Endocrine toxicants can interfere with hormone signaling through various mechanisms. Some of these mechanisms are interrelated in a manner that might result in synergistic interactions. Here we tested the hypothesis that combined exposure to chemicals that inhibit hormone synthesis and that function as hormone receptor antagonists would result in greater-than-additive toxicity. This hypothesis was tested by assessing the effects of the ecdysteroid-synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone on ecdysteroid-regulated development in the crustacean Daphnia magna. Both compounds were individually characterized for effects on the development of isolated embryos. Fenarimol caused late developmental abnormalities, consistent with its effect on offspring-derived ecdysone in the maturing embryo. Testosterone interfered with both early and late development of embryos, consistent with its ability to inhibit ecdysone provided by maternal transfer (responsible for early developmental events) or de novo ecdysone synthesis (responsible for late developmental events). We predicted that, by decreasing endogenous levels of hormone, fenarimol would enhance the likelihood of testosterone binding to and inhibiting the ecdysone receptor. Indeed, fenarimol enhanced the toxicity of testosterone, while testosterone had no effect on the toxicity of fenarimol. Algorithms were developed to predict the toxicity of combinations of these two compounds based on independent joint action (IJA) alone as well as IJA with fenarimol-on-testosterone synergy (IJA+SYN). The IJA+SYN model was highly predictive of the experimentally determined combined effects of the two compounds. These results demonstrate that some endocrine toxicants can synergize, and this synergy can be accurately predicted.

The role of ecdysteroids in crustacean embryo development and the susceptibility of the developing embryo to the antiecdysteroidal properties of an environmental chemical were evaluated. The agricultural fungicide fenarimol was shown to exhibit antiecdysteroidal activity to the crustacean Daphnia magna by lowering endogenous ecdysone levels and delaying molting in a concentration-dependent fashion that was mitigated by co-exposure to exogenous 20-hydroxyecdysone. Exposure of either gravid maternal organisms or isolated embryos to fenarimol resulted in embryo abnormalities ranging from early partial developmental arrest to incomplete development of antennae and shell spines. Developmental abnormalities were associated with suppressed ecdysone levels in the embryos and the abnormalities could be prevented by co-exposure to 20-hydroxyecdysone. Developmental abnormalities caused by the antiecdysteroid were associated with reduced fecundity of the parental organisms. These results demonstrate that ecdysteroids are critical to normal crustacean embryo development and environmental antiecdysteroids can disrupt normal embryo development and compromise the production of viable offspring. Antiecdysteroidal activity may provide a means by which environmental chemicals impact crustacean species while not affecting vertebrates.

Methyl farnesoate is a juvenoid hormone that regulates a variety of processes in crustaceans including male sex determination among daphnids (Branchiopoda, Cladocera). The synthetic juvenoids pyriproxyfen and fenoxycarb mimic the action of methyl farnesoate in daphnids. In the present study we tested the hypothesis that juvenoids also can regulate ecdysteroid activity in a crustacean (Daphnia magna). Methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. Exposure of ecdysteroid-responsive cells to 20-hydroxyecdysone reduced cell proliferation and increased mRNA levels of the ecdysone receptor and its partner protein ultraspiracle. Co-treatment of cells with the juvenoid pyriproxyfen attenuated all of these ecdysteroid mediated responses. While juvenoids functioned as anti-ecdysteroids in both intact embryos and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. The combined effects of pyroproxyfen with the ecdysteroid synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone were evaluated in an effort to discern whether the action of the juvenoids were additive with those of know anti-ecdysteroids. The anti-ecdysteroid effects of pyriproxyfen were non-additive with those of either anti-ecdysteroid. Rather, joint effects conformed to a model of synergy. These results demonstrated that juvenoids elicit anti-ecdysteroidal activity in a crustacean through a unique mechanism of action. A model involving receptor partner deprivation is proposed that explains the synergistic interactions observed.

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Bisphenol A is a key industrial chemical used in the manufacture of polycarbonate plastics and other products. Several recent reports ascribe toxicological properties to this compound that have been attributed to the disruption of endocrine-related processes. In the present study, the toxicity of bisphenol A was definitively characterized in the water flea (Daphnia magna) in an effort to discern whether this compound may elicit endocrine toxicity in an invertebrate species and to establish the mechanism by which this toxicity is elicited. The ability of bisphenol A to interfere with two ecdysteroid-dependent physiological processes--molting and embryonic development--was evaluated. Bisphenol A elicited antiecdysteroidal activity as indicated by its prolongation of the intermolt period and interference with embryonic development. This apparent antiecdysteroidal activity was not due to reduced availability of endogenous ecdysteroid nor due to ecdysteroid-receptor antagonism. The ability of bisphenol A to elicit antiecdysteroidal activity by functioning as a juvenoid hormone was next evaluated. Bisphenol A, alone, did not elicit juvenoid activity. However, bisphenol A did enhance the activity of the crustacean juvenoid hormone methyl farnesoate. A definitive assessment of the effects of bisphenol A on the reproductive capacity of daphnids revealed a concentration-response relationship that extended at least one order of magnitude below exposure levels that were overtly toxic to the maternal organisms. These results demonstrate that bisphenol A is chronically toxic to daphnids, probably through its ability to interfere with ecdysteroid/juvenoid regulated processes. However, effects are elicited at levels that are not likely to pose environmental concern.