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Extramural Research

Final 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: Turner, Vivian
Project Period: October 1, 1997 through September 30, 2000 (Extended to September 30, 2002)
Project Amount: $406,155
RFA: Endocrine Disruptors (1997)
Research Category: Economics and Decision Sciences , Endocrine Disruptors

Description:

Objective:

Significant evidence has accumulated over the past decade, indicating that some environmental chemicals can interfere with the endocrine system. This results in the disruption of various endocrine-regulated physiological processes. The observation that exposure of the embryo/fetus to some endocrine disrupting chemicals can alter development results in subsequent deficits in growth, maturation, or reproduction, is of particular concern. The ecological significance of this mode of toxicity has undergone significant investigation with various vertebrate species. Despite the fact that invertebrates constitute more than 95 percent of the animals on this planet, little research has focused upon chemical-induced endocrine disruption in invertebrates.

The masculinization of female gastropods (e.g., snails) and crustaceans (e.g., crabs, shrimp) has been documented worldwide. Among gastropods, this condition has been causally associated with the environmental contaminant tributyltin. Causality has not been conclusively established among crustaceans, though correlative evidence suggests that environmental pollution is contributing to this condition. The objectives of this research project were to: (1) elucidate the mechanism by which tributyltin elevates testosterone levels in the mud snail Ilyanassa obsoleta (elevated testosterone levels in females is considered responsible for the pseudohermaphroditism in these organisms); and (2) identify mechanisms by which environmental chemicals could masculinize the crustacean Daphnia magna.

Summary/Accomplishments (Outputs/Outcomes):

Mud Snails. The first objective of this research project was to elucidate the metabolic fate of testosterone in the snails. Once the normal processing of testosterone by these organisms was understood, we developed and tested hypotheses regarding the effect of tributyltin on these processes. Unlike all other species thus far evaluated in our laboratory, mud snails did not eliminate excess testosterone as polar metabolites. Rather, excess testosterone was stored in the tissues of the snail as fatty acid esters. This esterification was mediated by a microsomal acyl CoA:testosterone acyltransferase (ATAT) enzyme. Experiments were undertaken to evaluate whether this esterification process served to regulate testosterone levels in the snail. Snails were provided various treatments to modulate the level of total testosterone in their tissues. We evaluated the snails for esterified and free (unesterified) testosterone levels. Testosterone ester levels varied in direct proportion to the level of total testosterone in the organisms. In contrast, free testosterone levels remained remarkably constant, despite total testosterone levels in the tissues that ranged several orders of magnitude. These results demonstrated that testosterone esterification stores excess amounts of testosterone, and draws from this store when testosterone levels are depleted. Thus, snails have developed a remarkably simple, yet effective, means of regulating testosterone levels in their tissues.

These observations led to the hypothesis that tributyltin elevates testosterone levels in snails by interfering with the testosterone esterification process. Concordant with this hypothesis, tributyltin exposure decreased testosterone esterification in snails, resulting in an elevation of free testosterone. This effect was not due to the direct inhibition of the ATAT enzyme by tributyltin, nor due to a decrease in ATAT expression. Rather, tributyltin appeared to interfere with the ability of testosterone to regulate ATAT activity through a feedback mechanism.

We sampled snails from tin impacted and nonimpacted sites along the North Carolina coast at various times during the reproductive cycle, and we monitored for sexual development, reproduction, and testosterone homeostasis. Consistent with laboratory studies, snails collected from the tin-impacted site generally had lower testosterone ester levels and higher free testosterone levels as compared to snails from the nonimpacted site. Snails from the impacted site also exhibited abnormalities in sex differentiation and had reduced fecundity. Results also revealed that free testosterone levels normally increase at the time of sexual differentiation and at the end of reproduction. At these periods, testosterone levels were higher in males. These surges in free testosterone levels were due largely to decreases in the esterification of testosterone. Among snails sampled from the tin-impacted site, the surge in free testosterone levels associated with sexual differentiation was attenuated and occurred earlier as compared to the snails from the nonimpacted site. These field observations confirmed that tributyltin tin elevates free testosterone levels by interfering with testosterone esterification. Tributyltin also appears to disrupt seasonal surges in free testosterone that may be instrumental in regulating sexual differentiation and reproduction.

Daphnids. We determined mechanisms in which environmental chemicals could cause pseudohermaphroditic conditions in crustaceans characterized by the development of male sex characteristics in females. We also formulated a paradigm in which the development of male sex characteristics is dependent upon both organizational events during embryo development when the male sex of the embryo is determined and activational events during maturation in which male sex differentiation occurs.

