* Department of Biology, Samford University, Birmingham, Alabama 35229;
Laboratories for Reproductive Biology, Department of Pediatrics, and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599;
Biology Department and
Comprehensive Cancer Center Mass Spectrometry Shared Facility, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received August 29, 2002; accepted January 13, 2003
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ABSTRACT |
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Key Words: environmental androgens; phytosteroids; masculinized mosquitofish; Gambusia holbrooki; androgen receptor; androgen-dependent gene expression; Fenholloway River, Florida.
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INTRODUCTION |
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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 ß-sitosterol. Tremblay and Van Der Kraak (1999)
provided evidence that ß-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 ß-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.
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MATERIALS AND METHODS |
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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 x 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 17ß-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 17ß-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 x 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 x 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.
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RESULTS |
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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%).
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DISCUSSION |
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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 ß-sitosterol (72%), stigmastanol (11%), and campesterol (8%). Nagasawa et al.(1969)
demonstrated that cholesterol, ß-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).
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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 ß-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.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (205) 975-6097. E-mail: raangus{at}uab.edu.
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REFERENCES |
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Bortone, S. A., and Davis, W. P. (1994). Fish intersexuality as indicator of environmental stress. Bioscience 44, 165172.[ISI]
Bortone, S. A., and Drysdale, D. T. (1981). Additional evidence for environmentally induced intersexuality in poeciliid fishes. ASB Bull. 28, 67.
Carlstrom, K. (1974). Transformation of steroids by cell-free preparation of Penicillium lilacinum NRRL 895. IV. Enzyme catalyzed acyl transfer. Acta Chem. Scandinavia 28, 2328.
Colborn, T. (1995). Environmental estrogens: Health implications for humans and wildlife. Environ. Health Perspect. 103(Suppl. 7), 135136.[ISI][Medline]
Conner, A., Nagaoka, M., Rowe, J. W., and Perlman, D. (1976). Microbial conversion of tall oil sterols into C19 steroids. Appl. Environ. Microbiol. 32, 310311.[ISI][Medline]
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, 627632.[ISI][Medline]
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, 19731976.[CrossRef][ISI][Medline]
Guillette, L. J., Jr. and Craine, A. (2000). Environmental Endocrine Disruptors. Taylor & Francis, New York.
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, 4229342301.
Hegrenes, S. G. (1999). Masculinization of spawning channel catfish in the Red River of the North. Copeia 1999, 491494.
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, 676681.
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, 4351.[ISI]
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, 272277.[ISI][Medline]
Ikeuchi, T., Todo, T., Kobayashi, T., and Nagahama, Y. (1999). cDNA cloning of a novel androgen receptor subtype. J. Biol. Chem. 274, 2520525209.
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, 13251331.[ISI][Medline]
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, 968974.
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, 19992000: A national reconnaissance. Environ. Sci. Technol. 36, 12021211.[CrossRef][ISI][Medline]
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, 392395.[CrossRef]
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, 2998329990.
Larsson, D. G. J., Hallman, H., and Forlin, L. (2000). More male fish embryos near a pulp mill. Environ. Toxicol. Chem. 19, 29112917.[ISI]
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, 173179.
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, 297308.[CrossRef][ISI][Medline]
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, 16441650.[ISI]
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, 377378.[Medline]
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, 257267.
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, 46270.[CrossRef][ISI][Medline]
Stahlschmidtallner, P., Allner, B., Rombke, J., and Knacker, T. (1997). Endocrine disruptors in the aquatic environment. Environ. Sci. Pollut. Res. 4, 155162.[ISI]
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.
Takeo, J., and Yamashita, S. (1999). Two distinct isoforms of cDNA encoding rainbow trout androgen receptors. J. Biol. Chem. 274, 56745680.
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, 14561461.[CrossRef][ISI][Medline]
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, 21652170.[CrossRef][ISI][Medline]
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, 378383.[CrossRef][ISI][Medline]
Tremblay, L., and Van Der Kraak, G. (1999). Comparison between the effects of the phytosterol ß-sitosterol and pulp and paper mill effluents on sexually immature rainbow trout. Environ. Toxicol. Chem. 18, 329336.[ISI]
Tyler, C. R., Jobling, S., and Sumpter, J. P. (1998). Endocrine disruption in wildlife: A critical review of the evidence. Crit. Rev. Toxicol. 28, 31961.[ISI][Medline]