioactivity in the samples was quantified by scintillation counting for 2 min (Beckman LS 5000 TD, Irvine CA).
Statistical analyses.
Binding data were expressed as percent bound minus nonspecific binding, whereas the gene expression data were expressed as fold induction above the medium value, calculated for each replicate. When warranted, fold induction data were log-transformed to correct for heterogeneity of variance, typical for this sort of biological response. In addition, the CV-1 AR and GR assay data were recalculated as percent of the DHT (for AR) or dexamethasone (for GR) positive controls so the effects of PME could be compared to these potent inducers of gene expression. In vitro data and the number of segments in the anal fin were analyzed by ANOVA using PROC GLM available with SAS version 6.08 on the U.S. EPA's IBM mainframe. Statistically significant effects (p < 0.05, F statistic) were examined using the LSMEANS procedure (t-test) to compare the sites with the media control group and river water from the 2 reference/control sites. Site-related differences in the distribution of AR (nuclear versus perinuclear), detected using immunohistochemistry, were analyzed using 2 and Fisher's exact tests.
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RESULTS |
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When the data from the CV-1 AR- and GR-dependent assays are displayed as percent response of the appropriate positive control (DHT for AR and dexamethasone for GR), it is evident that PME has considerable AR agonist activity, but induces little or no gene expression via the GR (Fig. 5). The slight simulation of GR at Site 1, the highest value in the water samples, did not differ significantly from the medium control value (4.04 ± 2.83 for Site 1 versus 1.0 for medium control, p > 0.15).
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DISCUSSION |
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Consistent with our observations about the androgencity of PME, another research group also has presented data indicating that androgenic activity is displayed by Fenholloway River water (Howell and Angus, 1999). Jenkins et al. (2000, 2001) identified 2 fractions of Fenholloway River samples with androgenic activity. One of these peaks contained the steroidal androgen androstenedione at a concentration of 0.14 nM (also described 15 ng androstenedione per liter by Raloff, 2001). In female mosquitofish, it is possible that androstenedione would be converted in tissues to more androgenic steroidal androgens such as testostereone and dihydrotestosterone as in mammals (Labrie et al., 1989
). However, it is uncertain if 0.14 nM androstenedione alone would be of sufficient potency to produce the responses that we observed in vitro or if it would directly masculinize female mosquitofish in the field. There may be other androgens along with androstenedione present in the PME. In our study, testosterone was measured by RIA in the water samples from the 5 sites (3 replicates per sample), because it had been hypothesized that PME contains androgens, possibly including testosterone, produced from microbial metabolism of phytosterols (Conner et al., 1976
).
The data presented herein are derived from river water samples collected concurrently with mosquitofish, so the in vitro data could be compared with the data on the reproductive physiology and morphology of the male and female mosquitofish from Sites 3 (Fenholloway) and 5 (Econfina; Orlando et al., in preparation). Masculinization of the female mosquitofish was confirmed by measuring the number of segments in the anal fin rays, which is an androgen-dependent secondary sex characteristic. Male mosquitofish from the Econfina or Fenholloway Rivers display about twice as many segments in the longest ray on the anal fin as a normal, unexposed female (40 versus 18, Econfina male versus female, respectively ) while the anal fin is only about 25% longer in the male than in the normal female. Female mosquitofish from Site 3 on the Fenholloway River are masculinized, as indicated by an overall 55% increase in the numbers of segments in the longest ray of the anal fin as compared to females from the Econfina River (p < 0.001). Masculinization has been achieved in female mosquitofish and killifish in the laboratory with exposures to either PME or metabolites of microbial degradation of chemicals present in the PME by such microbacterium as Mycobacterium smegmata (Conner et al., 1978; Denton et al., 1985). These observations led to the hypothesis, confirmed in the current study, that PME contained androgenic substances because masculinization of the ray segments in the anal fin of female mosquitofish also can be induced with androgens like methyltestosterone (Rodriquez-Sierra and Rosa-Molinar, 1990).
In addition to the "androgenic" hypothesis, Orlando et al., (1999) hypothesized that inhibition of the ovarian aromatase by PME could result in masculinization of the female fish by causing an accumulation of testosterone (T), the precursor of estradiol an aromatized steroid hormone. Hence, until now, it was not certain if PME was directly androgenic or if the observed masculinization was caused by alterations in steroid metabolism.
Our data indicate that PME does not masculinize female mosquitofish by displaying cortisol-like activity, as no GR-like activity was detected in vitro, although GR agonists could induce luciferase activity, because hGR and hAR transcriptional complexes both bind the hormone response element (HRE) on the MMTV promoter gene when activated by a ligand. The HRE for these steroids is a 15-bp palindromic consensus hormone-response element that binds the highly conserved amino-acid sequence in the DNA-binding domain of the receptor homodimer complex. As these receptors, and the progesterone receptor as well, are all from the same steroid hormone superfamily, even though separate endocrine functions have evolved, they all bind the same HRE on DNA (Martinez and Wahli, 1991). The lack of any hGR-mediated induction of luciferase, along with the ability of the well characterized antiandrogen hydroxyflutamide to block the induction of luciferase caused by PME binding supports the hypothesis that PME is not acting via GR. Even if GR-like activity was present in PME, it is unlikely that this would induce masculinization of female fish. In general, the corticosteroids antagonize the action of androgens in vivo via nonreceptor-mediated mechanisms and are more likely to demasculinize males than masculinize females (Konagaya and Max, 1986
).