We evaluated activational effects of endocrine active compounds on sex differentiation by exposing immature male and female daphnids to endocrine active compounds and by evaluating the rate of development of sex-specific characteristics. The compounds that we evaluated did not cause the development of abnormal sex characteristics in either sex. However, several compounds altered the rate of development of sex characteristics in the appropriate sex. For example, the juvenoid hormone methoprene stimulated the development of the female-specific abdominal process, and the androgen androstenedione stimulated elongation of the first antennae, a male-specific characteristic.

Several endocrine active compounds also were evaluated for organizational effects during embryo development. These experiments initially focused upon chemicals that were postulated to be androgenic. Steroidal androgens (testosterone and androstenedione) and chemicals that inhibited the elimination of testosterone (4-nonylphenol and propiconazole) elicited common developmental abnormalities. However, these compounds did not alter sex ratios of offspring in favor of males, nor did they masculinize female offspring. Further analyses revealed that some of these compounds elicited developmental abnormalities by functioning as antiecdysteroids. Two mechanisms of antiecdysteroidal activity were discerned: lowering of ecdysteroid levels and ecdysteroid receptor antagonism. Mechanistic studies revealed that mixtures of chemicals that elicit antiecdysteroidal effects on developing embryos by these two differing mechanisms can result in synergistic toxicity with greater than additive decreases in population fecundity. Results also revealed that these synergistic effects could be modeled and accurately predicted.

Continued investigation into activational effects of chemicals on male sex determination revealed that the juvenoid pesticide methoprene significantly altered sex ratios of offspring in favor of males. This effect was confirmed with two additional juvenoids (methyl farnesoate and pyriproxyfen), but not in several other chemicals that elicit toxicity through various differing mechanisms. We concluded that the crustacean juvenoid hormone methyl farnesoate is the endogenous male-sex determining hormone in daphnids and environmental juvenoids (methoprene, pyriproxyfen) have the ability to disrupt sex ratios in daphnid populations by acting as methyl farnesoate agonists. Sexually ambiguous offspring (i.e., pseudohermaphrodites) were sometimes produced during exposure of maternal daphnids to the juvenoids, though the frequency was too low to ascribe any significance to the occurrences.

Results demonstrated that testosterone homeostasis is maintained in the snail by fatty acid esterification. High total testosterone levels result in increased esterification and low total testosterone levels result in decreased esterification. Tributyltin interferes with the regulation of the esterification process by total testosterone resulting in generally elevated free testosterone levels and disruptions in seasonal surges in free testosterone.

Data have provided insight into the regulation of testosterone homeostasis in the mud snail. The elucidation of major processes that contribute to the maintenance of testosterone homeostasis in this species represents a significant advancement in understanding the basic endocrinology of gastropods. Elucidation of the effect of tributyltin on normal testosterone homeostasis in snails has led to an increased mechanistic understanding of tributyltin as an endocrine disrupting compound in invertebrates. Demonstration of the inimical effects of tributyltin in laboratories has contributed to a ban on tributyltin-based paints that was ratified by the International Maritime Organization, effective January 1, 2003. Our observations have identified a target of tributyltin on testosterone regulation in the mud snail. More information on the precise mechanism of imposex remains elucidated and this understanding is necessary to assure that antifoulants can be developed in the future that do not have adverse impacts on nontarget organisms. Testosterone biotransformation in normal snails is a unique process and tributyltin modulation of this process has been demonstrated through this research project.

Dahpnids. Environmental chemicals interfered with two hormonal regulatory pathways in the crustacean Daphnia magna that would reduce fecundity of exposed populations. Ecdysteroid hormones were critical to normal crustacean embryo development, and several chemicals had antiecdysteroidal activity. Among the chemicals shown to have antiecdysteroidal activity were vertebrate androgen receptor agonists (testosterone, androstenedione), and steroid synthesis inhibitors (propiconazole, fenarimol). Other environmental chemicals (4-nonylphenol, bisphenol A) also were shown to exhibit apparent antiecdysteroid activity, though the mechanism of activity has not yet been established. Juvenoid hormones were male sex determinants in daphnids. Methyl farnesoate is the putative endogenous male sex determining hormone. We hope to evaluate changes in endogenous methyl farnesoate levels in maternal organisms in association with the production of male offspring. We also have determined in recent experiments that methyl farnesoate elevates hemoglobin levels in daphnids, and we are using the well characterized daphnid hemoglobin genes to elucidate the molecular mechanisms by which juvenoid hormones regulate gene activity.

