TY - JOUR
AU - Kirk, Marion
AB - Abstract The Fenholloway River near Perry, Florida, receives effluent from a paper mill and contains populations of masculinized female eastern mosquitofish, Gambusia holbrooki. A previous study identified the androgen precursor androstenedione at a low concentration (0.14 nM) in water samples from the river. The present study makes use of a toxicity identification and evaluation approach that includes solid phase extraction and high pressure liquid chromatography purification, androgen receptor transcription assays, and liquid chromatography mass spectroscopy to identify and characterize steroids in the Fenholloway River sediment. Androstenedione (2.4 nM) and progesterone (155 nM) were identified in the river sediment at concentrations greater than in the river water column (0.14 nM androstenedione, and 6.5 nM progesterone). Spring Creek, a comparison stream that does not receive mill effluent, contained low levels of progesterone (0.3 nM) but no androstenedione in the sediment. The data are consistent with the hypothesis that pine pulp-derived phytosteroids in the paper mill effluent accumulate in river sediment where they are converted by microbes into progesterone and this into androstenedione and other bioactive steroids. Equally important is that normal streams with much less organic matter still contain progesterone, but at dramatically lower levels. The presence of androgens and androgen precursors in the river water and sediment likely contributes to the masculinized phenotype of the female Gambusia holbrooki in the Fenholloway River. environmental androgens, phytosteroids, masculinized mosquitofish, Gambusia holbrooki, androgen receptor, androgen-dependent gene expression, Fenholloway River, Florida In recent years it has become evident that a variety of chemical compounds are being introduced into the environment that have the potential to disrupt endocrine activity in human and wildlife populations (e.g., Colborn, 1995; Guillette and Craine, 2000; Stahlschmidtallner et al., 1997; Tyler et al., 1998). Research on endocrine disruptors has predominantly focused on environmental estrogens since most compounds identified thus far have hormone-like activity of weak estrogens. However, environmental androgens have also been detected. Three coastal streams in Florida that receive effluent from paper mills contain populations of masculinized female mosquitofish, Gambusia holbrooki, and other fishes. These include Elevenmile Creek (Howell et al., 1980), Fenholloway River (Bortone and Drysdale, 1981; Howell et al., 1980), and Rice Creek (Bortone and Cody, 1999). Parks et al.(2001) used an androgen-dependent gene expression system to detect unidentified androgenic substances in the Fenholloway River water collected downstream from the paper mill. Using a toxicity identification and evaluation approach, we identified 0.14 nM androstenedione in the water of the Fenholloway River (Jenkins et al., 2001). Larsson et al.(2000) demonstrated the masculinizing effects of paper mill effluent on embryonic development of the viviparous fish, the eelpout (Zoarces viviparous). Thomas et al.(2002) identified six androgenic steroids in water and sediment samples collected from estuaries and sewage treatment effluents in the United Kingdom. The results suggest that androgenic compounds are more abundant in aquatic environments than previously suspected. The objective of the present study was to identify androgenic substances in the Fenholloway River, Perry, Taylor County, Florida, associated with paper mill effluent that were responsible for masculinizing female mosquitofish in the river. Androgenic steroids have been indirectly implicated as the causal agents for masculinization of fish downstream of paper mills in the southern United States. Conner et al.(1976) showed that pine pulp and paper mill waste effluent contain an abundance of phytosteroids, most notably β-sitosterol. Tremblay and Van Der Kraak (1999) provided evidence that β-sitosterol in the paper mill effluent has a role in masculinizing resident fish populations. Androstenedione detected in the Fenholloway River water column (Jenkins et al., 2001) could be derived from the pine pulp itself or from microbial degradation of abundant phytosteroids such as β-sitosterol, campesterol, and stigmastanol, which are present in wastes produced from the processing of pine tree into pulp. To test the hypothesis that androstenedione is derived by microbial transformation from complex plant phytosterols, we analyzed the sediment of the Fenholloway River for the presence of intermediate steroids in the biosynthetic pathway to androstenedione. The purpose of the present study was to determine whether the Fenholloway River sediment downstream of a paper mill effluent outfall contains additional steroids with androgen receptor mediated transcriptional activity that serve as precursors to