2000 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, 1999 through September 30, 2000
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 in the model crustacean, the water flea (Daphnia magna) and the model gastropod, the mud snail (Illyanassa obsoleta).

Progress Summary:

The following accomplishments were attained in this past funding period:

  • The mechanism of metabolic androgenization was discovered. We discovered that steroid androgens function as antiecdysteroids in crustaceans and inhibit both the molting process and embryonic development.

  • The agricultural fungicide fenarimol was found to act as an antiecdsteroid in crustaceans. Fenarimol elicits toxicity consistent with our previously described metabolic androgenization. We conclusively demonstrated that fenarimol is an antiecdysteroid in crustaceans.

  • The juvenile hormone analog methoprene interferes with the sequence of events involved in the sexual reproductive cycle of the crustacean (Daphnia magna). This previously undescribed toxicity of methoprene was demonstrated at environmentally-relevant levels of this insecticide.

  • Fatty acid conjugation is the primary mechanism of steroidal androgen inactivation utilized by the mud snail. This discover raises serious doubt regarding the primary hypotheses of how the biocide tributyltin causes imposex in gastropods, but identified new potential targets of tributyltin.
Metabolic Androgenization in Crustaceans

We had previously observed that steroidal androgens and chemicals that interfere with androgen clearance cause developmental abnormalities in the crustacean (Daphnia magna). Characteristic abnormalities included underdeveloped antennae, abnormalities of the carapace, and early arrest of certain developmental processes. Abnormalities were inevitably lethal to the neonate. We have referred to this toxicity as metabolic androgenization.

Mechanistic studies were undertaken during the past funding period to establish the mechanism by which androgens are embryotoxic to crustaceans. We discovered that, in addition to its effects on embryonic development, testosterone also interfered with normal molting. As molting is controlled by another steroid hormone, 20-hydroxyecdysone, we hypothesized that testosterone may interfere with the action of the ecdysteroid. We subsequently demonstrated that 20-hydroxyecdysone could protect daphnids against the effects of testosterone on both molting and embryonic development. Thus, both toxicities appeared to be a consequence of the antiecdysteroid properties of testosterone.

This conclusion was validated by conducting similar experiments with a compound known to have antiecdysteroidal activity in insects, the agricultural fungicide fenarimol. This compound lowers ecdysteroid levels in insects by inhibiting key enzymes in ecdysteroid biosynthesis. The physiological effects of fenarimol were identical to those of testosterone. The compound specifically interfered with the molting process and caused developmental abnormalities. Furthermore, daphnids could be protected against the effects of fenarimol by co-exposure to 20-hydroxyecdysone. These results revealed that fenarimol also functions as an antiecdysteroid in crustaceans and further confirms that steroidal androgens function as antiecdysteroids.

Additional Studies. Additional studies are underway or scheduled to establish the precise role of ecdysteroids in embryo development. Planned for the next year is the evaluation of the role of ecdysteroids in vitellogenin and fatty acid synthesis and transport to the oocytes. Also planned are the characterization of possible embryonic molting and the role of ecdysteroids in this putative process. Identification of the precise mechanism by which ecdysteroids regulate embryonic maturation should reveal sensitive biomarkers of antiecdysteroid exposure and effect that would have significant utility in both laboratory studies and field evaluations.

Studies are underway with chemicals that we had previously demonstrated caused metabolic androgenization, the alkylphenol 4-nonylphenol, the fungicide propiconazole, and the insecticide synergist piperonyl butoxide. Experiments are underway to determine whether these chemicals also function as antiecdysteroids in crustaceans. It is anticipated that results from these experiments will identify a unifying mechanism (anti-ecdysteroid) by which diverse environmental chemicals elicit endocrine-disrupting toxicity to crustaceans.

Experiments are planned to determine whether each identified antiecdysteroid elicits its activity by lowering endogenous ecdysteroid levels or by functioning as a ecdysteroid receptor antagonist. We are currently collaborating with the US EPA to generate a daphnid cDNA library. This library will be used to identify the daphnids ecdysteroid receptor and ultimately develop an in vitro screening tool for the evaluation of receptor antagonism by environmental chemicals.