Finally, environmental juvenoids, which have widespread application as insecticides, had significant effects on sex ratios of daphnid offspring. Concentrations of pyriproxyfen were evaluated that elicited no adverse effect on maternal organisms, but caused them to produce only male offspring. Clearly, such effects would have devastating consequences to similarly exposed field populations. This organizational effect of pyriproxyfen occurred at many orders of magnitude below the exposure levels known to be acutely toxic to daphnids or elicit effects to other species. We hope to evaluate other environmental juvenoids for such organizational toxicity and also evaluate common environmental pollutants for their ability to interfere with juvenoid activity resulting in male deficient populations.

In addition to the individual effects of the antiecdysteroids and juvenoids on crustacean fecundity, we observed several instances of synergistic interactions among the chemicals that we evaluated. Future studies will further elucidate such synergism, particularly between antiecdysteroidal chemicals and juvenoid chemicals. Research suggests that interactions occur between chemicals of these two mechanistic classes that would result in significantly impaired reproductive capacity of exposed crustacean populations.


Journal Articles on this Report : 25 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 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. R826129 (Final)
R826130 (1999)
  • Abstract from PubMed
  • Abstract: ScienceDirect
    Exit
  • 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 Gooding MP, Wilson VS, Folmar LC, Marcovich DT, LeBlanc GA. The biocide tributyltin reduces the accumulation of testosterone as fatty acid esters in the mud snail (Ilyanassa obsoleta). Environmental Health Perspectives 2003;111(4):426-430. R826129 (Final)
    not available
    Journal Article Gooding MP, LeBlanc GA. Seasonal variation in the regulation of testosterone levels in the eastern mud snail (Ilyanassa obsoleta). Invertebrate Biology 2004;123(3):237-243 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 LeBlanc GA. Crustacean endocrine toxicology: a review. Ecotoxicology 2007;16(1):61-81. R826129 (Final)
    R831300 (Final)
    R832739 (2007)
    R832739 (Final)
  • Abstract from PubMed
  • Abstract: Springer-Abstract
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  • Other: ResearchGate-Abstract
    Exit
  • Journal Article LeBlanc GA, McLachlan JB. Molt-independent growth inhibition of Daphnia magna by an anti-androgen. Environmental Toxicology and Chemistry 1999;18:1450?1455. R826129 (1999)
    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 Mu XY, LeBlanc GA. Cross communication between signaling pathways: Juvenoid hormones modulate ecdysteroid activity in a crustacean. Journal of Experimental Zoology Part A-Comparative Experimental Biology 2004;301A(10):793-801. R826129 (Final)
    R829358 (2004)
    R829358 (Final)
    R831300 (2004)
    not available
    Journal Article Mu XY, LeBlanc GA. Synergistic interaction of endocrine - disrupting chemicals: Model development using an ecdysone receptor antagonist and a hormone synthesis inhibitor. Environmental Toxicology and Chemistry 2004;23(4):1085-1091. R826129 (Final)
    R829358 (2003)
    R829358 (2004)
    R829358 (Final)
    not available
    Journal Article Mu XY, LeBlanc GA. Developmental toxicity of testosterone in the crustacean Daphnia magna involves anti-ecdysteroidal activity. General and Comparative Endocrinology 2002;129(2):127-133. R826129 (Final)
    R829358 (2003)
    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. 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 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 Olmstead AW, LeBlanc GA. Juvenoid hormone methyl farnesoate is a sex determinant in the crustacean Daphnia magna. Journal of Experimental Zoology 2002;293(7):736-739. R826129 (Final)
    R829358 (2003)
    not available
    Journal Article Olmstead AW, LeBlanc GA. Insecticidal juvenile hormone analogs stimulate the production of male offspring in the crustacean Daphnia magna. Environmental Health Perspectives 2003;111(7):919-924. R826129 (Final)
    R829358 (2003)
    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 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
    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:

    screening tests, ecological effects, sentinels, toxicity, ecosystem, environmental exposure, health, pesticides, adverse outcomes, animal models, biochemistry, bioindicator, biological effects, chemical mixtures, ecological effects, ecological exposure, exposure studies, hormone production, invertebrates, metabolic androgenization, invertebrates, natural hormones, propiconazole, reproductive processes, snails., 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, adverse outcomes, ecological effects, metabolic androgenization, natural hormones, bioindicator, ecological exposure, propiconazole, metabolic androgenization of invertebrates, exposure studies, chemical mixtures, trbutyltin, animal models, toxicity, reproductive processes, biochemistry, invertebrates, biological effects, hormone production

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    1999 Progress Report
    2000 Progress Report
    2001 Progress Report

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