2001 Progress Report: Metabolic Androgenization of Invertebrates by Endocrine-Disrupting Chemicals

EPA Grant Number: R826129
Title: Metabolic Androgenization of Invertebrates by Endocrine-Disrupting Chemicals
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Carleton, James N
Project Period: October 1, 1997 through September 30, 2000 (Extended to September 30, 2002)
Project Period Covered by this Report: October 1, 2000 through September 30, 2001
Project Amount: $406,155
RFA: Endocrine Disruptors (1997) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Health , Safer Chemicals , Endocrine Disruptors


The overall objective of this program is to definitively characterize the phenomenon of metabolic androgenization in invertebrates and to elucidate the putative relationships among chemical exposure, metabolic androgenization, and toxicological consequences such as pseudohermaphroditism, developmental abnormalities, and reproductive impairment. The studies are being conducted with the model crustacean, the water flea (Daphnia magna) and the model gastropod, the mud snail (Ilyanassa obsoleta).

Crustacean Model. We discovered earlier in this program that the effects of steroidal vertebrate androgens and chemicals that caused metabolic androgenization in daphnids were due to antiecdysteroidal properties of these compounds. Research was conducted during the past project period to better understand the mechanism by which these chemicals elicit antiecdysteroid activity.

Progress Summary:

The mechanism by which testosterone functions as an antiecdysteroid was elucidated. Using intact daphnids, we determined that testosterone had no significant effect on endogenous ecdysone levels. However, using a cell-based assay, we established that testosterone functions as an antiecdysteroid by acting as an ecdysone receptor antagonist.

The Mechanism by Which the Agricultural Fungicide Fenarimol Functions as an Antiecdysteroid Was Elucidated. Using intact daphnids, we determined that fenarimol significantly suppressed endogenous ecdysone levels. Using the cell-based assay, we established that fenarimol did not interact with the ecdysone receptor. Thus, unlike testosterone, fenarimol functions as an antiecdysteroid by lowering endogenous ecdysone levels.

Normal Ecdysone Levels in the Developing Daphnid Embryo Were Determined. Because the antiecdysteroids under study all interfered with daphnid embryo development, we hypothesized that ecdysone was critical to normal embryo development. Ecdysone levels were measured in the developing embryo using radioimmunoassay. We discovered that ecdysone levels are high in the early embryo and progressively decline through late organogenesis. Ecdysone levels then increase to the time of neonate release from the brood chamber of the mother. We surmised that the initially high levels of ecdysone in the embryo are due to maternal transfer of the hormone. As these stores are exhausted, the embryo develops the capability to synthesize ecdysone resulting in the late stage increase in hormone levels. These results suggest that either maternal or direct embryo exposure to antiecdysteroids can adversely impact embryo development. This hypothesis currently is being tested. We also established that during the molt cycle of neonatal organisms, ecdysone levels remain very low until approximately 3 hours before molting when ecdysone levels precipitously increase then decrease. Molting occurs following the peak in ecdysone levels. Thus, antiecdysteroids can interfere with growth and molting by blocking the molt-stimulating surge in ecdysone levels.

Structure Activity Analyses Is Underway as a Means To Screen Environmental Chemicals for Ecdysteroid or Antiecdysteroid Activity. A total of 72 chemicals have been evaluated for ecdysone receptor activity and physical-chemical characteristics. Using Molecular Modeling Pro software, seven molecular characteristics that describe physical structure and charge characteristics of molecules have been identified that are shared by ecdysone receptor agonists. Studies currently are underway to identify molecular characteristics that will differentiate between receptor agonists and antagonists as well as provide some quantitative assessment of receptor interaction potency.

Additional Studies. Combination structure-activity analyses, cell-based assays, and short-term whole organism screening assays are being developed and used to identify environmental chemicals having ecdysteroidal or antiecdysteroidal activity with adverse consequences to crustaceans (Tier I evaluation). Five putative ecdysone antagonists were identified in initial screens: bisphenol A, lindane, diethylphthalate, ketoconazole, and piperonyl butoxide. Bisphenol A, lindane, and diethylphthalate appear to function as ecdysone receptor antagonists, as observed with testosterone; while ketoconazole and piperonyl butoxide seem to function as ecdysone synthesis inhibitors, as observed with fenarimol. Experiments are underway to establish whether these compounds adversely impact fecundity at environmentally relevant exposure concentrations (Tier II evaluation).

Gastropod Model. We demonstrated earlier in this program that exposure of mud snails to the biocide tributyltin elevates testosterone levels and causes females to develop male external sex organs (imposex). Experiments were undertaken to establish the mechanism by which tributyltin elevates testosterone levels in the snail.

The Conversion of Testosterone to 17b-estradiol Is a Minor Testosterone Biotransformation Process in the Mud Snail. The predominant hypothesis for the mechanism by which tributyltin elevates testosterone levels in gastropods is that the biocide inhibits the enzyme responsible for the conversion of testosterone to 17b-estradiol (CPY19, aromatase). According to this hypothesis, the inhibition results in the accumulation of the 17b-estradiol precursor? testosterone. We were unable to detect significant conversion of testosterone to 17b-estradiol in the mud snail using a variety of in vivo and in vitro experimental approaches. Thus, while tributyltin may inhibit the aromatase enzyme, this inhibition would not result in a significant accumulation of testosterone.