androstenedione. MATERIALS AND METHODS Sediment collection and methanol extraction. River sediment was collected in the months of May 2002 and November 2002 from the Fenholloway River where it crosses county road 361A, east of Perry, Florida, and 3.9 river km downstream from the settling ponds of a large paper mill. No tributaries or other effluents enter the river between the collection site and paper mill. Above the paper mill, the Fenholloway River was dry and not a possible control site. Sediment from a comparison stream free of paper mill effluent was collected from Spring Creek, a tributary of the Fenholloway River, west of Perry, Florida, at the inlet of Pimple Creek. River sediment was collected with a 2.8 mm mesh dip net from the top 20 cm of sediment. Water was allowed to drain thoroughly from the dip nets, leaving only pore water in the sediment. River sediment (2 liters) was immediately mixed with 2 liters of 100% HPLC grade methanol (MEOH; Fisher Scientific Inc., Atlanta, GA). Immediately upon returning to the laboratory and within 24 h, 100% MEOH was added to bring the mixture to 80% MEOH by adding 2.4 liters of 100% MEOH to 4 liters of the 1:1 sediment-MEOH sample. Five 0.5-liter portions of the 80% MEOH mixtures from the Fenholloway River and five 0.5-liter portions of the Spring Creek sediment (equivalent to 0.156 liters of original sediment) were filtered through acid-extracted glass wool. The filtrate was then vacuum filtered through 0.8 μm and 0.1 μm cellulose filters (Varian, Walnut Creek, CA). The filtrate was passed through methanol-washed solid-phase extraction cartridges (Mega Bond Elut, 6 ml, C-18; Varian). The solid-phase eluant from 0.5-liter sediment −MEOH mixture was dried under N2 followed by lyophilization. Fractions were reconstituted in 1 ml 100% acetonitrile for high pressure liquid chromatography (HPLC) fractionation. HPLC fractionation of the solid phase eluant. Solid phase extracts were fractionated using a 30 min (1 ml/min) gradient HPLC system with 0.25% metaphosphate (solvent A) and 100% acetonitrile (solvent B) on a Varian 4.5 cm × 4 mm Microscorb-MV, C18 column with a Dynamax ultraviolet detector with detection at 210, 220, and 235 nm (Varian) as previously described (Jenkins et al., 2001). All HPLC solvent components were purchased from Fisher Scientific. From 100 μl sample injections, 30 1 – min fractions were collected. This was repeated for 10 sample injections, to give 10 ml of each of the pooled minute fractions. The 30 fractions were dried under N2 and reconstituted in 1 ml 100% ethanol (Sigma Chemical Co., St. Louis, MO) for transcription assays and HPLC purification. Efficiency of recovery of MEOH/solid phase extractions. To one 0.5-liter 80% MEOH fraction of Fenholloway River sediment, 100 nmol of 17β-estradiol cypionate was added and stirred for 12 h at room temperature. Three aliquots were filtered through glass wool, 0.8 μm, and 0.1 μm cellulose filters, and eluted through a solid phase extraction cartridge (Mega Bond Elut, 6.0 ml). HPLC resulted in an average recovery of 40% of the 17β-estradiol cypionate, which eluted at 23.5 min. Androgen receptor transcription assays. Androgen activity in river water sediment extracts was determined in transient cotransfection assays using a luciferase reporter gene assay (He et al., 2001; Jenkins et al., 2001; Kemppainen et al., 1992). Briefly, monkey kidney CV1 cells (0.425 × 106 cells/6 cm dish) were transfected using the calcium phosphate DNA precipitation method with the human androgen receptor expression vector pCMVhAR (25 ng/dish) and the luciferase reporter vector under control of the mouse mammary tumor virus promoter (MMTV-Luc, 5 μg/dish). Cells were incubated for 24 h at 37°C with the indicated concentrations of dihydrotestosterone (DHT) or 10 μl additions of purified river sediment extract fractions. Representative luciferase activity assays are expressed in optical units relative to the no hormone control. The MMTV-luciferase assay performed in CV1 cells demonstrates human androgen receptor mediated gene activation, which lacks absolute specificity for activation by androgen (Kemppainen et al., 1992). Liquid chromatography-mass spectrometry. The HPLC, C18 column fractions of the Fenholloway River sediment that induced androgen receptor mediated transcriptional activity were further purified using a 45 min gradient HPLC solvent system. In place of phosphate, solvent A contained 10 mM ammonium acetate (Fisher Scientific) to minimize interference in the mass spectrometry and solvent B was 100% acetonitrile. Individual chromatogram peaks were collected, dried under N2, and reconstituted in 100% methanol for liquid chromatography-mass spectrometry (LCMS) verification on a Hewlett-Packard 1050 system (Avondale, PA). A 10-cm × 2.1-mm, C-8 Aquapore column (Applied Biosystems, Foster City, CA) with a 12 min linear 0 to 100% methanol gradient in 10 mM ammonium acetate