Grantee Research Project Results
1999 Progress Report: A Short-Term In Vivo Screening System For Endocrine Disruptors Utilizing Mosquitofishes (Gambusia affinis and G. holbrooki)
EPA Grant Number: R826130Title: A Short-Term In Vivo Screening System For Endocrine Disruptors Utilizing Mosquitofishes (Gambusia affinis and G. holbrooki)
Investigators: Angus, Robert , Blanchard, Paul , Watson, R. Douglas , Howell, W. Mike
Institution: The University of Alabama at Birmingham , Samford University
EPA Project Officer: Aja, Hayley
Project Period: October 1, 1997 through September 30, 2000 (Extended to December 31, 2001)
Project Period Covered by this Report: October 1, 1998 through September 30, 1999
Project Amount: $320,982
RFA: Endocrine Disruptors (1997) RFA Text | Recipients Lists
Research Category: Environmental Justice , Human Health , Safer Chemicals , Endocrine Disruptors
Objective:
The primary objective of this project is to establish and document the use of the mosquitofish (Gambusia affinis) as a bioindicator organism for use in screening substances with suspected endocrine activity. Expression of the (normally female-limited) vitellogenin (VTG) gene in males is being used to test chemicals for estrogenic activity. Development of a normally male-limited morphological structure (a modified anal fin called a gonopodium) in females is being used to test for androgenic activity. A project to identify the androgenic components of kraft (i.e., paper) mill effluent also was started this year. Progress to date on the various components of the project is summarized below.Progress Summary:
Vitellogenin as a Biomarker for Estrogens. During 1997?1998, polyclonal antisera against vitellogenin (VTG) of G. affinis was identified, purified, and raised. Western blot analysis revealed that antibodies in the anti-VTG antiserum bound specifically to the protein previously identified as VTG. During the second year of the grant, the anti-VTG antiserum was used to develop an enzyme-linked immunosorbent assay (ELISA) for mosquitofish VTG. The ELISA yielded a standard curve that was linear from 46.9 to 1500 ng. Despite a number of methodological manipulations, the ELISA did not prove suitable for quantifying VTG in blood samples, leading to the tentative conclusion that a component of fish blood interferes with antibody-antigen binding.
The anti-VTG antiserum was used to develop a solid phase (western blot) immunoassay for VTG. When coupled with densitometric scanning, the assay appears to be specific, quantitative, and sensitive enough to detect VTG in less than 1 microliter of G. affinis blood. The assay has been used to demonstrate that the effect of 17 -ethynyl estradiol on vitellogenin synthesis in male G. affinis is both concentration- and time-dependent. These results will be reported in a manuscript that is in preparation.
The solid phase immunoassay also is being used in a continuing study of mosquitofish living immediately below a local sewage treatment plant (STP).
Although the solid phase immunoassay appears to be a reliable means of quantifying VTG in blood samples, the assay is time consuming, limiting the number of samples that can be tested; therefore, the development of an ELISA for VTG still would be desirable. To that end, a second antiserum has been generated, this time using vitellin purified from G. affinis eggs as the immunogen. The antiserum currently is being tested to determine its suitability for use in an ELISA.
Assay for Gonopodium Development in Females. A quantitative morphological study of gonopodium development in normal males and in females treated with 11-ketotestosterone has been completed and a manuscript submitted to General and Comparative Endocrinology. In the paper, the morphometric and meristic traits used to quantify the degree of gonopodial development, both in normal males and androgen-treated females is described. The results of a dose-response experiment in which female mosquitofish were masculinized by exposure to 11-ketotestosterone in their food also are presented.
Dose-response studies also have been completed for 17 -methyltestosterone and the nonsteroidal drug spironolactone. Manuscripts reporting on these studies currently are in preparation.
Known androgens mostly have been used to stimulate development of gonopodia in female mosquitofish to date. These studies demonstrate the usefulness of the chosen meristic and morphometric traits in characterizing the responses of female mosquitofish to substances with androgenic activity, and provide basic dose-response data on the masculinizing effects of various androgens on female mosquitofish. Currently, the group is actively engaged in a study to identify the androgenic substances in kraft mill effluent (see below). Once identified, female mosquitofish will be exposed to these environmental androgens, individually and in combinations, to investigate their effectiveness at producing masculinization and their effects on reproductive fitness.
Identifying the Androgenic Components of Kraft Mill Effluent. A Toxicity Identification and Evaluation (TIE) approach that consists of fractionation combined with in vitro assays is being used to identify the androgenic components of kraft mill effluent. This same approach was recently used to isolate and identify the major estrogenic chemicals present in treated sewage effluents in the United Kingdom. Briefly, a sample of the effluent water is filtered and put through a C18 solid-phase extraction cartridge. The cartridge then is eluted with a series of methanol/water mixtures, then eluted with solvents of low polarity to nonpolar solvents. The different fractions are then blown down to dryness, made up in methanol, and tested for androgenic activity in a cell assay. The assay uses monkey kidney cells transfected with DNA for the human androgen receptor DNA and a luciferase reporter. So far, two kraft mill effluent fractions containing substances that bind to the androgen receptor in the cell assay have been found.
