Publications (54) View all
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Article: A transgenerational endocrine signaling pathway in crustacea.
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ABSTRACT: BACKGROUND: Environmental signals to maternal organisms can result in developmental alterations in progeny. One such example is environmental sex determination in Branchiopod crustaceans. We previously demonstrated that the hormone methyl farnesoate could orchestrate environmental sex determination in the early embryo to the male phenotype. Presently, we identify a transcription factor that is activated by methyl farnesoate and explore the extent and significance of this transgenerational signaling pathway. METHODOLOGYPRINCIPAL FINDINGS: Several candidate transcription factors were cloned from the water flea Daphnia pulex and evaluated for activation by methyl farnesoate. One of the factors evaluated, the complex of two bHLH-PAS proteins, dappuMet and SRC, activated a reporter gene in response to methyl farnesoate. Several juvenoid compounds were definitively evaluated for their ability to activate this receptor complex (methyl farnesoate receptor, MfR) in vitro and stimulate male sex determination in vivo. Potency to activate the MfR correlated to potency to stimulate male sex determination of offspring (pyriproxyfen>methyl farnesoate>methoprene, kinoprene). Daphnids were exposed to concentrations of pyriproxyfen and physiologic responses determined over multiple generations. Survivial, growth, and sex of maternal organisms were not affected by pyriproxyfen exposure. Sex ratio among offspring (generation 2) were increasingly skewed in favor of males with increasing pyriproxyfen concentration; while, the number of offspring per brood was progressively reduced. Female generation 2 daphnids were reared to reproductive maturity in the absence of pyriproxyfen. Sex ratios of offspring (generation 3) were not affected in this pyriproxyfen lineage, however, the number of offspring per brood, again, was significantly reduced. CONCLUSIONS: Results reveal likely components to a hormone/receptor signaling pathway in a crustacean that orchestrates transgenerational modifications to important population metrics (sex ratios, fecundity of females). A model is provided that describes how these signaling processes can facilitate population sustainability under normal conditions or threaten sustainability when perturbed by environmental chemicals.PLoS ONE 01/2013; 8(4):e61715. · 4.09 Impact Factor -
Article: Tributyltin synergizes with 20-hydroxyecdysone to produce endocrine toxicity.
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ABSTRACT: One of the great challenges facing modern toxicology is in predicting the hazard associated with chemical mixtures. The development of effective means of predicting the toxicity of chemical mixtures requires an understanding of how chemicals interact to produce nonadditive outcomes (e.g., synergy). We hypothesized that tributyltin would elicit toxicity in daphnids (Daphnia magna) by exaggerating physiological responses to 20-hydroxyecdysone signaling via synergistic activation of the retinoid X receptor (RXR):ecdysteroid receptor (EcR) complex. Using reporter gene assays, we demonstrated that RXR, alone, is activated by a variety of ligands including tributyltin, whereas RXR:EcR heterodimers were not activated by tributyltin. However, tributyltin, in combination with the daphnid EcR ligand 20-hydroxyecdysone, caused concentration-dependent, synergistic activation of the RXR:EcR reporter. Electrophoretic mobility shift assays revealed that tributyltin did not enhance the activity of 20-hydroxyecdysone by increasing binding of the receptor complex to a DR-4 DNA-binding site. Exposure of daphnids to elevated concentrations of 20-hydroxyecdysone caused premature and incomplete ecdysis resulting in death. Tributyltin exaggerated this effect of exogenous 20-hydroxyecdysone. Further, exposure of daphnids to tributyltin enhanced the inductive effects of 20-hydroxyecdysone on expression of the 20-hydroxyecdysone-inducible gene HR3. Continuous, prolonged exposure of maternal daphnids to concentrations of tributyltin resulted in mortality concurrent with molting. Taken together, these results demonstrate that xenobiotics, such as tributyltin, can interact with RXR to influence gene expression regulated by the heterodimeric partner to RXR. The result of such interactions can be toxicity due to inappropriate or exaggerated hormonal signaling. The application of the in vitro/in vivo approach used in this study is discussed in relation to modeling of nonadditive interactions among constituents of chemical mixtures.Toxicological Sciences 06/2011; 123(1):71-9. · 4.65 Impact Factor -
Article: Regulation and dysregulation of vitellogenin mRNA accumulation in daphnids (Daphnia magna).