was used to separate the components. Elutants were passed into an electrospray interface of a PE Sciex API III triple-quadrupole mass spectrometer (Foster City, CA). Multiple reaction monitoring was used for the final comparison of unknown compounds with standards. In this procedure, the parent ion was selected with the first quadrupole and passed into the second quadrupole, containing argon gas. Collision of the parent ion with the argon produced fragment ions. Monitoring of a specific parent ion by the first quadrupole and ion fragments in the second and third quadrupoles constituted the multiple reaction monitoring method. Elution of the selected parent-fragment pair at the same chromatographic retention time as a standard confirmed the identity of the steroids. RESULTS Sediment collected from the Fenholloway River was a highly organic, strongly anaerobic, and noxious black sludge. In contrast, sediment from Spring Creek was largely sand with limited decayed leaf and other organic matter. The acetonitrile/phosphate HPLC separations of the Fenholloway River sediment revealed a large heterogeneous peak of tall oil between fractions 12 and 22 min, consisting of fat-soluble resinous byproducts from the manufacture of wood pulp (Fig. 1A). Smaller peaks were resolved on top of the tall oil peak with detection at 210, 220, and 235 nm. Sediment from Spring Creek revealed a single prominent peak (fraction 26) (Fig. 1B). Androgen Receptor-Mediated Transcriptional Assays of Initial HPLC Separations Figure 2 illustrates the androgen receptor (AR)-mediated transcriptional activity determined in CV-1 cells of HPLC fractions collected at 1-min intervals from C18 columns with increasing acetonitrile concentrations. The Fenholloway River sediment fractions at 6, 16, and 19 min induced androgen receptor-mediated transcriptional activity at levels that were significantly greater than background (p < 0.05, ANOVA with Tukey HSD post hoc comparisons). The HPLC gradient of the C18 columns separates compounds using increasing concentrations of acetonitrile, so that compounds eluting in the 6 min fraction are more polar than compounds eluting at 19 min. The 6 min fraction induced a 5- to 13-fold increase in the androgen-dependent androgen receptor NH2-terminal and carboxyl-terminal interaction (He et al., 2001; Langley et al., 1995) when assayed in Chinese hamster ovary cells (data not shown). Compounds in the 16 min HPLC fraction weakly induced the NH2-terminal and carboxyl-terminal interaction as previously reported (Jenkins et al., 2001) but the 19 min HPLC fraction was not detected in this assay. Because of the androgen specificity of the androgen receptor NH2-terminal and carboxyl-terminal interaction assay, the data suggest that compounds in the 6 and 16 min fractions are androgens whereas those in the 19 min fraction are not. The 16 min fraction was also obtained from the HPLC fractionation of Fenholloway River water in a previous study and was shown to be androstenedione (Jenkins et al., 2001). From the sediment of Spring Creek androgen-dependent mediated transcriptional activity in CV-1 cells occurred in the 19 min and later HPLC fractions (Fig. 2B). No binding was detected in the 6 min and 16 min fractions, as was observed in the Fenholloway River sediment. Liquid Chromatography Mass Spectroscopy Prior to LCMS, the 6, 16, and 19 min fractions were further purified by C18 reverse phase HPLC using a 45 min acetonitrile gradient system. A principle component of the 16 min fraction had a retention time equivalent to that of androstenedione and a principle component of the 19 min fraction had a retention time equivalent to that of progesterone (Fig. 3). The androgenic compound(s) in the 6 min fraction did not coelute with any known steroid standard tested and remains uncharacterized (see Jenkins et al., 2001, for a list of standards). The 16 and 19 min fractions were collected separately, dried under N2, and reconstituted in 100% MEOH for LCMS-multiple reaction monitoring. Androstenedione was verified as the primary component of the 16 min fraction based on the identical retention time as the androstenedione standard of the parent ion and two fragment ions: 287/97 and 287/109 (Figs. 4A and 4B, only the 287/97 shown). The ratios of parent ion and two fragment ions: 287/109 and 287/97 were 49% for the standard and 51% for the Fenholloway River sediment. The primary component of the 19 min HPLC fraction was confirmed as progesterone based on the identical retention time as the progesterone standard of the parent ion and two fragment ions: 315/97 and 315/109 (Figs. 4C and 4D, only 315/97 shown). The ratio of the counts of the 315/109 to 315/97 from the standard and the Fenholloway River sediment were identical (64%). Steroid Levels in the Fenholloway River Sediment The preparatory HPLC purification methods used prior to LCMS resolved androstenedione and progesterone. HPLC