Temporal Alterations in Sexual Reproductive Cycling by Methoprene

Cyclic parthenogens, such as daphnids, utilize both asexual (parthenogenetic) and sexual reproduction in order to maximize population fitness in variable environments. Parthenogenetic reproduction is the default strategy among Daphnia magna, while various environmental cues trigger cycles of sexual reproduction. EPA guidelines require that only effects on parthenogenetic reproduction be evaluated when assessing chemical toxicity to daphnids, which are considered to be representative freshwater crustaceans. Experiments were conducted with the juvenile hormone analog methoprene to test the hypothesis that members of the insect juvenile hormone/vertebrate retinoic acid family of transcription factors are involved in the regulation of sexual reproduction in daphnids. Neither methoprene, food reduction, or crowding independently stimulated entry into the sexual reproductive phase of the daphnids. However, the combination of food deprivation and crowding stimulated entry into the sexual reproductive phase characterized by an initial high production of males and the subsequent intermittent production of haploid egg-containing ephippia. Exposure to 160 nM methoprene along with food deprivation and crowding caused a significant reduction in the percentage of males produced during the early phase o the sexual cycle and significantly increased the percentage of males produced during the later stages of the cycle. Methoprene concentrations as low as 1.3 nM significantly reduced the number of resting eggs produced and proportionately increased the production of parthenogenetically-produced neonates. These experiments demonstrated that methoprene uncouples the coordinate production of males and resting eggs during the sexual reproductive period of daphnids. Methoprene stimulates male offspring production and defers their production to latter stages of the sexual reproductive period, while inhibiting the production of resting eggs and promoting the continuance of parthenogenetic reproduction. Finally, these results demonstrated that methoprene adversely affects sexual reproduction of daphnids at concentration significantly below those that affect asexual reproduction.

Additional Studies. Additional studies are underway to develop assays that could be routinely used by testing laboratories to evaluate the effects of environmental chemicals on the sexual reproductive cycle of daphnids. We will use these testing protocols to determine whether or not methoprene is unique in its ability to interfere with sexual reproduction at low, environmentally relevant concentration.

Tributyltin-Induced Imposex in the Mud Snail

Imposex is defined as the development of male secondary sex characteristics by female gastropods. Imposex has been causally associated with environmental levels of the marine biocide tributyltin. Current hypotheses on the mechanism by which tributyltin causes imposex propose that tributyltin inhibits the conversion of testosterone to estradiol or polar elimination products. The proposed consequence is an increase in testosterone levels resulting in masculinization (imposex).

Over the past funding period, we have completed our evaluation of the normal metabolic inactivation/elimination of testosterone by the mud snail (Ilyanassa obsoleta). This study was undertaken to identify potential targets by which tributyltin could alter endogenous testosterone levels. The mud snail converted testosterone to at least five apolar conjugates which we designated AP1 through AP5. All were retained by the organisms. No significant amount of administered testosterone was retained or eliminated as polar metabolites. Following enzymatic hydrolysis of the most abundant metabolite (AP1), free fatty acids and testosterone were liberated. Further, AP1 was produced when homogenized snail tissue was incubated with testosterone and fatty acid-coenzyme A. These results indicated that AP1, which represents over 70 percent of the testosterone biotransformation products, is a fatty acid ester of testosterone. The esterification of testosterone to fatty acids might be a mechanism by which steroid titers are regulated and could represent a target of tributyltin toxicity. It is noteworthy that we have never, to date, detected the conversion of testosterone to estradiol by the mud snail which raises questions as to whether the inhibition of this process could possibly be the cause of elevated testosterone levels associated with imposex.

Experiments are currently underway to establish relationships among tributyltin exposure, testosterone levels, testosterone-fatty acid esterification, and imposex. The following tentative results have been obtained.

  • Male snails have higher levels of free testosterone than do females.

  • The ratio of free to esterified testosterone varies in order to maintain the level of free testosterone within the sex-appropriate range.

  • Tributin causes imposex in the mud snail at aqueous concentrations as low as 1.0 n/L.

  • Tributyltin-exposed females that exhibit imposex have elevated testosterone levels; whereas, tributyltin-exposed females that do not exhibit imposex have normal testosterone levels.

  • Testosterone exposure elevates free testosterone levels in female to levels associated with imposex in tributyltin-exposed snails; however, these females did not exhibit imposex.

The latter two observations suggest that elevated testosterone levels may be a consequence of imposex rather than a cause of the abnormality. We postulate that imposex females have elevated testosterone levels due to the development of testosterone-producing glands.