Fatty Acid Conjugation Is the Primary Route of Testosterone Metabolism Utilized by the Mud Snail. Snails were found to regulate free endogenous testosterone levels by conjugating excess testosterone to fatty acids. These fatty acid esters are retained by the snails and presumably serve as a steroid reserve for subsequent use. This discovery has identified a new potential target for the action of tributyltin?the fatty acid esterification process.

Tributyltin Inhibits the Fatty Acid Esterification of Testosterone in the Mud Snail. Exposure of mud snails to environmentally relevant levels of tributyltin resulted in a decrease in the fatty acid esterification of testosterone and a commensurate increase in immunodetectable free testosterone. Thus, the inhibition of the fatty acid esterification process is a likely means by which tributyltin elevates free testosterone levels in the snail.

Fatty Acid Esterification of Testosterone by the Mud Snail Is Mediated by a Microsomal Enzyme?Acyl CoA:Testosterone Acyl Transferase (ATAT). Tributyltin exposure neither directly inhibited ATAT activity nor suppressed levels of the enzyme. Experiments are underway to determine whether tributyltin limits availability of cofactors required for ATAT activity (i.e., fatty acid:coenzyme A) resulting in decreased activity.

Field Collected Imposex Mud Snails Have a Reduced Testosterone-Fatty Acid Esterification Capacity. A population of snails were discovered in a marina along the North Carolina coast in which 100 percent of the females are experiencing imposex. Adjacent to the marina is a creek in which less than 5 percent of the female mud snails are imposexed. Comparative analyses of these two populations have revealed the following:

  1. The marina population contains approximately10 times higher levels of tin as compared to the creek population.
  2. The marina population contains higher free testosterone levels as compared to the creek population.
  3. Total testosterone levels (free + esterified) are comparable between the two populations.
  4. The marina population has a decreased capacity to cause fatty acid esterification of testosterone as compared to the creek population.

These observations are consistent with our hypothesis that tributyltin elevates free testosterone levels by impairing the ability of the snails to regulate free testosterone levels through fatty acid esterification and storage of excess testosterone.

Future Activities:

Currently, we are proceeding with a no-cost extension to this grant. We will continue efforts to complete the studies outlined above.

Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other project views: All 61 publications 38 publications in selected types All 26 journal articles
Type Citation Project Document Sources
Journal Article Gunderson MP, LeBlanc GA, Guillette LJ. Alterations in sexually dimorphic biotransformation of testosterone in juvenile American alligators (Alligator mississippiensis) from contaminated lakes. Environmental Health Perspectives 2001;109(12):1257-1264. R826129 (2000)
R826129 (2001)
R826129 (Final)
not available
Journal Article Mu XY, LeBlanc GA. Environmental antiecdysteroids alter embryo development in the crustacean Daphnia magna. ournal of Experimental Zoology 2002;292(3):287-292. R826129 (2001)
R826129 (Final)
R826120 (Final)
R829358 (2003)
not available
Journal Article Olmstead AW, LeBlanc GA. Low exposure concentration effects of methoprene on endocrine-regulated processes in the crustacean Daphnia magna. Toxicological Sciences 2001;62(2):268-273. R826129 (2001)
R826129 (Final)
not available
Journal Article Putz O, Schwartz CB, Kim S, LeBlanc GA, Cooper RL, Prins GS. Neonatal low- and high-dose exposure to estradiol benzoate in the male rat: I. Effects on the prostate gland. Biology of Reproduction 2001;65(5):1496-1505. R826129 (2001)
R826129 (Final)
R826299 (1999)
not available
Journal Article Putz O, Schwartz CB, LeBlanc GA, Cooper GL, Prins GS. Neonatal low- and high-dose exposure to estradiol benzoate in the male rat: II. Effects on male puberty and the reproductive tract. Biology of Reproduction 2001;65(5):1506-1517. R826129 (2001)
not available
Journal Article Wolf CJ, Hotchkiss A, Ostby JS, LeBlanc GA, Gray LE. Effects of prenatal testosterone propionate on the sexual development of male and female rats: A dose-response study. Toxicological Sciences 2002;65(1):71-86. R826129 (2001)
R826129 (Final)
R829358 (2003)
not available

Supplemental Keywords:

water, risk assessment, ecological effects, metabolism, steroids, terpenoids, hazard., RFA, Health, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Ecosystem/Assessment/Indicators, Health Risk Assessment, pesticides, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Environmental Microbiology, Molecular Biology/Genetics, Endocrine Disruptors - Human Health, ecological effects, ecological exposure, adverse outcomes, bioindicator, metabolic androgenization, natural hormones, metabolic androgenization of invertebrates, propiconazole, exposure studies, chemical mixtures, trbutyltin, toxicity, animal models, biochemistry, reproductive processes, invertebrates, biological effects, hormone production

Relevant Websites:

http://www.cals.ncsu.edu/toxicology/index.htm Exit EPA icon

Progress and Final Reports:

Original Abstract
  • 1998
  • 1999 Progress Report
  • 2000 Progress Report
  • Final Report