quantitation of androstenedione and progesterone was based on peak area from five parallel samples as compared to standards and an estimation of 40% recovery from MEOH extraction and elution from solid phase extract cartridges. Table 1 compares steroid concentrations in the sediment with previous data from the water of the Fenholloway River. Figure 5 illustrates the relative effectiveness of DHT, androstenedione, and progesterone in the transcription assay. DHT has a high affinity for the AR that result in a transcriptional response 10–100-fold greater than androstenedione based on the concentration of ligand needed to induce a transcriptional response. Similarly, there was a 100–1000-fold greater sensitivity to DHT compared to progesterone. While the steroid concentrations in the river sediment samples and the results of the transcription assay are in general agreement, the transcriptional assay was used primarily as a means to detect river sediment fractions that contain compounds that induce a transcriptional response through the AR using DHT as a standard. More precise measurement of steroid concentration was achieved by HPLC absorbance measurements of purified samples. DISCUSSION Androgens are beginning to be recognized as potential environmental endocrine disruptors (Thomas et al., 2002). Reports of environmental androgens have focused predominantly on paper mill effluent (Bortone and Cody, 1999; Bortone and Davis, 1994; Bortone and Drysdale, 1981; Denton et al., 1985; Howell et al., 1980; Larsson et al., 2000; Parks et al., 2001). However, other sources of environmental androgens have been identified. Plant-derived compounds have been associated with masculinization of female catfish collected downstream from the outfall of a sugar beet processing plant (Hegrenes, 1999). Effluent from a sewage treatment works was also reported as a possible source of chemicals that may have caused the masculinization of the female catfish. Estrogenic compounds have been widely reported in treated sewage effluent (e.g., Kuch and Ballschmiter, 2000; Matsui et al., 2000; Metcalfe et al., 2001; Rodgers-Gray et al., 2001; Thomas et al., 2001). Thomas et al.(2002) identified six androgenic steroids in the effluent from a plant where sewage received only primary treatment. These included dehydrotestosterone, androstenedione, androstanedione, 5β-androstane-3α,11β-diol-17-one, androsterone, and epiandrosterone. The concentration of androstenedione in the sewage effluent (0.37 nM) was greater than we found in the Fenholloway River water (0.14 nM; Jenkins et al., 2001) but less than that found in the Fenholloway River sediment (2.4 nM). In the present study, we determined that androstenedione is 17 times more concentrated in the sediment of the Fenholloway River downstream of a paper mill (2.4 nM) than previously found in the water column (0.14 nM). The presence of androstenedione in the Fenholloway River water column was recently confirmed (Durhan et al., 2002). In the Fenholloway River sediment, the concentration of progesterone (155 nM), a biosynthetic precursor of androstenedione, is 65 times greater than that of androstenedione. An assessment of unpublished HPLC data from the Fenholloway River water column (Jenkins et al., 2001) indicates a progesterone concentration of 6.5 nM, which is 46 times the concentration of androstenedione. In addition, we detected a substance (currently unidentified) with strong androgen receptor-mediated transcriptional activity in the 6 min HPLC fraction. This substance did not coelute with any of 23 tested standard vertebrate steroids (listed in Jenkins et al., 2001) but appears to be an active androgenic substance based on its ability to induce both transcriptional activity in the MMTV-luciferase transcription assay and the androgen-specific, androgen receptor NH2- and carboxyl-terminal interaction. Further studies are required to determine the identity of this compound. Durhan et al. (2002) reported an AR-binding compound in the water column of the Fenholloway River and determined that it is not androstenedione. There are obviously several potential androgens in paper mill effluent that are found at low concentrations in the water and at higher concentrations in the sediment. Interestingly, the sediment from Spring Creek, a stream not receiving paper mill effluent, also contained low levels of androgen receptor-mediated transcriptional activity. The 19 min fraction corresponded to the progesterone fraction of the Fenholloway River sediment and was quantitated at 0.3 nmol/l sediment by HPLC analysis. However, progesterone, which elutes in the 19 min fraction in this HPLC system, was not detected in the water column of Spring Creek (Jenkins et al., 2001). Progesterone has been detected at appreciable levels (maximum of 0.199 μg/l or 0.64 nmol/l) in water from streams that were subject to intense urbanization (Kolpin et al., 2002). Because Spring