Anti-Ecdysteroidal Activity and Associated Toxicity of Environmental Chemicals in the Crustacean Daphnia magna.

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Title: Anti-Ecdysteroidal Activity and Associated Toxicity of Environmental Chemicals in the Crustacean Daphnia magna.
Author: Mu, Xueyan
Advisors: Dr. Gerald A. LeBlanc, Committee Chair
Abstract: The focus of this research was to characterize potential mechanisms by which environmental chemicals may disrupt ecdysteroid-regulated processes in crustaceans and the consequences of these perturbations. Several chemicals that are likely to elicit anti-ecdysteroidal activities through different mechanisms were selected and used to establish mechanisms of anti-ecdysteroidal activity. The fungicide fenarimol was used to modulate ecdysone levels and evaluate the role of ecdysteroids in crustacean embryo development. Fenarimol lowered endogenous ecdysone levels, delayed molting, interfered with embryo development, and reduced fecundity in the crustacean Daphnia magna. The molting effect and embryotoxicity of fenarimol were abrogated by co-exposure to exogenous 20-hydroxyecdysone. These experiments demonstrated that ecdysteroids are critical to normal embryo development and chemicals that lower ecdysteroid levels induce developmental abnormality and adversely impact fecundity. In chapter two, we tested the hypothesis that the known developmental toxicity of testosterone is due to anti-ecdysteroidal activity. Two ecdysteroid-regulated processes in daphnids' molting and embryo development were disrupted by testosterone. These disruptions were mitigated by co-exposure to 20-hydroxyecdysone. However, unlike fenarimol, testosterone had no discernable effect on endogenous ecdysone levels. Testosterone significantly antagonized the action of 20-hydroxyecdysone in an ecdysone-responsive cell line. These results demonstrated that ecdysteroid receptor antagonism could be one mechanism by which testosterone elicits embryo toxicity. Based upon results of the first two studies, we concluded that fenarimol is an anti-ecdysteroid by reducing endogenous ecdysone levels and testosterone elicits anti-ecdysteroidal activity by functioning as ecdysteroid receptor antagonist. We next hypothesized that combined exposure to fenarimol and testosterone would result in greater than additive toxicity due to their different but interactive modes of action. Effects of fenarimol and testosterone on ex vivo embryo development were evaluated independently and in combination in order to detect and quantify any synergistic interactions. Synergy was observed between the two compounds. A mathematical model was developed called 'independent joint action with fenarimol-on-testosterone synergy' (IJA+SYN) that was highly predictive of the experimentally-determined combined effects of the two compounds. In chapter four, we investigated the possibility that juvenoid hormones can regulate the ecdysteroid activity in daphnids in an antagonistic fashion. Juvenoids disrupted ecdysteroid-regulated embryo development in daphnids. Juvenoids also antagonized the effects of 20-hydroxyecdysone on cell proliferation and gene expression of the ecdysone receptor and its partner protein ultraspiracle in a cultured cell line. While juvenoids functioned as anti-ecdysteroids both in vivo and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. We concluded that the ability of juvenoids to block ecdysteroid-mediated induction of the ecdysone receptor and ultraspiracle could partially explain the anti-ecdysteroidal activities of these compounds. However, demonstrated synergistic interactions between a juvenoid and fenarimol could not be explained based upon this mode of activity. We concluded additionally, unidirectional properties of juvenoids contribute to their anti-ecdysteroidal activity. Having identified and characterized several mechanisms by which chemicals can elicit anti-ecdysteroidal activity, we lastly investigated the mechanism by which the environmental contaminant bisphenol A elicits toxicity. We discovered that bisphenol A elicit juvenoid-like activity and likely interfere with ecdysteroid regulated processes via this activity. A definitive assessment revealed that bisphenol A is chronically toxic to daphnids, probably through its anti-ecdysteroidal/juvenoid activity. However, effects are elicited at levels that are not likely to pose environmental concern. Overall, results of this study provide conclusive evidence that diverse environmental chemicals can elicit anti-ecdysteroidal activity in crustaceans through several mechanisms and result in developmental abnormalities and reduced fecundity. Such effects of environmental contaminants should be considered when evaluating causes of population decline among economically-important crustacean populations.
Date: 2004-02-03
Degree: PhD
Discipline: Toxicology

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