Masculinization of female fish by Kraft mill effluent is not limited to Florida rivers. Morphological alterations consistent with exposure to androgens have been reported in other fish exposed to effluent from other pulp and paper mills and from other plants (sugar-beet processing plant; Hegrenes, 1999). On Jackfish Bay, Lake Superior, male and female white sucker fish (Catostomus commersoni) exposed to PME in the field displayed a variety of reproductive effects, including an increase in size and number of reproductive tubercles, which are androgen-dependent sex traits of males of this species (Wells et al., 1999). Wells and Van Der Kraak (2000) found that substances in PME competed for binding to the androgen receptor (AR) isolated in cytosolic preparations of fish testes, gonad, and brain. They also identified several known PME constituents that bound fish AR.
Due to the fact that some plant sterols and PME have been correlated with the masculinization of female fish, it is tempting to hypothesize that these substances could also be responsible for some of the reproductive impairments identified in other fish species (fathead minnow, brown trout, white sucker, and rainbow trout) exposed to PME (Lehtinen et al., 1999; Munkittrick et al., 1998
; NCASI, 1997
; Tremblay and Van Der Kraak, 1999
). However, PME contains thousands of toxicants, including dioxins, which likely alter reproduction and development via alternative (none AR-mediated) mechanisms. Future PME-exposure studies should incorporate an assessment of androgen-dependent traits appropriate for the fish species of concern, along with standard measures of reproductive fitness. Only carefully integrated laboratory and field studies will be able to sort out which toxicants in PME are responsible for the different reproductive effects in exposed populations.
It is informative to contrast the methods used by Wells and Van Der Kraak (2000), with those employed herein. Wells and Van der Kraak examined the ability of PME constituents to bind fish AR isolated from cytosolic preparations from several tissues, whereas we examined the ability of PME to bind human AR in a mammalian whole-cell assay. The approach of Wells and Van Der Kraak has advantages, because it allows for AR binding to be evaluated in the vertebrate class and even in the species of concern. This could be a significant advantage if fish AR display different affinities for toxicants as compared to mammalian AR, as has been suggested (Makynen et al., 2000; Wells and Van der Kraak, 2000
). However, cytosolic preparations from tissues contain many different receptors in addition to AR, so it is possible that other factors in the cytosol could affect the results. In addition, binding data alone do not allow one to discrminate androgens from antiandrogens. In contrast, our approach, using mammalian AR and cell lines, allows us to discriminate between AR agonists and antagonists, which is not possible at this time with AR assays for most lower vertebrates. However, both approaches have merit and should be utilized until in vitro binding and gene expression assays for nonmammalian vertebrates are developed. When sufficiently standardized and validated, they can replace or augment the methods used herein.
Identification of an environmental mixture with androgenic activity resulting from anthropogenic activity is a novel observation. Over the last few years, several environmental antiandrogens that act as AR antagonists have been detected, including the fungicides vinclozolin and procymidone, the herbicide linuron (Gray et al., 1999), the pesticide fenitrothion (Tamura et al., 2001
) and metabolites of DDT, especially p,p` DDE (Gray et al., 1999
; Kelce et al., 1997
). However, until now, based upon the preponderance of observed AR antagonism, it had been suspected that most, if not all, environmental toxicants that bound AR would be antiandrogenic. As we gather more data about endocrine-disrupting chemicals, unexpected effects exerted via additional mechanisms of action will almost certainly be identified.
The detection of chemicals in the environment with androgenic activity raises questions about the potential for these substance(s) to bioaccumulate and move through the food chain. Androgenic hormones are important for reproduction in all vertebrates. Since the biochemical and molecular mechanisms of hormone action are highly conserved among the vertebrates (Norris, 1996), exposure to significant levels of androgenic substances could adversely affect reproduction in all species, including humans. In fact, the adverse effects of androgenic drugs, taken during human pregnancy are well established, and are to be avoided (Schardein, 2000
). For these reasons, it is important that the androgenic chemicals in PME be identified and their potential to adversely affect other vertebrates determined. In summary, we report here that some component(s) in water collected from sites downstream from a Kraft pulp and paper mill on the Fenholloway River displays androgenic activity. It appears that the in vitro potency of PME from the Fenholloway River may be sufficient to account for masculinization of the female mosquitofish (90% affected) that we collected from Site 3 on this river (Fig. 1
).
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ACKNOWLEDGMENTS |
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NOTES |
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The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, and was approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.
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