Creek is not known to be severely impacted by pollution from Perry, Florida, it is likely that the progesterone in this creek is a natural product of breakdown of leaf and other plant material. The absence of masculinized mosquitofish in Spring Creek (which are common in the Fenholloway River) indicates that the concentrations of progesterone and other steroids are below the threshold for biological activity in this creek. We hypothesize that androstenedione previously reported in the Fenholloway River (Durhan et al., 2002; Jenkins et al., 2001) derives from bacterial metabolism of progesterone present in the river sediment. The source of progesterone in the sediment downstream of the paper mill is likely microbial degradation of pine phytosterols in the pulp waste. At the paper mill, pulp wastes are pumped into outdoor settling ponds where initial biological breakdown takes place. The settling ponds appear to function as steroid generators where bacteria transform phytosterols by the side chain cleavage reaction into a host of steroids prior to their release to the Fenholloway River where they become incorporated in the river water column and bottom sediment. Conner et al.(1976) determined that the most common phytosterols in paper mill effluent tall oil are β-sitosterol (72%), stigmastanol (11%), and campesterol (8%). Nagasawa et al.(1969) demonstrated that cholesterol, β-sitosterol, and stigmasterol are degraded to androstenedione and androstenedienedione by common soil bacteria, including Arthrobacter, Bacillus, Mycobacterium, and Nocardia. Owens et al.(1978) showed that steroid-synthesizing bacteria also include E. coli from human feces, which convert cholesterol to androstenedione and androstenedienedione. The microbial enzyme used in the side-chain cleavage of cholesterol or phytosterols was identified as a lyase (Carlstrom, 1974). The finding of a relatively high concentration of progesterone (155 nM) in the sediment of the Fenholloway River suggests it is an intermediate in paper mill effluent phytosteroid degradation as seen in this modification of the pathway proposed by Conner et al.(1976).   \[{stigmasterol_{{\beta}-sitosterol}^{cholesterol}}{\rightarrow}cholest-4-en,\ 3\ one{\rightarrow}progesterone{\rightarrow}androstenedione\] Denton et al.(1985) and Howell and Denton (1989) first suspected microbial degradation of phytosterols as the source of androgenic steroids responsible for masculinization of mosquitofish exposed to paper mill effluent. They demonstrated that Mycobacterium smegmatis converts β-sitosterol to a steroid (or steroids) that masculinize female mosquitofish. Both our previous (Jenkins et al., 2001) and present studies validate the original hypotheses of Howell and Denton to include androstenedione as a bioactive precursor and potentially masculinizing steroid in paper mill effluent and sediment. Progesterone as a sediment-associated compound likely serves as an intermediate in the biosynthesis of androstenedione and other biologically active androgens. The androgen receptor transcription assay utilizes a human androgen receptor. However, we feel that the compounds found to be active in this system are likely to be responsible for the masculinization of female mosquitofish. This is supported by the homology between fish and human androgen receptor ligand binding domains, which share ~70% sequence similarity based on sequence comparisons between the human, rainbow trout (Takeo and Yamashita, 1999), and eel (Todo et al., 1999) androgen receptors. Competitive steroid binding studies also support a similar specificity for androgen binding among these receptors (Ikeuchi et al., 1999; Todo et al., 1999). The results predict a susceptibility of fish androgen receptor to activation by biologically active androgens with the resulting masculinization of female eastern mosquitofish, Gambusia holbrooki, by exposure to androstenedione and other androgenic precursors dissolved in water (Hunsinger and Howell, 1991). Durhan et al.(2002) doubted that androstenedione could contribute to the androgenic responses of female mosquitofish in the Fenholloway River. They based this on (1) lack of activity by androstenedione in their androgen receptor transcription assay, and (2) an unpublished report by Stanko et al.(2001) that androstenedione administered in the diet was ineffective in masculinizing female mosquitofish. We support our previous conclusion that androstenedione is a bioactive constituent of the river water (but not the only one) with two observations: (1) androstenedione has detectable activity in our androgen receptor transcription assay, and (2) further studies by Stanko (personal communication) have shown that androstenedione added to water is much more effective at masculinizing mosquitofish than it is when administered via the diet. TABLE 1 Mean Levels of Androstenedione and Progesterone from the Water Column and Sediment of the Fenholloway River and Spring Creek Collection area  Androstenedione  Progesterone  Note. Spring Creek is a tributary not receiving paper mill effluent. Values are represented in nM and μg/l; n = 5.  