Additional Studies. The following additional studies are underway:

  1. Establish whether tributyltin alters testosterone esterification to fatty acids.

  2. Determine whether accessory male sex organs associated with imposex are responsible for the secretion of additional testosterone by these organisms.

  3. Definitively establish the cause-effect relationships among tributyltin exposure, testosterone levels, testosterone-fatty acid esterification, and imposex.

  4. Determine the precise role of testosterone in imposex.

  5. Determine whether or not environmental chemicals other than tributyltin have the ability to induce imposex.

  6. Identify biomarkers that could be used to screen chemicals for this unique endocrine-disrupting toxicity.

Future Activities:

We are currently proceeding with a no cost extension to this grant. Specific planned experiments are outlined above as Additional Studies. As many of these experiments will be conducted as funds allow.

Journal Articles on this Report : 11 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 Gooding MP, LeBlanc GA. Biotransformation and disposition of testosterone in the eastern mud snail Ilyanassa obsoleta. General and Comparative Endocrinology 2001;122(2):172-180. R826129 (2000)
R826129 (Final)
not available
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 Kast-Hutcheson K, Rider CV, LeBlanc GA. The fungicide propiconazole interferes with embryonic development of the crustacean Daphnia magna. Environmental Toxicology and Chemistry 2001, Volume: 20, Number: 3 (MAR), Page: 502-509. R826129 (2000)
R826129 (Final)
not available
Journal Article Korte JJ, Kahl MD, Jensen KM, Pasha MS, Parks LG, Leblanc GA, Ankley GT. Fathead minnow vitellogenin: Complementary DNA sequence and messenger RNA and protein expression after 17 beta-estradiol treatment. Environmental Toxicology and Chemistry 2000;19(4):972-981 R826129 (2000)
R826129 (Final)
not available
Journal Article LeBlanc GA, McLachlan JB. Changes in the metabolic elimination profile of testosterone following exposure of the crustacean Daphnia magna to tributyltin. Ecotoxicology and Environmental Safety 2000;45(3):296-303. R826129 (2000)
R826129 (Final)
not available
Journal Article LeBlanc GA, Mu XY, Rider CV. Embryotoxicity of the alkylphenol degradation product 4-nonylphenol to the crustacean Daphnia magna. Environmental Health Perspectives 2000;108(12):1133-1138. R826129 (2000)
R826129 (Final)
not available
Journal Article Olmstead AW, LeBlanc GA. Effects of endocrine-active chemicals on the development of sex characteristics of Daphnia magna. Environmental Toxicology and Chemistry 2000;19(8):2107-2113. R826129 (2000)
R826129 (Final)
not available
Journal Article Olmstead AW, LeBlanc GA. Temporal and quantitative changes in sexual reproductive cycling of the cladoceran Daphnia magna by a juvenile hormone analog. Journal of Experimental Zoology 2001;290(2):148-155. R826129 (2000)
R826129 (Final)
not available
Journal Article Wilson VS, LeBlanc GA. Petroleum pollution. In: Hodgson E, ed. Reviews in Toxicology. Volume 3. Environmental Toxicology. Amsterdam: IOS Press, 2000, pp. 77-112. R826129 (2000)
R826129 (Final)
not available
Journal Article Wilson VS, LeBlanc GA. The contribution of hepatic inactivation of testosterone to the lowering of serum testosterone levels by ketoconazole. Toxicological Sciences 2000;54(1):128-137. R826129 (2000)
R826129 (Final)
not available
Journal Article Wolf CJ, LeBlanc GA, Ostby JS, Gray LE. Characterization of the period of sensitivity of fetal male sexual development to vinclozolin. Toxicological Sciences 2000;55(1):152-161. R826129 (2000)
R826129 (Final)
not available

Supplemental Keywords:

water, risk assessment, ecological effects, metabolism, steroids, terpenoids, hazard., RFA, Scientific Discipline, Health, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Health Risk Assessment, Ecosystem/Assessment/Indicators, Endocrine Disruptors - Environmental Exposure & Risk, pesticides, Environmental Microbiology, endocrine disruptors, 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

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Progress and Final Reports:

Original Abstract
  • 1998
  • 1999 Progress Report
  • 2001 Progress Report
  • Final Report