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ABSTRACT: The induction of vitellogenin in oviparous vertebrates has become the gold standard biomarker of exposure to estrogenic chemicals in the environment. This biomarker of estrogen exposure also has been used in arthropods, however, little is known of the factors that regulate the expression of vitellogenin in these organisms. We investigated changes in accumulation of mRNA products of the vitellogenin gene Vtg2 in daphnids (Daphnia magna) exposed to a diverse array of chemicals. We further evaluated the involvement of hormonal factors in the regulation of vitellogenin expression that may be targets of xenobiotic chemicals. Expression of the Vtg2 gene was highly responsive to exposure to various chemicals with an expression range spanning approximately four orders of magnitude. Chemicals causing the greatest induction were piperonyl butoxide, chlordane, 4-nonylphenol, cadmium, and chloroform. Among these, only 4-nonylphenol is recognized to be estrogenic. Exposure to several chemicals also suppressed Vtg2 mRNA levels, as much as 100-fold. Suppressive chemicals included cyproterone acetate, acetone, triclosan, and atrazine. Exposure to the estrogens diethylstilbestrol and bisphenol A had little effect on vitellogenin mRNA levels further substantiating that these genes are not induced by estrogen exposure. Exposure to the potent ecdysteroids 20-hydroxyecdysone and ponasterone A revealed that Vtg2 was subject to strong suppressive control by these hormones. Vtg2 mRNA levels were not significantly affected from exposure to several juvenoid hormones. Results indicate that ecdysteroids are suppressors of vitellogenin gene expression and that vitellogenin mRNA levels can be elevated or suppressed in daphnids by xenobiotics that elicit antiecdysteroidal or ecdysteroidal activity, respectively. Importantly, daphnid Vtg2 is not elevated in response to estrogenic activity.Aquatic toxicology (Amsterdam, Netherlands) 01/2011; 101(2):351-7. · 3.12 Impact Factor -
Article: Interactions of the crustacean nuclear receptors HR3 and E75 in the regulation of gene transcription.
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ABSTRACT: Endocrine signal transduction occurs through cascades that involve the action of both ligand-dependent and ligand-independent nuclear receptors. In insects, two such nuclear receptors are HR3 and E75 that interact to transduce signals initiated by ecdysteroids. We have cloned these nuclear receptors from the crustacean Daphnia pulex to assess their function as regulators of gene transcription in this ecologically and economically important group of organisms. Both nuclear receptors from D. pulex (DappuHR3 (group NR1F) and DappuE75 (group NR1D)) exhibit a high degree of sequence similarity to other NR1F and NR1D group members that is indicative of monomeric binding to the RORE (retinoid orphan receptor element). DappuE75 possesses key amino acid residues required for heme binding to the ligand-binding domain. Next, we developed a gene transcription reporter assay containing a luciferase reporter gene driven by the RORE. DappuHR3, but not DappuE75, activated transcription of the luciferase gene in this system. Co-transfection experiments revealed that DappuE75 suppressed DappuHR3-dependent luciferase transcription in a dose-dependent manner. Electrophoretic mobility shift assays confirmed that DappuHR3 bound to the RORE. However, we found no evidence that DappuE75 similarly bound to the response element. These experiments further demonstrated that DappuE75 prevented DappuHR3 from binding to the response element. In conclusion, DappuHR3 functions as a transcriptional activator of genes regulated by the RORE and DappuE75 is a negative regulator of this activity. DappuE75 does not suppress the action of DappuHR3 by occupying the response element but presumably interacts directly with the DappuHR3 protein. Taken together with the previous demonstration that daphnid HR3 is highly induced by 20-hydroxyecdysone, these results support the premise that HR3 is a major component of ecdysteroid signaling in some crustaceans and is under the negative regulatory control of E75.General and Comparative Endocrinology 03/2010; 167(2):268-78. · 3.27 Impact Factor -
Article: Intracellular conversion of environmental nitrate and nitrite to nitric oxide with resulting developmental toxicity to the crustacean Daphnia magna.
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ABSTRACT: Nitrate and nitrite (jointly referred to herein as NO(x)) are ubiquitous environmental contaminants to which aquatic organisms are at particularly high risk of exposure. We tested the hypothesis that NO(x) undergo intracellular conversion to the potent signaling molecule nitric oxide resulting in the disruption of endocrine-regulated processes. These experiments were performed with insect cells (Drosophila S2) and whole organisms Daphnia magna. We first evaluated the ability of cells to convert nitrate (NO(3)(-)) and nitrite (NO(2)(-)) to nitric oxide using amperometric real-time nitric oxide detection. Both NO(3)(-) and NO(2)(-) were converted to nitric oxide in a substrate concentration-dependent manner. Further, nitric oxide trapping and fluorescent visualization studies revealed that perinatal daphnids readily convert NO(2)(-) to nitric oxide. Next, daphnids were continuously exposed to concentrations of the nitric oxide-donor sodium nitroprusside (positive control) and to concentrations of NO(3)(-) and NO(2)(-). All three compounds interfered with normal embryo development and reduced daphnid fecundity. Developmental abnormalities were characteristic of those elicited by compounds that interfere with ecdysteroid signaling. However, no compelling evidence was generated to indicate that nitric oxide reduced ecdysteroid titers. Results demonstrate that nitrite elicits developmental and reproductive toxicity at environmentally relevant concentrations due likely to its intracellular conversion to nitric oxide.PLoS ONE 01/2010; 5(8):e12453. · 4.09 Impact Factor