aData presented in Jenkins et al., 2001; 3.9 km below the paper mill.  bData calculated from HPLC chromatograms from Jenkins et al., 2001.  Water column      Fenholloway R.  0.14 nM ± 0.06 nM (0.04 ± 0.02 μg/l)a  6.55 ± 1.22 nM (2.06 ± 0.38 μg/l)b      Spring Creek  None detected  None detected  Sediment      Fenholloway R.  2.4 nM ± 0.8 nM (0.7 ± 0.2 μg/l)  155.1 ± 22.2 nM (48.8 ± 7.0 μg/l)      Spring Creek  None detected  0.3 nM ± 0.2 nM (0.09 ± 0.06 μg/l)  Collection area  Androstenedione  Progesterone  Note. Spring Creek is a tributary not receiving paper mill effluent. Values are represented in nM and μg/l; n = 5.  aData presented in Jenkins et al., 2001; 3.9 km below the paper mill.  bData calculated from HPLC chromatograms from Jenkins et al., 2001.  Water column      Fenholloway R.  0.14 nM ± 0.06 nM (0.04 ± 0.02 μg/l)a  6.55 ± 1.22 nM (2.06 ± 0.38 μg/l)b      Spring Creek  None detected  None detected  Sediment      Fenholloway R.  2.4 nM ± 0.8 nM (0.7 ± 0.2 μg/l)  155.1 ± 22.2 nM (48.8 ± 7.0 μg/l)      Spring Creek  None detected  0.3 nM ± 0.2 nM (0.09 ± 0.06 μg/l)  View Large FIG. 1. View largeDownload slide Crude reverse phase, C-18, 30 min HPLC chromatographs of sediment from Fenholloway River (A) and Spring Creek (B). Sediment was extracted in 80% methanol, vacuum filtered, and separated by C-18 solid phase extraction. HPLC solvent A was 0.25% meta-phosphate and solvent B was 100% acetonitrile with solvent A at 20% from 0 to 5 min, linearly increased to 100% solvent B to 20 min and held constant until 30 min. Detection in this chromatogram was at 235 nm. The full vertical scale constitutes 1 absorbance unit (106 μvolts). FIG. 1. View largeDownload slide Crude reverse phase, C-18, 30 min HPLC chromatographs of sediment from Fenholloway River (A) and Spring Creek (B). Sediment was extracted in 80% methanol, vacuum filtered, and separated by C-18 solid phase extraction. HPLC solvent A was 0.25% meta-phosphate and solvent B was 100% acetonitrile with solvent A at 20% from 0 to 5 min, linearly increased to 100% solvent B to 20 min and held constant until 30 min. Detection in this chromatogram was at 235 nm. The full vertical scale constitutes 1 absorbance unit (106 μvolts). FIG. 2. View largeDownload slide Androgen receptor-mediated transcriptional activity of HPLC Fenholloway River (A) and Spring Creek (B) sediment fractions (mean + SE, n = 2). Cotransfection assays were performed in monkey kidney CV-1 cells as described in Materials and Methods. Luciferase activity was measured in optical units. Minutes on the X axis refer to elution time of the 80% methanol fractions of Fenholloway River sediment after fractionation from solid-phase cartridges and C-18 HPLC shown in Figure 1. Dihydrotestosterone (DHT) was the positive control. The data are representative of at least three independent experiments. FIG. 2. View largeDownload slide Androgen receptor-mediated transcriptional activity of HPLC Fenholloway River (A) and Spring Creek (B) sediment fractions (mean + SE, n = 2). Cotransfection assays were performed in monkey kidney CV-1 cells as described in Materials and Methods. Luciferase activity was measured in optical units. Minutes on the X axis refer to elution time of the 80% methanol fractions of Fenholloway River sediment after fractionation from solid-phase cartridges and C-18 HPLC shown in Figure 1. Dihydrotestosterone (DHT) was the positive control. The data are representative of at least three independent experiments. FIG. 3. View largeDownload slide Reverse phase, C-18, 45 min HPLC chromatograph of Fenholloway River sediment of the previous 19 min HPLC chromatograph (B) and standards (A) (cortisol, androstenedione, deoxycortisol, and progesterone, respectively) following androgen receptor transcriptional assays. Solvent A was 0.1 mM ammonium acetate and solvent B was 100% methanol. The gradient was linear from 40% at 0 min to 100% at 30 min and onto 45 min. The full vertical scale constitutes 1 absorbance unit (106 μvolts). FIG. 3. View largeDownload slide Reverse phase, C-18, 45 min HPLC chromatograph of Fenholloway River sediment of the previous 19 min HPLC chromatograph (B) and standards (A) (cortisol, androstenedione, deoxycortisol, and progesterone, respectively) following androgen receptor transcriptional assays. Solvent A was 0.1 mM ammonium acetate and solvent B was 100% methanol. The gradient was linear from 40% at 0 min to 100% at 30 min and onto 45 min. The full vertical scale constitutes 1 absorbance unit (106 μvolts). FIG. 4. View largeDownload slide Liquid chromatography-mass spectrometry with multiple reaction monitoring (MRM) chromatograms of parent fragment 287/97. (A) Partially purified components of the 16 min HPLC fraction. (B) 