We are in the process of repeating the extraction procedures on a new sample of kraft mill effluent water to: (1) determine whether the fractions determined to contain androgenically active substances in the first sample will consistently contain these active compounds, and (2) produce highly concentrated extracts for analysis by gas chromatography/mass spectroscopy (GC/MS).
After the androgenically active components of kraft mill effluent have been positively identified, they will be used, individually and in combinations, in the in vivo mosquitofish anal fin assays. It will be important to determine whether compounds that bind to the human androgen receptor in the kidney cell assay also affect fish as androgens.
Future Activities:
In addition to continuing work on the projects already described, the following is planned:
Dose-Response Assays With Androgenic Chemicals. These studies to characterize the relative sensitivity of the female anal fin rays to a variety of steroidal and nonsteroidal chemicals will continue.
Reproductive Effects of Masculinization in Females. Female mosquitofish of reproductive age are being masculinized by exposure to methyltestosterone. Then, the effects of the androgen exposure on their reproductive ability will be measured to examine the effects on ovarian weight and morphology, histology, female fecundity (brood frequency and size), and survivorship of the young after birth.
Effects of Masculinizing Chemicals on Embryos. Pregnant females will be treated with masculinizing chemicals, exposing the embryos in utero. The young will be raised to maturity so that the effects of the chemical treatments on sex ratio and fertility can be assessed.
Journal Articles on this Report : 11 Displayed | Download in RIS Format
| Other project views: | All 20 publications | 12 publications in selected types | All 11 journal articles |
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Meleth AD, Angus RA. Age as a variable in the induced masculinization of female mosquitofish Gambusia affinis. Journal of the Alabama Academy of Science 2000;72. |
R826130 (1999) |
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Angus RA, Weaver SA, Grizzle JM, Watson RD. Reproductive characteristics of male mosquitofish (Gambusia affinis) inhabiting a small southeastern U.S. river receiving treated domestic sewage effluent. Environmental Toxicology and Chemistry 2002;21(7):1404-1409. |
R826130 (1999) |
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Angus RA, Stanko J, Jenkins RL, Watson RD. Effects of 17α-ethynylestradiol on sexual development of male Western mosquitofish (Gambusia affinis). Comparative Biochemistry and Physiology C: Toxicology & Pharmacology 2005;140(3-4):330-339. |
R826130 (1999) |
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Angus RA, McNatt HB, Howell WM, Peoples SD. Gonopodium development in normal male and 11-ketotestosterone-treated female mosquitofish (Gambusia affinis): a quantitative study using computer image analysis. General and Comparative Endocrinology 2001;123(2):222-234. |
R826130 (1999) R826129 (Final) |
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Estes ECJ, Katholi CR, Angus RA. Elevated fluctuating asymmetry in eastern mosquitofish (Gambusia holbrooki) from a river receiving paper mill effluent. Environmental Toxicology and Chemistry 2006;25(4):1026-1033. |
R826130 (1999) |
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Jenkins RL, Wilson EM, Angus RA, Howell WM, Kirk M. Androstenedione and progesterone in the sediment of a river receiving paper mill effluent. Toxicological Sciences 2003;73(1):53-59. |
R826130 (1999) |
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Jenkins RL, Wilson EM, Angus RA, Howell WM, Kirk M, Moore R, Nance M, Brown A. Production of androgens by microbial transformation of progesterone in vitro: a model for androgen production in rivers receiving paper mill effluent. Environmental Health Perspectives 2004;112(15):1508-1511. |
R826130 (1999) |
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Jenkins R, Angus RA, McNatt H, Howell WM, Kemppainen JA, Kirk M, Wilson EM. Identification of androstenedione in a river containing paper mill effluent. Environmental Toxicology and Chemistry 2001;20(6):1325-1331. |
R826130 (1999) |
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Miller CL, Angus RA. Preliminary studies on the use of Poeciliid fishes as sensitive bioindicators of environmental androgens. Journal of the Alabama Academy of Science 1997;68:137. |
R826130 (1999) |
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Thomas P, Rahman MS, Kummer JA, Lawson S. Reproductive endocrine dysfunction in Atlantic croaker exposed to hypoxia. Marine Environmental Research 2006;62(Suppl 1):S249-S252. |
R826130 (1999) R829458C005 (2005) |
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Tolar JF, Mehollin AR, Watson RD, Angus RA. Mosquitofish (Gambusia affinis) vitellogenin: identification, purification, and immunoassay. Comparative Biochemistry and Physiology C-Toxicology & Pharmacology 2001;128(2):237-245. |
R826130 (1999) |
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Supplemental Keywords:
water, ecological effects, dose-response, animal, sex, chemicals, toxics, effluent, aquatic, zoology, southeast, Alabama., RFA, Scientific Discipline, Health, Toxicology, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Biochemistry, Biology, Endocrine Disruptors - Human Health, mosquito-fishes, natural hormones, bioindicator, complex mixtures, aquatic ecosystem, anti-androgen, dose response, immunosorbent assay, in vivo model, in vivo screening system, animal models, developmental processes, biological effectsRelevant Websites:
http://www.uab.edu/uabbio/angus.htmProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.