100 pmol androstenedione standard. MRM chromatograms of parent fragment 315/97. (C) Partially purified component of the 19 min HPLC fraction. (D) 100 pmole progesterone standard. FIG. 4. View largeDownload slide Liquid chromatography-mass spectrometry with multiple reaction monitoring (MRM) chromatograms of parent fragment 287/97. (A) Partially purified components of the 16 min HPLC fraction. (B) 100 pmol androstenedione standard. MRM chromatograms of parent fragment 315/97. (C) Partially purified component of the 19 min HPLC fraction. (D) 100 pmole progesterone standard. FIG. 5. View largeDownload slide Relative effectiveness of DHT, androstenedione, and progesterone in the monkey kidney CV1 androgen receptor mediated transcription assay. Luciferase activity was measured in optical units of increasing standard steroid levels. FIG. 5. View largeDownload slide Relative effectiveness of DHT, androstenedione, and progesterone in the monkey kidney CV1 androgen receptor mediated transcription assay. Luciferase activity was measured in optical units of increasing standard steroid levels. This research was supported in part by the U.S. Environmental Protection Agency and by the National Institutes of Health, Public Health Service. It has not been subjected to government agency approval required for peer and policy review and therefore does not necessarily reflect the views of the Agency. No official endorsement should be inferred. 1 To whom correspondence should be addressed. Fax: (205) 975-6097. E-mail: raangus@uab.edu. The work was supported in part by the United States Environmental Protection Agency through grant number R826130 (R.A.A.), and by National Institutes of Health Public Health Service Grant HD16910 (E.M.W.). We thank Scott Linton for collection of some of the sediment samples. REFERENCES Bortone, S. A., and Cody, R. P. ( 1999). Morphological masculinization in poeciliid females from a paper mill effluent receiving tributary of the St. John’s River, Florida, USA. Bull. Env. Contam. Toxicol.  63, 150–156. Google Scholar Bortone, S. A., and Davis, W. P. ( 1994). Fish intersexuality as indicator of environmental stress. Bioscience  44, 165–172. Google Scholar Bortone, S. A., and Drysdale, D. T. ( 1981). Additional evidence for environmentally induced intersexuality in poeciliid fishes. ASB Bull.  28, 67. Google Scholar Carlstrom, K. ( 1974). Transformation of steroids by cell-free preparation of Penicillium lilacinum NRRL 895. IV. Enzyme catalyzed acyl transfer. Acta Chem. Scandinavia  28, 23–28. Google Scholar Colborn, T. ( 1995). Environmental estrogens: Health implications for humans and wildlife. Environ. Health Perspect.  103(Suppl. 7), 135–136. Google Scholar Conner, A., Nagaoka, M., Rowe, J. W., and Perlman, D. ( 1976). Microbial conversion of tall oil sterols into C19 steroids. Appl. Environ. Microbiol.  32, 310–311. Google Scholar Denton, T. E., Howell, W. M., Allison, J. J., McCollum, J., and Marks, B. ( 1985). Masculinization of female mosquitofish by exposure to plant sterols and Mycobacterium smegmatis. Bull. Environ. Contam. Toxicol.  35, 627–632. Google Scholar Durhan, E. J., Lambright, C., Wilson, V., Butterworth, B. C., Kuehl, D. W., Orlando, E. F., Guillette, L. J., Jr., Gray, L. E., and Ankley, G. T. ( 2002). Evaluation of androstenedione as an androgenic component of river water downstream of a pulp and paper mill effluent. Environ. Toxicol. Chem.  21, 1973–1976. Google Scholar Guillette, L. J., Jr. and Craine, A. ( 2000). Environmental Endocrine Disruptors. Taylor & Francis, New York. Google Scholar He, B., Bowen, N. T., Minges, J. T., and Wilson, E. M. ( 2001). Androgen-induced NH2- and COOH-terminal interaction inhibits p160 coactivator recruitment by activation function 2. J. Biol. Chem.  276, 42293–42301. Google Scholar Hegrenes, S. G. ( 1999). Masculinization of spawning channel catfish in the Red River of the North. Copeia 1999, 491–494. Google Scholar Howell, W. M., Black, D. A., and Bortone, S. A. ( 1980). Abnormal expression of secondary sex characters in a population of mosquitofish, Gambusia affinis holbrooki: Evidence for environmentally-induced masculinization. Copeia  1980, 676–681. Google Scholar Howell, W. M., and Denton, T. E. ( 1989). Gonopodial morphogenesis in female mosquitofish, Gambusia affinis affinis, masculinized by exposure to degradation products from plant sterols. Environ. Biol. Fishes  24, 43–51. Google Scholar Hunsinger, R. N., and Howell, W. M. ( 1991). Treatment of fish with hormones: Solubilization and direct administration of steroids into aquaria water using acetone as a carrier solvent. Bull. Environ. Contam. Toxicol.  47, 272–277. Google Scholar Ikeuchi, T., Todo, T., Kobayashi, T., and Nagahama, Y. ( 1999). cDNA cloning of a novel androgen receptor subtype. J. Biol. Chem.  274, 25205–25209. Google Scholar Jenkins, R., Angus, R. A., Mcnatt, H., Howell, W. M., Kemppainen, J. A., Kirk, M., and Wilson, E. M. ( 2001). Identification of androstenedione in a river containing paper mill effluent. Environ. Toxicol. Chem.  20, 1325–1331. Google Scholar Kemppainen, J. A., Lane, M. V., Sar, M., and Wilson, E. M. ( 1992). Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activity-specificity for steroids and antihormones. J. Biol. Chem.  267, 968–974. Google Scholar Kolpin, D. W., Furlong, E. T., Meyer, M. T., Thurman, E. M., Zaugg, S. D., Barber, L. B., and Buxton, H. T. ( 2002). Pharmaceuticals, hormones, and other organic wastewater contaminants in US streams, 1999–2000: A national reconnaissance. Environ. Sci. Technol.  36, 1202–1211. Google Scholar Kuch, H. M., and Ballschmiter, K. ( 2000). Determination of endogenous and exogenous estrogens in effluents from sewage treatment plants at the Ng/L-level. Fresenius’ J. Analyt. Chem.  366, 392–395. Google Scholar Langley, E., Zhou, Z. X., and Wilson, E. M. ( 1995). Evidence for an antiparallel orientation of the ligand activated human androgen receptor dimer. J. Biol. Chem.  270, 29983–29990. Google Scholar Larsson, D. G. J., Hallman, H., and Forlin, L. ( 2000). More male fish embryos near a pulp mill. Environ. Toxicol. Chem.  19, 2911–2917. Google Scholar Matsui, S., Takigami, H., Matsuda, T., Taniguchi, N., Adachi, J., Kawami, H., and Shimizu, Y. ( 2000). Estrogen and estrogen mimics contamination in water and the role of sewage treatment. Water Sci. Technol.  42, 173–179. Google Scholar Metcalfe, C. D., Metcalfe, T. L., Kiparissis, Y., Koenig, B. G., Khan, C., Hughes, R. J., Croley, T. R., March, R. E., and Potter, T. ( 2001). Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias latipes). Environ. Toxicol. Chem.  20, 297–308. Google Scholar Nagasawa, M., Bae, M., Tamura G., and Arima, K. ( 1969). Microbial transformation of sterols: Part II. Cleavage of sterol side chains by microorganisms. Agr. Biol. Chem.  33, 1644–1650. Google Scholar Owens, R. W., Tenneson, M. E., Bilton, R. F., and Mason, A. N. ( 1978). The degradation of cholesterol by Escherichia coli isolated from human faeces. Biochem. Soc. Trans.  6, 377–378. Google Scholar Parks, L. G., Lambright, C. S., Orlando, E. F., Guillette, L. J., Ankley, G. T., and Gray, L. E. ( 2001). Masculinization of female mosquitofish in kraft mill effluent-contaminated Fenholloway River water is associated with androgen receptor agonist activity. Toxicol. Sci.  62, 257–267. Google Scholar Rodgers-Gray, T. P., Jobling, S., Kelly, C., Morris, S., Brighty, G., Waldock, M. J., Sumpter, J. P., and Tyler, C. R. ( 2001). Exposure of juvenile roach (Rutilus rutilus) to treated sewage effluent induces dose-dependent and persistent disruption in gonadal duct development. Environ. Sci. Technol.  35, 462–70. Google Scholar Stahlschmidtallner, P., Allner, B., Rombke, J., and Knacker, T. ( 1997). Endocrine disruptors in the aquatic environment. Environ. Sci. Pollut. Res.  4, 155–162. Google Scholar Stanko, J. P., Dean, J., and Angus, R. A. ( 2001). Effects of exposure to a bioactive constituent of pulp mill effluent on the reproductive physiology of female mosquitofish, Gambusia affinis. Poster presented at the e.hormone 2001 meeting, Tulane University, New Orleans, LA, October 19, 2001. Google Scholar Takeo, J., and Yamashita, S. ( 1999). Two distinct isoforms of cDNA encoding rainbow trout androgen receptors. J. Biol. Chem.  274, 5674–5680. Google Scholar Thomas, K. V., Hurst, M. R., Matthiessen, P., Mchugh, M., Smith, A., and Waldock, M. J. ( 2002). An assessment of in vitro androgenic activity and the identification of environmental androgens in United Kingdom estuaries. Environ. Toxicol. Chem.  21, 1456–1461. Google Scholar Thomas, K. V., Hurst, M. R., Matthiessen, P., and Waldock, M. J. ( 2001). Characterization of estrogenic compounds in water samples collected from United Kingdom estuaries. Environ. Toxicol. Chem.  20, 2165–2170. Google Scholar Todo, T., Ikeuchi, T., Kobayashi, T., and Nagahama, Y. ( 1999). Fish androgen receptor: cDNA cloning, steroid activation of transcription in transfected mammalian cells, and tissue mRNA levels. Biochem. Biophys. Res. Commun.  254, 378–383. Google Scholar Tremblay, L., and Van Der Kraak, G. ( 1999). Comparison between the effects of the phytosterol β-sitosterol and pulp and paper mill effluents on sexually immature rainbow trout. Environ. Toxicol. Chem.  18, 329–336. Google Scholar Tyler, C. R., Jobling, S., and Sumpter, J. P. ( 1998). Endocrine disruption in wildlife: A critical review of the evidence. Crit. Rev. Toxicol.  28, 319–61. Google Scholar © 2003 Society of Toxicology
TI - Androstenedione and Progesterone in the Sediment of a River Receiving Paper Mill Effluent
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfg042
DA - 2003-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/androstenedione-and-progesterone-in-the-sediment-of-a-river-receiving-Jda5zjAXK7
SP - 53
EP - 59
VL - 73
IS - 1
DP - DeepDyve
ER -